Opioid Peptides:

Medicinal Chemistry

DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Alcohol, Drug Abuse, and Mental Health Administration
Opioid Peptides:

Medicinal Chemistry

Editors:

Rao S. Rapaka, Ph.D.

Gene Barnett, Ph.D.

Richard L. Hawks, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 69

1986

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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NIDA Research Monographs are prepared by the research divisions of the

National Institute on Drug Abuse and published by its Office of Science. The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and

integrative research reviews. Its dual publication emphasis is rapid and targeted

dissemination to the scientific and professional community.

Editorial Advisors

MARTIN W. ADLER, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic lnstitute

Troy, New York

RICHARD E. BELLEVILLE, Ph.D.

NB Associates, Health Sciences

Rockville, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

WashIngton, DC

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph.D.

The Johns Hopkins University School of

Medicine

Baltimore, Maryland

THEODORE J. CICERO, Ph.D.

Washington Universitv School of

Medicine

St Louis, Missouri

SIDNEY COHEN, M.D.

Los Angeles, California

MARY L. JACOBSON

National Federation of Parents for

Drug Free Youth

Omaha, Nebraska

REESE T. JONES, M.D.

Langley Porter Neuropsychiatric lnstitute

San Francisco, California

DENISE KANDEL, Ph.D.

College of Physicians and Surgeons of

Columbia University

New York, New York

HERBERT KLEBER, M.D.

Yale University School of Medicine

New Haven, Connecticut

RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

CHARLES R. SCHUSTER, Ph.D.

Director. NIDA

JEAN PAUL SMITH, Ph.D.

Acting Associate Director for Science, NIDA

Acting Editor

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Opioid Peptides:

Medicinal Chemistry
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the

technical review on the medicinal chemistry and molecular

pharmacology of opioid peptides and the opiates which took place on

September 4 - 6, 1984. at Bethesda. Maryland. The meeting was

sponsored by the Office of Science and the Division of Preclinical

Research, National Institute on Drug Abuse. The papers on medicinal

chemistry are presented in this volume; those on the molecular

pharmacology, biosynthesis, and analysis of opioid peptides appear

in NIDA Research Monograph 70.

COPYRIGHT STATUS

The diagram on the cover of this volume is reprinted by permission

from Science, Vol. 199. p. 1214, copyright 1978, The American

Association for the Advancement of Science. The National Institute

on Drug Abuse has also obtained permission from the copyright

holders to reproduce certain previously published material as noted

in the text. Further reproduction of this copyrighted material is

permitted only as part of a reprinting of the entire publication or

chapter. For any other use, the copyright holder's permission is

required. All other material in this volume except quoted passages

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reproduced without permission from the Institute or the authors.

Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the

National Institute on Drug Abuse or any other part of the U.S.

Department of Health and Human Services.

DHHS Publication No. (ADM)90-1454

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1986, Reprinted 1987, 1990

NIDA Research Monographs are indexed in the Index Medicus They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Foreword

The search for the molecular basis of drug addiction led, in the

mid-seventies, to the exciting discovery of opioid receptors and a

variety of endogenous and exogenous ligands. The fact that

thousands of publications have appeared already is astounding and

attests to the attention attracted by this area. The discovery of

multiple opioid receptors brings an additional dimension to research

in this area, and medicinal chemists have already succeeded in

synthesizing many highly specific ligands to these receptor subtypes.

The interest in this area is not only to develop nonaddictive

peptide analgetic substitutes for morphine, but also to understand

the role of these endogenous peptides in health and in disease.

Although several thousand analogs of enkephalins and other opioid

peptides have been synthesized, including some that are stable and

can be taken orally, a nonaddicting opioid has yet to be synthesized.

Hence, the National Institute on Drug Abuse felt that it was timely

to call together experts in the area to evaluate current research on

the development of new opioid peptides with potential value for

basic research and therapeutics.

In this monograph, emphasis is placed on structure-activity

relationships, conformational analysis using various spectroscopies,

computer assisted drug design, and molecular mechanics. It is hoped

that the discussions of current research by various experts

presented here will helpful in establishing future goals for

development of new molecular entities with desired biological

activity or receptor specificity. An effort has been made to cover

all aspects of medicinal chemistry pertaining to opioid peptides, so

that the volume can serve as a broadly useful reference text.

Marvin Snyder, Ph.D., Director

Division of Preclinical Research

National Institute on Drug Abuse

v

Contents

Foreword

Marvin Snyder v

Introduction

Rao S. Rapaka 1

Frontiers of Research in the Medicinal Chemistry and

Molecular Pharmacology of Opioid Peptides

Rao S. Rapaka 3

Synthesis of Opioid Peptide Analogs

John M. Stewart 21

Structure-Activity Relationships of Opioid Peptides

J. S. Morley and A. S. Dutta 4 2

Design Principles: Enkephalins With Predictable Mu/Delta

Receptor Specificity

Yasuyuki Shimohigashi 6 5

Structure-Activity Relationship and Pharmacology of the

Highly Selective μ-Opioid Agonist, Morphiceptin

Kwen-Jen Chang 1O1

Dermorphin: Autonomic Pharmacology and Structure-Activity

Relationships

Giora Feuerstein 112

Design of Conformationally Constrained Cyclic Peptides

With High Delta and Mu Opioid Receptor Specificities

Victor J. Hruby 128

Dehydro and Cyclopropyl Amino Acids and Peptides

Charles H. Stammer 148

Affinity Labels as Probes for Opioid Receptor Types and Subtypes

Phillip S. Portoghese and A. E. Takemori 157

Peptide Conformation and Biological Activity:

A General Review with Examples of Approaches Relevant

to Opioid Peptide Function

Dan W. Urry 169

vii
Opioid Peptides: Analysis of Specificity and Multiple

Binding Modes Through Computer-Aided Drug Design and

Structure-Activity Studies

Raoul D. Nelson; David I. Gottlieb; T. M. Balasubramanian;

and Garland R. Marshall 204

Mechanistic Structure-Activity Studies of Peptide and

Nonpeptide Flexible Opioids: An Interdisciplinary Approach

Gilda H. Loew; Lawrence Toll; Edward Uyeno; Alice Cheng;

Amrit Judd; John Lawson; Christopher Keys; Peter Amsterdam;

and Wilma Polgar 231

Conformations of Opioid Peptides as Determined by Nuclear

Magnetic Resonance and Related Spectroscopies

M. Abu Khaled 266

Conformational Analysis of Opioid Peptides and the Use of

Conformational Restriction in the Design of Selective Analogs

Peter W. Schiller 291

Stereochemically Constrained Enkephalin Analogs Containing

X-Aminoisobutyric Acid and 1-Amino-Cyclopentane-l-Carbolylic

Acid

Raghuvansh Kishore and Patinabhan Balaram 312

Enkephalin Conformation in Solution: A Perspective From

Vibrational Spectroscopic Studies

V. Renugopalakrishnan; T. W. Collette; L. A. Carreira;

and R. S. Rapaka 332

Conformational Features of the Opioid Peptides in the Solid

State: A Review of X-Ray Crystallographic Research

Arthur Camerman and Norman Camerman 351

The Search for Nonaddicting Strong Analgesics: Hopes and

Frustrations

Sydney Archer 364

viii
Introduction

Rao S. Rapaka, Ph.D.

The discovery of opioid peptides has opened up new scientific area

and posed questions which are still at an early stage of

exploration. This area is of major interest to the immediate and

long-range goals of the National Institute on Drug Abuse, and in

order better to focus the rapidly expanding research areas

associated with peptide chemistry and its potential application to

drug abuse research and therapeutic treatment, the Institute

sponsored a technical review in September 1984.

This monograph presents contributions both from the symposium

speakers and from other invited authors in the various aspects of

the medicinal chemistry of the opioid peptides and the opiates.

Highlights of these reviews are presented here. Biosynthetic,

analytical, and molecular pharmacology aspects are presented in NIDA

Research Monograph 70.

A number of presentations are on structure-activity relationships

(SAR) of the opioid peptides--Drs. Morley and Dutta on the SAR of

enkephalins, Dr. Chang on morphiceptins, and Dr. Feuerstein on

dermorphins. Conformational restriction has produced, to date,

highly active μ- and -selective enkephalin analogs. This feature

is described extensively by Drs. Schiller and Hruby. In addition,

conformational restriction by stereochemically constrained analogs

is described by Drs. Kishore and Balaram. Very recent work on the

synthesis of dehydro and cyclopropyl amino acids is presented by Dr.

Stammer; and a general review on enkephalin SAR, including the

design of peptides with predictable specificity and the

synthesis of enzymatically stable peptides, is presented by Dr.

Shimohigashi. An excellent and elaborate account of peptide

conformation and biological activity, with general examples from

such peptides as gramicidin analogs and elastin analogs, is given by

Dr. Urry.

A number of presentations are related to conformational analysis of

the opioid peptides, advanced methodologies, and design of

peptides. Nuclear magnetic resonance as a tool in determination of

conformations, along with a preliminary background on peptide

conformations (such as -turns and -sheets), 2-D NMR, and NOE, is

1
presented by Dr. Khaled. An update on X-ray crystallographic

research on enkephalins and enkephalin analogs, such as [p-bromo

Phe4]-leucine enkephalin and Tyr-D-Nle-Gly-Phe-NleS, is presented

bv Drs. Camerman and Camerman. Applications of the comolementarv

techniques FT-IR and laser Rama spectros copies to the determination

of conformational states of Leu5- and Met5- enkephalins are

described by Dr. Renugopalakrishnan and colleagues. An account on

computer-aided drug design is given by Dr. Marshall and colleagues,

and an investigation on SAR of opioids by an interdisciplinary

approach is discussed by Dr. Loew and her associates. An excellent

account of conformational analysis and conformational restriction in

the design of receptor-selective analogs is given by Dr. Schiller.

Great progress in research on opioid peptides has been made possible

by simultaneous advances in the techniques of synthesis,

purification, and analysis of peptides. In this volume, synthetic

techniques are discussed by Dr. Stewart.

In this monograph, one approach to understanding receptor types, by

affinity labels, is discussed by Drs. Portoghese and Takemori.

Receptor types and analysis of binding data are dicussed at greater

length in NIDA Research Monograph 70.

A review of the historical search for a nonaddicting

analgesic--starting from days of opium use to the development of

agonists, antagonists, and the advent of opioid peptides--and of

future hopes is presented by Dr. Archer.

Through the interdisciplinary exchanges that took place at this

conference and the resulting volumes, an effort has been made to

summarize the existing information in the area of opioid peptides

and to discuss the implications of that information for future

research objectives.

2
Frontiers of Research in the Medicinal

Chemistry and Molecular Pharmacology of

Opioid Peptides

Rao S. Rapaka, Ph.D.

The conquest of pain has been long sought by monks, medical

practitioners, quacks, and laymen, alike, and each of them has

attempted to achieve their goal in their own way. Opium was

used for centuries by the ancient cultures for its analgetic

effects. In 1806, Serturner discovered the analgetic effects

of morphine. However, the side effects of the opiates, such as

addiction liability and respiratory depression, were soon

realized. This further motivated research for other compounds,

either from naturally occurring plant drugs or synthetic

sources. A number of useful compounds--such as meperidine,

methadone, d-propoxyphene, nalorphine, phenazocine, etc.--were

synthesized. However, the goal to discover an ideal analgetic

without the undesirable side effects has not been achieved. In

the meantime, advances in the neurosciences, endocrinology,

protein synthesis, molecular biology, and other sciences have

resulted in a better understanding of brain function and its

modulation. The characterization of opiate receptors (Pert and

Snyder 1973; Simon et al. 1973; Terenius 1973) and later the

isolation and structure determination of the enkephalins (Hughes

et al. 1975) have ignited an explosive interest in the understanding of the role of the endogenous ligands in general, and

their role in brain function in particular. Subsequent

developments in recombinant DNA technology added an important

tool to research on precursors of the endogenous ligands.

The opiate receptor--the site where the opiates interact to

produce the biologic effect--was soon to be called the opioid

receptor, as the endogenous ligands are peptides. A number of

other opioid peptides were soon discovered. Following their

discovery, several hundred synthetic analogs were made and

tested. Extensive pharmacological testing and studies on

binding at the receptor site showed that the opioid receptors

are heterogeneous (Martin et al. 1976) and that at least four

types of receptors exist (Martin et al. 1976; Lord et al.

1977). As information accumulated on opioid peptide families

and their functions, it seemed that it would be useful to

discuss the pertinent questions on the medicinal chemistry and

molecular pharmacology of opioid peptides.

3
Research on opioids is progressing in several directions. Some

of the important topics that have attracted a great deal of

attention by researchers are: biosynthesis, processing of

precursors, endogenous antagonists, specificity in drug design,

conformational restriction in analog design, inhibitors of

enkephalin-degrading enzymes as analgetic drugs, conformational

features of peptides, conformation-receptor selectivity,

retro-inverse analogs, amphiphilic helices, receptor types and

their structure and organization, drug-receptor interaction, the

role of lipids in the interaction, and analytical methodology.

Some of these aspects are reviewed in a preliminary fashion

below. (For additional information, see reviews by Udenfriend

and Kilpatrick 1983; Coplov and Helme 1983; Hughes 1983; Rapaka

et al. 1985.)

Biosynthesis of opioid peptides is being actively investigated.

A number of translational and posttranslational events are

involved in the generation of active peptides, and these

processes occur in a well-defined order. Processing of the

opioid peptides appears to be tissue-specific, and different

sets of processing enzymes seem to be responsible for the

tissue-specific processing of the neuroendocrine peptides.

Pairs of basic amino acids serve as signals for the processing

enzymes. The enzymes appear to be trypsinlike and, quite often,

carboxypeptidase like activity is also involved (Fricker 1985;

Rapaka et al., in press). Other posttranslational modifications

include phosphorylation, acetylation, sulfation, methylation,

glycosylation, amidation, etc.

Processing of precursor peptides to bioactive peptides is

largely explored using synthetic peptides (Marks et al., this

volume; Rapaka et al., in press). In addition to the "dibasic

residues," X-Arg-Pro- appears to be another processing signal as

postulated by Matsuo (NIDA Research Monograph 70), or referred

to as the single arginine cleavage (Weber et al. 1983). This is

exemplified by the generation of metorphamide (Weber et al.

1983) or adrenorphin (Matsuo et al., NIDA Research Monograph

70) from proenkephalin (corresponding to 210-217). Another

example of single arginine cleavage is the generation of

dynorphin(1-8) from prodynorphin corresponding to 209-216

residues). Single arginine cleavage was also postulated for the

processing of dynorphin B-29 to dynorphin B(1-13) at the Thr-Arg

bond (Devi and Goldstein 1984). Here it is to be noted that Arg

is followed by Ser (not Pro).

Another interesting processing event is amidation. The

mechanism of C-terminal amidation was investigated by Bradbury

et al. (1982). C-terminal amidation involves the action of a

specific enzyme, and glycine is a mandatory amino acid in the

C-terminal position. The amide does not arise from amnonia or

by direct amidation or transamidation. The mechanism involves

removal of hydrogen from the C-terminal glycine and spontaneous

hydrolysis of the imino linkage. Amidation is well known for

neurohypophyseal peptides, oxytocin, and vasopressin; and

4
more and more amidated opioid peptides are being isolated.

Metorphamide or adrenorphin Tyr-Gly-Gly-Phe-Met-Arg-ArgVal-NH2 is an amidated peptide. Dermorphin, Tyr-D-Ala-GlyPhe-Tyr-Pro-Ser-NH2, is another amidated peptide and is

isolated from the skin of the South American frog of the

phyllomedusa species (Montecucchi et al. 1981). Recently,

another amidated peptide, amidorphin, was isolated from bovine

adrenal medulla (Seizinger et al. 1983). A very significant

aspect in the isolation of this peptide is that it was logically

deduced to be a processed product of the known bovine proenkephalin A sequence, as it has the necessary processing

signals, including a terminal glycine essential for amidation.

The other significant aspect of the paper is that the expected

product was first synthesized to establish the presence of the

proposed endogenous product.

An important aspect of amidation is its effect on receptor

selectivity. This is reflected in the decreased -receptor

affinity of Met5-enkephalinamide as compared to Met5-

enkephalin. However, amidation of Val-8, as in metorphamide,

causes an increased affinity at the k-receptor site with an

insignificant change at the μ and sites.

Li (NIDA Research Monograph 70) and colleagues have determined

the primary sequence for -endorphins from various species.

They all consist of 31 amino acids with Tyr-Gly-Gly-Phe-Met at

the N-terminal sequence. Although minor differences are noted

in the primary sequence for -endorphins isolated from various

species as compared to the human, it appears that the amino acid

sequence in general is highly conserved. These various

-endorphins isolated from several species, due to minor changes

in the primary sequence, may be considered as the naturally

occurring analogs of the -endorphin. Biological activity of

some of these endorphins was evaluated and it was found that the

ratio of binding activity to analgetic potency varied for these

endorphins. This might be due to the fact that once the peptide

was bound to the receptor, the analgetic potency might depend on

the efficacy of the agonists.

In addition, smaller fragments of -endorphin appear to be

present in the pituitary glands of several animals. In fact,

-endorphin (1-27) was isolated from porcine tissue (Smyth et

al. 1978) and was later synthesized (Zaoral et al. 1981) . This

naturally occurring -endorphin (1-27) inhibits the analgetic

properties of -endorphin. Other synthetic fragments of

-endorphin (1-28) and (1-26) also inhibit analgesia induced

by -endorphin (Nicolas and Li 1985). These observations

have led Li and colleagues to formulate the following hypothesis: "Inhibition of a peptide hormone by a naturally

occurring segment of the same hormone may be a general

phenomenon in biologically active peptides."

After the realization that the opioid receptor is heterogeneous

and that there are at least three types of receptors (μ, and

k) and possibly a fourth type several groups have attempted

5
to understand the features essential for receptor selectivity.

Several thousand analogs of enkephalins were synthesized and

receptor specificity was investigated (see Shimohigashi, this

volume; Fournie-Zaluski et al. 1981; Roques et al. 1982).

Following rational design, -receptor specific enkephalin

analogs, such as H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH (DSLET) and

H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH (DTLET), were developed. To

summarize briefly, the principal characteristics for -receptor

binding affinity for noncyclic (excluding the conformationally

restricted analogs) enkephalin analogs are:

a) hydrophilic side chain in position 2,

b) aromatic residue (4th position),

c) C-terminal carboxyl group in the fifth position, and

d) the lengthening of the peptide chain by Thr6 residue.

Here, it is to be noted that (D-Pen2, D-Pen5)

enkephalin, a conformationally restricted analog, is

the most selective -ligand available (See Hruby, this

volume and references therein).

Characteristics of high specificity for μ-binding sites are,

a) D-residue in the second position,

b) shortening of the enkephalin sequence,

c) removal of the terminal carboxyl group,

d) replacement of Phe4 residue with lipophilic alkyl

chain and stereo-orientation of the Phe4 residue, and

e) N-methylation of Phe4 residue.

Although these modifications produced significantly improved

receptor-specific ligands, alternate approaches were also

attempted.

One of the synthetic approaches that has resulted in the

development of highly receptor-selective ligands is conformational restriction or constriction. For an excellent

minireview on conformational restriction in peptides via amino

acid side chains, see Hruby (1982). Linear peptides are highly

flexible and exist in a conformational equilibrium. Moreover,

the conformational states may be influenced by the solvent and

the surrounding environment. Hence, if one of the various

conformers is the most active form, then dilution of the active

forms can be avoided by reducing the number of inactive forms.

Formation of cyclic peptides is one of the main modes. Some

other structural modifications include N-methylation of the

amide NH, replacement of the -H (to -CH3), formation

of disulphide bonds, and similar side-chain cyclizations. These

conformationally restrained analogs might have increased agonist

or antagonist properties due to enhanced conformational integrity, thus possessing increased specificity and biological

activity. These analogs may have increased enzymatic stability,

as well. This technique has already been utilized for developing analoqs with desired properties in the oxytocin and

bradykinin series. In the opioid peptides, a number of

conformationally restricted analogs were synthesized for the

6
enkephalin peptides. As the integrity of the Tyr residue has to

be maintained at position 1, and as manipulations are

permissible at positions 2 and 5, mast of these analogs were

modified at positions 2 and 5. One of the earliest syntheses

was by Schiller and colleagues using w-diaminobutyric acid

as the second residue and affecting the cyclization of w-amino

group to the C-terminal carboxyl group (DiMaio and Schiller

1980; DiMaio et al. 1982). Similarly, other cyclic peptides

were synthesized utilizing cyclizations between the side chains

of Lys and Orn, with those of Asp and Glu (Schiller et al.

1985). Generally, the cyclic peptides exhibited preference for

μ-receptors over -receptors and were biologically more active,

whereas the corresponding open chain analogs did not show

similar preference or enhanced activity. Another type of

side-chain to side-chain cyclization was attempted using Cys

residues in the 2 and 5 positions.

Using this technique, Hruby and colleagues (this volume)

syntesized a series of analogs. Among these analogs,

[D-Pen2, Cys5]-enkephalin, [D-Pen2, Pen5]-enkephalin and 

[D-Pen2, D-Pen5]-enkephalin are some of the very highly

selective -receptor selective compounds. (D-Pen2,D-Pen5)-

Enkephalin demonstrates beneficial effects of further conformational restriction of gem-dimethyl groups of the Pen moiety.

It also appears that the cyclic analogs and the linear compounds

have similar modes of binding to receptor sites.

Other types of sterically constrained enkephalins are those that

contain -amino isobutyric acid and cyclopentane-1-carboxylic

acid residues (see Kishore and Balaram, this volume).

Another mode of conformational restriction is the synthesis of

peptides incorporating -dehydroamino acids (referred to as

the dehydro- or -amino acids) or the cyclopropyl amino acids

-amino acids). These amino acid substitutions confer unique

chemical and stereochemical properties, as they effect both

chemical reactivity and conformation. For reviews, see Noda et

al. (1983) and Stammer (this volume, 1982). A substitution at

the second position, i.e., -Ala2-enkephalin, facilitated,

interaction at the μ-receptor site (due to enhanced lipophilicity), whereas substitution at 3-position in enkephalin may

have facilitated the interaction at the -receptor site.

Studies on -Phe4-enkephalins indicated that the Z-compound

(phenyl ring and C=O are trans) sterically favors interaction

with the -receptor site (Shimohigashi and Stammer 1983). An

additional advantage of substitution with amino acids is that

peptide linkages with -dehydroamino acid residues are highly

resistant to enzymatic degradation at the C-terminal side and

completely resistant at the N-terminal side (Shimohiqashi et al.

1982). Enkephalin analogs with cyclopropyl amino acid residues

seem to be resistant to enzymatic degradation as well (Stammer,

this volume).

To date, conformational restriction appears to provide the most

successful approach in the design of synthetic analogs with high

7
receptor selectivities.

Another original approach for improving the selectivity of

receptor binding, although less explored, is formation of

dimeric peptides (Shimohigashi, this volume; Shimohigashi et al.

1982; Rodbard et al. 1983). Dimeric penta- and tetra peptide

analogs of enkephalins were shown to exhibit enhanced affinity

and selectivity for the -receptor.

The biological activities of peptides generally depend on both

primary and secondary structure. For some cyclic peptides it

was shown that retro-isomers--in which the direction of the

amide bonds linking the residues is reversed--retain biological

activity. In these cyclic peptides, it may be assumed that

biological activity results from the three dimensional topology

and not from the peptide backbone. The concept of retropeptides has also been extended to linear peptides (Goodnan and

Chorev 1979). To obtain closeness in three-dimensional

structure, a further modification of chirality at each chiral

center was also investigated. These modified analogs are named

"retro-inverso-peptides." Several partially modified retroinverso-peptides were synthesized for a number of biologically

active peptides. These modifications helped in assessing the

relative importance of side-chain versus backbone structure in

conferring biological activity. An additional reason for synthesis of these analogs is the expected resistance to biodegradation. This concept was applied in developing analogs of

enkephalins (Berman et al. 1983), enkephalinase inhibitors such

as retrothiorphan (Roques and Fournie-Zaluski, NIDA Research

Monograph 70), and dermorphin (Salvadori et al. 1985).

Prediction of the formation of certain types of secondary

structures from the amino acid sequences is very helpful in the

rational design of biologically active peptides. The amphiphilic environment of the aqueous solution and lipid bilayar of

the membrane could impose a secondary structure on the peptide.

If the secondary structure formation is initiated by the membrane, it is likely to be characterized by the amphiphilic

distribution of the individual amino acid side chains; amphiphilic helical structures have been hypothesized to be important

in determining the biological activity of a number of peptides.

This approach was successfully utilized in the design of

-endorphin analogs (Blanc et al. 1983). For a review on

amphiphilic secondary structures, see Kaiser and Kezdy (1984).

One of the main problems in the bioassay of the peptides and in

their use as analgetics has been their ease of biodegradation

(NIDA Research Monograph 70: Marks et al.; Shimohigashi; Roques

and Fournie-Zaluski). Enzymes involved in the metabolism of

opioid peptides, as well as in their isolation, assay, and

specificity are described by Marks et al. Enkephalins are

easily metabolized; Tyr1-Gly2 bond is cleaved by

amniopepidases, Gly3-Gly4 bond by enkephalinase, and

Phe4

-Leu5 (Met5) by carboxypeptidases. Although extensive

research has been carried out on these enzymes, the specificity

8
of enzymess and the precise mechanisms of action are still not

established. In addition to the specific cleavage at a

particular amide bond, sequential processing has also been

proposed as an alternate mechanism of action (Fricker 1985;

Mains et al. 1983; Rapaka et al. in press).

In order to prevent the degradation of the enkephalins, a

variety of approaches were utilized, such as replacement of

D-amino acid for L-amino acid, N-methylation of the amide

linkages, replacement of amide linkages, or substitution with

unusual amino acids (dehydroamino acids, etc.). A closely

related but more innovative approach is the rational design of

inhibitors for the enkephalin-degrading enzymes (Roques and

Fournie-Zaluski, this volume). These compounds will prolong the

duration of action of the endogenously released enkephalins and,

hence, may be devoid of the major side effects of narcotic

analgetics. A number of enkephalinase inhibitors are derived

from Phe-Leu bearing carboxyl groups (to chelate Zn required for

this metalloenzyme) and are of the general formula: R-CH-

-CONH-CH[CH2CH(CH3)2]COOH with R=-COOH,-CH2COOH,

-NH-CH2-COOH and -NH-CH2-CH2-COOH. Highly potent

enkephalinase inhibitors were also obtained from L-Phe- -Ala

derivatives. Another series of enkephalinase inhibitors was

obtained from compounds that contained a thiol group, R-CH-

)CONH-CH[CH2CH-(CH3)2]-COOH, where R=-CH2SH and

-SH-CH2-CO. Although these are excellent enkephalinase

inhibitors, they are also angiotensin converting enzyme (ACE)

inhibitors. To enhance the specificity, the "retro" compound

HS-CH2CH(CH2 ) NH-CO-CH2-COOH, retrothiorphan, was

developed which, while retaining enkephalinase inhibitors

property, exhibited significantly less ACE inhibitor property.

In summary, this type of rational design has provided researchers with a number of inhibitors, such as thiorphan,

retrothiorphan, and kelatorphan. The clinical use of these

compounds, however, is not promising, due to the limited

bioavailability of some. Development of synthetic analogs that

are orally active or have good bioavailability may be of therapeutic use; and this is expected to be achieved in the near

future.

Conformational aspects of opioid peptides have been examined

extensively. Most of the work has been focused on enkephalins,

on morphiceptins, and to some extent on endorphins. The

presence of a -turn in enkephalins was proposed, from x-ray

crystallographic data and solution studies (Camerman and

Camerman, this volume; Schiller, this volume; Schiller 1984).

The number of hydrogen bonds (2 or 3), and the type of -turn

(type I or II or I' or II') are not yet agreed upon universally

and much more work is warranted in this area. Recently,

Yamashiro and Li (1984) reviewed the conformational features of

endorphins.

Some of the other opioid peptides of interest are dermorphin and

dynorphin. Few conformational studies are reported on dynorphin

in the literature (Schiller 1983; Maroun and Mattice 1981).

9
From circular dichroism studies on dynorphin(1-13), Maroun and

Mattice concluded that dynorphin does not have an ordered

structure in water. However, addition of a detergent, such as

dodecyl sulfate, enhances helical content to some extent. From

fluorescence studies on the biologically active Trp4-analogs

of dynorphin(1-13), Schiller observed that Tyr1-Trp4 are at

least 15 Ao apart (unlike the enkephalin Tyr1-Trp4

residues, which appears to have a folded conformation) and the

predominant conformation is where the N-terminal tetrapeptide is

almost completely extended. In addition, Schiller proposed that

the these conformational differences between dynorphin and

Leu5-enkephalin may play a role in preferences for various

opioid receptor subclasses.

To date, few conformational studies have been done on dermorphin

(Salvadori et al. 1983; Arlandini et al. 1985; Bhatnagar et al.,

in press). Salvadori et al. suggested the presence of a linear

extended structure from NMR studies and, due to lack of

significant dependence on PMR parameters over a concentration

range, the results indicated a monomeric peptide. Arlandini et

al. (1985), using various spectroscopic techniques, suggested

that dermorphin may preferentially exist in an extended,

flexible conformation and that an intermolecularly stabilized

form exists in DMSO. Recently, from FT-IR, laser Raman, CD,

conformational energy calculations, and molecular modeling

studies, it was proposed that dermorphin contains two -turns--a

type III -turn between residues one and four and a type II

-turn between residue five and the terminal amide group. This

conformation with two -turns is highly folded and mimics the

"morphine-type" conformation; and due to this conformation, it

may act as a potent μ-receptor aqonist. This highly folded

conformation and the hydrophobic environment may explain its

stability as well (Bhatnagar et al., in press).

Although earlier conformational studies mainly addressed the

correlation of structure between opioid peptides and rigid

opiates, the discovery of multiple opioid receptors and the

realization that different opioid receptor types may have

differing conformational requirements (Schiller and DiMaio 1982)

changed the direction of research in this area considerably.

Conformational analysis using theoretical methods has been

extensively reviewed by Gorin et al. (1978) and Schiller

(1984). In addition, computer-assisted drug design (Marshall,

this volume) and multidisciplinary approaches have also played a

significant role in the understanding of conformational

features. Recently, a computational procedure for determining

the energetically favorable binding sites on macromolecules has

been reported (Goodford 1985) and this may be of help in druq

design.

The role of conformation and its relation to biological activity

is not fully established. The peptide may exist in a number of

conformations and may continuously interconvert, especially in

the case of small peptides such as the enkephalins. It is a

question of whether a bioactive conformation preexists, such as

10
in the hydroxylation of protocollagen by proline hydroxylase

(Rapaka et al. 1978) or is adopted (such as the "zipper"-type

model, Burgen et al. 1975; See also Schiller, this volume).

Similar to the concept of Burgen et al. is the concept of

"orbital steering," where large rate accelerations are observed

in enzymatic reactions. In orbital steering, the reactive atoms

are constrained to react along selected pathways (Dafforn and

Koshland 1971). It is conceivable that the opioid receptors

have conformational requirements for interaction which differ

depending on the receptor type (Schiller and DiMaio 1982). A

folded -turn conformation, similar to morphine, was proposed to

be the bioactive conformation for morphine-like or μ-receptor

activity (Smith and Griffin 1978). On the contrary, Camerman et

al. (1983) proposed for μ-receptor binding that an extended

conformation is the preferred conformation. Doi et al. (1984)

found that Boc-Tyr-Gly-Gly-(4-bromo)Phe-Met-OH is composed of

antiparallel -sheets, that this structure is stabilized by

intermolecular hydrogen bonds, and that the Boc-peptide has

considerable analgetic activity (although less than the

zwitterionic peptide). From these observations, they proposed

that the dimeric extended conformation may be involved in

binding to the -receptor. This proposal is supported by the

finding that dimeric enkephalins exhibit enhanced binding to

-sites (Shimohigashi et al. 1982). Later, the same group

(Ishida et al. 1984) determined the crystal structures of

Leu5- and Met5-enkephalins and their (4-bromo)-Phe4-

analogs. From these studies they found that Leu5-enkephalins

have type I' -turn conformation and Met5-analogs possess an

extended dimeric conformation. They proposed that dimeric

extended forms interact with the -receptor, while the -turn

conformer interacts with the μ-receptor sites. Recently,

Renugopalakrishnan et al. (1985), from their conformational

studies using FT-IR and laser Raman spectroscopies, proposed

that -turn containing enkephalins interact with -receptor

sites and -sheet containing peptides interact with μ-receptor

sites. Future studies might help in determining the conformation-receptor selectivity in the opioid peptides.

So far, I have discussed the ligand-receptor interaction as if

these were the only components involved. The situation is much

more complex. Interaction between the opioid peptides and lipid

bilayers might be a very important event in the molecular

mechanisms of biological activity of these peptides (Loh and Law

1980; Schwyzer et al. 1982). "The lipid phase of the membrane

acts as a matrix for the receptors and is essential for the

functionality and biological arrangement of the receptor

proteins" (Gysin and Schwyzer 1983). This does not mean that

lipids are the peptide receptors, but rather that they are a

means to capture peptides (either by hydrophobic or electrostatic interactions or both) and facilitate interaction with the

receptor (Berg and Purcell 1977). Gysin and Schwyzer investigated the head group and structure specific interaction of

dynorphin and enkephalin liposomes. -Agonists interacted more

with phosphatidylserine (PS) and a mixture of lecithin and

phosphotidic acid (PC/PA), than with cerebroside sulfate (CS).

11
μ-Agonists interacted more strongly with CS liposomes than with

PS and PC/PA, and k-agonist dynorphin (1-13) interacted more

strongly and more equally with all the systems. This makes it

possible to distinguish ligand types by lipid head group

specificity. Several other groups studied the interaction of

lipids and detergents with hormonal peptides (Gremlich et al.

1984), methionine enkephalin (Spirtes et al. 1978), and

dynorphin(1-13) (Maroun and Mattice 1981). The significance of

these investigations has yet to be explored by further research.

An area of growing interest and need is the quantitative

analysis of opioid peptides. Although RIA methods are largely

being used at present, instrumental methods such as HPLC and

LC/MS that are accurate, specific, sensitive, and reproducible

are required. These methods are essential to provide specific

analysis of low levels of endogenous peptides (active components, precursors, and metabolites) and to understand

pathological states mediated through either lower or higher

levels of these peptides. These methods should be available in

conjuction with immunoassay methods to determine the levels of

synthetic peptides if they are used as druqs. These methods

should also be available for the pharmacokinetic evaluation of

the various bioactive moieties in biofluids.

Current research on opioid receptors has been reviewed

extensively (Simon, NIDA Research Monograph 70; Robson et al.

1983). In earlier studies, Pert and colleagues (Bowen et al.

1981; Olgiati 1982;) characterized type I and II receptors. The

receptor types are now understood better. For reviews see

Simon, this volume; Goldstein and James 1984; Herz 1984;

Paterson et al. 1984; and Kosterlitz 1985. Now sub-types are

already identified for some receptors. In the near future, the

receptor types will be mapped and the subtle differences

involved in their structure and organization will be

understood. At present, it may be speculated that endogenous

ligands might exist for μ-, and k-receptors. A recent report

indicated the existence of morphine as an endogenous compound in

the bovine CNS and in the skin of the toad and other animals

(Oka et al. 1985). This raises the question of whether there

exists an endogenous peptide ligand for the μ-receptor and

whether it is peptidic in nature. A PCP receptor has been

identified (Quirion et al. 1981) and evidence sugqests that the

endogenous ligand is present and is peptidic in nature (Quirion

et al. 1984). One might speculate that this will be an area of

research in drug abuse in the near future.

Other developments include synthesis of peptide antaqonists such

as ICI 174,864 (Cotton et al. 1984) and photoaffinity agents

such as azido DTLET (Garbay-Jaureguiberry et al. 1983; Morley

1983) and [D-Ala2,Leu5]-enkephalin N-[(2-nitro-4-azidopheny)-

amino]ethylamide [Fujoka et al. 1984).

Attempts are also under way to develop clinically useful druqs

and evaluate tnem. One example, FK 33-824, was shown to be

useful for detoxifying hard-core heroin abusers (Wen 1982).

12
Extensive research has been carried out on naturally occurring

and synthetic opiates in order to obtain more useful analqetic

agents. Opioid peptides offer more promise than the opiates in

drug development because:

1. These compounds are endogenous and, on metabolic

degradation (unlike the opiates), break down to amino

acids. Hence, the metabolites are nontoxic and do not cause

kidney and liver damage (unlike the opiates).

2. Since the peptides are made up of several subunits, the

amino acid residues, a large number of analogs can be

synthesized from a few basic building blocks. Moreover,

simple modifications, such as substitution of unnatural

amino acids, sterically constrained amino acids, or other

modifications, may be attempted to develop analogs with a

desired biological activity.

3. The peptide analogs (unlike the opiates) are flexible

conformationally and are ideal molecules to investigate

relationships between 3-D structure and biological

activity. They may be cyclized or subjected to other

synthetic modifications, so that analogs of varying and

controlled flexibility can be synthesized and studied

(unlike the opiates).

4. As these molecules are substantially polar, solution

studies can be performed with different solvents and ions

(calcium, sodium, etc.) to understand their influences on

peptide conformations.

5. Technology for the synthesis of peptides is readily

available and solid-phase peptide synthesis is already

automated to a considerable extent, so that even complex

molecules can be synthesized in large quantities. Even the

ultimate goal of synthesizing pro-enkephalin (about 250

residues) and pro-dynorphin to provide scientists with

adequate material for studies on processing mechanisms seems

possible (see Bodanszky 1985).

6. To date, the compounds that are highly selective for

-receptors have been opioid peptides.

7. Due to the unique character of some of the peptide drugs,

i.e., their inability to cross placental barrier (due to

placental enzymatic deactivation of the peptides), these

drugs have a distinct advantage for being used as analgetics

in pregnant females. They are definitely superior to

meperidine in this respect.

At present, research in opioid peptides is progressing at a

rapid rate. Some of the important aspects follow:

Analgetic peptides are to be designed that will show minimal

drug dependence potential; the peptides of potential interest

13
are enkephalins, endorphins, dynorphins, dermorphins,

morphiceptins, and related opioid peptides. Some of the goals

are: a) the development of peptide analogs that can be

administered orally, that have long duration of action, and that

are without the potential for abuse; and b) synthesis of

inhibitors of enkephalin-degrading enzymes as potential

analgetics. Extensive SAR studies on the opioid peptides may be

of great value in the rational design of these analogs. It is

further anticipated that SAR studies on opioid peptides might

result in the development of antagonists and mixed agonistantagonists that are clinically useful in the treatment of

opiate addiction.

Several hundred analogs of the opioid peptides have been

evaluated to aid in delineating the essential features of the

μ-and -receptors. The synthesis of even more specific analogs

for all types of receptors can be anticipated.

The receptor types are well differentiated on the basis of

binding studies and/or pharmacological studies. However,

considerable information on sites and functions is still

lacking. Particularly, the component peptides of the different

receptors must be isolated and characterized, and receptors or

receptor models must be synthesized or reaggregated to

understand mechanism(s) of action. It may be possible to study

the conformation of the receptor in response to environmentmediated conformational changes (i.e., lipids, ions, solvents,

ionic strength, polarity, etc.) and the conformation of the

drug-receptor complex itself.

A number of conformational studies have been conducted on opioid

peptides; however, most of them are performed on enkephalin

peptides as they are relatively small pentapeptides and are

easily accessible. Conformational studies using theoretical

solid-state and solution methods have to be extended to all the

pertinent opioid peptides and their analogs. The effects of the

surrounding environment (solvent, pH, ionic effects, etc.) must

be examined; and, finally, the receptor-bound conformation of

the drug must be determined. Advanced methods such as X-ray

crystallography, FT-IR, laser Raman, nuclear magnetic resonance,

C.D., and computer-assisted drug design will play an increasing

role in these studies.

Research in the opioid peptides is progressing at a rapid rate.

In the near future, we may look forward to still more exciting

discoveries, and there is room for optimism to believe that

therapeutically useful peptide analgetics may soon be

developed.

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brain homogenate. Proc Natl Acad Sci USA 70:1947-1949, 1973.

Smith, G.D., and Griffin, J.F. Conformation of [Leu5

]-

enkephalin from X-ray diffraction: Features important for

recognition. Science 199:1214-1216, 1978.

Smyth, D.G.; Snell, C.R.; and Massay, D.E. Isolation of the

C-fragment and C'-fragment of lipotropin from pig pituitary

and C-fragment from brain. Biochem J 175:261-270, 1978.

Spirtes, M.A.; Schwartz, R.W.; Mattice., W.L.; and Coy, D.H.

Circular dichroism and absorption study of the structure of

methionine-enkephalin in solution.

81:602-609, 1978.

Biochem Biophys Res Comnun

Stammer, C.H Dehydroamino acids and peptides. In:

Weinstein, B., ed. Chemistry and Biochemistry of Amino Acids

Peptides and proteins. Vol 6. New York: Dekker, 1982. pp.

33-74.

Terenius, L. Characteristics of the "receptor" for narcotic

analgesics in synaptic plasmas membrane fraction from rat

brain. Acta Pharmacol Toxicol 33:372-384, 1973.

Udenfriend, and Kilpatrick, D.L. Biochemistry of the

enkephalins and enkephalin-containing peptides. Arch Biochem

Biophys 221:309-323, 1983.

Weber, E.; Esch, F.S.; Bohlen, P.; Paterson, S.; Corbett, A.D.;

McKnight, T.A.T.; Kosterlitz, H.W.; Barchas, J.D.; and Evans,

C.J. Metorphamide: Isolation, structure, and biologic

activity of an amidated opioid octapeptide from bovine brain.

Proc Natl Acad Sci USA 80:7362-7366, 1983.

Wen H.L. Clinical trial of Met-enkephalin analog (DAMME, FK

33-824) on heroin abusers: Costa, E., and Trabucchi, M.,

eds. In: Regulatory Peptides: From: Molecular Biology to

Function. New York: Raven Press, 1982. pp.397-403.

Yamashiro, D., and Li, C.H. -Endorphin: Structure-activity

relationships. In: Udenfriend, S., and Meienhofer, J., eds.

The peptides. Vol. 6. New York: Academic Press, 1984. pp.

191-217.

Zaoral, M.; Yamashiro, D.; Hammonds, R.G., Jr.; and Li, C.H.

-Endorphin: Synthesis and radio-receptor binding activity of

-endorphin (1-27) and its analogs. Int J Pept Protein Res

17:292-296, 1981.

ACKNOWLEDGMENTS

The author acknowledges Dr. H. W. Kosterlitz, University of

Aberdeen, Aberdeen, Scotland for the review of the manuscript

and for his valuable suggestions. This article is modified from

the minireview to be published by Pergamon Press, in Life

Sciences.

19
AUTHOR'S NOTE

This review is not comprehensive. Only a few selected

references and topics are included, as it is impossible fully to

review an extremely active area such as this, and other authors

have presented pertinent topics and references. For further

information on the topics or references, the reader is referred

to the following chapters in this monograph, NIDA Research

Monograph 70, and The peptides: Analysis, Synthesis, Biology.

Vol. 6, Opioid Peptides: Biology, Chemistry, and Genetics (S.

Underfriends and J. Meienhofer. eds. New York: Academic Press

1984).

AUTHOR

Rao S. Rapaka, Ph.D.

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

20
Synthesis of Opioid Peptide Analogs

John M. Stewart, Ph.D.

INDTRODUCTION

Elucidation of the structures of the first opioid peptides, the

enkephalins, in 1975, stimulatad an unprecedented wave of synthetic activity. Fortunately, peptide synthetic methods had been

develoqed to the point where synthesis of opioid peptides was

easily accomplished, particularly the small ones. In this chapter, contemporary methods of peptide synthesis will be discussed,

especially as they apply to the cpioid peptides.

Peptides may be synthesized either in solution, using classical

organic synthetic procedures, or by the newer method of solid

phase peptide synthesis introduced by Merrifield. Solution synthesis may be practical for small peptides, particularly if large

quantities are to be synthesized. Successful application of solution methods generally requires that the person performing the

synthesis be skilled and experienced in peptide synthetic procedures. On the other hard, solid phase peptide synthesis (SPPS),

introduced twenty years ago, allows rapid synthesis of a wide

variety of peptides and doss not require a high degree of chemical shill on the part of the person doing the synthesis. It has

been applied sucessfully to the synthesis of many longer peptides, such as ß-endorphin and its analogs. The availability of

commercial automatic peptide synthesizers sakes SPPS a rapid and

convenient procedure. It is particularly useful for synthesism of

a wide variety of analogs, such as has been done in the case of

the cpioid peptides.

PRINCIPLES OF PEPTIDE SYNTHESIS

Successful peptide synthesis rests upon two maajor factors: 1)

application of a suitable set of selectively removable blocking

groups for the reactive functions of the amino acids to be incorporated into the peptide, and 2) use of suitable activating

reagents to perform the coupling reactions involved. Synthesis of

a tripeptide is depicted schematically in figure 1. During every

coupling step, only those reactive groups which are intended to

21
FIGURE 1

Schematic Synthesis of a Tripeptide

S=stable blocking groups; L=labile blocking group

react are left unprotected. Other reactive groups are blocked

either by stable blocking groups (indicated with S),which remain

intact throuh out the process of assembly of the desired peptide,

or by means of a labile blocking group (indiated above as L),

which will be removed at each step of the synthesis. Synthesis

usually begins at the carboxyl end of the peptide. The most commonly used procedure for synthesis of peptides of the size range

of the opioid peptides is that of stepwise synthesis, in which a

single amino acid is added at each coupling reaction. It is extremely important that optical purity of all the amino acid residues be maintained throughout the synthesis. For all the naturally occuring amino acids, the L isomer is used. However, in

synthesis of enzyme-resistant opioid analogs, D-amino acids are

frequently used. Coupling reactions must be chosen with care to

avoid racemization of the amino acid residue which is activated

during the coupling reaction. The most reliable method for avoiding such racemization is coupling of single amino acids, each

having its -amino group protected with a urethane-type protecting group.The most commonly used such urethane group at the

present time is the t-butylaxycarbonyl (Boc) group: its structure

is indicated in figure 3. The Boc protecting group is commonly

22
FIGURE 2

Mechanism of coupling Reactions Mediated by DCC

removed at each step of the synthesis by treatment of the protected peptide with anhydrous acid, much as trifluoracetic acid

(TFA) in an inert solvent such as dichloramethane (DCM).

When the Boc group is used for the labile -protecting group, the

stable blocking groups are usually derived fran benzyl-related

blocking groups. Thus, the stable blocking group for the Cterminal carboxyl of the peptide may be a benyl ester, which can

also be used for blocking side chain carboxyls of aspartic and

glutamic acid residues. The aliphatic hydroxyl groups of serine

and threonine may be blocked as benzyl ethers. The hydroxyl group

of tyrosine and the amino group of lysine are usually blocked

with a group related to the benyloxycarbonyl blocking group,

which has been in use for many years in peptide chemistry.

Arginine and histidine side chains are most commonly blocked with

the p-toluene sulfonyl (Tos) group. All these stable blocking

groups can be removed from the peptide at the end of the synthesis by treatment with a strong anhydrous acid, such as liquid

anhydrous hydrogen fluoride.

23
More labile systems of blocking groups have been developed for

peptide synthesis. For example, the labile blocking group can be

the biphenylylpropoxycarbonyl (Bpoc) or the phenylisopropyloxycarbonyl (Poc) group. When these very labile groups are used

for -protection, side chains can be blocked with functions

related t-butyl groups, such as t-butyl esters and Boc groups.

Very dilute TFA is used to remove the labile protecting group at

each stage of the synthesis, while more concetrated TFA can be

used to remove the stable blocking groups at the end of the

synthesis.

These two synthetic schemes outlined above depend for their

success on the differential lability to anhydrons acid of the

labile and stable blocking groups. Some reaserch has been done on

developement of orthogonal systems of blocking groups where the

labile and stable blocking groups are removed by completely different types of reagents. Such a systen can use the fluorenylmethoxycarbonyl (Fmoc) -blocking group; it is removed by treatment with an amine such as piperidine. The stable blocking groups

can be t-butyl derived functions, which are stable to the mine

used to remove the labile blocking group. This systen has received only limited application to synthesis of opioid peptides,

principally due to the greeter cost of Fmoc amino acid derivatives. Furthermore, yields have usually been quite satisfactory

using selective acidolysis procedures.

Given a set of blocking groups suitable for the synthesis at

hand, ate needs then to choose appropriate coupling reactions. By

far the most commenly used coupling reagent is dicyclohexylcarbodiimide (DOC). Use of DOC is particularly convenient, since it

can usually simply be added to the solution containing the amine

and carboxyl component to be coupled. It reacts rapidly with the

free carboxylic acid present to form an "active ester" internediate (the isaurea),which may react with the amine component directly or may proceed through an intermediate symetrical anhydride (see figure 2). Rearrangement of the isourea to the N-acyl

urea is an undesired side reaction. Coupling reactions mediated

by DOC can also be modified by addiction of other reagents, such as

as 1-hydroxybensotriazole (HOBt). This leads to intermediate formation the HOBt active ester, which may minimize racemization

and may also facilitate some coupling reactions. If glutamine is

being incorporated into the peptide, it can also minimize dehydration of the omega amide group to a nitrile. Mixed anhydrides

have also been frequently used to promote coupling reactions,

particularly in solution synthesis methods. However, a discussion of such reagents is beyond the scope of this article. A

good introduction to peptide synthesis in general is given by the

book of Bodanszky et al. (1976).

SYNTHESIS OF PEPTIDES IN SOLUTION

Peptide chmists working during the last fifty years have developed methods suitable for effective synthesis of peptides in

24
solution, applying the principles outlined in the previous

section. Solution methods have been used mainly for synthesis of

shorter opioid peptides, such as analogs of the enkephalins.

Solution synthesis is usually used for production of large

quantities of peptides, particularly if the peptide chain is

short. Solution synthesis also offers the opportunity for use of

segment coupling methods, in which a small block peptide is

synthesized and then coupled to the reminder of the chain in me

step. A major problem with segment synthesis methods is that of

racemization of the carboxy-terminal residue in the segment when

it is activated for coupling. However, this is not a problem with

enkephalin synthesis if the fragment is properly chosen, since

glycine residues cannot racemize and the presence of two glycine

residues in the enkephalin sequence allows for convenient

breaking of the chain into segments. This approach can offer

practical advantages for synthesis of a series od analogs

modified at the carboxy end. For example, variations in the

structure of enkephalins at the 4 and 5 positions (phenylalanine

amd leucine or methionine) could utilize a blocked fragment

consisting of tyrosyl-glycyl-glycine or tyrosyl-D-alanyl-glycine

for coupling to various C-teminal dipeptides. In solid phase

synthesis (SPPS), the entire sequence must usually be repeated in

a stepwise fashion for each of such analogs.

Various combinations of the blocking groups and coupling reagents

described in the previous section have been used in the synthesis

of opioid peptides. Syntheses of these peptides described in the

literature may appear very simple, and in principle they are.

However, successful results in solution synthesis of peptides

generally requires considerable experience. The novice will frequently waste a great deal of time and money attempting to synthesize even small peptides by these methods, and is advised to

use solid phase techniques instead.

Amides ob enkephalin peptides have been found to have high

potency, particularly for mu receptors. In solution synthesis,

peptide amides are synthesized by beginning the synthesis with

the mide of the C-terminal residue to be incorporated into the

peptide, e.g., methionine amide. They can also be synthesized

fran peptide esters by ammonolysis, provided there are no

aspartic or glutamic residues in the peptide (these would be

concomitantly changed to asparagine and glutamine residues).

Certain enkephalin analogs in which the C-terminal methionine

carboxyl group has been reduced to an alcohol have been found to

be very potent. Such peptide alcohols can be synthesized by

beginning the synthesis with a benzyl ether of the C-terminal

alcohol and using HF for final deblocking of the finished

peptide. If there are no aspartic or glutamic acid residues in

the peptide, the synthesis my be carried out with the methyl

ester of the C-terminal residue. Treatment of the finished

blocked peptide with lithiumborohydride will then yield the Cterminal alcohol.

25
SOLID PHASE PEPTIDE SYNTHESIS

Solid phase peptide synthesis was introduced by Bruce Merrifield

in 1963 in the hope of overcoming many of the problems inherent

in solution methods of peptide synthesis. The experience of the

past twenty years has shawn that hope to be amply justified.

Autmatic instruments have been develcped and are available

ambercially which will carry out SPPS rapidly and efffectively.

Many thousands of peptides have been synthesized with SPPS. In

contrast to the relatively high degree of technical sophistication required for successful peptide synthesis in solution,

successful SPPS of many peptides can be carried out by persons

with no prior experience in peptide synthesis and only limited

competence as organic chemists. As one imnunolcgist said, "solid

phase synthesis is so simple than even an inmunologist can do

it!" The comprehensive review of Barany and Merrifield (1980)

provides an encyclopedic coverage of the solid phase literature,

and the laboratory manual of Stewart and Young (1984) provides

specific laboratory directions for synthesis of peptides by SPPS.

FIGURE 3

The Merrifield Scheme of Solid PHase Peptide Synthesis.

The polymer is 1%-crosslinked polystyrene; TFA=trifluoroacetic

acid; TEA=triethyl amine; DCC=dicyclohexylcarbodiimide.

26
Solid phase peptide synthesis is a special case of the general

scheme of stepwise synthesis of peptides fran the carboxy end,

in which the stable blocking group for the C-terminal carboxyl

of the peptide being synthesized is an insoluble polymer (figure

3). In the Merrifield system of SPPS, the C-terminal amino acid

residue is esterified to a functionalized cross-linked polystyrene resin, which serves as the carrier. Mast SPPS has been

done with Boc amino acids. Follwing attachment of the first Boc

amino acid to the resin, the resin is analyzed for the degree of

substitution of the amino acid cm the polymer. A batch of Bocamino acid resin is then placed in the reaction vessel of a

manual or automatic synthesis apparatus; the resin need never

be rmoved from the vessel until the assembly of the peptide is

finished. By avoiding any transfer of material from one vessel to

another during the synthesis, much loss of material is eliminated. The Boc group is removed from the amino acid on the resin

by treatment with an acidic reagent, usually TFA in DCM. HCL in

dioxane has also been used successfully for my syntheses, but

is less covenient to use. The amino acid-resin is then treated

with a tertiary amine base to neutralize the acid salt and

liberate the free amino group for coupling with the next Bocamino acid. The next Boc-amino acid can be coupled to the amino

acid-resin by use of DCC or any other oonvenient method for

coupling. Use of DOC: is rapid and convenient, and usually gives

satisfactory results. In those cases where complete coupling

cannot be achieved in one or two cycles, use of an active ester

(either formed in situ by addition of HOBt or used pre-formed

HOBt active ester) or use of a Boc-amino acid symetrical

anhydride can be tried. Symmetrical anhydrides are very effective

for promoting complete coupling reactions, but their use involves

additional work and is frequently not necessary. Repetition of

the deprotection, neutralization and coupling steps with each Bocamino acid to be introduced into the peptide sequence will then

yield the blocked peptide an the resin. If the peptide being synthesized is to have a free carboxyl group, treatment of the peptide-resinwith liquid anhydrous HF will cleave and deprotective

peptide in me operation.

If all deprotection and coupling reactions go fully to completion, a homogeneous peptide will be assembled on the resin.

If the cleavage reagent does no harm to the peptide, then the

crudepeptidewill be essentially pure. Using the best of modern

techniques, this level of success can frequently be achieved

in practice. In such cases purification of the final peptide is

very simple. After the initial Boc-aminoacylresin is synthesized

and analyzed, autamatic synthesizers can carry out assembly of

the peptide at the rate of one residue every two to fair hours.

Cleavage of the peptide fran the resin with HF will require an

additional half day of time. If the synthesis procdures were

carried out effectively, purification can then frequently. be a

simple short operation like chromatography or countercurrent

distritution. The speed of SPPS carried out in this way makes

it particularly practical for synthesis of analogs of opioid

peptides, especially those analogs in which variation in the

sequence will be toward the anino end of the peptide. In that

27
case, a large batch of aminoacyl-resin can be carried through the

synthesis up to the point where the different substitutions will

be made. Individual syntheses can then be carried out with the

desired amino acids on a smaller scale, using portions of the

peptide-resin previously prepared.

Synthesis of peptide amides is also convenient by SPPS. If the

peptide chain does not contain aspartic or glutamic acid residues, the sequence can be assembled on Merrifield resin and

cleaved by amonolysis. However, if the sequence contains these

residues (ß-endorphin contains glutamic acid residues), this

approach is not satisfactory, because the glutamic residue will

be banged to glutamine if the ammnolysis is conducted on a

peptide resin in which the glutamic side-chain carboxyl is

blocked with a benzyl ester. A hydroxymethyl resin support can

be used for a synthesis, however, in which the labile system of

blocking groups or the orthogonal systen (as described above) is

used. In those cases, the glutamic carboxyls can be protected as

t-butyl esters, which should not be converted to glutamine

residues during ammonolysis. A simpler approach uses for the

insoluble support a resin which will yield a peptide amide

directly upon cleavage of the peptide with anhydrous HF. Such

resins are the benzhydrylamine (BHA) and methylbenzbydrylamine

(MBHA) resins (see figure 4). If an aminanethyl resin analogous

to the hydroxymethyl Merrifield resin is used for synthesis of

peptides, HF will not cleave the peptide fran the resin, as that

group is not sufficiently labile. If the aminomethyl resin is

FIGURE 4

Synthesis of peptide Amides via Amine Resins

28
labilized by addition of another phenyl or p-methylphenyl residue

as in the case of the BHA and MBHA resins, the nitrogen-carbon

linkage is now sufficiently labile that treatment of peptide-BHA

(or MBHA) resins with HF will yield peptide amides directly. The

linkage of peptides to the MBHA resin is somewhat more labile

than in the case of the BHA resin, and the MBHA is more

satisfactory for the synthesis of peptides whose C-terminal

residue is me which makes the peptide relatively resistant to

acicdolysis, such as phenylalanine or leucine. Miny analogs of [DAla1

]-methionine-enkephalin amide have been synthesized on these

resins. Directions for synthesis and use of these resins are

given in the laboratory manual of Stewart and Young (1984).

Peptide alcohols,whichhavebeen found useful in the enkephalin

series, can also be synthesized satisfactorily by solid phase

methods if the amino acid sequence does not contain glutamic or

aspartic residues. The peptide sequence is assembled on the hydroxymethyl Merrifield resin, and the blodced peptide-resin is

treated with lithium borohydride to effect reductive cleavage to

an alcohol of the ester linking the peptide to the resin.This

procedure is also described in the Stewart and Young manual.

SIDE REACTIONS DURING PEPTIDE SYNTHESIS

If an appropriate set of blocking groups and reaction conditions

is chosen for synthesis of peptides, undesirable side reactions

during assembly and deblocking of the peptide can be minimized.

If the conditions for solid phase synthesis recommended in the

Stewart and Young manual are followed, little difficulty can be

expected However, if less than ideal condtions and derivatives

are used, some problems can be expected. Methionine-enkephalin

contains two residues which can cause problems: methionine and

tyrosine. The sulfur in methionine residues can be readily alkylated under appropriate conditions (see figure 5). Such conditions can cccur during deblocking of the peptide. When benzyl

blocking groups are removed from side-chain functions during HF

cleavage, the benzyl oation, if allowed to persist for any significant length of time,will alkylate methionine to the sulfonium derivative. The sulfonium derivative of methionine-enkephalin

has very low biological activity. This undesirable alkylation can

FIGURE 5

Formation of Methionine Sulfonium Peptides

29
be prevented by inclusion of a large excess of scavenger reagent

in the clevage reaction mixture. Such reagents as anisole and

methylethylsulfide are commonly used for this purpose.Methionine

can also be oxidized easily to the sulfoxide derivative. Indeed,

the sulfoxcide has been found highly potent the case of certain

methionine-enkephalin derivatives. However, in this case, the

synthesis can be done with Boc-methionine sulfoxide, which should

be stable throughout the synthesis and HF cleavage. If sulfoxide

is not desired, syntheses can usually be carried out with Bocmethionine without significant oxidation to the sulfoxide. However, if it is found that in a particular sequence or under particular reaction conditions being used this undesirable oxidation

takes place, any sulfoxide in the peptide-resin can be reduced

conveniently to methionine by inclusion of 2-mecaptopyridine in

the HF cleavage reaction mixture (see Stewart and Young 1984).

If the hydroxyl group of tyrosine is blocked during synthesis as

the benzyl ether, HF treatment can yield some of the undesired 3-benzyl tyrosine (see figure 6). This Friedel-Crafts type

reaction is promoted by anhydrous HF. The alkylation can be

prevented if large quantities of scavenger are included in the

cleavage reaction mixture. Most peptide chemists currently practicing solid phase synthesis do not use O-benzyl tyrosine for

this reason. The 2,6-dichlorobenyl and the 2-bromocarbobenzoxy

derivatives have been found to yield very little of the undesired

alkylated derivate. Bath the 3-benzyl tyrosine and the S-benzyl

methionine analogs of enkephalin have been found to have very

little biological potency. One should normally take pains, there

fore, to suppres these side reactions. Other possible side

reactions which may be encountered during solid phase synthesis

are disscused in the Stewart and Young laboratory manual.

If the directions given in the Stewart and Young manual are

followed even novices could be able to synthesize opioid

peptides without serious problems. Only general procedures are

given in that manual. If the chemist wishes to follow a set of

directions specific fox methionine-ankephalin, such directions

FIGURE 6

Alkylation of the Tyrosine Ring by Benzyl Groups

30
can be found in the volume of "Methods of Enzymology" dealing

with synthesis of neuropeptides (Stewart 1983).

PURIFICATION AND ANALYSIS OF PEPTIDES

Following assembly and deblocking of a desired synthetic psptide,

the product must be purified essentially to homogeneity and

analyzed for identity before being used. While some ideas of

biological potency my be obtained in rapid screening programs by

use of new analogs not fully purified, accurate quantitative data

can only be obtained with fully purified peptides. Physical

studies on peptides also require synthetic wterials of the

highest purity.

When peptides are synthesized in solution, the intermediate

products are usually pirified frequently during the various steps

of the synthesis. In that case, final purification of the peptide

is frequently quite simple. However , even if the final protected

peptide is fully purified, same by products may be introduced

during deblocking, and these must be removed. Some small peptides

can be satisfactorily synthesized in solution by repetitive

procedures without full purification at each stage. In those

casses, final purification of the product can be quite difficult.

In solid phase synthesis there is normally no opportunity for

purification ob the peptide at intermediate stages, and the only

purification possible cones after removal of the peptide fran the

resin support. In order to achieve a homogeneous product, every

deprotection and coupling reaction must go fully to completion.

If the best available techniques are used for synthesis, this

god can be practically achieved, or at least very nearly so.

Every assembly of opioid peptides on solid phase resins should

finish with a deprotection step, in order to remove the

protecting group fran the chain to minimize alkylation side

reactions during cleavage. This is equally true if the final

cleavage will be done by HP or on labile resin systems by use of

trifluorcacetic acid. As noted above, adequate scavengers must

always be used during the cleavage to minimize side reactions.

Procedures using partition methods, such as countercurrent

distribution, partition chromotography or reversed-phase high

performance liquid chromotography (HPLC) are particularly

appropriate for purification of opioid peptides synthesized by

solid phase methods. The crude synthetic peptide usually contains

some of the scavengers used in the cleavage reaction, and these

materials are frequently very different in polarity fran the

desired peptide. Moreover, if alkylation of methionine or

tyrosine residues has occurred, these by products will also differ

significantly in polarity and partition coefficiant. The benzyl

tyrosine derivative will be found to be sore hydrcphobic while

the methionine sulfonium derivative will be more hydrophilic, and

thus readily separated fran the desired product by partition.

Quantities greater than 100 mg of peptide can usually be

purified by an overnight automatic run in a Craig countercurrent

31
distribution apparatus. Preparative HPLC is even more rapid, but

the apparatus is quite expensive and the resins used will also

represent a significant expenditure, especially since the packing

material can reasonably be used only for one peptide. Peptides

frequently adsorb quite tightly to the reversti-phase material

and the final traces are gradually eluted in subsequent runs. In

commercial Or very large-scale operations, where large amounts

of peptide or repeated syntheses will be made, this may be very

practical. For the investigator synthesizing large numbers of

analogs, the economics may be a problem. Partition chromotography

has long been used for purification of synthetic peptides, and

while very large columns will be needed for purification of

significant quantities of peptide, this an be done rapidly.

All these procedures are discussed in the laboratory manual of

Stewart and Young.

All purifications mist be accompanied by suitable analytical

techniques which will reveal the presence of any impurities in

the synthetic product and indicate their nature. Paper electrophoresis is uniquely appropriate for this purpose. Thin-layer

electrcphoresis can also be used but is not quite as convenient.

Gel electrophoresis is not appropriate for small peptides of the

size of the opioids because of excessive diffusion of the

peptide in the gel matrix. If suitable amino acid markers are

included as standards in an adjacent channel, in paper or thinlayer electrophoresis, quite accurate information on the size and

charge of the peptide product can be obtained. Inpurities

differing in charge and differing significantly in size are

readily resolved. Electrophoresis can be done in buffers of

different pH in order to enable appropriate jud regarding

inpurities to be made. Thin layer chranatcgraphy (TLC) is also a

valuable analytical technique. It is particularly useful for

indicating the presence of impurities which differ in hydrophobicity from the desired product.

If meaningful infomation on purity of synthetic peptides is to

be obtained, it is critical that the procedures used for analysis

be based upon different phenomena than the procedures used for

purification. For example, if the peptide WaS purified by a pertition method such as countercurrent distribution or reversedphase partition HPLC, then similar HPLC or TLC should not be used

to assess purity. Rather, a method utilizing charge and/or size

of the peptide should be used, such as electrcphoresis or ionexchange TLC. In any event, once homogeneity has been acheived by

a suitable purification procedure or procedures, and has been

demonstrated by appropriate analytical methods, an aliquot of the

peptide should be hydrolyzed with acid and the ratio of constitutent amino acids determined by amino acid analysis. Since all

opioid peptides contain tyrosine, the acid hydrolysis mixture

should contain phenol to prevent loss of tyrosine by chlorination

due to contamination of the HCL by chlorine. If tryptophan-containing analogs are synthesized, reasonable values for this acidlabile amino acid can often be obtained by including marceptoethanol in the hydrolysis mixture. Since many opioid peptides

32
contain methionine residues, anaslysis of the oxidation state of

this residue is quite important. The biological potency of a

peptide can frequently change drastically depending upon whether

the methionine residue is present per se or is oxidized to

the sulfaxide. Rydrolysis with 6 N HCL converts methionine

sulfoxide largely to methionine, so the original state of that

residue cannot be determined in this way. Rather the peptide must

be hydrolyzed with methanesulfonic acid, which does not disturb

the oxidation state of methionine residues. Precise details of

these procedures are given in the Stewart and Young (1984)

handbook.

Accurate amino acid analysis also provides a reasonable estimte

of the "working molecular weight" of the peptide. Samples of

carefully purified and lyophilized peptides contain varying

amounts of non-peptide material. Peptides which have a net acidic

or basic charge will also contain a counter-ion to neutralize

these excess charges. These salts must be taken into account in

deriving the theoretical molecular weight. One must also remember

that the final salt form will depend upon the acidic or basic

strength of the final purificaticn system. Far example, a peptide

containing a free amino and carboxyl group in addition to a

single arginine residue will be found to contain one mole of

acetic acid if it is purified fran an acetic acid-containing

system. On the other hand, if the peptide is purified finally

from a system conaining trifluorcetic acid, two moles of TFA

will be bound to the peptide since TFA is a stronger acid than

the carboxyl group of the peptide chain. This same consideration

alsoapplies to the side-chain carboxyl groups of aspartic and

glutamic acids. The theoretical molecular weight calculated in

this way (containing bound salt) is useful for determining the

purity of the synthetic peptide. The most practically useful

value, however, is the "working molecular weight," which is the

reciprocal of the average micromoles per milligram of peptide

analyzed. In calculating this ratio, one should not include

values found for residues known to be destroyd during hydrolysis, such as marine, theronine, and tryptophan. This

"working molecular weight" should be applied by the person

synthesizing the peptide to whomever will use the peptide, 80

that accrate molar potencies can be calculated.

STORAGE AND HANDLING OF PEPTIDES

Most synthetic opioid peptides will be found to be quite stable

if stored absolutely dry and in the freezer. When the peptide

container is removed from the cold, the contianer should be

allowed to warm to room temperature in a desiccator before the

container is opened; other wise atmspheric moisture will condense

on the cold peptide particles. When the peptide is again stored,

this moisture will accelerate degradation of the peptide. Peptides should be stored in relatively small aliguots in separate

containers, so that a large batch of peptide is not repeatedly

warmed and chilled.

33
Amino acid analysis frequently reveals that lyophilized peptides

contain considerable quantities of solvent. If these peptides

were lyophilized from acetic acid, as is often the case, cold

storage is particularly important to avoid degradation of the

peptide by the acetic acid in the sample. Peptide bonds adjacent

to aspartic acid residues are particularly labile to this type of

degradation.

Amide bonds, of the C-terminal amide if it is present, and of

glutamine and asparagine side chains, are also a problem. Glutamine and asparagine amides have been found to be particularly

labile when they occur in the peptide sequence adjacent to the

basic residues, such as arginine or lysine; asparagine amides are

the more labile. This typed hydrolysis can occur during storage

of the "dry" peptide as well as after the peptide is dissolved in

aqueous solution. This hydrolysis is also more rapid if the "dry"

peptide contains excess acid or base, or if it is dissolved in a

solution that is significantly removed fran neutrality. The surface of glass is alkaline, particularly if the glass is aged. All

glass containers to be used for storage of peptides or peptide

solutions should be rinsed with molar acetic acid and dried care

fully. For most careful work, they should then be silanized.

Many peptides, particularly those that are basic and/or hydrophobic, adsorb avidly to surfaces of glass and some plastics.

The extremely high potency of some opioid peptides causes them to

be used in very dilute solutions in biological experiments. In

some cases, these experiments may be totally invalid due to

adsorption of peptide from solution onto the surfaces of test

tubes, pipettes, and syringes. Among the plastics, polystyrehe is

particularly bad for peptide adsorption and should not be used.

Polyethylene and polypropylene are probably the best plastics for

use with peptides,although some peptides will stick to these

surfaces. If low concentrations of peptides must be used, adsorption can be prevented by such measures as making all the dilutions in 0.01 M acetic acid, or by including a low concentration of protein in the solution, in addition to silanizing the

glass surface. The method chosen will depend upon the requirements of the experiments. In certain cases, a difference of 1,000-

fold in potency of a peptide solution has been ovserved when

dilute acetic acid was included in the solution.

Oxidation of methionine residues in dilute solutions of peptide

is a serious problem. All solvents to be used for dissolving such

peptides should be thoroughly bubbled with nitrogen before use.

some very potent enkephalin analogs have contained disulfide

rings formed from two residues of cysteine or penicillamine in

the peptide chain. Such disulfides are extremely labile in solution if the pH is above neutrality. At higher pH, disulfide interchange occurs, and the monomeric peptide may be rapidly convertd to dimers and polymers, which are usually biologically

inactive. Experience with oxytocin and vasopressin solutions has

shown that they retain their potency best when slightly acidified

(pH 5) and stored just above the freezing point. These conditions

34
may also be most appropriate for storage of solutions of disulfide-containing opioid peptides.

When dissolving peptides for use, only fairly concentrated solutions (such as 0.1 mg/ml) should be frozen for storage. These

solutions should be divided into small aliquots containing the

amount to be used for one day's experiments. The tubes should

be flushed with nitrogen, closed tightly to prevent spillage or

lyophilization in the freezer, and frozen. The ease of dissolution of peptides depends strongly upon the physical state of

the material. If the peptide was lyophilized from fairly dilute

solution, it will be finely divided and will usually dissolve

readily, except in the case of some hydrophobic peptides, which

are difficult in any case. If the peptide was dried by evaporation or lyophilized fron a very concentrated solution, the

material may be in the form of hard particles which dissolve

only very slowly. Such particles may have an index of refraction

similar to that of the aqueous medium, and their failure to

dissolve may not be noticed unless the solution is examined with

a magnifier. Very hydrophobic peptides may need to be dissolved

first in a mall amount of a powerful solvent such as dimethyl

sulfoxide and then diluted with hater or saline. In this case,

appropriate controls must be run. Bubbling air through the

pipette or vortexing should not be used for mixing if there is

any possibility of oxidation of the peptide.

DESIGN OF OPIOID PEPTIDE ANALOGS

NEW analogs of opioid peptides are synthesized for a variety of

reasons. A major goal has beeu to find analogs which will have

absolute selectivity for the various classes of opioid receptors.

When such selectivity is achieved, further modifications may well

be undertaken to improve the resistance of the peptide to enzymatic degradation in order to increase potency and prolong

activity in vivo and in vitro. Such selective, potent and longacting analogs will find many practical applications. Additional analogs may need to be designed in order to delineate more

precisely the nature of peptide receptor interactions, as well as

for certain physical chemical studies. Analogs containing easily

detectable "reporter" functions are particularly useful in such

studies. Potent selective antagonists are much needed for a

variety of reasons, both practical and theoretical. Since there

are no universally applicable rules for design of peptide antagonists, this letter search has been particularly frustrating.

Conformational restriction has been a very valuable tool in

design of selective opioid analogs. The ancestral opiate

compound, morphine, and many of its analogs synthesized by

medicinal chemists were the years have led the way for more

recent peptide work. Use of N-methyl amino acids in peptide

analogs not only restricts the conformtions available to

the peptide, but frequently confers enzyme resistance as well.

Some -methyl amino acids have been incorporated into opioids

35
with startling results. Aditional work needs to be done with

other -alkyl amino acid residues. Conformtional restriction by

cyclization of all or part of the peptide chain has also been

very useful in opioids.

Examination of the lists of opioid analogs in this volume and

in other publications will show the degrees of selectivity,

enzyme resistance and antagonist that peptide chemists have been

able to incorporate into analogs at the present time. These

developments will not be discussed again here. Details of two

groups of peptides not covered elsewhere will be discussed in

somewhat more detail. Several aspects of the wisdan gaind from

three decades of peptide analog work are sumnarized in a recent

article (Stewart, 1982).

MINIMUM STRUCTURE ENKEPHALINS

One of the earliest types of structure modification applied to

the enkephalin field was shortening of the peptide chain, either

by elimination of residues fran the ends or by removal of

residues within the sequence. From those experiments it was found

that while significant potency remained if the C-terminal

methionine or leucine residue was remved, the phenylalanine

residue in position 4 was critical for activity. It was also

found that activity was reduced to less than 1% of that of the

pentapeptide if one of the glycine residues was removed fran

within the chain. Fran this last work it was propsed that the

spacing between the essential phenolic side chain of the tyrosine

in position me and the phenylalanine side chain was critical for

biological activity. This last proposal was somewhat difficult to

understand in light of the fact that morphine combines well with

the opiate mu receptors and that considerable folding of the

enkephalin chain was required to make it conform roughly to the

morphine structure.

After it had been learned that enkephalin potency could be

greatly enhanced by replacing the glycine in position two with a

Dalanine residue and converting the C-terminals to an amide

(Marley, this volume), we foumd (Chipkin et al. 1981) that the

tetrapeptide Tyr-DAla-Phe-Met-amide was more potent than Metenkephalin and nearly as potent as Tyr-DAla-Gly-Phe-Met-amide.

Evidently the problem with the earlier experiments was not that

the Tyr-Phe spacing is so critical but rather that the shortened

peptide Was being degraded very rapidly, even in the in vitro

assay system. This result led to a systematic investigation

enkephalin analogs having even shorter chains (vavrek et al.

1981) (see table 1). The most surprising result of this

investigation was that Tyr-DAla-phenylpropyl amide had nearly the

full potency of Met-enkephalin on stimulated guinea pig ileum and

produced analgesia in vivo. It is interesting that in this compound the spacing along the peptide chain from the tyrosine to

the aromatic ring is not identical to the spacing in Tyr-DAlaPhe-Met-amide, which we had previously found to be quite potent.

36
TABLE 1.

Relative Potencies on Guinea Pig Ileum of Minimum

Structure Enkephalins

Tyr-Gly-Gly-Phe-Met 100

Tyr-Gly-Gly-Phe-Met-amide 160

Tyr-DAla-Gly-Phe-Met-amide 140

Tyr-DAla-Phet-Met-amide 120

Tyr-Pro-Phe-amide 19

Tyr-DAla-Phe-amide 25

Tyr-DAla-phenylethylamide 6

Tyr-DAla-phenylpropylamide 77

Tyr-DAla-phenylbutyl amide 13

The same spacing as in the tetrapeptide is found in Tyr-DAlaphenylethyl amide, which has very low potency (table 1). In our

potent dipeptide the separation is me carbon atom further. The

reason for this difference is not understood at this time.

Perhaps when the mu receptor structure is known will will have

answers to these questions. Resistance to enzymatic degradation

is doubtless an important factor in the high potency of Tyr-DAlaphenylpropyl amide. The only amide bonds in this compound involve

the amino or carboxyl group of D-alanine, and they should be

quite stable to most enzymes.

Sane work done with Stammer (see Stamner, this volume) on

enkephalins containing -unsaturated amino acids is also of

interest in this regard. When a double bond is introduced between

the alpha and beta carbons, the resulting amino acid is quite

rigid, and the phenyl ring is held in a position toward the

carboxyl in the case of the isomer away from the carboxyl

in the case of the Z isomer.When these two isomers were

incorporated into the potent pentapeptide enkephdlin Tyr-DAla-GlyPhe-Met-amide in place of the phenylalanine residue, the

potencies were dramatically different (see table 2). The analog

containing Z-Phe, in which the aromatic ring is held toward the

tyrosine, was super-potent, while the analog containing E-Phe,

in which the phenyl ring is held away from the tyrosine, had

TABLE 2

Relative Potency on Ileum of Some Altered Enkephalins

The symbol indicates the direction of the phenyl ring in the

analog .

Tyr-Gly-Gly-Phe-Met

Tyr-DALa-Gly-Phe-Met-amide

Tyr-DALa-Gly- ZPhe-Met-amide

Tyr-DALa-Gly- Phe-Met-amide

Tyr-DALa-Phe-Met-amide

Tyr-DAla- ZPhe-Met-amide

100 

150

500

low

130

low

37
FIGURE 7

Solution Synthesis of the Dipeptide Enkephalin

negligible activity on ileum. This result suggests that the

spacing between the binding sites for the aromtic rings on the

receptors is rather closely specified, and that the flexibility

of the normal pentapeptide allows sufficient bending of the

molecule to bring the two aromatic rings into the correct

spacing. Since we had found Tyr-DAla-Phe-Met-amide to be very

potent, we then examined this analog in which the phenylalanine

had been replaced by Z-Phe; this analog was essentially

inactive on ileum. unfortunately, the corresponding E-Phe

analog was not made, but it probably would have had more potency.

Evidently in these compressed analogs the phenyl ring must bend

somewhat away fran the tyrosine ring - or at least not be held

rigidly toward it.

38
The dipeptide amide enkephalins are mainly mu receptor agonists;

they have quite low potency on the stimulated mouse vas differences

and for binding to delta receptors (Vavrek et al. 1983). The

receptor selectivity (and relative potency for guinea pig ileum

versus muse MS deferens) could be enhanced by addition of a

lysine residue to the amino terminus of these short-chain

enkephalins. Surprisingly, addition of the lysine residue caused

only a alight decrease of ileum potency; the increased

selectivity was due to a great loss of vas deferens potency.

The potent dipeptide derivative Tyr-DAla-phehylpropyl amide can

be synthesized readily by either solid phase or classical

solution methods of peptide synthesis. For solid phase synthesis

(Vavrek et al. 1981), the dipeptide Boc-Tyr(BrZ)-DAla was

assembled on Merrifield resin. Brz designates the 2-

bramobenzyloxycarbonyl protecting group used for the hydroxyl

group of tyrosine. The dipsptide-resin was treated for ah

extended time with methanol and Methyl amine to remove the

peptide as the methyl ester. This ester was then treated with

phenyl-n-propyl amine to convert it to the amide. For synthesis

of large quantities of the peptide, the solution synthesis scheme

shown in figure 7 was used. Boc-D-alanine was converted to the

amide with 3-phenyl-n-propyl amine, using DOC as coupling agent.

Following renewal of the Boc group with TFA and neutraliation of

the salt, Boc-Tyr(BrZ) was coupled, again using DOC. Treatment

with TFA for removal of the Boc group and then removal of the BrZ

group with HF in the presence of scavengers yielded the desired

dipsptide amide.

DYNORPHIN-REALTED PEPTIDES

Of the three major classes of opioid peptides, the dyhorphin

group were the last to be characterized (see the chapters in this

volume by Chavkin and Kasterlitz). These appear to be the

endogenous ligands for the kappa class of opioid receptors. Among

the peptides available at this time, dynorphin(1-9) appears to be

a very elective ligand for kappa receptors. It has the amino

acid sequence Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg. Unfortunately,

this peptide is rapidly degraded by enymes, and is thus not

practical for in vivo work. Even for receptor binding studies on

isolated membranes, a "cocktail" of enzyms inhibitors must be

used. One rapid cleavage is at the bond following the leucine

residue; this cleavage yields Leu-enkephalin, which is

principally a delta receptor agonist. This cleavage thus destroys

the kappa selectivity. It would be extremely useful if enzyme

resistance could be built into this peptide to yield a stable,

selective kappa agonist.

The aualog containing D-alanine in position 2 was synthesized

quite soon after the dynorphin sequence was known; that peptide

was found to be mainly a delta agonist. It was suggested that the

peptide was probably being cleaved to yield [DAla2]-Leuenkephalin, a potent delta agonist. In order to investigate this

question, we synthesized several analogs of dynorphin(1-9) which

39
TABLE 3

Potencies of Dynorphin(1-9) Analogs for Binding to

MU, Delta and Kappa Receptors in Guinea Pig Brain

Peptide Structure Relative Potency

Mu Delta Kappa

Tyr-Gly-Gly-Phe-LeU-Arg-Arg-Ile-Arg 1 1 1

Tyr-Gly-Gly-Phe-Leu-DArg-Ile-Darg 42 1.6 0.2

Tyr-Gly-Gly-Phe-Leu-DArg-DArg-Ile-DArg-NH2 77 1.3 0.6

Tyr-DAla-Gly-Phe-Leu-Arg-Arg-Ile-Arg 29 1.9 0.1

Tyr-DAla-Gly-Phe-Leu-DArg-DArg-Ile-DArg 43 7.7 0.04

Tyr-DAla-Gly-Phe-Leu-DArg-Ile-DArg-NH2 81 5.6 0.1

incorporated D-amino acids at various places and in addition

examined the effects of making the C-terminal amide of these

peptides. All of these changes should impart added enzyme

resistance to the peptides. Table 3 gives the structures of the

peptides synthesized and compares their binding to guinea pig

brain receptors. Fran these experiments (Paterson et al. 1984),

done in the cold to minimize enzymatic degradation, it is clear

that all the modifications caused loss of potency at the kappa

receptor while enhancing binding potency at both mu and delta

receptors. Other experiments done at roan temperature or in the

presence of enzyme inhibitors showed that the D-alanine

substitution conferred enzyme stability on the peptides, while

the D-arginine substitutions were not effective. The effect of

the D-alanine substitution in dynorphin peptides then is clearly

to change receptor specificity. Additional work is needed for

develqment of stable, selective kappa agonists.

40
REFERENCES

Barany, G., Merrifield, R.B. Solid phase peptide synthesis. In:

Gross, E., Meienhofer, J., eds. The Peptides. Vol. 2. New

York: Academic Press. pp 3-284.

Bodanszky, M., Klausner, Y.S., Ordetti, M.A. Peptide Synthesis.

NeW York: Wiley, 1976.

Chipkin, R.E., Morris, D.H., English, M.G., Roeamond, J.D.,

Stammer, C.H, York, E.J., Stewart, J.M. Potent tetrapeptide

eukephalins. Life Sci 28:1517-1522, 1981.

Paterson, S.J., Koeterlitz, R.W., Vavrek, R.J., Stwart, J.M.

Effects of D-amino acid substitution in dyhorphin A(1-9) on

binding at the mu- delta- and kappa sites. Neuropeptides (in

press).

Stewart, J.M. Design of peptide hormone analogs. Trends

Phamacol Sci 3:300-303, 1982.

Stewart, J.M. Synthesis and use of nsuropeptides. In: Conn,

M.P., ed. Methods in Enzymology. Vol. 103. New York: Academic

Press, 1983. pp. 3-32.

Stewart, J.M., Young, J.D. Solid Phase Peptide Synthesis.

Rocford,IL: Pierce Chemical Co., 1984.

Vavrek, R.J., Cui, R-L., York, E;J., Stewart, J.M., Paterson,

S.J., Kosterlitz, H.W. Lysine substitution increases muselectivity of potent di- and tri-peptide enkephalin analogs.

Life Sci 33 (Sup 1): 451-454, 1983.

Vavrek, R.J., Hsi, L.-H., York, E.J., Hall, M.E., Stewart, J.M.

Minimum structure opioids - dipeptide and tripeptide analogs

of the enkephalins. Peptides 2: 303-308, 1981.

AUTHOR

John M. Stewart, PhD., Professor of Biochemistry

university of Colorado School of Medicine

Denver, Colorado 80262.

41
Structure-Activity Relationships of

Opioid Peptides

J. S. Morley, D.Sc., and A. S. Dutta, Ph.D.

INTRODUCTION

Since our last review (Morley 1980). several new aspects of the structure-activity

relationships (SAR) of enkephalinlike peptides have been explored. Most are

covered in separate chapters of this volume: SAR of conformationally restricted

opioid peptides Schiller; Hruby); SAR of active di-, tri-, and tetrapeptide

analogs (Stewart); SAR of dehydro-, cyclopropyl-, and multichain analogs

(Stammer); SAR of dermorphin analogs (Feuerstein/Faden), SAR of morphiceptin

analogs (Chang, and SAR enkephalin analogs in relation to specificity (Hruby;

Roques). We have therefore decided to restrict our own coverage to an update of

subject matters contained in our last review--general principles of SAR, the

effect of single substitution in [Met/Leu] enkephalin on activity in isolated tissue

preparations, and selected in vivo effects--and the outstanding additional topics

not covered by other authors.

We discard use of the popular names Met-enkephalin and Leu-enkephalin, since

these contravene nomenclature rules (Morley 1981). Instead, the forms

[Met]enkephalin and [Leu] enkephalin. now recommended by the International

Union of Pure and Applied Chemistry (IUPAC) and the International Union of

Biochemistry (IUB), are employed. Nomenclature proposals for describing

backbone-modified enkephalins (see Morley 1981) are also recommended.

GENERAL PRINCIPLES

“Activity,” as measured in in vitro, “receptor” binding, and even in vivo studies,

continues too frequently to be interpreted as “activity at opiate receptor(s).” This

has often led to speculation as to the nature of opiate receptors based on the

results of SAR studies. In our last review (Morley 1980), which analyzed the

molecular processes involved before activity is expressed, we stressed that such

speculation is unjustified. The interplay of two of these processes--the events at

the receptor, and proteolysis--has now been convincingly demonstrated by

McKnight et al. (1983). By adding inhibitors of peptidases, the observed potency

of [Met]enkephalin, for example, in the guinea pig myenteric plexus-longitudinal

muscle (GPI), mouse vas deferens (MVD), and rat vas deferens (RVD) tests was

increased 18-, 13-, and 200-fold, respectively. The potencies of

[Leu]enkephalin, and hexa-, hepta-, and octapeptide C-terminal extensions of

[Met] and [Leu]enkephalin, were likewise increased in the presence of peptidase

inhibitors, with the greatest effects being seen in the RVD test; whereas the

4 2
potencies of more metabolically stable opioid peptides (e.g., beta-endorphin) were

little affected.

If we can assume that the combination of peptidase inhibitors used by McKnight

et al. (1983) specifically eliminates the capacity of the tissues to inactivate

enkephalins, interpretation of results in isolated tissues becomes more secure.

For example, the 25-fold greater potency of [D-Ala2, MePhe4, Gly-ol5]

enkephalin, as compared with [Met]enkephalin in the GPI test without inhibitors

(which some had previously interpreted as reflecting greater activity of the

analog at the opiate receptor), reduces to near equipotency when inhibitors are

present. If the assumption is correct, we may now conclude that the receptor

interaction of the two peptides is very similar. More instructively, two patterns

of activity remain on after comparing the activities of metabolically unstable

analogs in the ‘inhibited’ GPI and MVD tests, and more stable analogs in the two

normal tests. An enkephalinlike pattern, observed with [Met] and

[Leu]enkephalin,[D-Ala2, L and D-Leu5] enkephalin, and most endogenous opioid

peptides, is characterized by greater potency in the MVD as compared with the

GPI test. In contrast, a morphine like pattern, characterized by greater potency in

the GPI as compared with the MVD test, is seen with some synthetic peptides,

e.g., [D-Ala2, MePhe4, Gly-ol5]enkephalin. This finding is compatible with the

evidence for two distinct receptors--predominantly mu in the GPI and delta in

the MVD--in the tissues, but the dangers of drawing conclusions as to the nature

of receptors based on work with agonists should be recognized.

Use of cocktails of peptidase inhibitors in receptor binding assays has not yet

been described, but we can assume that similar shifts of potencies will be

observed.

RESULTS USING ISOLATED TISSUE PREPARATIONS

The tables given in our last review (Morley 1980) showing the effect of single

substitution (any one amino acid position varied, but not two) on the potency of

[Met] or [Leu]enkephalin, or their methyl esters or amides, are now updated

(tables 1 to 7). We also include data on peptide backbone modifications (table 8);

in these cases, the [D-Ala2]enkephalin series is included since [D-Ala2]enkephalin

has often served as reference compound.

All potencies have again been related to [Met]enkephalin = 1. When

[Leu]enkephalin has been used as standard in the original paper, the conversion

factor [Leu]enkephalin = 0.37 x [Met]enkephalin in the GPI test (Morgan et al.

1976) has been applied. When values for neither [Met] nor [Leu]enkephalin have

been quoted, the IC50 values for analogs have been compared with the following

IC50 values for [Met]enkephalin (McKnight et al. 1983): 125 nM (GPI)50. 17.7

nM (MVD).

Limitation of space permits only cursory discussion of the new results. At the

Tyr1 position, restriction in the degree of conformational freedom of the residue

as in the tetralin analog (table 2, third compound listed) leads to a sevenfold to

eightfold increase in potency in the GPI test, and a thirtyfold decrease in the

MVD test, as compared with the reference compound, [Leu]enkephalin (Deeks et

al. 1983). The inactivity of the indanyl analog (table 2, fourth compound listed)

illustrates the differential effect of restricting conformational flexibilit on

potency in the two tests. Requirement of the p-hydroxy group of Tyr1 for

activity is questioned from the finding that a melphalan analog (in which the phydroxyl group is replaced by bis-2-chloroethylamino) of [D-Ala2

, Leu5]

4 3
TABLE 1

N-Terminlly Substituted Derivatives. In Vitro Activitya of Analogs of

Type X-Tyr-Gly-Gly-Phea

potencies in the eletrically stimulated guinea pig and mouse vas deferences and receptor binding relative to

[Met]enkephal = 1.

bAbbreviations for amino acids and their use in the formulation of derivatives follow the various recommendations of the

IUPAC-IUB Commision Biochemical Nomenclature.

c

comparism of data obtained by different investigators seldom justified.
TABLE 2

Variation of Tyrosine1 Residue. In Vitro Activitya of Analogs of X-Gly-Gly-PheX =

Analogb Guinea Mouse Receptor Reference

pig deferens binding

Y = ileun c

(-) isomer

(+) isomer

(-) isomer

(+) isomer

a

- 

c

 See footnotes to table 1.

Met-Ome 0.005 0.001

Leu-OH

Friedmann et al. 1982

Amar et al. 1983

Leu-OMe 1.3 0.052 Deeks et al. 1983

Leu-OMe 0.028 0.00036

Met-NH2 inactive inactive

Met-NH2 inactive inactive

Met-NH2

inactive inactive

Met-NH2 inactive inactive

Deeks et al. 1983

Ramakrishman & Portoghese

1982

Ramkrishman & Portoghese

1982
TABLE 3

Variation of Glycine2 Residue. In Vitro Activitya of Analogs of Type Tyr-X-Gly-Phea

-

c

 See footnotes to Table 1
TABLE 4

Varaiation of Glycine5 Residue. In Vitro Acivitya of Analogs of Type

Tyr-Gly-X-PheTABLE 5

variation of Glycine2,5 residue. In Vitro Activitya of Analogs of Type Tyr-X-Phea

-

c

 See footnotes to Table 1.
TABLE 6

Variation of Phelylalanine4 Residue. In Vitro Activitya

 of Type Tyr-Gly-Gly-Xa

-

c

 See Footnotes to Table 1

a

-

c

 See Footnotes to table 1
Variation of Met/Leu5

 Residue In Vitro Activity of Analogs of Type Tyr-Gly-Gly-Phe-X

TABLE 7

(Table 7 continued next page)
Table 7 (continued)a

a

-

c

 See footnotes to table 1.
enkephalin methyl ester has high affinity for mu and delta binding sites (IC60 50

and 10 nM respectively) (Szucs et al. 1984), and activity in the GPI test

(Medzihradszky 1984). Judgment on this issue must be suspended since cleavage

of the bis-2-chloroethyl-amino group to a hydroxy group (via ethyleneimmonium

intermediate), under the conditions of the tests, seems possible, offering an

alternative explanation of the results. However, in support of the conclusion of

Szucs et al., it will be recalled that the (tyrosine) O-methyl ether of

[Met]enkephalin, presumably metabolically stable at the p-position, is claimed to

have significant activity in the GPI test (see Morley 1980). The Azgly3 (-NHNH-CO-) analog still remains the only 3-substituted enkephalin with appreciable

activity in any test, suggesting exploration of planar 2, 3-substituted analogs.

Such analogs have not yet been described (see table 5). At the Met/Leu5

position, substitution of the carboxyl group of [Nle]enkephalin by a phosphonic

or sulphonic acid group causes, respectively, a 5- or 12-fold increase in potency

in the GPI test, and a 26- or 14-fold increase in the MVD test (table 7, 16th and

17th compounds listed) (Bajusz et al. 1980). The increased acidity and bulkiness

of the phosphonic acid group apparently favors interaction at the delta binding

site. In contrast, similar replacements in [D-Nle6]enkephalin result in substantial

loss of activity.

Turning to the backbone modifications (table 8). significant activity in one or

more of the tests results from replacement of the Tyr1-Gly2 peptide bond by

CH2-NH or CH2 -CH2 (Szelke et al. 1979; Hudson et al. 1980), by CH=CH (Hann

and Sammes 1980; Hann et al. 1982; Cox et al. 1980), but not by CS-NH (Clausen

et al. 1983. 1984); replacement of the bond by an aminoxy linkage (CO-NH-O)

provides a weakly active analog (Salvadori et al. 1981). Replacement of the

Gly2-Gly3 peptide bond by CS-NH leads to a 1.48-fold increase in potency in

the GPI test and a 15- to 22-fold increase in the MVD test (Clausen et al. 1983,

1984); other modifications of the bond (CH=CH, CH2S, CH2-NH, CH2

-CH2, CONH-O) lead to inactive analogs (see table 8 for references). Replacement of the

Gly3-Phe4 peptide bond by CH2-NH leads to slightly increased potency in the

GPI test, and to decreased potency in the MVD test (Szelke et al. 1979); other

modifications lead to almost inactive analogs (CH2-S. CH2-SO, CO-NH-O).

Finally, all replacements of the Phe4-Met/Leu5 peptide bond (CS-NH, CH2-NH,

CO-NH-O. CH2-S, NH-CO, CO-O) lead to analogs having potencies similar to,

or slightly greater than. those of the reference compound (analogs in the D-Ala2

series should be compared with [D-Ala2]enkephalin).

ENKEPHALIN ANTAGONISTS

A significant development in the past 2 years has been the emergence of

antagonists which are structurally related to the enkephalins. They are of two

main types--one based on N,N-diallyenkephalin with delta binding site

specificity, and the other based on enkephalin (1-4) tetrapeptide dialkyl amides

with mu binding site specificity. The former type has probably been explored

more extensively, but little of the work has yet been published. All published

work on both types is summarized in table 9.

Dealing first with the N,N-diallyl type, it has been known for some years that

substitution of the (N) methyl group of morphine and other opiates by certain

lipophilic groups (e.g., allyl, cyclopropylmethyl) leads to loss of agonist activity,

and may provide analogs with potent antagonist activity (e.g., naloxone,

naltrexone). Monosubstitution of such groups at the amino terminus of

enkephalins provides only weak agonists or partial agonists, but Shaw et al.

(1982) found that substitution of diallyl at the amino terminus provided an

51
TABLE 8

In Vitro Activitiesa of backbone Modified Analogs of Tyr-Gly-GLy-Phe-Met and Tyr-Gly-Gly-Phe-Leu

(Table 8 continued next page)
Table 8 (continued)
Table 8 (continued)

(Table 8 continued next page)
Table 8 (continued)

a

- 

c

 see footnotes to table 1.
TABLE 9

Enkephal in Antagonist and Their Potency
analog, N,N-diallyl-[Leu]enkephalin methyl ester (ICI 139462) (table 9, , 2nd

compound listed), devoid of agonist activity, which effectively antagonized the

action of [Leu]enkephalin in the MVD, but was much less effective in

antagonizing the action of normorphine. Based on this lead, the metabolically

more stable N,N-diallyl-Tyr-Gly-Gly-psi(CH2S)-Phe-Leu-OH (ICI 154129)

(table 9, 8th compound listed) evolved, which is approximately 30 times more

potent against [Leu]enkephalin than against normorphine in the MVD, and which

is effective in vivo in antagonizing a response mediated by delta-type opiate

agonist (Gormley et al. 1982). No simple relationship was found to exist between

the agonist activity of a given enkephalin analog and antagonist potency of its

diallyl congener (Belton et al. 1983). Thus, N,N-diallylation of the potent and

relatively mu selective agonists [D-Ala2Leu5]enkephalin. [D-Met2,

Leu5]enkephalin methyl ester. and (D-Met2, Pro-NH2

5

]enkephalin gave rise to

weak nonspecific antagonists (table 9; 4th, 6th, and 7th compounds listed). In

these analogs, the delta antagonist potencies are severely reduced relative to N,Ndiallyl-[Leu]enkephalin. More surprisingly, the N,N-diallyl analog of the potent

and relatively delta selective agonist [D-Ala2, D-Leu5]enkephalin was found to be

a weak delta partial agonist, with little or no mu antagonist activity (table 9, 5th

compound listed). Replacement of the Gly3-Phe4 peptide bond by a

methylenethio (CH2-S) linkage in [Leu]enkephalin or [D-Phe4, Leu5]enkephalin

gave analogs (table 9; 8th and 9th compounds listed) which were both moderately

potent and highly selective delta antagonists as diallyl congeners. The effect of

replacing the Gly3 residue by D- and L-Ala was also explored by Belton et al.

(results not shown in table 9). Replacement of Gly3

 by L-Ala led to sevenfold

reduction in potency, but the D-Ala3 derivative was equipotent with N,Ndiallyl-[Leu]enkephalin. This result contrasts markedly the results of 3-

substitution in enkephalin agonists.

The tetrapeptide N,N-diallylamide type of antagonists arose from an investigation

of structural substituents which may interact with complementary structural

features adjacent to the opiate receptor (Bower et al. 1981). In this series,

antagonist activity was found to be restricted to a relatively limited range of

structures; the nitrogens of Phe4 and C-terminal amide must be fully substituted,

and the latter must bear a 2-phenylethyl group. Greater structural flexibility is

tolerated with respect to the other obligatory C-terminal amide substituent;

isoamyl, phenethyl, and 3-methylthiopropyl (table 9; 10th, 12th, and 13th

compounds listed) substituents provided essentially pure antagonists of

approximately equal activity, which reversed the effect of morphine in the MVD

at least 10 times more easily than they reversed the effects of [Met]enkephalin.

When the second substituent on the terminal amide was methyl (table 9; 11th

compound listed), antagonism in the MVD test was weak, and the analog was a

weak agonist in the GPI test.

IN VIVO TESTING

Attention continues to be mostly directed to assessment of the antinociceptive

activity of analogs, using the mouse tail flick, hot plate, or writhing tests.

Results with the most potent analogs, administered by various routes, have

recently been summarized (Morley 1983). Results with a more comprehensive

range of analogs are given in table 10.

57
TABLE 10

Molar Potencies of Enkaphalin Analogs Relative to MetE (In Vitro Tests) or Morphine (In Vivo Tests)

(Table 10 continued next page)
TABLE 10 (continued)

Mouse writhing test (algesic agant phanylquinone or unspecified).

aResults obtained using electrically-stimulatad guinea pig myenteric plxus-longitudinal muscle test (GPI), or mouse vas deferens (MVD) teat.

bAnalog administered intracerabroventricularly (icv). subcutaneously (sc), intravenously (iv), intraperitoneally (ip), or orally (po);

usually in mice (SC, iv, ip, po), or rata (icv).

cAbbreviation for amino acids and, their use in the formation of deravatives follow the various recommendations of the IUPAC-IUB Commission

on Biochemical Nomenclature. Pip = L-pipecolic acid; HCys = L-homocysteinethiolators; Nfe = (R) 2-amino-4-phenylbutanoic acid; Ia = 3-

methylbutyl.
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63
AUTHORS

J.S. Morley, D.Sc.

Pain Relief Foundation

Walton Hospital

Liverpool L9 1AE

United Kingdom

A.S. Dutta. Ph.D.

I.C.I. Pharmaceuticals Division

Alderley Park

Macclesfield, Cheshire SK 10 4TG

United Kingdom

64
Design Principles: Enkephalins With

Predictable Mu/Delta Receptor Specificity

Yasuyuki Shimohigashi, Ph.D.

INTRODUCTION

Since the discovery of enkephalins in 1975 (Hughes et al.), numerous

studies have been done during the last 10 years to elucidate the

structure-activity relationships (SAR) of enkephalins and related

peptides by scientists belonging to various disciplines. A number

of questions have been raised, such as, What determines the receptor

affinity? What determines the biological activity? What determines

mu/delta receptor selectivity? etc. Considerable activity has been

directed toward answering these questions, and by now a reasonable

amount of infomation is available to answer the above questions.

Table 1 shows the number of publications in enkephalin and related

areas, as listed in Biological Abstracts and chemical Abstracts.

It is interesting to note that publications in this area of research

have shown a steady growth both in biological and chemical abstracts;

however, there appears to be a major growth in chemical abstract

publications, particularly in the medicinal chemistry area. This

indicates the interest of chemists in important research topics such

as structure elucidation. conformational similarity of enkephalin to

morphine, the finding of multiple types of opiate receptors, and the

recent finding of various enkephalin-containing peptides in precursor

proteins and mechanisms for processing. Among them, the most important turning point on the SAR studies of enkephalins is the postulation Martin et al. 1976; Lord et al. 1977) and confirmation

(Robson and Kosterlitz 1979; Chang and Cuatrecasas 1979) of a multiplicity of opiate receptors: mu receptors to which morphine binds

preferentially, and delta receptors which prefer natural enkephalina.

Although before that point most of the synthetic efforts were centered

on obtaining highly bioactive peptides (Coy and Kastin 1980; Morley

1980), it is obvious that the accumulated information by such SAR

studies has greatly helped us to better understand the receptor

specificity to design more specific and more selective ligands for

each receptor.

Recent research highlights in the genetic structure elucidation of

protein precursors have suggested the presence of a number of

65
TABLE 1

Numbers of Enkephalin Publications Listed in

Biological and Chemical Abstracts

Year Biological Chemical

Abstracts Abstracts

1975 1 3

1976 14 35

1977 143 174

1978 224 291

1979 412 201

1980 580 535

1981 692 326

1982 752 494

1983 522 587

(Searched by NIH Library Medline)

enkephalin like pept ides having the enkephalin sequences in their Nterminal portion. These include elongated-enkephalins (Noda et al.

1982; Gubler et al. 1982; Comb et al. 1982), neo-endorphins and

dynorphins (Kakidani et al. 1982), and endorphins (Nakanishi et al.

1979). It is not clear yet whether these peptides have different

functions and receptors. On the other hand, sane studies have suggested that the opiate receptors designated as “delta” may not be

so closely related in the central and peripheral nervous systems

(Brantl et al. 1982; Shimhigashi et al., unpublished). The elucidation of such a further heterogeneity of the receptors and their

functions must be one of the research directions of future SAR studies on enkephalins, and thus it becomes more important to have an

insight into the interaction between the enkephalins and their

receptors.

A new generation of the SAR studies has also been developed recently

by the bioevaluation of various conformationally restricted analogs

such as dehydro- (Shimohigashi et al. 1981a. 1982b). cyclopropyl-

(Kimura et al. 1983). cyclic- (Schiller and DiMaio 1982; Mosberg et

al. 1983). and thioamide- (Clausen et al. 1984) enkephalins, and of

dimeric enkephalins (Shimohigashi et al. 1982b, 1982c). Such more

dynamic structure-activity studies are certainly useful as probes of

specific receptor interactions.

In this article, the classical structure-activity studies of enkephalins will be reviewed briefly and reevaluated in order to better

interpret the new SAR data specific for delta and mu opiate receptor

Structural elements in the enkephalin molecule essential to the specific interaction with these receptors will be analyzed as character

istics for the predictable receptor specificity.

66
ENZYMOLYSIS OF ENKEPHALINS

Enzymatic Degradation of Enkephalins

The in vivo inactivation of opioid peptides is due to their susceptibility to enzymatic degradation during passage to their receptors

(Schwartz et al. 1981). Studies of 3H-enkephalins in mice and rats

have shown that their half-life is extremely short (Meek et al. 1977;

Stine et al. 1980). Enzymatic hydrolysis is possible at all the

peptide linkages in the eknkaphalin sequence. A shown in the figure 1. 

i.e., Tyr1-Gly2 by amniopeptidases (Hambrook et al. 1976; Marks et

al. 1977): Gly2-Gly3 by dipeptidylaminopeptidase (enkephalinase B)

(Gorenstem and Snyder; 1979), Gly3-Phe4 by enkephalinase (Malfroy

et al. 1978), and Phe4-Leu5 (or Met5) by carboxypeptidases (Marks

et al. 1977; Meek et al. 1977). The most important cleavage is the

removal of the Tyr1 residue by aminopeptidases, which have been found,

besides in blood (Coletti-Previero et al. 1981; Turkall et al. 1982),

as a membrane-bound form in brain (Schnebli et al. 1979; Hui et al.

1983). neuroblastoma x glioma hybrid cells (NG-105-18) (Deslauriers

et al. 1980), and guinea pig ileum (GPI) (Geary et al. 1982). Enkephalinase, a membrane-bound carboxydipeptidase; seems responsible

for the metabolism of enkephalins, and its distribution in brain has

been demonstrated to be in good agreement with that of enkephalin

and opiate receptors (Schwartz et al. 1981).

For the prevention of these enzymatic degradations, potent and specific inhibitors of enkephalin-degrading enzyms have been developed

especially to protect the peptide in the bioassay systems. Puromycin (Barclay and Phillipps 1978; Vogel and Altstein 1979) and

bestatin (Wagner and Dixon 1981) have been shown to be potent inhibitors of aminopeptidases. Thiorphan (Roques et al. 1980), some hydroxmic acid derivatives of peptides (Hudgin et al. 1981) and amino

enkephalinase B carboxypeptidases

H—Tyr 1—Gly2—Gly3—Phe4—Met 5(or Leu5)—OH

aminopeptidases enkephalinase

FIGURE 1

Enzymatic Degradations of Enkephalin

67
acids (Coletti-Previero et al. 1982), and phosphoryl-Leu-Phe

(Altstein et al. 1982) strongly protect the enkephalins fran the

action of enkephal inase. Bacitracin, a nonspecific inhibitor of

a variety of peptidases, inhibits the aminopeptidases and enkephalinase equally well (Schwartz et al. 1981; Hudgin et al. 1981) and

it is utilized most frequently for protection of enkephalin assay

systems.

Blood-Brain Barrier

Another important key to in vivo biological activities after systemic

administration of the enkephalin is their passage through the bloodbrain barrier. After administration of enkephalin analogs by both

the intracranial and intraperitoneal routes and then monitoring the

general activity in goldfish, Olson et al. (1978) suggested that

enkephalin analogs might cross the blood-brain barrier. Rapoport et

al. (1980) have also shown, by using radioactive opioid peptides,

that they have a moderate and significant cerebrovascular permeability in rats. However, Pardridge and Mietus (1981) have suggested

that a high-affinity receptor or transport mechanism for Leu-enkephalin does not exist in the blood-brain barrier, and the barrier

contains active aminopeptidase and enkephalinase components. Thus,

the interaction of enkephalins with the barrier have been characterized by both a very low permeability and a high enzymic degradative

activity. Such a low permeability (2% to 3%) was also demonstrated

by Conford et al. (1978). The reported results regarding the enkephalin transport through the blood-brain barrier are somewhat

conflicting.

HIGHLY BIOACTIVE ENKEPHALIN ANALOGS RESISTANT TO ENZYMOLYSIS

Effect of General Modifications on Enkephalin Enzymolysis

Since enkephalins are immediately degraded by a variety of peptidases

before they reach their receptors, one of the synthetic approaches to

more active analogs of enkephalins has been directed toward the prevention of these enzymatic degradations to prolong their circulatory

life. This has been done by various chemical modifications of the

peptide molecule. In general, the replacement of L-amino acids by

D-amino acids and N-methylation of amide linkages are ordinary procedures to prevent the enzymatic hydrolysis of the peptides. The

results of these modifications of enkephalins are clear, as shawn in

table 2. Enkephalin has three optically active amino acid residues:

Tyr, Phe, and Leu (or Met). The Tyr and Phe residues cannot tolerate the L- and D-amino acid substitution, destroying almost all of

its activity (Coy et al. 1976; Beddell et al. 1977a). Even the

combination of D-Tyr and D-Phe completely destroys the receptor

affinity (Shimohigashi et al., unpublished data). These results

may be related to the importance of the stereo-orientation of aromatic groups in enkephalins to opiate receptor recognition (Shimohigashi et al. 1984b). However, the Leu5

 residue is quite insensitive to the replacement by D-Leu, which retains almost complete

activity.

68
The N-methylation of the Gly2

 and Gly3 residues interferes with the

interaction of the peptide with the receptors

while the N-methylation of the Tyr1

 and Phe4 residues preserves con-

(Beddell et al. 1977b).

siderable activity (Beddell et al 1977b; Dutta et al. 1977). The

substitution of MeLeu for the Leu5 residue has been shown to enhance

the activity (Dutta et al. 1977). It should be noted that such

small structural modifications alter dramatically the activity of

enkephalins.

Classical Structure-Activity Studies of Enkephalins

Numerous chemical modifications for each amino acid residue in the

enkephalin sequence have been done as a structural variation for the

elucidation of dynamic structure-activity relationships. A number

of SAR studies have clarified the mine acid residues that are essential

Phe4 residues, as shown in figure 2. In general, chemical modifito biological activities. These include the Tyr1

, Gly3 and

cation of these residues resulted in loss of activity. In order to

obtain a highly bioactive analog, other residues like Gly2 and Leu5

or Met5 have therefore been the main target of modifications for

prevention of enzyrmolysis and for increased affinity with the

receptors.

Tyrosine-l. For the Tyr1

 residue, all of its structural elements

are required for the biological activity (table 3). The p-hydroxy

group (Morgan et al. 1976; Beddell et al. 1977a), aromatic ring,

with only one methylene spacer between the ring and -carbon (Beddell et al. 1977b; Dutta et al. 1977). asymetry of the -carbon

(Coy et al. 1976). and amino group (Morgan et al 1976; Gacel et al.

1979) are all structural requirements of the Tyrl residue for the

TABLE 2

Relative Activitieis of Leu-enkephalin Analogs

Raving D-Amino Acid or N-Methylamino Acid Substitutionsa

Substitutions Relative Potency

(MVD)

D-amino acid

D-Tyrl

D-Phe4

D-Leu5

N-methylamino acid

MeTyr1

Metyr1

Sar3

MePhe4

MeLeu5

0.4

0.03

118

63

0.3

0.3

0.3

150b

a) Calculated relative to Leu-enkephalin (=100) using data

of Beddell et al. (1977a) and Dutta et al. (1977).

b) Relative activity in GPI.

69
FIGURE 2

Chemical Structure of Enkephalin and Its Structural Elements

Essential for Biological Activities (With Shadowing)

Leu-enkephalin: R = CH(CH3)2, Met-enkephalin: R = CB2SCH3

TABLE 3

Relative Activities of Met-enkephalins

Having Chemical Modifications on Tyrosine-1 Residue

Modifications Relative Potency”)

GPI MVD

References

Amino Group

des-amino

NH2CH2CONH2

(CH2)2-5COCH3COCH3

-

CH3(CH2)1-7-

p-Hydroxy Group

des-hydroxy

CH3OTyr-substitution

des-Tyr

D-Tyr

homo-Tyrb)

p-OH-pheGlyc)

Inactive inactive Morgan et al. 1976

19 30 Gacel et al. 1979

1.3 0.3 Gactl et al. 1979

0.1 Dutte et al. 1977

145 23 Somers & Hayes 1981

4-67 0.8-2.7 Summers & Hayes 1981

0.2

inactive

0.2

0.03 Morgan et al. 1976

2.3 Beddell et al. 1977b

0.1 Dutta et al. 1977

0.1 Coy et al. 1976

3.7 Beddell et al. 1977b

Dutta et al. 1977

a) Relative to Met-enktphalin (-100)

b) Substituted by Leu5

c) Substituted by Leu-OCH3

5

70
fundamental activity of the enkephalin. The removal of the residue,

per se, eliminates canpletely the activity (Hambrook et al. 1976).

Thus, tyrosine is an essential constituent of the enkephalin, and

there appears to be no way at present to modify it chemically for

an increased receptor affinity. The only exception, however, is

N-alkylation by methyl or pentyl of the amino group, which gives a

fairly good activity in an isolated and electrically stimulated

smooth muscle preparation of the GPI (Sumners and Hayes 1981).

Glycine-2. Since any chemical modifictaion of the Tyr1 residue

destroys the activity, the prevention of degradation by aminopeptidases should be achieved by modification of the Gly2

 residue.

Fortunately, the replacement of this residue with various D-amino

acids leads to a preservation or even enhancement of potency. For

example, D-Ala (Coy et al. 1976; Beddell et al. 1977b; Walker et al.

1977), D-Met (Dutta et al. 1977; Shaw et al. 1978), D-Ser (Shaw et

al. 1978), and D-Thr (Dutta et al. 1977) in position 2 give a fairly

active enkephalin analog.

isolated a novel opioid

Surprisingly, Montecucchi et al. (1981)

which contains the D-Ala2

peptide “dermorphin” fran the skin of frogs,

Gly-Tyr-Pro-Ser-NH2.

residue in its sequence: H-TYr-D-Ala-PheGlycine-3. The Gly3

 residue is essential for the enkephalin activity. Almost all the modifications of this residue eliminate the

activity. The exceptions are substitutions with azaglycine (Dutta

et al. 1977) and dehydroalanine (Shimohigashi and Strammer 1982b)

which exhibit a considerable potency in the GPI and in receptor

binding, respectively.

Phenylalanine-4. In order to overcome the enzyme barrier by enkephalinase at the Gly3

-Phe4 linkage, N-Methylation of the Phe4

residue results in retention of some activity (table 2) and seems

synergistic with further modifications in other positions for a

higher activity. Whilst, placement of D-Phe or any other D-amino

acid in position 4 results in a total loss of activity. The bulkiness or hydrophobicity of the side-chain may be one of structural

requirements at this position, since a considerable retention of

potency has been observed when the aromatic ring is saturated

(Dutta et al. 1977) or when it is substituted by carboranylalanine

containing boron atoms (Eberle et al. 1977) or by tryptophan

(Schiller et al. 1978). The stereo-orientation of the phenyl group

is apparently important in receptor recognition, as evidenced by

synthesis of dehydrophenylalanine4-enkephalins (Shimohigashi et al.

1982d, 1984b) and cyclopropylphenylalanine4-enkephalins (Kimura et

al. 1983). in which the phenyl ring is conformationally restricted

to each particular arrangement.

Leucine or Methionine-5. The native enkephalins, Leu- and Metenkephalins. possess a difference only at this position in their

amino acid sequence. Leu-enkephalin is slightly more active in isolated smooth muscle of the mouse vas deferens (MVD) and less active

(20% to 40%) in the GPI (Beddell et al. 1977b; Dutta et al. 1977)

(table 6). Although it is generally accepted that the MVD contains

predaninantly delta receptors, and that the GPI contains

71
predaninantly mu receptors, it is not clear yet that even these two

natural enkephalins have different receptors and functions.

For prevention of degradation by carboxypeptidases, modifications

of the C-terminal COOH group by esterification, amnidation, or reduction to alcohol increase the activity considerably in the GPI, but

not in the MVD. This fact is one of the important lines of evidence

for postulation of a multiplicity of opiate receptors and is also

important information regarding the structural requirements of this

residue for synthesis of the receptor selective analogs (Ronai et

al. 1979, 1981). Replacement of leucine by an amino acid having a

relatively smaller side chain, like alanine, leads to a drastic drop

in potency, while use of a similar sized mine a id,

or norleucine, results in fair activity. Met(O)5-enkephalin exhiblike isoleucine

its 40% to 70% activity of Met-enkephalin (Beddell et al. 1977b;

Schiller et al. 1977). Even the removal of the terminal COOH group

or the Leu5 (or Met5) residue itself results in the retention of

considerable activity when compared with the removal of the Tyr1

residue (Beddell et al. 1977b; Dutta et al. 1977). The elongation

of mine acid residues fran the carboxyl end of the enkephalin sequence does not alter potency and sometimes even elicits a higher

potency (Beddell et al. 1977b; Terenius et al. 1976). This observation seems rather reasonable after the discovery of various potent

elongated-enkephalins in nature.

Highly Bioactive Enkephalin Analogs

Ineffectiveness due to short duration of action of Leu- and Met-enkephalins in most physiological and pharmacological systems is mainly a function of their susceptibility to enzymatic degradation.

The bioevaluation Of the enkephalins has thus required same special

steps to be taken in assay systems: the direct injection into a target tissue, temperature adjusmmt (0°C). or addition of proteolytic

enzyme inhibitors. It was evident that further elucidation of their

biological roles and functions would be greatly facilitated by the

availability of opioid peptides resistant to enzymatic hydrolyses

and having greater activity. This line of study has, of course,

aimed at the development of more effective analgesics than at the

usual alkaloid opiates. The term "highly bioactive" must be satisfied by the following requirements: (1) an increased receptor affinity, (2) an increased enzyme stability, (3) an increased transportability, and (4) an increased permeability of the blood-brain barrier.

It is important for the SAR study to evaluate the extent to which

the above requirements play a role in the molecular mechanism of

action of enkephalin analogs. Classical structure-activity studies

have clarified some such components essential to enkephalin activity

(figure 2). The design for the synthesis of highly potent analogs

has been carried out by a canbination of chemical modifications of

portions not included in the essential structural elements.

Pentapeptide enkephalin analogs. Table 4 lists sane representative

examples of highly bioactive enkephalin analoga and their activities

in vivo and in vitro. The first reported analog was [D-Ala2

,MetNH2

5

]-enkephalin,having modifications at positions 2 and 5

72
TABLE 4

Relative Potencies of Highly Bioactive Pentapeptide Enkephalin Analogs

Enkephalins

Inhibitory Activitiesa) Analgesic Activitiesb)

GPI MVD Reference c) I.C.V. I.V. Referencesc

)

D-Ala2,Met-NH2

5

D-Ala2,MePhe4,Met(0)-ol5

D-Ala2,D-Leu5

D-Ala2

rMeMet-NH2

5

D-Ala2,F5Phe4,Met-NH2

5

D-Met2,Pro-NH2

5

D-Met2,Thz-NH2

5

Leu-enkephalin

Met-enkephalin

morphine

780

2,120

330

2,920

380

9 4

2,720

500

500

9 7

2 0

100

220

119

100

3

Shav 1978

Kosterlitz 1980

Rosterlitz 1980

Frederickson 1981

Coy 1978

Shav 1978

Kosterlitz 1980

Kosterlitz 1980

100 3Pert 1978

1,000 640 Roemer 1977

1 5 8 Belluzzi 1978

23,800 Rederickzon 1981

1,000 Coy 1978

7.800 590 Bajusz 1977

2,700 480 Yamashiro 1977

weak Inactive Beddell 1977a

0.01 Inactive Beddell 19778

100 100

a) Relative to Met-enkephalin (-100)

b) Relative to morphine (-100)

c) Only shows top authors listed in references.
simultaneously (Pert et al. 1976b; Coy et al. 1976; Shaw et al.

1978). This analog binds to opiate receptors with almost the same

affinity as Met-enkephalin and causes profound and long-lasting

analgesia when injected into rat brain (Pert et al. 1976b). After

a systematic SAR study, Roemar and coworkers (Roemer et al. 1977;

Roemer and Pless 1979) achieved the synthesis of [D-Ala2, MePhe4

Met(O)-ol5]enkephalin (FK 33-824), utilizing a series of modifications with D-amino acid at position 2, N-ethylation or p-nitration

of Phe and with amino acid alcohol at position 5. This compound

is extremely bioactive and produces analgesia even when administered

orally. Many other analogs, some of which are listed in table 4,

have been reported as highly bioactive enkephalins (see previous

reviews by Coy and Rastin 1980; Morley 1980). However, their receptor affinity is not necessarily improved drmatically and most

of the analogs invariably lose the receptor specificity for the

delta and mu receptors to result in nonselective or mu-preferential

1 igands . Thus, their high potency has been suggested to be due to

the combined effects of enzyme stability in the circulation and

tissues and due to favorable transport properties.

Enkephalin analogs with shortened sequence. McGregor et al. (1978)

have shown that the tetrapeptide amide resulting from the removal

of the amino acid in position 5 is as potent as morphine in vivo

and suggested that the tetrapeptide sequence would be a minimal

structural requirement essential for the enkephalin activity. Such

reported highly active analogs with shortened sequences are shown

in table 5. Some of tetrapeptide acyl-hydrazide analogs were reported to be several times more active than morphine in the in vivo

assay (Fujino et al. 1979). One of the most successful synteses

of an analog with potent and long-lasting peripheral and central

opioid activity is that of H-Tyr-D-Met(O)-Gly-MePhe-ol by Kiso et

al. (1981), which is 23,300 times and 9 times as active as morphine

in the GPI and tail-flick analgesic assays, respectival

be noted that the removal of the C-terminal Leu5 or Met5 followed

y. It should

by chemical modifications enhances the opioid agonist activity in

the GPI and in vivo, but leads to a considerable drop in activity

in the MVD. This fact indicates at least that the high potency of

shortened enkephalins is due to their retention or increase in affinity for mu receptors, accompanied by a decrease in affinity for

delta receptors.

Enkephalin Analogs Containing Unusual Amino Acids

Modifications of peptide bond with regard to the direction or electrical properties have also been attempted for prevention of biodegradation by proteolytic enzymes. These include the retroinverso

isomers (Chorev et al. 1979), isosteric analogs (Cox et al. 1980;

Hudson et al. 1980; Kawasaki and Maeda 1982), and depsi-analogs

(Gesellchen et al. 1981). Analogs containing so-called fatty amino

acids, such as carboranylalanine and t-butylglycine, in position 4

or 5 were reported to be almost as active as Met-enkepha in in the l

isolated muscle assays (Schwyzer et al. 1981; Do and Sohwyzer 1981;

Fauchere 1982).

74
TABLE 5

Relative Potencies of Enkephalin Analogs Having Shortened Sequences

Inhibitory

Enkephalins Activitiesa

)

GPI MVD

H-Tyr-D-Ala-Gly-Phe-NH2

800 7

H-Tyr-D-Ala-Gly-EtPhe(pF)-NH2 550

H-Tyr-D-Ala-Gly-NHCH2CH2C6H5 440 2

H-Tyr-D-Met(O)-Gly-MePhe-ol 5.1x106

H-Tyr-D-Met(O)-Gly-MePhe-NHNHCOEt

H-Tyr-D-Met-Gly-Phe-NH2 3,760 12

H-Tyr-Ala-Phe-Met-NH2 120

Met-enkephalin 100 100

morphine 220 3

a) Relative to Met-enkephalin (=100)

b) Relative to morphine (=100)

c) Only shows top authors listed in references.

Analgesic

Activity b)

Referencesc)

i.c.v. or i.v.

250 McGregor 1978

Fournie-Zalueki 1981

1.2x106

Shuman 1981

100 Morgan 1917

900 Kiso 1981

400 Fujino 1981

1,810 Ronai 1979

5 0 Chipkin 1981

0.01 Beddell 1977a

100 Kosterlitz 1980
Shimohigashi et al. (1981a) have synthesized a series of dehydroenkephalins containing -dehydrogenated amino acid residues

(-NHC(=CHR)CO-) in each position of [D-Ala2, Leu5]enkephalin and

have shown that dehydrogenation of the enkephalin molecule can

afford a peptide with full receptor and biological activity having

unchanged delta-selectivity. Thus, simple dehydrogenation of an

amino acid residue in a peptide sequence may elicit at least one

or all of three changes in the properties: (1) physicochemical and

conformational, i.e., rigidity, increased hydrophobicity restricted

orientation of ß-substituents, and possible ß-turn type folding;

(2) chemical, i.e., a new site of electrophilicity at the -double

bond; and (3) biological, i.e., an increased resistance to enzymatic

degradation providing a longer in vivo lifetime. Some of these

properties have been demonstrated for dehydro-enkephalins (Shimohigashi et al. 1981b. 1982d. 1983, 1984b).

Enzyme Stability of Synthetic Enkephalin Analogs

Stability of enkephalin analogs synthesized for prevention of proteolytic breakdown has been examined by means of brain hanogenates

and various purified ezymes. and it was demonstrated that the

enhanced in vivo activity of some analogs can be explained by their

greater resistance to enzvmatic hvdrolysis. Met-enkephalin is

completely degraded after 30 minutes by incubation with mouse brain

homogenates, while [D-Ala2, Met-NH2

5

]enkephalin analog is stable

for at least 3 hours (Marks et al. 1978) The Tyr1-D-Ala2 linkage

in FK 33-824 is also quite stable, since 50%. of the compound can

survive similar conditions even after 6 hours (Huguenin and Maurer

1980). Fauchere and coworkers (Fauchere and Petermann 1980; Eberle

et al. 1977) observed a considerable resistance of carboranylalanine-containing enkephalin analog to various purified enzymes such

as aminopeptidase M, -chymotrypsin, and pepsin. When mined with

carboxypeptidase Y, the saturated Leu-enkephalin is rapidly hydrolyzed from the COOH-terminus. However, it was demonstrated that

peptide linkages with -dehydromino acid residues placed in the

enkephalin molecule are strongly resistant to the enzyme at the

carboxyl side and completely resistant at the amino side of the

dehydro residue (Shimohigashi et al. 1982a).

RECEPTOR-SPECIFIC ENKEPHALIN ANALOGS

Multiple Opiate Receptors

Following the postulation of three distinct types of opiate receptors (mu, kappa, and sigma) (Martin et al. 1976), the possible

existence of another type of opiate receptors was proposed for the

endogenous peptides of enkephalins and endorphins (Lord et al. 1977).

Since then, several lines of pharmacological and biochemical evidence have confirmed the heterogeneity of opiate receptors. In

the assay to evaluate the inhibitory activity for the electrically

stimulated contraction of isolated smooth muscle preparations, it

was demonstrated that morphine is more potent than enkephalins in

the GPI by interacting with the mu receptors. However, enkephalins

are more potent than morphine in the MVD, and the receptor

7 6
responsible for the opiate action in this tissue was designated as

“delta.” Although these tissues contain predominantly mu and delta

receptors, it was found that the GPI contains additional kappa and

sigma receptors (Su et al. 1981), and the MVD contains additional

mu and kappa receptors (Lord et al. 1977).

The presence of multiple opiate receptors was also demonstrated in

brain by examining the independent receptor binding affinity using

low concentrations of radiolabeled mu and delta ligands (Chang and

Cuatrecasas 1979; Robson and Kosterlitz 1979). The ratio of receptors in the guinea pig brain was calculated as 25% (mu), 45% (delta),

and 30% (kappa) (Kosterlitz et al. 1981). Radioautographical studies have also elucidated regional differences in the distribution

of the three receptor subtypes in the brain (Pert et al. 1976a;

Goodman et al. 1980; Goodman and Snyder 1982).

The fact that the specific receptor for enkephalins is different

fran that for morphine dramatically altered the research direction

of the structure-activity studies of enkephalins. The classical

SAR studies based on somewhat opportunistic bioevaluations must thus

be reevaluated to find the structural characteristics for discriminative recognition of delta and mu receptors by enkephalin analogs.

Also, a new direction in the synthesis of analogs has been undertaken to obtain more specific and more selective ligands for each

receptor (Ronai et al. 1981; Wüster et al. 1980).

Delta-Receptor Specificity: Enkephalinlike Characteristics

The substantiation of opiate receptor multiplicity has inevitably

required the reevaluation of the specificity and selectivity of

enkephalins for at least two distinct receptors: mu and delta.

Eventually, it became clear that most of the enkephalin peptides,

which bear various chemical modifications for enzyme stability and

favorable transport in the circulation, are nonspecific or mu-preferential and that their pattern of activity resembles that of morphine rather than that of the naturally occurring peptides. There

was an indication that Met-enkephalin is mu-preferential, while Leuenkephalin is delta-preferential , as evidenced by both uncle and

binding assays (Lord et al. 1977; Kosterlitz et al. 1980). Especially for the elucidation of functional roles of the delta receptors, it becomes urgent to unravel the important determinants of

delta specificity and selectivity and then to synthesize more enkephal inlike peptides.

The Tyr1 residue, per se, is essential to the display of enkephalinlike activity via delta receptors.

Replacement of the Gly2 residue with D-alanine increases the potency

5 to 10 times in the muscle assays of both GPI and MVD. When the

delta selectivity is calculated, however, Met- and Leu-enkephalins

show a different profile due to their different extent of potency

increments in each tissue. [D-Ala2]-Met-enkephalin shows almost

unchanged selectivity of Met-enkephalin because of simultaneous gain

in mu and delta activities, whereas delta-preferential Leu-enkephalin

7 7
drastically loses its delta selectivity by D-Ala2

-substitution due

to a marked increase in mu activity (table 6). These results indicate that a hydrophobic subsite for the specific interaction with

the D-Ala2 residue may exist in the mu receptors (Fournie-Zaluski

et al. 1981). -Dehydrogenation of D-Ala2 in Leu-enkephalin provides further evidence that a hydrophobic character in position 2

facilitates the interaction with the mu receptors (Shimohigashi and

Stammer 1982a). On the other hand, a nonhydrophobic character in

position 2 appears favorable for interaction with the delta receptors.

As for the Gly3 residue, it seems that the presence of this amino

acid itself in enkephalin is important for delta, but not mu, receptor interaction. The evidence supporting this postulate are as

follows: (1) Chipkin et al. (1981) reported that the tetrapeptide

analogs of des-Gly3-enkephalin are potent agonists in the GPI; (2)

dermorphins , having an N-terminal amino acid sequence very similar

to enkephalin but missing the Gly3 residue, also display high mu

activity (Broxardo et al. 1981); and. similarly, (3) morphiceptin or

beta-casomorphin-4-amide (H-Tyr-Pro-Phe-Pro-NH2). which lacks Gly3,

is highly selective for the mu site in rat brain membranes (Chang

et al. 1981; Brantl et al. 1982). These results suggest that the

presence or absence of glycine at position 3 in these enkephalin like

peptides influences the interrelationship of the Tyr1 and Phe residues, and such a relationship between the two aromatic groups appears

to be of great importance in discriminative receptor recognition

(Chang and Cuatrecasas 1981). Thus, the delta receptors may require

the Gly5 residue for enkephalins as an adequate spacing element

between the Tyr1 and Phe residues, as one of the conformational

determinants of the enkephalin molecule, or as a structural factor

to interact directly with a particular subsite located in the delta

receptors.

The phenyl ring of the side chain in Phe4

 plays a crucial role in

delta-receptor recognition. The importance of its existence in position 4 was demonstrated by Rogues et al. (1979) by replacement of

the phenyl ring by an aliphatic hydrophobic moiety, which causes a

total loss of activity in the MVD but retention of some activity in

the GPI. Whilst, the importance of the stereo-orientation of aromatic groups (Tyr and Phe) in enkephalins to opiate receptor recognition has been emphasized by the synthesis of dehydrophenylalanine4

-

enkephalins having the Z- (phenyl and C=O, trans) and E- (phenyl

and C=O, cis) configurations (figure 3) (Shimohigashi et al. 1982d,

1984b). The Z-isomer exhibits an enhanced or unchanged receptor

affinity over the saturated analogs, showing a greater delta selectivity. However. the E-isomer is dramatically decreased in its

affinity for both receptors, which is almost equal to that of inactive [D-Ala2

, D-Phe4]-Leu-enkephalin. Due to the difference in the

orientations of the phenyl groups in the Z- and E-isomers of Phe,

the phenyl ring in the zPhe4-enkephalin is held rigidly toward the

amino end of the enkephalin sequence, to which the other aromatic

amino acid (Tyr1) residue appears. In contrast, the phenyl group

in the E-isomer is restricted to the opposite arrangement. These

results clearly show that the molecular shape, restricted and

78
FIGURE 3

Chemical Structures of Dehydrophenylalanine4-enkephaline

Having the Z- (1) and E- (2) Configurations

stabilized by the Z-configuration of Phe4, enhances discriminative

recognition between the opiate receptors. Recent work leading to

the incorporation of cyclopropylphenylalanines into enkephalin has

shown similar results (Kimra et al. 1983; unpublished data). Collectively, a subtle discriminative recognition between delta and mu

receptors by these conformationally restricted analogs may be due

to the different stereochemical requirements of each receptor at the

binding regions corresponding to the two aromatic groups in the enkephalin ligand.

Leu-enkephalin is delta-preferential (table 6), indicating that an

isobutyl side-chain of Leu5 is an appreciable receptor-discriminating

component. Placement of D-Leu in position 5 results in a marked

gain in delta selectivity in muscle assays, as shown in table 6. The

COOH-terminal modifications, such as amidation and esterification,

invariably cause a change in delta versus mu selectivity, arid at

least partially eliminate interaction with the delta receptors.

Therefore, the COOH group can be recognized as one of the most important enkephalinlike characteristics. It should be noted, however,

that the mechanism of a selectivity shift by COOH modifications may

79
TABLE 6

Receptor Selectivity of Enkephalin Analogs with Substitutions at Position 2 and 5

a

)

Amino Acids Inhibitory Activities b

)

position 2 position 5 GPI MVD

Delta c ) Selectivity

Gly

D-Ala

D-Ala

D-Ala

D-Ala

Gly

D-Ala

D-Ala

D-Ala

morphine

Met-OH

Met-OR

D-Met-OH

D-Met-OCH3

D-Met-NH2

Leu-OH

Leu-OH

D-Leu-OH

D-Leu-OCH3

100 100 10.7

560 500 9.5

180 590 35.5

300 300 10.7

130 60 4.9

35 170 51.6

550 900 17.6

330 2,720 88.5

990 160 1.8

220 3 0.14

a) Calculated using data of Beddell et al. (1977a) and Kosterlitz et al. (1980).

b) Relative to Met-enkephalin (=100).

c) Selectivity ratio defined as IC50(GPI)/IC50(MVD).
be different between the binding assays in rat brain and the isolated muscle assays (Shimohigashi et al. 1982d). For the muscle assays,

the existence of the COOH group is distinctly effective for obtaining a high delta potency, while it causes a decrease in the mu

affinity in the receptor binding assays. Sane possibilities may

account for this difference: (1) in the muscle assays a degradation

of the carboxyl-free peptides may occur; (2) in the MVD both mu and

delta receptors are present; (3) the ligands may have same agonistantagonist properties; and (4) the delta receptors in rat brain and

in the MVD may have a structural subtlety in interacting with enkephalin peptides. By characterizing the acidic function of the COOH

group, Bajusz et al. (1980) synthesized a series of analogs containing amino sulfonic acid and amino phosphonic acid residues at position 5. Those analogs had little effect on their receptor selectivity.

In summy, structural elements for each residue that result in

greater specificity and selectivity for the delta receptors in the

enkephalin sequence, or the so-called “enkephalin like characteristics,”

are as follows (figure 4):

(i) the Tyr1

 residue,

(ii) non hydrophobicity in position 2,

(iii) the Gly3 residue,

(iv) existence of the phenyl group and its stereo-orientation in

position 4,

(v) the isobutyl side-chain of Leu5

, and

(vi) the C-terminal COOH group.

FIGURE 4

Enkephalin like Characteristics

Essential for Specific Interactions with Delta Opiate Receptors

81
Leu-enkephalin, per se, which possesses all of these characteristics, can be an ideal ligand for the delta receptor under physiological conditions. Among these characteristics, (i), (iii), (iv),

and (vi) appear to

ence of Tyr1, Gly3,

be particularly important. However, the existand COOH group is inevitable in Leu-enkephalin,

and thus factor (iv), which is related to the phenyl ring in position 4, seems a very important determinant for eliciting and

enhancing delta specificity and selectivity.

The most utilized delta ligand to date is [D-Ala2, D-Leu5]enkephalin

(DADLE), which exhibits a selectivity ratio, defined as the ratio

of (IC50s in mu/IC5 0 in delta), of 50 to 100 in muscle assays

(GPI/MVD), and of about 10 in binding assays in using rat brain

(

3H-naloxone/3H-DALE). Taking a nonhydrophobic character at position

tion 2 into consideration, Roques’ group has synthesized Leu-enkephalin analogs containing ß-hydroxyamino acid, D-Ser, or D-Thr at position 2, with a Thr extension at position 6 (Gacel et al. 1980; Zajac

et al. 19831. Their selectivity ratio was reported to be 1,000 to

3,000 in muscle assays, but only 7 to 20 in rat brain binding assays. A selectivity ratio of 170 to 330 in similar muscle assays,

however, has been reported by another group (Mosberg et al. 1983),

and thus the design and synthesis of a highly delta-specific ligand

still appear to be of great importance and urgency. It is noteworthy that these reported delta ligands possess little restriction

or rigidity around the Phe4 residue.

Mu-Receptor Specificity

One way to increase mu-receptor specificity is to eliminate sane of

the enkephalinlike or delta characteristics from the peptide molecule without introducing any chemical modifications at the Tyr1

residue. It should be noted that such a synthetic sequence seems

to coincide with the design for highly bioactive enkephalins as

described above. In fact, the peptides having these types of modifications have been proved to be nonselective or mu-preferential

due to their decrease in delta specificity. Highly potent FK 33-824

is mu-selective with a selectivity ratio of about 7 in both muscle

and binding assays, mainly due to a pronounqced gain in mu potency

(Kosterlitz et al. 1980). Similarly, a shortened peptide analog

synthesized by Kiso et al. (1981) was reported to be several thousandfold more selective for mu than for delta receptors in binding

assays (Quirion et al. 1982). This extreme mu selectivity is apparently caused by a drastic loss of delta affinity, but not by a great

gain in mu affinity. This may be related to the removal of the

amino acid residue at position 5 followed by a possible conformational alteration. The most utilized mu-specific ligand to date is

[D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO), an analog of FK 33-824.

This peptide lacks only the side chain of Met(O)-ol5 residue in FK

33-824, and its mu selectivity ratio is almost 100 in rat brain

binding assays Wanda et al. 1981).

Ligand characteristics for mu-receptor specificity can be summarized

as follows: (1) the Tyr1 residue, (2) hydrophobicity in position 2,

and (3) the phenyl ring arranged in a specific array in position 3

82
or 4. It seems that N-methylation of Phe4

, for example, in FK 33-

824 and DAGO, may elicit a favorable interaction with mu receptors

which is presumably due to restriction of sane conformational parameters for suitable spacial arrangement of the phenyl group,

together with an increased resistance to enkephalinase.

Conformationally Restricted Enkephalin Analogs

For the elucidation of more dynamic and definitive structure-activity relationships, the incorporation of conformational constraints

into enkephalin peptides has been recognized as a useful structural

variation to permit an assessment of the stereochemical consequences

on the resulting biological profiles, especially on receptor specificity and selectivity. Since a different conformational feature

required for interaction with different subtypes of opiate receptors

can be obtained fran conformational flexibility, the restriction of

this flexibility may lead to an analog having the conformation to

interact preferentially with only one class of receptors. One

approach toward such a restriction involves cyclization of enkephalin peptides, and it has proven to be adequate to increase mu-or

delta-receptor selectivity (Schiller and DiMaio 1982; Mosberg et al.

1983).

A potent cyclic enkephalin was first reported by DiMaio and Schiller

(1980); cyclfzation was performed between the COOH-terminal carboxyl

group of Leu and the -amino group of D- -diamino butyric acid

placed in position 2. The resulting cyclic peptide was found to be

mu-preferential in both muscle and binding assays, while its openchain analog was almost nonselective (Schiller and DiMaio 1982).

By canparison of the potency and selectivity of cyclic and open-chain

peptides, it is suggested that different receptors have different

structural preferences for each complementary ligand. Schiller et

al. (1981) also synthesized cyclic analogs containing a cystine

bridge between positions 2 and 5, which exhibit moderate selectivities for the mu receptors (figure 5a). Quite interestingly, extremely high delta selectivity has been obtained for analogs of cystine

enkephalin only by virtue of substitutions of cysteine by penicillamine (ß,ß-dimethyl cysteine) at both positions 2 and 5, as shown

in figure 5b (Mosberg et al. 1983). Its delta

3,200 in GPI/MVD assays and 175 in 3H-naloxone/3H-DADLE rat brain

selectivity is almost

binding assays. The structural difference between these two S-S

bridged cyclic enkephalins lies in the existence or absence of a

dimethyl group on the ß-carbon at positions 2 and 5 and amide modification at the COOH terminus. The effect of dimethyl substitutions

seems to cause a dramatic selectivity shift in receptor recognition.

This indicates that the mu and delta receptors have a more subtle

conformational variation for discrimination and optimal interaction

with each specific ligand. It is interesting to note that the Phe4

residue, which impacts an important enkephalin like character, exists

within the constraint of cyclic structure. Another interesting

cyclic analog, synthesized by Siemion et al. (1981). is a semi-rigid

Leu-enkephalin peptide having an azo-bridge between Tyr1 and Phe4

residues (figure 5c). By observing a long-lasting morphine like

analgesic effect in vivo, they suggested that the biologically

83
FIGURE 5

Chemical Structures of Conformationally Restricted Cyclic Enkephalins

(a) Mu-preferential [D-Cys2-D-Cys5]enkephalin Amide,

(b) Delta-Selective [D-Pen2-D-Pen5]enkephalin (Pen: Penicillamine),

and (c) Azo-bridged Enkephalin

active conformation of enkephalin interacting with the opiate receptor must be such that both aromatic rings of Tyr and Phe would

appear close to each other.

Local conformational restriction by incorporation of such unusual

amino acids as dehydro or cyclopropylamino acids, can also affect

strongly the overall molecular conformation of enkephalins and their

receptor interactions. This has been well demonstrated by synthesis

of the Z- and E-isomers of dehydrophenylalanine4-enkephalins, as

mentioned previously.

Camerman et al. (1983) and Karle et al. (1983) have recently elucidated the crystal structure of Leu-enkephalin, in which four

8 4
conformers with extended backbones form an infinite antiparallel ßsheet. An intriguing feature is the interrelationship between the

aromatic rings of Tyr1 and Phe4 residues located on opposite sides

of the backbone. Two of the four conformer are similar and somewhat puckered, having a distance of 8.2-8.9Å between the two aromatic rings, while the other two conformers are also similar in

having a flat backbone (13.3-13.9Å). Such a steric difference in

Leu-enkephalin might be related to the differential recognition of

mu and delta receptors (Shimohigashi et al. 1984b).

Receptor-Specific Enkephalin like Antagonists

For the agonist-antagonist profile, the structure-function relationships of alkaloid opiates are well established. Substitution of the

methyl group by a typical antagonist-inducing pharmacophore, allyl

or cyclopropylmethyl, on the mine nitrogen of opiates usually increases narcotic antagonist activity. However, application of these

modifications to the enkephalin peptides does not result in generation of analogs having an appreciably pure antagonist property (Hahn

et al. 1977; Morgan et al. 1977; Pert et al. 1977) Unfortunately,

the structure-activity studies of enkephalins have shed little light

on how to prepare peptidic antagonists. Specific enkephalin antagonists that have been reported possess very weak receptor affinity

in toto.

Shaw et al. (1982) reported a delta-selective pure enkephalin antagonist having modifications such as diallylation of the amino group

of Tyr1 and replacement of the peptide bond of Gly3-Phe4 by a CH2-S

linkage (figure 6a). Its antagonist activity in reversing the effects of normorphine or Leu-enkephalin on the MVD indicates that the

peptide acts predominantly on the delta receptors. However, its

potency seems considerably poor (Shaw et al. 1982), and its extremely

weak receptor affinity has also been found to be 340 times less than

that of agonist DADLE in rat brain binding assays (Zajac et al. 1983).

On the other hand, Bower et al. (1981) synthesized mu-selective enkephalin antagonists by furnishing additional groupings to the enkephalin molecule, based on the idea that antagonists would bind to

the receptor enviromnent adjacent to the agonist receptor (figure

6b). Their binding to opiate receptor labeled by 3H-naloxone was

almost unaffected by sodim ion addition, indicating a pure antagonist property, although their receptor affinity was 10 to 70 times

less than that of naloxone.

MIF-I, H-Pro-Leu-Gly-NH2, the alleviative of Parkinson’s disease,

has been described as a blocker of the analgesic effects of enkephalins and morphine in the tail-flick test in vivo (Kastin et al.

1979). Furthermore, Dickinson and Slater (1980)demonstrated that

MIF-I weakly and selectively inhibits mu receptors and suggested its

possible role as an endogenous antagonist. Maurer et al. (1982)

showed that a somatostatin analog, H-D-Phe-Cys-Phe-D-Trp-Lys-ThrCys-NH-CH(CH2OH)CHOHCH3

, is an opiate mu antagonist having a receptor affinity of 10% that of naloxone.

8 5
FIGURE 6

Chemical Structures of Enkephalin like Antagonists

(a) Delta-selective N,N-Bisallyl-[Gly 3(CH2S)Phe4]-Leu-enkephalin,

and (b) Mu-selective Des-Leu5

- D-Ala2, MePhe4 enkephalin-

(3-methylthiopropyl)-(2-phenylethyl)amide

DIMERIC ENKEPHALINS AND THEIR RECEPTOR SPECIFICITY

Delta Receptor-Specific Dimeric Enkephalins

A comprehension of the molecular mechanism of interaction between

the receptor and its complementary ligand would facilitate the better

design of more specific and more selective ligands in receptor action. Efforts in clarifying receptor organization and dynamics on

the membrane have somewhat affected the synthetic and biochemical

studies of enkephalins. Among them, the suggestion by Hazum et al.

(1979) that delta receptors might be extremely closely clustered in

the membrane has provided anideato synthesize a newtype of deltaspecific ligand. It is likely that the overall macroscopic affinity

8 6
of a ligand for its receptor is due to multiple interactions between a nunber of chemical elements in the ligand and corresponding

subsites present in the receptor. Structural variations by incorporation of modified groupings or constraints may enhance such

interactions, thereby increasing the receptor affinity. Also,

increased affinity can be attained by an increase in the number of

interactions or in binding energy obtainable with the resulting

multiple attachements to the receptors. In this rationale, multivalent ligands would offer a fair possibility, provided each peptide

unit in a ligand sustains parent active interactions with receptors.

Even a bivalent ligand in which two active molecules are cross-linked

by a spacer is expected to produce at least a twofold enhancement

in affinity, because of the statistical advantage in association.

Furthermore, if the connecting spacer is long enough to permit simultaneous binding or bridging between two adjacent receptor molecules,

the magnitude of enhancement in affinity should be much larger. I t

is likely that such bridging of receptors might be selective for

only a single type of receptor.

A systemic study on dimerization of enkephalin molecules has been

performed by Shimohigashi et al. (1982b, 1982c) on the basis of such

a subsite-interaction hypothesis (Delean et al. 1979) and of the

suggestion of receptor topological organization on the membrane

(Hazum et al. 1979). Earlier studies on enkephalin dimers has involved their synthesis by Coy et al. (1978), Hazum et al. (1982),

and Lipkowski et al. (1982). In our case, the general strategy for

the synthesis of a series of enkephalin dimers was based on traditional SAR studies as described (figure 7): (1) Tyr-D-Ala-Gly-PheLeu sequence was chosen as parent backbone for stability; (2) dimerization was carried out at the COOH-terminal, since the NH2

-terminal

has been established as essential for activity; and (3) methylene

bridges of varying length, (CH2)n with n = 2-22, was utilized as

spacers to provide simplicity, flexibility, and chemical stability.

Dimerization was performed at the carboxyl side of each amino acid

residue successively from the COOH terminus, producing dimeric

pentapeptide- (DPEn), tetrapeptide- (DTEn), and tripeptide- (DIREn)

enkephalins (figure 7). The examenation of the optimal spacer length

among these series would help to characterize receptor organization.

In the first series of DP the monamer for dimerization was a

pentapeptide H-Tyr-D-Ala-Gly-Phe-Leu-NH2 (DALEA), which cannot discriminate well between delta mu receptors. In the mu assays

(GPI and binding assay using 3H-naloxone) , the dimer with spacer of

n = 2. DPE2. showed no significant changes in relative potency compared with the monaner DALEA (Shimohigashi et al. 1982c; Costa et

al. 1985). Increases in the chain length resulted in a progressive

loss of potency In contrast, in the delta assays (MVD and binding

assay using 3H-DADLE) the pentapeptide dimers were considerably

more potent than monomer DALEA. The most active compound was DPE2,

which was eightfold and twentyfold more potent than DALEA in the

binding and MVD assays, respectively. Spacers of 4, 6, and 8 methylene units caused a consistent increase in potency. Further extension of the methylene bridge to n = 10 and 12 resulted in a progressive loss of activity. These results reveal several distinct

87
FIGURE 7

Chemical Structure of Dimeric Enkephalins

Strategy and Expected Variation Following Dimerisation

features about the receptor action of dimers: 1) Nonselective monomer becomes "delta-selective" by dimerization, and the selectivity

ratio of the most specific DPE2 is compatible with that of most

utilized delta ligand,DADLE, in both binding and muscle assays. I t

should be noted that DPE dimers lack the importantenkephalinlike

characteristic of the COOH-terminal free carboxy group, yet they

exhibit delta selectivity. 2) Relative potencies are higher in the

MVD biological assay than expected on the basis of binding affinities

in rat brain or muroblastoma-glioma hybrid cells. 3) The GPI activity and mu-receptor affinity, however, are highly correlated: as

the methylene chain length increases from 2 to 12, there is a progressive drop in potency in both muscle and binding assays. The

fact that the linkage of two molecules of a nonselective ligand

(DALEA) results in a very delta-selective compound with enhanced

biological activity has elicited an attractive hypothesis that the

dimeric enkephalins might cross-link the delta receptors, presumably

clustering to ether with appropriate intermolecular distances (Hazum et al. 1979) or they might cross-link copies of several enkephalin binding sites in the delta receptors (Costa et al. 1985).

Several lines of evidence supporting these postulations have also

been presented by various kinds of kinetic studies (Costa et al.

1982; Krumins et al. 1982).

8 8
The second series involves dimeric tetrapeptides of DTEn. The monomer, H-Tyr-D-Ala-Gly-Phe-NH2

 (DAPHA), showed little difference in

potency in the GPI and MVD assays, but had a tenfold increase in

selectivity for the mu receptor in the binding assays. A comparative study between muscle and binding assays has also been performed

for the DTE dimers (Costa et al. 1985). DTEn with spacers of n = 2,

4, 6, 8, and even 10 methylene units were nearly equipotent with

monaner DAPHA in both GPI and binding assays. In contrast, a spacer

of n = 12 produced a dramatic drop in GPI activity and mu-binding

affinity, having only 8% to 9% of monaner activity (table 7). This

is in contrast with the marked enhancement of potency by DTE 2 in

the MVD and delta-binding assays. Thus, DTE12 becomes a "deltaselective” ligand with a significant selectivity ratio of 20 to 90

(Shimohigashi et al. 1982b; Zajac et al. 1983; Costa et al. 1985).

Again, it should be noted that dimerization causes a striking selectivity shift, although DTE dimers “lack” two enkephalin like characteristics: the carboxyl group and Leu5

 side-chain. In the delta

assay, the DTE dimers are consistently more potent than the DAPHA

monamer . However, the increase in MVD activity is significantly

less than that expected on the basis of delta-binding affinity (Costa

et al. 1985). Such a discrepancy has been seriously discussed as a

function of the spacer length, which produces hydrophobicity (Costa

et al. 1985; Shimhigashi et al. 1984a).

Bifunctional interaction of the most potent dimers of DPE2 and DTEl2

with delta receptors has also been demonstrated by evaluation of

activities of alkylmidated monamers having corresponding spacer

molecules: DALEA-C2 and DAPHA-Cl2 (Shimhigashi et al. 1984a; Costa

et al. 1985). Ethylamide substitution of DALEA, the spacer addition

which creates DALEA-C2, produces a threefold increase in MVD activity, but an insignificant (1.3 times) increase in delta binding

(table 7). When another enkephalin molecule is attached to DALEA-C2

,

TABLE 7

Relative Activities of Enkephalin Monmers and Dimers

for Delta and Mu Opiate Receptors

Delta-activity Mu-activity

Enkephalins MVD 3H-DADLE GPI 3H-NAL

Penta-series

DALEA 100 100 100 100

DALEA-C2 300 130 160 82

DPE2 2,300 800 75 75

Tetra-series

DAPHA 100 100 100 100

DAPHA-Cl2 6 32 8 8

DTEl2 320 1,300 9 8

DALEA-C2 : H-Tyr-D-Ala-Gly-Phe-Leu-NH-CH2CH3

DAPHA-Cl2: H-Tyr-D-Ala-Gly-Phe-NH-(CH2)11-CH3

89
creating a dimer DPE2

, the increment in potency is about sevenfold

in both the muscle and binding assays. This increment is clearly

the net increase due to the bivalency of DPE2. Such an effect of

bivalency, however, cannot be observed in the mu assay systems.

Similarly, the net relative effect attributable to bivalency of the

DTE12 dimer is calculated as a fortyfold to fiftyfold increase in

delta, but not in mu, assays (table 7). It is now evident that most

of the increase in delta activity and affinity obtained by dimers

is the result of the simultaneous presence of the two enkephalin

peptides in one molecule.

In the third series, dimerization was performed using a nearly inactive tripeptide, H-Tyr-D-Ala-Cly-NH2

, with the same synthetic

strategy. The resulting dimeric tripeptide enkephalins, DTREn (n =

2-22), exhibited up to a several hundredfold increase in binding to

delta and mu receptors (Lutz et al. 1985). Delta affinity increased

progressively fran n = 2 to a broad plateau at n = 16. It is interesting to note that if one amino acid residue were approximately as

long as a methylene chain with n = 3, then chain lengths of n = 2-6

for DPEn dimers--the most delta-active dimers in the series--would

correspond to n = 8-12 for DTEn, and to n = 14-18 for DTREn, dimers.

Such optimal spacer lengths have been found and must be related to

a characteristic feature of membrane organization of the delta receptors. Although all the DTRE dimers are mu-preferential or nonselective, the almost 200-fold increase of DTRE16 in delta affinity

is quite notable, since it “lacks” the additional enkephalin like

characteristic of the Phe4 residue.

Several possible mechanisms have been proposed to explain theoretically the gain in delta activity and selectivity by enkephalin

dirmers (Costa et al. 1985). Also, much effort has recently been

centered around the isolation and structure elucidation of the opiate

receptors. In combining these research efforts, it will become feasible to obtain a novel receptor-specific enkephalin ligand, and,

clearly, the study of the dimeric enkephalins appears to be a guidepost.

Mu Receptor-Specific Dimeric Enkephalins

Unexpectadly, the dimeric tripeptide enkephalin with a spacer length

n = 2, DTRE2, showed a dramatic increase in mu-selectivity (Lutz

et al. 1985). Its selectivity ratio of, mu versus delta receptors

was almost 150-fold, with a fair mu affinity (IC50 = 29 nm). DTRE2

is 10 times as potent as its monomer in delta receptor binding, but

almost 400 times in mu binding. Its agonistic activity was also

demonstrated in the GPI assay (Shimohigashi et al., unpublished data).

Although the mechanism of interaction between DTRE2 and mu receptors

is not clear at this manent, a considerable synthetic study is in

progress in our laboratory to elucidate the receptor action of such

shortened enkephal in dimers.

CONCLUDING REMARKS

With numerous structure-activity studies, it is now becoming more

90
evident what determines mu and delta receptor specificity and selectivity. Conspicuous structural elements necessary for the delta

receptor interaction have been analyzed and defined as enkephalinlike characteristics. The characteristics of peptides for the mu

receptor interaction are also praninent in part. Emphasizing or

reinforcing these characters by chemical modifications would offer

a kind of “fully” receptor-specific and selective enkephalin-ligand.

At this moment, two distinct synthetic sequences seem promising for

the design of such ligands: the synthesis of conformationally restricted peptides and the synthesis of multivalent peptides.

Little has been done in the way of SAR studies on elongated-enkephalins and even in kappa receptor-specific dynorphins (Corbett et al.

1982). The natural ligand(s) for the mu receptors is still in question. It is a fact that newly elucidated biological or biochemical

aspects of opiate and opioid peptides have affected dramatically the

structure-activity studies of enkephalins. If the characteristic

membrane feature of each opiate receptor, and then the structural

picture of binding sites, were specified for each corresponding

ligand, even a graphical &sign of the receptor-fitted

be possible. With burgeoning interest in elucidating heterogeneous

ligands would

receptor functions of the enkephalin peptides, the design and synthesis of fully receptor-specific ligands will unequivocally became an

increasingly important requisite.

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ACKNOWLEDGMENTS

The author acknowledges Dr. Dsvid Rodbard of the Natinol Institutes of Health and Dr. Tomaso Costa of the Max-Planck Institute

for their encouragemnt in writing this review and for all of their

supports in the full-scale study on the enkephalina. His sincere

appreciation is also due to Prof. Charles H. Stamaer of the University of Georgia, Dr. Michinori Waki and Rof. Nobuo Izumiya of

Kyushu University, Dr. Hao-Chia Chen of the National Institutes of

Health, and Dr. Yasmasa Kurono and Prof. Kunio Yagi of Institute

of Applied Biochemistry for their collaborative supports.

AUTHOR

Yasuyuki Shimohigashi, Ph.D.

Laboratory of Biochemistry

Faculty of Science

KyuShu University 33

Fukuoka 812, Japan

100
Structure-Activity Relationship and

Pharmacology of the Highly Selective

μ-Opioid Agonist, Morphiceptin

Kwen-Jen Chang, Ph.D.

Morphiceptin is an amidated tetrapeptide with the

sequence of Tyr-Pro-Phe-Pro-NH2 (Chang et al. 1981).

This sequence is contained in ß-casein, which is a

major milk protein. Morphiceptin is structurally

related to the opioid peptide ß-casomorphin, a

heptapeptide (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) originally

isolated from bovine casein peptone (Brantl et al.

1979). The amino acid composition and receptor selectivity of morphiceptin differ from those of

enkephalins. Morphiceptin is a highly selective

opioid peptide agonist for μ-receptors (Chang et al.

1981; Zhang et al. 1981). This property is contrasted

to that of enkephalins which are selective for

-receptors (Lord et al. 1977; Chang and Cuatrecasas

1979). Because of its unique high selectivity for

μ-receptors, a number of morphiceptin analogs have

been synthesized in order to gain insight into the

structural requirements for μ-receptor activity and to

obtain more potent and metabolically stable μ-opioid

peptide. Structure-activity relationship (SAR) of

morphiceptin analogs will be first discussed below in

detail. Several potent analogs have also been

obtained. Some pharmacology of morphiceptin will be

summarized here. One morphiceptin analog, [NMePhe3

,-

D-Pro4]morphiceptin (PL017), shows better receptor

affinity than morphiceptin and is the most potent

μ-opioid morphiceptin analog in in vivo studies (Chang

et al. 1983). The pharmacology of this morphiceptin

analog will be discussed in detail and compared with

classical μ-agonist, morphine, as well as -agonist,

[D-Ala2,D-Leu5]enkephalin (DADLE).

Structure-Activity Relationship

The SAR is based on the μ-receptor binding affinity

determined by the IC50 value, the concentration of a

compound required to decrease 50% of the specific

101
binding of 125I-labeled μ-agonist, FK33824 [Tyr-D-AlaGly-NMePhe-Mel(O)ol], to rat brain membranes (Chang

et al. 1979). The bioactivity of μ-receptors was also

examined to determine their ability to inhibit the

electrically stimulated muscle contraction of isolated

tissues.

TABLE 1

Relative Receptor Binding Activity of Morphiceptin

Analog with Various Substitutions at Fourth Amino

Acid Residue

Tyr-Pro-Phe-X-NH2

X = Pro (Morphiceptin)

D-Pro

Thz

Thz(o)

Tyr-Pro-NMePhe-Y

Y = D-Pro-NH2 (PL017)

D-Pro

D-Pro-ol

-OH

D-Pro-NHNH2

D-Pro-Gly-NH2

μ-Receptor Relative

Affinity” Affinityb

(IC50, nM) (%)

63 100

4.3 1,470

12 530

40 160

5.5 1,150

186 30

5 1,260

1,500 4

2,000 3

8.7 720

a

μ-Receptor binding affinity was determined by the

concentration which inhibited the binding of 125IFK33824 to the μ-binding sites of rat brain

membranes by 50% (IC50) (Chang et al. 1979).

b

The relative affinity is expressed as percent of the

parent compound (100%). Thz = thiazolidine-4-

carboxylic acid; Thz(o) = thiazolidine-4-carboxylic

acid sulfoxide; Pip = pipecolinic acid; Pro =

3,4-dehydroproline; Hyp = 4-hydroxyproline.

Morphiceptin contains two prolines at the second and

fourth positions. Levorotary configuration of proline

at the second position, but not the fourth position,

is essential for μ-receptor activity (Chang et al.

1981) Replacement of L-proline at second position

with D-proline produced an analog with little

activity. In contrast, replacing fourth L-proline

with D-proline, or various cyclic analogs such as

thiazolidine-4-carboxylic acid and thiazolidine-4-

carboxylic acid sulfoxide, yielded peptides of greater

102
activity (table 1). The D-proline substitution is

most effective. This analog, [D-Pro4]morphiceptin, is

about 15 times more active than morphiceptin (Chang

et al. 1983).

As shown in table 1, methylation of amino group of

[D-Pro4]morphicept in produced an analog, [NMePhe3,-

D-Pro4)morphiceptin (PLO17), of comparable μ-receptor

binding affinity but greater in vivo activity (Chang

et al. 1983). Removal of thea amide group of PLO17

attenuates activity. However, the activity is

retained when the amide is changed to the carbinol.

Deleting the fourth amino acid or replacing the amide

with the hydrazine markedly reduces activity.

Activity is almost fully retained when glycineamide is

added to the fifth residue on the carboxyl terminus.

TABLE 2

Relative Receptor Binding Activity of Morphiceptin

Analogs with Various Amino Acids at Third Position

μ-Receptora Relativeb

Affinity Affinity

(IC50, nM) (%)

Tyr-Pro-X-D-Pro-NH2

X = Phe 4.3 100

N-Me-Phe 5.5 78

D-Phe 200 2

Trp 100 4

Pheg 1,500 0.3

Tvr 4,000 0.1

0-Me-Tyr 5,000 0.08

His 25,000 0.017

a

μ-Receptor binding affinity was determined by the

concentration which inhibited the binding of 125IFK33824 to μ-binding sites of rat brain membranes by

50% (IC50) (Chang et al. 1979).

b

The relative affinity is expressed as percent of the

parent compound (100%).

Phenylalanine at the third position is essential for

activity (table 2). Substitution of phenylalanine by

histidine, phenylglycine, tyrosine, or 0-methyltyrosine dramatically reduces the affinity. Replacement of phenylalanine with D-phenylalanine or tryptophan yielded peptides of considerably lower affinity.

Schiller et al. (1983) have also shown that the

addition of a nitro-group to phenylalanine of

103
morphiceptin produces a drastic potency drop which is

sharply contrasted to the enkephalin analogs. The

Phe(pN03) substitution of enkephalins always yields

more potent enkephalin analogs.

The second amino acid can be substituted with other

cyclic amino acid analogs, such as thiazolidine-4-

carboxylic acid, pipecolinic acid, or 3,4-dehydroproline, with only a slight reduction in activity

(table 3) (Chang et al. 1983). Substitution with

other hydrophilic cyclic amino acid analogs, such as

thiazolidine-4-carboxylic acid sulfoxide or 4-hydroxyproline, greatly abolishes the activity. Unlike

enkephalins, the insertion of a glycine between second

and third amino acid virturally abolishes activity.

TABLE 3

Relative Receptor Binding Activity of Morphiceptin

Analogs with Various Amino Acids at Second Position

μ-Receptor Relative

Affinity Affinity

(IC50, nM) (%)

Tyr-X-NMePhe-D-Pro-NH2

X = Pro

Thz

Pip

Pro

Thz(O)

Hyp

Pro-Gly

5.5 100

25 22

16 34

21 34

80 6.9

800 0.69

12,000 0.06

All peptide analogs of morphiceptin that have been

synthesized so far exhibit extremely low affinity

toward -binding sites. The IC50 values determined in

the binding assay against the binding of 125I-labeled

-ligand, DADLE, to -sites are greater than 10 μM

(Chang et al. 1981, 1983). All active morphiceptin

analogs mentioned above retain very high selectivity

for μ-sites. The μ-site selectivity of (NMePhe3,-

D-Pro4]morphiceptin is superior to that of other

μ-agonists, such as morphine, fentanyl, FK33824, and

[D-Ala2,NMePhe4,gly-ol5]enkephalin (DAGO) (Chang 1984).

However, Brantl et al. (1982) have shown that

substitution of D-Ala at the second position leads to

an increased binding affinity to -sites, whereas the

affinity to μ-sites is unchanged; thus, the

selectivity is greatly reduced. Neither -receptors

104
nor -receptors exhibit significant affinity for

morphiceptin and its analogs.

IN VITRO PHARMACOLOGY

Morphiceptin and most analogs can inhibit the

electrically stimulated smooth muscle contraction of

the guinea pig ileum (GPI), a μ-receptor dominated

tissue, as well as the mouse vas deferens (MVD), a

-receptor dominated tissue. The potency in MVD is

about five- to tenfold lower than that in GPI (Chang

et al. 1983). The correlation studies show that the

bioactivities in both tissues are highly correlated

with their μ-site binding affinities. Further studies

measuring the antagonist potency of naloxone revealed

that morphiceptin and analogs interact with

μ-receptors in the MVD despite the predominance of

-receptors in this tissue (Chang et al. 1983).

Naloxone competitively antagonizes PL017 in the MVD.

Schild plots yield a pA2 value of 8.7 for naloxone

against PL017,

of naloxone

which is very similar to the pA2 value

against μ-agonist morphine in the

μ-receptor dominated tissue of the GPI and is of an

order of magnitude greater than that against DADLE in

MVD (Chang et al. 1983). This result raises the

question on the validity of using the relative potency

ratio in GPI and MVD as a means to assess the μ- and

-receptor selectivity.

It was also recently shown that morphiceptin and

PL017, like enkephalin, can induce excitatory effect

on CA1 neurons in superfused hippocampus slices,

suggesting that μ-receptors can participate in the

excitatory effect in the hippocampal formation

(Dingledine et al. 1983 Bostock et al. 1984).

As a highly selective μ-opioid, morphiceptin was shown

to decrease the somatic calcium-dependent act ion

potential of cultured mouse dorsal root ganglion (Werz

and MacDonald 1982, 1983), suggesting that μ-receptors

are involved in the regulation of Cal2+-dependent action

potential in dorsal root ganglion.

It is interesting to note that although the affinity

of PL017 for μ-sites is lower than that of morphine,

PL017 is more potent than morphine in all in vitro

studies (table 4), suggesting a difference in the way

in which peptide and alkaloid activate μ-receptors.

Another difference between alkaloid morphine and

peptide agonist is that desensitization occurs to. the

excitatory effect of morphiceptin but not to morphine

in the hippocampus slices (Dingledine et al. 1983).

105
TABLE 4

Summary of the In Vitro Studies of [NMePhe3,-D-Pro4]-

Morphiceptin and Comparison with Morphine

[NMePhe3,

D-Pro4]-

Morphiceptin Morphine References

Receptor binding

Ki value, nM 5.5 0.4 Chang et al.

1983

GTP sensitivity + +

Na+ sensitivity + +

Isolated tissues,

ED50

 Guniea pig

ileum

Mouse vas

def erens

Hippocampus

slices, ED50

34

240

134 Chang et al.

1983

1,300 Chang et al.

1983

0.4

μM

3 Bostock

et al. 1984

IN VIVO PHARMACOLOGY

Morphiceptin produces transient bradycardia

intravenous (i.v.) administration (Wei et al. 1980)

and elicits analgesia after intracerebroventricular

(i.c.v) injection (Brantl et al. 1982; Chang et al.

1982; Zhang et al. 1981: Barrett and Vaught 1982).

The analgesic activities of morphiceptin and ß-casomorphins correlate well with the μ-site binding

activities (Chang et al. 1982). [D-Pro4

]Morphiceptin

also produces analgesic action (Matthies et al. 1982).

Morphiceptin produces a pressor response following

third, but not fourth, ventricular injection in

unanesthetized rats (Holaday 1982). In pentobarbitone

anesthetized rats, morphiceptin caused hypotension and

bradypnea after injection into the periventricularis

hypothalamus (Feuerstein and Faden 1982); it also

caused tachycardia at the lower doses, as well as

hypotension and bradypnea at the higher doses, after

injection into hypothalamic nucleus preopticus

medialis (Faden and Feuerstein 1983). Morphiceptin

produces catalepsy by i.c.v. administration (Chang

et al. 1982; Holaday 1982). All of these effects are

reversible by naloxone.

106
In vivo studies have also been carried out for PL017

and compared with the classical μ-agonist morphine as

well as -agonist DADLE. These are summarized in

tables 5 and 6.

TABLE 5

Summary of the In Vivo Pharmacology of [NMePhe3,-

D-Pro4]Morphiceptin and Comparison Comparison with Morphine

(NMePhfe3,

D-Pro4]-

Morphiceptin Morphine References

Analgesia, ED50,

nmole/animal

i.c.v. route

i.t. route

Tolerance &

physical

dependence

Depressor &

bradycardia

i.t., nmole/

animal

Respiratory

depression

V t / T tot

nmole/kg/

dog

Abolition of

conditioned

responses

0.23 8.4 Chang et al.

1983

1 ~30 Han et al.

1984

+ + Chang et al.

1983

~5 >100 Li and Han

1984

~50 ~800 Haddad et al.

1984

+ + Mauk et al.

1982 ; Lavond

et al. 1983

PL017 injected centrally produces analgesia (Chang

et al. 1983), respiratory depression (Haddad et al.

1984), and abolition of conditioned responses (Mauk

et al. 1982; Lavond et al. 1983. By intrathecal

(i.t.) injection, PL017 potently produces analgesic

(Han et al. 1984), depressor, and bradycardia effects

(Li and Han 1984). After chronic administration,

PL017 produces tolerance and physical dependence

(Chang et al. 1983). Like the above in vitro studies,

PL017 is far more potent than morphine by the same

route of administration in inducing these effects

107
(table 5). Since similar results are obtained from

the in vitro studies in which the metabolism is not a

likely explenation for the higher potency for PL017,

we propose that the efficacy of peptide morphiceptin

or its analogs for activating the μ-receptor system is

higher than that of alkaloid morphine.

TABLE 6

Comparison of the Respiratory Effects of [NMePhe3

,-

D-Pro4]Morphiceptin and [D-Ala2,-D-Leu5]-Enkephalin

[D-Ala2,-

D-Leu5

]-

Enkephalin

[NMePhe3,DPro4

]Morphi-

(8) ceptin (μ)

Instantaneous minute

ventilation

Vt/Ttot

Tidal volume, Vt then normal

Respiratory cycle time,

T tot

Expiratory duration, Te,

Inspiratory duration, No change

Ti

Respiratory duty cycle

Ti/Ttot

Slight

Like morphine, PL017 can induce respiratory depression

after intracisternal injection (Haddad et al. 1984).

In conscious dogs, strikingly different effects on the

ventilation and ventilatory pattern were observed

between PL017 and DADLE (table 6). PL017 induced a

fast and shallow breathing pattern, while DADLE

produced deep breathings separated by a period of

aprea (Haddad et al. 1984). Detailed analysis reveals

that DADLE induces a prolongation in expiratory time

(Te) and a decrease in instantaneous minute ventilation (Vt/Ttot), which is a result of a decrease of tidal

volume (Vt) and an increase

(Ttot) In contrast, PL017 induces a decrease in. the

of respiratory cycle time

inspiratory time (Ti), Te,

in Vi/Ttot. The respiratory effects of μ- and

and Vt, with a net increase

-agonists can be reversed by naloxone, but larger

doses are required after DADLE than after PL017.

108
These results suggest that μ- and -receptors in

conscious dogs participate in different regulatory

functions in breathing, and involve different neuronal

pathways.

CONCLUSION

Morphiceptin analogs with various substitutions at

positions 2, 3, and 4 have been synthesized and

studied for their receptor activities. Substitutions

with D-proline at fourth position and N-Me-Phe at

third position yield a potent and highly selective

μ-peptide agonist, PLO17. PL017 and morphiceptins

interact with μ-receptors in GPI and MVD. They elicit

analgesia by i.c.v. and i.t. injection, and evoke

depressor and bradycardia by i.t. injection. PL017

produces tolerance and physical dependence after

chronic administration. PL017 is more potent than

morphine in all pharmacological tests in spite of its

lower μ-receptor binding affinity. In conscious dogs,

PL017 and DADLE produce a profound difference in

regulating ventilation.

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Opposite effects of the delta- and mu-opioid

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AUTHOR

Kwen-Jen Chang, Ph.D.

Department of Molecular Biology

The Wellcome Research Laboratories

Research Triangle Park, North Carolina 27709

111
Dermorphin: Autonomic Pharmacology and

Structure-Activity Relationships

Giora Feuerstein, M.D.

INTRODUCTION

In recent years, several biologically active peptides, such as

neurotensin, thyrotropin-releasing hormone, bombesin, and other

tachykinins, have been identified in the gastrointestinal tract

as well as in central and peripheral elements of the nervous

system (Dockray and Gregory 1980). Many of these neuroendocrine

peptides (e.g., bradykinin, CCK-8, substance P, bombesin) were

originally isolated from the skin of various

(Erspamer 1981) .

amphibian

Dermorphin is a hepta

species

tide recently

isolated from the skin of the south American frog of the genus

Phyllomedusae (Brocardo et al: 1981; Montecucchi, et al 1981)

and has the following amino acid sequence: H-Tyr1-D-Ala2-Phe3-

Gly4-Tyr5-Pro8-Ser7-NH2. In addition to demorphin, Hyp6-

demorphin was also isolated from the skin of same of these

frogs. Dermorphinlike inmmoreactivity was also demonstrated in

rat brain, but the final identification of dermorphin in

mammalian tissue (central or peripheral) has not yet been made

(Negri et al. 1981). The structure of these new tides is

unique since they contain a D-amino acid residue in molecule

which is critical for the biological activity (L-Ala2-demorphin

is only 1% as active as dermorphin). The structure proposed for

the extracted dermorphin from the natural source has been

confirmed by synthesis (de Castiglione et al. 1981a).

Dermorphin has already been tested extensively for a variety of

opiate like activities. Moreover, analog of dermorphin were

already synthetized in order to clarify structure-activity

relationships for the multiple biological activities of these

opioid peptides.

This chapter summarizes the biological effects of dermorphin,

its site of action, the opiate receptors mediating its effects,

and the structure-activity relationships of this unique opioid

peptide.

112
DERMORPHIN--OPIOID PEPTIDE

In Vitro Tests

Dermorphin displays a potent depressive action on electrically

stimulated contractions of the guinea pig ileum (GPI) and mouse

vas deferens (MVD)--two classical reparations for opioid

activity assay. In these actions, dermorphin is much more

potent than the endogenous enkephalins, -endorphin, and the

opiate prototype, morphine. In the GPI preparation, the only

peptide which is more potent (three-to- five-fold) than

dermorphin is dynorphin A-(1-13) (Erspamer et al. 1981). In

the MVD, dynorphin6 A-(1-13) enkephalins are eqiupotent to

dermorphin, Hydro -dermorphin is 80% to 90% as potent

as dermorphion in these tests. These preferential actions of

dermorphin on GPI suggest preferential u-receptor mediated

action as also indicated from in vitro binding studies (Glaser

et al. 1981).

Analgesia

Dermorphin is an extremely potent anal analgesic agent in most

species tested so far (rat, mice, rabbit). Surprisingly,

dermorphin does not produce analgesia in the chick at low doses

(Erspamer et al. 1981). Analgesia can be produced by micramole

doses injected ripherally. No known opiate or opioid peptide

can compete with dermorphin in analgesia tests (hot-plate, tailflick and even ß-endorphin is only one-twentieth as potent as

dermorphin. Actually, replacement by dermorphin or the Nterminal sequence 1-7 of ß-endorphin results in increased

analgetic potency of the [denrmorphin1-7]-ß-endorphin as

compared to ß-endor phin (Yamashiro et al. 1983). In the hotplate test (rat, intracerebroventricular injection), dermorphin

is 2000-fold more potent than morphine (Erspamer 1981; Erspamer

et al. 1981; Broccardo et al. 1981; Yamashiro et al. 1983).

GASTROINTESTINAL EFFECTS OF DERMORPHIN

Sensory input from the gastrointestinal (GI) tract is conveyed

to several brain regions by afferent vagal nerves. Many of

these regions, e.g., the solitarius nucleus in the brain stem,

limbic areas, contain high levels of opiate receptors as well as

opioid peptides (Mar 1978; Snyder 1977). In addition, opiates

and opioid peptides were shown to have potent effects on various

GI functions.

Dermorphin was also tested for its effects on the GI tract by

both peripheral and central routes of administration. Dermorphin was shown to inhibit basal gastric secretion or to

stimulate gastric secretion induced by various stimuli-- e.g.,

water distension of the stomach and insulin-induced gastric acid

secretion; however, histamine-induced gastric acid secrction was

resistant to inhibition by dermorphin (Improta et al. 1982). In

addition, dermorphin was also shown to affect the motility of

113
both the stomach and intestine.

transit time are substantially increased by dermorphin

Gastric emptying and intestinal

(Broccardo et al. 1982.; Rossi et al. 1983). Intestinal transit,

however, is only partially blocked by dermorphin (Broccardo et

al. 1982).

The subcutaneous (s.c.) or intracerebroventricular

administration of dermorphin produces all of these GI effects;

however, the dose of dermorphin necessary to produce GI

inhibition by the central route of administration was

one-hundredth of the dose necessary to produce comparable

effects by peripheral administration of dermorphin, indicating

that the opioid peptides act through a centrally mediated

mechanism

All of the GI effects of demorphin could be completely prevented by naloxone, suggesting an opiate receptor mediated

effect. Furthermore, the inhibition of intestinal propultion

induced by central administration of dermorphin can be blocked

by a quaternary naloxone analog injected into the cerebroventricular system in a dose which has no effect when administered peripherally (Parolaro et al. 1983); thus., it seems that

the GI effects of demorphin are primarily medicated through a

centrally located opiate receptor.

Since the studies cited above were conducted on rats, which

differ from guinea pigs and other species in the type and

distribution of opiate receptors in the central nervous system,

it also would be important to investigate the GI effects of dermorphin in other species. Moreover, the type of opiate receptor

and the specific sites (nuclei) in the brain where GI regulation

by dermorphin commences remain to be explored.

CARDIOVASCULAR AND RESPIRATORY EFFECTS OF DERMORPHIN

Opiates and opioid peptides were long known to affect blood

pressure, cardiac functions, and the respiratory system. Therefore, it has been postulated that dermorphin, a potent opioid

peptide, might also significantly affect the cardiovascular and

respiratory systems. Indeed, administration of dermorphin to

human subjects causes a brief increase in blood pressure and

heart rate without affecting urinary electrolyte excretion

(Degli-Uberti et al. 1983b, 1983c). In the anesthetized dog,

systemic administration of demorphin elicits pressor and

cardiac acceleration responses (gander and Giles 1982).

In the conscious rat, intracerebroventricular injection of

dermorphin causes complex cardiovascular responses; while low

doses (picomoles) cause elevation of blood pressure and heart

rate, higher doses will cause hypotension and bradycardia

(Feuerstein and Faden 1983a). The cardiodepression produced by

higher doses of demorphin injected into the cerebroventricular

system is accompanied a respiratory depression as evident by

hypoxemia, elevated carbon dioxide, and reduction of blood pH

(respiratory acidosis) these cardiorespiratory changes are

114
accompanied by a marked increase in plasma catecholamines

(Feuerstein and Faden 1983a). However, the cardiac responses to

central administration of dermorphin are more complex: vagal

activation seems to underly the bradycardia produced by high

doses of dermorphin since the peripheral acting muscarinic

blocker, N-methyl-atropine, can prevent or reverse the bradycardia produced by dermorphin (Feuerstein and Faden 1983a.

Thus, dermorphin activates both the sympathetic and parasympathetic components of the autonomic nervous system. Also,

naloxone reverses all of the central cardiovascular effects of

dermorphin.

The anterior hypothalamus is probably the site for the central

cardiovascular effects of dermorphin since the pressor/cardiac

acceleration effects can be produced by picomole doses of

dermorphin microinjected into discrete nuclei of the hypothalamus (Diz and Jacobowitz 1984; and Diz, Vitale, and

Jacobowitz 1984; Feuerstein and Faden 1983a). The role of the

sympatho-adrewmmdullary system in mediation of the cardiac

acceleration and pressor responses elicited by hypothalamic

injections of dermorphin is also indicated by the blockade of

the cardiac responses with the -adrenergic blocking agent,

propranolol. Yet, it should also be pointed out that other

brain nuclei besides the hypothalamus are involved in dermorphin

effects since dermorphin induces bradycardia, probably by

activation of efferent vagal neurons in the nucleus ambiguus and

the dorsal motor nucleus of the vagus. This suggestion,

however, awaits further clarification.

The cardiovascular and respiratory effects of dermorphin resemble those of [D-Ala2

-MePhe4-Gly-ol3] enkephalin (DAGO), an

enkephalin analog which is 200-fold more selective for μversus -opiate receptors (Kosterlitz et al. 1980; Pfeiffer et

al. 1982). Since all of the dermorphin effects on the cardiovascular system are produced at a much lower concentration than

DADLE, [D-Ala2-D-Leu3] enkephalin, a relatively selective

-receptor agonist, it is pertinent to suggest that μ-opiate

receptors mediate the central cardiovascular responses to

dermorphin. These conclusions are summarized in table 1.

ENDOCRINE AND METABOLIC EFFECTS OF DERMORPHIN

Many opiates and opioid peptides have been shown to affect the

regulation of glucose metabolism and the release of pituitary

hormones (Dupont et al. 1977; Van-Vugt and Meites 1980;

Locatelli et al. 1978). Opioid peptides and opioid receptors

are found in both central (brain, pituitary) and peripheral

organs involved in endocrine and metabolic regulation (Grandison

et al. 1980; Rivier et al. 1977).

115
TABLE 1

1.

2.

3.

4.

5.

Central Cardiorespiratory Effects of Dermorphin

Complex Cardiovascular Responses

low doses: presser/cardiac acceleration

high doses: depressor/cardiac deceleration

site and receptor specific

Respiratory Depression

respiratory acidosis (low pH, low p02, high pC02)

Activation of the Sympatho-Adrenomedulary System

increases circulating epinephrine and norepinephrine

Activation of the Vagus (High Doses)

atropine reverses the bradycardia

μ-Opioid Receptors Mediate Dermorphin’s Effects

similarities to DAGO (highly selective μ-agonist)

highly sensitive to naloxone

Prolactin

When dermorphin (0.1 to 5 mg/kg, s.c.) is administered to the

rat, it produces a dose-dependent increase in plasma prolactin

(Rossi et al. 1983). This effect can also be produced by

subnanomole doses of dermorphin injected into the

cerebroventricular space, and can also be seen in castrated male

rats. However, in vitro experiments show no direct effect of

dermorphin on prolactin release from rat pituary cells,

suggesting a hypothalamic site for dermorphin-induced prolactin

release (Giudici et al. 1984).

dermorphin was also shown to increase plasma levels of prolactin

in human subjects when infused systemically (Degli-Uberti et al.

1983a). The increase in prolactin response was more pronounced

in females than males. In both the rat and human studies,

naloxone effectively blocked the dermorphin effects, suggestion

an opiate receptor mediated response. However, Güllner and

Kelly (1983) failed to increase plasma prolactin by

116
low doses of dermorphin in jected intravenously into the

anesthetized rat. This discrepancy might be related to the

different rat species, the type of anaesthesia (Ketamine),

and/or the relatively low doses of dermorphin which might have

failed to reach sufficient levels in the brain.

Growth Hormone

Opiates and opioid peptides have been shown to release growth

hormone (GH) in rat and man (Von Graffenried et al . 1978;

Stubbs et al. 1978; Van-Vugt and Meits 1980). Dermorphin was

also shown to produce an increase in GH levels within minutes

after intravenous administration to the anesthetized rat

(Gullner and Kelly 1983) or to human subjects (Degli-Uberti et

al 1983a). The increase in plasma GH by dermorphin could be

blocked by naloxone (Degli-Uberti et al. 1983a).

Calcitonin

Dennorphin administration to normal human volunteers (5μg/

kg/min over 30 min) does not change the plasma levels of

calcitonin over a 3-hour period. In addition, the circulating

levels of this hormone are not changed by naloxone infusion

(Degli-Uberti et al. 1983b). These data suggest that opiates

may not play a significant role in regulation of calcitonin

release. However, the role of opiates and dermorphin was not

examined during state where endogenous calcitonin release is

stimulated (e.g., hypercalcemia).

Thyroid Stimulating Hormone (TSH)

Intravenous injection of dermorphin to rats caused a

dose-dependent increment in plasma TSH levels (Gullner and

Kelly 1983). The increase in plasma TSH produced by dermorphin

in opposite to the effect of endogenous opioid peptides on this

anterior pituitary hormone (Bruni et al. 1977).

Luteinizing Hormone (LH)

Opioid peptides were previously shown to reduce plasma levels

of LH (Bruni et al. 1977). Dermorphin administration to adult

male rats causes a marked decrease in plasma LH at extremely

low doses (Gullner and Kelly 1983). The mechanism of this

effect has not been studied as yet.

ENDOCRINE AND METABOLIC EFFECTS OF DERMORPHIN

Several neuropeptides, (e.g., somatostatin, ß-endorphin) and

peptides isolated from frog skin (bombesin, substance P) have

been shown to affect glucose metabolism (Gullner et al - 1983;

Brown and Vale 1976). Injection of dermorphin to the rat at

doses up to 10 g did not effect plasma insulin or

somatostatin; however, plasma glucose was slightly reduced

while plasma glucagon was significantly increased (Gullner et

al. 1983).

117
dermorphin's effect of reducing blood glucose is opposite to the

increase in plasma glucose induced by ß-endorphin. Also, it is

still unclear whether dermorphinexists in the endocrine cells

of the pancreas or in any of the peripheral tissues involved in

these endocrine functions.

dermorphin Effects on the Renal-Adrenocortical Endocrine System

Opiates and opioid peptides have been shown to affect the

pituitary-adrenocortical functions in several species (Smythe et

al. 1980; Gaillard et al. 1981; Volavka et al. 1979). Dermorphin infusions to normal volunteers caused elevation of

plasma renin activity (PRA) and a small decrease in plasma

cortisol which was accompanied by a similar decrease in plasma

adrenocorticotropic hormone (ACTH). Plasma aldosterone levels

were also slightly increased by dermorphin infusion (DegliUberti et al. 1983c).

accord with the

The increase in PRA by dermorphin is in

known effect of other opioid peptides to

increase PRA (Cantalamessa et al. 1982) and such an increase in

PRA might generate sufficient angiotensin II to account for the

pressor response observed shortly after dermorphin

administration (Degli-Uberti et al. 1983c). The reduction in

plasma cortisol and ACTH by dermorphin is also in accord with

the same response of plasma cortisol to enkephalin analog and

other opiates (Gold et al. 1980; Stubbs et al. 1978; Gaillard et

al. 1981). A role for opioid peptides in regulation of ACTH and

cortisol release is also indicated by the elevation of the

circulatory levels of these hormones by naloxone administered to

normal human subjects.

Catecholamines

Several opioid peptides were shown to stimulate the sympathoadrenomdullary axis and increase the circulatory levels of

norepinephrine, epinephrine, and dopamine (Feuerstein et al.

1983; Pfeiffer et al. 1982; Van-Loon and Appel 1981). Intracerebroventricular administration of dermorphin to conscious

rats was likewise shown to produce a marked increase in plasma

levels of epinephrine (over twentyfold)

(sixfold) (Feuerstein et al. 1983). The site of the

and norepinephrine

sympatho-adrenal activation seems to lie within the

hypothalmus, since microinjection of dermorphin to the

anteroventral region of the hypothalamus produces similar

incraments in plasma catecholamines and, primarily, epinephrine

(Feuerstein and Faden 1983a). The increase in peripheral sympathetic activity is considered to underlie the increase in heart

rate produced by dermorphin since adrenalectomy or propranolol

(a ß-adrenoceptor blocker) prevents the increase in heart rate

by dermorphin (Diz and Jacobowitz 1984). In addition, the

increase in sympathetic tone is probably the main stimulus for

the observed increase in PRA (Degli-Uberti et al. 1983c).

Table 2 summarizes the endocrinological effects dermorphin

obtained from animal and clinical research.

118
TABLE 2

Endocrine and Metabolic Effects of Dermorphin

Epinephrine

Norepinephrine

Renin

Aldosterone

Glucagon

TSH

Growth Hormone

Increase No Change Decrease

Prolactin

Galcitonin

Insulin

Sumtostatin

Glucose

Cortisol

ACTH

TSH = thyroid stimulating hormone; ACTH = adrenorticotropic

hormone; * anesthetized rat; number of (+) or (-) denotes intensity of increase or decrease in plasma levels, respectively.

BEHAVIORAL EFFECTS OF DERMORPHIN

Effects of dermorphin on the Electroencephalogram (EEG)

Injection of low doses (few nanomoles) of dermorphin into the

cerebroventricular system of the rabbit produces synchronous

patterns in the cortical EEG and disruption of hippocampal theta

waves. Higher doses reduce spike wave lexes (Aloisi and

Scotti De Garolis 1981). In the chick, dermorphin administration into the third ventricle also caused total voltage power

increase and an EEG shift toward lower frequencies (Erspamer et

al. 1981). The EEG changes are all reversed by naloxone.

In rats, doses of dermorphin ranging from 5 to 2,500 ng produced

electroencephalographic seizures associated with grand mal motor

119
manifestations: twitches and tremor with low doses; tonicclonic convulsion by higher doses. The convulsive patterns were

followed by high voltage slow waves and other abnormalities

(Aloisi et al. 1982).

Demorphin produces behavioral responsesn known for many opiates,

e.g., catatonia, rigidity, sedation. In the chick, “squating"

behavior was also observed. In mice, doses as low as 10 ng

produce the “Straub Tail Reaction” (tail elevation 906), restlessness , and increased motor activity (runing fits); hi her

doses produce catalepsy. All these effects are also produced by

morphine at higher dose range. In the rat, higher doses of

dermorphin result first in reduced spontaneous movement, and

later in rigidity and catatonia. Exophthalmos, meiosis, and

hypothermia are also observed. In the rabbit, muscular

rigidity, catatonia, and loss of righting reflexes are always

seen at higher doses along with hypothermia, exophthalms, and

mydriasis. In the rabbit, high doses of dermorphin will result

in flaccid paralysis of the hindlimbs and, ultimately, death

(Aloisi et al. 1982). Topical administration of dermorphin over

the sensorimotor cortex in this species results in highly

disorganized EEG activity, characterized by exclusively slow

waves but no motor or EEG convulsive manifestations. Thus, it

appears that in the rat, dermorphin causes EEG; and behavioral

epileptic phenomena but is devoi of convulsant activity in the

STRUCTURE-ACTIVITY RELATIONSHIPS OF DERMORPHIN

The minimal peptidic sequence of dermorphin which possesses

biological activity is respresented by the N-terminal H-TyrD-Ala-Phe-Gly-NH2

 tetrapeptide (Broccardo et al. 1981; de

Castiglione et al. 1981b; Salvadori et al. 1982). However,

dermorphin (1-4) is only 2% to 5% as active as dermorphin in inhibiting GPI or MVD contraction in response to electrical

stimulation and is practically devoid of analgetic and

cataleptic activity. dermorphin (1-3) has less than 0.5% of any

of the biological activities of dermorphin

However, modifications of the C-terminal of the dermorphin by

lipophilic substituents, e.g., Tyr-b-Ala-Phe-Gly-NH-CH2-C6H5,

markedly increase the opioid activity of the dermorphin tetrapeptide, and the biological activity; H-Tyr-D-Ala-Phe-Gly-NHAdamantyl results in over twentyfold increased potency in GPI

and MVD preparation, which strongly suggests a μ-receptor rather

than -receptor mediated effect (Broccardo et al. 1981;

Kosterlitz et al. 1980). A quantitive dependence of biological

activity on the lipophylic character of the C-terminus was also

1978

reported for a series of enkephalin like peptides (Do et al.

Profound changes in the biological activity of dermorphin can

also be seen by relatively minor changes in position 1 (H-Tyr).

120
Replacement of Tye by D-Phe1

 or Ala1 causes a 6-order decrease

in potency on the GPI; substitution of au Acetyl Tyr1 also

profoundly decreases the relative potency in GPI inhibition

test. The analog also display reduced capacity to displace

naloxone from binding sites on striatum homogenates (Darlak et

al. 1983). However, the capacity to displace naloxone from

opiate receptors might not alwayslcorrelate with the biological

activity of the analog; Tyr (Me)1-dermorphin for example, is

not less active in displacing naloxone from rat striatun

homogenates as compared to dermorphin but is >thirty fold less

potent than dermorphin in inhibiting of GPI contraction and

almost a hundredfold less active in producing analgesia in mice

(Darlak et al. 1983). Modifications of position 1 of dermorphin

analog also reveal some differential changes in the various

tests of biological activities. Phe1-dermorphin is only about

0.0001% as active as dermorphin on GPI, but has thus still 5%

analgetic activity as compared to the natural peptide. It is

also noteworthy lthat the introduction of a sulphate group on the

hydroxyl of Tyr1-residue of dermorphin completely destroyed all

of the opioid activity of dermorphin (de Castiglione and Perseo

1983).

Modification of enkephalins at position 2 by introducing D-Arg2-

residue produces analog with potent analgetic

(Takagi et al. 1982; Kubota et al. 1980). Similar modification

in the dermorphin tetrapeptide has also proved to produce potent

opioid tide analog (Sasaki et al. 1984). Thus, D-Arg 2-

dermorphin (1-4) is tenfold more potent than dermorphin tetra

peptide in the mice tail-flick test; replacing Gly4 by Sar4-

residue in the D-Arg2-dermorphin (1-4) further increases the

analgesic potency fourfold. Thus, it is clear that D-Ala2-

residue in dermorphin is not always necessary for the opioid

activity, as originally thought. Further modifications at the

C-terminus which increase the lipophilic character of the

peptide also increase the potency. This is due, in part, to an

increased resistance a against carboqieptidases. Resistance

against enzymatic breakdown might account for the prolonged

(several hours) action at relatively low doses injected

subcutaneously.

Several other modes of amino acid substitutjons yield various

potencies of the dermorphin molecules. Substitution of Tyr3 by

aromatic amino acid is well tolerated, but Gly dermorphin is

devoid of must activities. Substitution of Pro8

 has little

effect on the overall opioid activity of dermorphin and this

applies also for position 7 (de Castiglione et al. 1981b). An

interesting finding is that the deaminated dermorphin maintains

its peripheral opioid activity but is devoid of effect on the

central nervous system.

Of special interest in the structure-activity relationships of

dermorphin analog are the differential activitiy displayed

by some analog. Thus, dermorphin (1-5) and Leu5 dermorphin

(1-5) demonstrate augmented potency (about twofold) in the GPI

121
bioassay test but reduced activity (-80%) in the MVD bioassay

test; these dermorphin pentapeptides are virtually

inactive in the common analgesia and catalepsy tests. Another

example of differential preservation of opioid activity is

demonstrated by the benzylated-Tyr5-dermorphin which presents

about 50% of its inhibitory action on MVD bioassay but only 5%

in the GPI bioassay. In no case so far (of over 100 dermorphin

analog synthesized) has analgesia been preserved as a primary

property of a dermorphin analog. The main structure-activity

relationships of dermorphin are presented in table 3.

TABLE 3

Differential Opioid Activity of dermorphin Analog

GPI MVD ANALGESIC CATALEPSY

Dermorphin 100 100 100 100

Bzl-Tyr5-Dermorphin <5 50 <1 <0.5

Des-Tyr5-Dermorphin <0.1 <0.1 <1 <l

Dermorphin (1-6) 50 40 20 <1

Dermorphin (1-5) 20 200 <5 <5

Leu5-Dermorphin (1-5) 15 175 <1

Dermorphin (1-4) <2 <5

Dermorphin (1-3) <0.3 <0.5

Numbers represent percent activity as compared to dermorphin

(100%). GPI = guinea pig ileum bioassay test; MVD = mouse vas

deferens bioassay test.

DERMORPHIN AS A μ-RECEPTOR AGONIST

The type of opiate receptor which mediates the biological

actions of dermorphin is still obscure. However, several

pharmacological properties of dermorphin effects in vitro and

in vivo strongly suggest the μ-opiate receptors are primarily

involved in dermorphin effects. The evidence underlying this

suggestion is: 1) Dermorphin is much more potent in blocking

the contraction of the electrically stimulated GPI rather than

the MVD; this bioassay test is considered to be a prototype for

morphine (μ) rather than enkephalin effect. 2) Dermorphin

is less potent than enkephalins in inhibiting the PGE1-stimulated adenylate cyclase (accumulation of c-AMP) in neuroblastoma

x glioma hybrid cells (Glaser et al. 1981). 3) Dermorphin

122
effects were shown to be easil and completely reversed by low

doses of naloxone administered peripherally or centrally. 4)

Dermorphin effects on the cardiorespiratory system are similar

to those of DAGO, which is a highly selective μ-receptor

agonist; the cardiovascular effects of dermorphin (pressor/

cardiac acceleration) are produced by doses which are onehundredth of those necessary to produce the same responses by

the relatively selective -receptor agonist DADLE (Feuerstein

and Faden 1983a, 1983b; Feuerstem et al. 1983).

SUMMARY

The biological activities of dermorphin summarized in this

chapter indicate that dermorphin possesses all known effects of

opiate/opioid peptides. It seems that dermorphin effects are

primarily mediated through a μ-receptor of high affinity. Since

none of the known endogenous opioid peptides displays high

potency and selectivity for μ-receptors, dermorphin like peptides

might be the endogenous selective ligands for such μ-receptors.

This hypothesis, however? awaits further investigation. The

structure-activity relationships of dermorphin and the unique

D-alanine at position 2 clearly indicate that the N-terminus of

the first four amino acids is crucial for the complete

biological activity. Systematic modifications of the dermorphin

sequence also reveal the potential for developing selective

agonists (or antagonists) for discrete opioid functions by

mdifying receptor selectivity.

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Int J Pept Protein Res

ACKOWLEDGMENTS

This review was supported by Uniformed Services University of the

Health Sciences grant R09211.

AUTHOR

Giora Feuerstein, M.D.

Neurobiology Research Division

Department of Neurology

Uniformed Services University of the Health Sciences

4301 Jones Bridge Road

Bethesda, Maryland 20814-4799

127
Design of Conformationally Constrained

Cyclic Peptides With High Delta and Mu

Opioid Receptor Specificities

Victor J. Hruby, Ph.D.

INTRODUCTION

Efforts to understand the conformational structure-biological

activity relationships for the endogenous opioid peptides, such as

methionine enkephalin, leucine enkephalin, and dynorphin, have

been difficult. Despite the preparation of at least a few thousand peptide analogs (for reviews, see Miller and Cuatrecasas

1978; Morley 1980; Bajusz 1982; Olson et al. 1982, 1983), highly

receptor-selective and potent peptide analogs have been obtained

only recently. The reasons for these difficulties are many, but

basically they arise fran the intrinsic physical-chemical properties of most endogencus opioid peptides, the comlexity of the

biochemical events related to their biological activities, and the

existence of several different classes of receptors for these

molecules, each of which my mediate different biological responSeS.

Most of the endogenous cpioid peptides that have been isolated

thus far are small or moderate size linear peptides which have a

high degree of conformational flexibility. These properties do

present certain advantages (table 1), many of which are undoubtedly utilized by the cpioid peptides in vivo. However, fran the

standpoint of understanding the physical-chemical basis for information transfer and biological activity, these properties pose

several problems for many peptide hormones and neurotransmitters

which we (Meraldi et al. 1977; Hruby 1981a, 1982, 1984; Hruby et

al. 1983) and others Narshall et al. 1978; Veber 1979) have

discussed. For the purpose of this chapter, suffice it to say

that the high conformational flexibility of the opioid peptides

such as methionine enkephalin, H-Tyr-Gly-Gly-Phe-Met-OH, makes it

difficult to determine which of the multitude of possible conformations are of biological significance.

A similar problem exists with regard to the complexity of biological information transfer in the hormone-receptor complex. We have

pointed out Hruby 1981a, 1981b, 1984; Hruby et al. 1983; Hruby

and Hadley, in press) that binding, transduction, and reversal

128
TABLE 1

Some Advantages of Conformational Flexibility

in Biologically Active Peptides

1 .

2 .

3 .

4.

5 .

Many Conformations Accessible at kT.

Different Conformational Structures Can Readily Be Utilized

at Different Receptors.

Easily Fit into Enzymes; Biological Degradation Facile.

“Zipper Model” Is Possible, Providing a Thermodynamically

Accessible Route to Peptide-Receptor Interaction.

Different Conformational Structures Available for Binding,

Transduction, and Reversal at Receptors.

probably each utilize different conformational structure properties of a peptide hormone or neurotransmitter. Thus, appropriate

ligands and biological assays are needed to help sort out this

complexity

Finally, as with most peptide hormones and neurotransmiters, the

endogenous opioid peptides appear to interact with several different receptors at different cell types. These include the delta

mu (μ), kappa epsilon and possibly other receptors

(Martin et al. 1976; Lord et al. 1977; Chang et al. 1979). These

different receptor types have biological significance, and in

several cases there is good evidence that they have different

structural requirements for cpioid peptides. Thus, the pharmacophore for each receptor type is different, a fact which has

been largely ignored in previous attempts to understand the

“biologically active conformation” for opioids.

In an effort to resolve some of these problems, we have sought in

our studies to develop receptor-specific peptide analogs. Our

approach to obtaining such analogs has been via conformational

restriction (Marshall et al. 1978; Veber 1979; Meraldi et al.

1977; Hruby 1981a, 1982, 1984). In this chapter, we review how

this approach has led our laboratory to the design and synthesis

of cyclic, conformationally constrained peptides with high delta

and mu opioid receptor specificities.

CYCLIC DELTA RECEPTOR-SELECTIVE PEPTIDES

Oue starting point in 1981 for the development of receptor-specific enkephalin analogs was several observations related to

enkephalin structure-function studies. First, substitution of

Gly2 in methionine- or leucine-enkephalin by several D-amino acid

residues was compatible with high biological activity (e.g., see

Beddel et al. 1977). Second, substitution of Met5 or Leu5 by a

wide variety of L or D amino acid residues was compatible with

high potency (e.g., see Morley 1980). Third, the compact

129
structural nature of many nonpeptide opioids suggested that, on

interacting with a receptor, the opioid peptides may assume more

compact conformations involving a turn conformation. With this in

mind, we decided to apply the concept of pseudoisosteric cyclization which we had applied successfully in the development of

superpotent cyclic melanotropins (Sawyer et al. 1982). In this

regard, the Gly2, Met5 residues in enkephalin were highly reminiscent of the Met4,

of Gly2,

GlylO residues in -MSH.

Met5 by D-Cys2, D(or L)-Cys5 to produce a 14-membered

Hence, substitution

cyclic structure appeared to be a reasonable starting point for

conformational restriction.

On examining the literature, we found that Sarantakis (1979) had

already prepared a [D-Cys2, L-Cys5]enkephalin analogue, and

though the compound was active, the bioassay results provide no

data on receptor specificity. We, therefore, decided to prepare

even more rigid analogs by utilizing geminal dimethyl groups for

restricting the conformation of these medium-sized peptide rings,

which we had demonstrated to occur in oxytocin antagonist analogs

(Meraldi et al. 1975, 1977; Mosberg et al. 1981; Hruby 1981b).

Thus, we prepared the enkelinamide (EA) analogs[D-Pen2,

Cys5]EA and [D-Pen2, D-Cys5]EA, and examined (Mosberg et al. 1982)

their delta versus mu receptor selectivity using the mouse vas

deferens (MVD) and guinea pig ileum (GPI) as the putative delta

and mu receptor-selective assays (Kosterlitz and Watt 1968;

Henderson et al. 1972). Fortunately, as we began our studies,

Schiller et al. (1981) also prepared the enkephalinamide compounds

of Sarantakis and examined their biological activities in the GPI

TABLE 2

Biological Activities (IC50) of Cyclic Enkephalinamide Analogs in the Guinea Pig Ileum and Mouse

Mouse Vas Defrens (MVD) Assays

Compounda

IC50(nM)b

IC50(GPI)

GPI MVD IC50(MVD)

118 ± 19 3.6±0.67 32.4

117 ± 21 16.8±3.1 6.9

1.51 ± 0.03

0.78 ± 0.01

2.20 ± 0.40 3.75±0.04 0.59

Normorphinec91 ± 19 540±113 0.17

aEA = enkephalinamide

bValues are the mean (± s.e.m.) of 3 to 4 determinations.

cMosberg et al. 1982 dSchiller et al. 1981

130
assay and membrane binding assay system. The results from the

studies of Schiller and our studies are given in table 2. An

interesting result was observed. While the cyclic [D-Cys2, D(or

L)-Cys5]EA analogues of Schiller et al. and Sarantakis are

slightly μ receptor-selective (schiller et al. 1981), the [D-Pen2,

D(or L)-Cys5]EA analogs we prepared were somewhat delta selective

(Bosbsrg et al. 1982). Examination of the data in table 2

indicates that this delta receptor selectivity is primarily a

result of continued high potency of the D-Pen2 analogs at the MVD

receptor system but much lower potency at the GPI receptor

system (μ). Conformational analysis of the [D-Pen2, D(or L)-

Cys5]EA analogs and the D-Cys2, D(or L)-Cys5]EA analogs(Mosberg

and Schiller 1984) suggests that, whereas the D-Pen2 analogs are

the more conformationally constrained analogs, as expected, the

differences in biological activity may result from conformational

difference at the C-terminal portion of the peptides (possibly a

transanular effect of the geminal dimethyl groups of D-Pen2 in a

l4-membered ring).

These interesting results led us to examine this receptor specificity of the carboxylate terminal analogs of D-Pen2-substituted

cyclic enkephalins (E) since previous structure-activity studies

(Morley 1980; Kosterlitz et al. 1980; Ronai et al. 1981) had indicated that carboxylate terminal enkephalins were relatively more

reactive in the MVD assay ( receptor), while carboxamide terminal

enkephalins were relatively more reactive in the GPI assay (μ receptor). Indeed, as shown in table 3, [D-Pen2, L-Cys5]E and

[D-Pen2, D-Cys5]E are considerably more potent than their enkephalynmnide counterparts in the MVD assay and, interestingly, they

are considerably less active in the GPI assay. Thus, these analogs have exceptional delta receptor selectivity, and indeed

appeared to be the most delta-selective enkephalin analogs which

had been obtained up to that time.

TABLE 3

Biological Activities (IC50) of Cyclic Enkephalin

Analogs in the GPI and MVD Assays

aEa = enkephalinamide, E = enkephalin.

bvalues are the mean (± s.e.m.) of 3 to 4 detenninations.

cMosberg et al. 1982

dMosberg et al. 1983a

131
TABLE 4

Receptor Binding Affinities for Cyclic Enkephalin

Analogs in Rat Brain Preparation

Compounda

IC50(nM)b

[

3H]NAL [

3H]DADLB

IC5O(NAL)

IC5O(DADLE)

73.4 ± 14.9 3.35 ± 0.15 21.9

178. ± 15.8 11.7 ± 1.20 15.2

16.2 ± 34.8 7.20 ± 1.80 22.6

157 ± 736 26.0 ± 0.50 6.0

23.3 ± 2.4 27.2 ± 1.2 0.9

53. ± 2.3 5.4 ± 0.10 9.8

22. ± 2.8 3.5 ± 0.8 6.3

aEA = enkephalinamide, E = enkephalin. bValues are the mean (± s.e.m.) of 3 to 6 determinations, each

done in triplicate. cMosberg et al. 1982

dMosberg et al. 1983a

eMosberg et al. 1983b

In order to further evaluate the receptor specificity of these

cyclic enkephalin analogs, we (Mosberg et al. 1982) also have

examined their receptor binding to enkephalin (opiate) receptors

in rat brain membrane preparations in competition with the putative μ receptor-selective radioligand [

3H]naloxone and the putative receptor-selective radioligand [

3H]DADLE ([3H]-D-Ala2

,

D-Leu5]E). Through neither of these ligands have particularly high

μ and receptor selectivity, respectively, they have served as

the prototypical ligands for these receptors. Thus, though the

results from these binding assays must be interpreted with

caution, they can provide insight into the structural and conformational trends for receptor-specific ligands.

Interestingly, in these binding assays, the very high delta

selectivity for the cyclic D-Pen2 enkephalin analogs was less

striking. In fact, the enkephalinamide cyclic analogs appeared to

be slightly more delta selective than the corresponding enkephalin

analogs in this assay system. Thus, a lack of quantitative, and

in some cases qualitative, agreement between the muscle contraction assays and the receptor binding assays is observed. This

lack of agreement could be the result of one or more of the

following: a) species differences; b) different assay conditions;

c) lack of discrimination of receptor subtypes by competing

ligands; and d) heterogeneity of receptor subtypes. Nonetheless,

the high potency and significant selectivity of

132
these compounds in all assay systems prompted us to attempt to

further increase this receptor selectivity.

Examination of possible conformational models for the D-Pen2

-

and the D(or L)-Pen5-analogs suggested that the ß-hydrogens on the

half-cystine residues, L(or D)-Cys5 and D-Cys2, respectively, were

not sterically hindered, and that it thus might be possible to

prepare the more conformationally constrained bis-penicillamine

cyclic enkephalin analogs. Placing two geminal dimethyl substituents in a 14-membered ring would generally be considered very

difficult due to energetically unfavorable transannular interactions. However, we felt that if our conformational model was

correct, and if the linear sulfhydryl peptides would assume a

similar conformation, ring closure might be possible. Therefore,

we decided to prepare [D-Pen2, Pen5]E (DPLPE) and [D-Pen2,

D-Pen5]E (DPDPE) (Mosberg et al. 1983c). The synthesis proceeded

well, and a good yield of the desired monomer was obtained after

purification by partition chromatography and gel filtration (Hurst

1984). The biological activities in the MVD, GPI, and binding

assays are shown in tables 5 and 6, and the results are compared

with those obtained for the D(or L)-Cys5 analogs and notmorphine.

Examination of the MVD and GPI bioassay data (table 5) and the

binding data (table 6) provide convincing evidence that DPLPE and

TABLE 5

Biological Activities (IC50) of Cyclic Enkephalin

Analogs in the GPI and MVD Assays

Compounda

(IC50)

b

IC5O(GPI)

GPI MVD IC5O(MVD)

2720 ± 50.1 2.50 ± 0.03 1088

213 ± 63 0.32 ± 0.03 666

6930 ± 124 2.19 ± 0.30 3164

1350 ± 340 6.27 ± 1.2 215

234 ± 85.6 0.70 ± 0.08 334

100 ± 19.6 0.58 ± 0.06 172

228 ± 52 0.60 ± 0.06 380

a

E = enkephalin

bValues are the mean (± s.e.m.) of 3 to 4 determinations, each

done in triplicate.

cMosberg et al. 1983c

dMosberg et al. 1983b

eHurst 1984

133
TABLE 6

Receptor Binding Affinities for Cyclic Enkephalin

Analogs in Pat Brain Preparations

Compounda

IC50(nM)b

[

3H]NAL

IC50(NAL)

[

3

H]DADLB IC50(DADLE)

3710 ± 740 10.0 ± 2.2 371.

178 ± 15.8 11.7 ± 1.2 15.2

2840 ± 670 16.2 ± 0.9 175.

157 ± 73.6 26.0 ± 0.5 6.0

Morphinec 23.3 ± 2.4 27.2 ± 1.2 0.86

H-Tyr-D-Ala-Gly-Phe-NHe

(CH2)12 96.3 1.06 91

H-Tyr-D-Ala-Gly-Phe-NH

[D-Ser2

,Leu5,Thr6]Ec88 ± 6.0 5.7 ± 0.4 15.4

[D-Thr2,Leu5,Thr6]Ec 36.3 ± 3.8 6.4 ± 0.6 5.7

146 ± 25 5.2 ± 0.70 28.1

aE = enkephalin

bValues are the mean (± s.e.m.) of 3 to 6 determinations, each

done in triplicate.

cMosberg et al. 1983c

dMosberg et al. 1983b

eShimohigashi et al. 1982 fHurst 1984

DPDPE are exceptionally delta receptor-specific peptides.

Interestingly, the DPDPE is more delta selective based on the MVD

versus GPI assays, while DPLPE is more selective based on the

binding assays. The reasons for this are that whereas both Pen5

diastereimeric analogs are much less potent in the GPI assay

than the corresponding D(or L)-Cys5-containing analogs, DPDPE is

more potent (almost threefold) than the corresponding D-Cys5 analogs, and DPLPE is much less potent (about eightfold) than the

corresponding L-Cys5 analogs. Thus, the relative receptor specificity becomes much greater for DPDPE than for DPLPE in these

assays. On the other hand, while DPLPE has slightly better

binding potencies than DPDPE (table 6) against [

3H]DALE (putative

ligand), the opposite relative binding potencies are seen versus

[

3H]NAL (putative μ ligand). Thus, the DPLPE appears to be

slightly more delta selective than DPDPE in this assay system.

These results have recently been confirmed by James and Goldstein

(1984). Using what different assays and assay conditions,

134
TABLE 7

Receptor Binding Affinities of Selected Enkephalin

Analogs in Neuroblastosm X-Glioma NG 108-15

Membrane Preparations

Compounda

Ki (nm)b Hill Coefficient

1.95 ± 0.65 1.00 ± 0.08

1.78 ± 0.58 1.01 ± 0.06

[D-Ser2, Leu5, Thr6]E 1.59 ± 0.20 1.01 ± 0.06

[D-Thr2, Leu5, Thr6]E 1.42 ± 0.33 1.03 ± 0.04

aE = enkephalin

bvalues are the mean (± s.e.m.) of 3 to 6 determinations, each

done in triplicate.

they have examined the receptor selectivities of DPDPE, DPLPE,

and several other putative -selective analogs, and have found

that on the basis of their stringent requirements for selectivity,

only DPDPE and DPLPE are truly receptor specific. They also

found that these peptides have little or no kappa receptor activity. The bis-penicillamine cyclic enkephalin analogs also appear

to have considerably higher delta receptor selectivity than the

dimeric enkephalin analogs of Shimohigashi et al. (1982).

To further examine the delta selectivities and the receptor potencies of our highly -selective cyclic analogs, we have done

several additional studies. We have examined their binding

affinities for receptors in neuroblastoma X-glioma NG 108-15 cell

membrane preparations (Mosberg et al. 1983d) using [

3H]DADLE.

These opioid receptors have been suggested to be a single class of

receptors. The results given in table 7 clearly show that cur

-selective analogs are indeed highly potent at these receptors

(nmolar range). We have also prepared the delta-selective analogs

of Roques and colleagues, [D-Ser2, Leu5, Thr6]enkephalin (DSTLE)

(Gacel et al. 1980) and [D-Thr2, Leu5, Thr6]enkephalin (DTTLE)

(Zajac et al. 1983), for direct comparison in the two different

binding assays. As shown in table 7, these linear compounds have

similar binding properties as the cyclic bis-penicillamine

analogs. However, as seen in tables 5 and 6, the Thr6 analogs of

Rogues and colleagues are not highly delta selective based on

these assays, and similar results have been reported by James and

Goldstein (1984) using their assays.

Finally, we have made a number of analogs to test various aspects

of structure-function relationships in cur cyclic series using the

[D-Pen2, Cys5]E as the "standard" compound. First, we examined

whether other D-amino acid-containing diastereoisaner might be

more potent or selective. As seen in table 8, both the D-Tyr1 and

the D-Phe4 analogs lose considerable potency in all assay systems

135
TABLE 8

Biological Activities of the Cyclic Enkephalin Analogs

in the GPI, MVP, and Rat Brain Binding Assaysa,b

aAssay results were determined as in tables 2 and 4.

bData from M.S. thesis of R. Burst (1984).

cE = enkephalin

and, in addition, their receptor specificities are not changed.

Second, Rogues et al. (1982) have suggested that hexapeptide enkephalin analogs may have higher delta receptor selectivity. We

first prepared [D-Pen2, Cys5, Thr6]enkephalin and found that

though it did retain high potency in delta receptor assays, its

selectivity was poorer than several other analogs.. We then

prepared [D-Pen2, Leu5, L-Pen6]enkephalin, a cyclic 17-membered

ring analog which is pseudoisosteric to the previously reported

-selective analog MTLE. However, this compound was quite

inactive (though it did retain some 6 receptor selectivity) and,

thus, it appears that the conformational restrictions impose by

the disulfides and geminal dimethyl groups do not permit this

analog to interact effectively with opioid receptors. Finally, we

increased the ring size one CH2 group (to 15-membered) by

pre ring [D-Pen2, ß-Ala3, Cys5]enkephalin, and we deleted the

Tyr1 residue in the cyclic 14-membered ring series. Both of these

compounds had little or no activity.

In summary, we have prepared a series of cyclic penicillaminecontaining l4-membered ring analogs of methionine enkephalin.

All have delta opioid receptor specificity, and some are the most

selective delta receptor ligands available to date. Based on our

results and the results of James and Goldstein (1984), [D-Pen2,

Pen5]E and [D-Pen2, D-Pen5]E would appear to be the ligands of

choice for exploring the physiological and pharmacological roles

of delta opioid receptors in the central nervouss system and in

peripheral tissue. Indeed, though it is not the subject of this

chapter, in collaboration with Prof. Tom Burks, Dr. Frank Porreca,

Dr. James Galligan, and Mr. Lane Hirning, we have examined several

aspects of this problem (Porreca et al. 1983, 1984; Galligan et

al., 1984; Burks et al., in press). Suffice it to say, these

studies suggest that highly delta-selective opioid peptides have

unique biological properties which make them candidates for

therapeutic applications.

136
CYCLIC MU RECEPTOR-SELECTIVE PEPTIDES

Our success in designing -selective peptides prompted us to examine the possibility of preparing μ-selective cyclic peptides. At

the time we began this work (early 1983), the most μ-selective

peptide agonist analog appeared to be Tye-D-Ala-Gly-(Me)Phe-Gly-ol

(DAGO) (Handa et al. 1981), and this still appears to be the case

(James and Goldstein 1984). Schiller and coworkers (DiMaio and

Schiller 1980; Schiller and DiMaio 1982; DiMaio et al. 1982) have

prepared a series of cyclic enkephalin analogs of which

H-Tyr-cyclo-[N D-A2bu-Gly-Phe-Leu-] is the paradigm example.

These peptides all have μ receptor selectivity with an optimum

μ-selectivity value of 38.8 (as measured for H-Phe-cyclo[-N -DLys-Gly-Phe-Leu]. However, by the criteria of James and Goldstein

(1984), this is not a sufficiently μ-selective analog for many

studies. Undoubtedly, Schiller and his coworkers will continue

to further develop this cyclic series for higher μ selectivity and

it is certain such will be learned in the process regarding the

conformational requirements for the μ receptor (Schiller 1984).

We have taken another starting point for the development of a μselective peptide. Several years ago, it was reported (Terenius

1976; Razek et al. 1978) that sanatostatin can weakly inhibit the

binding of [

3H]NAL and [3H]DADLE to rat brain homogenates and also

can give an analgesic response in mice. More recently, it was

reported that the much modified somatostatin analog

had an IC50 of 38 nM at a

putative μ receptor, and was capable of antagonizing the excitory

effects of a stable enkephalin analog in electrophysiological

experiments (Maurer et al. 1982). The compound also was a potent

antagonist of insulin release. We became interested in utilizing

this lead compound to develop conformationally constrained somatostatin analogs with high potency and high receptor selectivity

with regard to glucagon release, growth hormone release, insulin

release, or μ opioid receptor interactions. We report here our

progress in developing a potent μ receptor-selective conformationally constrained sunatostatin analog (Pelton et al. 1985).

We began our studies by examining two parameters in the structure

of Maurer et al. We replaced the carbinol terminal group with a

carboxylate, and placed a geminal dimethyl group in the 20-membered ring by substituting either the Cyst or Cysll residue (the

numbering schema used in sanatostatin analogs) for a penicillamine residue. The biological activity at mu and delta receptors

was assessed by examinating the binding of the analogs to opioid

receptors in rat brain membranes in competition with [

3H]naloxone

([3H]NAL) and [

3H] [D-Ala2, D-Leu5]enkephalin ([3H]DADLE), respectively. The results are shown in table 9 along with the data

for sanatostatin (control), morphine-HCl (control), and the [Cys6,

Cys11]-analogue in which

only the carboxylate residue change is made (control). The Pen6

substitution greatly reduces potency relative to the cyclic somatostatin5-12 analog and even relative to sanatostatin at both

137
TABLE 9

Receptor binding Affinities of Samatostatin Analogs

in Rat Brain Membrane Preparations

Compounda,c IC5O(nM)b IC5ODADLE

[

3H]NAL [

3H]DADLE IC5ONAL

27,400 ± 4,200 16,400 ± 8,500 0.60

23 ± 2.4 27 ± 0.9 1.17

51,500 ± 4,100 5,800 ± 1,000 0.11

2,600 ± 260 3,100 ± 720 1.21

61,000 ± 17,500 38,100 ± 3,000 0.62

930 ± 72 5,400 ± 800 5.8

aS = somatostatin

bAssay values are the mean (± s.e.m.) of 5 to 6 determinations, each done in

cPelton et al. 1985
the mu and delta receptor sites. On the other hand, the Pen11

cyclic analog [D-Phe5, Cys6, D-Trp8, Pen11]somatostatin5-12 shows

increased binding potency at the μ receptor and slightly decreased

binding potency at the receptor and, hence, is slightly μ selective. It is interesting to note that the putative "active site"

peptide for sanatostatin, Ac-Phe-D-Trp-Lys-Thr-OH (Ac-D-Trp8-

somatostatin7-l0-OH), is somewhat less potent than sanatostatin at

the μ binding site, but somewhat more potent than somatostatin at

the delta binding site. Thus, it is quite delta selective, while

sanatostatin has very little selectivity for either the delta or

the mu binding site.

In an effort to further increase the selectivity and potency of

these somatostatin-based cyclic analogs, three different strategies were followed. First, we further conformationally constrained the cyclic 20-membered ring by preparation of the bispenicillamine analog [D-Phe5, Pen

6, D-Trp8, Pen11]-somitostatin5-12.

Second, we substituted the Phe7 residue for a tyrosine residue to

give and

Third, we replaced the

carboxylate terminal group with a carboxamide group. The bispenicillamine analog was intermediate in binding potency

between the Pen6 and Pen11 analogs at both the μ and receptor

(table 10), but had little discrimination for the μ or receptor.

Replacement of the Phe7 for a Tyr7 residue greatly increased the

binding potency of the compound at the μ receptor in both the

Pen6 and the Pen11 series and slightly increased potency at the

receptor as well. The differential effect was qreatest in the

Pen11 series and is now

quite μ receptor selective.

However, the most dramatic effect came by converting the carboxylate terminal to a carboxamide terminal group. This simple

change in the Pen11 series led to an analog H-D-Phe-Cys-Phe- DTrp-Lys-Thr-Pen-Thr-NH2 which was over 90 times more potent in

binding at the μ receptor, but only about 5 times more potent in

binding at the receptor. As a result, this compound now has an

EC50 of 10 nM at the μ receptor and is 110-fold selective for this

receptor versus the receptor. Addition of the Tyr7 residue in

place of the Phe7 residue led to an even more selective analog

which is our most potent

(EC50 3.5 nM) and most selective (270-fold) analogue for the μ

receptor system as measured in this assay system. Preliminary

studies show that these analogs act as μ receptor antagonists in

in vitro and in vivo experiments.

Careful analysis of the competitive

[3H]NAL gave Hill numbers near unity

binding experiments against

, suggesting that these analogues were interacting with the receptor in a manner similar to

that of the labeled antagonist. However, the Hill number obtained

from binding experiments with [

3H]DADLE were substantially less

than one, indicating that the somatostatin octapeptide analogs

were interacting with either multiple receptor sites or multiple

139
TABLE 10

Receptor Binding Affinities of Somatostatin Analogs in Rat Brain Membrane Preparations

Compounda

IC50(nM)b

[

3H]NAL [3H]DADLE

IC50DADLE

IC50NAL

27,400 ± 4,200

61,000 ± 17,500

8,300 ± 1,000

470 ± 10

930 ± 72

290 ± 58

9.9 ± 1.6

3.5)± 0.20

2,500 ± 400

$6,400 ± 8,500

38,100 ± 3,000

11,000 ± 2,000

2,600 ± 410

5,400 ± 800

3,800 ± 610

1,100 ± 120

950 ± 210

13,500 ± 3,000

0.60

0.62

1.3

5.5

5.8

13.

110.

270.

5.4

aS = somatostatin

bAssay values are the mean (± s.e.m.) of 5 to 6 determinations, each done in duplicate.

cPelton et al. 1985

dPelton et al., unpublished results
TABLE 11

Inhibition of [

3H]DADLE Binding to Bat Brain Homogenates by

Various Analogs Fitted to a Two-Receptor Site Model

Compounda [

3H] DADLE

IC50(1) IC50(2) BMax1 BMAX2

aS = Somtostatin

conformational states. The results of a computer analysis of the

latter data are given in table 11. The data can be fitted to a

two-site model with high- and low-affinity sites and approximately

equal nunbers of receptors (Pelton et al. 1985).

In an effort to establish the differential binding characteristics

of our analogs to somtostatin as compared to opiate receptors,

we have examined the ability of these analogs to inhibit the

binding of the biological1y stable 125I,labeled somtostatin analogue GCP 23,996, [des-Ala1, Gly2] [desamincCys3, [Tyrll], dicarba3,

14]somtostatin. The results (table 12) show that the

TABLE 12

Binding Affinities of Somtostatin Analogs to

Somtostatin Receptors in Rat Brain Membrane Preparations

Compounda

IC50(nM)b,c

3.3 ± 0.30

170. ± 99

400. ± 200

690. ± 220

800. ±300

980. ± 480

1500. ± 470

1600. ± 460

4000. ± 780

aS = somatostatin

bDetermined by inhibitor 125I-labeled OGP 23,996.

cPelton et al. 1985

141
selectivity of the cyclic octapeptide analogs extend to their

interaction with the sanatostatin receptor system in the brain.

However, the selectivities are much different than seen in the

opioid receptor systems. Indeed, our most potent μ receptorselective analogs, [D-Phe5, Cys6, Tyr7, D-Trp8, Pen11]-

somatosting5-12-NH2 and [D-phe5, Cys6, D-Trp8, Pen11]-

somatostatin5-12-NH2, are respectively 210 and 450 times less

potent than sanatostatin in this somatostatin receptor binding

assay, but about 8,000 and 2,700 times more potent, respectively,

than sanatostatin in the μ receptor binidng assay. A discussion

of the data in the sanatostatin binding assay is outside the scope

of this chapter. Suffice it to say, these studies show that the

opiate and somatostatin receptor systems, at least in the rat

brain, have substantial differences in the structural and conformational requirements of ligands for strong receptor interactions.

The structural and conformational features which lead to the high

and specific μ opioid receptor binding for the much modified somatostatin analogs reported here are not clear as yet. The relatively small effect of replacing Phe7 with Tyr7 in cur analogs

suggests that the increases seen may not be directly related to

the well-known requirement for a phenolic hydroxyl moiety in the

opiates and enkephalins. The much larger effect of substituting

the carboxylate terminal for a carboxamide terminal might suggest

that the increased potencies and selectivities are a result of a

general increase in lipophilicity. However, octanol-water partition coefficients of the sanatostatin analogs do not correlate

well with receptor affinity or selectivity. Hence, other features

such as conformational changes or specific peptide-receptor inter

actions may be involved. Work is in progress in cur laboratory to

examine these questions.

C0NCLUSIONS

We have utilized principles of conformational restriction in conjunction with careful examination of structure-biological activity

relationships to develop a series of highly receptor-selective,

potent cyclic peptide analogs of enkephalin and somatostatin

which have exceptionally high delta and mu receptor selective

respectively. Two of these compounds [D-Pen2

, D-Pen5]enkephalin

and [D-Phe5, Cys6, Tyr7, D-Trp8, Pen11]somatostatin5-12-NH2 appear

to be, respectively, the most receptor-specific delta receptor

agonist and mu receptor antagonist reported thus far. These compounds appear to have sufficient receptor selectivity, so that

they should be excellent tools for examining the physiological

roles of and μ receptors in peripheral and central nervous

system tissue. Indeed, considerable progress in this direction

has already appeared in the case of the delta-selective agonist.

In addition, since these compounds are cyclic, conformationally

constrained peptides, it may be possible to establish their preferred solution conformations and from this develop a model for

conformation-biological activity relationship of enkephalins and

sanatostatin at and μ opioid receptors, respectively. Conformationally constrained peptides provide a more valid approach to

142
utilizing solution conformations for assessing the conformational

and topological properties of a peptide ligand which may be important to biological activity, since in such constrained molecules

the overall conformational properties found in solution are more

likely to have relevance to the peptide-receptor complex. Thus,

insight into conformation-biological activity relationships are

more readily obtained using such analogs. Such insight should

lead to a rational design of more potent, more receptor-selective,

more biologically stable, etc., peptide analogs. There is still

much to be done utilizing this approach, and it is expected that

many important new insights regarding the physical-chemical basis

for the biological activity of peptide hormones and neurotransmitters will follow fran these studies.

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ACKNOWLEDGMENTS

This study represents a multidisciplinary effort. The author

especially acknwoledges Drs. Henry I. Mosberg and John T. Pelton,

and Robin Hurst, M.S., who designed, synthesized, and purified

most of the compounds discussed in this report. He also

acknwoledges his colleagues who skillfully performed the biological assays, especially Drs. Thomas Burks, Henry Yamamura, Frank

Porreca, James Galligan, Kelvin Gee, and Karoly Gulya, and Mr.

Lane Hirning. Without their complete biological studies, the

146
development of the receptor-specific analogues would not have been

possible. The partial support of the U.S. Public Health Service

Grant NS 19972-01A1 is acknowledged.

AUTHOR

Victor J. Hruby, Ph.D.

Department of Chemistry

University of Arizona

Tucson, Arizona 85721

147
Dehydro and Cyclopropyl Amino Acids

and Peptides

Charles H. Stammer, Ph.D.

In the recent past, there have been several excellent

reviews covering the synthesis of dehydro amino

acids and peptides (Stammer 1982; Schmidt et al.

1979; Noda et al. 1983). This review will cover

only the most recent literature on this subject

and is not intended to be exhaustive. The work

on cyclopropyl amino acids is quite new, coming

mostly from the author's laboratory, and the literature can be covered completely.

DEHYDRO AMINO ACIDS

Some of the latest work on dehydro amino acid synthesis will be summarized here. -Phosphoryl glycine

esters have recently been synthesized in two different laboratories (Schmidt et al. 1982; Kober

and Steglich 1983). One procedure used an -methoxy

glycine derivative, previously prepared in Israel

(Zoller and Ben-Ishai 1975), in which the -methoxyl

function is replaced with the diethoxyphosphoryl

group. The other process replaces an -bromine

atom with the diethoxyphosphoryl group to give

the same intermediate. The N-acyl -phosphoryl

glycine exter was then used in a Wittig condensation

with aryl or aliphatic aldehydes to give, in one

step, the dehydro amino acid derivative. Both

of these methods appear to be imminently useful.

A new kind of specific synthesis of dehydro tryptophan has also appeared (Moriya et al. 1982). A

readily prepared pyrrolidino enamine of 3-indole

aldehyde was treated with N-fornyl glycine ester

to give the dehydro tryptophan derivative which

is easily N-deblocked with ethanolic HCl. In 1983,

a stereospecific synthesis of dehydro amino acids

was described (Somekh and Shanzer 1983) which proceeded through the dehydration of ß-hydroxy amino

acids synthesized by a previously reported method

148
(Shanzer et al. 1979). The dehydration was accomplished using (diethylamino)sulfur trifluoride (DAST)

in pyridine.

DEHYDRO PEPTIDES

Only a few new ways to prepare dehydro peptides

have been reported recently. A series of communications describing the use of dehydro N-carboxy anhydrides (NCA'S) has appeared (Shin et al. 1981).

The synthesis of the NCA directly from an N-Cbz

dehydro amino acid using thionyl chloride proceeds

normally to give the desired product. This compound

undergoes alcoholysis to give N-free dehydro esters

or can be condensed with amino acid esters or peptide

esters to give N-terminal dehydro peptides.

Another novel approach to the synthesis of dehydro

peptides incorporates 2-aziridine carboxylic acids

(Azy) into a peptide chain followed by base-catalyzed

rearrangement of the aziridine ring to give a dehydro

amino acid moiety (Nakajima et al. 1982). The

method has been used to make several actinomycin

analogs.

Some years ago in the author's laborator, the

first dehydro peptide hormone, D-Ala2 ZPhe4,Met5-

enkephalin amide, was synthesized and shown to

be bioactive and stable to enzymatic attack (English

and Stammer 1978).

dehydro Phe4-

The in vivo activity of this

enkephalin was confirmed later (Chipkin

et al. 1979), and various other dehydro Phe4-enkephaline were prepared and their binding affinity to

the various opiate receptors were compared

(Shimohigashi et al. 1982b). The dehydro Phe moiety

in these compounds was prepared by two methods,

i.e., dehydrogenation of an azlactone with dicyanodichlorobenoquinone (DDQ) (Konno and Stammer 1978;

Shimohigashi et al. 1983b) and by the more convenient dehydration of an azlactone formed from

-phenylserine. In all cases, the presence of

a dehydro amino acid was confirmed by double beam

ultraviolet measurements in which the peak(s) characteristic of the dehydro moiety was isolated

(Shimohigashi et al. 1981b).

Ala3-, Leu5-enkephalins

The dehydro Ala2-,

and were all synthesized

and tested for binding and in vivo activity (Shimohigashiet al. 1981a; Shimohigashi Stammer 1982a).

In general, these compounds were of somewhat reduced

biological activity, but showed a preference for

the delta receptor over the mu.

Leu5-enkephalin

The dehydro Ala3

,

was particularly skewed in this

direction even though its activity was below that

of the naturalcompound (Shimohigashi and Stammer 1982d).

149
Most impressive was the dehydro Leu5

-enkephalin

in this respect, since it was almost four times

more active than its D-Leu5 analog in the radioligand

binding assays and showed good delta selectivity.

More interestingly, it was very resistant to hydrolysis by carboxypeptidasey, showing that the presence

of the dehydro moiety again caused the peptide

to be incompatible with the enzyme active site

(Shimohigashi and Stammer 1983). An attempt to

prepare Tyr1-enkephalin was successful up to the

last step when the N-Boc group was removed and

gel chromatography in aqueous acetic acid was attempted (Shimohigashi et al. 1983a). Hydrolysis

of the N-terminal enamine function was apparently

responsible for the loss of ninhydrin positive

material. Difference spectroscopy showed the

presence of the dehydro tyrosine residue in the

N-blocked peptide with a strong peak just above

300 nM. Later, quantitative amino acid analysis

was used to measure the stability, relative to

the saturated compound, of all the dehydro enkephalins toward carboxypeptidase. They showed very

high stability to this enzyme (Shimohigashi et

al. 1982a).

The configurations of dehydro peptide moieties

have been determined in various ways (Stammer 1982;

Schmidt et al. 1979 ; Noda et al. 1983). X-ray

crystallography has been used to prove the configuration of dehydro Phe and dehydro Leu (Chauhan et

al. 1979), but nuclear magnetic resolution (NMR)

methods have been used to infer the configurations

of the other unsaturated amino acids. When a derivative of dehydro E-Phe became available (Nitz et

al. 1981), nuclear Overhauser effect measurements

were carried out in which the N-H and the vinyl

protons were irradiated using a Nicolet NMC-500

NMR spectrometer (Shimohigashi et al. 1982c). Only

the E-form showed any effect at all.

Some recent work has been done to determine the

effect of a dehydro amino acid moiety on peptide

conformation. An NMR study of a di- and tri-peptide

containing a dehydro Phe residue showed that this

residue can occur in the i+2 position of a ßor -turn without greatly affecting the stability

of the turn (Bach et al. 1983).

More recently, dehydro E-Phe4

-enkephalin has been

synthesized in the author's laboratory. Compared

with the Z-isomer, the E-compound showed a drastically reduced potency in the radioligand binding assays,

i.e., 260- and 150-fold loss of affinity toward

the and μ receptors, respectively (Shimohigashi

150
et al. 1984). It is interesting to note that the

recently reported crystal structure of Leu5-enkephalin (Camerman et al. 1983) showed the phenylalanine

side chain oriented toward the amino end of the

sequence, just as it appears in the Z-isomer of

the dehydro Phe residue.

A dehydro cyclic hexapeptide analog of somatostatin

containing a dehydro Z-Phe residue in the 2-position

has recently been prepared. The NMR spectra of

the saturated and unsaturated cyclic compounds

were very similar, even though the dehydro peptide

had only one-tenth the biological activity of the

saturated material (Brady et al. 19841. The dehydro

E-Phe isomer may have the preferred configuration

in this case.

CYCLOPROPYL AMINO ACIDS

The synthesis and incorporation of cyclopropyl

amino acids into various peptides is a new area.

These amino acids have a cyclopropane ring superimposed upon the bond of the amino acid moiety

just as the dehydro amino acids have a double bond

in this position. Similarly, the information carrying ß-group of the cyclopropyl amino acid is fixed

in space, just as it is in the dehydro compounds

(figure 1). These two kinds of residues differ,

Z

Amino Acid E

Amino Acid

FIGURE 1

however, in that dehydro amino acid residues

are achiral while cyclopropyl moiety has two chiral

centers at the and ß-carbon atoms. This, of

course, means that all cyclopropyl amino acids

(except Ala, Val, and Pro) exist as four stereoisomers, i.e., two diastereoisomeric pairs of racemates.

Thus, in order to determine the effect of such

a residue on the bioactivity of a peptide,

peptides must be prepared and tested.

four

It is clear,

also, that these rigid amino acids will have profound

effects on the overall conformation of a peptide,

especially in the space proximal to the rigid moiety.

It seems quite probable that one of the four stereo151
isomers may cause the peptide to approximate a

shape which is most compatible with the enzyme

or receptor site normally occupied by the natural

peptide in question. Thus, at least one of the

four possible cyclopropyl peptides should be bioactive and, most important, stable to indiscriminate enzymatic cleavage. Very stable peptide hormones and/or enzyme inhibitors should then result.

We have recently published the synthesis of both

racemic cyclopropyl Phe isomers and have now resolved

these racemates and incorporated them into D-Ala2,

Leu5-enkephalin (King et al. 1982; Ximura et al.

1983). The amino acid synthesis requires the preparation of known azlactones followed by cyclopropanation with diazomethane. The deblocking of this

material to give the free amino acids follows relatively easily depending upon which amino acid is

being prepared. Cyclopropyl Phe requires relatively

mild deblocking conditions due to the ease with

which the cyclopropane ring is opened in hot acid.

To overcome this problem, another N-blocking group

has been developed (Suzuki et al. 1983b) in which

an incipient phthaloyl group is incorporated into

the azlactone molecule. A completely different

and more general approach was later used in the

synthesis of coronamic acid (Suzuki et al. 1983a).

In this case, a substituted diasomethane is added

to a dehydro alanine derivative followed by pyrolysis

or photolysis of the resulting pyrazoline. A halo

substituted cyclopropyl amino acid has also been

prepared by the azlactone method, but this is not

an analog of a protein amino acid (Bland and Stammer

1984).

CYCLOPROPYL PEPTIDES

Incorporation of a cyclopropyl amino acid residue

into a peptide chain will strongly affect the conformation of the peptide. Examination of extended

conformations (figure 2) shows that the cyclopropyl

Z-residues will be most restrictive (+90° -90°)

Z-peptide

FIGURE 2

E-peptide

152
of the (N-C ) angle while the E-residues restrict

the (C -C') angle to small values. These effects

will most probably promote the formation ß- and

-turns in that area.

A preliminary account of the synthesis of the first

cyclopropyl peptide hormone has been published

by Kimura et al. (1983). This cyclopropyl enkephalin

incorporated cyclopropyl E-Phe in the 4-position.

As in the case of the dehydro Phe4

 enkephalin,

the E-configuration of this rigid phenylalanine

residue was extremely deleterious to the binding

affinity of the peptide, with the more active isomer

being less active than its counterpart by a factor

of 3000. Most recently, both cyclopropyl Z-Phe4

-

enkephalins have been prepared. These compounds

are more active by a factor of about 8000 than

the E-isomers, and they bind to rat brain receptors

with about the same affinity as the natural compounds

do. All of these compounds are stable in vitro

to enzymic attack and have not yet been assayed

in the muscle assays or in vitro. The synthesis

of cyclopropyl Tyr is under way and we hope soon

to see the effect of a rigid tyrosine residue on

the bioactivity of enkephalins.

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ACKNOWLEDGMENTS

This work was supported by the National Institute

of Drug Abuse (grant DA 02938A), the National Science

Foundation (grant CHE 8122011), and the Vice

President for Research and the Department of

Chemistry, University of Georgia.

AUTHOR

Charles H. Stammer, Ph.D.

University of Georgia

Department of Chemistry

Athens, Georgia 30602

156
Affinity Labels as Probes for Opioid

Receptor Types and Subtypes

Phillip S. Portoghese, Ph.D., and A. E. Takemori, Ph.D.

Progress in most areas of pharmacology and medicinal chemistry depends upon

the availability of highly selective molecular probes. The opioid field is a case in

point. With the burgeoning evidence that the actions of opioids are mediated

through multiple receptor types and subtypes (Martin 1983). it has become

apparent that more selective ligands are required as tools. Thus, the narcotic

antagonist naloxone, which proved to be extremely useful in determining opioid

receptor involvement, is now considered to be inadequate for sorting out actions

mediated by different receptor types. This presentation reviews our approach to

addressing this problem through the design of highly selective, nonequilibrium

molecular probes for opioid receptors. Because such ligands become bound

covalently to opioid receptors, they are preferred where firm attachment of a

ligand is desirable. In this connection, they are finding utility in receptor

isolation, in mapping receptor distribution, in receptor binding paradigms, and in

determining the relative involvement of different opioid receptor types in

pharmacologic studies.

DESIGN RATIONALE

The original impetus for pursuing affinity labels for opioid receptors stemmed

from conclusions (Portoghese 1965, 1966) drawn from our stereochemical studies

on opioid ligands. In this connection, the divergent stereochemical requirements.

Convergent pharmacologica studies (Martin et al. 1976; Ward and Takemori 1976;

Lord et al. 1977) subsequently complemented and provided a detailed framework

for this concept. During that period, we had begun to investigate electrophilic

affinity labels as an alternative to stereochemical probes to explore opioid

receptors (Portoghese et al. 1971, 1977; Takemori et al. 1974).

The rationale for our approach was based upon studies of Baker (1967) who

pioneered the area of active-site, irreversible enzyme inhibitors. In theory, when

the affinity label contains an electrophilic center, high selectivity for opioid

receptors should be dependent on four parameters. These are: a) affinity of the

receptor for the ligand; b) equilibrium-controlled receptor selectivity; c) location

of electrophilic center in the ligand, and d) reactivity and chemical selectivity of

the electrophile.

In considering these parameters, it is evident that an electrophilic affinity label

may be involved in two consecutive recognition processes which lead to covalent

attachment to the receptor (figure I). The first recognition step is manifested by

157
receptor affinity and the second recognition step involves proper alignment of

the electrophilic center of the ligand with a compatible receptor-based

nucleophile.

FIGURE 1

A schematic illustration of the principle of recognition amplification in the

covalent binding of receptor type A by affinity label containing a group-selective

electrophile X. Note that receptor types A-C have similar topographic features

which lead to reversible binding (1° recognition). However, the receptor types

differ with respect to the reactivity (G1

and C) of nucleophiles. Only in A is the nucleophile G1, reactive with respect to

vs G1 in A and B) and location (G1 in A

X and within covalent binding distance (2’ recognition).

Because two recognition steps rather than one lead to covalent binding, enhanced

receptor selectivity (recognition amplification) is possible, particularly with

chemically selective electrophiles. On the other hand, if an affinity label

contains a highly reactive electrophilic group, such recognition amplification is

minimal and covalent binding selectivity is determined primarily by the first

recognition step. For example, since the promiscuity of the aziridinium ion

enables it to alkylate almost any nucleophile within covalent bonding distance.

the selectivity of a ligand which contains the active electrophilic group will be

determined primarily by its relative affinity for different receptor populations.

The concept of recognition amplification may be utilized for the design of

affinity labels that are highly selective for a single opioid receptor type among

multiple types if each receptor type contains a unique array of nucleophiles that

differs with respect to nucleophilicity and accessibility. This is illustrated

schematically in figure 1 where three receptor types with similar topographic

features (A, B, C) all are capable of associating reversibly with the affinity label.

However, the second recognition step takes place only with receptor type A

because the other receptor types contain nucleophiles that either are

insufficiently reactive (type B) or are not within covalent bonding distance (type

C) of the electrophile (X).

158
EXPLORING THE FIRST RECOGNITION STEP

An important consideration in the design of opioid receptor affinity labels is the

nature of a ligand which will be modified with an electrophilic moiety. Since

our prior efforts and those of others with the modification of agonist ligands did

not yield compounds that have had wide utility, we turned our attention to the

antagonist pharmacophore as the recognition unit. A consideration in this choice

was based on the fact that antagonists, especially those without agonist activity,

have been extremely useful in pharmacologic investigations and in characterizing

opioid receptor involvement.

We therefore selected naltrexone 1 (table 1) for modification, as this pure

antagonist possesses high affinity for opioid receptors and contains convenient

functionality at the C-6 position for molecular modification. Moreover, C-6 was

chosen as a point of attachment for the electophilic group because it was

reported previously (Hahn et al. 1975) that a variety of C-6 substituents did not

destroy receptor affinity (first recognition step). In this connection, the 6-amino

derivative of naltrexone, beta-naltrexamine 2, possessed an affinity equal to that

of naltrexone (Jiang et al. 1977). This was relevant to our approach in that 6

beta-amino group was employed as a handle to attach various electrophilic

moieties. Our preference for the 6 beta-isomer rather than the 6 alpha-isomer

was based on the greater affinity of the former in the binding assay.

THE SECOND RECOGNITION STEP: RANGE-FINDING STUDIES

After it was established that the first recognition step was unimpaired by the

presence of a 6 beta-amino group, our design strategy involved the introduction

of a highly reactive electrophilic moiety (i.e., aziridinium ion) to assess the

availability of a proximal receptor-based nucleophile. In this case, it was

expected that the selectivity for different receptor types would be conferred

largely by the relative affinity (first recognition step) of the ligand at these sites.

Once nonequilibrium blockage was demonstrated, then less reactive and more

selective electrophilic groups were introduced in an effort to “fine-tune” the

ligand to allow a second recognition step to occur at one receptor type in a

population of multiple opioid receptors.

This rationale led first to the development of a nitrogen mustard derivative of

naltrexone, beta-chlornaltrexamine 3 (beta-CNA) (table 1) (Portoghese et al.

1978). Beta-CNA was found to be highly selective for opioid receptors and it

inhibited irreversibly the specific binding of opioids to brain membranes

(Portoghese et al. 1979). In both the guinea pig ileal (GPI) longitudinal muscle

and mouse vas deferens (MVD) preparations, beta-CNA produced irreversible

antagonism to opioids (Ward et al. 1982a; Caruso et al. 1979). This antagonism,

which was characterized by a shift of the concentration-response curve to the

right, followed by a decline in the maximum effect, is typical of classical

nonequilibrium antagonism involving spare receptors. The antagonism could be

prevented but not reversed by naloxone. Since nonopioid nitrogen mustards had

no selectivity for opioid sites, the data suggested that beta-CNA exerts its

sustained antagonism by alkylating opioid receptor nucleophiles. The selectivity

of its action was demonstrated by the fact that beta-CNA failed to block the

effect of norepinephrine in the GPI or block other binding sites (cholinergic,

prostaglandin, benzodiazepine) (Fantozzi et al. 1981). As beta-CNA was found to

be highly reactive toward nucleophilic reagents, it presumably reacts with

receptor nucleophiles via its aziridinium ion 4 (table 1).

159
Table 1

Naltrexone and Related Compounds

R

1

 = CH2CH(CH2)2 unless

otherwise indicated

1 = 0 (naltrexone)

2 NH2

11

3 N(CH2CH2Cl)2(ß-CNA)

4 N(CH2CH2)CH2CH2Cl 12 NHCOCH2N

5 N(CH2CH2Cl)2,R1= CH3 (ß-COA)

6 NHCOC=CCOOMe (ß-FNA)

7 NHCOC=COOMe, R

1

-CH3 (ß-FOA) 15 NHCOCH2SS

8

9 NHCOCCl=CH2

13 N-C-S

14 NHCOCH2I

16 NHCOCH2HgCl

17 NHCOCOC6H5

18 NHCOCH2CH2COOMe

10 NHCOC-CCOOMe

160
The ease with which beta-CNA blocked different opioid receptor types was mu >

kappa > delta, which paralleled its reversible binding affinity for these sites

(Sayre et al. 1983c; Goldstein and James 1984). This is in harmony with the idea

that little or no distinction between receptor-based nucleophiles occurs with a

highly reactive and promiscuous electrophile such as aziridinium ion.

The pharmacologic profile of beta-CNA in mice also was consistent with the

alkylation of opioid receptors, in that it produced ultralong antagonism (> 3 days)

of morphine-induced analgesia after a single (i.c.v.) injection (Caruso et al.

1980b). By comparison, naltrexone antagonism lasted less than 2 hours. It was

determined that the opioid binding capacity of brain membranes of beta-CNAtreated mice was significantly decreased during this 3-day period, which

suggested covalent binding had occurred in vivo. When administered

intrathecally (i.t.), beta-CNA produced even longer antagonism (> than 13 days)

against it. morphine antinociception (Larson and Armstrong 1980).

In view of the potent nonequilibrium opioid antagonism displayed by beta-CNA,

we investigated the consequence of attaching a nitrogen mustard group to the

same position in oxymorphone. a potent agonist. This derivative, designated as

beta-chloroxymorphamine 5 (beta-COA) (table 1) (Caruso et al. 1979). was of

interest as a tool to test the generality of the rate theory of drug action proposed

by Paton (1961). According to this hypothesis, a characteristic feature of

antagonists is their slow dissociation from receptors when compared to agonists.

Beta-COA behaved as an irreversible agonist in the GPI. The fact that naloxone

protected against irreversible agonism, but could not reverse it, implicated the

involvement of opioid receptors. These results suggested that, at least in the

GPI, ligand receptor occupation rather than rate of dissociation is important for

agonist activity. Beta-COA administered i.c.v. in mice produced analgesia that

lasted 4 times longer than oxymorphone (Caruso et al. 1980a). The brains of

beta-COA-treated animals exhibited a decreased capacity to bind opiates,

suggesting covalent binding had occurred. Interestingly, beta-COA given i.c.v.

displayed a long-lasting antagonism (6 days) after the agonism had disappeared.

The antagonism was more pronounced (> 21 days) when beta-COA was

administered i.t. (Larson and Armstrong 1980).

BETA-CNA AS A TOOL

The irreversible nature of beta-CNA has made it useful as a research tool. [3H]-

beta-CNA has been employed to isolate opioid receptor components from brain

membranes (Caruso et al. 1980b). The elution profile suggested four covalently

bound fraction, two of which migrated as single-peak calibrated to be 590,000

daltons. These complexes may represent multiple forms of opioid receptors.

Since beta-CNA alkylates all opioid receptor types, it has been used in protection

studies. For example, in the MVD it has been used in the presence of a

protecting concentration of a delta agonist to irreversible block mu and kappa

receptors (Ward et al. 1982b). Such a preparation contains the highly enriched

population of delta receptors. Other protecting ligands have been employed with

beta-CNA in the smooth muscle preparations (Chavkin et al. 1982; Chavkin and

Goldstein 1981). Using the protection procedure, beta-CNA has been used to

enrich specific binding sites in brain membranes (Goldstein and James 1984;

James and Goldstein 1984). Beta-CNA has been employed to progressively block

dynorphin receptors in the GPI and MVD. It was concluded that the GPI has

more spare kappa receptors than the MVD (Cox and Chavkin 1983). An effort

161
to estimate the dissociation constant of the normorphine-receptor complex in the

GPI using beta-CNA has been reported (Porreca and Burks 1983).

Beta-CNA also has been employed as a pharmacologic tool in vivo. These

include studies on the opioid nature of delta-tetrahydrocannabinol (Tulunay et

al. 1981) in rats and behavioral studies in mice (Quack and Lucas 1981), rats

(Messing et al. 1982), and chicks (Panksepp et al. 1982). The inhibition of

tolerance development to, and withdrawal from, morphine after i.t.

administration of beta-CNA also was investigated (DeLander and Takemori

1983).

THE SECOND RECOGNITION STEP: FINE TUNING

Since the pharmacologic characterization and binding studies of beta-CNA and

beta-COA suggested that these ligands selectively and irreversibly bind to opioid

receptors, it seemed likely that neighboring receptor-based nucleophiles were

involved in covalent bond formation with the aziridinium ion that was generated.

The reactivity and promiscuity of this electrophile minimize the importance of

the second recognition step. Consequently, the facility with which beta-CNA

alkylates different opioid receptor types is determined by the first recognition

step (affinity of reversibly bound ligand for the receptor type). Thus, based on

the assumption that different opioid receptor types contain different arrays of

nucleophiles, our strategy for enhancing selectivity of the second recognition step

was to employ electrophilic groups with a reactivity that is limited to only one or

two different nucleophiles. In this regard, Michael acceptors appeared to fulfill

this requirement because they react readily with sulfhydryl and sluggishly or not

at all with other nucleophiles. Another reason for selecting a Michael acceptor

groups was that studies with N-ethylmaleimide implicated the presence of a

receptor-based sulfhydryl (Simon et al. 1973).

BETA-FUNALTREXAMINE (BETA-FNA)

The fumarate methyl ester group was one of several Michael acceptor moieties

that were attached to the C-6 position of naltrexone (Portoghese et al. 1980).

This compound, which has been named beta-funaltrexamine (beta-FNA), was

the most selective ligand in the series (table 1).

In the GPI, beta-FNA is a reversible kappa agonist and irreversible blocks mu

receptors in a concentration- and time-dependent manner (Takemori et al. 1981).

The lack of inhibition of kappa agonists (e.g., ethylketazocine) revealed that this

antagonism was selective for mu receptors. It is noteworthy that beta-FNA did

not antagonize its own agonist effect (mediated through kappa site). The

inhibition of morphine antagonism was manifested as a parallel shift of the

morphine concentration-response curve with no diminution in the maximal

response. Using naloxone as an antagonist, PA2 analysis revealed that beta-FNA

depleted the GPI of functional mu receptors and that the residual opioid receptor

population was of the kappa type (Ward et al. 1982b). Thus, the presence of a

maximal response to morphine after mu receptor blockage was due to the fact

that morphine can act as a full agonist at kappa receptors. This pharmacologic

profile was in contrast to beta-CNA, which produced both a rightward shift and

a decline in the concentration-response relationship of morphine as a

consequence of blockage of both mu and kappa receptors (Caruso et al. 1979). In

the MVD, beta-FNA displayed all of the reversible kappa agonist and

irreversible mu antagonist features it exhibited in the GPI, and it did not

significantly inhibit delta agonists at concentrations employed for the blockage of

162
mu receptors (Ward et al. 1982a). The binding characteristics of beta-FNA

binding appeared to be consistent with its profile in the GPI and MVD in that

the irreversible portion of beta-FNA binding demonstrated selectivity for mu

over delta binding sites, while the reversible component of beta-FNA binding

exhibited selectivity for kappa over mu or delta binding sites (Ward et al., in

press).

In mice, beta-FNA exhibited a pharmacologic profile similar to that observed on

smooth muscle preparations. It produced an antinociceptive effect of short

duration which appeared to be mediated by kappa receptors. In contrast. the

antagonist action was of remarkably long duration (> 4 days) (Ward et al. 1982c).

BETA-FNA AS A TOOL

Because of the high mu selectivity of beta-FNA, it has many applications as a

pharmacologic tool and opioid receptor probe. One of the initial uses was in

depleting the GPI of functional mu receptors, thereby affording a more

homogeneous population of kappa receptors (Ward et al. 1982b). This

preparation was useful in evaluating the kappa activity of so-called mu agonists.

It has been found that even mu agonists that were presumed to be highly

selective exhibited kappa activity at high concentrations (Takemori and

Portoghese, unpublished data). Since the high selectivity of beta-FNA obviates

the necessity of protection experiments involving mu receptors, the beta-FNAtreated GPI has been employed to characterize dynorphin as a kappa agonist

(Huidobro-Toro et al. 1982) and to demonstrate the presence of cross-tolerance

to opioids in the GPI (Seidl and Schulz 1983).

A number of studies in vivo have made use of beta-FNA to study the relative

involvement of mu receptors in various opioid actions. These include opioidinduced respiratory depression (Ward and Takemori 1983a). decrease in

gastrointestinal transit (Ward and Takemori 1983b). cardiovascular effects

(Holaday and Ward 1982; Pfieffer et al., in press), spinal analgesia (Hylden and

Wilcox 1983a. 1983b), antidiuresis (Zimmerman et al. 1984) and prolactin release

(Holaday et al. 1983). Beta-FNA also has been used to demonstrate the noninvolvement of mu receptors in certain opioid actions such as endotoxic shock

(Holaday et al. 1983) and post-ictal analgesia (Belenky et al. 1983). The mu

receptor involvement in analgesia (Hynes et al. 1384; Dystra 1984; Frederickson

et al. 1984; Leander et al. 1984; Schafer et al. 1984) produced by kappa and delta

agonists and the phenomena of tolerance and physical dependence (Aceto et al.

1984; DeLander et al. 1984; Gmerek and Woods, in press) also have been

investigated using beta-FNA.

An interesting application of the high mu selectivity of beta-FNA stemmed from

the finding that its N-methyl analogue 7 (table 1) derived from oxymorphone,

beta-FOA (Portoghese et al. 1980; Takemori et al. 1981). is reversibly acting mu

agonist in the GPI. No irreversible blockage of opioid receptors was observed.

The possibility that mu agonists and antagonists interact with separate recognition

sites was studied by evaluating a series of opioid agonists and antagonists for

their ability to protect against the irreversible mu antagonism by beta-FNA

(Portoghese and Takemori 1983). Antagonists afforded excellent protection

against irreversible blockage by beta-FNA, whereas most agonists were relatively

poor protectors. Moreover, the ability of the compounds to protect against betaFNA appeared to correlate with their antagonist potencies (Ke

) but not their

agonist potencies (IC50). These results suggested that mu agonists and antagonists

interact at separate sites on the mu receptor system. It was proposed that the

163
antagonist recognition site is a regulatory site which controls the agonist effect

mediated by mu receptors.

STRUCTURE-ACTIVITY RELATIONSHIP STUDIES

A number of Michael acceptor analogues of beta-FNA were synthesized in order

to delineate the steric requirements of covalent bond formation with opioid

receptors (table I) (Sayre et al. 1983a. 1984). In the beta-series, it was found

that the ketone 8 and acrylamide 9 exhibited irreversible blocking activity against

morphine but not ethylketazocine. This antagonism was in the range of that

displayed by beta-FNA. It is noteworthy that dihydro-beta-FNA 18 is a

reversibly acting compound, as would be expected for an unreactive ligand. The

fact that the Michael acceptors with cis double bonds (10, 11) were not active as

irreversible antagonists suggests that steric factors do play an important role in

the second recognition step. This is supported by kinetic studies which show that

the reactivities of the Michael acceptors toward cysteine do not parallel the rank

order of irreversible activities. Thus, it is plausible that proper alignment of the

electrophile with a receptor-based nucleophile is critical for covalent binding

with mu receptors in the GPI preparation. In support of this idea it was reported

that, even at high concentrations, alpha-FNA was incapable of irreversible

blockage of opioid receptors (Sayre et al. 1983b). The fact that irreversible

antagonism of beta-FNA could be blocked by protection with alpha-FNA

established that the electrophilic moiety is not properly oriented in the alphaseries. Significantly, the C-6 stereospecificity observed for beta-FNA was not

found among the CNA C-6 epimers. as both beta-CNA and alpha-CNA (Sayre et

al. 1983c) are irreversibly acting ligands. This indicates that the second

recognition step is important only with relatively selective electrophilic groups.

It was noted that the placement of a spacer between the opiate and the Michael

acceptor group dramatically changed the pharmacologic profile. While the

maleimide 11 was devoid of irreversible antagonist activity, its N-acetyl analogue

12 selectively blocked my receptors in the MVD without a significant effect on

the GPI preparation (Sayre et al. 1984). The rationale for the synthesis of 12 was

based on the assumption that different receptor subpopulations contain a

different array of nucleophiles. Thus, by altering the distance of the electrophile

from the pharmacophore, opioid receptor selectivity can be altered to distinguish

opioid receptor subtypes.

Electrophiles other than Michael acceptors exhibited varying degrees of

selectivity in irreversibly blocking the effects of different agonists (Sayre et al.

1983c, 1984). Most notable was the isothiocyanate 13 which appeared to block

mu receptors in the GPI and mu and delta receptors in the MVD. Interestingly,

the alpha-isomer of 13 did not irreversibly block mu receptors in the GPI. Thus,

the stereoselectivity is similar to that of beta-FNA and other active Michael

acceptors in this preparation. The iodoacetamide 14 showed considerably less

cross-reactivity with different receptor types by selectively blocking the mu

receptors in the MVD. The pharmacologic profiles of 12 and 14 indicate that the

mu receptors in the MVD are different from those in the GPI.

Other compounds in this series (15 to 17) are electrophilic but produced no

significant blockage of morphine, ethylketazocine. or [D-Ala2-D-Leu6]enkephelin

(DADLE) in the smooth muscle preparations. The reason for the inability of

these compounds to act as irreversible antagonists is not well understood at this

time. However, it may be related to a deficient secondary recognition step.

164
CONCLUSIONS

The extraordinary selectivity of beta-FNA for mu opioid receptors can be

viewed as arising from two sequential recognition steps leading to covalent bond

formation. While reversible binding to the mu receptor system is important, the

proper alignment of the electrophiles with a compatible receptor-based

nucleophile is critical for the selectivity of irreversible blockage. In this

connection, the fumarate moiety appears to possess a narrow spectrum of

reactivity and a geometry which permits facile access to the receptor-based

nucleophile. Refinement of this approach has led to development of subtypeselective blockers of mu receptors. The enhanced selectivity of these probes is

proving to be very useful in the pharmacologic and biochemical characterization

of opioid receptors.

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ACKNOWLEDGMENT

This research was supported by the National Institute on Drug Abuse.

AUTHORS

Phillip S. Portoghese, Ph.D.

A.E. Takemori, Ph.D.

Departments of Medicinal Chemistry

and Pharmacology

University of Minnesota

Minneapolis, MN 55455

168
Peptide Conformation and Biological

Activity: A General Review With Examples

of Approaches Relevant to Opioid Peptide

Function

Dan W. Urry, Ph.D.

INTRODUCTION

In this International Conference on the Medicinal Chemistry and

Molecular Pharmacology of the Opioid Peptides and the Opiates,

it is a challenge to respond to the invitation to give a review

of a general nature with examples of both methodological

approaches and conformational analyses of peptides, and to do so

in a manner that could have some relevance to the theme of the

conference. This is particularly so as it has been a number of

years since, working with my colleague M.A. Khaled, we were

directly concerned with the solution conformation of Met- and

Leu-enkephalins (Khaled et al. 1977, 1979). Nonetheless, it is

satisfying that the fundamental perspective of enkephalin conformational flexibility is retained and that the two conformations considered in that work, a -chain conformation capable

of antiparallel association and a monomeric -turn conformation,

continue to be relevant to current considerations in solution

and in the crystalline states (Schiller 1984; Karle et al.

1983a). It is the conformational flexibility of the enkephalins

and the opioid peptides in general, coupled to the objective of

determining a receptor bound state, that makes the conformationfunction studies of opioid peptides so challenging.

An attractive approach is to impose structural restrictions on

conformational freedom which would allow probing of receptors

and which could lead to more selective binding at one of the

several receptors now recognized for the opioid peptides (Atweh

and Kuhar 1983; Paterson et al. 1983; Barnard and Demoliou-Mason

1983). Accordingly, amino isobutyric acid (Aib) and D•Ala analogs have been synthesized with interesting results (Gorin et

al. 1978; Sudha and Balaram 1983; Beddell et al. 1977).

Enkephalins with restricted side chains have been prepared and

studied with delineation of different receptor activities and

with increased potency at particular receptors (English and

169
Stammer 1978; Shimohigashi and Stammer 1982a, 1982b;

Shimohigashi et al. 1982; Deeks et al. 1983). Additionally,

cyclic enkephalin analogs have been synthesized and their activities determined. sometimes with strikina increase in activity

in one assay and decrease in activity in another (DiMaio and 

Schiller 1980; Schiller et al. 1981). It has been concluded

that "Activity profiles obtained with a cyclic enkephalin analog

and its linear correlate revealed that μ and -receptors differ

in their conformational requirements" (Schiller 1984). Such

approaches, coupled with graphic volume analyses such as those

of Marshall and colleagues (Gorin and Marshall 1977; Gorin et

al. 1980), can be expected to provide significant progress on

the conformation-activity problem of the opioid peptides and

receptors.

Much progress has been made in the characterization of the

opioid receptors and of the actions of opioid peptides. It has

been found that opioid receptors reside in the plasma membrane

of nerve cells and that they are protein in nature with a significant phospholipid component (Barnard and Demoliou-Mason 1983).

The opioid peptides alter membrane conductance (West and Miller

1983) with both pre- and post-synaptic actions in mammalian

parasympathetic ganglia (Katayama and Nishi 1984). It has been

reported that they hyperpolarize cells by means of increasing

potassium ion membrane conductance in the central nervous system

(Henderson 1983) and specifically by opening potassium ion channels in peripheral nerve cells (North and Egan 1983; Morita and

North 1981). A significant action in dorsal root ganglion cells

and peripheral nerve cells is the inhibition of neurotransmitter

release (Hughes and Kosterlitz 1983; North and Egan 1983;

Wouters and van den Bercken 1980; Werz and Macdonald 1982).

Additionally, opioid peptides stimulate hormone release

(Grossman and Rees 1983) and there are yet other actions

reported, including some which relate to the development of

tolerance and addiction (West and Miller 1983). What clearly

emerges, of course, is action at a membranous site with dominant

effects being on channel conductance and neurotransmitter

release.

This conformation-activity review with concern for methodological approaches will attempt to address peptide channel activity

at a membranous site with some effort to note the wide-ranging

set of methodologies that are required to attack such a problem

effectively. Disciplines involved in a comprehensive approach

to the understanding of opioid peptide function include: 1)

synthesis of peptide analogs (Peptide Chemistry); 2) characterization of molecular conformation by physical methods and by

molecular mechanics calculations (Molecular Biophysics); 3) isolation and reconstitution of receptors and associated transport

components (Membrane Biochemistry); and 4) characterization of

opioid peptide and analog activities (Molecular Pharamacology,

Membrane Biophsyics. and Electrophysiology). These components

170
for a comprehensive attack on the mechanism of action of opioid

peptides all have analogous components in determination of the

molecular structure and ionic mechanism of Gramicidin A channel

transport.

Accordingly, this chapter reviews a general conceptual and

experimental approach of considering cyclic conformations and

linear conformational correlates with use of the physical

methods of nuclear magnetic resonance (NMR), circular dichroism,

and dielectric relaxation. This will allow a conceptual derivation of the Gramicidin A channel structure, an experimental

development of a dynamic molecular structure wherein molecular

flexibility is an integral part of function, and the conceptual

derivation of a voltage-dependent channel structure. There will

be a brief consideration of channel transport phenomenology combined with the utilization of an array of disciplines, with

similarity to those noted above for approaching the opioid peptide structure-activity problem, to derive the molecular structure and mechanism of Gramicidin A channel transport. Finally,

there will be a few comments on the patch clamp methodology of

Neher and Sakmann and their colleagues (for an excellent review

of the subject, see Sakmann and Neher 1983) which is being used

in the Gramicidin A channel tranport study and which appears to

be particularly well-suited for the detailed study of opioid

peptide activity. This approach provides a means of studying

single whole cells or small membrane patches from cells, or of

reconstituted protein-membrane systems. It can be expected to

give rise to more detailed and less ambiguous demonstration of

activities and mechanisms related to ion transport across cell

membranes. It could also be used to follow, by means of cell

membrane capacitance changes, the fusion of neurotransmitter

loaded vesicles with plasma membrane in this mechanism of

neurotransmitter release (Neher and Marty 1982).

CYCLIC CONFORMATIONS WITH LINEAR CONFORMATIONAL CORRELATES

(CCLCC): AN ANALYTICAL CONCEPT AND AN EXPERIMENTAL APPROACH

The analytical concept of a cyclic conformation with a linear

conformational correlate (Urry 1972) derives from the realization that, if a cyclic peptide with an adequate number of residues forms a definable conformation with reasonable stability,

then one can anticipate that a linear polymer comprised of the

repeating sequence of the cyclic structure could exist in a

related conformation. This is because, with two torsion angles

per peptide and with a substantial number of residues in the

cycle, only small changes in the torsion angles could result in

a left- or right-handed helical structure comprised of the

repeating unit. The resulting helical structure is particularly

favorable when the conformation gives rise to favorable interturn interactions. This concept is relevant to the channel

structure of Gramicidin A (see below): it was originally

described in the first enunciation of voltage-dependent channel

formation using only dipole moments, in this case the dipole

171
moment of the peptide moiety (Urry 1972); it will be used as an

analytical concept below to derive two different channel structures; and it will be used as an experimental approach to derive

a particularly interesting helical conformation referred to as a

-spiral.

The Channel Conformation of Gramicidin A

If one considers the L•amino acid-Doamino acid (L-D) repeating

dipeptide unit contained in a cyclic hexapeptide, the relative

orientation of the peptide moieties would be as depicted in

figure 1A, pointing alternately up and down with respect to the

mean plane of the cyclic hexapeptide.

FIGURE 1

Representation of a cyclic conformation A, and a linear conformational correlate B, comprised of repeating L-D dipeptides.

Part B is from Urry et al. 1971. Copyright 1971, National

Academy of Science.

The side chains (R groups) would be oriented in a radial manner.

This is closely the structure of the ionophore enniatin B

(Plattner et al. 1963), wherein the cation is held centrally by

peptide carbonyl oxygens that are librated inward with three

carbonyl oxygens above and three below the plane to achieve

coordi-nation of the cation. To convert this to a linear conformational correlate, the cyclic structure is ruptured; small

torsion angle changes are made to give a helical pitch of about

5A; the hexapeptide is repeated; and it is found that hydrogen

bond formation readily occurs between turns of the helix, as

schematically shown in figure 1B. The hydrogen bonding pattern

between turns exhibits the same pattern as between chains in the

parallel -pleated sheet. For this reason, the structure is

referred to as a single stranded -helix. In fact, the structure is also readily derived from a single -chain by

172
FIGURE 2

A. and B. Spacefilling model of the Gramicidin A transmembrane channel in channel view (A) and in

side view (B). C. Wire model of the backbone and -carbons of the channel seen in side view as in B.

From Urry et al. 1975. Copyright 1975, New York Academy of Science.
interchanging backbone and side chain (i.e., converting from an

L-residue to a D-residue) at every other residue in the -chain.

The primary Structure of Gramiciden A is HCO-L•Val1-Gly2-L•Ala3-

D•Leu4-L-Ala5-D•Val6-L•Val-D-Val8-L•Trp9-D•Leu10-L•Trp11-

D•Leu12-L•Trp13-D•Leu14-L•Trp15-NHCH2CH20H (Sarges and Witkop

1964); that is, Gramicidin A could be a linear conformational

correlate of enniatin B which is a cyclohexapeptide with a

repeating L-D dipeptide sequence. The result is the channel

conformation of Gramicidin A (Urry et al. 1971) which is shown

in figure 2 (Urry et al. 1975). Note the pattern of hydrogen

bonding between turns of the helix in figure 2C. If two

chains were in either parallel or antiparallel association and

the same conversion of alternate residues from L to D were

carried out, the result would be double stranded -helices--a

structure proposed by Veatch et al. (1974). As will be noted

below, delineation of channel structure as between single and

double stranded -helices can be demonstrated in a simple way

using carbon-13 NMR of carbonyl carbon cation induced chemical

shifts.

The Polypentapeptide of Elastin --An Experimental Use of CCLCC

The precursor protein of the elastic fiber contains as a

repeating peptide sequence, L•Val1-L•Pro2-Gly3-L•Val4-Gly5

(Sandberg et al. 1981), referred to simply as VPGVG. When this

pentameric sequence is synthesized and the pentamer polymerized

and cross-linked either by chemical means (Urry et al. 1976) or

by y-irradiation (Urry et al. 1984c), the resulting matrix is

found to have elastomeric properties strikingly similar to those

of the native elastic fiber (Urry et al. 1985). The polypentapeptide forms a relatively complex and dynamic structure. One

particularly interesting way in which its conformation was

approached involved a search for a cyclic analog which, by means

of various physical characterizations, exhibited a similar conformation to that of the linear high polymer. The solution conformational approaches are in large part the same as those that

have been used to study the opioid peptides (Schiller 1984),

particularly the enkephalins, and the result provides a clear

demonstrations of the sensitivity of the approaches to conformation.

Comparison of physical characterizations of the cyclic and

linear structures: Figure 3 contains the circular dichroism

curves in water for the cyclopentapeptide, i.e., cyclo(VPGVG);

the cyclodecapeptide, i.e., cyclo(VPGVG)2; the cyclopentadecapeptide, i.e, cyclo(VPGVG)3; and the linear polypentapeptide, i.e., H-(VPGVG) V-OMe. It is seen that the cyclopentapeptide exhibits a dramatically different conformation with the

right-hand ordinate being for this curve. The cyclodecapeptide

exhibits a significantly different conformation, and the cyclo174
FIGURE 3

Circular dichroism spectra of cyclic analogs and the linear high

polymer. From Urry 1985a. Copyright 1985, Elsevier Science.

pentadecapeptide exhibits a conformation which is more similar

to that of the linear polymer. Thus, the cyclopentadecapeptide

is a candidate for the cyclic conformational correlate of the

linear polymer.

Using proton NMR, the temperature dependence of peptide NH proton chemical shift in dimethylsulfoxide was determined and is

shown in parts a, b, and c of figure 4 (Urry et al. 1981) for

the cyclic analogs. The dotted curves give the data for the

linear polymer. The cyclopenta- and cyclodecapeptides exhibit

very different patterns of temperature dependence of chemical

shift, whereas the patterns are very similar for the cyclopentadecapeptide and the linear polypentapeptide. Since in each

case the sequence is the same, i.e., each residue has the same

nearest neighbor and next nearest neighbor etc., the differences

that appear must be solely due to differences in conformation.

This demonstrates that the approach is very sensitive to conformation and that the cyclopentadecapeptide is a cyclic conformational correlate of the linear polypentapeptide. The small

175
FIGURE 4

Temperature dependence of amide proton in dimethylsulfoxide of

cyclic analogs of the elastin pentamer. Adapted with permission from Urry et al. 1981. Copyright 1981, American

Chemical Society.

FIGURE 5

Solvent dependence of peptide carbonyls in dimethylsulfoxide

and water of cyclic analogs of the elastin pentamer. Adapted

with permission from Urry et al. 1981. Copyright 1981, American

Chemical Society.

176
chemical shift in the Val1NH proton, which is essentially

constant with temperature, correlates with a small difference in

the Val1 coupling constant. Rotation of the 1 torsion

angle by a few-degrees in a direction that would make the

coupling constants similar would convert the cyclic structure to

a right-handed helical structure. This would also move the

Val1NH peptide proton toward the edge of a peptide plane in a

manner that would lead to a downfield chemical shift due to the

magnetic anisotropy of the peptide moiety. Thus, the data not

only argues that the conformations of the cyclopentadecapeptide

and linear polymer are similar, but it also suggests that the

linear polymer forms a right-handed helical structure.

In figure 5, carbon-13 NMR is used to compare the solvent dependence of peptide carbonyl carbon chemical shifts of the cyclic

and linear structures (Urry et al. 1981). The solvent pair is

dimethylsulfoxide and deuterated water and, again, dotted curves

are used for the linear polymer data to facilitate comparison

with each of the cyclic structures. The peptide C-0 chemical

shift dependence on solvent also shows the cyclopenta- and

cyclodecapeptides to be in conformations very different from

each other and very different from the linear polymer, whereas

again the cyclopentadecapeptide conformation is very close to

that of the linear polypentapeptide.

FIGURE 6

Solvent dependence of amide proton in dimethylsulfoxide and

water of cyclic analogs of the elastin pentamer. From Urry et

al. 1981. Copyright 1981, American Chemical Society.

177
It is also interesting to look at the solvent dependence of the

peptide NH chemical shifts. This is shown in figure 6, using

the same Solvent pair as in figure 5 but for the six cyclic

analogs with n=1, 2, 3, 4, 5, and 6. As with the data of

figures 4 and 5, the data for the linear polypentapeptide is the

dotted set of curves superimposed on each of the sets of curves

for the cyclic analogs. Again, the patterns for the cyclopentaand cyclodecapeptides are dramatically different from each other

and from that of the linear polymer. The data for the cvclopentadecapeptide, as before, closely follow that of the linear 

polypentapeptide. The data for the cycloicosapeptide (n=4) and

the cyclopentaicosapeptide (n=5) do not follow so closely. But

the data for the cyclotricosapeptide (n=6) follow extraordinarily closely, even more so than for the cyclopentadecapeptide. This is thought to be due to a figure 8 type of structure

comprised of two pentadecapeptide moieties related by twofold

symmetry and differing from the cyclopentadecapeptide primarily

by the introduction of a helical pitch resulting from chain

crossing.

These results demonstrate the validity of such approaches as

sensitive monitors of conformation. The use of these methods is

particularly favorable for a sequence such as this pentamer,

where all side chains are aliphatic--having neither hydrogen

bonding capability nor magnetic anisotropies that can complicate

interpretation of temperature and solvent dependence data. The

interpretation of such data for the enkephalins is complicated

by the ring current effects of the Phe and Tyr side chains and

the hydrogen bonding capabilities of the Tyr OH and the amino

and carboxyl end groups.

Conformations of the Cyclic and Linear Structures of the

Polypentapeptides of Elastin

The solution derived conformations are shown in figure 7A, B,

and C for the cyclopentapeptide, the cyclodecapeptide, and the

cyclopentadecapeptide, respectively (Khaled et al. 1981, 1982;

Venkatachalam et al. 1981). These were derived using the above

type of NMR data to identify secondary structure, with coupling

constants to limit Hi-NHi torsion angles, and with the nuclear

Overhauser effects to limit the Hi-NHi+1 distances, all in

combination with conformational energy calculations to limit

further the allowed torsion angle ranges. Essentially the same

conformation for the more rigid cyclopentapeptide has been

determined in the crystal by X-ray diffraction (Einspahr et al.

1980), as has that of the more flexible cyclopentadecapeptide

which is shown in figure 8 (Cook et al. 1980). The molecular

structure of the cyclopentadecapeptide is analogous to a threefooted stool with the Pro2-Gly3 -turns functioning as the feet

of the stool. The crystal structure of the cyclopentadecapeptide is particularly interesting since the threefold symmetry is

retained (see figure 8A); the intermolecular interactions are

178
FIGURE 7

Crystal structures of cyclic analogues of the elastin pentamer.

A. The cyclopentapeptide; B. the cycle-decapeptide; C. the

cyclopentadecapeptide (stereo view). Reproduced with permission

from Khaled et al. 1981(A). Copyright 1981, Munksgaard,

Copenhagen. Khaled et al. 1982(B). Copyright 1982, Elsevier

Biomedical Press. Venkatachalam et al. 1981(C). Copyright

1981, American Chemical Society.

hydrophobic, and the substantial water in the crystal is within

the stack of cyclic molecules that are stacked exactly on top of

each other (see figure 8B).

It is a relatively simple conceptual process to take a stack of

cyclopentadecapeptide molecules and to convert them to a linear

left-handed or right-handed helix as described above for forming

the Gramicidin A channel from an enniatin E-like cyclic structure. When this is done by means of molecular mechanics, using

the potential functions of Scheraga and colleagues (Momany et

179
FIGURE 8

Crystal structure of the cyclopentadecapeptide. A. molecular

structure; B. crystal packing. From Cook et al. 1980.

Copyright 1980, American Chemical Society.

al. 1974, 1975), a right-handed helical structure is found to be

preferred (see figure 9), whereas going left-handed disrupts the

conformational features of the cyclic structure (Venkatachalam

and Urry 1981). This is consistent with the analysis of the

small 3J( coupling constant difference and the small difference in chemical shift of the Val1NH as discussed relative to

figure 4 above. [It may be noted that the same potential functions have been utilized in conformational energy calculations

of Met- and Leu-enkephalins and their analogs (Manavalan and

Momany 1979, 1980, 1981)]. The resulting linear structure

depicted in figure 9 is termed a -spiral, as the -turn is the

dominant repeating secondary structural feature. The term

spiral is used instead of helix to emphasize in the structure

that there is not the obligatory hydrogen bonding between

repeating units as in the classical helical structures of polypeptides. The interturn interactions are hydrophobic; there is

water within the -spiral; and the -turns function as spacers

between the turns of the -spiral. This is shown as stereopairs

for an all atom depiction in figure 9A and B and for an -carbon

only representation in figure 9C and D. The upper structures

180
FIGURE 9

Representations of the proposed spiral for the polypentapeptide of elastin. A, B, C, and D from

Venkatachalam and Urry 1981. Copyright 1981, American Chemical Society. E and F from Urry 1983.

Copyright 1983, McGraw-Hill.
give the helix axis view. Figure 9E schematically shows the

helical nature and figure 9F shows the -turns functioning as

spacers between turns.

-Spiral Temperature Dependence and the Source of Elasticity

While the -turns are present at temperatures below 20 °C, they

are less stable at that temperature and become more stabilized

as the temperature is raised. Also, the hydrophobic Val-Pro

side chains come into closer association and the interturn

hydrophobic interactions develop. These hydrophobic interactions are responsible for the -spiral formation. Particularly

on slight extension of the structure, as occurs on stretching,

the -turns are joined by a suspended segment, Val4-Gly5-Val1,

which is allowed a great deal of motional freedom as it is

largely surrounded by water and as the central residue is a glycine without a sterically restricting side chain. The peptide

moieties of this suspended segment are able to librate, or to

rock, thereby providing motion and entropy. It is thought that

the damping of this motion is the decrease in entropy that

occurs on stretching and this is responsible for the entropic

elastomeric retracting force. This has been termed a peptide

librational entropy mechanism of elasticity (Urry et al. 1982b,

1983a). This proposed mechanism has been substantiated by

FIGURE 10

Temperature dependence dielectric relaxation spectrum of

polypentaprptide of elastin. Adapted from Henze and Urry 1985.

Copyright 1985, American Chemical Society.

182
selected synthetic analogs (Urry et al. 1983a, 1983c), and by

the development of a motional mode on raising the temperature

that can be detected by means of dielectric relaxation studies.

As shown in figure 10, at 20 °C no resolvable relaxation is

observed in the real part of the dielectric permittivity in the

3 MHz to 1000 MHz range; but on raising the temperature, an

intense relaxation develops at 25 MHz as the -spiral forms

(Henze and Urry, unpublished data). This motional mode is due

to the motions of a dipole moment with the indicated frequency.

As the peptide is the only source of a dipole moment, this is

due to peptide motion. As the material is very viscoelastic,

the change is not whole molecule reorientation but rather a peptide rocking motion. This experimental result provides direct

evidence for a structure-dependent peptide librational entropy

mechanism of elasticity. Whereas structural flexibility is a

problem in characterizing opioid peptide conformations, this

does not mean the absence of conformation and, as in the case of

the polypentapeptide of elastin, flexibility could be an essential aspect of function.

The Helical Rack Model and a Proposed Voltage-Dependent Channel

The concept of CCLCC described above was used to derive the

Gramicidin A channel conformation and, in the elastin study, it

was used experimentally to determine which of a series of cyclic

analogs had a conformation resembling the linear structure. In

the process of comparing cyclic analogs, the experimental

methods used--temperature and solvent dependence of peptide NH

and C-O nuclear magnetic resonance chemical shifts--were showii

to be very sensitive to conformation. In this section, the concept of CCLCC is used to develop a structural motif for a

membranous protein which would function as a voltage-dependent

channel.

The classical helices, e.g., the helix and 310-helix, have

large dipole moments which could be used to orient in an

electric field or to induce helix formation on the application

of such a field. In 1975 (Urry 1975; Urry et al. 1975),

extending the use of peptide dipole moments to form voltagedependent channels (Urry 1972). a helical rack model was proposed utilizing a classical helix cyclized head to tail by a

-chain segment continuing in sequence as shown in figure 11.

The dipole moments of the individual peptide moieties of the

helix sum to make a large dipole moment which, since the dipole

moment of a -chain is small, cause the cyclic molecules to

align across a membrane on application of a potential. A schematic representation of the structure from a view perpendicular

to the membrane surface is given in figure 12 (Urry 1982).

Since all the -helices cross the membrane in the same direction

and the -chains do so in the opposite direction, all the

helices and -chains run in parallel and it is a simple matter

to convert from a series of cyclic subunits to the linear conformational correlate as shown in figure 13. This results in a

183
protein structural motif of a -barrel surrounded by -helices.

This has been recognized by Richardson (1981) as the structural

motif for triosephosphate isomerase, the pyruvate kinase domain

I, and aldolase. A related structured motif has been derived by

Marshall and colleagues (Hall et al. 1984) from studies on alamethicin voltage-dependent channel formation. Obviously, such

a structural motif and the insiahts it provides aive rise to

numerous experimental approaches. Thus; we have a proposed

molecular structure for a voltage-dependent channel which could

possibly provide a point of reference for the consideration of

the effect of opioid peptides on increasing potassium ion conductance.

b.

FIGURE 11

Helical rack model for voltage dependent channel formation.

From Urry 1975. Copyright 1975, John Wiley & Sons, Inc.

MOLECULAR STRUCTURE AND MECHANISM OF MEMBRANE CHANNELS: THE

GRAMICIOIN A CASE

As noted in the Introduction, the set of disciplines required in

an approach to the mechanism of action of opioid peptides is

closely similar to that required for a comprehensive approach to

the molecular structure and mechanism of Gramicidin channel

transport. Peptide synthesis is required to make analogs for

testing structural concepts and to prepare selectively isotopelabeled molecules for determining elements of mechanism.

Characterization of conformations, interactions, and rate processes by physical methods--such as circular dichroism, NMR,

ultraviolet, infrared, and fluorescence spectroscopies;

dielectric relaxation; etc.--provide an essential second component. Molecular mechanics calculations are required to test

and refine proposed elements of mechanism and to add information

and insight not directly available from experiment. In addition, characterization of peptide or protein effected ion

transport across lipid bilayer membranes is by methods that provide the greatest sensitivity and time resolution, such as the

planar bilayer approach of Mueller and Rudin (1967) and the

184
single channel recording techniques of Sakmann and Neher (1983).

The interplay and essential information exchange between each of

these components is extensive and the loss of any component

would mean a less well-characterized and less certain mechanism.

As an action of opioid peptides is to increase the opening of

potassium ion channels, a complete understanding of this mechanism of action would require analogous approaches.

FIGURE 12

Voltage dependent channel formed from cyclic subunits with each

subunit containing an -helical and a -chain component. This

is the same structure as given in figure 11 from a different

perspective. The perspective of figure 11 is the cross section

indicated by the line labeled 3.

1.

2.

-barrel at center comprised of parallel -chains.

Right-handed -helices with positive end of dipole at top.

3. Horizontal line indicates cross section for figure 11.

4.

5.

Side chains on -helices are hydrophobic.

Alternate side chains on -chain determine character of

channel. Others may be hydrophobic.

6. Arrow indicates direction of chain

From Urry 1982. Copyright 1982, Alan Liss, Inc.

Phenomenology of Channel Transport

In its simplest terms, characterization of channel transport

involves a pair of electrodes separated by a small lipid bilayer

membrane of high resistance. Commonly, the salts in the aqueous

solutions on each side are equivalent and a potential is applied.

The presence of peptides or proteins that can form channels

across the lipid bilayer result in conductance events as a chan185
FIGURE 13

Cyclic conformational correlate of the structures shown in

figures 11 and 12. Protein model for a voltage dependent channel.

1

2

-barrel at center.

Right-handed -helix with positive end of dipole at top.

3. Parallel aligned -chains.

4. Hydrophobic side chains of -helix.

5. Alternating side chains on -chains determine character of

the channel.

6. Arrow indicates direction of chain.

From Urry 1982. Copyright 1982, Alan Liss, Inc.

nel forms and disappears. This was first observed by Hladky and

Haydon (1970, 1971, 1972) with the Gramicidin peptides isolated

from bacillus brevis. Such single channel events have now been

observed for many different channels from higher organisms in

their natural cell membranes and reconstituted in model membranes

(see, for example, Sakmann and Neher 1983). The two basic channel characteristics are their single channel conductance given in

Siemens (channel current in coulombs/sec divided by applied

potential in volts) and channel mean lifetime, where a single

conductance event can last from milliseconds or less to seconds

or even minutes. Analgesics and anesthetics can be expected to

act indirectly or directly on a channel to alter one or both of

these two characteristics.

Figure 14 shows single channel current traces for Gramicidin A in

the upper trace and for N-acetyl desformyl Gramicidin A in part B

(Szabo and Urry 1979). The difference between these two channel

186
FIGURE 14

Single channel current traces in the presence of 1M RbCl with an

applied potential of 1OOmV for Gramicidin A (A) and for N-acetyl

desformyl Gramicidin A (B). The step increase and decrease is

the turning on and off of a single channel. From Szabo and Urry

1979. Copyright 1979, American Association for the Advancement

of Science.

structures is simply the replacement of a formyl hydrogen by a

methyl moiety (i.e., acetyl for formyl), yet the effects on channel conductance and lifetime are dramatic. For the potassium ion,

the single channel conductance is reduced from a most probable

value of 45 pico Siemens (pS) to 27 pS and the channel mean lifetime is reduced from 3.3 seconds to 56 milliseconds. On a

macroscopic basis, this has the effect of reducing conductance to

1%. As will be briefly argued below, even the reorientation of

an amino acid side chain removed by some distance from the channel itself can have a similar dramatic effect in varying conductance.

This allows that even a most subtle binding effect of an opiate

or other effector could either greatly increase or decrease

effective conductance.

187
Mechanism of Gramicidin A Channel Transport

Once structure is known, the major components of determining the

ionic mechanism of channel transport are to locate binding sites

in the channel, if indeed they exist, to determine binding

constants, and to estimate rate constants for each of the major

rate processes. When this is adequately achieved, a complete

free energy profile can be plotted and, when this is achieved

for different temperatures, complete enthalpy and entropy profiles should be obtained. With such information, the single channel currents would be calculable over wide ranges of ion

concentration, of applied potential, and of temperature. As

this is achieved, all of the obvious and subtle control mechanisms will become apparent.

Location of Ion Binding Sites

From the conformation of the channel (see figures 1 and 2), it

is apparent that, as the cation passes through the channel, it

is coordinated laterally by the peptide carbonyl oxygens.

Accordingly, if an appreciable occupancy occurs, it should be

observable--using carbon-13 NMR--as an ion-induced carbonyl carbon chemical shift. This had previously been observed for the

cyclic conformation correlate, the enniatin B-K+ complex (Urry

1976). However, to determine the ion-induced carbonyl carbon

chemical shift for the Gramicidin A channel in lipid bilayers,

it is necessary to synthesize Gramicidin A molecules wherein

only the carbonyl carbon of a single residue is enriched with

carbon-13. This has been done in 10 syntheses, i.e., for 10

different peptide carbonyl. For each synthesis, the channel is

then packaged in lipid bilayers at 3 mM concentrations and the

presence or absence of a cation-induced carbonyl carbon chemical

shift is determined for one residue. The results, plotted as a

function of carbonyl location in the left-handed single stranded

6

-helix, are given in figure 15 (Urry et al. 1982c). These

cation-induced carbonyl carbon chemical shifts, when plotted

with respect to other proposed structures, e.g., the double

stranded -helices (Veatch et al. 1974), can also be used to

determine unequivocally the channel structure (Urry et al.

1983b) and even to determine the helix sense (Urry et al.

1982c). The axiom utilized in delineating structures is that

helically (or structurally) equivalent carbonyls should show

similar ion-induced chemical shifts when equally proximal to the

ion.

188
Wire Model and Ion Binding Sites of the

Gramicidin A Transmembrane Channel

FIGURE 15

Location of ion binding sites using carbon-13 NMR to observe

ion-induced carbonyl carbon chemical shifts. Adapted with permission from Urry et al. 1982c. Copyright 1982, SpringerVerlag, Inc.

Determination of Binding Constants

The ion-induced carbonyl carbon chemical shift can be used to

estimate binding constants and also to clarify the nature of the

binding process. Shown In figure 16 is the thallium ion-induced

carbonyl carbon chemical shift followed with carbonyl carbons of

the Trp and Trp residues and plotted as a function of log

[Tl+]. What is apparent is that there are two binding

constants--one for entry of the first ion (a tight binding process) and one for entry of the second ion which, due to

repulsion from the first ion in the channel, is a weaker process. A tight and a weak binding constant can also be estimated

using quadrupolar ion NMR and longitudinal relaxation time studies (Urry et al. 1980). Thus, in this twofold symmetric channel with two structurally equivalent binding sites, there two

binding constants--a tight one for single occupancy and a weak

one for double occupancy.

189
FIGURE 16

Plot of thallium ion-induced carbonyl carbon chemical shifts as

a function of the logarithm of the ion concentration. This

clearly shows tight and weak binding processes, respectively,

for the entry of the first and second ions in the channel. From

Urry et al., unpublished data.

Estimation of Rate Constants

Ion NMR linewidths plotted versus chemical shift for spin l/2

ions or for situations that can so approximated and traverse

relaxation time studies of the spin 3/2 quadrupolar ions can be

used to estimate rate constants for ions leaving the channel

(Bull 1972; Venkatachalam and Urry 1980). The latter approach,

in particular, utilizes approximate expressions and arguable

assumptions to convert experimental relaxation times into correlation times and finally to rate constants. Such estimates have

been made for the sodium ion, and the obtained rate constants

have been shown with the other data on binding constants and

site location to calculate single channel currents over wide

ranges of ion activity and applied voltage (Urry et al. 1980).

190
FIGURE 17

Dielectric relaxation spectrum of a suspension of malonylGramicidin A channels. The solid curve showing the additional

relaxation beginning just below 10 MHz is the case where there is

one Tl+ ion in the channel. From Henze et al. 1982. Copyright

1982, Springer-Verlag New York, Inc. Inset: Representation of

the dipole moment change attending an intrachannel ion translocation from one binding site to the other. From Urry 1985b.

Copyright 1985, D. Reidel Publishing Company.

A particularly notable means, when useable, for obtaining a rate

constant relevant to transport through the channel is dielectric

relaxation. There is a large change in charge distribution when

an ion jumps from one binding site to the other in the channel.

This is depicted in the inset of figure 17. When the frequency

at which the field reverses is faster than the ion can move, it

does not contribute to the dielectric permittivity of the medium

containing the channels. When the frequency is slower than the

rate for ion translocation, the ion can jump back and forth as

the field reverses and, as such, can contribute to the

dielectric permittivity of the medium. This is seen for

thallium ion in the channel in figure 17 where a relaxation is

observed giving a rate constant for jumping between sites of

191
about 4 x 106

/sec. (Henze et al. 1982). A particularly attractive aspect of this approach is that it requires no complicated

theory with arguable assumptions to estimate the rate constant.

Thus, dielectric relaxation has been seen to provide critical

information above in the elastin structure-function problem and

here in the development of the ionic mechanism of channel

transport. It could also be used to provide information on the

dipole moment of enkephalin molecules in solution.

Modulation of Single Channel Conductance and Lifetimes

Single channel conductance magnitudes and channel lifetimes are

readily modulated. The dramatic effect of replacing a hydrogen

by a methyl moiety, i.e., conversion N-formyl to N-acetyl, was

noted above in relation to fiaure 15. This resulted in a 99%

decrease in conductance. There are many examples of the marked

sensitivity of the amino (formyl) end to modification, but there

is a relative insensitivity to derivitization at the ethanolamine

OH. For example, the 0-pyromellityl derivative with its triple

negative charge has only a small counterion cloud effect (Apell

et al. 1977), whereas the N-pyromellityl derivative completely

destroys activity (Barnberg and Janko 1977). More subtly, the

D•Leu2-GA analog results in a marked decrease in lifetime with

essentially no change in conductance (Bradley et al. 1981a). As

external effects. addition of the anesthetic halothane at the

physiological dose reduces lifetime from 25% to 30% of normal

(Bradlev et al. 1981b); raising calcium ion concentration

decreases conductance (Bamberg and Laüger 1977) by competitive

binding of calcium ion at the binding site even though divalent

cations are not conducted (Urry et al. 1982c); and increasing

membrane thickness decreases channel lifetime with no change in

conductance (Hladky and Haydon 1972).

An interesting analog is the des L•Va17-D•Val8-Gramicidin A which

is shorter by 3A due to the removal from the channel of two L-D

dipeptide units, one in each monomer of the dimeric channel. The

result, quite expectedly, is a shorter lifetime, but also, quite

instructively, the conductance is decreased to almost one-half

(Urry et al. 1984a). Since, by a diffusion-limited process, conductance would be expected to be increased by 30% for this extent

of shortening, the result indicates the essential correctness of

an Eyring barrier-type description and indicates further that the

dominant conducting state is one with double ion occupancy.

Of particular interest with respect to control of conductance is

the multiplicity of conductance states. When single channel conductance was mentioned above, the reference was actually to the

most probably conductance state. As shown in the histogram of

figure 18 by the dashed enclosure, while there is a most probable

conductance state for Gramicidin A at 100 mV, 20 °C, and 1M KCl

of 17 to 18 pS, there are other less probable states with lower

conductance (Urry et al. 1984c; Venkatachalam et al. 1984).

192
FIGURE 18

A. Conductance histograms for Gramicidin A (...) and L•Leu5-Gramicidin A (-) at 100 mV, 1 M KCl and

20 °C in diphtanoyl lecithin/n-decane membranes. From Venkatachalam et al. 1984. Copyright 1984,

John Wiley & Sons, Inc. Single channel conductance traces for Gramicidin A (B) and

L•Leu5-Gramicidin A (C) under these conditions.
FIGURE 19

Stereo views of the sideview of the Gramicidin A channel (A) and

of a channel view taken from the solution end of a monomer (B).

For crosseye (close) viewing, cover the right-hand structures and

use the left-hand pair; for walleye (distance) viewing, cover the

left-hand structures and use the right-hand pair. These stereo

views show the orientation of side chains for a favorable in

vacuo distribution. This is one of a class of states with similar energy but with different side chain distributions. Adapted

from Venkatachalam and Urry 1983. Copyright 1983, John Wiley &

Sons, Inc.

This multiplicity of conducting states is a challenging issue to

resolve in connection with abundant data arguing that the same

channel backbone structure is responsible leading to the proposal

that the differing conductance states arise from different side

chain distributions with lifetimes on the order of seconds. One

preferred side chain distribution is seen in figure 19

(Venkatachalam and Urry 1983). In

mobility, it was found that the Trp

an analysis of side chain

side chain was especially

free to occur in different rotameric states, partly because it is

at the end of the helix but also because the residue on the

adjacent turn of helix,

small side chain.

residue-5, is an alanine residue with a

This allows the Trp side chain to reside

194
over the Ala side chain, much as the Trp side chain is seen in

the distribution of figure 19 to lay up over the Ala side chain.

Thus, it was reasoned that replacement of Ala by a residue with

a bulkier side chain should restrict to a significant extent some

of the rotameric states available to the Trp side chain and

thereby decrease the multiplicity of states. To test this

perspective, the Leu -GA analog was synthesized. As shown in

figure 18, the probability of the most likely conductance state

has been doubled. This provides a demonstration of a most

intriguing and subtle means of modulation of conductance. Simply

by altering or restricting the orientation of a side chain not

directly involved in cation contact can markedly alter conductance. The mechanism is thought to involve the energetics of

peptide libration. As the ion enters the channel, the peptide

carbonyls must librate inward toward the channel center in order

to coordinate adequately the cation. The energetics of this peptide libration are viewed as being dependent on the orientation

of the side chain of the involved carbonyl. Accordingly, there

are many ways to modulate the effective conductance of channels

and it will be interesting to see if any of those which are exhibited by the Gramicidin A channel system will be analogous to the

manner in which opioid peptides increase potassium ion conductance in nerve tissue.

Further Comments on Mechanisms at a Membranous Site

The single channel conductance traces in figure 18B and C were

obtained using a cell with a teflon partition between two chambers and with a small hole in the partition over which a planar

bilayer is formed. This is basically the Mueller and Rudin

(1967) approach. In order to improve signal to noise and

membrane stability to voltage ranges very small membrane areas

are desirable. These small areas can be obtained by using a

glass pipette drawn to a fine diameter of a few microns and by

further preparing the tip to decrease noise and make it suitable

for lipid bilayer coverage. This approach has been used by

Andersen (1983) in studies on Gramicidin A and is essentially

what has become known as the patch clamp technique so well developed by Sakmann and Neher (1983). This approach, which can

carry the Gramicidin A problem to the important refinement of

understanding side chain distribution issues, has found its

greatest application to cells and to small membrane patches from

cells. The mechanics of the process are schematically shown in

figure 20 (Hamill et al. 1981).

This means that channels of suitable nerve and other cells can be

studied much as the Gramicidin A channel has been studied and

that the response of these channels to neurotransmitters and

other effectors, such as the opioid peptides, can be assessed.

It is not unreasonable to expect in the future that the patch

clamp approach will provide more specific and informative assay

systems for opioid peptides and will also provide simpler systems

on which to dissect ionic transport mechanism.

195
FIGURE 20

The patch clamp experiment (schematic representation). From

Hamill et al. 1981. Copyright 1981, Pflügers Arch.

A particularly intriguing use of the patch clamp pipette technique, which has relevance to the neurotransmitter release activity of opioid peptides, is the study of chromaffin cells by

Neher and Marty (1982). Patch clamp pipettes were used to

measure the cell membrane capacitance of small adrenal chromaffin

cells. Cell membrane capacitance was observed to exhibit steplike changes (2 to 6 x 10-14+F) under conditions of enhanced

release of chromaffin granules. Quoting directly from the Neher

and Marty paper, "The size distribution of step-like capacitance

changes is well compatible with the idea that steps of capacitance increase reflect individual events of exocytosis of chromaffin granules, whereas steps of the opposite polarity reflect

the formation of vesicles or vacuoles by endocytosis."

196
It appears over the entire range, starting with the synthesis and

conformational studies of analogs and spanning to the emerging

means for studying mechanisms at membranous sites, that the study

of the mechanism of action of the opioid peptides and opiates is

entering a most challenging and productive period.

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ACKNOWLEDGMENTS:

This work was supported in part by the National Institutes of

Health grant nos. GM 26898 and HL 29578, and the manuscript was

written during the tenure of a Senior U.S. Investigator Award

(Reinvitation Program) from the Alexander von Humboldt Foundation. The author would also like to acknowledge many past and

present members of the Laboratory of Molecular Biophysics who

have contributed so significantly to the work reviewed here.

AUTHOR:

Dan W. Urry, Ph.D., Director

Laboratory of Molecular Biophysics

School of Medicine

University of Alabama at Birmingham

University Station/P.O. Box 311

Birmingham, Alabama 35294

203
Opioid Peptides: Analysis of Specificity

and Multiple Binding Modes Through

Computer-Aided Drug Design and

Structure-Activity Studies

Raoul D. Nelson, Ph.D.; David I. Gottlieb, Ph.D.;

T. M. Balasubramanian; and Garland R. Marshall, Ph.D.

INTRODUCTION

Analysis of the structure-activity data in the area of opioid

peptides and opiates is severely compromised by the presence of

multiple receptors, by incomplete pharmacological

characterization of compounds in the literature, and by

oversimplification of possible drug-receptor interactions. We

will use our efforts to determine the receptor-bound

conformation of morphiceptin and its relationship to enkephalin

to illustrate both the approaches we have developed (Marshall et

al. 1979) and the pitfalls which we have encountered.

Morphiceptin is a synthetic opiofd peptide, Tyr-Pro-Phe-Pro-NH2,

with moderate opioid agonist activity and extreme selectivity

for the mu opiate receptor. A mu receptor pharmacophore was

proposed for morphfceptin based on the structural homology with

enkephalin and other nonpeptide opioids. In order to

simultaneously test the pharmacophore hypothesis and increase

the potency and selectivity of morphiceptin, morphiceptin

analogs were synthesized with potentially important chemical

groups modified or eliminated, as well as conformationally

constrained analogs. Mu receptor agonist and antagonist potency

was determined in the stimulated guinea pig ileum (GPI)

bioassay. Mu receptor affinity and delta receptor affinity were

determined in receptor binding assays. The mu receptor agonist

potency and affinity correlated well.

The structure-activity relationships of the morphiceptin analogs

refined the pharmacophore hypothesis. The activity of the

analogs with potentially important chemical groups, modified or

eliminated, supported a three-group pharmacophore hypothesis.

There was a strict requirement for the Tyr-1 nitrogen group,

Tyr-1 phenolic group, and Phe-3 phenyl group. These groups are

similarly required for activity in enkephalin and nonpeptide

opiates. However, the Phe-3 phenyl of morphiceptin must either

bind to a different subsite on the mu opiate receptor or to a

similar subsite of the mu opiate receptor in a different

orientation than the corresponding group (Phe-4) of

enkephalin. Furthermore, there are strict conformational

requirements for the activity of morphiceptin.

204
The study of the structure-activity relationships of

morphiceptln by computer analysis of conformation and computer

graphics further refined the pharmacophore hypothesis. The

active and conformationally constrained analogs of morphlceptin

can produce a common pharmacophore In three dimensions.

Morphiceptin cannot interact with the mu opiate receptor when

the pharmacophore groups are in the same relative orientation as

the corresponding groups in the nonpeptide opiates.

Morphfceptin can interact with the opiate receptor with the

pharmacophore groups in the same relative orientation as the

corresponding groups in enkephalin, but with a very different

backbone arrangement. The results of these computer studies

show that opioids with different overall constitution may

interact with the mu opiate receptor in very different modes.

METHODS

Molecular Modeling

The molecular modeling system consisted of a VAX-11/750 and an

NEC advanced personal computer as a color raster graphics

terminal. The system was equipped with SYBYL (Trfpos Assoc.,

St. Loufs, MO), a computer-aided drug design software package.

The peptide models were assembled with standard amino acid

fragments which were obtained by using the Empirical

Conformation Energy Program for Peptides (ECEPP) (Momany et al.

1975; Nemethy et al. 1983) and a standard amide bond geometry

Bendetti 1977). which represented averaged crystallographic

data. Unusual amino acids and nonamino acid groups were

constructed from standard fragments of chemical groups derived

from crystallographic data, and their geometry was optimized

using a molecular mechanics program with a standard force field

(White 1978; Labanowskf and Marshall, in preparation).

The computer program SEARCH systematically determined all

possible sterically allowed conformations of a molecule by

incremently changing the torsfonal angles of freely rotatable

bonds and discarding the conformations for which there were

repulsive van der Waals interactions (Bosshard et al. 1972). A

hard sphere potential model, with the van der Waals radii

calibrated against protein crystallographic data, was used to

calculate the repulsive van der Waals interactions (Marshall et

al. 1974). The program SEARCH also simultaneously determined

the specified distances that define each relative orfentatfon of

specified chemical groups for each sterically allowed

conformation of a molecule. The set of distances that define

the relative orientations of the specified chemical groups

produced by all the sterfcally allowed conformations of

molecules was defined as an orientation map. The program SEARCH

also determined the mathematical intersection of the orientation

maps for a set of different molecules with the corresponding

chemical groups, which represented the common orientations of

the specified chemical groups for the set of molecules. The

205
orientation map was represented by a table of the observed

maximum and minimum distances, a table of vectors which

described the set of all possible distances, or threedimensional plots of three of the possible distances.

Conformations of molecules that produced the entries in

orfentation map were retrieved and examined by molecular

graphics.

The same SEARCH program also was able to determine all the

sterically allowed conformations of a molecule, as described

previously, and simultaneously output vectors produced by a

specified pair of atoms relative to a fixed aggregate of atoms

for each conformatfon of a molecule (Marshall et al. 1983). The

vector map common to a set of different molecules could also be

determined and it indicated that sterically allowed

conformations existed where the specified aggregate and pair of

atoms can be superimposed.

A molecular mechanics program MAXIMIN was used to minfmfze the

energy of a set of molecules with the options to simultaneously

superimpose specified aggregates of atoms common to a set of

molecules; and it also was used to fix the relative orientation

of aggregates of atoms within molecules (Labanowski and

Marshall, in preparation). The parameters of MAXIMIN were

derived from a literature valence force field (White 1978).

Radioligand Binding Assay

The saturation and competition binding assays for opiate

receptors were performed by a modification of literature methods

(Pert and Snyder 1973a; Chang et al. 1979; Chang and Cuatrecasas

1979; Gilian et al. 19791. The binding reaction was Initiated

by the addition of freshly thawed homogenate to a solution of

radfolfgand in the presence or absence of cold ligand in 50 mM

Tris-HCl, pH 7.5 at 24 °C with a total reaction volume of 1

ml. The reaction was incubated for 60 minutes at 24 °C and was

terminated by the addition of 4 ml of 50 nM Tris-HCl, pH 7.5 at

4 °C, and rapid filtration through a Whatman glass-fiber filter

(GF/F). The filters were washed twice with 4 ml 50 mM Tris-HCl,

pH 7.5 at 4 °C. The fflters were shaken in 5 ml scintfllatfon

cocktail and the radioactivity determined with an efficiency of

38% to 42%. The assay was performed using rat brain homogenate

(0.5 mg protein per ml assay volume), NMB cell homogenate (1.0

mg protein per ml assay volume), or NG108-15 cell homogenate

(0.25 mg protein per ml assay volume).

Saturation binding isotherms

homogenate and the radiolfgand [

3

H]-DADLE at eight

were performed using NMB cell

concentrations, from 0.1 to 20 nM, in the presence and absence

of 2.5 μM levorphanol. Saturation binding isotherms were also

performed using NG108-15 cell homogenate and the radioligand

[

3

H]-DADLE at efght concentrations. from 0.1 to 20 nM, in the

presence or absence of 2.5 μM DADLE. Similarly, saturation

binding isotherms were performed using rat brain homogenate and

206
the radfoligand [

3

Hl-DADLE at eight concentrations. from 0.1 to

20 nM, in the presence and absence of 2.5 μM levorphanol.

Competition binding isotherms were performed using NMB cell

homogenate with the radfolfgand [3H]-DADLE at a concentration of

1.0 nM in the absence or presence of 6 to 10 concentrations of

cold competing lfgand and 2.5 μM levorphanol. Competition

binding isotherms were also performed using NGl08-15 cell

homogenate wfth the radfolfgand [3H]-DADLE at a concentration of

1.0 nM In the presence and absence of 6 to 10 concentrations of

cold competing lfgand and 2.5 μM DADLE. Sfmflarly, competition

binding isotherms were performed using rat brain homogenate and

the radfolfgand [

3

H]-DHM at a concentration of 0.24 nM in the

presence and absence of 6 to 10 concentrations of cold competing

lfgand and 2.5 μM levorphanol.

Nonspecific binding was defined as the binding in the presence

of 2.5 μM levorphanol or DADLE, and specific binding was defined

as the total binding minus the nonspecific binding. The

receptor binding assays were performed wfth each combination of

radfoligand and cold competing lfgand concentration in

triplicate within an experiment, and experiments were performed

in triplicate.

Receptor Binding Assay Data Analysis

Binding data from experiments wfth rat brain and NG105-15 cell

homogenate were analyzed with LIGAND (Munson and Rodbard

1980). This program fits a mass action-based mathematical model

for receptor binding, involving one or more lfgands and

receptors, by nonlinear regression to radfoligand binding

data. Binding affinity (Kq3) and capacity (Bmax) for [

3

H]-DADLE

and NG1D8-15 cells, or [

3

H]-DHM and rat brain, were first

calculated for each saturation binding isotherm individually.

Similarly, Kd for each cold competing lfgand was calculated for

each conpetition isotherm, using the previously calculated

binding affinity of the radfolfgand. Nonspecific binding was

treated as a fftted parameter. The variance model for weighting

was for constant percent error. The data are reported for fits

with a single binding site model. The arlthmatfc mean and

standard error of the mean of Bmax and the geometric mean and

standard error of the mean of Kd were calculated for replicate

binding isotherms.

The data for the binding isotherms using the NMB cell homogenate

were treated in the traditional manner. Kd and Bmax were

calculated by linear regression of Scatchard plots for each

saturation binding isotherm (Scatchard 1949). The IC50 for each

cold competing lfgand was calculated for each competition

binding isotherm using SIGMOID (DeLean et al. 1978). SIGMOID is

a program which fits a four parameter logistic function to

radfoligand displacement data by nonlinear regression. and

interpolates the IC50. The (Ki) for each competing lfgand, in

each competition binding isotherm, was calculated according to

207
the Prusoff equation, from the IC50 of the competing ligand. the

concentration of the radioligand, and the affinity of the

radioligand (Cheng and Prusoff 1973).

Guinea pig ileum bioassay

.

Opiate activity of potential opioid compounds was determined in

the coaxially stimulated GPI bioassay (Gyang and Kosterlitz

1966; Pert and Snyder 1973a, 1973b). Male albino guinea pigs

with an average weight of 350 g were killed by a blow to the

head and the small intestine was quickly removed. The distal 10

cm of the ileum and the duodenum were discarded and 3 cm strips

of ileum were hung via silk in a 15 ml organ bath from an F50

myograph microdisplacement transducer with a 1 g resting

tension. The organ bath contained Krebs-Ringer bicarbonate

solution (120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4 2.5 mM CaCl2

1.2 mM KH2P04, 25 mM NaHC03. 10 mM dextrose) with 20 μM choline

chloride and 0.125 μM mepyramine maleate at pH 7.4 and 37 °C

bubbled with 95% 02/5% C02. The ileum was stimulated via silver

electrodes transmurally using a Grass SD9 stimulator with

supramaximal voltage (0.2 Hz, 1.5 ms). and the contractions were

monitored with a Physiograph DMP-4B recorder. The tissue was

allowed to equilibrate in the bath for 15 to 30 minutes.

The compounds to be evaluated were added to the bath in 10 to

100 μl aliquots, and were allowed to stay in the bath until

maximal inhibition of contraction occurred. Then the bath

contents were removed and the tissue was washed four times over

15 to 30 minutes. For a typical dose-response study of a

compound, a minimum of four doses which produced 10% to 90%

inhibition of contractions were used. Potency was defined as

the IC i.e the concentration of an agonist which produced

50% inhibition of contractions. The IC was calculated by

linear regression of the LOGIT transformation (Rodbard and

Frazier 1972) of percent inhfbftion of contraction

[LOGIT(%inhibition) = Ln(%inhibitionl(l%inhibition))] versus the

log of the concentration of the compound, and interpolation to

the concentration of the agonist that produces 50% inhibition of

contraction. The IC for DADLE was determined in each tissue

so that the %relative potency could be calculated for each

compound tested in the same tissue. The % relative potency was

defined as 100 x IC50 DADLE/IC50 compound. The geometric mean

and standard error of the mean of the IC50 and %relative potency

for each compound were calculated from dose-response studies

with at least three tissues. For each compound that an IC50

could be determined, a supramaximal dose was added to the ba

to demonstrate complete inhibition of contractions (i.e., full

agonist activity) and 200 nM naloxone was added to the bath to

demonstrate that the inhibitory activity was completely

reversed. In addition, compounds which exhibited little or no

inhibitory activity were checked at concentrations at which

little or no inhibitory activity was observed for their ability

to block or antagonize the inhibitory activity of DADLE.

208
The sensitivity of the opiofd agonist, morphiceptin, to naloxone

antagonism was examined according to a modification of a method

described by Kosterlitz and Watt (1968). A dose-response study

was performed, and the IC50 calculated, for each agonist. both

without and with three concentrations of naloxone (4.12, 20.6,

103, and 515 nM). The pA2 Ke, and slope were calculated by

linear regression of a Schild plot of the log (dose ratio - 1)

versus the log ([naloxone]/nM), for each agonist. The

arfthmatfc mean and standard error of the mean of the slope and

pA2. and the geometric mean and standard error of the mean of

the Ke, were calculated for replicate experiments. The opiofd

agonists used were Met5-enkephalin, ethylketocyclazocine, and

morphiceptin.

Chemistry of morphiceptin analogs

All solvents were HPLC grade and chemicals were reagent grade.

Free and protected amino acids were purchased except for 2-

aminoindane-2-carboxylate (Mauger and Ross 1962), trans-3-

phenylproline (Cox et al. 1964; Sarges and Tretter 1973), and 2-

methylproline (Ellington and Honigberg 1974) which were prepared

by methods reported in the literature. Benzhydrylamine

polystyrene 1%-crosslinked divinlybezene resin was synthesized

by the method of Pietta et al. (1973); p-methylbenzhydrylamine

polystyrene 1%-crosslinked divinylbenzene resin was purchased.

Melting points were determined on a Hoover capillary apparatus

and microanalyses were performed by Robertson Laboratory of

Florham Paris, New Jersey. Optical rotations were performed

using a Perkin-Elmer 241 automatic polarimeter with a 1 dm

cell. Proton magnetic resonance spectra were recorded at 60 MHz

using a Perkin-Elmer T60 spectrometer or at 100 MHz using a

Varian XL100 spectrometer. The IR spectra were recorded on a

Perkin-Elmer 7108 spectrometer for compounds as a KBr pellet.

Fast atom bombardment mass spectra were recorded on a Finfgan

3200 mass spectrometer equipped with a FACS fast atom bombardment gun using Xe gas at 6 to 8 Kev. Two to ten μg of each

sample in 1 μl MeOH was applied to a glycerol matrix of the

probe for analysts. Twenty five to fifty μg of each peptide was

examined for homogeneity by thin-layer chromatography (TLC)

using precoated silica plates. The peptides were visualized

with iodine vapor, chlorox/starch-KI, and/or ninhydrin. All

peptides showed a single spot on TLC. Hydrogenations were

carried out on a low pressure Parr hydrogenator at room

temperature. Selected protected peptides were purified by

medium pressure liquid chromatography on Lobar silica columns

(E. Merck) with various solvent systems.

For amino acid analysis, the peptides were hydrolyzed at 110 °C

for 24 hours in deaerated tubes in 6N HCl and 0.5% phenol with

Nle as an internal standard. Amino acid analysis was performed

using one of the following methods: a procedure described by

Benson and Hare (1975) and modified to include a Glenco MM amino

acid analyzer equipped with a dual beam photometer (570 nm and

209
440 nm), and Hewlett-Packard 3390A and Spectra-Physics

Minigrator integrators; or the procedure of Bohlen and Mellet

(1979) using a Waters amino acid analyzer equfpped with a filter

fluorometer optimized for o-phthalaldehyde amino acid

derivatives, and a digital integrator; or a modified PTC

procedure (Henrickson and Meredith 1984).

Selected analogs of morphfceptin were synthesized by the solid

phase method. Some analogs were synthesized by this method

because of the side reactions encountered in the initial

solution phase synthetic strategies. The peptides were

synthesized on the benzhydrylamine (Pietta et al. 1973) and pmethylbenzhydrylamine (Matsueda and Stewart 1981) polymers,

which afford a C-terminal amide. Standard initial attachment

and elongation protocols were used, with DCC for coupling, BOC

groups for N-terminal protection, Bzl groups for side-chain

protection of tyrosine, and TFA for BOC removal. Extended

coupling times and double coupling cycles were used in the

syntheses with the sterically hindered amino acids: 2-

methylproline, N-methyl-D-alanine, N-methylphenylalanine, 2-

aminoaminoindan-2-carboxylate, and cycloleucine. The final

peptide was deprotected and cleaved from the polymer by either

the high HF or high/low HF (Tam et al. 1983) procedures, and was

worked up by standard procedures.

Selected analogs of morphiceptin were synthesized by solution

methods, including those substituted wfth sterfcally hindered

amino acids and C-terminal phenyl alkyl amides that are not

easily incorporated by solid phase methods. Multiple synthetic

strategies were chosen to take advantage of common fragments and

afford the peptides in high yield and purity. The synthetic

strategies used common coupling reagents and protecting

groups. DCC with HOBT (Konig and Geiger 1970). EDCI with HOBT

(Sheehan et al. 19651, PPA (Wissmann and Kleiner 19801, and DPPA

(Hamada et al. 1977) were the coupling reagents used. EDCI and

PPA were preferable because no purification other than the

standard workup was usually necessary.

Preparative RP-HPLC is becoming a standard analytical and

preparative technique that is extremely valuable in purification

and analysis of synthetic peptides. The preparative techniques

at the time this work was begun were either for large gram scale

purifications using the Waters Prep-500 HPLC equipment (Rivier

et al. 19841 and very expensive columns, or for the smaller mg

scale purifications using the conventional HPLC equipment and

small semipreparative columns. This prompted the development of

an intermediate scale preparative RP-HPLC methodology. Twenty

to eighty mg of each deprotected peptide was chromatographed on

a 22 x 250 mM Zorbax 5 μM ODS column. The peptides were eluted

with isocratic conditions at 5% B for 10 minutes, a concave

gradient between 5% and 20% to 35% B over 10 to 30 minutes, and

isocratic conditions at 20% to 35% B for 1 to 2 hours, at 8.0

ml/minute while monitoring absorbance at 280 nM. A was 1.1 N

HCOOH in H20 with the pH adjusted to 3.0 with TEA, and B was

210
MeOH. The homogenous fractions demonstrated by analytical RPHPLC were subsequently desalted by chromatographing on a Waters

4.6 x 250 mM μBondapack ODS column. The peptide was applied to

the column, the salts eluted with isocratfc conditions of 100%

A, and the peptide eluted with a sharp linear gradient of 0% to

75% B, where A is 0.1 N CH3COOH and B is MeOH. Lyophflization

from dilute CH3COOH afforded homogeneous peptides as acetate

salts with yields of 30% to 90%.

RESULTS

Structure-Activity of Morphiceptin Analogs

The analogs prepared are shown in figures 1 and 2. Figure 1

concentrates on analogs useful in testing the pharmacophore

hypotheses. Figure 2 shows compounds designed with different

conformational constraints necessary to determine the receptorbound conformation. The opiate activity of the synthetic

morphiceptin analogs was measured in the stimulated GPI bioassay

FIGURE 1

Selected Structures of Morphiceptin Analogs The numbering

scheme corresponds with that in the tables of biological data.

211
for each morphiceptin analog, DADLE and morphine. The analogs

of isorphiceptin produced parallel dose-response curves in the

bioassay, consistent with their interaction at a common receptor

and full agonist activity (table 1).

The sensftfvfty of morphiceptin to naloxone antagonfsm was

studied in order to determine whether the agonist activity of

morphfceptin was mediated by the μ or opiate receptor.

Typically, μ agonists are more sensitive to naloxone than

agonists. but both are antagonized by high doses of naloxone, in

the GPI bioassay (Lord et al. 1977). Dose-response studies were

performed with Met-enkephalin, a μ agonist in this bfoassay;

ethylketocyclazocine. a agonist in this bioassay; and

morphiceptin, with and without three concentrations of

naloxone. Parallel shifts in the dose-response curves were

observed for morphfceptin with increasing concentrations of

naloxone, i.e., the same slope and maxima, as well as Metenkephalin and ethylketocyclazocine, and a Schild plot of the

data were linear with a slope that was not significantly

different from unity.

These results are consistent with naloxone acting as a

competitive antagonist of morphiceptin (Lord et al. 1977). The

pA2, and Ke for naloxone antagonism of the agonist activity of

morphiceptin were not significantly different-from that of Metenkephalin (P<O.O5), a μ agonist in this bioassay, but were

significantly different from that of ethylketocyclazocine

(P<O.O5), a agonist in this bioassay. The activity of

morphiceptin is mediated by a receptor that has a similar

affinity for naloxone as the receptor that mediates the activity

of Met-enkephalin, but a different affinity from the receptor

that mediates the activity of ethylketocyclazocine.

A competition binding assay was used to determine the affinity

of the synthetic morphfceptin analogs for the μ and 8 opiate

receptors. The appropriate combinations of opiate receptor

selective radioligands and sources of opiate receptor were used

to obtain the most accurate measure of affinity for each

receptor. μ-Receptor affinity was measured in a competition

binding assay using [

3

H]-DHM, a relatively specific μ ligand

(Chang and Cuatrecasas 1979; Gillan et al. 1979), and rat brain

homogenate, which contains a mixed populatfon of opiate

receptors (Chang and Cuatrecasas 1979; Chang et al. 1981).

Receptor affinity has often been measured in a competitive

binding assay using [3H]-DADLE, a moderately -specific lfgand

(Kosterlitz et al. 1981), and rat brain homogenate.

However, since [

3

H]-DADLE has an affinity for the receptor

that is only fivefold to tenfold greater than the affinity for

the μ receptor at concentrations required for a reasonable

signal in a competition binding assay, a significant proportion

of the ligand occupies the μ receptor as well as the receptor

at the concentrations required for the competition

212
binding-assay. As a result, competition binding assays with

highly μ- specific competing ligands, such as morphfceptin.

produce shallow or biphasic displacement curves and afford

inaccurate estimates of the true -receptor affinity (Chang et

al. 1981a, 1981b). Since more selective radiolfgands are not

yet commercially available. an alternative technique was

chosen. [

3

H]-DADLE was used, but with NG108-15 murine

neuroblastoma x rat glioma and NMB human neuroblastoma cell

homogenates, both homogeneous sources of receptors (Chang et

al. 1978; Simantov and Snyder 1976; Ard and Gottlfeb, in

preparation).

FIGURE 2

Selected Structures of Morphiceptin Analogs The nunbering

corresponds with that in the tables of biological data.

213
TABLE 1

Summary of Biological Activity of the Morphiceptin Analogs as

Determined in the Stimulated Guinea Pig Ileum Bioassay

Compound IC50/nMa SEM %Rel.Pot.b

SEM

a

All compounds for which the IC was determined were full agonists, and

their activity was fully reversible by 200 nM naloxone.

b

%Relative potency (%Rel.Pot.) was normalized to morphiceptin.

c

Compounds for which an IC could not be determined were also

inefiective in antagonizing DA The IC E. 50 and %Rel.Pot. are expressed

as the geometric mean and standard error of the mean (SEM). The IC50

and %Rel.Pot. were determined according to the described experimental

methods.

214
Competition binding isotherms were performed with each

morphiceptin analog, DADLE and morphine, using each combination

of radfolfgand and receptor preparation. The Kd was calculated

with the computer program LIGAND (Munson and Rodbard 1930) for

each analog from the competition binding assays using [

3

H]-DHM

and rat brain homogenate, and using [3H]-DADLE and NG108-15 cell

homogenate. The competition binding isotherms were monophasic

and were best fit by the single site model (data not shown).

The structure-activity relationships for morphiceptin analogs in

the GPI bioassay and the μ-receptor binding assay will be

discussed together since there was such a strong correlation

between agonist potency and affinity. The correlation of the

log of the %relative potency and the log of the %relative

affinity for the morphiceptin analogs produced a correlation

coefficient of 0.93. This suggests that the changes in agonist

potency for this series are due to changes in affinity. In no

cases were partial agonists or antagonists encountered in the

bioassay. A partial agonist would be expected to show a

decrease in potency without a decrease in affinity, and would

lie off the line of correlation of affinity and potency. This

correlation also suggests that the receptor in the bioassay and

binding assay are very similar, even considering that they are

from different species and different parts of the nervous

system.

The structure-activity relationships for morphiceptin at the mu

opiate receptor support the μ-receptor pharmacophore hypothesis

stated previously for morphiceptin. The smallest fully active

fragment of morphiceptin is Tyr-Pro-NH(phenethyl) [11],

suggesting that the terminal Pro-NH2 is not required for

activity. This indicates that pharmacophoric groups are limited

to this fully active fragment of morphiceptin.

The Tyr1 tyramine group of morphiceptin is very important for

activity. The elfmination [2] or methylation [5] of the

phenolic hydroxyl and the N-acetylation of the nitrogen [4]

results in a dramatic loss in activity. The effects of these

modifications suggest that the phenolfc hydroxyl could be

interacting with the receptor via a hydrogen bond or ionic

interaction.

The Phe3 phenyl group of morphiceptin is also very important for

activity. Reduction of the phenyl group to a cyclohexyl

derivative [6] results in a dramatic loss of activity,

suggesting an important role for a flat hydrophobic and,

perhaps, aromatic group. Nitratfon of the phenyl group [7] also

results in a dramatic loss in activity, possibly as a result of

negative steric or electronic interactions with the receptor.

In additfon, nitration of the D-Phe [9] and truncated phenethyl

amide [13] analogs of morphfceptin also resulted in a dramatic

loss in activity, suggesting that even analogs with different

possible conformational freedom can not fold to accommodate the

nftro group on the phenyl ring. Clearly, the activity of

215
morphiceptin is particularly sensitive to modificatfons of the

Phe3 phenyl group.

The remainder of the morphiceptin structure besides these three

critical groups, the phenol group of Tyr1, the amine group of

Tyr1, and the phenyl group of Phe3, probably serves as a linker

and orients these three pharmacophoric groups relative to each

other. The loss of activity for the D-Tyr1 analog of

morphiceptin [3] is interesting and probably represents a form

of stereospeciffcity much like that seen with the classical

opioids (Pert and Snyder 1973a, 1973b; Cox and Weinstock 1966;

Kosterlftz et al. 1973). The relative diastereomeric

selectivity for the -carbon of the morphiceptin Tyr1 is about

50. With a minimum three receptor subsites, the receptor would

be expected to be able to recognize the chfralfty in a ligand.

The remainder of the backbone may interact with the receptor

directly, but the direct interaction is not necessary for

activity. This is difficult to prove, but a few analogs are

consistent with this conclusion. The high activity of the DNMeAla2

 [22], D-Ala2 [21]. NMePhe3 [19], and tPhpro3 [17]

analogs of morphiceptin suggests that the μ receptor is somewhat

tolerant of changes in steric volume of the backbone of

morphiceptin. Furthermore, the high activity of the NMePhe3

[l9] and tPhpro3 [17] analogs of morphiceptin suggests that the

amide nitrogen is not functioning as an important hydrogen bond

donor in the interaction with μ opiate receptor. There is a

requirement for a nontyramine phenyl group for the activity of

morphiceptin similar to enkephalin and selected nonpeptfde

opiates (Morley 1980; Fournie-Zaluski et al. 1981; Gacel et al.

1981; Simon et al. 1981; Ophefm and Cox 1976), suggesting that

the nontyramine phenyl groups are also interacting with a common

subsfte of the opiate receptor. However, the loss of activity

upon nitration of the phenyl group in morphiceptin, as opposed

to the enhancement of activity upon nitration of the

corresponding phenyl group in enkephalin (Fauchere et al. 1983;

Schiller et al. 1983), suggests that either the phenyl group of

morphiceptin binds to a different subsfte of the μ opiate

receptor than does the corresponding phenyl group of enkephalin,

or the phenyl group binds in an entirely dffferent orientation

to the same subsite of the μ opiate receptor. In addition, the

nitration of the D-Phe3 [9l and phenylamfde [13] analogs of

morphiceptin also decreases activity. Since these analogs might

be expected to have different conformational possibflfties that

would allow a unique orientation of the phenyl group relative to

the tyramine group, and a decrease of the repulsive interactions

between the receptor and the nftro group. the inactivity of the

nitrated analogs of morphfceptin is more likely a result of

interaction with a unique receptor subsite. It would be

interesting to examine the activity of the meta-NO2 Phe3 and

ortho-N02Phe3 analogs of morphiceptin and enkephalin to further

test the possibility that the phenyl group of morphiceptin is

interacting with a unique opiate receptor subsite.

216
Many constraints in conformation that were imposed in

morphiceptin analogs by substitution for the Phe3 also

maintained or enhanced activity at the μ opiate receptor. Most

importantly, the 2-aminoindanyl amide analog of morphiceptin

[15] was much more active than morphiceptin. It is possible

that this increase of activity is a result of the favorably

constraint of the torsional angle equivalents of the x1 and x2

of the Phe3 of morphiceptin, since the indan ring has limited

flexibility. It is interesting that the 2-indanylamide analog

of morphiceptin [15] was active, but the homologous Ain3 analog

of morphiceptin [20] was inactive. Ain would be expected to be

able to mimic the 2-indanylamide. However, the extra Pro-NH2 in

the Ain3 analog [20], which was not present in the 2-

indanylamide analog [15], could be fixed in a region of space

that sterically disallows interaction the opiate

receptor. The trans-2-phenylcyclopropyl amide analog of

morphiceptin [23] was also as active as morphiceptin. In this

analog, the torsion angle equivalent to the x

1

 of the Phe of

morphiceptin is constrained by the cyclopropyl ring.

The tPhpro3

analogs of morphiceptin turned out to be very

interesting. One of the diastereomers was 200-fold more active

[l7] than the other [18]. Again, it is likely that the

difference in the activity of these diastereomers is a result of

the conformational constraints imposed. By analogy with

proline, the

Phe3, and x

1

torsional angle equivalents of morphiceptin at

should be relatively fixed (Balasubramanian et

al. 1971; Madison 1977; Delaney and Madison 1982). and tPhpro is

likely to restrict the freedom of preceding torsional angle of

Pro2 (Marshall and Bosshard 1972; Tonelli 1976). The two

isomers should differ significantly in the position of the

phenyl group relative to the tyramine group. Perhaps, the

different allowed positions of the phenyl relative to the

tyramine in the two isomers is the factor which determines

activity. It is also possible that one isomer is inactive

because of another repulsive interaction of a nonessential group

with the receptor. Unfortunately, the absolute configuration of

tPhpro in the active or inactive isomers is not yet known.

Certainly, the active diastereomeric tPhpro analog of

morphfceptin will be most important in defining the active

conformations of morphiceptin by conformational analysis.

Only selected analogs of morphiceptin were examined for

receptor binding affinity using the two different sources of

receptors. The major objective was to determine if the analogs

of morphiceptin that had a high affinity for the μ receptor had

an increased or decreased -receptor affinity, and therefore,

decreased or increased μ selectivity, relative to morphiceptin.

The tPhpro3analog of morphiceptin [17]. which had hfgher

affinity for the μ receptor, had lower affinity for the

receptor than morphfceptin [1], and potentially increased

selectivity for the μ receptor, although an exact measure could

not be determined because of the extremely low affinity for the

217
receptors. It is important to recognize that few opiofds

exhibit the extreme ectfvfty shown in table 2 for the mu

receptor as the tPhpro3 analog of morphiceptin [17], without

the sacrifice of affinity and potency at the μ receptor. It

will be important to demonstrate that this analog also exhibits

a very low affinity for the opiate receptor. as well as low

agonfst potency for the and receptors. This analog of

morphfceptin could prove to be most valuable as a selective tool

in the study of the role of the mu opiate receptor in mediating

the biological responses to opiofds.

TABLE 2

Summary of Morphiceptin Analog Binding Selectivity

for the μ and Opiate Receptor Sites

Compound Ki /Kd(μa) Kd /Kd(μa) Ki /Kd

1. Tyr-Pro-Phe-Pro-NH2 >49 >2,210

15. Tyr-Pro-NH(2-indanyl) 366 338 1.083

17. Tyr-Pro-Pro(trans phenyl)- >558 >7,480 --

22. Tyr-D-NMeAla-Phe-Pro-NH2

N(CH3)2 *HPLC fraction 1

25. Tye-D-Ala-Gly-Phe-D-Leu2

251 --

0.196 0.358 0.547

26. Morphine 155 155 0.998

a

DHM in rat brain homogenate.

b

DADLE in NG108-15 cell homogenates

c

DADLE in NMB cell homogenates

Molecular Modeling

Opioid agonists and antagonists are recognized and bound by the

opiate receptors via important chemical groups, and in addition,

agonists activate the opiate receptors. There is a particular

bound conformation of an agonist or antagonist that is the most

thermodynamically stable for the receptor complex as a whole, with

noncovalent interactions between the receptor and the agonist or

antagonist optimized. Binding energy may be consumed in the

deformation of the agonist or antagonist conformation from the most

stable solution conformation. Therefore, it would seem to be

important to define this receptor-bound conformation in order to

design agonists and antagonists with the receptor-bound

conformation prestabilized in solution. The result should be an

increase in the thermodynamic stabflity of the receptor-bound

agonist or antagonfst complex and, thus, an increase in potency and

affinity. Furthermore, it should be possible to design agonists

and antagonists which take advantage of the subtle differences in

the conformational requirements of the subtypes of opiate receptors

and exhibit receptor-subtype selectivity.

218
It is also important to realize that it is difficult to compare

noncongeneric molecules in terms of conformation, because their

chemical constitution is so different. For this reason, it is

important to define the chemical groups that are dominant in the

interaction with the receptor, i.e., the pharmacophore. Then the

noncongenerfc molecules can be compared in terms of the possible

orientations of the pharmacophore groups, rather than their

possible conformations. Again, it is likely that the different

subtypes of receptors may preferentfally interact with different

pharmacophoric groups and/or different relative orientations of the

pharmacophoric groups.

For rigid compounds, the relative orientation of the pharmacophoric

groups is envfronmentally independent, and, therefore, the relative

orientation of the pharmacophoric groups in states for which

conformational analysis can be performed (NMR, X-ray, etc.) will

not differ from that in the receptor-bound state. However, for

flexible compounds, e.g., opioid peptides and many nonpeptide

opioids, there are many possible orientations of the pharmacophorfc

groups populated to varying degrees, and the possible orientations

are also environmentally dependent. Without knowledge of the

environment or structure of the receptor site, it is difficult to

choose from the many possible receptor-bound orientations of the

pharmacophore groups, if, indeed. there is a single one. The most

likely orientation(s) of the pharmacophoric groups in states where

the conformational analysis is performed may not resemble that when

bound to the receptor.

It was originally thought that structure-activity studies of

morphiceptin would provide further insight into the recognition and

activation requirements at the μ opiate receptor involving

enkephalin, Tyr-Gly-Gly-Phe-(Leu,Met). Structural homology to

enkephalin suggested that morphiceptin interacted with the μ

receptor in the same way as enkephalin. If this was the case, the

structure-activity studies of morphiceptin could be combined with

those of enkephalin, further refining the pharmacophore model for

the highly flexible enkephalin analogs. Alternatively, if

morphiceptin did not interact with the opiate receptor in the same

way as enkephalin, structure-activity studies would have to proceed

independent of enkephalin.

Initially, a pharmacophore was proposed by considering the

structural homology of morphiceptin to enkephalin, and possibly to

the representative nonpeptide opioids, morphine, GPA1657 and PEO.

The Tyr1tyramine of morphiceptin could potentially be equivalent

to the corresponding Tyr1

tyramine of enkephalin and the tyramine

of morphine, GPA1657, and PEO. In addition, the Phe3

 phenyl of

morphiceptin could be equivalent to the Phe4phenyl of enkephalin

and perhaps the nontyramine phenyl of GPA1657 and PEO. The Pro2

probably functions as a linker to orient the tyramine and the

phenyl relative to each other in the same way as the Gly2-Gly3 of

enkephalin, or the D-Ala2-Gly3

of the most potent enkephalin

analogs--perhaps the same way as the core structures of GPA1657 and

PEO. In order to determine if morphiceptin was simply an

219
enkephalin analog and whether the Pro2

 of morphiceptin was a

surrogate for the D-Ala2-Gly3segment of enkephalin. the model

fragments N-acetylproline methylamide [I] and N-acetyl-D-alanine

methylamide [II] were examined using SEARCH. Sterically allowed

conformations were determined and, simultaneously, the vectors

produced by the methylamide nitrogen and carbon of [I] and [II]

were plotted relative to the fixed acetyl group. There was clearly

no intersection of the vector maps produced by the model peptides,

indicating that the N-acetyl and methyl amide groups of the model

peptides [I] or [III] were not superimposable in any of the

sterically allowed conformations. Clearly, the Pro of morphiceptin

is not a surrogate for the D-Ala-Gly segment of enkephalin. This

is consistent with the idea that if the tyramine and phenyl groups

of morphlceptin and enkephalin are interacting with the μ opiate

receptor in the same relative orientation, the backbones must be

very different, or the phenyl and tyramine groups are interacting

with the μ opiate receptor in a very different relative

orientation.

To further address the issue of whether the tyramine and phenyl of

morphiceptin and enkephalin can interact with the μ opiate receptor

in the same relative orientation, SEARCH was used in the same way

as for morphlceptin by creating orientation maps. Preliminary

results suggest that it may be possible for enkephalin to adopt a

conformation in which the relative orientation of the tyramine and

phenyl are similar to the corresponding constrained morphiceptin

analogs [15] and [l7/18]. However, further studies are required,

especially with the more rigid analogs of morphiceptin.

The conformationally constrained analogs of morphlceptin. Tyr-ProNH(2-indanyl) [15], and Tyr-Pro-tPhpro-N(CH3)2 [17/18] exhibit high

activity at the μ opiate receptor. It was assumed that the

tyramine and phenyl groups of these analogs, like their parent

compound, morphlceptin [1] were similarly important for activity

and interact with the same subsites of the μ opiate receptor as

morphiceptin [1]. In other words, it was assumed that these

compounds have a common μ-receptor pharmacophore.

SEARCH was used to determine the common relative orlentations of

the tyramine and phenyl groups produced by the sterically allowed

conformations of [15] and [17/18]. The studies followed four

possible pathways in order to consider the possibility for the

cis/trans isomerization for the Tyr-Pro amide bond in both [15] and

[17/18] (Liakopoulou-Kyriakiades and Galardy 1979; Deslauriers et

al. 1979). As a result, there were four possible orientation maps

describing the common relative orientations of the tyramine and

phenyl groups produced by the sterically allowed conformations of

[15] and [17/18].

The conformational analysis was simplified by assuming that the

tyramine of morphiceptin interacts with the μ opiate receptor with

the same relative orientation of the amine and phenol groups as for

the rigid opioids, morphine, GPA1657, and PEO. This conformation

of tyrosine does not invoke any strong repulsive van der Waals

220
contacts. A similar assumption was made in molecular modeling

studies of enkephalin (Gorin and Marshall 1977) and was actually

supported by the high activity of enkephalin analogs in which the

tyrosine was substituted with conformationally rigid subpieces of

morphine (Shaw and Turnbull 1978; Deeks et al. 1983; Cardinaux and

Pless 1984). According to molecular mechanics calculations and

molecular graphics comparisons, these constrained tyrosine

substitutes do constrain the tyramine to a low energy conformation

which places the phenol and amine in the same relative orientation

as observed in morphine, GPA1657, and PEO.

Using SEARCH, four orientation maps were determined according to

the four strategies. Six distances were used to define the

relative orientations of the phenyl relative to the tyramine

(figure 3). All four pathways produced orientation maps that were

circumscribed by maximum and minimum distances that differed by one

to five angstroms (table 3). The orientation maps were visualized

by graphical plots of dimensions 1-3 and 4-6 where the axes

represent the defined distances (figure 4). These orientation maps

demonstrate that these analogs of morphiceptin may interact with

the opiate in four possible sets of common relative orientations of

the tyramine and phenyl groups produced by conformations that were

energetically reasonable, i.e., no repulsive intramolecular van der

Waals contacts. It is important to observe that the superimposed

orientation maps 1 and 2, and similarly 3 and 4, for the different

tPhpro3

diastereomeric morphiceptin analogs did not intersect

significantly, suggesting that the difference in activity between

these analogs is a result of the different allowed orientations of

the phenyl group relative to the tyramine group. Identification of

the active configuration of tPhPro will determine which of the

orientation maps describes the relative orientations of the

tyramine and phenyl groups of morphfceptin required for activity at

the μ opiate receptor.

.

Conformations of [l51] and [17/18] derived from each of the

orientation maps were selected, and MAXIMIN was used to determine

four possible conformations of [l5] and [17/18] in which the

tyramine and phenyl groups were superimposed with the least energy

of deformation. This is desirable because of the increment used in

SEARCH which may not optimize overlap of pharmacophoric groups.

Again, there were four unique possibilities because of the

cis/trans Tyr-Pro isomerism and the ambiguity in the absolute

configuration of the tPhpro of [17/18] (figure 5). Identification

of the absolute configuration of tPhpro in [17/18] and the amide

configuration which is required for activity at the μ receptor will

determine which conformation or relative orientation of the

tyramine and the phenyl is most important for activity at the μ

opiate receptor.

221
FIGURE 3

The Opiofd Pharmacophore and the Defined

Orientation Map Distances 1-6

TABLE 3

Orientation Map Distance Ranges

Defined Distance

Number

Distance Range

(Minimum and Maximum in Angstroms)

Orientation Map*

1 2 3 4

1 9298 10 4-11 1 6.9-87 7291

2 10.1-10.7 11.1-12.2 9.2-11.7 8.2-10.9

3 10.4-11.8 12.0-13.1 7.9-10.8 9.3-10.7

4 7.9-9.7 10.5-11.5 5.6-9.4 7.7-9.8

5 9.2-12.0 11.5-13.1 6.0-12.4 7.8-11.4

6 9.6-12.0 12.2-15.9 6.2-11.8 9.2-11.4

*Map 1 = Pro2-trans, L-tPhpro3; Map #2 = Pro2-trans, D-tPhpro3; Map #3 =

Pro2

-Cis, L-tPhpro3; Map #4 = Pro2-Cis, D-tPhpro3.

222
FIGURE 4

Orientation Maps for Morphiceptin Analogs

The orientation maps are plotted as distances #1-3 (left) and distances

#4-6 (right). The top set of maps correspond to Map 2 of table 3 and

the bottom set to map 1.

223
FIGURE 5

Conformations of Tyr-Pro-NH(2-indanyl) and

Tyr-Pro-L-tPhpro-N(CH3)2, with the Tyr-Pro

Amide in the Trans Configuration Superimposed

If one examines the four possible conformations of [15] and [l7/l8]

previously calculated in relation to GPA1657 and PEO, it is

possible to conclude that morphfceptin may not interact with the

opiate receptor in the same way as enkephalin. Gorin and Marshall

(1977) proposed that enkephalin interacted with the μ opiate

receptor in a conformation in which the tyramine and phenyl groups

were superimposed over the corresponding groups of GPA1657.

Clearly, the constrained analogs of morphiceptin cannot adopt a

conformation in which both the tyramine and phenyl are superimposed

over the corresponding groups of GPA1657. This suggests that the

tyramine of morphfceptin and enkephalin interact with the same

subsites of the μ opiate receptor, and that the phenyl of

morphiceptin and enkephalin may interact with a different subsite

of the μ opiate receptor.

224
The suggestion that the corresponding tyramine groups of

norphiceptin and enkephalin interact with a common primary binding

site and that the corresponding phenyl groups interact with different secondary sites of the μ opiate receptor is also consistent

with the previously described structure-activity relationships of

morphiceptin and enkephalin. The activity of morphiceptin and

enkephalin at the μ opiate receptor is generally sensitive to the

chemical modification of the tyramine and phenyl groups. However,

the activity of morphfceptin decreases dramatically with the

nitration of the phenyl group, whereas the activity of enkephalin

increases with the nitration of the phenyl group. It will be

important in the future to actually see if the nitration of the

constrained analogs of morphiceptin also decreases activity at the

μ receptor in order to insure that these analogs themselves

interact with the receptor in the same way as morphfceptin, an

underlying assumption in all these molecular modeling studies.

Also, it will be important to determine if nitration of the ortho

and meta positions of Phe3 or morphiceptin, perhaps, increases the

activity of morphfceptin, as does paranftration of Phe4 of

enkephalin. This would suggest that the phenyl groups of

morphiceptin and enkephalin interact with the same sites, but in a

different orientation.

CONCLUSIONS

The structure-activity studies of morphfceptin clearly demonstrated

that the minimal active structure of morphfceptin for μ-receptor

activity, i.e.. binding affinity and agonist potency, was Tyr-ProNH(phenethyl). Furthermore, unmodified tyramine and phenyl groups

were required for activity at the μ receptor. These requirements

paralleled the similar requirements for the unmodified tyramine and

phenyl groups in the enkephalin. the requirements for the

unmodified tyramine in the nonpeptide opioids PEO and GPA1657, and

the requirement of the phenyl group of PEO for superpotency.

However, the activity of morphiceptin would not tolerate the

nitration of the phenyl group, whereas the activity of enkephalin

was enhanced by the nitration of the phenyl group.

The possibility of multiple divergent modes of interaction of

molecules with opiate receptors has been described as accounting

for the structure-activity relationships of the multitude of

structurally dissimilar nonpeptide opiofds. It is possible that

the phenyl group of these different opioids may bind to a variety

of subsites or accessory sites of the μ opiate receptor, in order

to confer significant activity through sufficient binding energy.

Thus, a μ-receptor pharmacophore for morphiceptin analogs

consisting of tyramine and phenyl groups oriented in one of several

specific orientations relative to each other was suggested from the

computer modeling studies of morphiceptin structure-activity

relationships.

The computer-modeling studies of morphfceptin, although in the

preliminary stages, helped to refine the pharmacophore hypothesis

in three dimensions and suggested some possible relationships

225
between morphiceptin, enkephalin, and selected nonpeptide

opiates. The systematic analysis of the conformations of the most

conformationally constrained analogs of morphiceptin demonstrated

that there were a limited number of possible common relative

orientations of the phenyl and tyramine groups in which there were

no intramolecular repulsive van der Waals contacts. The common

orientations of these groups as determined by the conformational

analysis are candidates for the receptor-bound orientation, i.e.,

the μ-receptor pharmacophore for morphiceptin.

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229
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ACKNOWLEDGMENTS

Dr. Duane Goldman synthesized and made available the trans

phenyl-proline analog used in this study. This work was supported

in part by a grant from the Monsanto Fund and by grants GM24483 and

2-T32-GM07200 from the National Institutes of Health.

AUTHORS

Raoul D. Nelson, Ph.D.

David I. Gottlieb, Ph.D.*

T.M. Balasubramanian, Ph.D.

Garland R. Marshall, Ph.D.

Department of Physiology and Biophysics, and

Department of Anatomy and Neurobiology

Washington University School of Medicine

St. Louis, Missouri 63110

230
Mechanistic Structure-Activity Studies of

Peptide and Nonpeptide Flexible Opioids:

An Interdisciplinary Approach

Gilda H. Loew, Ph.D.; Lawrence Toll, Ph.D.; Edward

Uyeno, Ph.D.; Alice Cheng, Ph.D.; Amrit Judd, Ph.D.;

John Lawson, Ph.D.; Christopher Keys, B.S.; Peter

Amsterdam, B.A., B.S.; and Wilma Polgar, M.S.

SUMMARY

In this chapter, results obtained using an interdisciplinary

approach to investigate opioid narcotic analgesic agonism and

antagonism are presented. In particular, this approach is

illustrated by studies conducted on two specific classes of

flexible opioids: peptides and 3-phenylpiperidines. For each

class, the work discussed is a continuation of the two major themes

of our past theoretical studies complemented by more recent

experimental efforts involving medicinal chemistry and receptor and

animal pharmacology.

One major focus of our past activities has been the use of theoretical chemistry techniques to characterize common molecular features among diverse classes of fused-ring and flexible opioids

leading to high μ-receptor affinity and analgesic activity. A

second major theme has been the identification of molecular determinants which modulate the extent of analgesic agonist and

antagonist activity in a given opioid, and the design of analogs

with mixed agonist/antagonist activity.

Continuing these themes, we report here theoretical calculations,

analog synthesis, receptor binding, and in vivo animal studies of a

series of 3-pheoylpiperidines and peptide opioids. In addition, we

report, for the first time, preliminary attempts to ideotify

peptide conformers leading to high affinity at -receptors.

231
THEORETICAL AND EXPERIMENTAL STUDIES OF 3-ARYLPIPERIDINES:

DETERMINATION OF MOLECULAR FEATURES LEADING TO μ-RECEPTOR SITE

AFFINITY AND MODULATION OF ANALGESIC AGONIST/ANTAGONIST ACTIVITY

The discovery of opioid analgesic properties in meperidine aroused

interest in other classes of phenylpiperidines. In particular, 3-

arylpiperidins congenere have been synthesized and tested in

several laboratories (Kugita et al. 1964, 1965; Iorio and Casy

1978; Jacoby et al. 1981; Cheng et al. 1984). The known structureactivity data for two representatives of this class of compounds,

3R=CH3 and COOC2H5-( meperidines),are summarized in table 1

(Kugita et al. 1965; Iorio and Casy 1978; Jacoby et al. 1981). As

seen in this table, this class of opioids exhibits structureactivity profiles markedly different from those of the rigid

opiates, with antagonist activity absolutely requiring a phenolicOR, and being further modulated by 2-CH3

, and 3-R groups, without

the requirement of N-substituent variation.

Using the semiempirical quantum mechanical method Perturbative

Configurstion Interaction using Localized Orbitals (PCILO) (Diner

et al 1969), we have calculated the energy-conformation behavior

and electronic structure of analogs in this class of opioids in an

attempt to explain their structure-activity profile in terms of

fundamental opiate-receptor interactions.

The results of these calculations, also briefly summarized in table

1, together with their known activity profiles, led to postulated

pharmacophores, i.e., conformations and orientations at the μreceptor site that could initiate analgesic agonist and antagonist

activity.

TABLE 1

Calculated Low Energy Isomers and Observed

Pharmacological Activity for N-CH3-3-

Phenylpiperidines

Activity (Agonist/Antagonist)

Low Energy

3-R 2-R Conformer No OH (m+H) (p-OH)

CH3 H ND wk ag/ND N D

-CH3 N D inact/ant N D

-CH3 E Q N D inact/ant N D

COOEt H

-CH3

EQ ag/inact ag/ant wk ag/inact

ND N D N D

-CH3

EQ

EQ N D N D ND

232
Specifically, for all the -aeperidines studied, only one low

energy conformer was found. This conformer is a piperldine-chair,

with the pheoyl group equatorial. No stable conformer with the

pheoyl axial or piperidine boat form could be found. Thus, as

shown in figure 1, we have proposed that this conformer could

initiate both analgesic agonist and antagonist activity by binding

to a μ-receptor in two diffareot (bimodal) orleotations. In this

figure, the proposed antagonist pharmacophore (I) satisfies our

hypothesized minimum requirement for antagonism: an orientation in

which its (m-OH)-pheoyl group can interact at the μ-opiate receptor

in a manner similar to that of fused-ring opiates (shown in dotted

lines for comparison). The agonist pharmacophore II satisfies the

minimum proposed requirement for analgesic activity: interaction

of the protonated piperidine amine group with an anionic receptor

subsite as in fused ring opiates. In both of these pharmacophores,

the ester chain can provide additional polar interactions with

cationic receptor sites.

I . Antagonist II. Agonist

FIGURE I

Calculated Molecular Electrostatic Potential Minima

(kcal/mol) Around Meperidine Oxygen Substituents in

Proposed Agonist and Antagonist Pharmacophores

233
To model receptor interaction in the agonist and antagonist modes,

molecular electrostatic potentials (Weinstein et al. 1973) in the

vicinity of the (m-OH) and ester substituents were calculated with

and without a protonated amine interaction with a model anionic

receptor site. The negative potentials calculated in the plane of

the phenol ring and in the ester group are a measure of the

electrostatic energy of interactions with cationic receptor

sites. The results shown in figure 1 indicate that without the

initial protonated amine-model anionic receptor site interaction,

postulated as crucial to agonist activity, the (m -OH)-phenyl group

has the larger minimum negative potential and could, therefore,

more effectively bind at cationic receptor sites. This result is

consistent with the hypothesis that pharmacophore I leads to antagonism and that the (m-OH)-phenyl group contributes important

receptor interactions for such antagonist activity.

The results also show that, in the presence of an initial interaction of the protonated amine with a model anionic receptor site,

(OH), while both polar groups are activated, the augmented

negative potential around the two ester oxygen atoms is larger than

that around the phenol group, thereby favoring their binding to

cationic receptor sites. These results are consistent with the

hypothesis that pharmscophore II binding of meperidines at the

receptor site could be responsible for their agonist activity.

Substitution of a 3-CH3 group for the 3-COOC2H5 substituent of Bmeperidine yields more flexible analogs. Specifically, extensive

energy-conformation studies indicated the presence of three low

energy conformers: two piperidine chair conformers, one with the

phenyl-group equatorial and the other with the phenyl-group axial;

and a piperidine boat conformer. While the two chair forms had the

lowest energy, a boat form was also a stable conformation, with

calculated activation energies of 9.5 to 16.5 kcal/mol and

conformational energies of 5.5 to 6.5 kcal/mol, low enough to be a

candidate conformation at the receptor site. Experimental values

of chair-boat activation energies and conformational energy

differences for the related ring system cyclohexane are in the same

range as the calculated values, i.e., 9 to 11.5 kcal/mol and 4.8 to

6.0 kcal/mol, respectively (Komornicki and McIver 1973), confirming

the validity of the calculated values.

Thus, for the 3-CH3,3-phenylpiperidines, but not for B-meperidines,

results of energy-conformation studies imply two possible modes of

receptor binding which could initiate mixed agonist and antagonist

activity. As indicated in figure 2, one is a bimodal interaction,

similar to that proposed for the -meperidines, in which antagonist

activity is initiated by pharmscophore I and agonist activity is

234
initiated by pharmacophore II binding to the receptor. If this

bimodal interaction is responsible for mixed agonist/antagonist

activity, then N-substituent variation should not modulate such

activity as it does in fused-ring opiates.

Pharmacophore I Pharmacophore II Pharmacophare III

Possible Mixed

Antagonist Agonist Agonist/Antagonist

FIGURE 2

Postulated 3-Phenyl Piperidine Modes of Initiating

Agonist/Antagonist Activity

Alternatively, for these 3-methyl-3-phenyl compounds, the boat

conformation of the piperidine ring in pharmacophore III can have

simultaneous overlap of both the protonated amine group and (m -OH)-

phenyl group with fused-ring opiates. If this is the mode of

interaction at the receptor, N-substituent variation should modulate agonist/antagonist activity in a manner similar to fused-ring

opiates.

To determine the effect of varying N-substituents, we undertook an

experimental effort involving the synthesis, optical resolution, in

vitro receptor binding and in vivo pharmacological activity of four

N-R-substituted 3-methyl-3(m-hydroxyphenyl)piperidine analogs shown

in table 2. The synthesis of racemic analog 1 followed a modified

procedure of Kugita et al. (1964). The resolution of the

enantiomers and their N-alkylation were reported for the first time

by our laboratory and are described elsewhere (Cheng et al.

1984, 1985).

235
Opioid-receptor binding assays on rat brain homogenates were performed essentially as described by Pasternak et al. (1975). In the

present studies, self- and cross-competition experiments were conducted for four labeled ligands: [H]naloxone, D-Ala2

-D-Leu5-

enkephalin (DADL), ethylketocyclarocine (EKC), and dihydromorphine

(DHM) at two different concentrations of labeled ligand. This

yielded a 4 by 4 “matrix” of competitive inhibition behavior. For

each pair of enantiomers, inhibition of binding of each of the four

labeled ligands was performed.

TABLE 2

Resolved Isomers of 3-Phenylpiperidines Studied

Compound R R

(+) and (-)

1 H

2

m-OCH3

CH3 m-OH

3 Allyl m-OH

4 Cyclopropylmethyl m-OH

5 Phenethyl m-OH

Data obtained from binding studies of each enantiomeric pair were

analyzed simultaneously with that from the 4 by 4 matrix using a

modified version of the program LIGAND (Munson and Robard 1980),

which predicts the receptor binding affinities and capacities

using weighted nonlinear, least squares regression analysis. The

five-receptor-site model, shown in table 3, yielded a selfconsistent set of receptor binding affinities and capacities that

was statistically much more significant than a one-, two-, three-,

or four-site model. A six-site model was not statistically more

significant than the five-site model.

As can be seen in table 3, a site was identified which has high

affinity for all the ligands, and was labeled μl. Sites “μ2” and

were labeled as those of high affinity for DHM and DADL,

respectively. The fourth site with high affinity for EKC and

naloxone, but low affinity for DHM and DADL, was labeled The

fifth residual site with large capacity has apparent low affinity

for all labeled ligands and could be a composite site.

236
Also seen in table 3, all 3-phenylpiperidine analogs have highest

receptor affinity at the μl site and in general are n-selective

compounds. While there are not dramatic differences in affinity at

μ between pairs of enantiomers, the N-allyl and N-cyclopropylmethyl

analogs differ most in this respect. Relative affinities at the μ)

site vary from about 2% to 10% of the values for DHM and

naloxone.

TABLE 3

Receptor Affinities and Maximum Binding Capacities

for a live-Receptor-Site Model

“μl”

KD(nM)

"μ2" 5th Site

Naloxone 0.4 5.0

DADL 0.9 22

EKC 0.5 6.7

DHM 0.2 10

3-Phenylpiperidines

(-)N-CH3

(+)N-CH3

(-)N-ally1

(+)N-ally1

(-)N-CPM

(+)N-CPM

(-)N-phenethyl

(+)N-phenethyl

Bmax(pmol/g)

114 1,170

132 1,000

24 110

100 370

32 43

120 256

13 130

5.0 6.2

3.0 30.0

18 1.0 154

2.1 625 10,000

1 4 0.3 4,160

100 830 200

30,000

15,900

4,000

4,000

4,350

2,700

2,170

588

7.8

625 8,330

714 19,200

370 1,720

137 3,700

122 1,700

83 833

625 1,390

179 26,300

4.2 233

The similarity in receptor-site affinities between the enantiomeric

pairs is in contrast to the striking differences obtained in in

vivo activities. As noted in table 4, determination of agonism as

shown by inhibition of mouse acetic acid writhing (Koeter et al.

1959), and antagonism of morphine-induced tail-flick analgesia

(D’Amour and Smith 1941; Harris et al. 1969), revealed that all (-)

isomers are- pure agonists, while (+) isomers are mixed

agonist/antagonists.

237
TABLE 4

Analgesic and Narcotic Antagonist Potencies of

Resolved 3-Methyl-3-m-hydroxypheaylpipcrldine

with N-Substituent Variation

Mouse Writhing Testa Mouse Tail-Flick TestD

ED50

(95% Con. limits) AD50 (95% Con. limits)

Compound μmol/kg i.p μmol/kg i.p.

N-Methyl

(-)-2 25.69 (14.68 - 44.96) c

(+)-2 41.36 (21.55 - 79.42) 101.34 (71.35 - 143.90)

N-Ally1

(-)-3 14.34 (8.96 - 22.94) c

(+)-2 160.94 (106.58 - 243.02) 112.O2 (69.58 - 180.36)

N-Cyclopropylmethyl

(-)-4 12.23 (2.63 - 21.41) c

(+)-5 d 139.32 (83.93 - 231.27)

N-Phenethyl

(-)-5 18.38 (11.08 - 30.52) c

(+)-5 11.53 (7.21 - 18.44) 13.07 (8.71 - 19.58)

Morphine Sulfate

0.81 (0.51 - 1.29)

N-Allylnormorphine

Hydrochloride

2.04 (1.44 - 2.8 7)

a

Inhibition of acetic acid induced writhing (Koster et al 1959)

bAntagonism of tail-flick inhibition induced by morphine sulfate

(21.08 μmol/kg, s.c.) (D’Amour and Smith 1941, Harris et al

1969).

cCompounds showed no significant antagonist activity up to dose

of SO mg/kg.

d

Showed no significant agonist activity at a dose of 707.21

μmol/kg.

In general, the relative affinities of the analogs at the μl-

receptor site correspond to their relative agonist activity,

lending credence to the hypothesis of Pasternak et al. (1980) of a

common high-affinity site for all types of opioids initiating

analgesic activity.

238
The results presented are most consistent with the hypothesis that

different receptor binding modes lead to agonism and antagonism.

All (-) isomers are pure agonists and thus would seem to bind to nreceptors only in an orientation without a (m-OH)-phenyl overlap

(pharmacophore II), preventing antagonist activity. By contrast,

the (+) isomers appear to bind in a bimodal fashion with

agonist activity initiated by binding in a pharmacophore II mode

and antagonist activity initiated by binding in a pharmacophore I

mode.

The N-R aubatituents do not modulate agonist/antagonist activity as

in fused-ring opioids. In fused-ring opiates, N-methyl compounds

rarely show antagonist activity, and N-phenethyl compounds have

never been shown to possess antagonist activity. Also, the N-allyl

and cyclopropylmethyl substitutions in this series do not increase

antagonist activity as they do in the fused-ring opiates. Thus,

pharmacophore III with simultaneous cationic amine and hydroxyphenyl overlaps is probably not involved in the binding.

The surprising finding of strict etereoapecificity for antagonism,

but not agonism, provides clues to additional spatial requirements

for antagonist activity of 3-phenylpiperidines, other than (m -OH)-

phenyl overlap with morphine at the μ-opioid receptor. Comparing

the two different enantiomers in a pharmacophore I orientation

(figure 3). it is seen that the amine-N of the piperidine ring

differs in its spatial relationship to the crucial (m -OH)-phenyl

group. Apparently, only one of the amine positions is compatible

with local receptor subsite interactions.

(+) Enantiomer (-) Enantiomer

FIGURE 3

Proposed Antagonist Pharmacophore for the Two 3-Phenylpiperidine Enantioners (m-OH)-phenyl Overlap with Morphine

239
Preliminary X-ray structure determination of the (+)-enantiomer of

the m-OCH3 nor-analog, 1 (Cheng et al. 1984), reveals that the

series of active antagonists derived from this compound have an Sconfiguration, as shown in figure 3. Moreover, it was found, as

shown, that the piperidine ring has a chair conformation and the

phenyl ring is equatorial. These X-ray results support the conclusion that bimodal piperidine chair pharmacophores (I and II),

rather than a boat conformation (pharmacophore III), are involved

in receptor interactions. The X-ray determination of the absolute

configuration of the active enantiomer further defines the relative

position of the crucial amine and phenyl groups in the antagonist

binding mode.

IDENTIFICATION OF OPIOID PEPTIDE CONFORMERS LEADING TO HIGH

AFFINITY AT μ-RECEPTORS

Although the μ-site is traditionally associated with morphine and

the fused-ring opiates, a number of peptides have also been found

to show specificity for binding to the μ-site as measured by

tissue-specific assays and in vitro receptor binding assays (Morley

1980). Of these peptides, the first found were analogs of enkephalins which had been synthesized in an attempt to produce

opioids that were protected against proteolytic breakdown and thus

increase in vivo activity. The structural modifications made in

these analogs included substitution and modification of residues as

well as lengthening and shortening of the peptide chain. A

decrease in in vivo degradation did result, but at the same time,

the μ-6 binding specificity of the peptide was often altered.

Initial energy-conformational studies made in our laboratory were

designed to identify peptide conformations leading to μ-receptor

affinity and focused on three of the first Met-enkephalin analogs

synthesized as shown in table 5A (Pert et al. 1976, Walker et al.

1977). Subsequent studies were made of tetrapeptide analogs shown

in table 5B. The tetrapeptide analogs were found to be even more

potent than morphine as in vivo analgesics (Terenius et al. 1976,

McGregor et al. 1978).

Of more recent interest are structure-activity studies of a series

of analogs in which modifications have been systematically made in

order to explore the structural requirements for specificity. In

this regard, morphiceptin, an amidated tetrapeptide (Tyr-Pro-PhePro-NH2

) fragment of casomorphin, a naturally occurring peptide

in milk (Henschen et al. 1980), is of interest because it was found

to have morphinelike physiological activity, as well as bind with

high specificity to the μ-receptor (Chang et al. 1981). A series

of morphiceptin analogs has subsequently been synthesized in which

the second, third,, and fourth residues were modified, leading to

240
more potent, highly μ-selective analogs (Chang et al. 1983).

In a continuing effort to identify μ-selective opioid peptide conformers, energy conformation studies of the morphiceptin analogs,

shown in table 5C, have begun in our group. Specifically, in this

series we have examined the conformational effects of substitution

of D-Pro for L-Pro in the second and fourth positions, and the

addition of a methyl group to the backbone nitrogen of the

phenylalanina residue at the third position.

TABLE 5

Three Types of μ-Selective Peptide Opioids Studied

241
In all of these studies, we have used the methods and procedures

outlined in table 6 to identify μ-selective peptide conformers.

Underlying these procedures are the assumptions that: (1) different

conformations are associated with optimum binding at μ- and -receptor

sites; and (2) differences in affinity between peptide analogs at each

site are related to the relative energy required to obtain a favorable

conformation.

TABLE 6

Methods and Procadures Used to Identify μ-Peptide

conformations

I . SELECI A SET OF μ-ACTIVE/INACTIVE ANALOGS.

USE: Receptor affinity

Guinea pig ileum (G.P.I.) activity

Antinociceptive activity

II. PERFORM ENERGY-CONFORMATION CALCULATIONS.

USE: Different methods

Quantum mechanical

Empirical energy

Different search strategies

Aufbau: From single amino acids

Use standard secondary structures

III. SELECT A SET OF PUTATIVE ACTIVE CONFORMERS. CRITERIA:

Low energy in active analogs/High energy in

inactive analogs

Relative energy parallels relative affinity in a

series of analogs

IV. REFINE SELECTIONS USING RESIDUE SUBSTITUTION/ENERGY

CALCULATIONS:

Active analogs "accommodated"

Inactive analogs "not accommodated"

V. COMPARE CANDIDATE CONFORMERS WITH μ-SELECTIVE, FUSED-RING

OPIOIDS (MORPHINE-LIKE). LOOK FOR:

Crucial tyramine overlap

Other regions of overlap

242
At the time of our first study, extensive energy conformation

studies had already been made of Met-enkephalin using the

Empirical Conformational Energy Program for Peptides (ECEPP)

(Isogai et al. 1977, Momany et al. 1975). In addition, a number

of conformational comparisons with rigid opiates had been made by

model building and comparison of overlaps of different assumed

critical regions of Met-enkephalins and rigid opiates. Of

particular interest were the p-OH phenethylamine (tyramine) moiety

formed by the terminal amino group and the tyrosine residue

(Bradbury et al. 1976; Horn and Rodgers 1976).

While valuable in describing numerous low and medium energy conformers of Met-enkephalin, none of the optimized conformers

previously reported had significant spatial overlap with several

functional groups in morphinelike opiates. On the other hand, the

conformers proposed by overlap with rigid opiates were based on

model building with no estimate of their relative energies.

Deviations from minimum energy conformations of the isolated

molecule could occur by an induced fit at the receptor site,allowing enhanced resemblance to rigid opiates, but with some energy

required.

In order to determine the most likely conformers for interaction

at the receptor site, we have made systematic energy-conformational studies by both empirical and quantum mechanical methods.

The aim was to determine the energy of Met-enkephalin conformers

with varying degrees of similarity to rigid opiates and select as

most likely those conformers with modest energy expenditure and

significant overlap with rigid opiates. In keeping with the

protocol outlined in table 6, an additional requirement in our

selection strategy was the accommodation of D-Ala2, but not LAla , in the place of the Gly residue. This second criterion is

consistent with the retention of activity when Gly is replaced by

D-Ala (Walker et al. 1977), and with a large decrease of receptor

affinity upon replacement by L-Ala (Pert et al. 1976).

The conformational energy calculations were performed using the

ECEPP and the PCILO methods. While the empirical and quantum

mechanical energy calculations did not give identical results, a

number of likely candidates common to both methods were

obtained.

As shown in table 7, both methods showed a trend toward higher

energies for conformers with increasing resemblance to rigid

243
opiates. A conformer constructed to have maximum overlap with 7-

[l-phenyl-3-hydroxybutyl-3]-endothenotetrahydrothebaine (PET), and

not energy optimized, gave extremely high energies by both the

ECEPP and PCILO methods. These high energies are due to the

crowding of the terminal nitrogen and hydrogen atoms with the Gly2

carbonyl carbon and oxygen atoms. Relaxation of the Tyr1 and Gly2

backbone angles to a local minimum decreased the energy

significantly. Step-wise relaxation of a starting structure with

w angles ~180° yielded a series of lower and lower energy

structures by both methods.

TABLE 7

Energy of Met-Enkephalin Analogs with Increasing

Overlap with Fused-Ring Thebaine Opiate (PET)

Description

(ECEPP) (PCILO)

(kcal/mol)

Gly2, no tyramine overlap 0 0

D-Ala2, no tyramine overlap 1.5 -0.5

D-Ala2, tyramine overlap 9.6 2.3

D-Ala2, minimal PET overlap 14.55 3.8

Increased PET Overlap

1 = 180°) 25.2 14.4

Maximum Constrained

PET overlap 1 = 0°) 135.0 25.0

In another approach, it was assumed that a minimum viable overlap

of Wet-enkephalin with rigid opiates should involve the tyramine

0H)moiety of both opiates. Imposition of this constraint on optimized Met-enkephalin conformers resulted in 22

conformers with relative energy 9 to 20 kcal/mol by ECEPP and 1.0

to 8.5 kcal/mol by PCILO, above the lowest energy conformer

without the imposed overlap. The further condition that D-Ala2

be accommodated more readily than L-Ala in place of Gly reduced

the number of viable conformers from 22 to 12. These 12

conformers were optimized with the constraint of accommodating

both the Gly and D-Ala residues and the tyramine overlap with

morphine.

The results of this procedure were that the minimum energy con244
formers calculated by both the ECEPP and PCILO methods did not

have any overlap with rigid opiates beyond the imposed tyramine

overlap. On the other hand, the lowest energy conformer with

significant additional rigid opiate overlap was the same by both

methods: a bend with a hydrogen bond between the carbonyl

group of tyrosine and the amine group of phenylalanine. This is a

particularly viable candidate for interaction at the receptor

site since it is only 3 kcal/mol above the minimum energy form as

calculated by the PCILO method.

Figure 4 shows the overlap of this structure with the potent

agonist PET. Not only do the important phenethylamine moieties

overlap, but the phenylalanine side chain overlaps with the

phenethyl C19 substituent of PET. In addition, the methionine

backbone C=O group and side chain are in the region of the C6

rethoxy group of PET. This conformer is quite similar to the one

previously proposed by model building (Bradbury et al 1976) and is

confirmed by these studies as a very likely candidate at the

receptor site.

FIGURE 4

A Candidate Active Conformer for Met-Enkephalin

Analogs: Turn

interact with the opiate receptor is provided by the first

published X-ray crystal structure determination of Leu-enkephalin

(Smith and Griffith 1978), shoving a G-G bend stabilized by

hydrogen bonding between the tyrosine and phenylalanine

residues. While a subsequent crystal structure determination

yielded extended structures (Karle et al. 1983), indicating the

conformational flexibility of these peptides, a turn remains

245
a viable candidate conformer.

In the second study, the conformational behavior of the five

tetrapeptide enkephalin analogs given in table 5B were examined to

continue identification of peptide opioid conformations leading to

high and low affinity at the μ-receptor site.

Using the same empirical energy method (ECEPP) and semi-empirical

quantum mechanical method (PCILO), conformational energies vere

obtained for a large number of optimized geometries of each

tetrapeptide. Two methods of selecting candidate active

conformations from low-energy conformers mere used. In the first

method, inactive conformers were designated as those having low

energy in the very weak tetrapeptide, Tyr-Gly-Gly-Phe-OH, and high

energy in the more potent ones. These candidate inactive conformers had geometries resembling or “random” peptide

conformations. Conversely, candidate active conformers selected

were low-energy conformations for both Tyr-D-Ala-Gly-Phe-NH2 and

Tyr-D-Ala-Gly-(NMe)Phe-NH2, but not low-energy conformers for TyrGly-Gly-Phe-OH. In the second method of selection, conformers

with relative energies in the active and inactive peptides that

followed the potency order Tyr-Gly-Gly-Phe-OH << Tyr-D-Ala-GlyPhe-NH2 < Tyr-D-Ala-Gly-(NMe)Phe-NH2

 were chosen as candidate

active conformers. By using both methods of selection, a

bend geometry, shown in figure 5, was found as the active

conformer.

FIGURE 5

Candidate Active Conformer for Enkephalin like

Tetrapeptides: A II' Bend (---) Superimposed on PET

246
While very similar in structure to that found for the pentapeptides, this conformer is not stabilized by a 1-4 hydrogen

bond, but instead is stabilized by hydrogen bond between the

tyrosine amine hydrogen atom and the phenylalanine carbonyl oxygen

atom. Thus, our results show that such conformers can exist

as relatively low-energy forms for active peptides with an N(CH3)

group on the fourth residues of the bend.

The candidate active forms have a greater degree of spatial overlap with the fused ring opiate PET than do the inactive ones,

suggesting that the extent of this overlap may be important in

high-affinity μ-receptor binding and in analgesic activity. The

identification of a -type conformer as an inactive form of these

peptide opiates is in agreement with proposed inactive conformation of Tyr-Gly-Gly-Phe based on crystallographic (Prange and

Paecard 1979) and NMR data (Fournie-Zaluski et al. 1977).

The morphfceptin analogs under current study, listed in table 5C,

are among the most μ-specific linear peptides found and should,

therefore, add considerable insight into the continued search for

μ-selective opioid peptide conformers. In these energy conformation studies, we have used only the empirical energy method ECEPP

and began with an “aufbau” search strategy. Starting with low

energy derivitized single amino acid conformers, these were

optimized and combined to form dipeptidee which were in turn

optimized. A number of unique low energy conformers of the

dipeptides were then combined to form tetrapeptide conformers

which were then optimized. The results obtained for these analogs

are summarized in table 8A-C.

Despite two proline residues, 38 different conformers of morphiceptin (Tyr-Pro-Phe-Pro-NH2

) vere found within 10 kcal/mol of the

lowest energy conformer (table 8A). However, addition of a methyl

group to the nitrogen backbone atom of the phenylalanine residue

greatly diminished the flexibility, yielding only 12 conformers in

this same energy range (table 8B). Moreover, while the low energy

conformers found for morphiceptin varied widely, those for TyrPro-NMe-Phe-Pro-NH2

 (NMePheMorphiceptin) were very similar to each

other, falling into two basic classes: one class roughly equivalent to a turn, and the other shoving backbone and

angles similar to those in the repeating C7 structure.

Since both of these analogs have similar μ-receptor affinities, it

is reasonable to assume that similar peptide conformers would be

involved in such receptor binding. Yet, there was no significant

overlap between any of the low energy < 10 kcal/mol) morphiceptin and NMePheMorphiceptin analogs found by the aufbau search

strategy. Thus, a different strategy was used to find common

conformations for the two peptides.

247
TABLE 8

Calculated Energies of Optimized Conformers for Three

Morphiceptin Tetrapeptides Using an Empirical Energy

Method (ECEPP)

In the second procedure, low energy conformers of each analog were

examined for their ability to accommodate each other. The result

was that no low energy conformer of morphiceptin could accomodate

the N-methyl analog. The energy required to maintain the N-methyl

analog in these conformers was of the order of hundreds of

248
kcal/mol and no constrained conformer vas a local minimum. By

contrast, when morphiceptin was constrained to overlap with the

low energy conformers of NMePheMorphiceptin, the energy required

ranged from 4 to 14 kcal/mol. Some of these conformations were

additional local minima for morphiceptin not found by the aufbau

search strategy, while others were not local minima at all.

Figure 6 shove the

conformation (33’)

energy for each.

two analogs in an overlapping

which is a local minimum with

II-type

relatively low

FIGURE 6

Overlap of Similar Optimized Conformers of

Morphiceptin = 6.5 kcal/mol) and N-Me-Phe3-

Morphiceptin = 2.8 kcal/mol)

The morphiceptin analog Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) is the

most potent and μ-selective analog and can be considered the

template for μ-specific morphiceptins. Thus, D-Pro4 was

eyetematically substituted into all the lov energy conformers of

Tyr-Pro-N(Me)Phe-L-Pro-NH2, given in table 8B. The relative

energy of the D-Pro4 conformers, optimized from this initial set

and labeled by their initial similarity to the L-Pro analogs from

which they were formed, are given in table 8C.

The effect of D-Pro4 substitution was large on some conformers and

small on others. Because the effect on activity was small, we

eliminated as candidates those conformers which would not incorporate D-Pro in the fourth position without a large energy cost.

The -type conformer 33’ best accomodated D-Pro4 with very

little geometry or energy change (figure 7) although it was not

the conformer with lowest energy for NMePhe-Morphiceptin or its DPro4 analog.

249
FIGURE 7

Overlap of Similar Optimized Conformers of N-Me-Phe3

L-Pro = 2.8 kcal/mol) and N-Me-Phe-D-Pro -

Morphiceptin = 2.4 kcal/mol)

As indicated in figure 6, it was also one of the conformers

which best accommodated morphiceptin, and thus is our candidate

conformation for morphiceptin like compounds.

Finally, we examined the effects of substituting a μ-Pro for an LPro in the second position of morphiceptin. In contrast to the

fourth position, a D-Pro substitution produces an inactive analog

(table 5C). Consistently, we found that forcing the Tyr-D-ProPhe-Pro-NH2

 analog to conform to any low energy conformer of NMe-Phe-Morphiceptin required an energy of 25 to 400 kcal/mol

compared to its lowest energy conformer. Moreover, none of these

constrained conformations were local minims (figure 8A), nor did

local minima of the D-Pro overlap with L-Pro2

-morphiceptin

(figure 8B).

In contrast to destruction of activity by substituting a D-Pro2

residue in morphiceptin, a number of modified L-Pro2 residues can

be substituted in the 2 position without significant effect on μaffinity and in vitro activity. Among these analogs, the most

significant decrease in bioactivity was found with the substitution of a 4-hydroxy-proline into the second position of PL017.

Conformational analysis revealed that there was virtually no

energy cost for adding a 4-OH group to proline in any candidate

conformers of PL017. This result indicates that the decrease in

the observed bioactivity results from adverse electronic

interactions at the receptor site rather than from conformational

differences in these analogs.

250
A. D-Pro2morphiceptin conformer constrained to overlap with

L-Pro2 analog = 249 kcal/mol)

B. Optimized D-Pro2

 conformer = 8.9 kcal/mol). Little

overlap with L-Pro2

Comparison of L-Pro2 and D-Pro2 Morphiceptin Conformers

FIGURE 8

251
Conclusions and Future Directions

The three types of studies made thus far indicate a “universal”

type of μ-selective opioid peptide conformer involving a o r

turn. In enkephalin-type analogs, a conformer is a

likely candidate, while μ-selective morphiceptin analogs, appear

to prefer a turn. As shown in figure 9, these two types of

turns have a considerable overlap.

Studies are currently under way to investigate the effects of

other subatitutions in the second position of morphiceptin

analogs. Work is also currently proceeding on conformational

analysis of the cyclic μ-selective compounds of Schiller et al.

These authors have synthesized Leu-enkephalin analogs in which the

aliphatic side chain (CH2)n of the second residue has been

extended to various lengths (n=1,4) and cyclized with the Cterminal carboxyl group through an amide linkage (DiMaio et al.

1982). While the the degree of n-specificity varied in the four

cyclic analogs of

this type synthesized, all shoved greater μ-specificity than their

linear counterparts in binding assays, although diacrepanciea with

tissue-specific assays did exist. The increased μ-specificity

appeared to be due generally to a decrease in -receptor

affinity.

FIGURE 9

Tyr-D-Ala2-Gly-Phe-NH2 Shown in

and Conformations

252
Comparing the candidate conformers of the most μ-selective and

highest potency morphiceptin analog (PL017) with the lowest energy

conformers of the most μ-specific cyclic compounds (with n=2,4),

should provide a further probe of μ-selective opioid conformers.

It will also be interesting to compare these conformers with the

-selective linear and cyclic analogs which we are also currently

studying.

PRELIMINARY STUDIES OF -SELECTIVE OPIOID PEPTIDE CONFORMERS

LEADING TO HIGH AFFINITY AT -RECEPTOR SITES

Although the -receptor site was first identified by its preference for peptide compared to nonpeptide opioids, moat opioid

peptidea synthesized have been found to bind and act with

comparable or higher affinities at the μ-receptor.

Among the first molecules rationally designed for specificity

were the hexfpeptide Tyr-D-Set-Gly-Phe-Leu-Thr (DSLET), and its

related DThr2 (DTLET) analog (David et al. 1982). It was thought

that this sequence would lead to -specificity since structure

activity studies of peptidea had previously shown that: (a) Phe

at position 4 plays a crucial role in μ- versus -selectivity;

(b) lengthening of Met-enkephalin by Thr enhances MVD potency

while decreasing GPI activity; (c) Leu-enkephalin shows increased

MVD potency relative to Met-enkephalin; and (d) replacement of

Gly2 by a D-amino acid inhibits degradation and reinforces μselectivity if the residue is hydrophobic.

As summarized in table 9, GPI versus MVD studies indicate that

DSLET and DTLET are 4 to 10 times more -selective than DADL,

while binding studies indicate that they are only somewhat more

selective than DADL.

Thus, while the linear peptides DSLET and DTLET seem to be somewhat more -selective than DADL, the extent of this selectivity is

unclear from the existing data. Since these linear peptidea are

quite flexible, it is not surprising that they would not be overwhelmingly selective for a particular receptor.

As discussed above, Schiller and coworkers (DiMaio et al 1982)

demonstrated that reduction of flexibility by cyclization could

modulate the extent of μ- and -selectivity and lead to μselective Leo-enkephalin type peptides. These investigators have

also synthesized another series of cyclic analogs baaed on a TyrCys-Gly-Phe-Cys pentapeptide cyclized by a diaulfide bridge

253
TABLE 9

Receptor Binding and In Vitro Activity of

-Selective Opioid Peptides

Enkephalin

Analog

D-Ser2-Leu5Thr6a

D-Thr2-Leu5-Thr6a

[D-Pen2-L-Pen5]

a

[D-Pen2-D-Pen5]

a

[D-Pen2-L-Cya5]

b

[D-Pen2-D-Cys5]

b

[D-Cys2-L-Cya5]

[D-Cys2-D-Cys5]

[L-Cys2-L-Cys5]

IC50 nM

[

3H]Nalox [

3H]DADL

DADLa

Morphinea(normorphine)

88 5.7

36 6.4

3710 10

2840 16

178 12

157 26

2.0 c0.8 c

3.5 c1.6 c

16 4

23 27

ED50 nM

G.P.1 MVD

234 0.70

100 0.58

2720 2.5

6930 2.2

213 0.32

1350 6.3

1.5 d

0.80d

0.80d0.30d

210d

950d

24 0.72

91 541

aMosberg et al. 1983a; bMosberg et al. 1983b, c

Schiller et al. 1981;

d

Schiller and DiMaio 1983.

between the second and fifth reaiduaa (Schiller et al. 1981,

Schiller and DiMaio 1983). As shown in table 9, of thrae such

analogs synthesized, all were relatively unselective. By analogy

with oxytocin analogs, Mosberg and coworkers (1983a. 1983b)

reasoned that gem-dimethylation of the cysteine -carbons would

greatly rigidify this cyclic structure. As shown in table 9, the

resulting two analogs [D-Pen2-L-Pen5]enkephalin and [D-Pen2-DPen5

]enkephalin, while retaining affinity and activity at

receptors, had greatly diminished activity at μ-receptors,

yielding the most -selective peptides yet found.

We have begun energy conformation studies of both the linear

peptide DTLET and the cyclic peptide [D-Pen2-L-Pen5]enkephalin, in

order to identify, for the first time, peptide opioid conformers

leading to high affinity at but not μ-receptors.

254
For the hexapeptide, DTLET, the aufbau search strategy was used

with the empirical energy program ECEPP. Each of approximately 10

low energy derivatized single amino acid conformers was combined

to give about 100 initial conformations for the Tyr-D-Thr and PheLeu dipeptides. Each of these dipeptide conformers were then

optimized. To the optimized Tyr-D-Thr peptides were added low

energy conformers of Gly to form tripeptides. A similar procedure

was used to obtain Phe4-Leu5-Thr6 tripeptidea. Finally, optimized

tripeptide fragments were combined to form 64 initial hexapeptide

conformers which were once again optimized. These 64 conformers

spanned an energy range of 14 kcal/mol relative to the lowest

energy conformer obtained. Fifty conformers had energies in the

range of < 10 kcal/mol.

This considerable effort resulted in characterization of the

energy-conformational profile and indicated a high degree of

flexibility of this linear peptide. This flexibility makes it

difficult to identify a probable selective conformer baaed

solely on studies of linear peptides.

To help alleviate the problem of extensive flexibility, we have

begun a systematically study of the more conformationally restricted

[D-Pen2-L-Pen5] cyclic enkephalin. In order to perform this study,

we have recently modified our structure-generating and empirical

energy programs to incorporate and recognize diaulfide

bonds between cyateinelike residues. The search strategy we are

using involves:

1) Starting with the linear analog formed by breaking the

diaulfide bond, and constructing a number of standard

secondary structures as initial geometries.

2) Generating an initial cyclic structure by rotating side

chain and backbone torsion angles to form a diaulfide

bond with an S-S distance of 2.04 A.

3) Optimizing all initial structures.

Using this strategy, torsion angle optimization of an -helical

initial conformation was made, leading to a low energy minimum,

the first geometry-optimized cyclic structure of this analog to be

reported. This conformation of D-Pen -L-Pen was then used as a

template to which DTLET was maximmly overlapped. Maximum overlap

includes exactly matching the Tyr1 and Phe4 side chain angles of

both the cyclic and linear analogs. Partial optimization of the

DTLET conformation was then performed, allowing only torsion

angles in DTLET, with no counterparts in the cyclic analog to

vary. The resulting structure (figure 10) had an energy only 11

255
FIGURE 10

Overlap of Optimized (D-Pen2-L-Pen5)Enkephalin

Conformer with Partially Optimized DTLET Conformer

DTLET = 11 kcal/mol)

kcal/nol higher than the lowest energy DTLET conformer obtained

from the previoua aufbau search. Moreover, unconstrained optimization of this conformer caused only small departures from

maximal overlap, as shown in figure 11, and resulted in a gain of

4 kcal/mol. Thus, the maximumly overlapped conformer of DTLET is

near a local minimum of the potential energy surface, only 7

kcal/mol above the global minimum. Therefore, the conformer of

DTLEI and [D-Pen2-L-Pen5]enkephalin shown in this figure are

plausible candidates for a -selective peptide opioid conformer.

This conformer of DTLET was not among the minima found from the

aufbau search strategy and illuatratea the usefulness of a cyclic

template in sorting out candidate -selective conformers.

We are continuing these studies, searching for additional low

energy conformers of this and other cyclic analogs, to further aid

in selecting the most probable conformer responsible for highaffinity binding at -receptors.

DESIGN OF μ-SELECTIVE OPIOID PEPTIDE ANTAGONISTS

The synthesis of opioid peptide antagonists has been repeatedly

attempted since the initial discovery of the enkephalina. Moat

previous attempts have, by analogy to fused-ring opiates, relied

upon the addition of one or two N-alkyl moieties to Met- or

256
FIGURE 11

Overlap of Optimized DTLET with Optimized [D-Pen2-LPen5

]Enkephalin Conformer DTLET = 7 kcal/mol)

Leu-enkephalin analogs. Initial studies with Met- or Leuenkephalina and their N-allyl analogs revealed alight antagonism

to morphine’s inhibition of amooth muscle contraction (Hahn et al.

1977), but no antagonism to analgesia after intracerebral

ventricular (i.c.v.) administration (Pert et al 1977). The

addition of N,N-diallyl to Leu-enkephalin analogs has produced

potent -selective antagonists. ICI 154,129 (Shaw et al. 1982)

and, more recently, ICI 174,864 (Cotton et al. 1984) have been

shown to be Nghly selective in their inhibition of agonist

actions in the field-stimulated MVD preparation. ICI 154,129 was

also shown to inhibit the slowing of the head-turn time induced by

etorphine, the model uaed to investigate -receptors in vivo by

Gormley et al. (1982). These compounds, however, being

selective, showed no inhibition of morphine-induced analgesia.

Conformational studies of enkephalin analogs and comparisons with

flexible, rather than fused-ring, nonpeptide opiates led us on a

different path to peptide antagonists, and produced compounds with

μ-selectivity. Previous energy calculations, described above,

indicated that, in low energy conformations of Met-enkephalin, the

amino-terminal phenethylamine moiety could overlap with the 3-

phenylpiperidine class of flexible opiates (figure 12A).

Moreover, these two types of flexible opioida share only a

phenethylamine overlap with fused-ring opioida (figure 12B). Also

257
as diacuaaed in detail above, our own and previous atudiea of the

3-phenylpiperidinea indicate that they are moderately potent μselective opioid agonists, in which addition of an -OH group to

the phenyl ring induces antagonist activity, and thus generates

mixed agonist/antagonist compounds. A p-OH group produces very

weak antagonism and an m-OH group greatly increases antagonist

activity (Jacoby et al. 1981). Furthermore, in the 3-CH3-3(mOH)phenylpiperidine series, N-aubatitutiona did not influence

relative agonist/antagonist activity as in fused-ring opiates

(Cheng et al. 1984, 1985).

A. L-Tyr superimposed on

3-phenylpiperidine

B. L-Tyr superimposed on portion

of morphinan nucleus

FIGURE 12

Comparison of 3-Phenylpiperidine and Peptide Opioids,

A) With Each Other and B) With Morphinan

Thus, the two enkephalin analogs with modified tyroaine residues,

showo in figure 13, were chosen for synthesis and testing along

with D-Ala -Met-enkephalin amide. The synthesis of these peptides

is described elsewhere (Loaw et al. in press). As shown in

this figure, the modified tyroaine residues consisted of a m-OH

tyroaine and a (m-OH) -CH3 tyroaine chosen to correspond to the

( m-OH) and 3-CH3

 groups of the 3-methyl-3-(m-OH)phenylpiperidines,

respectively. Binding affinities as well as in vivo, i.c.v.

agonist and antagonist activity were determined for the three

peptides.

As seen in table 10, the parent compound D-Ala2-Met-enkephalin

amide (p-Tyr) is a potent agonist in the tail-flick teat when

administered i.c.v. in mice, being about one-third as potent as

258
morphine. This compound also showed significant antagonist

activity in inhibiting a morphine-induced increase in tail-flick

latency, but was only one-tenth as potent an antagonist as an

agonist. Displacement of the hydroxy group on the tyrosine to the

meta position produced striking changes in the relative activities

TABLE 10

Analgesic and Narcotic against and Antagoniat

Potencies of (D-Ala2, Met5)Enkephalin Amide Analogs

Evalueted in the Mouae Tail-Flick Teat

COMPOUND

p -Tyr

m-Tyr

-Ch3-m-Tyr

Nalorphine

Morphine

AGONISMa ANTAGONISMb

K D 5 0

μmol/kg

95% Conf. Lia. Ant.AD50 95% Conf. Lia.

μmol/kg μmol/kg μmol/kg

0.214 0.074 ± 0.616 2.283 1.512 ± 3.453

10.302 5.630 ± 18.854 1.684 0.785 ± 3.596

2.394 1.203 ± 4.764 3.530 2.263 ± 5.507

19.320 7.963 ± 46.754 0.322 0.184 ± 0.563

0.063 0.026 ± 0.151

aA modification of tail-flick teat of Li et al. (1978).

bAntagonism against 21.08 μmole/kg (s.c.) of morphine sulfate was

measured 2, 5, 10, 20, 30, 45, 60, 90, and 120 min. after i.c.v.

administration of enkephalin amide analogs.

FIGURE 13

Peptides Selected for Synthesis

259
of the compound. The (m-OH)Tyr analog is 50 times weaker as an

agonist, but slightly stronger as an antagonist, than its p-OH

counterpart. This produces a compound six times more potent as an

antagonist than as an agonist, and corresponds well with relative

changes in potencies when 3-phenylpiperidines are modified from pOH to m-OH (Jacoby et al 1981). The -CH3

- m-Tyr analog has an

activity profile between that of the other two compounds with

approximately equal agonist and antagonist activity.

Competitive binding studies of these compounds were made using the

four labeled ligands [H] naloxone, [E]DADL, [E]EKC, and

[H]DHM. Also conducted were self- and cross-competitive binding

of the labeled ligands themselves, as decribed above. The

resulting 4 by 7 matrix of competitive inhibition experiments was

analyzed simultaneously using the computer curve fitting program,

LIGAND.

As shown in table 11, all of the peptides exhibited high affinity

at the site designated as μl. Furthermore, the relative analgesic

potency of the three peptides corresponds to their relative

affinities at “μl-” The affinity at μ1 and analgesic activity of

the p-Tyr analog are 20 to 50 times higher then either of the two

novel peptide analogs. This observation lends support to the

evidence presented by Pasternak that the μ1 receptor is

responsible for opiate-induced analgesia (Pasternak et al.

1980). Also evident from table 11 is the n-selectivity induced by

the (m-OH)Tyr. While the p-Tyr analog is very potent at

receptors, both m-Tyr and -CH3

-m-Tyr have quite low affinities at

consistent with the 3-phenylpiperidine templates.

Thus, it appears that (m-OH)Tyr and methyl-(m-OH)Tyr-Metenkephalin-amide analogs are the first μ-selective enkephalin-type

peptides with demonstrated antagonism to whole animal analgesia.

Moreover, requirements for potent antagonism of Met-enkephalin

analogs appear similar to that for 3-phenylpiperidines, possibly

indicating similar binding conformations.

To continue to explore this similarity, we are in the process of

synthesizing two more novel tyrosine analogs, an N-phenethyl-(m -

OH)-tyrosine and a dimethyl-(m-OH)-tyrosine to incorporate

into Met-enkephalin-amide peptides. If they behave as 3-phenylpiperidines do, then both of these modifications should enhance

antagonist potency.

260
TABLE 11

Receptor Affinities and Maximum Binding Capacities of

(D-A1a2-Met5)Enkephalin Amide Analogs Assuming a

5-Receptor-Site Model

Site 1

"μ1"

KD(nM)

Site 2 Site 3 Site 4 Site 5

“μ2"

Naloxone 0.60 3.0 20.8 0.5 100

DADL 1.80 14.9 1.3 312 0,333

EKC 0.60 4.3 15.0 0.003 588

DHM 0.30 12.7 119 1.515 83

p-Tyr 0.53 5.0 0.40 1600 476

m-Tyr

-CH3-m-Tyr

Bmax pmol/g

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ACKNOWLEDGMENT

This work was supported by grant DA-02622 from the National

Institute on Drug Abuse.

264
AUTHORS

Gilda B. Loew, Ph.D.

Lawrence Toll, Ph.D.

Edward Uyeno, Ph.D.

Alice Cheng, Ph.D.

Amrit Judd, Ph.D.

John Lawson, Ph.D.

Christopher Keys, B.S.

Peter Amsterdam, B.A.,B.S.

Wilma Polgar, M.S.

Life Sciences Division

SRI International

333 Ravenswood Avenue

Menlo Park, California 94025

265
Conformations of Opioid Peptides as Determined by Nuclear Magnetic Resonance and

Related Spectroscopies

M. Abu Khaled, Ph.D.

INTRODUCTION

The peptides that possess opiate like biological properties are

called “opioid peptides.” Known members can be divided into

three main groups according to their sources. 1) Endorphins:

These peptides have been found to occur naturally in the central

nervous sys tern. -Endorphin is the largest in this group,

followed by dynorphin, neoendorphins, and the enkephalins which

are the smallest pentapeptides in the family (Hughes and Kosterlitz 1977). 2) Exorphins: These peptides are obtained as a

result of partial enzymatic digestion of proteins derived from

food stuffs (Loukas et al. 1983). For example, -casomorphins

have been isolated from the bovine milk protein, -cesein. 3)

Kyotorphins: These are synthesized small peptides (Takagi et

al. 1979) whose opiate like properties do not arise from direct

interaction vith opiate receptors,but perhaps act by releasing

enkephalina and/or by inhibition of enkephalin degradation

(Morley 1983).

Since the discovery of enkephalins, much attention has been

dravn to this area. The enkephalins are distributed over wide

areas of the CNS, specific parts of the brain, spinal cord, and

in certain nerve plexi of intestines. Because of the interaction of enkephalina at the same receptor site as that of

morphine, numerous structural analosa of nkephalins have been

prepared and tested for their opioid activity, their propenaity

to cause tolerance and physical dependence,their susceptibility

to degradation by brain enzymes,and for their behavioral effects

on laboratory animals and on schizophrenic patients. The isolation, identification, characterization, and structure-activity

relationships of two naturally occurring enkephalins, namely

H-L. Tyr-Gly-Gly-L•Met-OH([Met5]-Enk) and H L•Tyr-Gly-Gly

L•Phe-L•Leu-OH ([Leu5]-Enk), are covered in detail in some

recent reviews (Hughes and Koaterlits 1977; Miller and Cautrecasas 1978; Morley 1980; and Schiller 1984).

266
Structural similarities between the morphine derivatives and the

enkephalins must exist if they share common receptors. Extensive research efforts have, therefore, been made to predict the

enkephalin structures compatible with the rigid morphine structure. The main approaches utilized in these studies are: 1)

speculation as to the conformation of the enkephalins at the

opiate receptor resulting from model building and comparison

with models of narcotic drugs, 2) conformations of enkephalins

in solution as derived from various spectroscopic data, and 3)

conformational energy calculations.

Recently, in an elegant review article, Schiller (1984) has

exhaustively summarized all the previously reported conformationactivity relationships of enkephalin molecules. Data from

various spectroscopic methods, nuclear magnetic resonance (NMR)

in particular, have been discussed. In order to avoid repetition of the similar subject matters, a different approach will

be taken in this review article as outlined in the folloving

sections.

1. Definitions of various conformational states of a

peptide/polypeptide backbone.

2. Descriptions of various NMR methods that are used in

obtaining conformational information.

3. Use of such NMR methods in relation to A) backbone and

B) side-chain conformations of enkephalin molecules.

4. Concluding remarks.

PEPTIDE CONFORMATIONS

The backbone of a polypeptide or protein is formed from a

repeating peptide unit, as depicted in figure 1.

It can be seen that the overall conformational characteristics

of the peptide unit are determined by rotations about the

C'-N, and bonds (R is the side-chain of an amino

acid). A convenient description of the conformation about each

bond is in terms of the torsion angles -C'), (N- (C'

-N), and -R) as shown in figure 1: refers to the torsion

angle of sequence of atoms to the sequence N1

, C'1

, N2; and to the sequence

negative(- )according as when the system (figure 1) is viewed

Torsion angle is considered positive (+ )or

along the central bond N2- in the direction N2 C ' 2

quires to be rotated to the right or to the left, respectively,

(or C'2 N2), the bond to the front atom C'1

 (or C'2) rein order that it may eclipse the bond to the rear atom C'2

 (or

C'1). Similarly, and can be determined by viewing along

the central bond Torsion angle is either zero (for

cis peptide bonds) or 180 (for trans peptide bonds).

267
Side-chain torsion angles designated by are specified by two

(or three) superscripts, the first one (or two) indicating the

central bond about which the angle is measured and the last one

indicating whether the angle measured is relative to any

branched chain atom. By the sequence rule (IUPAC-IUB 1970),

when =0, is in the eclipsed position relative to

N. Positive or negative angles can be generated by viewing

along the central bond in the direction and

rotating right or left, respectively,in order to

eclipse with N. A fully extended polypeptide chain is characterized by +180° (IUPAc-IUB 1970).

Rotations at the above mentioned bonds give rise to various conformations that are stabilized by intra- and/or inter-molecular

hydrogen bond (H-bond) formations between the C=0 and N-H groups,

as shown in figure 2.

An H-bonded ring between the N-H of an amino acid sequence

number m and the C=0 of another residue of the sequence number n

is designated as m n which encompasses a certain number of

atoms of the backbone chain. The possible H-bonded structuree

in a system of 4 linked amino acids are shown in figure 2. A 2

2, 3 3, or 4 4 H-bond encompassee 5 atoms; the 3 1 or 4

2 contains 7 atoms; similarly, 2 3 or 3 4 and 4 1 H-bonds

contain 8 and 10, respectively; while a 2 4 H-bonded ring consists of 11 atoms. These aforementioned intramolecular H-bonded

rings are also indicated by C5, C7, C8, Cl0, and Cl1

conformations, respectively.

FIGURE 1

A Peptide Backbone Showing Various Torsion Angler

268
The conformation is extended and usually occurs in conjunction

with an antiparallel -pleated sheet structure that is formed by

intermolecular H-bonds, as shown in figure 3.

FIGURE 2

Possible Intramolecularly H-Bonded

Rings in a Peptide System with Four

Linked Amino Acids

Both the C7 and C8 conformations are also known as shortrange interactions and usually occur with other dominant conformational features, such as helices or higher ring structures.

For example, C7 always coexists with C11, as shown in figure

4. Following the -turn, which is a 10-membered H-bond, chronologically, an 11-membered H-bonded ring (figure 4) should be

termed as -turn (Nemethy and Printz 1972; Matthews 1972).

Unfortunately, however, controversy remains in literature regarding the designation of this structure. The structure in

figure 4 happens to contain one 7-membered (C7)ring which some

research workers prefer to call -turn, while the C11 is

referred to as -turn.

269
FIGURE 3

Antiparallel -Sheet Structure with C5 H-Bond

FIGURE 4

A -Turn Conformation in Peptides

270
The -turn, a 10-membered intramolecular H-bonded ring, just

mentioned above, will be further discussed here since the occurrence of this conformational feature in enkephalin analogs is

most abundant. The reader should, therefore, be adequately

familiar with conformational properties of a -turn. The main

characteristic of a -turn is to reverse the backbone chain, containing a suitable dipeptide moiety, in the same plane where it

thus differs from a 310-helix. It was, therefore, previously

termed as 4 1 intramolecularly H-bonded nonhelical conformation (Venkatachalam 1968; Benedetti et al. 1976). Conformstions related to -turns have also been called earlier as: 3

(or 3-10) bond (Kuntz 1972), folded -conformation or -fold10

(Shields et al. 1968), -turn (Urry and Ohnishi 1970), -bend

Chandrasekaran et al. 1973), hairpin bend (Kuntz 1972;

Chandrasekaran et al. 1973), -loop (Kuntz 1972), 1 4 bend

(Nemethy and Prints 1972), chain reversal (Lewis et al. 1973),

U-bend (Chandraeekaran et al. 1973), lo-membered H-bonded ring

(Deber 1974), 1,4 turn (Hruby 1974), -twist (Benedetti et al.

1976), and tight turn (Richardson et al. 1978).

Various foldings of a peptide backbone chain into different

forms or types of -turns are depicted in figure 5. Venkatachalam (1968) classified the -turns into conformational types:

I, II, III, and their mirror images I', II', III'. The torsion

angles and that define these types are given in table 1.

It can be seen in table 1 that the -turn type III is actually a

helix. Because of sterical hindrance, it is necessary that

the two residues within the H-bonded ring should be with L-configuration, i.e., L-L arrangement is preferred for a type I

-turn, while an L-D arrangement is preferred for a type II

-turn. The mirror images, D-D and D-L, are obviously required

to form type I' and II' -turns, respectively. There configurational arrangemente are clearly shown in figure 5 by the different projections of R2

 and R3 on side-chains. A glycine

residue can be conveniently accommodated in place of a D-substituted amino acid , particularly in type II and II' -turns. A

convenient way of distinguishing type I and type II -turns is

to consider the orientation of the peptide moiety between

residues 2 and 3 (see figure 5). In type I -turn, the -H

proton of residue 2 is oriented opposite to the N-H proton of

residue 3, as indicated by a double-headed arrow in figure 5A.

In type II -turn, however, the same two protons are on the same

side which is also indicated by a double-headed arrow in figure

5B. These are the simple descriptions of intra- and/or intermolecular H-bonded structures that will be mentioned frequently

in section 3, which deals with the backbone conformations of

various enkephalin analogs.

NUCLEAR MAGNETIC RESONANCE METHODS

NMR is the most powerful technique which can provide all the

necessary information needed to describe a peptide conformation.

271
A. TYPE I B. TYPE. II

Type

I

I '

II

II'’

III

III'

FIGURE 5

-Turn Structures in Peptides

TABLE 1

Torsion Angles of Various Types of -Turns

Residues

2 3

2 2

-60 - 30 -90 0

60 30 90 0

-60 120 80 0

60 -120 -80 0

-60 - 30 -60 -30

60 30 60 30

3 3

272
Resolution of NMR signals and their assignment to individual

amino acid residues are the first steps in conformational

analysis of peptides and proteins. Usually, the assignments are

carried out from the inspection of the side-chain spin pattern

of a particular amino acid residue by using spin decoupling technique. This is true when a molecule contains different amino

acid residues occurring only once in the backbone with wellresolved NMR signals. However, it becomes more complicated when

a molecule contains a recurring amino acid residue such as in

enkephalin molecules where two glycyl residues are involved in

positions 2 and 3. Selective isotope enrichment of one of such

residues either by C, H, or N is the usual way for

unequivocal assignments of NMR resonances. The two glycyl

residues in [Leu5]enkephalin were thus assigned by the partial

C

13 enrichment (Tancrede et al. 1978). Recently, however, a

combined use of 1H and 13C resonances has been made to

both 1H and 13C signals of a particular amino acid in

peptide system (Khaled and Urry 1981; Khaled et al. 1981;

Khaled and Watkins 1983). The basic principle of this technique

is to selectively irradiate the -H. and N-H. protons,

across a peptide bond. These two protons are coupled to

hauser enhancement (NOE) for this carbonyl carbon nucleus.

=O carbon which also provide almost all the nuclear OverOnce

two such protons are located and one of the amino acid residues

is identified, either from its side-chain spin pattern or by

perturbing the end groups in case of a linear peptide, the rest

of the residues in the molecule can be assigned by stepwise

irradiation of the 1H resonances. Thin method ultimately

allows the unequivocal NMR signal assignments without involving

an expensive and time-consuming synthetic iaotope enrichment.

After having assigned all the resonances, conformational

analyses are carried out by using the well-established NMR

methods as described next.

Identification of H-Bonds

The intra- and inter-molecular H-bonds are usually delineated

from the temperature dependence of peptide N-H proton chemical

shifts. An intramolecularly H-bonded peptide N-H proton has

less temperature dependence compared to an intermolecularly

H-bonded or an exposed N-H proton. The temperature coefficient

of an intramolecularly H-bondgd N-H proton typically

varies from 2 x 10-3 to 5 x 10-3 ppm/°C depending on the

stability of the H-bond (Kopple et al. 1969; Ohnishi and Urry

1969). However, there exists a controversy regarding the temperature dependence of peptide N-H protons. Stevens et al. (1980)

reported value of the order of 2.4 ppm/°C which they

attributed to the N-H proton that is exposed to nonpolar

solvents, such as chloroform. The values higher than 2.4

ppm/°C are interpreted as the indications of change of N-H

protons from bound to free state, i.e., from H-bonded to exposed

to nonpolar solvents.

273
The rates of peptide N-H proton exchange with the labile hydrogens or deuteriums in the medium can also provide information on

the H-bonded and nonbonded N-H protons (Molday et al. 1972;

Stern et al. 1968). This can be achieved either by adding a controlled quantity of D2O into a protonated medium or by the

saturation transfer of the labile proton of the solvent by a

double resonance NMR method (Redfield 1978). The N-H protons

which are exposed to the solvent experience reduced signal intensity according to the extent of their exposure to solvent. The

difficulty with this method is that the proton exchange rates

are the kinetic parameters which reflect the properties of the

transition state of rate determining step in the protolysis

reaction i.e., a state which may not resemble the preferred

equilibrium state of the peptide. However, it is generally

desirable to confirm intramolecularly H-bonded peptide N-H

protons by as many methods as possible. There is, therefore,

another method where the peptide N-H protons show differential

dependence on the composition of a solvent mixture (Pitner and

Urry 1972). The advantage of this method is that both the peptide N-H protons and peptide C=O carbons can be classified as an

appropriate H-bonded pair, i.e., N-H...O=C pairing can be established. There is, however, one very essential requirement in

this method which requires that the molecule should retain the

same conformation(s) in both the solvents, which can be verified

by coupling constants and temperature studies as stated above.

Such an experiment is performed by monitoring the chemical shift

changes of peptide N-H protons and peptide C=O carbons from a

weak basic solvent, such as dimethyl sulfoxide, to a protic

solvent, such as trifluoroethanol. Very recently , Khaled and

Watkins (1983) have developed a new 13C(1H) NOE method to

detect simultaneously an H-bonded pair, i.e., N-H...O=C, in

peptides.

Estimation of Torsion Angles and

A conformation is completely described when the angle of rotation about each bond of a peptide is specified (see figure’ 1 and

section 1). In peptides and amino acids, vicinal coupling between N-H and H protons provides information about the peptide

torsion angle using a Karplus relationship (1959) of the type

(1)

where is the dihedral angle formed between the H-Nand planes.

The dihedral angle is related to the peptide torsion angle

by the equations

= -60 for L-amino acids

= +60 for D-amino acids (2)

274
Empirically, Bystrov et al. (1969) have established the

following equations:

(3)

and for glycine residues

(4)

The above expressions give several possible angles for each experimental coupling constant. One of the possible angles would

be approximately correct, if there were no torsional motion.

Since, virtually in every case for a biological peptide, there

would be some toreional flexbility, it is important to realize

that the observed value is obtained from a summation of states

properly weighted according to its energy dependent probability.

Therefore, if the values ranging from 5.5 to 7.5 Hz were observed, then this could imply either a single state, , or

nearly unrestricted rotation about the N-E bond. However, if a

value of 4 Hz or 8 Hz is obrerved, then these values indicate

substantially restricted angles. Similarly , if the residue is a

glycine, then the greater the nonequivalent the

NB-NHX coupling constants, the stronger the argument is for

limited rotation about the N- bond. If the coupling constants

are equivalent, it may be due to essentially unrestricted rotation or, looking along the N- bond, due to the N-H line segment

bisecting the H- -H bond angle. The same considerations may

also apply to H-CbH2 for the determination of angle X, as

described below.

For the torsion angle of glycine residues, Barfield et al.

(1976) have determined an expression for the geminal coupling,

2

J, which is also a function of the of equation 4.

= -13.91-1.55 (5)

For L- and D-amino acid residues, NOE can be used as long as the

peptide moiety remains approximately planar, becaure the H i

to NHi+1 interproton distance is a function of the angle

(Leach al. 1977).

Side-Chain Conformations (X)

The conformational properties of amino-acid side-chain sometimes

restrict the allowed range of and angles if there is an

intramolecular interaction with the adjacent amino acid. For a

complete understanding of the conformation of peptides it is

important to determine the conformation about the bond. A

correlation has been observed between 3J(HH) coupling constant

and the dihedral angle for the CH-CH system using again a

Karplus-like relation: 3J = B cos2-C, were B + 10.4 for

0< <90, and B = 13.5 for 90< <180 and C = 0.

275
FIGURE 6

Possible Side-Chain Conformations of Amino

Acids Rl= R2 = H for Ala, R1 = R2

= CH3 for Val, and Rl = H, R2 = aryl or

alkyl group representing Phe, Tyr, Leu, etc.

For most amino acid residues,there is considerable rotational

freedom about the bond. It is assumed that the three

staggered conformations (I, II, and III depicted in figure 6)

have relative populations, PI, PII, and PIII such that

PI + PII + PIII

(6)

For rotation about the bond that is rapid on the NMR time

scale

3

J(HH)obs = JtPII+J8PI+J8PIII (7)

assuming Jg has the same magnitude for conformers I and III.

For free rotation (PI - PII = PII the observed 3J is

given by

3

J(HH)free = 1/3 (Jt + 2Jg)

(8)

From experimental coupling values and assuming J = 13.6 Hz

and J

rotamer

= 2.56 Hz, obtained by Pachler (1964), the relative

populations can be calculated for different amino acid

side-chains, such as valine, leucine, phenylalanine, aspartic

acid, and tyrosine. The torsion angles can also be estimated by

using the equation of Abraham and McLauchlan (1962):

10.5 cos2 X1 - 0.28 for X

1

 = 0 - 90°

(9)

10.7 cos2 X1- 0.29 for X

1

 = 90 - 180°

The relative population of trans and gauche, isomers can be

calculated, but it is not possible to distinguish between the

two possible gauche conformers (I and III in figure 6) unless

Rl and R2 have been unequivocally assigned, e.g., for valine

276
(R1

 = R2 = CH3) and phenylalanine (Rl or R2 = H). For

phenylalanine and tyrosine, the assignments of the two -CH2

protons could be made by stereospecific deuterium labeling.

This has been accomplished in enkephalin molecules by Kobayashi

et al. (1979, 1980). As mentioned above, a large or small

coupling constant or nonequivalence, involving a CH2 moiety ,

provides direct evidence for restricted torsion angles. Where

it becomes important to resolve the ambiguity of midrange

coupling constants and equivalent coupling to CH2 moieties.

synthetic enrichment with 13C and/or 15N can be carried out

and the additional heteronuclear coupling constants can be used

to resolve the ambiguity experimentally. This has recently been

done for the H2 torsion angles of enkephalins (Stimson

et al. 1979; Garbay-Jiurequiberry et al. 1982) and will be

discussed further in section 3.

Nuclear Magnetic Relaxation Times (Tl and T2)

The longitudinal or spin-lattice relaxation time (Tl) and the

trsnsversal or spin-spin relaxation time (T2), particularly the

Tl, have been used to study the dynamic behavior of enkephalin

molecules by many research workers.

In practice, T2 is measured from the line width of a single

signal by using the equation

(10)

where W is the line width at half height of a signal.

Various molecular dynamics can be well characterized by a time

factor called correlation time,which serves as a measure of the

average time that two nuclei remain in a given orientation and is

designated as Tc. For molecular rotation, the Tc is the average

length of time that the molecule requires to rotate through an

angle of a radian, while for translation, the Tc is the time it

takes to move the molecule to one molecular diameter. The rotational process is related to the intramolecular relaxation,

while the translational process is related to the intermolecular

relaxation. In general, both processes depend on the viscosity

of the medium and vary critically on the size and shape of the

molecule involved. If the molecule being studied tumbles isotropically and behaves like a spherical rigid rotor (having

only rotational motion),

given by (Levy and Nelson 1972).

the equation to derive Tc from T1 is

(11)

Where is the gyromagnetic ratio, w is the angular frequency,

and r is the distance between the two coupled nuclei. This is

277
the relsxstion for one IH attached to a I5C; for I3C-IH,

the right-hand side of the equation will have to be multiplied

by n (n is the number of protons attached to a carbon atom).

Nuclear Overhauser Effect

As mentioned in the preceding section, besides providing the

estimate of torsion angle NOE can, in principle, be used in

determining the spatial relationships between nuclei that are

not covalently linked. It is the fractional change, fi (j),

in intensity by cross-relaxation of one resonance, i, when

another resonance, j, is perturbed (Noggle and Schirmer 1971).

Simple mathematical derivations of fi (j) are given by Roques

et al. (1980) and, therefore, the repetition of such expressions

here is unwarranted. The change in intensity of the signal of

spin i on saturation of spin j reflects the relative proximity

of i and j. The NOE, fi(j), is, therefore, a function of both

the molecular geometry if a molecule and the molecular dynamics.

Two-Dimensional (2-D) NMR

In the event that the NMR spectra are too complex to be resolved

in normal NMR experiments, 2-D NMR in such cases appears to be an

invaluable tool. The basic principle of the experiment involves

two successive precession periods, t1 and t2. The first

time tl, is called the evolution period an the second time, d

t2, is called the detection period. The spectral resolution

is characterized by two frequency coordinates, one with chemical

shift information and the other with the coupling constant information. A large variety of experiments is possible, depending

on the phase or frequency of Rf irradiation, decoupling level,

and so on, that are applied to the nuclear spins during the

intervals t1 and t2.

These experiments include 2D J-resolved spectroscopy, 2D correlated spectroscopy (COSY) and 2D cross-relaxation spectroscopy. In enkephslin systems both the 2D-J-resolved and COSY

have been utilized by a few research workers (Gidley et al.

1981; Kessler et al. 1983, in press).

CONFORMATIONS OF ENKEPHALINS

Various physical methods, such as NMR, UV, CD, IR, and Laser

Raman spectroscopy have been used to derive the conformations of

enkephalin molecules. NMR, however, remains the most informative, as discussed. In this section, therefore, most of the NMR

work will be discussed along with other spectroscopic methods.

Since it is desirable to describe the backbone and side-chain

conformations separately, this section is divided into two

subsections.

270
Backbone Conformations

From the structural modifications of enkephalin,the stereospecificity and the structure-activity relationships of each

residue have been established. Tyrosine at position 1 is the

most sensitive one; a slight alteration, even in its aromatic

side-chain, drastically reduces the biological activity.

Clycine at position 2 can conveniently be replaced only by any

D-substituted amino acid residue. Positions 3 and 4 are fixed

for the glycine and L-phenylalanine residues, respectively,

while position 5 is quite open to any amino acid residue with

either L or D configurations. Based on these findings, Beddell

et al. (1977) have suggested various H-bonded conformations of

enkephalin whose general definitions and terminologies have

already been described. All such possible H-bonded conformations of enkephalin molecules are described below. Each of

these structures as observed by NMR and either supported or

refuted by other physical techniques has also been discussed.

Phe4 N-H 0=C Tyr1 (4 1 -turn, Bradbury et al (1976)

first predicted this -turn, formed between the Phe4 N-H and

Tyr1 C=0 groups from the application of the empirical rules

for predicting secondary structure from amino acid sequence.

Smith and Griffin (1978) also found this H-bond in crystalline

state of [Leu5] enkephalin grown from an aqueous methanol

solution. In all the previously reported studies in solution,

the occurrence of such H-bond could not be found. Only very

recently Beretta et al. (1984), for the first time, have predicted the formation of this -turn in complexes of [Met5

] and

[Leu5] enkephalin amides with 18-crown-6-ether in chloroform

using high temperature dependence of Phe4 N-H proton

resonance, measured at 500 MHz, as the indication of H-bond formation. As mentioned previously , there exists the controversy

of using parameter; accordingly, this report is one such

example. The lowest values reported in this study are for

Leu5 and Met5 N-H protons which, based on the other criterion

(see the indentification of H-bonds), should give rise to a

-turn involving the [Met5] N-H and Gly2 C=0 groups. This

conformational feature is discussed next.

Met5 or Leu5 N-H 0=C Gly2 (5 2 -turn), This is the

most commonly observed conformational feature of enkephalins

reported by many research groups. Roques et al. (1976) and

Jones et al. (1976) were the first to report the occurrence of 5

2 turn in DMSO-d6 based on the coupling constants and the

low values of Met5 or Leu5 N-H proton. Almost at the

same time , Bleich et al. (1976) reported a normal value

for the Met5 N-H proton and suggested that a folded conformation might be stabilized by a nonbonded interaction. Jones et

al. (1977) later came out with an explanation for this discrepancy showing that the cationic and switterionic forms of

enkephlin gave different NMR parameters. In all NMR studies

involving 1H, 13C and 15N nuclei in DMSO, the same 5 2

279
-turn was observed (Khaled et al. 1977; Garbay-Jaureguiberry et

al. 1977; Stimson et al. 1979; Marion et al. 1981; Zetta and

Cabasai 1982). The controversy, however, remaineder for sometime

on the type of the turn. This -turn contains Gly3

 - Phe4

peptide moiety within the 5 2 H-bonded ring. Since a glycine

residue could be substituted for a D-amino acid residue, thus

forming a D-L peptide moiety, this D-L configurational arrangement should then be designated by a type II' -turn (see section

1) The 13C NMR studies by Stimson et al. (1979), however,

vere found to be beat compatible with a type I -turn vith presumably uncharged terminal groups. Laser Raman studies (Han et

al. 1980; Rapaka et al. 1984) supported a compact -turn structure, but the exact type could not be delineated. The proton

T1 measurement by Niccolai et al. (1980) also revealed the

occurrence of a type I -turn. The unequivocal determination of

the type of -turn by NOE (Khaled and Urry 1976) was impeded by

the complex spin pattern of the Gly3 methylene protons. More

recently, however, mearurement of 3J1H(Gly3)1 5N(Phe 4 )

by Garbay-Jaureguiberry et al. (1982) finally indicated the

occurrence of a type II' -turn which is also energetically more

favorable, as previously proposed by Isogai et al. (1977).

Associated form. The associated form with an antiparallel

cross -structure was, first reported by Khaled et al. (1977)

from combined 1H and 13C NMR data. In this structure, four

intermolecular H-bonds were found to occur bet

and Gly3 C=0 groups and between the Met5

ween the Gly3 NH

or Leu5 NH and

Tyr1 C=O groups. A “head-to-tail” interaction was also proposed and later supported by Anteunis et al. (1977). Occurrence

of such associated form in crystalline state was first noted by

Blundell and Wood (1982) but later completely described by Karle

et al. (1983).

Conformational equilibrium. Enkephalins are small molecules

and, therefore, it is likely that there could be a number of conformations that these molecules may adopt in solution. The

variety of conformers discuessed above confirms the existence of

such molecular flexibility. Using 1H, 13C, CD, and UV spectroecopies, Khaled et al. (1977) demonstrated that different conformations may occur, depending on the nature of solvents and

concentration. They even proposed (Khaled et al. 1979) a conformation resulting from the inversion at central Gly3

 carbon

atom which is similar to a V-conformation (Beddell et al. 1977).

The results of subsequently performed 13C and 15N T1

studies (Tancrede et al. 1978; Marion et al. 1981) supported

such folding since Gly3 residue was found somewhat more restricted than the Gly2

 residue. Using sterochemically deuterated Gly2

 and Gly3 residues Fischman et al, (1978) also

showed the averaging of Gly2 and Gly3 angles in both

DMSO and D2O solvents. In addition, Higashiiima et al. (1979)

suggested that the zwitterionic form of [Met5] enkephalin in

DMSO at low concentration may exist in an equilibrium of

280
extended and folded conformations. Later, Miyazawa and Higashijima (1981) used IR absorption spectroscopy to distinguish

cationic, zwitterionic, and anionic forms of N- and C-terminal

free enkephelins in DMSO and once again supported the existence

of a folded = extended conformational equilibrium. Although a

folded form was stabilized by an electrostatic interaction between

the free N- and C-terminals, the results from an NOE experiment

ruled out the possibility of a 5 2 -turn formation (Higashijima et al. 1979).

Many different conformational equilibria vere also predicted

from the CD results as a function of temperature and pH (Spirtes

et al. 1978; Rollosi et al. 1980). Soos et al. (1980) used the

CD spectra of enkephalin to determine structure-activity relationship. Based on the increased negative ellipticity at 225 nm

as indicative of folded conformation, they obtained a good correlation betveen the degree of folding and the potency in the

GPI but not in the MVD, thus reflecting the requirement of folded

form at the μ-receptor sites. Conformational flexibility of

[Met5] enkephalin amide was also observed in methanol and trifluoroethanol by using CD spectroscopy (Sudha and Balaram 1981).

The Laser Raman spectroscopic results reported by Ran et al.

(1980) also showed different conformers of [Leu5] enkephalin

in D20 in zwitterionic form.

(1982) used Raman spectroscopy to show that the occurrence of

Most recently, Rapaka et al.

-turn in [Leu5] enkephalin in vater is more predominant than

in [Met5] enkephalin. This interpretation was

the observation of a strong band at 1263 cm-1 for [Leu5]

derived from

enkephalin in comparison with a shoulder at 1265 cm-1 for

[Met5] enkephalin.

In an effort to minimize the conformational freedom, several

cyclic analogs of enkephalin have been Synthesised with rertric ed

D+Cys5] and [D-Pen2, D+Cys5] enkephalinamides, cyclired

backbone. Two sush cyclic analogs are: [D-Cys2

,

via S-S bond. Pen stands for penicillamine and is equivalent to

-dimethylcyeteine. The former peptide showed the preference for

the μ-receptor (Schiller et al. 1981), while the latter

displayed significant -receptor specificities (Hosberg et al.

1982). The only difference between these two peptides is the

side-chain of the amino acid at position 2. Recently the 1H

NMR studies by Mosberg and Schiller (1984) revealed a common conformational characteristic for both peptides. Although they did

not indicate any particular conformational feature, it appears

from the parameter of the amide protons that the NH proton

of the L or D-Cys5 is the most shielded one, thus indicating

the formation of a 5 2 -turn. Another interesting group of

cyclic analogs of enkephalin reported by Kessler et al.

(in press) are the Tyr-Cyclo (-N-Xxx-Gly-Phe-Leu-) where Xxx =

L-Orn, L-Lys, or D-Lys. The D-Lys2 analog is 10 times more

active in the GPI assay, i.e., μ-receptor specific, than the

D-Orn2-compound. Use of 2D 1H and 15N-1H COSY, 3J, and

281
parameters have all been used in order to understand the

conformational properties of these peptides. The D-Orn2-

analogs showed a Leu5 N-H O-C Gly3 H-bond which they

designated as -turn (please see the first section and note the

discrepency) and an intramolecularly bifurcated H-bond between

the Orn2 N-H and Orn2 C=0 groups. The D-Lys compounds, on

the other hand, have been found to possess another bifurcated

10-membered intramolecular H-bond between the D-Lys N-H and the

Gly3 C=O groups which they called a -turn (See the definition

o f -turn that appeared above and note again the discrepancy).

The D-Lys2

compound is rather flexible and the μ-receptor

specificity has been attributed to this reduced rigidity. However, contrary to this finding, Mosberg and Schiller (1984)

correlated the increased flexibility of [D-Pen2, L-Cys5]

enkephelinamide with the stronger preference for the -receptor

sites.

Side-Chain Conformational (X)

An extensive effort has been made to elucidate the side-chain

conformations of Tyr1, Phe4, and Met5/Leu5 amino acid

residues in enkephalin molecules. NMR remained to be the sole

spectroscopic method for providing information in this area.

However, various ways of interpreting NMR data have made it difficult to reach any concensus on the ride-chain conformetions.

As mentioned earlier (see the section on side-chain conformations), there are two ways of analyzing 3

J couplings. The

usual way is to obtain the weighted average of three populations

(see figure 6) in case of free rotation along the bond.

If , however, there is any indication of restricted rotation

either from the nonequivalent appearance of H2 protons or

from the relaxation time measurements of the side-chain, the

interpretation becomes more complicated. Some of these complications wil now be discussed in reletion to Tyr1

, Phe4, and

Met5/Leu5 side-chain conformations in enkephalins.

Tyr1 Side-Chain

Since an unsubstituted tyrorine residue at position 1 is absolutely necessary for opioid activity in enkephalinlike molecules) the determination of its side-chain conformations drew

considerable attention. Horn and Rodgers (1976) first suggested

that the N-terminal of enkephalins may be serving the same role

as found in several narcotics, such as levorphenol, metazocine,

and profadol. All such drugs contain en important hydroxyl (OH)

group and a tertiary amino group within the molecule. The distances between these two groups in various opiate agonist and

antagonist molecules varies from 6.89 to 7.08 A. Loev and Burt

(1978) attempted to gain insight into this problem by comparing

enkephalin conformations vith rigid opiate 7-[1-Phenyl-3-bydroxybutyl-3] endoetbano-tetrahydrotbebeine (PET) and found that

there is an excellent overlap of tyramine moiety of PET with the

282
Tyr1

 side-chain when = -90°, although it was not the

lowest energy conformation. In the first crystgllographic study

by Smith and Griffin (1978), tvo values (-43° and -86°)

for Tyr1 were reported. al.

(1983), hovever,

In a second X-ray study,Karle et

reported four sets of values, i.e.,

177° 70°, 53°, and 169°. The = -86 reported by

Smith and Griffin (1978) for [Leu5] enkephalin in its folded

form seems to bear a good resemblance with the postulation of

Loew and Bert (1978).

The solution conformations of Tyr1side-chain remained most

controversial and confusing. Following a Pachler-like (1964)

analysis of the 1H NMR data obtained in

DHSO-d6 (Jones et al. 1976; Garbay-Jaureguiberry et al. 1976,

1977; Robayashi et al. 1980; Zetta and Cabassi 1982) and in

D20/DMSO-d6 (Anteunis et al. 1977) shoved that all three

ride-chain rotamers are equally populated; in another word, the

bond is freely rotating. The problem associated in this

kind of NMR data analysis in the averaging of the two

and 3J values. For example,

one of the Tyr1values is ca. 5 Hz and the other is

ca. 8 Hz; the mean value is 6.5 Hz, which is essentially an indication of free rotatio Whereas, as discussed previously, this

nonequivalency, i.e., 3JAX = 5 Hz and 3JBX= 8 Hz,

should be considered as indicative of some kind of restricted

motion at the bond. Bleich et al. (1976, 1979) reported

the 1H and 13C relaxation parameters, confirming such

restricted rotationlof the Tyr1 ride-chain. They even concluded that the Tyr1

 side-chain alternates between the g+ and

states at a slow rate of 400 sec. Careful analysis of

values of 2.7 and 1.0 Hz obtained by Stimson et

gave a number of values, out of which = -60 or

-90 has been considered most favorable. It appears from all the

NMR studies that the Tyr1

side-chain prefers a more populated

tg conformation, reflecting one of the above mentioned values

ranging from -60° to -90°. Interestingly, however this

side-chain conformation of tyroeine is more compatible with the

tyramine moiety of an opiate.

Phe4 Side-Chain Conformation

Folloving again the method of Pachler (1964), the three rotamer

populations of Phe4

side-chain were found to be equally preferred (Jones et al. 1976; Carbay-Jaureguiberry et al. 1976,

1977; Anteunis et al. 1977) both in DMSO-d6

 and D2O. Kobayashi et al. (1979) and Zetta and Cabassi (1982), however, suggested the predominance of a tg conformation which is also

strongly supported by Stimson et al. (1979). The 1H and 13C

T1 values, on the other hand, indicted a rapid internal motion

relative to the peptide backbone (Bleich et al. 1976; Niccolai et

al, 1980). It appears, therefore, that there still exist disagreements in the case of for L-Phe4

 residue in enkephalin

molecules.

283
Met5

/Leu5 Side-Chain

This is the only residue in all enkephalin molecules that seems

to enjoy a common agreement on its side-chain conformation,

1H NMR data analysis using the Pachler (1964) method gave a

free rotating bond both in DMSO-d6 and D3O (Jones et

al. 1976; Garbay-Jaureguiberry et all

1977; Zetta and Cabasei 1982). The 1H and 13C relaxation

1976; Anteunis et al.

parameters in this case are also in excellent agreement, i.e.,

they shov a rapid internal rotation (Bleich et al. 1976; Combrisson et al. 1976; Niccolsi et al. 1980). Leu5

 side-chain, to

the contrary, adopts a conformation that exists predominantly in

the tg state (Garbay-Jaureguiberry et al. 1977, 1982; Stimson

et al. 1979).

CONCLUDING REMARKS

Numerous applications of theoretical and physical methods have

at least established the fact that the enkephalins are extremely

flexible molecules. By introducing the cyclization constraint

in the backbone, it was possible to define a particular conformation. The question remains, hovever, why one molecule prefers a

μ- and the other a -receptor vhile both the molecules seem

to adopt a similar backbone conformation? Recently, Deek et al.

(1983) have synthesized tvo analogs of [Leu5] enkephalin,

namely [(2-Amino-5-hydroxy-2-indanyl)carbonyl] and [(2-Amino-6-

hydroxy-2-tetralinyl)carbonyl]Gly-Gly-Phe-Leu-OMe, with restricted Tyr1

 side-chain where the latter has been found to be

7 to 8 times more active than the [Leu5] enkephalin at the μreceptor site. Although no conformational studies have been

reported, the Dreiding model of this constrained side-chain of

Tyr1 favors = -90, which is compatible with the tyramine

moiety of an opiate (vide supra). Possibly, this means that the

tyroeine alone can mimic the opioid activity if appropriately

carried to the receptor site(s). To test this hypothesis, L Tyr

was linked with dodecalamine and its activity vas tested using a

mouse seizure model (Khaled and Beaton 1985). Surprisingly,

this molecule was found to possess analgesic activity similar to

other opiates, as previously reported (Snead and Bearden 1982).

These findings, therefore, resffirm the notion that the derivation of the actual bioactive conformation(s) of enkephalins will

have to wait until such time as the receptor sites are identified,

isolated, and characterized.

284
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ACKNOWLEDGMENTS

The author wishes to acknowledge Drs. J.A. Pittman, Jr.. C.E. Butterworth, Jr.,

and C.L. Krumdieck for their support of this work. The work was supported in

part by grants from the NIH SO7-RR05349 and PO1-CA28103.

AUTHOR

M. Abu Khaled, Ph.D.

Associate Professor

Department of Nutrition Sciences

and Neurosciences Program

University of Alabama Medical Center

Birmingham, Alabama 35294

290
Conformational Analysis of Opioid Peptides

and the Use of Conformational Restriction

in the Design of Selective Analogs

Peter W. Schiller, Ph.D.

INTRODUCTION

The discovery of the enkephalins by Hughes et al. (1975) was

almost imediately followed by efforts to determine the

conformation of there pentapeptider and to correlate it with the

three-dimemlonal structurer of the more rigid opiates. The

conformational analysis of small linear peptides like the

enkephelins is not without problems. There is a strong

possibility that rather than assuming a single preferred

conformation in solution, they may exist in a conformational

equilibrium. It is quite likely that their conformation(s) may

be determined to a large extent by the molecular environment.

Furthermore, the bioactive conformation may not preexist in

solutlon, but may only be adopted when the peptide interacts

with the receptor, perhaps in a step-wise binding process

similar to the “zipper”-type model propored by Burgen et al.

(1975). In the part 8 years, the conformation of the

enkephalins and of other opioid peptides has been studied

extensively by means of nearly all theoretical and experimental

methods available to date. A wealth of information about

conformational features of there peptide in the isolated state,

in cryrtalline form, and in solution has been obtained and will

be reviewed in the first part of this chapter. Ideally, the

bioactive conformation should be studied with the peptide bound

to a purified receptor. Since purified opioid receptors are

still unavailable at this time, insight into the receptor-bound

conformation of opioid peptides ten only be obtained indirectly

through analysis of conformationally constrained analogs which

are biologically active. Moreover, in the case of the opioid

peptides, the situation is further complicated through the

existence of multiple opioid receptors etc.) which

differ from one another In their structurel requirements.

Therefore, it will be necessary to determine a distinct

receptor-bound conformation for each type of receptor capable of

interacting with a particular opioid peptide. The pharmacologic

and conformational characterization of conformationally

rertricted opioid peptide analogs will be dercribed in the

second part of this chapter.

291
A review covering the early literature on conformational studies

of enkephalin has been published by Gorin et al. (1978). A more

recent comprehensive review dealing with the conformational

analysis of the enkephalins and with conformation-activity

relationship has been written by Schiller (1984).

Confonnational features of the endorphins have been reviewed by

Yamashiro and Li (1984).

HYPOTHETICAL MODELS OF THE BIOACTIVE CONFORMATION OF ENKEPHALIN

Structural comparisons between opioid peptides and opiates

require first of all the identification of structural elements

which may play analogous roles in the interaction with opioid

receptors. Subsequently, a pharmacophoric conformation of the

peptide has to be postulated such that spatial overlap between

corresponding functional groups in the peptide and in the more

rigid opiates is achieved. The fact that corresponding

modifications of the tyramine moiety in morphine and of the Nterminal tyrosine residue in enkephalin produce similar changes

in the activity profile (cf. Schiller 1984) strongly suggeste

that the tyramine moieties contained in the peptide and in the

alkaloid are functional correlates. This correspondence formed

the basis for nearly all hypothetical models of the bioactive

conformation of enkephalin which have been proposed to date.

Essentially two different proposals have been made concerning

the functional role of the Phe4

residue in enkephalin in

relation to structural elements of morphine-related analgesics.

Bradgury et al. (1976) suggested that the Phe4side-chain of

[Met5]enkephalin and the phenethyl substituent attached to

carbon-19 of the potent morphine derivative PEO1 might bind to

an identical receptor subsite. Consequently, an appropriate

pharmacophoric

accossaodate

conformation of enkephalin would have to

the tyramine segment of Tyr1 and the Phe4aromatic

ring in a relative spatial disposition analogous to that

existing between the corresponding moieties in PEO (phenol-phenyl

separation of about 10 Å). Proposed confonnational models which

fulfil this condition include a 4 1 hydrogen-bonded -bend

conformation (Bradbury et al. 1976), the 5 2 hydrogen-bonded

II'-bend (Clarke et al. 1978), and a bent conformation centered

on Gly2-Gly3 lacking an intramolecular hydrogen bond (Hudson et

al. 1980). In an alternative proposal, the atoms in para and

meta position of the Phe4 residue in enkephalin were considered

as functional correlates of atoms C5 and C6 contained in the Cring of morphine, again under the assumption of functional and

spatial correspondence between the tyramine moieties contained

in the two molecules. A systematic computer search performed

with the active tetrapeptide H-Tyr-D-Ala-Gly-Phe-OH resulted in

a single sterically allowed conformation in which the meta

position of the Phe4 aromatic ring nearly coincided with the

spatial position of the carbon-6 atom in morphine. In the

latter conformation, the intramolecular distance between the two

aromatic rings is much shorter (about 5) than in the -bend

models (see above). Aside from these two major proposals, a

292
number of other suggestions concerning corresponding functional

moieties in enkephalin and opiates have been made and, obviously,

no general consensus has been reached to date.

CONFORMATIONAL ANALYSIS OF OPIATES

Theoretical Energy Calculations

The first conformational energy study of [Met5]enkephalin

performed by Isogai et al. (1977) using the ECEPP program

resulted in a lowest energy conformation characterized by a type

II-bend centered on Gly3-Phe4 and stabilized by a hydrogen bond

between the tyrosyl hydroxyl group and the carbonyl group of

Gly3. This conformer permits substitution of Gly2 by L-Ala but

not by D-Ala and, therefore, it is unlikely to represent the

receptor-bound conformation, since it is well known that D-Ala2

analogs of enkephalln retain high potency and L-Ala analogs are

only weakly active. Performing a different type of

conformational energy calculation, DeCoen et al. (1977) obtained

a number of different olded and extended structures as low

energy conformers of [Met5

]enkephalin and it was suggested that

an equilibrium of such different conformations might also exist

in solution. The results of a study based on the use of the

Monte-Carlo method in conjunction with a clustering analysis

al so indicated that a limited number of different structural

classes might best represent the conformational behavior of

enkephalin in solution (Maigret and Premilat 1982). In a

subsequent study dealing with enkephalin analogs, Humblet and

DeCoen (1977) found a folded structure with a turn centered on

residues 2 and 3 as lowest energy conformer of [D-Ala2

,Met5]enkephalin which was considered as a likely candidate for the

bioactive conformation of enkephalln.

Ala2,Met5]enkephalin

Based on the results of a

semi-empirical conformational analysis of [Met5]enkephalin, [Dal. (1978) proposed a 4 1 hydrogen-bonded I-turn as the

and [D-Ala3,Met5]enkephalin, Balodis et

receptor-bound conformation. Using the ECEPP program, Manavalan

and Momany (1981) performed an analysis of seven enkephalin

analogs and found that several different low energy conformers

common to the group of analogs studied exist. Furthermore, it

became obvious that conformers with significantly different

backbone structures may still have a very similar spatial

disposition of the side-chains in positions 1, 4, and 5 and, in

fact, the existence of only three major categories of side-chain

configurations was indicated by the results of this study. Loew

and Burt (1978) used the ECEPP program as well as a quantum

chemical method to determine the energy content of enkephalin

conformers which display various degreea of spatial overlap with

PEO. A II'-bendlike structure showing spatial overlap of the

Tyr 1 tyramine segment and the Phe 4 side-chain with the

corresponding moieties in PEO was found to be only 3.5 kcal/mol

higher in energy than the lowest energy conformer and thus

represents a plausible candidate for the active conformation.

More recently, Loew et al. (1982) used a similar approach to

study N-terminal tetrapeptide analogs of enkephalin and obtained

293
two structures of the II'-bend type, stabilized by a hydrogen

bond between the N-terminal amino group and the Phe4carbonyl

group, which showed good rpatial overlap with PEO. In a similar

approach, Maigret et al. (1981) determined minimum energy

conformers of H-Tyr-D-Ala Gly-Phe-OH by imposing a somewhat

different rpatial overlap with structural elements of morphine

and obtained a putative pharmacophoric conformation which

differed significantly from that described by Loew et al.

(1982).

Grystal Structure Determinations

In a first X-ray study. the interpretation of the diffraction

pattern obtained with crystals of [Leu5

]enkephalin grown from

aqueous methanol led to the proposal of a -bend structure

stabilized by two antiparallel hydrogen bonds between the amino

group of Tyr and the carbonyl oxygen of Phe and between the

amide proton of Phe and the carbonyl group of Tyr (Smith and

Griffin 1978). Moreover, the data indicated the existence of

two different conformations of the Tyr1 reasidue. Essentially

the same crystal structure was recently obtained with the

analog [4'-Br)Phe4,Leu5]enkephalin as the result of an X-ray

study carried out by Ishida et al. (1984). In the meantime.

examination of improved diffraction patterns obtained with the

original [Leu5]enkephalin crystals led to the detection of

additional weak spots. indicating that the crystals balonged to

space group A2 rather than C2, as had originally been assumed.

The recently completed analysis of there patterns revealed the

existence of four conformationally distinct [Leu5]enkephalin

molecules per unit cell. Whereas the four molecules have very

similar backbone conformations closely resembling the originally

proposed -bend structure, they differ from one another

significantly in the orientation of the ride-chains (G.D. Smith,

personal communication, 1983). Karle et al. (1983) performed an

X-ray analysis with [Leu5]enkephalin crystals grown from DMF in

the presence of water which turned out to belong to space group

P 2 1. Again, the unit cell contained four molecules of

[Leu5]enkephalin. each having a distinct conformation, but

all of them showing similar extended backbones, in sharp

contrast to the folded conformations existing in the crystals

belonging to apace group A2. The major differences between the

four molecules are again observed in the orientation of the

side-chains. Preliminary results of an X-ray study recently

performed with [Met5]enkephalin also provided evidence for an

extended backbone structure (Ishida et al. 1984). The folded

and extended structures observed with the enkephalins in the

crystalline state moat likely represent conformers of comparably

low energy; however. there is no compelling reason to assume

that they represent bioactive conformations (cf. Schiller 1984).

Conformational Studies in Solution

Numerous studies on the solution conformation of the

enkephalins have been performed by spectroscopic techniques

under various solvent conditions. These experiments provided

294
information concerning the aggregation behavior of enkephalin,

the backbone conformation(s) of the peptide, intramolecular

distances in analogs. and side-chain orientations.

Both Khaled et al. (1977) and Higarshijima et al. (1979) observed

a concentration dependence of Shemica shifts in NMR studies

performed with [Met5]- and [Leu5]-enkephalin in DMSO which was

interpreted to indicate a molecular association process. In

contrast to these results, Bleich et al. (1976), Stimson et al.

(1979). and Marion et al. (1981) found no evidence for selfaggregation in DMSO. On the other hand, there is general

agreement that enkephalin does not self-associate in aqueous

solution (Bleich et al. 1976; Levine et al. 1979; Higashijima

et al. 1979; Schiller et al. 1978).

Baaed on the results of NMR and other studies, several propoeals

for a preferred backbone conformation of the enkephalins have

been made. NMR data obtained with [Met5]enkephalin in DMSO were

interpreted to indicate the existence of a 5 2 hydrogenbonded I-turn stabilized by a hydrogen bond between the amide

proton of Met5 and the carbonyl oxygen of Gly2 (Roques et al.

1976; Jones et a1. 1976). This type of conformation can

accommodate the Tyr1 residue and the Phe4 side-chain in a

relative spatial disposition similar to that existing between

the corresponding moieties in PEO and, therefore, it was

suggested that it might represent the bioactive conformation of

[Met5]enkephalin. However, the fact that the analog

[Leu(NMe)5]enkephalin is active indicates that a hydrogen bond

involving the amide proton in position 5 is not an absolute

requirement for the receptor-bound conformation (Schiller and

St.-Hilaire 1980). Performing an NMR study with the cationic

form of [Met5]enkephalin in DMSO, Bleich et al. (1976) found no

evidence for a 5 2 hydrogen bond and suggested that a

preferred conformation might be stabilized by solvent

interactions. Based on the results of yet another NMR study in

DMSO, Khaled et al. (1977) proposed a 5 2 hydrogen-bonded

turn model of the monomeric form of enkephalin containing a

second hydrogen bond between the amide proton of Gly3 and the

carbonyl oxygen of Tyr1 and a third one between the Tyr1

hydroxyl group and the Gly3 carbonyl group. Similar results

were obtained by Zetta and Cabassi (1982). The conformation of

[Leu5]enkephalin in DMSO was also investigated by NMR

spectroscopy. Originally obtained 1H NMR data (GarbayJaureguiberry et al. 1977) led again to the proposal of a 5 2

hydrogen-bonded -bend model which was subsequently revised in

favor of a 2-5 II'-bend structure on the basis of 15N NMR data

obtained by the same group (Garbay-Jaureguiberry et al. 1982)

The results of a 13C NMR study performed by Stimson et al.

(1979)

centered on Gly3-Phe4. Based on NMR data obtained in D20,

were interpreted to be compatible with a type I -bend

several proposale for a preferred conformation of the

enkephaline in aqueous solution were made. Roquee et al. (1976)

and Jones et al. (1976) suggested that the 2-5 I-bend model

they proposed for [Met5]enkephalin in DMSO might also represent

295
the predominant conformer in water. The studies performed by

Bleich et al. (1976). Levine et al. (1979), and Zetta et al.

(1979) also suggested the existence of a preferred conformation

in aqueous solution but did not provide any evidence for

intramolecular hydrogen bonding. Finally, 1H and 13C NMR data

obtained by Higashijima et al. (1979) were interpreted to

indicate that [Met5 lenkephalin in D20 predominantly exists in an

extended conformation devoid of intramolecular hydrogen bonds.

In contrast to the proposals in favor of a single preferred

back bone conformation of enkephalin discussed so far, several

other studies produced results which were more in tune with a

conformational equilibrium involving a number of different

conformers. Fishman et al. (1978) measured 3J(H’-H ) coupling

constants at positions 2 and 3 of the peptide sequence with two

isomers of [Leu5]enkephalin containing stereochemically

deuterated glycine residues. The obtained results indicated

that averaging of the and angles is taking place both in

2H6

-DMSO and in D20 and, therefore, provide strong evidence

against a single preferred backbone conformation in the central

part of the peptide. NMR data obtained by Higashijima et al.

(1979) with the zwitterionic form of [Met5]enkephalin in 2H6-

DMSO were taken to indicate an equilibrium of extended and

folded conformations. Analysis of CD spectra obtained with

[Met5]enkephalin in H20 and trifluoroethanol (Spirtes et al.

1978) and of a Raman spectrum recorded with [Leu5]enkephalin in

D2 0 (Han et al. 1980) also led to the conclusion that the

enkephal in molecules exist as an ensemble of different

conformers.

In a number of Studied, intramolecular distances between sidechains in enkephalin analogs were determined. A fluorescence

energy transfer experiment performed with the active analog

[Trp4,Met5]enkephalin in H20 resulted in an average

intramolecular distance between the phenol ring of Tyr1 and the

indole moiety of Trp4 of 9.3 ± 0.4 Å (Schilier 1977). This

distance would be compatible with the Tyr1-Trp4 separation

observed in the various -bend models and with the distance

between the two aromatic rings in PEO. However, it is quite

possible that the measured value represents an average of

distances present in several different conformers participating

in a conformational equilibrium and, in fact, the results of a

Monte-Carlo type calculation were found to be in agreement with

the latter interpretation (Demonté et al. 1981). In further

energy tranefer studies, no gross difference in the average

Tyr1-Trp4 intramolecular distance was observed within a series

of nine [Trp4lenkephalin analogs which differed widely from one

another in their μ-receptor affinity (Schiller et al. 1978;

Schiller and St.-Hilaire 1980). Interestingly, a much larger

Tyr1-Trp4 intramolecular distance (> 15 Å) was determined with

the potent dynorphin analog [Trp4]dynorphinI(1-13) (Schiller

1983a). This marked difference in the Tyr1-Trp4 separation

between the [Trp4]analogs of enkephalin and dynorphin may be

related to the different receptor selectivity profiles of the

two peptides. A close proximity between the methyl groups of the

296
Ala2

 and Met5 side-chain in [D-Ala2,Met5]enkephalin was

indicated by the results of an NOE1 experiment (Niccolai et al.

1980).

Numerous studies on side-chain conformations in enkephalin have

been performed (for a detailed review, see Schiller 1984).

Side-chain rotamer populations were mainly determined by Pachler

analysis of 1H NMR data. There is some agreement that the tgrotamer may represent the favored conformation of the Tyr1

 sidechain under a variety of conditions (Kobayaehi et al. 1979;

Zetta and Cabassl 1982; Stimson et al. 1979). Interestingly.

the tgrotamer shows closer conformational resemblance with the

tyramine segment in morphine than the other two side-chain

rotamera (tg+ and gg+). Furthermore, the results of most spinlattice relaxation time measurements indicated a fixed

conformation of the Tyr1 residue relative to the peptide

backbone (Bleich et al. 1976; Combrisson et al. 1976; GarbayJaureguiberry et al. 1977). Evidence to indicate that the Tyr1

hydroxyl group of enkephalin in H20 or DMSO/H20 mixtures does

not participate in an intramolecular hydrogen bond with a

backbone carbonyl group was obtained from a fluorescence study

(Schiller 1977). a UV titration etudy (Spirtes et al. 1978).

laser Raman experiments (Han et al. 1980). and a4

 1H NMR study

(Zetta and Cabassi 1982). In the case of the Phe4 side-chain,

all three rotamers were found to be significantly populated

according to the results of reveral studies, with the exception

of that performed by Stimson et al. (1979) which indicated that

in 2H6-DMSO the Phe4 tg- rotamer is exclusively populated.

Furthermore, it has been well established that the Phe4 sidechain in the natural enkephalins undergoes rapid internal

rotation relative to the peptide backbone. In 2H6-DMSO, all three

rotamers of the Met5 side-chain in [Met5]enkephalin were found

to be populated to some extent, where as in [Leu5]enkephalin the

Leu 5 side-chain appears to exiat exclusively in the tg -

conformation (Garbay-Jaureguiberry et al. 1977;

1979). With [Met5]enkephalin, rapid internal motion of the Met5

Stimson et al

side-chain was again indicated by the reaults of spin-lattice

relaxation time measurements.

CONFORMATIONALLY RESTRICTED OPIOID PEPTIDE ANALOGS

Insight into the receptor-bound conformation can be obtained

from structural analysis of conformationally restricted analogs

that retain biological activity. However, unambiguous

conclusions cannot be drawn in the case of conformationally

constrained analogs which are inactive, since the loss of

activity could be due either to an incompatible conformation or

to a lack of the necessary molecular flexibility required in a

step-wise binding process. A further interesting aspect of

introducing conformational constraints into biologically active

peptides is the possibility of obtaining selective analogs in a

situation of receptor heterogeneity.

297
Conformational constraints have been built into the peptide

backbone of enkephal in by substitution of and

methylamino acids in various portions (for a review, see Gorin

et al. 1978). Local conformational restrictions of the peptide

backbone through insertion of 5- or 6-membered cyclic structuree

resulted in mostly inactive analogs (for a review, see Schiller

1984). Several enkephalin analoge containing conformationally

restricted side-chains have been reported. Restriction of the

rotational freedom of the Tyr 1 side-chain in enkephalin

substitution of 2-amino-6-hydroxy-tetralincarboxylic acid in

position 1 resulted in an active compound, whereas an analog

with 2-amino-5-hydroxy-2-indancarboxylic acid in that same

position was found to be inactive (Deeks et al. 1983). The

relatively flexible tetralin residue can assume conformations

corresponding to the tg- or tg+ rotamer of tyrosine but not to

the gg+ rotamer, whereas the latter rotomer is the only one

accessible to the rigid indan structure. This observation is of

interest in view of the fact thst the tg - rotamer shows

resemblance with the conformation of the tyramine moiety in

morphine. Conformational restriction of the Phe4 side-chain was

achieved through substitution of l-aminoindan in H-Tyr-D-AlaGly-Phe-OH (Gorin et al. ,1980) and of dehydrophenylalanine with

Z-configuration in [D-Ala2,Met5]enkephalimide (Chipkin et al.

1979). In both cases, the obtained analog showed good activity.

More drastic restrictions of the overall conformation of opioid

peptides has been achieved through synthesis of cyclic analogs.

Cyclic enkephalin analogs obtained through direct head-to-tail

cyclitation between the terminal amino and carboxyl group

(Kessler and Holzemann 1981) or through linkage of the termini

via a glycyl residue or a 2-aminoethyl group as bridging element

(Hudson et al. 1980) were found to be either insoluble or

inactive.

In an alternative approech. cyclic opioid peptide analogs were

synthesised through ring closure between a side-chain group and

the C-terminal carboxyl group or through covalent linkage of two

side-chains. A common characteristic of these analogs is an

exocyclic Tyr1 residue. A first family of cyclic enkephalin

analogs was obtained through substitution of a D- -diamino

acid residue in position 2 of enkephalin and cyclication of the

-amino group to the C-terminal carboxyl group (DiMaio and

Schiller 1980; DiMaio et al. 1982a) (figure 1, compounds I-IV).

Analogs were tested in bioassays based on inhibition of

electrically evoked contractions of the GPI and of the MVD1,

which are representstive for μ- and -opioid receptor

interactions, respectively (table 1). In comparison to

[Leu5]enkephalin, the prototype of this series, H-Tyr-c[-N -DA2bu-Gly-Phe-Leu-] (II) was shows to be 17 times more potent in

the GPI assay and 7 times less potent in the MVD assay.

Therefore. this cyclic analog shows preference for μ-receptors

over -receptors, as indicated by its IC50(MVD)/IC50(GPI) ratio

of 5.77. No such preference is shown by the corresponding

open-chain analog, H-Tyr-D-Abu-Gly-Phe-Leu-NH2 (IIa), which is

distinguished from its cyclic counterpart II merely by the

298
FIGURE 1

Structural Formulas of Cyclic Opioid Peptide Analogs

299
TABLE 1

Potencies of Cyclic Enkephalin Analogs in the GPI and MVD-Assaya

Compound

GPI MVD

ICSO [nM] IC50 [nM]

MVD/GPI

IC50-ratio

I

II

IIa

IIb

IIc

III

IV

IVa

IVb

IVc

IVd

IVe

IVf

H-Tyr-c[-N -D-A2pr-Gly-Phe-Leu-] 23.4 ± 4.2

H-Tyr-c[-N -D-A2bu-Gly-Pht-Leu-] 14.1 ± 2.9

H-Tyr-D-Abu-Gly-Phe-Leu-NH2 28.7 ± 1.3

H-D-Tyr-c[-N -D-A2bu-Gly-Phe-Leu-] 3'230 ± 760

H-Tyr-c[-N -L-A2bu-Gly-Phe-Leu-] 1'450 ± 350

H-Tyr-c[-N -D-Orn-Gly-Pht-Leu-] 48.0 ± 4.3

H-Tyr-c[-N -D-Lye-Gly-Phe-Leu-] 4.80 ± 1.79

H-Tyr-D-Nle-Gly-Phe-Leu-NH2 24.6 ± 1.6

H-Tyr-c[-N -D-Lye-Gly-Phe-D-Leu-] 2.39 ± 0.57

H-Tyr-c[-N -D-Lye-Gly-Phe-Met-] 1.20 ± 0.26

H-Tyr-cc-N -D-Lye-Gly-Phe(pN02)-Leu-] 0.504 ± 0.138

H-Tyr-c[-N -D-Lye-Gly-Leu-Phe-] 3.36 ± 0.04

H-Phe-c[-N -D-Lye-Gly-Phe-Leu-] 105 ± 19

[Leu5]enkephalin 246 ± 39

73.1 ± 14.5

81.4 ± 5.8

45.6 ± 9.1

330 ± 102

475 ± 99

141 ± 28

25.2 ± 7.9

17.5 ± 5.7

34.3 ± 5.5

13.4 ± 6.3

41.6 ± 9.4

4'070 ± 200

11.4 ± 1.1

3.12

5.77

1.59

0.102

9.90

29.4

1.02

7.32

28.6

26.6

12.4

38.8

0.0463

a

 Mean of three determinations ± SEM.
opening of a single carbon-nitrogen bond. This result indicates

that the μ-receptor selectivity of cyclic analog II is

exclusively due to the conformational constraint introduced

through ring closure, and, furthermore, permits the unambiguous

conclusion that μ- and -opioid receptors differ in their

conformational requirements (Schiller and DiMaio 1982). Within

the series of cyclic analogs I-IV, the number of methylene

groups in the 2-position side-chain increases from 1 to 4 and,

therefore, subtle variation in the degree of conformational

restriction is achieved. All members of this series were found

to be μ-receptor selective and the IC50(MVD)/IC50(GPI) ratio

increased gradually with lengthening of the side-chain in

position 2, reaching a value of 29.4 with H-Tyr-c[-N -D-Lys-GlyPhe-Leu-] (IV) (table 1). The latter analog was 51 times more

potent than [Leu5]enkephalin in the GPI assay and 12 times less

potent in the MVD assay. The corresponding open-chain analog

(IVa) again showed no selectivity. Structure-activity studies

with linear enkephalin analogs had shown that L-configuration in

position 1 and D-configuration in position 2 are required for

activity, whereas in position 5 either L- or D-configuration is

tolerated. The results of potency determinations performed with

cyclic analogs IIb, IIc, and IVb (table 1) indicate that the

cyclic analogs have the same configurational requirements in

positions 1, 2, and 5 as linear enkephalins. Furthermore,

introduction of a nitro substituent in para position of the

Phe4 aromatic ring in cyclic analog IV (compound IVd) results in

approximately a tenfold potency increase in both essays, as is

the case with noncyclic enkephalins (Schiller et al. 1983).

Based on these results, it can be assumed that the mode of binding to opioid receptors is the same for cyclic and linear enkephalin analogs. Using cyclic analog IV as parent compound,

further structure-activity studies were undertaken. In most

cases, corresponding modifications in the cyclic analog and in

linear enkephalins had the same effect on potency (cf. analogs

IVc and IVd). However, two interesting exceptions were noticed.

Transposition of the Phe and Leu residues in the cyclic peptide

(compound IVe) resulted in a small potency increase in both

assays, whereas that same modification in the corresponding

open-chain analog lowered potency quite drastically (DiMaio of

al. 1982b). Even more interestingly, omission of the Tyr1

hydroxyl group in the cyclic analog results in a compound (IVf)

still more than twice as potent as [Leu5]enkephalin, whereas

linear enkephalin analogs containing Phe in position 1 show very

low potency. These two observations may reflect a difference in

the binding process between cyclic and linear analogs. It is

possible that the semi-rigid cyclic analogs may bind to the

receptor according to Fischer's "lock-and-key" model, whereas a

zipper-type interaction may take place in the case of the more

flexible linear analogs. Certain moieties may no longer be

absolutely required for efficient binding in a lock-and-keytype interaction. Whereas in a zipper-type interaction certain

modifications (e.g., Phe 4-Leu5 transposition) may increase

the step-wise binding process. The results obtained with the

cyclic enkephalin analogs in binding assays based on

301
displecement of [

3 H]naloxone as a μ-receptor-selective

radioligand and of [

3H][D-Ala2

,D-Leu5]enkephalin as a

-receptor-selective radiolebel from rat brain membrane

preperations were in general agreement with the activity

profiles determined in the GPI- and MVD-asssys. However,

unexpected results were obteined in a comparison of the cyclic

analogs with their linear correlates in the GPI bioassey and the

[

3H]naloxone binding assay, which are both representative for μreceptor interactions. For example. cyclic analog IV is five

times more potent than its corresponding open-chain analog IVa

in the GPI assay. whereas en almosf txectly reversed potency

relationship is observed in the [

3H]naloxone binding essay

(DiMaio et el. 1982a). This discrepancy can be interpreted to

indicate that the conformational restriction introduced in IV

through ring closure slightly reduces μ-receptor affinity, but

significantly increases the ability of the bound peptide to

activate the receptor ("efficacy" enhancement). High resistance

of cyclic analog II to enzymatic degradation was demonstrated in

vitro (DiMeio and Schiller 1980), and intracerebroventricular

administrstion of compound IV (20 nanomoles) produced a longlasting analgesic and catatonialike effect in rats (Schiller et

al. 1981a). The rigid 13- and 14-membered ring structures

contained in cyclic analogs I and II exclude formation of a 4-

1 or 5 2 hydrogen-bonded -bend and are also incompatible

with the crystal structures described by Smith (personal

communication. 1983) and by Karle et al. (1983). Kessler et al.

in press) investigated the conformations of cyclic

analogs III and IV by performing a lH NMR study in [

2H6

]DMSO.

In the case of compound III, the obtained data indicated a rigid

conformation stabilized by two transannular hydrogen bonds

(Leu5-NH OC-Gly3 and Orn2-NH OC-Orn2). On the other hand,

cyclic peptide IV showed a somewhat more flexible conformation

than III,and evidence for only a siqnle intramolecular hydrogen

bond between the group of D-Lys2 and the carbonyl group of

Gly3 was obtained. Since analogs III and IV are both μreceptor selective, these interesting results suggest that at

least two different backbone conformations of enkephalin are

tolerated et the μ-receptor, provided that conformational

changes of these semi-rigid analogs do not occur upon binding.

It should be noted, however, that compound IV is about 10 times

more potent then analog III in the GPI assey (DiMaio et al.

1982a).

A family of side-chain to side-chain cyclized opioid peptide

analogs was obtained through substitution of an -diamino acid

and a glutamic or aspartic acid residue in appropriate positions

followed by amide bond formation between the side-chein amino

and carboxyl groups (e.g., compound X, figure 1) (Schiller and

Nguyen 1984). Potencies of such annalogs are presented in table

2. Compound V WAS found to be 250 times more potent then

[Leu5]enkephalin in the GPI assay, but turned out to be nonselective since it showed high potency on the MVD as well.

Inversion of the configuration in position 2 (compound Va) again

produced a drastic drop in potency. Transposition of the Glu

302
TABLE 2

GPI and MVD Assay of Side-Chain to Side-Chain Cyclised Analogs

Compound

GPI MVD

IC50 [nM] IC50 [nM]

MVD/GPI

IC50-ratio

V 1.13 ± 0.14 0.648 ± 0.132 0.573

V A 1'580 ± 420 10'600 ± 300 6.71

VI 42.7 ± 6.5 699 ± 146 16.4

VII 24.1 ± 2.8 256 ± 36 10.6

VIII 2.93 ± 0.30 5.21 ± 0.88 1.78

IX 7.99 ± 0.78 101 ± 18 12.6

X 43.8 ± 4.4 4'690 ± 1'020 107

XI 631 ± 123 10'400 ± 4'300 16.5

XII 8'750 ± 2'520 > 40'000 > 4

XIII 55'000 20'600 ± 4'700 0.375

246 ± 39 11.4 ± 1.1 0.0463

a Mean of three determinations ± SEM.
and Lye residue in V resulted in an analog (VI) which was 6

times more potent then [Leu5]enkephalin on the GPI but 60 times

less potent on the MVD and, therefore, is μ-receptor selective.

The difference in potency and receptor selectivity between

compounds V and VI may be due to the different position and

direction of the side-chain connecting amide bond which is

likely to produce dissimilar transannular hydrogen bonding and

thereby different ring conformations. Shortening of the sidechain in position 2 of analog VI results in a twofold to

threefold potency increase with little change in selectivity

(compound VII). The des-Gly3 analogs of V and VI (analogs VIII

and IX) show activity profiles similar to those of their perent

peptides. Extraordinary μ-receptor selectivity is displayed by

H-Tyr-D- -NH2 (X), which is 6 times more potent then

[Leu5]enkephalin on the GPI and 400 times less potent on the

MVD. Its IC50(MVD)/IC50(GPI) ratio of 107 is comparable to that

of the highly μ-receptor selective ligand H-Tyr-D-Ala-Gly-MePheNH(CH2

)2OH (DAGO) (Kosterlitz and Peterson 1981). Transposition

of the Orn and Asp residues results in a less potent and less

selective analog (XI). Compound XII showed week potency, in

contrast to the significant activity which had been reported for

the related linear analog H-Tyr-D-Met-Gly-NH-CH(CH3)-CH2-

CH(CH3)2 (Roques et el. 1979). Analog XIII was also foun to be d

weakly active, which is surprising in view of the relatively

high potency observed with 1 linear [D-Ala2]enkephalin analogs

containing a side-chain with L-configuration in the 3-position.

A second type of side-chain to side-chain cyclized enkephalin

analog was prepared by substitution of a D-Cys residue in

position 2 and of a D- or L-Cys residue in position 5 followed

by oxidative disulfide bond formation (figure 1; anologs XIV,

XV, XXII, and XXIII) (Schiller et al. 1981b). The cyclic

enkephalinamide analogs XIV and XV were found to be highly

potent in both the GPI and MVD assays and, therefore, showed no

significant receptor selectivity (table 3). The receptor

binding mode of these analogs again seems to be similar to that

of linear enkephalins, since they showed the same

configurational requirements et the individual residues (cf.

analogs XV, XVI, XVII, and XIX). This assumption was again

supported by the potency increase observed upon substitution of

p-nitrophenylalenine in position 4 of XV which resulted in a

compound (XVIII) having five times the potency of dynorphin A-

(1-13) in the GPI assay and an IC50 value of about 20 picomolar

in the MVD assay. Substitution of Phe in position 1 and of Trp

in position 4 of compound XV produced Active analogs (XX and

XXI) showing considerable μ-receptor selectivity. In contrast

to the nonselective cystine-bridged enkephalinamide analogs (XIV

and XV), the corresponding analogs with a free C-terminal

carboxyl group (XXII and XXIII) were shown to be moderately

receptor selective. The same difference in receptor selectivity

is in general observed between linear enkephalins and

enkephalinamides. Thus, cyclic enkephalins XXII and XXIII

display about the same -receptor selectivity as

[Leu5]enkephalin but are about two orders of magnitude more

304
TABLE 3

Potencies of Cystine-Containing Enkephalin Analogs in the GPI and MVD Assaya

Compound

GPI MVD

IC50 [nM] IC50 [nMl

MVD/GPI

IC50-ratio

XIV

XV

XVI

XVII

XVIII

XIX

XX

XXI

XXII

XXIII

1.51 ± 0.03

0.780 ± 0.010

210 ± 58

135 ± 13

0.0351 ± 0.0100

483 ± 112

48.6 ± 6.05

0.842 ± 0.185

3.06 ± 0.52

1.48 ± 0.18

246 ± 39

0.760 ± 0.086 0.503

0.298 ± 0.037 0.382

950 ± 160 4.52

891 ± 139 6.60

0.0187 ± 0.0023 0.533

582 ± 113 1.20

456 ± 159 9.38

14.1 ± 1.1 16.7

0.190 ± 0.035 0.0621

0.122 ± 0.016 0.0824

11.4 ± 1.1 0.0463

a Mean of three determinations ± SEM.
potent then the netural peptide. Substitution of a halfpenicillamine (Pen) residue for the helf-cystine residue in

position 2 and/or 5 of the cystine-bridged enkephelin analogs

resulted in compounds with high preference for -receptors over

μ-receptors (Momberg al. 1983). A conformational comparispn

of corresponding Pen2- and Cys2-containing cyclic analogs by 1H

et

NMR spectroscopy in D20 indiceted a somewhet enhanced rigidity

of the ring structure contained in the [Pen2

]-analogs as

compared to the [Cys2]-analogs, which may be the cause of the

observed selectivity difference (Mosberg and Schiller 1984).

The results of fluorescence studies performed with analogs XV

and XXI in H20 indiceted a close interaction between the Tyr1

aromatic ring and the disulfide bridge, and an average

intramolecular distance between the Tyr1 and Trp4 aromatic rings

in XXI of 9.7 ± 0.2 Å, which is close to the corresponding

distance of 9.5 ± 0.3 Å determined with the linear analog H-TyrD-Ala-Gly-Trp-Met-OH (Schiller 1983b).

CONCLUSIONS

The results from theoretical energy calculations, crystal

structure determinations, and spectroscopic studies in solution

indicate that the enkephelins art flexible molecules which can

exist in several different conformations of comperatively low

energy. It appears that the molecular environment is likely to

have an important influence on the conformation(s) of these

peptides and there is good evidence to indicate that at least in

aqueous solution they exist in a conformational equilibrun. The

molecular flexibility of the enkephalins complicates the task of

determining their bioactive conformation(s) and presumably is

the cause of their lack of specificity in the interaction with

different opioid receptor types. In view of this situation. the

principle of conformational restriction has emerged as a

promising new tool in opioid peptide analog design. In

particular, synthesis of cyclic enkephelin analogs during the

pest few years has resulted in potent, stable, and highly

selective μ- and -receptor agonists. More extensive studies

on the conformation of these cyclic analogs can be expected to

lead to a better definition of the conformational requirements

of the different opioid receptor types in the near future.

FOOTNOTES

1 Abbreviations : CD, circular dichroism; ECEPP, empirical

conformational energy program for peptides; GPI, guinea pig

ileum; MVD, mouse vas deferens; NMR, nuclear magnetic resonance;

NOE, nucleer Overhauser enhancement; PEO, 7- -(1-(R)-hydroxy-1-

methyl-3-phenylpropyl)-6,14- endo-ethenotetrehydrooripavine.

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enkephalin: Comparison of fluorescence parameters at different

pH. Biochem Biophys Res Commun 79:493-498, 1977.

Schiller. P.W. Fluorscence on the solution conformation

of dynorphin in comparison to enkephalin. Int J Pept Protein

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309
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side-chain side-chain cyclized opioid peptides.

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1978.

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H., ed. Advances in Endogenous and Exogenous Opioids. Tokyo:

Kodansha, 1981a. pp. 383-386 

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1981b.

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Comparison of μ-, and -receptor binding sites through

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310
ACKNOWLEDGMENTS

The work of the author described in this chapter was supported

by operating grants of the Medical Research Council of Canada

(MT-5655) and the Quebec Heart Foundation.

AUTHOR

Peter W. Schiller. Ph.D.

Director, Laboratory of Chemical Biology and Peptide Research

Clinical Research Institute of Montreal

110 Pine Avenue West

Montreal, Quebec, Canada H2W 1R7

311
Stereochemically Constrained Enkephalin

Analogs Containing -Aminoisobutyric Acid

and I-Amino-Cyclopentane-I-Carboxylic Acid

Raghuvansh Kishore and Padmanabhan Balaram

INTRODUCTION

Small biologically active acyclic peptides like enkephalins are conformationally very flexible. As a consequence, It is difficult to establish unambiguously the

"biologically active" conformation which is recognized

by the i n vivo receptor. The relationship between stable

solution conformations and structures bound to receptors

is often not clear. The synthesis of conformationally

restricted analogs and an evaluation of their receptor

affinities or biological activity afford a possible

approach to the deli neation of bioactive conformations.

Imposition of backbone and side-chain comformational constraints must, in principle, be accomplished by covalent

modifications, which do not directly influenced interactions with receptors, but modulate peptide-receptor binding indirectly, by limiting the range of accessible

tide conformetions (Spatola 1963; Marshall et al. 1976:

This chapter briefly reviews an approach to restriction

of peptide backbone conformational flexibility, by introduction of achiral dialkyl residues (figure 1) in

enkephalins la place of Gly residues at positions 2 and 3.

Conformational Restrictions by -Allsylation

Two degrees of torsional freedom about the N-C

and bonds determine the backbone conformations

of individual residues in peptides. The peptide bond is

generally limited to a trans geometry = 180°). As a

consequence, peptide conformations are conveniently mapped

on the two-dimensional Remachandran plot (Ramachandran and Sasisekharan 1968). Figure 2 illustrates

the theoretically computed maps,for L-ALa and D-Ala

residues. The map for the Aib residue is shown in

figure 3. It is evident that substitution at limits

the energetically accessible regions of space. In

the case of symmetrical -dialkyl residues, exemplified by Aib, only a very limited region of conformational

space is, indeed, sterically and energetically favored

(Marshall and Bosshard 1972: Prasad and Balaram 1984).

312
FIGURE 1

Structures of -dialkylated residues having acyclic

and cyclic sidechains.
FIGURE 2

Pamachandran (Steric) map for N-acetyl-L-Ala-N'-methylamide and N-acetyl-D-Ala-N'-methylamide. The continous

and broken lines enclose regions allowed by normal and

extreme contact limits, respectively.
FIGURE 3

a ) Potential energy map for N-acetyl-Aib-N'-methylamide.

The contours are drawn with respect to the innrmost

contour enclosing the minimum. The ideal 3 1 0 and

helical confomations are marked. These conformations are compatible with Aib residues in Type I or III

-turns. Crystal structure observations of Aib

residues in nonhelical regions are marked. Cryrtallographic observations for Aib residues in peptides in the

right (b) and left (c) handed helical regions of the

map. Tips of arrow indicate ideal 3 1 0 and -helices.

N-terminal residues in protected peptides Nonterminal

residue x C-terminal Aib present as ester or acid.

Extensive conformational studies of Aib-containing peptides in the solid state and in solution have been reported. The conformational angles determined by X-ray diffraction for Aib residues in a large number of peptides

are summarized in figure 3. With very few exceptions,

Aib residues adopt conformations in the region of

±60° ±20° and = ±30 ±20°. These values of are

compatible with the presence of Aib residues at positions

i+1 or i+2 of Type I(I') or III (III') -turns or at

position i+2 or a Type II (II') -turn (Venkatachalam

1968; Smith and Press 1980). -Turn conformations in

peptides are generally, though not always, stabailized

by intramolecular 4 1 hydrogen bonding between the C=0

group of residue i and the NH group of residue i+3

(figure 4). The conformational parameters for the idealized Type I-III -turns are listed in figure 4. The

introduction of Aib resides into Acylic peptides thus

enhances the probabilty of -turns formation involing

Aib-X or X-Aib sequences. This tendency is amply demonstrated in crystal structures of several Aib-containg

315
FIGURE 4

Schematic representation of a -turn conformation stabilized by a 4 1 (C10) hydrogen bond. Conformational

angles for idealized Type I, II and III -turns are

indicated. The enantiomeric -turns (Types I, II, and

III') are generated by reversing the signs of al

angles.

model peptides (Nagarj and Balaram 1981; Prasad and

ance (NMR) studies of proteceted peptides in organic

Parlaram 1983. 1984) and also in nuclear magnetic resonsolvents (Nagaraj et al. 1979; Iqbal and Balaram 1981).

The presence of successive Aib residues in a sequence,

i.e., an Aib-Aib-X segment, or alternated Aib residues,

316
i.e., an Aib-X-Aib-X segment, generally leads to formation of consecutive B-turn structures of the Type

III-III or III'-III' astegories. Such conformations

are incipient right- or left-handed 310-helical structures

(Iqbal et al. 1981; Vijayakumar and Balaram 1983a, l983b).

The 1-aminocycloalkanecerboxylic acids (figure 1) are

structuolly analogous to Aib. Theoretical studies of

the cyclopentane (Acc 5) and cyclohexane (Acc b) containing

N-acetyl-aminoacid-N-methyl amides suggest that these

residues also strongly prefer right- or left-handed

3 1 0 /x-helical conformations (Prased, unpublished

results). The extended regions of space are, in

fact, even less favorable than in the case of Aib. These

conclusions are borne out by two recent crystal structrures of model peptides containg Acc 6

 residues. In

Aib-Acc 6-NHMe 6, a type III (III')B-turn conformation was observed with = +68.4°, = ±15° for the Acc6

residue. Molecules of both-handedness were present in

the achiral crystals. In Boc -Aib-Acc6-OMe, where an

intramolecular 4 1 hydrogen bond is not possible, a

semiextended conformation is observed

 = ±143°), but the Acc6

residue had

at Aib = +62.2°

= ±48°, = ±42.6°.

strongly supporting the notion that the cyoloalkyl sidechain imposes even greater restrictions on conformational

freedom (Bardi et al., in press). Intristingly, recent

studies by Italian and English groups suggest that the use

of -dialkyl residues like -diethylglycine (Deg)

or -di-n-propylglycine (Dpg) can result in a stabilibation of extended peptide conformations (Barone et al., in

press [1984a, 1984b]) may thus be possilble to modulate the

folding tendency of acyclic peptides by introduction of

Aib or 1-aminocycloalkanecarboxylic acids on the one hand

or the higher -dialkyl amino acids on the other.

The intrduction of each -dialkyl residues into

biologically active peptides could then provide a means

of selectively stabillzing certain backbone conformations.

In principle, the use of schiral amino acids could be most

appropriate when Gly residuee present in the natural

sequence are replaced in the analog structure.

Two complicating factors must be considered when analyzing structure-activity data generated by using such

analogs.

1) Enhanced hydrophobicity of the analog peptides may

influence binding to apolar receptor surfaces.

2) The alkyl substituents may affect peptide-receptor

interact ions by virtue of the new stereochemical

contacts involved.

The replacement of Gly residues by Aib imposes a considerable restriction on backbone flexibillty. The replacement

317
of hydrogen atoms by methyl groups at may be viewed

as a minimum steric perturbation. Steric effects at

receptor binding sites may be more significant when

bulky cycloalkyl groups like Acc 5 or Acc 6 are used

If the analog peptides retain full biological activity

or exhibit enhanced activity, then it is likely that the

conformations possible in the stereochemioally constrained analogs are indeed recognized by the receptor.

Furthermore, any direct effect of the substituent (noted

above) is minimal or promotes receptor-peptide association. If the analog peptide is inactive, then the conformations favored in the analog are not recognized by the

receptor. Alternatively, the substituent has a direct

inhibitory effect an receptor interactions by impeding

close contact of the peptide with the binding site.

STUDIES ON ENKEPHALIN ANALOGS

In the case of the enkephalins (Tyr-Gly-Gly-Phe-Met/

Leu), the presence of the Gly-Gly segment results in

considerable conformational flexibility (Fishchman et al.

1978). Solution spectral studies have, therefore, generally failed to provide evidence for a dominant conformtion in the native sequences, although evidence for folded

(Bleich et al. 1977; Khaled et al. 1977) structures have

Roques et al. 1976; Jones et al. 1976) and extended

been presented. The backbone conformational flexibility

of the enkephalins is also dramatically illustrated by

X-ray diffraction studies, where one crystal form has

yielded a Gly2Gly5 Type I' -turn structure (Smith and

Griffin 1978; Blundell et al. 1979), while another is composed of enkephalin molecules in an extended B-sheet conformation (Karle et al. 1933: Carmeman et al. 1983).

Conformations Determined by NMR

The Gly residues at positions 2 and 3 in Met- and Leuenkephalin amides have been separately or similtaneously

replaaed by Aib or Acc 5 residues and the conformation of

the resulting enkephalin analogs have been inferred from

NMR studies in (CD3)2SO (Sudha and Balaram 1983; Kiahore

and Balaram 1984). The presence of intramolecularly

hydrogen-bonded conformations is established by delineating

solvent-exposed and -shielded NH groups, using temperature

coefficiant of NH of chemical shifts (d8/dt) in (CD 3) 2SO as

a diagnostic criterion. The /dT values <0.003) ppm/k may

be generally considered to represent strongly solventshielded NH groups, while values <0.005 ppm/K are characteristic of solvent-exposed NH groups. Intermediate /dT

values (0.003 to 0.005 ppm/K) are harder to interpret

definitively, but are often indicative of moderately

solvent-shielded NH groups (Ravi et al. 1933; Iqbal and

Balaram 1981).

The chemical shifts and temperature coefficients

/dT) of nine enkephalin analogs incorporating Aib or

Acc 5 residues are summarized in table 1. The corresponding

318
TABLE 1

NMR Parameters of NH Groupsa in Enkephalinamide Analogs in Dimethylsulfoxide

Chemical Shifts (8 ppm) /dT x 103

 ppm/K

Peptides X

2

 Y 3 Phe Met 5/Leu 5 Y

3

 Phe Met 5/Leu 5

Tyr-Aib-Gly-Phe-Met NH2

Tyr-Gly-Aib-Phe-Met NH2

Tyr-Aib-Aib-Phe-Met NH2

Tyr-Alb-Gly-Phe-Leu NH2

Tyr-Gly-Aib-Phe-Leu NH2

Tyr-Aib-Aib-Phe-Leu NH2

Tyr-Aco5-Gly-Phe-Leu NH2

Tyr-Gly-Aco5-Phe-Leu NH2

Tyr-Aco5-Aco5-Phe-Leu NH2

Tyr-Gly-Gly-Phe-Met NHd

2

Tyr-Gly-Gly-Phe-Leu NH2

7.15 8.21 7.93 7.98 5.5 2.8 4.6

8.50 8.43 7.86 7.63 6.5 4.6 1.8

b 8.28 7.78 7.81 6.5 1.8 1.8

b 8.24 7.94 7.76 4.5 1.9 3.9

b 8.42 7.78 7.62 5.9 3.5 1.8

b 8.13 7.78 c7.71 c

6.3 2.00 2.3

b 8.12 7.80 7.85 4.5 1.8 4.9

b 8.44 7.78 7.57 5.9 3.7 1.9

b 8.09 7.67 c

7.61 c

6.2 1.9 2.1

8.24 8.13 8.15 8.05 4.6 4.9 5.6

8.20 8.05 8.10 7.90 a 3.7 4.6

a ) The NH of residue 2 generally broadene on heating in (CD3

) 2SO; /dT was therefore

not measured.

b ) NH resonance obscoured due to overlap with aromatic protons or due to broadening.

c) Not assigned due to overlap.

d ) Values taken front Higashijima et al. 1979.
Parameters for Met 5

/Leu5 enkephalinamide are also listed.

the following features of the NMR data are noteworthy.

1) The analogs with Aib/Acc 5 residues at position 2 show

low /dT values for the Phe NH, oharectieristiac of a

solvent -shielded proton. All other NH groups appear

to be solvent exposed.

2) In the position 3 analogs, the Met NH has a very low

/dT value, indicative of its sting shielding from

the solvent. Other NH groups appear to be solvent

exposed; although in the Aib and Acc 5 analogs of Leuenkephalinamide, the Phe NH has a moderate /dT value,

suggestive of a moderate degree of shielding from

solvent.

3) In the analogs vhere both Gly residues are replaced by

Aib/Acc 5 residues, both Phe and Met NH resonsnces have

very low /dT values, characteristic of solvent-shielded NH groups.

4) All NH group in the Gly-Gly analogs have relatively

high /dT values.

In small acylic peptides, involvement of NH groups in

intramolecular hydrogen bonding generally results in

their shielding from the solvent. Direct steric shielding

without hydrogen bonding is unlikely. The NMR results

therefore lead to the following inferences.

1) The Phe NH is intramolecularly hydrogen bonded in

position 2 analogs, incorpoating Aib or Acc 5

residues.

2 The Met NH is hydrogen bonded in the position 3

analogs.

3) Both Phe and Met NH groups are hydrogen bonded in

analogs substituted simultaneously with Aib or Acc 5

residues at positions 2 and 3.

The strong tendency of Aib residues to promote -turn

formation, together with the NMR results, suggests that

intramolecularly hydrogen-bonded conformations are stabilized in the Aib analogs. The conformations compatible

with HMR data and the known stereochemical preferences of

Aib residues are illustrated in figure 5. It thus appears

that conformations involving a -turn centered at positions

2 and 3 (residues i+1 and i+2, respectively) are highly

populated in (CD 3) 2SO for the Aib(2) analogs, while a

-turn at positions 3 and 4 is sugested for the Aib(3)

analogs. In the Aib(2)-Alb(3) analogs, a consecutive

Type III-III -turn structure is compatible with the

spectral data. Such conformations are also likely to be

favored in the Acc 5 analogs, in view of the similarities

in the spectral characteristics of the Aib and Acc 5

analogs. Furthermore, model building and theoretical

energy calculations suggest that Aib and AC C 5 residues

have similar stereochemical preferences.

320
FIGURE 5

Proposed backbone conformations of Aib analoga of Leuenkephalinamide a) Tyr-Aib-Gly-Phe-Leu+NH 2

 b) Tyr-GlyAib-Phe-Leu-NH 2

, c) Tyr-Aib-Phe-Leu-NH2.

The crystal structures of the protected fragments

Boc-Aib-Phe-Leu-OMe (Prasad, unpublished) and Boc-Aib-Aib-PheMet-NH 2

 (Prasad et al. 1983) provide further support for

theee conclusions. In the former, a Type I Aib-Phe -turn

is observed (figur 6), whlle the latter adopts a consecutive Type III-III (inciplent 3 1 0 helical) conformation

in the solid state (figure 6).

Circular Dichroism (CD) Studies

CD studies of Met(5)-enkephalinamide, Boc-Tyr-Gly-GlyPhe-Leu-NH2

, and the fragment Boc-Gly-Gly-Phe-Met-NH2

demonstrate a pronounced solvent dependence of spectra

in solvents like 2,2,2-tri-fluorethanol (TFE) and methanol, indicative of structural transitions (figure 7).

Replacement of the Gly residues by Aib results in an abolition of the solvent-dependent sign reversal of the CD

band at 215 to 220 nm (Sudha and Balaram 1981). These

observations have been interpreted by the authors as

indicaitve of the role of Aib residues in stabilizing

specific folded conformations in these sequences. In the

above study, protected tetrapeptide fragments were used

to examine the effect of Aib residues on solvent-dependent

conformational transitions. This approach has subsequently

been extended to a study of the enkephalin analogs themselves. The CD spectra of the three Aib analogs of Leu(5)-

enkephalinamide in MeOH, H20, and dioxane are shown in

figures 8 to 10, respectively. The spectra of the corresponding Acc 5

 analogs in MeOH and dioxane are shown in

figure 8 and 9, respectively.

The following CD spectra classes may be recognized:

A) Positive CD band at ~220nm,

B) Negative CD band at ~,215 to 217 nm,

C) Two negative CD bands at 230 nm and 220 nm.

321
FIGURE 6

Perspective view of the molecular conformations or two

peptide fragments related to Aib analogs of enkephalins.

Left: Boc-Aib-Aib-Phe-Met-NH 2 (Prasad et al. 1983).

Note 3 1 0 helical structures formed by repetitive 4 1

hydrogen bonds. Right: Boc-Aib-Phe-OMe (Presad

unpublished). A Type I -turn with Aib = -58.3°,

Aib = -29.8°, = -84.7°, Phe = 1.3°

Class A CD spectra are exhibited by the position 2

analogs (Aib or Acc 5) in MeOH and H 2O. These spectra

are characteristic of a -turn conformation, tentatively

assignable to either Type I' or Type III' categories,

consistent with the class C' spectra of Woody (Woody 1974;

Smith and Peace 1980; Crisms et al. 1984). Such spectra

have been assigned to Type II B-turns in model peptides

with Pro-X sequences (Criama et al. 1984; Rao et al.

1983). However, the occurrence of Aib or Acc 5 residues

at position i+1 of a Type II -turn = -60°, = 120°)

is sterically unfavorable. Is is, therefore, more

appropriate to aasign the class A spectra to Type I' or

Gly(2)-Aib/Acc5(3) or Aib/Acc5 (2)-Aib/Acc5(3) analogs in

III' -turns. The class B ectra obtained for the

MeOH can be assigned to the consecutive -turn (Type

III-III' structure). For the Gly-Aib (Acc 5) analogs in

H 2O both the negative band at ~217nm and the positive

band at ~224 nm are observed, suggesting an equlibrium

mixture of conformations yielding both class A and class

B spectra. interestingly, in the Aib-Aib-(Acc 5-Acc 5)

322
analogs, there is a rather dramatic change in CD patterns;

a class B spectrum is observed in MeOH, whereas a class A

spectrum is seen in H 2O (figures 8 and 10). The NMR

evidence suggests that a consecutive -turn conformation

(figure 5c) is favored in polar solvents like dimethyl

sulfoxide. This conformation is expected to be adopted

in MeOH also and could account for the class B spectrum

In H 2 O, one of the -turns--probably Aib(3)-Phe(4)--

may be destabilized, resulting in a single Type I' (III')

Aib(2)-Aib(3) -turn. This would be similar to the

structures proposed for the Aib (2) (Acc 5(2)) analogs and

may account for the observed class A CD pattern. In the

relatively apolar solvent, dioxane, all six analogs

exhibit disinctly different CD spectra as compared to

MeOH or H 2 0. An unusual feature of these spectra is the

230 nm. A definative conformational asignment in dioxpresence of a rather long wavelength negative band at

ane is not feasible at pressnt. Aggrgation effects may

also play a role in this solvent. The CD studies sugest

that even in the Aib or Acc 5 analogs of Leu-enkephalin

there are solvent dependent structural changes. It is

likely that well-defined conformational states like Type

I' (III') or consecutive -turn structures are populated

for them peptides, in solution. However, equlllbria

between them conformations must be considered.

The biological activity of all these analogs has not

yet been determined. However, an i n vivo behavioral

assay is mice following intracerebral injection of

peptide has been carried out on the three Aib analogs

of Met-enkephalinamide. This study established the

following order of activity: Aib-Aib Aib-Gly Gly-Gly

Gly-Aib (Nagaraj et al. 1970; Sudha and Balaram 1983).

while resistance to enzymatic degradation (Pert et al.

1976) likely to enhanoe the activity of the Aib analogs, relative to the native sequence in an i n vivo

assay, the observed activity does suggest that folded

enkephalin conformations are in fact, recognized by

appropriate receptors. This conclusion is based on the

assumption that extended conformations are unlikely to

be populated in these analogs seven at the receptor site,

since favorable binding interactions are unlikely to

offset unfavorable conformational energies.

This chapter has described an approach used in the

authors' laboratory to restrict backbone conformational

flexibility of the enkephalins. The use of alkylation

at as a means of limiting peptide backbone freedom

(Spatola 1983) is a promising approach to the develoment of conformationally constrained analgos of biologically active peptides. The use of 1-aminocyoloalkane

carboxlyic acids of varying ring size may permit altereations in the steric bulk and hydrophobicity of the residue

being used to limit conformational flexibility. The potential of this approach is indicated by the observation that

replacements of Leu(2) in the chemotactic peptide (FormylMet-Leu-Phe) by Aib, Acc5

, and Acc6 leads to biologically

323
FIGURE 7

Left: CD spectra of Boc-Gly-Phe-Met-NH 2 (1.96) in

a) TFE b) MeOH c) TFE MeOH, 1:1 (v/v). Right: CD spectra

of Tyr-Gly-Gly-Phe-Met-NH2 (1.74) in a) Me OH, b) TFE

Boc-Tyr-Gly-Phe-Lue-NH2 (1.53) in c) MeOH and d) TFE.

(From Sudha and Balaram 1981, Copyright 1981, Elsevier/

North-Holland Biomedical Press).
FIGURE 8

CD spectra of Tyr-X-Y-Phe-Leu-NH2 in MeOH. Left: -Aib-Gly-

-Gly-Aib- , -Aib-Aib- O. Right: -Ace5-Gly-

-Gly-Acc 5- , Acc5-Acc5- O.
FIGURE 9

CD spectra of Tyr-X-Y-Phe-Leu-NH2 in dioxane. Left:

-Aib-Gly- -Gly-Aib- -Aib-Aib- O. Right:

-Aoc 5

-Gly- -Gly-Acc 5- -Acc5-Acc5- O.
FIGURE 10

CD spectra of Tyr-X-YPhe-Leu-NH 2 in H 2O. -Aib-Gly-

-Gly-Aib- -Aib-Aib- 0.

327
active analogs. The Acc 6

 analog is a hundred fold more

active than the parent peptide (Iqbal et al. 1984;

Sukumer et al. 1985).

The studies described in this chapter outline a means

of imparting specific folded conformations to analogs of

enkephalins. Aditional restrictions improed on sidechain flexibility should complement these studies and lead

to a better definition of the structra requirements

for recognition by biological receptors.

REFERENCES

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). Crystal

structure of two model peptides. Int J Pept Protein

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Barone, V.; Bavoso, A.; Benedetti, E.; DiBlasio, B.;

Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora,

G.M.; Toniolo, C.; Lingham, T. and Hardy, P.M. Folded

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-dialkylated -amino acids. A conformational

energy computation and X-ray diffraction study. J A m

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Baraone; V.; Ba V oso, A.; Grimaldi, P.; Lelj, F.; Pavone,

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360 MHz proton NMR spectra of active and inactive

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derived parameters, conformational implications.

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Blundell, T.L.; Hearn, L.; Tickle, I.J.; Palmer, R.A.;

Morgan, B.A.; Smith, G.D.; and Griffin, J.F. Crystal

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Camerman, A.; Mastropaolo, D.; Karle, J.; Karle, J.; and

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Crisma, M.; Fasman, G.D.; Balaram, H.; and Balaram, P.

Peptide models for -turns. A circular dichroism study.

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Fischman, A.J.; Riemen, M.W.; and Cowburn, D. Averaging

of and in 5-Leucyl-enkephalin. NMR study of two

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AUTHORS

Raghuvansh Kishore

Pedmanabhan Balaram

Molecular Biophysics Unit

Indian Institute of Science

Bangalore 560 012, India

331
Enkephalin Conformation in Solution: A

Perspective From Vibrational Spectroscopic

Studies

V. Renugopalakrishnan, Ph.D.; T. W. Collette, Ph.D.;

L. A. Carreira, Ph.D.; and R. S. Rapaka, Ph.D.

The conformations of enkephalins have been assiduously explored

since their discovery by Hughes et al. (1975). Numerous

studies, often resulting in conflicting conclusions, have been

reported in the literature (see Schfller 1984 for a review). A

broad spectrum of physical methods has been utilized in

derivin the solid and solution phase conformations of

enkephalins. The central goal of the numerous studies has been

to determine the bfoactfve conformation of enkephalins in their

free state and, from the results obtained, derive the plausible

conformation at the receptor site whose topography can at best

be inferred. Many spectroscopic studies have been performed in

organic solvents (Schiller 1984), which have provided much

useful information but are of little direct relevance to the

conditions likely to prevail at the receptor site. Of the

numerous studies reported in the literature, only theoretical

conformational analysis and molecular graphics have enabled

researchers to postulate the receptor site conformations of

enkephalins (Gorin et al. 1978).

In this study, FT-IR, Raman, and 2D-NMR spectroscopic methods

were chosen to investigate the enkephalin conformations in

solution, as these methods are suitable for obtaining fresh

insight in the somewhat tedious and frustrating search for the

elusive bfoactfve conformations of enkephalins. 2D-NMR studies

and, in particular, 2D-NOE studies are still in progress; therefore, a perspective of enkephalin conformations from FT-IR and

Raman spectroscopic studies (Renugopalakrishnan et al. 1985) is

presented in this review. Previously, a Raman study of

Leu5-enkephalin was reported in the literature (Han et al.

1980). The present study differs in many respects from the

previous Raman study of Leu5-enkephalin. In the present

study, the conformations of both Leu5- and Met5-enkephalins

are investigated in aqueous and dimethyl sulfoxfde (DMSO)

solutions. The latter solvent was used in order to compare the

results of the present study with earlier studies (Schiller

1984). The FT-IR study of enkephalins in aqueous solutions

332
discussed here is the first of its kind and has utilized

state-of-the-art advancements In this field. These studies

were performed typically with millimolar concentrations of

enkephalins in solution and, at this concentration level, the

molecular aggregation of enkephalins can be expected to be

minimal. The final answers to the bioactive conformations of

enkephalins must await the isolation and purification of

receptors in whose presence the conformations of enkephalins

have to be determined using physical methods.

VIBRATIONAL SPECTROSCOPY AS A WINDOW ON SECONDARY STRUCTURE ON

POLYPEPTIDES AND PROTEINS

Recent developments in vibrational spectroscopy have placed

this technique in the forefront of physical methods for the

elucidation of secondary structures of polypeptides and

proteins. IR and Raman spectroscopic methods are based on the

principles of vibrational spectroscopy, but differ on the

fundamental mechanisms by which they arise: IR absorption

occurs only when the dipole moment changes during the

molecular vibration, whereas the Raman effect occurs through

the induced dipole moment, namely the polarizability term when

the incident light Interacts with the molecular system.

Therefore, the two methods provide complementary information on

a molecular system. For a general review of the application of

FT-IR and Raman spectroscopy to biological molecules, see

Parker (1983) and Tu (1982). Recently, the two methods and

their variants have been applied to the elucidation of

secondary structures of polypeptides and proteins

(Renugopalakrishnan et al. 1984; Renugopalakrishnan and

Bhatnagar 1984; Diem et al. 1984).

MID-IR AND RAMAN SPECTRA AS A BLUEPRINT OF THE PDLYPEPTIDE

BACKBONE CONFORMATION

Most of the IR and Raman spectra of polypeptides and proteins

reported in the literature contain much useful information in

the mid-IR and Raman frequency region of 1000 to 2000 cm-1

,

which can be broadly divided into the following three regions:

I. 1600 to 1700 cm-1

II.

III.

1500 to 1600 cm-1

1200 to 1300 cm-1

These regions are usually designated as amide I, II, and III

regions since they contain the three important vibrational

modes of the peptide moiety, as shown in figure 1. The amide I

vibrational mode consists of carbonyl bond stretching with a

minor contribution from NH in-plane bending; the amide II mode

consists of C'-N and N-C bond stretchings with a minor

contribution from NH in-plane bending; whereas the amide III

mode consists of C'-C and C'-N bond stretchings with a

minor contribution from NH in-plane bending. The amide II mode

333
is usually either Raman inactive or weak in intensity. The

three amide modes are generally sensitive to the secondary

structures of the polypetide backbone, but it has been

difficult to derive reliable secondary structural information

from the observed frequencies. Part of the difficulty in

deriving useful secondary structural information has been due

to the absence of accurate IR and Raman data on polypeptides

with well-characterized secondary structures. Much of the IR

data reported in the literature before the introduction of the

FT technique contained broad bands which lacked sufficient

resolution to derive precise frequencies for secondary

structural interpretation.

The introduction of lasers in the late sixties as the source of

excitation in Raman spectroscopy (Lord and Yu 1970) has made

it relatively easier to record Raman spectra of polypeptides

and proteins, which was quite difficult with the older

generation Raman spectrometers equipped with mercury arc

lamps. Therefore, in recent years, a wealth of data on

polypeptides and proteins has been reported in the literature

setting the stage for a qualitative and quantitative

interpretation of their IR and Raman spectra.

VIBRATIONAL FREQUENCIES OF SOME PROTEIN STRUCTURES RELEVANT TO

ENKEPHALIN CONFORMATIONS: AMIDE I AND III FREQUENCIES OF

-SHEET AND -TURN STRUCTURES

Synthetic polypeptfdes and proteins containing -sheets,

determined by other physical methods, manifest characteristic

amide I frequencies ranging from 1669 cm-l to almost 1690

cm-1. For example, phosvitin (a phosphoglycoprotein from hen

egg yolk containing more than 50% O-phosphoserines) assumes

largely a -sheet

band at 1690 cm-1 and a Raman active band at 1672 cm-1

structure at pH 2 and manifests an R active

(Renugopalakrishnan et al. 1985b). The amide I bands

typical of -sheets cluster at the higher frequency range,

while those characteristic of -helical structures cluster at

the lower frequency range of the amide I region. The amide III

frequencies characteristic of -sheet and -helical structures

occur in the lower and higher frequency ranges of the amide III

region respectively.

Amide I and III frequencies characteristic of -turns have been

calculated from normal vibration analysis of a tetrapeptfde,

CH3CO-(Ala-Ala-Ala-Ala)-NH-CH3 by Bandekar and Krimm

(1979). For effective transition dipole coupling perturbation,

eff between 0.35 and 0.45D, characteristic frequencies

around 1640 to 1645 cm-1 and 1685 to 1690 cm-1 for type I

-turns and around 1660 to 1665 car and 1685 to 1690 cm-1

for type II -turns were observed from these calculations.

Antiparallel -sheet structures manifest amide I frequencies

near 1690 cm-1 (Miyazawa and Blout 1961) which partly overlap

with the type I -turn frequencies (Bandekar and Krimm 1979).

Therefore, caution must be exercised in the interpretatlon of

334
Amide I

Amide II

Amide III

FIGURE 1

Amide I, II, and III vibrational modes of a peptide

moiety. From Tu 1982. Copyright 1982, John Wiley and

Sons.

335
the amide I frequencies. The amide II modes of the type I

-turn structures occur near 1550 to 1555 cm-l and 1567

cm-1, whereas type II e-turn structures exhibit amide II

bands around 1545, 1555, and 1560 cm-1 respectively,

corresponding to eff=0.27D (Bandekar and Krimm 1979). From

the calculations, the amide III modes were found to occur in

the 1290 to 1330 cm-1 region.

In general, a number of IR and Raman studies on -turn

structures have reaffirmed the theoretical calculations of

Bandekar and Krimm for amide I and II frequencies. However,

their observations on amide III frequencies, in general, are

not corroborated by experimental observations (Tu 1982). Since

the amide I frequencies of -turn and -sheet structures

sometimes conflict with each other by occurring in the same

region of IR and Raman spectra of polypeptides and proteins,

caution must be exercised in drawing secondary structural

interpretations.

From this wealth of information, the frequency range for

different types of secondary structures commonly present in

polypeptides and proteins is presented in figure 2, which was

adapted from Tu (1982).

As may be observed from figure 2, the region designated as

"random coil" is relatively smaller in area than the -helical,

-sheet, and -turn structures. In fact, this region contains

the regions where -turns, -turns, and collagenlike

polyproline II structures manifest characteristic vibrations

and, therefore, its definition remains quite arbitrary.

Interferences in the Amide I Region

The amide I region is usually obscured by bands arising from

the solvents comnonly used in vibrational spectroscopic

studies. Althouah diaital subtraction of solvent bands is

possible, in reality it becomes difficult to completely

subtract the solvent backaround. Therefore. sufficient caution

must be exercised in assigning secondary structural

significance to amide I bands. Fortunately, the introduction

of the Fourier self-deconvolution technique (Kauppinen et al.

1981) has made possible the resolution of complex overlapping

bands and has initiated a new era in the application of Raman

and FT-IR spectroscopic methods to the determination of

secondary structures. The interference of solvent in the amide

II and III regions is less severe than in the amide I region,

but the former vibrational modes are mixed modes involving the

stretching of multiple bonds, making them less dependable as

conformational probes.

336
FIGURE 2

The range of observed amide I and III frequencies for

-helical, -turn, -sheet, and "random coil"

structures. From Tu 1982. Copyright 1982, John Wiley

and Sons.

337
RELATIONSHIP BETWEEN AMIDE I, II, AND III FREQUENCIES WITH THE

SECONDARY STRUCTURE OF POLYPEPTIDE BACKBONE

The secondary structural dependence of amide I, II. and III

vibrational modes are based on the concept that the vibrational

frequencies of the peptide moiety are dampened to an extent

dependent on their spatial orientation in the polypeptide

backbone; peptide moieties with similar spatial constraints

give rise to somewhat similar vibrations. For example, the

amide I vibrational mode, figure 1, can be expected to depend

on the orientation of the it peptide unit with respect to

the preceding polypeptide backbone, figure 3, described by

Ramachandran angle. The -helical and -sheet structures

differ in, their value, and if the amide I frequency for these

two structures depend on , then one would expect to see marked

differences in their respective frequencies, as observed from

figure 2. It is at present difficult to distinguish between

and structures, as well as between e-sheet and -turn

structures, and -sheet and collagenlike polyproline II

structures based on amide I frequencies. It was recently

suggested that the low-frequency vibrational modes where

torsional interactions dominate are much more useful in

providing a clearer distinction between various polypeptide

structures (Renugopalakrishnan et al., in press [1985a]). It

was demonstrated by Lord (1977) that the amide III frequencies

of -helical and -sheet structures differ markedly and, hence,

a correlation between amide III frequencies and, was

proposed. From numerous observations, this proposal has been

further substantiated, although the amide III frequency is

sensitive to the nature of side chain which makes Its use as a

conformational probe quite difficult (Hsu et al. 1976). Raman

and IR spectroscopic studies can provide a qualitative estimate

o f , which at best can complement a time averaged value for

from NOE studies in solution phase (Leach et al. 1977; Urry et

al. 1978).

With the introduction of FT technique in IR spectroscopy, FT-IR

method is slowly gaining increasing popularity in protein

structural studies. The previous disadvantage of utilizing

aqueous solutions in IR studies has been largely overcome. The

technical problem of fluorescence overshadowing the weak Raman

lines has also been resolved by the introduction of the

coherent anti-Stokes Raman technique (CARS), the use of pulsed

lasers with electronic time gating to capture Raman photons,

and the prolonged exposure of the sample to the laser beam

which presumably bleaches the fluorescent moiety (Hudson

1977). The two methods, i.e., FT-IR and Raman spectroscopy,

are well suited for structural investigations of the solid and

solution phase of polypeptides and proteins and, therefore,

offer a rapid method for the determination of their secondary

structures.

338
FIGURE 3

Ramachandran angles about and

bonds for two peptide units linked at the atom.

From Tu 1982. Copyright 1982, John Wiley and Sons.

339
A PERSPECTIVE OF ENKEPHALIN CONFORMATION FROM FT-IR AND RAMAN

SPECTROSCOPIC STUDIES IN SOLUTION PHASE

Conformation of Leu5-Enkephalin in Aqueous Solution

The FT-IR deconvoluted spectrum of Leu5-enkephalin in aqueous

solution is presented in figure 4. The amide I and II regions

only are shown in figure 4 where the amide I bands occur at

1663 cm-1 with two shoulders at 1 58 cm-1 and 1675 cm-1

in addition to a peak at 1632 cm-1. We have assigned the

1663 cm-1 band to a type II -turn, whereas it is somewhat

difficult to assign conformational significance to the

shoulders at 1658 cm-1. The shoulder at 1675 cm-1 and the

peak at 1632 cm-1 can be assigned to the -sheet structure.

The amide I frequencies for the -sheet structures also exhibit

satellite bands in the 1630 to 1640 cm-1 region (Renugopalakrishnan et a1. 1985b), which is not shown in

figure 2. The IR active band at 1663 cm-1 for Leu5

-

enkephalin is quite close to the 1665 cm-1 band observed in

the Raman spectrum of Leu5-enkephalin presented in figure 5,

and its frequencies are tabulated in table 1. Raman spectrum

of Leu5-enkephalin in aqueous solution shows a multiplet

structure in the amide I and III regions with the amide II mode

being weak in this case. The most prominent amide I band

occurs at 1665 cm-1, whereas the amid

1263 cm-1 with a shoulder at 1255 cm-1 and a second peak at

III band occurs at

1275 cm-1. The amide III band at 1263 cm-1 is

characteristic of -turns, probably a type II -turn (Bandekar

and Krimm 1979). The shoulder at 1275 cm-1 is in the range

observed for -helical structures.

-turn structure for Leu5-enkephalin

Further support for the

in aqueous solution is

provided by the intense amide II band at 1547 cm-1 in the

FT-IR spectrum of Leu5-enkephalin (figure 4). The FT-IR

spectrum of Leu5-enkephalin (figure 4) shows somewhat less

secondary structure than the Raman spectrum of

the same (figure 5). but the frequencies of the observed bands

in both are reasonably close to one another. The Raman bands

at 1648 cm-1 and 1275 cm-1, which are typical of -helical

structures, have no counterpart in the FT-IR spectrum.

Conformation of Leu5-Enkephalin in Dimethyl Sulfoxide

The Raman spectrum of Leu5-enkephalin in DMSO solution

(figure 6) shows very few bands in the amide I and III regions,

unlike the Raman spectrum of the same in aqueous solution,

which was shown in figure 5. The amide I band occurs at 1616

cm-1 and 1686 cm-1. While the origin of the band at 1616

cm-1 is not certain, the 1686 cm-1 is typical of the

-sheet structure (Renu opalakrishnan et al. 1985b).

The conformation of Leu5enkephalin in DMSO is

quite different from that observed in aqueous solution.

340
FIGURE 4

The FT-IR deconvoluted spectrum of Leu5-enkephalin

solution at a concentration of 0.37 mg/ml in water. The

spectrum was recorded at room temperature. From

Renugopalakrishnan et al. 1985. Copyright 1985,

Academic Press.

341
FIGURE 5

Raman spectrum of Leu5-enkenphalin solution at a

concentration of 0.95 mg/50 μl in water (top) and of

Met5-enkephalin solution at a concentration of 1.05

mg/50 μl in water (bottom). The spectra were recorded

at room temperature using the 488 nm excitation line of

an argon ion laser at a laser power of 600 mW.

From Renugopalakrishnan et al. 1985. Copyright 1985,

Academic Press.

342
FIGURE 6

Raman spectrum of Leu5-enkephalin solution at a

concentration of 1.2 mg/50) μl of DMSO (top) and of

Met5-enkephalin solution at a concentration of

1.55 mg/50 μl of DMSO (bottom). The spectra were

recorded at room temperature using the 488 nm excitation

line of an argon ion laser at a laser power of 600 mW.

The amide I and III regions only are shown. From

Renugopalakrishnan et al. 1985. Copyright 1985,

Academic Press.

343
The amide III region shows one intense peak at 1210 cm-1 with

a shoulder (on DMSO peak) at 1274 cm-1. The amide III peaks

are hard to interpret in terms of the secondary structures and

provide little conformational information.

Conformation of Met5-Enkephalin in Aqueous Solution

The FT-IR spectrum of Met5-enkephalin in aqueous solution is

presented in figure 7 and the frequencies of amide I, II, and

III mode

1636 cm-1 and 1678 cm-1. The 1678 cm-1 is typical of

are listed in table 1. The amide I mode occurs at

-sheet structure (Renugopalakrishnan et al. l985b).

In contrast, the amide III exhibits a triplet structure with

bands occurring at 1246, 1273, and 1296 cm-1 respectively.

The 1246 cm-1 band arises from the -sheet structure

the 1273 cm-1 band is typical of -helical structure.

whereas

Bandekar and Krimm (1979) have predicted the 1290 to 1330

cm-1 region as characteristic of type I -turns, but none

have been reported to occur at this high frequency.

Further work is necessary to assign the 1296 cm-1 band.

From the Raman studies, a somewhat different perspective of its

conformation in aqueous solution emerges (figure 5) The amide

I mode exhibits a multiplicity of bands: 1613 (sh) 1623, 1633

(sh), 1649, and 1665 (sh) cm-1. An unequivocal assignment of

the bands is certainly quite difficult. The 1633 (sh) cm-1

could well be a satellite band characteristic of -sheet

structure, but if this is true, its higher frequency component

in the 1669 to 1690 cm-1 region is not observed in figure 5.

The 1665 cm-1 band which is only a shoulder, unlike a peak at

1665 cm-1 in the case of Leu5-enkephalin, is in the range

typical of type II -turn structures. The 1649 cm-1 band is

against typical of

(sh) cm-1 and the 1623 cm-1 peak are hard to interpret.

-helical structures. The shoulder at 1613

The amide III region shows three bands at 1252, 1265 (sh), and

1276 cm-1; whereas the 1252 (sh) cm-1 is too high for

μ-sheet structure, the other two can be assigned to the -turn

and -helical structures.

1265 cm-1 is only a shoulder in contrast to Leu5-enkephalin.

But, again, the -turn frequency of

Conformation of Met5-Enkephalin in Dimethyl Sulfoxide

As mentioned earlier, the Raman spectrum of Met5-enkephalin

in DMSO solution is shown in figure 6. The spectrum manifests

a doublet in the amide I region with bands occurring at 1616

cm-1 and 1686 cm-1. The band at 1686 cm-1 is typical of

-sheet structure. The conformational flexibility of Met5-

enkephalin is greatly reduced in DMSO solution, which is

similar to the observations on Leu5

-enkephalin in DMSO.

344
FIGURE 7

The FT-IR deconvoluted spectrum of Met5-enkephalin

solution at a concentration of 0.17 mg/ml in water. The

spectrum was recorded at room temperature. From

Renugopalakrishnan et al. 1985. Copyright 1985,

Academic Press.

345
TABLE 1

The Amide I, II. and III Frequencies of Leu5- and Met5-enkephalins in Solution Phase

From FT-IR and Raman Spectroscopic Studies

FT-IR RAMAN ASSIGNMENT

H2O H20 DMSO

Leu5

 EnkEphalin

1217 cm-1

1255(sh)

1263

1275

1210 cm-1

1274(sh on DMSO

peak)

1516 cm-1

1547

1632

1658(sh)

1675(sh)

1623

1648

1655

AMIDE III

AMIDE II

1616

1686

AMIDE I

Met5-Enkephalin

1246

1373

1296

1217

1252

1265(sh)

1276

1210

1274(sh on DMSO

peak AMIDE III

1512 AHIDE II

1551

1578

1636

1678

1613(sh)

1623

1633(sh)

1649

1665(sh)

1616

1686 AMIDE I
CONCLUSIONS

Numerous studies on enkephalins using theoretical conformational analysis, X-ray diffraction studies on crystalline

Leu5- and Met5-enkephalins, and spectroscopic studies of

enkephalins in solution have led to widely divergent perspectives on the bioactive conformations of enkephalins. Unfortunately, none of the studies could be directed toward the

bioactive conformations of enkephalins at the receptor site 

except for the theoretical conformational analysis of Gorin et

al. (1978) and the 500 MHz 1H NMR study of Beretta et al.

(1984).

From the earlier studies, enkephalins have been postulated to

be flexible molecules (Schiller 1984). This conclusion should

not be surprising if one considers the absence of long-range

stabilizing interactions in the pentapeptide. Hence, its

conformation is likely to be determined by short-range interactions. It is plausible that the peptide exists in a

conformational equilibrium modulated by the nature of the

solvents, ionic strengths, etc. Among the structures commonly

encountered for pentapeptide are and -turns; and, If

molecular aggregation occurs, larger structures can be

envisaged.

The FT-IR and Raman spectroscopic studies of enkephalins are

Indicative of a conformational equilibrium prevailing in

aqueous solution, whereas in DMSO, the conformational

flexibility is greatly reduced. In DMSO, enkephalins form

either -turn or -sheet structures or a combination of both;

while, in aqueous solutions, both structures definitively

coexist. While Leu5-enkephalin in aqueous solution shows a

-turn structure, Met5-enkephalin shows a less marked

tendency to form turn structures. The occurrence of amide I

bands at 1648 cm-1 in Leu5-enkephalin and at 1649 cm-1 in

Met5 enkephalin (typical of -helical structures) can

probably be explained by the occurrence of values at the

N- or C-terminal ends characteristic of -helical structures.

The peptides are too short to cause nucleation of -helical

structures (Chou and Fasman 1974). An equally valid

explanation for the above bands is the likely presence of a

type I -turn structure which, according to the calculations of

Bandekar and Krimm (1979). have low amide I frequencies in the

region 1640 to 1645 cm-1. At the present time, the results

from this FT-IR study are more conclusive since it was possible

to deconvolute the bands in the amide I and II regions. Han et

al. (1980) concluded that Leu5-enkephalin forms a -turn

structure in DMSO-d6 with an amide I band occurring at 1691

cm-1. This is slightly different from the frequency observed

in our study, namely 1686 cm-l. While an ambiguity probably

exists in the interpretation of -turn and

with frequencies of 1686 cm-1 and 1691 cm-1, we would like

-sheet structures

to interpret this band as arising from s-sheet structure based

on numerous observations (Renugopalakrishnan et al. 1985b).

347
Unfortunately. the earlier Raman study of Han et al. (1980)

indicated a broad band centered around 1640 cm-1 for

Leu5-enkephalin in aqueous solution from which little

conformational information could be derived. From their

observations, Han et al. concluded that an ensemble of

conformations existed in aqueous solution which is

qualitatively similar to the conclusions

study. The IR spectrum of Leu5-enkephalin in DMSO-d6 (Han

reached in the present

et al.) showed an intense band of 1666 cm-1 which was typical

of a type II -turn structure. This IR band at 1666 cm-1 is

quite close to the 1663 cm-1 band observed in the present

study of Leu5-enkephalin in aqueous solution.

For Met5-enkephalin, the propensity of the -turn is reduced

and evidence points to the presence of -sheet structure as one

of the prominent conformations. To date the observed

difference in biological activity of Leu5- and

Met5-enkephalins has not been explained based on

conformational requirements. Even though the present study

points toward differences in conformational populations in

solutions of Leu5- and Met5-enkephalins, further studies

are required to draw any definitive conclusions on the

relationship between the conformational requirements and

biological activity, such as μ/ specificity.

It is tempting to speculate that each opioid receptor subtype

obviously interacts with a single conformation from an ensemble

of enkephalin conformations based on the topography and

stereochemistry of both the receptor and the ligand. The rules

which govern this selectivity are as yet unknown. However,

Met5-enkephalin, as compared to Leu5-enkephalin, has higher

affinity for binding at μ-receptor sites as compared to receptor

sites, and, from our studies, it appears to preferentially

exist as a -sheet structure, over the -turn. It might be

that the -sheet conformation interacts at the μ-receptor site,

whereas as a -turn structure (as in Leu5-enkephalin), it

interacts at the -receptor site. The present progress in the

isolation and purification of opioid receptors (Demoliou and

Barnard 1984), and the degree of sophistication in physical and

theoretical studies attained, will pave the way in the future

for understanding the rationale of enkephalin structure

dynamics function relationships, in particular, and for

polypeptides and proteins, in general.

REFERENCES

Bandekar, J., and Krimm, S. Vibrational analysis of peptides,

polypeptides, and proteins: Characteristic amide bands

of -turns. Proc Natl Acad Sci USA 76:774-777, 1979.

348
Beretta, C.A.; Parrilli, M.; Pastore, A.; Tancredi, T.; and

Temusi, P.A. Experimental simulation of the environment of

the opioid receptor. A 500 MHz study of enkephalins in

CDCl3. Biochem Biophys Res Commun 121:456-462, 1984.

Chou, P.Y., and Fasman, G.D. Prediction of protein conformation. Biochemistry 13:222-245, 1974.

Demoliou, C.D., and Barnard, E.A. Solubilization in high yield

of opioid receptors retaining high affinity

binding sites. FEBS Lett 170:378-382, 1984.

Diem, M.; Bhatnagar, Druyan, M.E.; and Renugopalakrishnan, V. Solution phase Raman spectroscopic studies

on synthetic collagen analogs: Prolyl-prolylqlycine and

(prolyl-prolyl-glycine)lO. Biopolymers 23:2955-2961 1984.

Gorin, F.A.; Balasubramanian, T.M.; Barry, C.D.; and Marshall,

G.R. Elucidation of the receptor-bound conformation of the

enkephalins. J Supramol Structure 9:27-39, 1978.

Han, S.-L.; Stimson, E.R.; Maxfield, F.R.; and Scheraga, H.A.

Conformational study of [Leu5] enkephalin by laser Raman

spectroscopy. Int J Pept Protein Res 16:173-182, 1980.

Hsu, S.-L.; Moore; W.H.; and Krimm, S. Vibrational spectrum of

the unordered polypeptide chain: A Raman study of feather

keratin. Biopolymers 15:1513-1528, 1976.

Hudson, B.S. New laser techniques for biophysical studies.

Annu Rev Biophys Bioeng 6:135-150, 1977.

Hughes, J; Smith, T.W.; Kosterlitz, H.W.; Fothergill, L.A.;

Morgan, B.A.; and Morris, H.R. Identification of two related

pentapeptides from the brain with potent opiate agonist

activity. Nature 258:577-579, 1975.

Kauppinen, J.K.; Hoffatt, D.J.; Mantsch, H.H.; and Cameron,

D.G. Fourier self-deconvolution: A method for resolving

intrinsically overlapped bands. Appl Spectroscopy

35:271-276. 1981.

Leach, S.J.; Nemethy, G.; and Scheraga, H.A. Use of proton

nuclear Overhauser effects for the determination of the

conformations of amino acid residues in oligopeptides.

Biochem Biophys Res Commun 75:207-215, 1977.

Lord, R.C. Strategy and tactics in Raman spectroscopy of

biomolecules. Appl Spectroscopy 31:187-194, 1977.

Lord, R.C., and Yu, N.-T. Laser-excited Raman spectroscopy of

bianolecules. I. Native lysozyme and its constituent amino

acids. J Mol Biol 50:5O9-524, 1970.

Miyazawa, T., and Blout, E.R. The IR spectra of polypeptides

in various Conformations: Amide I and II bands. J Am Chem

Soc 83:712-719, 1961.

Parker, F.S. Applications of IR, Raman, and Resonance Raman

Spectroscopy in Biochemistry. New York: Plenum Press, 1983.

Renugopalakrishnan, V., and Bhatnagar, R.S. Fourier transform

infrared photoacoustic spectroscopy: A novel conformational

probe. Demonstration of helical conformation of poly

-benzyl glutamate). J Am Chem Soc 106:2217-2219. 1984.

Renugopalakrishnan, V.; Kloumann, P.H.B.; and Bhatnagar, R.S.

L-Alanyl glycylglycine: FT-IR and Raman spectroscopic

evidence for tripeptide packing in a collagen-like

arrangement. Biopolymers 23:623-627, 1984.

349
Renugopalakrishnan, V.; Rapaka, R.S.; Collette, T.W.; Carreira,

L.A.; and Bhatnagar, R.S. Conformational states of Leu5-

and Met5-enkephalins in solution. Biochem Biophys Res

Commun 126:1029-1035, 1985.

Reniiakrishnan, V.; Collette, T.W.; Carreira, L.A.; and

Bhatnagar, R.S. Low frequency Raman spectroscopy as a

conformational probe. Macromolecules in press (1985a).

Renugopalakrishnan, V.; Horowitz, P.M.; and Glimcher, M.J.

Structural studies of phosvitin in solution. J Biol Chem,

260:11406-11413, 1985b.

Schiller, P.W. Conformational analysis of enkephalin and

conformation-activity relationships. In: Udenfriend, S. and

Meienhofer, J., eds. The Peptides Vol. 6. New York:

Academic Press, 1984. pp. 219-268.

Tu. A.T. Raman Spectroscopy in Biology: Principles and

Applications. New York: Jon Wiley and Sons, 1982,

Urry, D.W.; Khaled, M.A.; Renugopalakrishnan, V.; and Rapaka,

R.S. Proton magnetic resonance and conformational energery

calculations of repeat peptides of tropoelastin. The

hexrpeptide. J Am Chem Soc 100:696-705, 1978.

ACKNOWLEDGMENTS

FT-IR experiments were performed at the National Center for

Biomedical IR Spectroscopy at Battelle Columbus Laboratories,

Columbus, Ohio, supported by NIH grant 1-P41 RR 0137. The help

of Ms. Katrina B. Smith with FT-IR experiments is gratefully

acknowledged.

AUTHORS

V. Renugopalakrishnan, Ph.D.

Laboratory of Skeletal Disorders and Rehabilitation

Department of Orthopaedic Surgery

Harvard Medical School

Children's Hospital, Enders-1220

300 Longwood Avenue

Boston, MA 02115

T.W. Collette, Ph.D. and L.A. Carreira, Ph.D.

Department of Chemistry

University of Georgia

Athens, Georgia 30602

R.S. Rapaka, Ph.D.

National Institute on Drug Abuse

Rockville, Maryland 20857

350
Conformational Features of the Opioid

Peptides in the Solid State: A Review of

X-Ray Crystallographic Research

Arthur Camerman, Ph.D., and Norman Camerman, Ph.D

INTRODUCTION

lsolation and characterization of the enkephalins led very

quickly to attempts to elucidate their three-dimensional

conformations. A variety of approaches, including model

bullding experiments (Gradbury et al. 1976), high resolution

nuclear magnetic resonance (NMR) (Roques et al. 1976; Jones et

el. 1976), and empirical energy calculations (Isogal et al.

1977) were interpreted to suggest a folded conformation with a

-bend between either Gly2-Gly3 or Gly3-Phe4 as the most

stable enkephalin structure. However, more comprehensive

studies utilizing NMR, ultraviolet spectroscopy (UV) and

circular dichroism (CD) data (Khaled et al. 1977), and

sophisticated energy considerations (De Coen et al. 1977)

demonstrated that fully extended enkephalin conformations are

also consistent with spectroscopic data and with energy

minima. From these studies, the existence of both folded and

extended enkephal in molecules in solution was postulated.

Seversl X-ray crystallographic investigations of leucine end

methlonine enkephalins and enkephal in analogs in the solid

state have recently been described. They have confirmed the

occurrence of both types of conformation and have al loved us

to observe directly the intra- and inter-molecular bondings

which help to stablize them.

FOLDED CONFORMATIONS

Three crystal structure determinations--of an enkephalin, a

substituted enkephalin, and an enkephalin analog--all have

pentapepide molecules which exhibit folded conformations in

the solid state. Despite the differences between the

molecules themselves and in crystal environments (see table

1), the three molecular conformations are remarkably similar.

351
TABLE 1

a

b

c

z

Space

Group

Crystal Data for Enkephalins and Analogs whIch Have Been Crystallized

Leucine Methionine

1

a

2

b

3

c

4

d

5

e 6

c,d

7

d

31.87 (31.93) 18.72 11.46 31.58 8.59 11.61 11.55

8.54 (17.08) 24.73 15.59 8.63 30.27 17.99 11.58

12.47 (24.86) 20.31 16.73 12.58 15.02 16.52 12.95

90.0 90.0 90.0 90.0 90.0 90.0 90.0 93.77

96.53 (95.54) 115.86 92.20 97.74 96.94 91.20 95.96

90.0 90.0 90.0 90.0 90.0 90.0 90.0 86.96

4 (16) 8 4 4 4 4 2

C 2( A 2) C2 P 1

aSmith and Griffin 1978; (Blundell et al. 1979). Leucine enkephalin; folded conformation.

bCamerman et al. 1983 Leucine enkephalin; extended conformation.

cBlundell and Wood 1982 Leucine and methionine enkephalins; unsolved but

.

likely both are extended.

d

Ishida et al. 1984. [p-bromoPhe4] Leu-enkephalin) folded conformation. Methionine

and [p-bromoPhe4] Met-entephalins unsolved but likely both are extended.

eEckle et al. 1984. [D-Nle2

,L-NleS5] enkephalin; folded conformation.
Leucine Enkephalin

The first enkephalin X-ray diffraction structural information

came from a crystal structure determination of Leu-enkephalin

monohydrate in crystals grown from aqueous methanol (Smith and

Griffin 1978). The molecular conformation is characterized

a Gly2-Gly3-bend stabilized by two intrmnolecular hydrogen

by

bonds, Tyr1(CO)-Phe4(NH) and Tyr1(NH)-Phe4(CO). The tyrosine

side chain is disordered and two positions of approximately

equal occupancies are found for the hydroxyphenyl atoms. The

Tyr and Phe side chains are located on the same side of the

molecular backbone "plane" and are roughly coplanar with it,

while the Leu side chain is on the opposite side and oriented

perpendicular to the backbone.

FIGURE 1

Molecular structure of leucine enkephalin in crystals grown from

aqueous methanol Smith and Griffin 1978. Copyright 1978,

American Association for the Advancement of Science

Subsequent to publication of this structure, Blundell et al.

(1979) discovered that the unit cell of the Leu-enkephalin

monohydrate crystals had been incorrectly characterized; the

true unit cell (see table 1) contains four independent peptide

molecules and the results of the structure analysis can be

interpreted as dercribing a conformation averaged over four

nearly identical molecules. The apparent disorder of the

tyrosine side chains Iikely reflects two distinct tyrosine

orientations in the four independent enkephalins.

353
[p-bromoPhe4

] Leucine Enkephalin

A Ieucine enkephalin with bromine substituted at position four

of the phenylalanine phenyl ring was recently synthesized and

crystallized from an ethanol/water mixture for an X-ray

crystallographic structure investigation (Ishida et al. 1984).

Reasons for bromine substitution were twofold: to add a

"heavy" atom which makes the structure solution easier, and to

see what effects slight modifications in chemical structure

would have on enkephalin solid-state conformation. Results of

the crystal structure determination show that even both the

substituted bromine and crystalization from ethanol/water

rather than methanol/cater, the conformation of [p-bromoPhe4]

Leu-enkephalin is almost identical to that of the previously

reported Leu-enkephalin.

FIGURE 2

Molecular conformation of [p-bromoPhe4]leucine enkephalin. Ishida

et al. 1984. Copyright 1984, The Biochemical Society, London.

The near identity of the the structures can be visualized by

comparison of figures 1 and 2. In this crystal from, the

[p-bromoPhe4] Leu-enkephalin molecule exhibits a -turn

between Gly2 and Gly3 that is stabilized by two Tyr1-Phe4

hydrogen bonds exactly as found in Leu-enkephalin.

Furthermore, just as initially described for Leu-enkephalin,

the tyrosine side chain is disordered between two equally

occupied positions. Torsion angles in the backbones and side

chains of the two structures match very closely; the largest

difference, 14°, is found in the leucine side chain and the

average difference over 21 angles is less than 4° Since the

354
conformations of these two molecules are essentially

identical, including disordered tyrosine positions, it is

natural to question whether the space group reported for

[p-bromoPhe4] Leu-enkephalin may also be misinterpreted with,

as found for the folded Leu-enkephalin, doubling of the b and

c axes necessary to characterize the correct unit cell. As of

this writing, lshida and coworkers have not reported observing

the weak diffraction intensities that could identify such a

circumstance.

Tyr-D-Nle-Gly-Phe-NIeS

X-ray crystallographic structure determination of a synthetic

analog of leucine enkephalin in which glycine 2 is replaced

by D-norleucine end leucine 5 by the sulfonic acid of

L-norleucine has been reported very recently (Eckle et al.

1984). The compound was crystalized from an ethanol/water

solution and the crystal asymmetric unit contains two

enkephalin analog molecules, one ethanol molecule and five

water molecules.

Despite the amino acid substitutions at positions 2 and 5, the

conformations of both analog molecules are strikingly

similar to that of folded leucine enkephalin. Again, each

molecule exhibits a -bend between amino acids at the 2 and 3

positions with intramolecular hydrogen bonds between

Tyr1(CO)-Phe4(NH) and Tyr1(NH)-Phe4(CO).

FIGURE 3

Steroscopic projections of the two independent molecules of

Tyr-D-NLe-Gly-Phe-NLeS. Eckle et al. 1984. Copyright 1984.

International Union of Crystallography.

355
The major differences between the two molecules in the

asymetric unit occur at the tyrosine, a situation analogous

to the two different tyrosine orientations found in the

previously discussed enkephalins. In contrast to those two

enkephalins, however, the side chains in this enkephalin

analog are oriented with the tyrosine and phenylalanine on

opposite sides of the peptide backbone.

It is notewrthy, in light of the fact that [D-Ala2]

Leu-enkephalin is boiologically active, that insertion of a

D-amino acid at position 2 does not alter the basic enkephalin

folded backbone conformation.

EXTENDED CONFORMATIONS

Four enkephalin crystal forms have been reported in which the

molecules either display or are strongly suggested to display

extended -pleated sheet conformations. Three of the four

crystal structures have not yet been solved so that the

molecular structures cannot be described with absolute

certainty, but extremely strong indications in all three

instances point to extended conformations. One completed

crystal structure determination of leucine enkephal in with

four independent pentapeptides in the asymmetric unit has

found all four enkephalins to display extended backbone

conformations.

Unsolved Structures

The first indications that both leucine and methionine

enkephalins could have extended backbones in the solid state

came from X-ray investigations of crystals of the two

compounds obtained from ethanol/water solutions (BlundelI and

Woods 1982). Although these studies did not lead to

successful structure elucidations, the X-ray data did allow

determination of the pentapeptide backbone orientations. In

both the leucine and methlonine crystals, the backbones appear

to be extended and to form hydrogen-bonded -pleated sheets.

A simllar situation involving X-ray data sets for methlonine

enkephalin (the same crystal form as above) and [p-bromoPhe4]

Met-enkephalin was reported very recently (lshida et al.

1904). These investigators also were unable to solve the

crystal structures but were able to conclude that the

methionine and bromo-substituted methionine enkephalins have

extended conformations.

Leucine Enkephalin

A crystal form of leucine enkephal in grown from a water-DMF

mixture was found from measurment of unit cell volume and

density calculations to contain four independent enkephalin

molecules plus a large amount of solvent in the asymmetric

356
unit. Elucidation of the crystal structure has shown that the

four enkephalin pentapeptides exhibit slightly differing

molecular conformations but all have extended linear backbones

(Camerman et al. 1983; Karie et al. 1983).

The extended molecules form a slightly irregular antiparallel

-pleated sheet with the amino

and below the backbone plane.

hydrogen bonds largely between

groups.

acid side chains oriented above

The sheet is held together by

peptide backbone NH and CO

Conformations of the four indepandent peptides in Lmaiuo

FIGURE 4

enkephalin crystals grown from water/DMF. Oxygen and nitrogen

atom are darkened. Camerman et al. 1983. Copyright 1983.

Macmillian Journals Ltd.

Comparison of me molecular conformations of me four

independent enkephalins reveals that the greatest differences

are the orientations of the tyrosine and leucine side chains;

the phenylalanine side chain orientation, in contrast, differs

only slightly from molecule to molecule.

The peptide backbones of molacules A and D are similar and are

more puckered than those of B and C, which also resemble each

other. In all four molecules, the regular alternation of ride

chain positions causes the Tyr and Phe side chains to be

located on opposite sides of the backbone with the Leu side

chain on the same side as the Tyr.

357
Conformational variations in the peptide backbones of leucine

enkephalins are illustrated in table 2 which lists the

backbone torslon angles in the four independent extended

conformers and, for comparison, those in the folded molecule.

Analysis of the and angles for glycine residues 2 and 3

reveals that at these positions molecules A and D display

conformations characteristic of L-amino acids, while in B and

C the values are closer to those which would be favored by

D-amino acids. The only other angles which differ appreciably

between enkephalin molecules are those at leucine. These data

are compatible with, and may provide an explanation for, the

observations that substitution of D-alanine at position 2 and

D-leucine at position 5 yield biologically active enkephalin

analogs, while substitution of D-phenylalanine at residue 4

destroys activity.

Extended Conformation and Biological Activity

Determining which enkephalin conformations are responsible for

binding to receptors and effecting the physiological actions

of these compounds would greatly aid our understanding of the

nature of opiate receptor binding and the molecular mechanisms

of analgesia. A sinificant step in this direction has been

the synthesis of cyclic Leu-enkephalin analogs with enhanced

opiate μ receptor affinity (DiMaio and Schiller 1980; Schiller

and DiMaio 1982; DiMaio et al. 1982). Substitution of D

diaminobutyric acid for glycine in position 2 of the

enkephalin sequence and cyclization of the -amino group to

the leucine C-terminal carboxyl group has produced a compound

that is twice as potent as its uncyclized analogue and 20

times as potent as Leu-enkephalin in inhibiting electrically

evoked contractions of the guinea pig ileum.

A rationale for this enhanced activity is that cyclization

produces a relatively rigid, fixed conformation in contrast to

Leu-enkephelin which displays both extended and folded shapes

in solution and in the solid state. Additionally, this fixed

conformation is similar to the "active" conformation adopted

by enkephalins when binding to μ receptors.

358
FIGURE 5

The four leucine enkephalin extended backbone conformations

aligned in comparable orientations, Camerman et al. 1903.

Copyright 1983. Nacmilian Journals Ltd.

TABLE 2

Torsion Angles (deg) in the Enkephalin Backbonesa

Residue Angle A B

Tyr1 1 135 154

1 172 177

Gly2 2 -144 151

2 114 -155

2 -177 180

Gly3 3 -122 154

3 132 -151

3 -179 -170

Phe4 4 -122 -128

4 139 130

4 168 174

Leu5 5 -79 -72

5

176 167

C D F

b

155 137 126

177 173 173

141 -131 59

-157 142 25

-178 179 179

174 -144 9 7

-170 131 -7

179 178 -174

-119 -147 -136

149 152 145

179 171 180

-141 -141 -105

137 151 -4

aEstimated standard deviations are 1.8-2.5°

b

Folded leucine enkephalin.

359
It has been demonstrated with molecular models that the

cyclized compound cannot retain the structural features of any

of the folded enkephalin conformations; thus, the μ receptor

active conformation almost certainly does not contain an

intramolecular bend (DiMaio and Schiller 1980). We have

investigated whether the crystal structure extended

conformations are compatible with the cyclized enkephal in

analog by constructing a model of the latter compound

derived from an extended Leu-enkephalin molecule with the

approprlate position 2 substituti and cycliatin added.

The model is shown figure 6. Analysis of the model reveals

that, in order to form the cyclic analog, the linear peptie

backbone has to be made somewhat more pleated, with the

largest changes in torsion angles occurring at Gly3.

FIGURE 6

Molecular model of Tyr-cycol-[- -DA2-bu-Gly-Phe-Leu-]

from leucine enkephalin extended conformation coordinated. A2bu:

diaminobutyric acid. Peptide linkage are shown in white:

-carbons and side chains are dark. (Camerma et al. 1983.

Copyright 1983, Macmillan Journals Ltd.

Comparison of figures 5 and 6 illustrates that although the

molecular fit is not exact, the main structural features of

the Leu-enkephalin extended conformation can be retained in

the cyclized analogue: the peptide backbone is linears

hydrogen bonding atoms are retained in similar positions and

orientations: Tyr and Leu side chains are located on the same

side of the backbone, while Phe is on the opposite side.

Quantitatively, the Tyr and Phe rings are, respectively, 8.2,

13.3, 13.9, and 8.9 Å apart in the extended molecules A, B, C,

and D and the same range of distances is permitted in the

360
cyclized model. In terms of arrangement of hydrogen bond

acceptors and donors, the distance between Tyr1 and Gly3

carbonyl oxygen atoms ranges from 6.8 to 7.2 in the extended

molecules, while Gly2 and Phe4 nitrogen atoms are 6.6 to 7.1 Å

6.2 Å. It appears, therefore, that if the enhanced activity

apart. These distances in the cyclized compound are 7.1 and

of the cyclized analog is due to forcing of the otherwise

flexible muIticonformationaI enkephalin molecule into a

conformation similar to that adopted when bound to μ

receptors, then conformations resembling the extended

molecules are suitable models for μ receptor active enkephalin

stereochemistry.

Apart from the extra atoms introduced by the substitution of

diaminobutyric acid for glycine, the biggest change to the

enkephal in structure on forming the cyclic analog is the

movement of Leu due to its Iinkage to the side chain at

position 2. It is of interest that for a series of cyclic

compounds differing in the identity of the linkage

substituent, inhibitory potencies in both the guinea pig ileum

bioassay and the rat brain 3H-naloxone binding assay increase

with increasing length of the linking side chain (DiMaio et

al. 1982). As the length of this side chain is increased, we

would expect the change in position of the leucine from its

location in the extended enkephal in conformation to be

lessened in formation of the cyclic compounds. Thus, these

biological data are also compatible with and support the

concept of an extended enkephalin conformation as a primary

candidate for binding to opiate μ receptors.

CONCLUSIONS

X-ray crystallographic inveqtigations of enkephalins and

enkephalin analog have to date provided four complete

three-dimensional structure determinations and three other

data sets from which structural inferences can be draw.

These investigations have enabled us to view directly

enkephalinolecular stereochemistry in the solid state under

a variety of envirormental conditions and to formulate

conclusions about conformational preferences and stabilities

(1) The enkephalins are flexible molecules and when the whole

pentapeptide is considered (side chains as well as

backbone), several conformationaI arrangements are

equally probable.

(2) There appeer to be two favored backbone conformations,

the folded one characterized by a -bend between residues

2 and 3 stabilized by two Tyr1-Phe4 hydrogen bonds, and

the extended conformation stabilized by -pleated sheet

intermolecular hydrogen bonding.

361
(3) D-amino acids can be substituted for glycine 2 without

changing either the folded or the extended backbone

conformations. This correlates significantiy with the

fact that enkephalins with D-amino acids substituted at

position 2 retain biological activity.

(4) As it is possible for conformationaIIy restricted

cyclized enkephal in analogs with greatly enhanced

receptor-mediated activity to maintain extended backbone

stereochemistry but not folded conformations, It is

Iikely that the enkephal in μ receptor active conformation

is one with an extended peptide backbone.

It is not known at present what factors are responsible for

the binding of enkephalins to each of the dlfferent classes of

opiate receptors. One possibility, of course, is that

dlfferent enkephalin conformations are required for

interaction with the different receptors. If this were the

case, then It would be reasonable to speculate from the

structural data that extended enkephalin molecules might bind

preferentially to the μ receptor and folded ones to the 8

receptor. Further structure analyses, particularly of

conformationaIIy restricted enkephalin analogs and other

peptides that bind selectively to one class of receptors, will

help to prove or disprove such speculations. They may also

help to eventually identify the factors that govern all types

of enkephal in-receptor interactions.

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and dynamics of polypeptide hormones. Annu Rev Biochem

51:123-154, 1982.

Blundell, T.L.; Hearn, L.; Tickle, I.J.; Palmer, R.A.;

Morgan, B.A.; Smlm, G.D.; and Griffin, J.F. Crystal

structure of Leu5-enkephalin. Science 205:220, 1979.

Bradbury, A. F., Smyth; D.G.; and Snell, C.R. Biosynthetic

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Synthesis and pharmacoIogicaI characterization in vitro

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molecular structure of the enkephalin analog

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Tomita, K.; Kimura, T.; and Sakaklbara, S. X-ray

diffraction studies of enkephalins. Biochem

210:677-689, 1984.

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Conformational analysis by means of empirical energy

calculations. Proc Natl Acad Sci USA 74:414-418, 1977.

Jones, C.R. ; Gibbons, W.A.; and Garsky, V. Proton magnetic

resonance studies of conformation and flexibility of

enkephalin peptides. Nature 262:779-762, 1976.

Karle, I.; Karle, J.; Mastropaolo, D.; Camerman, A.; and

Camerman, N. [Leu5

] Enkephalin: Four co-crystalizing

conformers with extended backbones that form an

antiparallel -sheet. Acta Cryst B39:625-637, 1983.

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Brown, G.B.; and Urry, D.W. Conformational states of

enkephalins in solution. Biochem Biophys Res Commun

76:224-231, 1977.

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Anteunis, A.; and Lala, A.K. Conformation of Met5

-

enkephalin determined by high field PMR spectroscopy.

Nature 262:778-779, 1976,

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differ in their conformational requirements. Nature

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ACKNOWLEDGMENTS

Supported by grant DA-03532 from the National lnstitute on

Drug Abuse. Dr. A. Canerman is a Klingenstein Senior Fellow

in the Neurosciences.

AUTHORS

Arthur Camerman, Ph.D.

Departments of Neurology and Pharmacology

University of Washington

Seattle, WA 98195

Norman Camerman,, Ph.D.

Department of Biochemistry

University of Toronto

Toronto, Canada M55IA8

363
The Search for Nonaddicting Strong

Analgesics: Hopes and Frustrations

Sydney Archer, Ph.D.

INTRODUCTION

The pleasures of the poppy have been known to man for many centuries and

have been glorified in literature by DeQuincy, among others. It was not until the

middle of the last century that the use and abuse of opium derivatives became a

serious cause for concern. Chemical examination of opium led to the isolation of

morphine by Serturner in 1803. Continued research on Papaver somniferum

resulted in the isolation and characterization of other alkaloids, and processes

were developed to make pure morphine readily available. The unrestricted

availability of this compound and the development of the hypodermic needle led

to widespread abuse of this alkaloid. The recognition of the addiction potential

of morphine stimulated attempts to find a substitute that would not be burdened

by this undesirable property. The first example was heroin, the diacetyl

derivative of morphine. This compound was indeed a satisfactory substitute in

the sense that it is an excellent analgesic and, on administration to morphinedependent subjects, prevented the appearance of the characteristic morphine

withdrawal syndrome. It was soon discovered that heroin was just as insidious as

its precursor. Two important lessons were learnt from this experience: first,

analogues of morphine may be clinically effective analgesics and, second, there is

the phenomenon of cross-dependence to be reckoned with.

There is very little relationship between the therapeutic dose of an opioid

analgesic and its potential for abuse. Some factors which are important in

determining overall abuse liability are: (1) the ability of a drug to induce

tolerance and physical dependence; (2) whether abrupt withdrawal of the drug

from a dependent individual will result in drug-seeking behavior; (3) whether

administration of the drug or a congener will relieve withdrawal symptoms

promptly; (4) whether the drug can be self-administered easily; and (5) whether

high doses of the drug will produce unpleasant side effects.

INITIAL STUDIES

A systematic search for a nonaddicting, strong analgesic was begun in 1929 when

the National Research Council and the National Academy of Sciences formed the

Committee on Drug Addiction whose charge was to develop a program involving

chemical synthesis, pharmacological evaluation, and clinical methodology to

364
determine analgesic potency and to assess addiction liability. The chemical group

was headed by Dr. Lyndon Small, who at that time was a young assistant

professor at the University of Virginia. Small investigated derivatives of the

main phenathrene alkaloids in opium. He was joined by Dr. Erich Mosettig, a

student of Professor Spaeth, from the University of Vienna, who concentrated on

synthetic partial structures related to morphine. One of Mosettig’s students, Dr.

Everette L. May, joined the group in 1935, and continued this work which led to

many significant advances.

The pharmacology program was started in 1930 under the leadership of Dr.

Nathan B. Eddy, who later became Secretary of the Drug Addiction Committee.

In order for this fruitful collaboration to be even more effective, the two groups

were transferred to the National Institutes of Health (NIH) in 1938. About this

time, a primate facility was established at the University of Michigan under the

leadership of Professor Maurice Seevers, who developed techniques for measuring

dependence liability in monkeys.

During the first decade of this program, over 200 morphine analogues were

synthesized and evaluated. During the course of the next three decades, this NIH

facility tested over 9,000 compounds, most of which were submitted by

laboratories engaged in analgesic research with the same goals in mind. About

1,000 of these compounds were examined in Dr. Seevers’ laboratory, of which

approximately 10% were tested in the Addiction Research Center in Lexington,

Kentucky.

The Addiction Research Center was established in 1935 to develop methods to

quantitatively assay the addiction potential of new analgesics. The first Director

was Dr. Clifton K. Himmelsbach. One of the first methods perfected under

Himmelsbach’s direction was the direct substitution procedure. Prisoner addicts

were stabilized on a given dose of morphine for about 1 week. Then a putative

analgesic synthesized in either Dr. Small’s laboratory or elsewhere was substituted

for the morphine, using doses suggested by Dr. Eddy’s assessment of analgesic

potency in mice. When suitable doses were used, in every instance the addicts

were not aware that a drug had been substituted for morphine. It was judged

that drugs which could substitute successfully had a high addiction potential.

It was well known that when morphine is withdrawn from dependent individuals,

a syndrome develops which is characterized by a number of signs and symptoms.

Himmelsbach developed a method to quantitate the morphine abstinence

syndrome as shown in table 1. By plotting these Abstinence Syndrome Intensity

scores against time, the entire clinical process can be seen graphically as in

figure 1.

By the time World War II started, methodologies were available to determine the

potency of strong analgesics in rodents and their addiction potential in primates

and man. A major gap remained: methodology for the clinical evaluation of

analgesic potency. Henry Beecher was a pioneer who led the way to close this

gap. Two of his students, Louis Lasagna and Arthur Keats, as well as Raymond

Houde, made major contributions to solving the problem of determining analgesic

potency in man (Houde et al. 1965). A time-effect curve measuring the

analgesic potency of oral and intramuscular morphine is shown in figure 2.

365
TABLE 1

Point System for Measuring Abstinence Syndrome Intensity by the Day (D) or

Hour (H)

Total abstinence syndrome intensity per day or per hour is the sum of the points scored

in the (D) or (H) columns respectively, with due attention to the limits.

From Himmelabach, C.K., and Andrews, H.L. 1943. Copyright 1943 by The American Society for

Pharmacology and Experimantal Therapeutics.

FIGURE 1

Morphine Abstinence Syndrome

Perhaps the only clinically useful compound from the research efforts of Small

and his group during the 1929 to 1939 decade was metopon,l , prepared in several

steps from the enol acetate of dihydrocodone (Small et al. 1939).

366
FIGURE 2

Time-Effect Curves For Oral (P.O.) And Intramuscular (I.M.) Morphine

From Houde, Wallenstein and Beaver 1965. Copyright 1965 by Academic Press.

This drug is about 3 times as potent as morphine and is reported to be more

effective orally than morphine. It readily substitutes for morphine in stabilized

addicts and is no longer available in the United States.

SYNTHETIC OPIOIDS

In the late 1930s. Schaumann discovered that meperidine (Demerol), 2, prepared

by Eisleb (Eisleb and Schaumann 1939). as an anticholinergic agent was in fact a

strong analgesic, a laboratory result that was confirmed in man. At first, it was

thought that meperidine produced less physical dependence than morphine, but

this was before it was appreciated that meperidine has a shorter duration of

action than morphine and tolerance develops more slowly. Continued

administration of high doses of the drug does produce physical dependence.

Abrupt withdrawal from dependent subjects results in a withdrawal syndrome

resembling that of morphine with somewhat less autonomic effects. Drugseeking behavior does occur. The finding that a totally synthetic agent could

mimic the effects of morphine led to the beginning of many research programs

devoted to the synthesis of analgesics.

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2, Meperidine (Demerol)

One of the more successful efforts was that of Bockmuhl and Ehrhart (1948),

who synthesized methadone. 3, and many of its congeners. Methadone is an

effective strong analgesic in man when administered either parenterally or orally.

3, Methadone

The drug substitutes for morphine in addicts and this property is the basis of the

methadone maintenance program. The abuse potential of methadone is

comparable to that of morphine.

d-Propoxyphene (Darvon), 4, an analogue of methadone, in which a methylene

group is inserted between the phenyl ring and the quaternary carbon, was

prepared in 1953 (Pohland and Sullivan 1953). In clinical trials, it was difficult

to show that the 32 mg oral dose was superior to a placebo. It was estimated that

90 to 120 mg of d-propoxyphene was equivalent to 60 mg of codeine (Beaver

1966). Combinations with aspirin give greater pain relief than either agent alone

(Lasagna 1964). Because d-propoxyphene does not substitute for morphine as

well as codeine, the drug was not classified as a narcotic, despite the fact that

when administered the subjects recognized it as an opioid.

4, d-Propoxyphene (Darvon)

However, the drug was so irritating when administered parenterally. that its

parenteral abuse was limited. Extensive experience with the drug led to the

368
conclusion that at equianalgesic doses the abuse potential is the same as codeine.

Huge doses of d-propoxyphene resulted in deaths of some individuals. For these

reasons, the drug has been placed in Schedule IV of the Controlled Substances

Act.

5, Phenazocine

Phenazocine, 5, was synthesized in NIH laboratories by Everette May (May and

Eddy 1959). It is about 10 times as potent as morphine in rodent tests and does

not substitute well for morphine in dependent monkeys. This encouraging result

prompted extensive clinical studies which showed that the drug was about 3 to 4

times as potent as morphine in man. Human studies at the Addiction Research

Center showed that. in contrast to the findings in monkeys, 1 mg of phenazocine

substituted adequately for 8.5 mg of morphine, and direct addiction studies

showed that phenazocine produced a morphinelike physical dependence.

These are just a few examples which illustrate how the hopes of divesting strong

analgesia from addiction liability were dashed on the rocks of experience. All

the synthetic opioids which emerged from the laboratory as strong analgesics in

rodent tests are, in modern terminology, mu agonists and as such were found to

have morphinelike properties. The situation apppeared to be so bleak that

Schaumann was prompted to write:

It is therefore not correct to say that the depression of respiration

and the constipating effect of the analgesics are side effects.

They are inseparable from their analgesic action. This

unfortunately is also true for the liability to cause addiction. It

will, therefore, not be possible to find morphine-like analgesics

without this undesirable addiction and, in fact, all efforts in this

direction have been unsuccessful. (Schaumann 1956)

ANTAGONISTS AND AGONIST-ANTAGONISTS

A major development in opioid pharmacology was the finding by Unna (1943)

that N-allylnormorphine, nalorphine, 6, was a morphine antagonist. Nalorphine

precipitated a withdrawal syndrome in morphine-dependent subjects. Early

studies in postaddicts (Isbell 1956) led to the conclusion that the drug had no

dependence potential. In later investigations. it was found that after chronic

administration of high doses of nalorphine, abrupt withdrawal resulted in a

syndrome characterized by “electric shocks to the head,” and light headedness. as

well as some other signs found in the morphine withdrawal syndrome, but the

intensity of these effects were far less. There was no drug craving or drugseeking behavior.

369
6, Nalorphine

Lasagna and Beecher (1954) reported that nalorphine was an analgesic in man.

This finding was confirmed by Keats (Keats and Telford 1956). who found that

6 was equal in milligram potency to morphine. Psychotomimetic effects occurred

in about 20% of the patients. The high incidence of unpleasant CNS side effects

precluded the use of nalorphine as a clinically acceptable analgesic. The Baylor

group (Telford et al. 1961) investigated a short series of narcotic antagonists as

analgesics. None of these showed agonist activity in the usual rodent tests, in

fact, they were antagonists. Some of the drugs proved to be potent analgesics in

man, but those that were also produced undesirable CNS side effects.

Some of us who were experiencing the frustrations in dealing with the problem

of separating analgesia from addiction were following these new developments

with keen interest. A critical analysis of all the pharmacology and addiction

studies carried out in my laboratory in 1959 led to the conclusion that the rodent

analgesic assays in use at that time were better predictors of addiction liability

than of analgesic potency. At the time we initiated our studies, there were no

laboratory tests which could evaluate the analgesic action of antagonists. A series

of benzomorphans of varying antagonist potency were prepared and several of

them were evaluated as analgesics in man by Keats (Keats and Telford 1964) and

Lasagna (Lasagna and deKornfeld 1963). The results are summarized in table 2.

Cyclazocine and pentazocine were chosen for further investigation because in

these early studies they produced less CNS side effects at analgesic doses than

any of the other antagonists. Further studies revealed that the morphine

equivalent dose of pentazocine was nearer to 30 mg than 20 mg, and that at doses

of 60 to 90 mg naloxone-reversible dysphoric effects occurred. Early studies at

the Addiction Research Center with pentazocine suggested that there was little, if

any, abuse potential for this drug, but more extensive investigations showed that

this was too optimistic a conclusion (Jasinski et al. 1970).

After chronic administration of 60 to 90 mg of pentazocine every 4 hours,

postaddicts develop a withdrawal syndrome possessing some features of both the

nalorphine and morphine withdrawal syndromes. The effects are milder in

intensity, but drug-seeking behavior is present.

370
TABLE 2

Benzomorphan Antagonists In Man

Instances of abuse of the parenteral form were reported soon after the drug was

made available commercially, but for many years the drug was not seriously

abused by heroin addicts. About 1977, a combination of pentazocine (extracted

from crushed tablets) and the antihistamine tripelennamine taken intravenously

became a popular form of drug abuse in a few urban centers. In order to

counteract this abuse, the manufacturers introduced an oral formulation of

pentazocine and naloxone. Since the latter is a very poor antagonist when given

orally but a powerful one when administered parenterally, there would be no

interference with the oral efficacy of the drug but the parenteral actions would

be antagonized by the naloxone. As a result, there has been a substantial

reduction in the abuse of oral pentazocine.

Cyclazocine is a potent opioid antagonist which, as an analgesic, is about 40

times as potent as morphine and is highly effective when given orally. Abrupt

withdrawal after prolonged administration resulted in a nalorphinelike mild

withdrawal syndrome without drug-seeking behavior. Extensive clinical studies

showed that although the incidence of dysphoric effects at clinically useful doses

was not as high as with nalorphine, they occurred too frequently to allow the

introduction of cyclazocine into clinical medicine.

The realization that agonist-antagonist opioids can be clinically effective, strong

analgesics with reduced dependence liability and limited dysphoric effects,

prompted other investigators to enter the field. Naloxone, and naltrexone, the

cyclopropylmethyl analog, were developed in the Endo Laboratories (Lowenstein

1964; Lowenstein and Fishman 1967; Blumberg et al. 1967).

371
7, Naloxone

Both naloxone and naltrexone are potent opioid antagonists essentially devoid of

agonist properties. Naloxone is used clinically and in the laboratory as a “pure”

opioid antagonist. Nalbuphine, an analog of naltrexone differing only in having

an N-cyclobutylmethyl group instead of the N-cyclopropylmethyl and an alcohol

rather than a keto function at C-6, is a mixed agonist-antagonist. It has about

the same milligram potency as morphine. At these clinical doses, dysphoric

effects are minimal or absent and it is only when doses of 70 mg are used that

unpleasant CNS symptoms appear. Chronic administration of nalbuphine

followed by naloxone precipitated a mild abstinence syndrome with drug-seeking

behavior. It is estimated that its abuse potential is similar to that of pentazocine

(Jasinski and Mansky 1972).

Butorphanol, 8, is a synthetic analog of nalbuphine (Monkovic et al. 1972, 1975)

and resembles the latter pharmacologically as well as clinically. As in the case of

nalbuphine, the presence of the 14-beta hydroxyl group lowered the incidence of

CNS side effects. Direct addiction studies at the Addiction Research Center

revealed that the drug produced a withdrawal syndrome of mild intensity similar

to other mixed agonist-antagonists.

8, Butorphanol

372
Buprenorphine, 9 (Lewis et al. 1971) is a derivative of thebaine. It is an agonistantagonist whose parenteral analgesic potency is about 25 to 50 times thatof

morphine. Doses of 0.4 to 0.8 mg given sublingually produce satisfactory

analgesia in postoperative patients.

9, Buprenorphine

When naloxone was given to subjects who were receiving doses of 8 mg of the

drug daily, no withdrawal syndrome was precipitated as in the case of other

mixed agonist-antagonists. Nevertheless, abrupt withdrawal did elicit a syndrome

which included drug-seeking behavior (Jasinski et al. 1978).

The original hope that mixed agonist-antagonists could be clinically acceptable

strong analgesics with no abuse potential remains unfulfilled. What has been

accomplished is more modest. Drugs in the potency range equal to or greater

than morphine with few dysphoric effects and reduced abuse potential are now

available for clinical use. From a pharmacological standpoint, this class of drugs

was of immense value in leading to a far better understanding of how opioids

behave at a cellular and receptor level. Their diverse actions led to the

formulation of the concept of multiple opiate receptors (Martin 1967). He

postulated the presence of mu, kappa, and sigma receptors to which at least one

more, the delta receptor, has now been added (Kosterlitz et al. 1980). There are

no highly specific ligands for each of these receptors, but compounds of varying

degrees of selectivity are known. It is almost certain that a mu-specific ligand

would resemble morphine so closely that the chances are virtually nil for finding

a compound of this type with no addiction liability. The clinically available

agonist-antagonists are mixed mu-kappa agonists. It would be of great interest

to have specific kappa and delta agonists available for study.

FUTURE OUTLOOK

The seminal discovery of Hughes and Kosterlitz and their associates (Hughes et

al. 1975) that the brain produces its own opioids ushered in a new area of

research. Soon thereafter several new polypeptides from brain and pituitary were

isolated, and a host of synthetic peptides having opioid properties were prepared

in many laboratories throughout the world (Morley 1982). Much of the efforts,

particularly in industrial laboratories, were directed toward finding a drug with

no addiction liability, based on a tenuous hope that the human body would not

synthesize compounds which would be addictive. Unfortunately, this proved to

be another unfulfilled hope. Many of the opioid peptides are ineffective

373
analgesics when administered parenterally. In order to demonstrate opioid effects

in mammals, they must be administered intraventricularly. When this was done

in morphine-dependent rodents, the analgesic peptides prevented the appearance

of a withdrawal syndrome.

Addiction liability is not the only problem to be solved in the development of a

peptide analgesic. Intraventricular administration is not a clinically acceptable

method of giving drugs. The putative peptide drug must be effective when

given parenterally, preferably by the subcutaneous or intramuscular routes. If

the drug were not effective when given orally or sublingually, it would be

limited in value. Effectiveness is only one part of the story. Side effects, if

present, must be acceptable to the patient. Finally. there is a cost factor which

must be considered. In order to avoid diastereomeric mixtures, optically active

amino acids must be used. In order for the cost of manufacture to be acceptable,

the drug must be very potent so that the price of a dose would be economically

competitive. For all these reasons, it is difficult to be optimistic that the solution

to the opioid addiction problem will be found in an opioid peptide.

It is not generally appreciated that the structure and conformation of ligands are

extremely important for recognition by an opioid receptor, but postreceptor

activation events are independent of these factors. A great deal is known about

the pharmacological events following the activation of the mu receptor because,

at pharmacologically effective concentrations in vivo, morphine and some of its

congeners are essentially pure mu agonists. On the other hand, very little is

known about the pharmacological sequelae of activating the delta or kappa

receptors because of the lack of pure kappa or delta agonists. It is essential to

have such compounds because postreceptor events following activation of kappa

or delta receptors probably would be different from those following mu-receptor

activation. Although the optimism of my younger days has been eroded, it is for

the considerations just discussed that I do not share Schaumaoo’s extreme

pessimism that addiction liability and analgesic action are not separable.

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AUTHOR

Sydney Archer, Ph.D.

School of Science

Rensselaer Polytechnic Institute

Troy, New York 12180

376

U.S. GOVERNMENT PRINTING OFFICE: 1990-276-458/20885
DHHS Publication No. (ADM)90-1454

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1986, Reprinted 1987, 1990