Conformationally restricted biologically active peptides, methods for their production and uses thereof

Electrochemical methods, preferably the Kolbe coupling reaction, are utilized to create intramolecularly bridged peptides, segments or peptide isosteres which are conformationally restricted and preferably, biologically active. Preferably, the peptide analogues contain methylene groups bridging particular amino acid side chains. Analogues of a variety of peptide hormones, including insulin, insulin-like growth factors, somatostatin, melanocyte stimulating hormone, and the like are prepared by the above methods. Such peptides are useful as agonists or antagonists for treatment of diseases associated with deficiency of the hormone or dysregulation of hormone activity, as well as for mechanistic studies to understand the interactions between peptide hormones and cells.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention in the field of peptide chemistry and medicine relates to 
novel conformationally restricted biologically active peptides and 
isosteres, methods for producing such peptides based on oxidative or 
reductive electrochemical coupling reactions, and methods of using such 
peptides as hormone agonists or antagonists in treating disease. 
2. Description of the Background Art 
A. Chemical Stabilization of Peptides 
New methods for restricting the secondary structure of peptides and 
proteins are highly desirable for (1) basic structure-function studies, 
(2) the elucidation of mechanisms, and (3) the rational design of 
therapeutically useful conformationally-restricted (or "locked") 
pharmacophores. 
These applications are exemplified by an analogue of eel calcitonin, 
[Asu.sup.1,7 ]-eel calcitonin, in which .alpha.-aminosuberic acid (Asu) 
replaces the cysteine residues at positions 1 and 7 (Morikawa, T. et al., 
Experientia 32:1104-1106 (1976)). This analogue had significant biological 
activity, leading the authors to conclude that the disulfide bond in 
calcitonin is not essential for biological activity as long as the 
specific conformation of the peptide is maintained by an intramolecular 
bridge. 
The purely chemical approaches for restricting secondary structure often 
requires extensive multistep synthetic work (Olson, G. L., J. Am. Chem. 
Soc. 112:323 (1990)). An alternative approach involves installing covalent 
bridges in peptides. However, due to the sensitivity of the peptide 
backbone and side chains, this method necessitates careful 
protection/deprotection strategies. For example, this problem occurs in 
the preparation of polymethylene analogues of [Arg.sup.8 ]vasopressin in 
which .alpha.-aminosuberic acid (Asu) replaces the cysteine residues at 
positions 1 and 7 and in which the N-terminal amino group is removed (S. 
Hase et al., Experientia 25:1239-1240 (1969); S. Hase et al., J. Amer. 
Chem. Soc. 94:3590 (1972)), yielding deamino-dicarba-Arg.sup.8 
-vasopressin. 
Covalent linkages can, in selected instances, be established using other 
chemical methods, for example, by lactam formation between carboxylic acid 
and amine side chains (J. S. Taylor, Acc. Chem. Res. 23:338 (1990)), or by 
incorporation of pairs of Cys residues which form a disulfide bridge. 
However, these approaches suffer from disadvantages which the present 
invention has been designed to overcome in the creation of new 
cross-linked peptides. 
B. Promotion of Peptide Secondary Structures 
A number of approaches have attempted to induce .alpha.-helical secondary 
structures by the introduction of covalent bridges (Taylor, supra). Most 
of these procedures require an extensive synthetic effort as they involve 
constructing several intermediates typically, and require orthogonal 
protection strategies. The potential of intramolecular bridging in peptide 
design has become evident from recent studies on lactam-bridged 
amphiphilic structures (Taylor, supra). 
For example, after synthesizing a protected peptide linked to an oxime 
polystyrene-resin through a glutamyl gamma-carboxyl side chain, an 
internal nucleophilic cleavage step generates the lactam ring and 
simultaneously frees the protected peptide. The utility of the 
lactam-bridging approach is exemplified by a 21-residue peptide prepared 
from three lactam-bridged units in series which exhibits unusually high 
.alpha.-helicity. This method is, however, cumbersome, and only allows 
covalent bridging on the hydrophilic side of an amphiphilic structure. 
Another method, involving the metallation of an [i,i+4]-bishistidyl or 
[i,i+4]-histidyl-cysteinyl peptide, raises the .alpha.-helicity of the 
unmetallated sequence from about 54% to about 90% in 5 mM sodium borate 
buffer, pH 6.7 at 4.degree. C. (M. R. Ghadiri et al., JACS 112:9633 
(1990); S. Marqusee et al., Proc. Natl. Acad. Sci. USA 84:8898 (1987)). 
The use of toxic or heavy metals (e.g., Cu.sup.+2, Ru.sup.+2, Cd.sup.+2) 
to induce helicity however makes the method less likely to find 
application in the design of clinically useful peptides. 
C. Electrochemical Methods in Organic Chemistry 
Electrochemical methods have a long history in organic chemistry and have 
recently found preparative application in the field of peptide synthesis 
(S. Coyle et al., J. Chem. Soc., Chem. Commun. 980 (1976)), largely 
limited to the installation or cleavage of protecting groups. 
Using electrochemical methods, it is possible to convert selectively 
compounds with functional groups which differ in half-wave potential by 
approximately 200 mV (M. Baizer, ed., Organic Electrochemistry, 2nd ed., 
Dekker, New York (1983)). Chemical methods are not readily capable of the 
precision and extent of variability of redox potential that is essential 
for this selectivity. 
Electrochemical methods generate reactive intermediates in concentration 
gradients which are highest near the surface of the electrode, whereas 
solution methods produce low steady-state levels of the intermediate 
species in the bulk medium (Baizer, supra). The high concentration of 
reactive intermediate at the electrode results in greater yields of 
coupling versus reaction with solvent or electrolyte (L. Eberson et al., 
In: M. Baizer, supra, p. 889; J. H. P. Utley et al., J. Chem. Soc., Perkin 
Trans. 2:395 (1978)). 
One of the classic methods of organic electrochemistry is the Kolbe 
synthesis (Eberson et al., supra). In this reaction, the anodic oxidation 
of carboxylic acids produces hydrocarbon coupling products with the loss 
of carbon dioxide, often with excellent efficiencies. The reaction 
proceeds under acidic conditions to give free radical intermediates, which 
react to form either coupling products R--R, 
EQU 2 RCO.sub.2 H.fwdarw.2 R..fwdarw.R--R 
proton transfer, or disproportionation, while under basic conditions the 
anodic process generates carbenium ion intermediates, which yields 
nucleophilic addition products (H. -J. Schafer, Top. Cur. Chem., 152:91 
(1990)). 
By its nature, an electrochemical process involves molecules interacting at 
an electrode interface at which heterogeneous electron transfer takes 
place. After becoming loosely associated or perhaps adsorbed on the 
surface and being exposed to a sufficiently oxidizing or reducing 
potential, the most reactive functional group present in the molecule will 
form a reactive intermediate, such as a radical cation, radical anion, or 
free radical. Proton transfer may then follow, affecting the net charge of 
the reactive intermediate. 
If a second reactive moiety is available at an appropriate distance and 
located on the same sterically accessible face of the molecule, coupling 
of the reactive intermediates will lead to bond formation. In the Kolbe 
method, two carbon free radicals join to form a new macrocyclic ring 
containing a carbon-carbon single bond. As long as the current density at 
the electrode is maintained at a sufficiently high level, the probability 
of forming two reactive intermediates in the same molecule within the 
lifetime of the first intermediate is reasonably high (Baizer, supra). 
D. Electrochemical Reactions of Amino Acids and Peptides 
Few precedents exist for the electrochemical transformation of protected 
amino acids and peptides under typical conditions common to other kinds of 
molecules (Schafer, supra). Suberic acid derivatives, from which dicarba 
analogues of cystine-containing peptides have been obtained chemically 
(for example, oxytocin, calcitonin and somatostatin) have been prepared by 
Kolbe electrolysis of protected D-Glu and L-Glu (R. F. Nutt et al., J. 
Org. Chem. 45:3078 (1980)). There, the electrochemical reaction was 
performed on amino acids, not on a peptide. Electrochemical reactions of 
amino acids and peptides have been carried out at platinum, lead or glassy 
carbon electrodes, in solvents routinely used for organic electrochemistry 
such as methanol, acetonitrile, tetrahydrofuran, and N,N-dimethylformamide 
(Baizer, supra). 
Classical studies by Takayama of the oxidation of simple amino acids in 
aqueous acid solutions, revealed that anodic electrolysis yields aldehydes 
resulting from loss of the carboxylic acid and primary e-amino groups. At 
alkaline pH, oxidation of amino acids yields nitriles as well as 
aldehydes, depending on the electrode used (Baizer, supra). These side 
reactions may have prevented the more widespread use of electrochemical 
reactions in peptide chemistry. 
E. Peptide Hormones and Growth Factors 
Peptide hormones are central to the regulation of metabolism, 
differentiation, proliferation, and growth. The relationship between 
peptide structure and activity remains best understood from studies of 
synthetic analogues designed to model biologically functional regions of 
the peptide. Direct structure-function correlation is rare due to 
difficulty in preparing crystals of intermediate-sized peptides of quality 
adequate for X-ray study. 
There is a long-standing need in the art for a better understanding of how 
the conformational structure of a peptide modulates its regulatory role, 
in order to improve the prospect of treating or ameliorating diseases 
associated with defects or dysregulation of proteins such as growth 
factors and peptide hormones. A wide range of diseases, including cancer, 
osteoporosis, diabetes, and other metabolic defects, await the development 
of rationally designed agonists and antagonists to native polypeptide 
hormones. As chemical and electrochemical manipulation of peptide 
structure becomes a more precise science, the likelihood improves for 
major strides in the prevention and management of a multitude of diseases 
related to metabolism, cell development and differentiation. 
1. Insulin and Insulin-Like Growth Factors 
The development and application of insulin for the treatment of diabetes 
mellitus, the first example of a peptide pharmaceutical, is one of the 
great medical achievements of the twentieth century. Insulin is a 6 kDa 
peptide hormone made up of two chains, A and B, linked by a pair of 
disulfide bonds, and is derived biosynthetically from proinsulin which 
consists of A and B chains coupled by a third segment, designated C. 
Glucose regulation, which is tightly correlated with normal development, is 
known to be associated also with other insulin-like peptide hormones (P. 
D. Gluckman, Oxford Rev. Reprod. Biol. 8:1 (1986)). Specific polypeptide 
hormone growth factors, like insulin, are now known to be critical in 
development (S. Heyner et al., In: Growth Factors in Mammalian 
Development, I. Y. Rosenblum et al. (eds), CRC Press, Boca Raton, Fla., 
1989, pp. 91-112). The insulin-like growth factors, IGF-I (A. Ullrich et 
al., EMBO J. 5:2503 (1986)) and IGF-II (E. Rinderknecht et al., FEBS Lett. 
89:282 (1978))), have extensive sequence homology with proinsulin. 
Computer modelling purports a similarity in the tertiary structures of 
IGFs and insulin (T. L. Blundell et al., Nature 287:781 (1980); T. L. 
Blundell et al., Feder. Proc. 42:2592 (1983)). X-ray crystal data are not 
yet available for IGF-I or IGF-II. All of these peptides have overlapping 
functions with differing activities at each other's receptor. 
Insulin has also been found to play a central role in growth regulation as 
well (D. S. Straus, Endocrinol. Rev. 15:356 (1984)). Receptors which bind 
insulin and IGFs have been detected in early mammalian embryos. Both 
deficits and excesses of insulin have been correlated with birth defects 
(D. E. Hill, In: The Diabetic Pregnancy: A Perinatal Perspective, R. 
Merkatz et al., (eds) Grune & Stratton, New York, 1979, pp. 155-156). 
Nanomolar concentrations of insulin and IGF-I induce myoblast 
differentiation in chick embryos (C. Schmidt et al., FEBS Lett. 116:117 
(1983)). Insulin also enhances neuronal proliferation (Garofalo, R. et 
al., Molec. Cell. Biol. 8:1638 (1988)). IGF-I and insulin are both able to 
stimulate RNA and protein synthesis (U. Widmer et al., Acta Endocrinol. 
108:237 (1985)). Abnormally high levels of insulin or proinsulin have been 
found to cause abnormal growth, teratogenic effects, and death in chick 
embryos, possibly by interaction at the IGF receptor (F. DePablo et al., 
Diabetologia 28:308 (1985)). 
2. Melanocyte Stimulating Hormone 
Melanocyte stimulating hormone (MSH) is produced in the pituitary, and 
controls skin melanin dispersion (T. K. Sawyer et al., Am. Zool. 23:529 
(1983); E. Schroder et al., In: The Peptides: Synthesis, Occurrence, and 
Action of Biologically Active Polypeptides, vol. 2, Academic Press, New 
York, pp. 165ff (1966)). The alpha, beta, and gamma forms of MSH are 
derived from the precursor proopiomelanocortin, which is also the source 
of adrenocorticotrophic hormone (ACTH) and the opioid peptide 
.beta.-endorphin. MSH is thought to play a role in fetal growth and 
development (Sawyer et al., supra). For example, MSH has been detected in 
human and other mammalian fetal pituitary tissue (A. J. Kastin et al., 
Acta Endocrinol. 58:6 (1968)), and postulated to be central to the timing 
of human birth (R. E. Silman et al., Nature 260:716 (1976)). Fetal MSH is 
also important for prenatal growth (G. J. Boer et al., Applications of 
Behavioral Pharmacology in Toxicology, Zbinden et al. (eds), Raven Press, 
New York, p. 251, 1983). Removal of the fetal rat pituitary prevents the 
normal growth spurt between days 19 and 21, which can only be restored by 
exogenous administration of MSH. In addition, treatment with antibodies to 
MSH stunts the growth during this same interval. The importance of 
restricted conformation to function in MSH, as has been shown for other 
cyclic lactam analogues (see below), makes it a good system in which to 
apply the approaches of the present invention. To date, effective MSH 
receptor agonists and antagonists have depended on the use of D-amino 
acids, as in [Nle.sup.4,D-Phe.sup.7 ]-.alpha.-MSH (Schroder et al., supra; 
W. M. Westler et al., J. Amer. Chem. Soc. 110:6256 (1988)). 
3. Cholecystokinins 
The C-terminal peptide of cholecystokinin, known as CCK-8 (residues 26-33), 
is a hormonal regulator of pancreatic secretion and gallbladder 
contraction, as well as a neuropeptide (Charpentier, B. et al., Proc. 
Natl. Acad. Sci. USA 85:19681972 (1988)). Cyclization of the CCK-8 
analogue, Boc-[2-aminohexanoic acid]CCK-(27-33), has been carried out 
using a fragment condensation method (Charpentier, B. et al., J. Med. 
Chem. 30:962-968 (1987)) in conventional chemical peptide synthesis. Two 
cyclic compounds having an internal amide bond between the side chain 
amino group of D-Lys-29 and either the .beta.-carboxyl group of a D-Asp-26 
residue or the .alpha.-carboxyl group of a D-Glu-26 residue were shown to 
have high affinity and selectivity for guinea pig brain CCK receptors. 
SUMMARY OF THE INVENTION 
A major objective of the present invention is the development of a general 
and practical method for restricting the secondary structure of peptides 
by introducing covalent bridges, and uses of these methods for the design 
and production of stable, highly active, therapeutically useful peptide 
hormone analogues. 
In a preferred embodiment, it is an objective of the present invention to 
provide conformationally stable secondary or tertiary polypeptide 
structures, such as alpha helix, beta turn reverse turn, etc., by means of 
building blocks which are conformationally restricted to the desired 
structure by means of specific covalent bonds produced using 
electrochemical reactions. 
The purely chemical approaches to installing covalent bridges in peptides 
often involve conditions which are too harsh for maintaining the integrity 
of the peptide backbone and side chains without special protection 
schemes. The present invention was conceived in part to overcome these 
difficulties. Because of the intrinsic redox-directed specificity of the 
method, the present inventor appreciated that organic electrochemistry 
offers great promise in the preparation of modified peptides. 
A rapid general method for generating conformationally defined peptides, as 
described herein, allows the efficient production of a vast array of 
compounds which are useful in determining the molecular structures 
essential for signaling or promoting cell differentiation and development. 
Congenital abnormalities or other disease states associated with a 
molecular defect in a hormone receptor resulting in too high or too low 
affinity for the hormone would be treatable using peptide hormone 
analogues according to the present invention wherein the binding 
affinities or biological activities are determined and finely tuned by the 
engineered secondary structures. 
The present inventor has utilized electrochemical methods to create 
intramolecularly bridged peptides or peptide segments for incorporation 
into the synthesis of a wide range of bioactive molecules. By removing a 
relatively small number of degrees of freedom from the biologically active 
peptide, it is possible to retain considerable levels of activity while at 
the same time reducing susceptibility to proteolytic degradation. Such 
stability to proteolysis is important for an effective half-life in the 
bloodstream upon in vivo administration. 
The present invention is directed to a method for producing a 
conformationally restricted peptide or peptide isostere, comprising 
subjecting a peptide or peptide isostere having at least two amino acids 
or amino acid derivatives, the amino acids or amino acid derivatives 
having side chains which can be coupled by means of an electrochemical 
coupling reaction, to conditions sufficient to form a covalent bond 
between the side chains by means of an electrochemical coupling reaction. 
Where necessary, labile functional groups which are not intended to be 
involved in the electrochemical reaction are first protected to avoid side 
reactions. 
In a preferred embodiment the electrochemical coupling reaction is an 
oxidative coupling reaction. In a more preferred embodiment of the above 
method, the peptide or isostere has at least two available carboxylic acid 
functional groups and the oxidative coupling reaction is the Kolbe 
reaction. 
The present invention includes a method as above wherein the peptide or 
peptide isostere has at least two cysteine residues, wherein, prior to the 
forming step, two cysteine residues are replaced with amino acids or amino 
acid derivatives having side chains capable of undergoing the 
electrochemical coupling reaction such that the covalent bond is formed 
between the two replacement amino acids or derivatives. 
In an embodiment useful for producing a stabilized alpha helical structure, 
the peptide or peptide isostere has at least five amino acids or amino 
acid derivatives, wherein two amino acids or derivatives at position i and 
position i+4 have side chains capable of undergoing an electrochemical 
coupling reaction such that a covalent bond is formed between them. 
Preferably, the amino acids or amino acid derivatives at positions i+1, 
i+2 and i+3 are selected so as to permit a well-defined alpha helical 
structure to be obtained upon the coupling reaction. 
In another embodiment of the above method useful for producing a stabilized 
beta turn structure, the peptide or peptide isostere has at least four 
amino acids or amino acid derivatives, wherein two amino acids or amino 
acid derivatives at positions i and i+3 have side chains capable of 
undergoing an electrochemical coupling reaction such that a covalent bond 
is formed between them. Preferably, the amino acids or amino acid 
derivatives at positions i+1 and i+2 are selected so as to permit a 
well-defined beta turn structure to be obtained upon the coupling 
reaction. The amino acids at positions i+1 and i+2, respectively, are 
preferably selected from the group consisting of Gly-Gly, Gly-Pro and 
Pro-Gly. 
In another embodiment, the peptide or isostere may have at least two 
available aromatic groups and the oxidative coupling reaction is the 
oxidative coupling of two aromatic rings. 
In yet another embodiment, the peptide has an available aliphatic amine 
group and a second amine group and the oxidative coupling reaction is the 
oxidative coupling of an aliphatic amine with the second amine forming a 
diazo linkage. 
In one embodiment, the electrochemical coupling reaction is a reductive 
coupling reaction. 
In a preferred embodiment of the reductive reaction method, the side chains 
comprise available halo groups or available hydroxyl groups which are 
first converted into halo groups, and the coupling reaction is the 
reductive coupling of the two halo groups. 
In another embodiment, side chains comprise hydroxyl groups which are first 
substituted with alkylsulfonyl or arylsulfonyl groups, preferably tosyl 
groups, and the coupling reaction is the reductive coupling of the two 
alkyl- or arylsulfonyl groups through an ether cross-linked bridge. 
In another embodiment of the reductive method, the side chains comprise two 
nitrophenylalanine residues and the coupling reaction is the reductive 
coupling of the nitrophenylalanine residues into a diazo linkage. 
The present invention provides a conformationally restricted peptide or 
peptide isostere having at least two amino acids or amino acid derivatives 
the side chains of which are linked by a covalent bond, wherein the 
covalent bond is other than a disulfide bond or a lactam formation between 
carboxylate and amine side chains, and the linked amino acid derivatives 
are not aminosuberic acid. Preferably, the peptide has biological 
activity. 
In one embodiment, a peptide or peptide isostere comprises a tetrapeptide 
or tetrapeptide isostere having a conformationally restricted beta turn 
secondary structure, wherein the side chains of the amino acids or amino 
acid derivatives at positions 1 and 4 of the tetrapeptide or tetrapeptide 
isostere are linked by a covalent bond. Preferably, *the amino acids or 
amino acid derivatives at positions 2 and 3 are Gly-Gly, Gly-Pro or 
Pro-Gly, or derivatives thereof. 
In another embodiment a peptide or peptide isostere comprises a 
pentapeptide or pentapeptide isostere having a stabilized alpha helical 
structure, wherein two amino acids or derivatives at positions 1 and 5 or 
the pentapeptide or isostere have side chains linked by a covalent bond. 
Preferably, the amino acids or amino acid derivatives at positions 2,3, 
and 4 of the pentapeptide or isostere are selected so as to permit a 
well-defined alpha helical structure. 
In other embodiments, the peptide is any of the following: formula IV in 
FIG. 2 (SEQ ID NO:2); formula IV in FIG. 3 (SEQ ID NO:3); formula IV in 
FIG. 4 (SEQ ID NO:4 SEQ ID NO:10). formula II in FIG. 5 (SEQ ID NO:5), 
formula II in FIG. 6 (SEQ ID NO:6) and formula II in FIG. 7 (SEQ ID NO:7).

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present inventor has conceived of a method for producing a 
conformationally restricted peptide or peptide isostere which preferably 
retains biological activity of the native peptide. 
Such a peptide or isostere may be used to create a stable conformationally 
restricted structure, such as a helix or turn, or to otherwise restrict 
conformational mobility, where desired in the course of basic research 
into the structure and activity of polypeptides. Such a peptide may also 
be mass produced and used as a building block for incorporation in the 
synthesis of larger peptides and polypeptides for the same purpose. 
Such a peptide or isostere, or larger polypeptides containing same, may 
also be used as an agonist to mimic the action of the peptide, or an 
antagonist to inhibit the action of the peptide. Thus, peptides and 
peptide isosteres produced according to the methods of the invention are 
useful as therapeutic agents for a variety of disease states associated 
with abnormal levels or actions of various biologically active peptides 
such as hormones. Thus, the peptides or isosteres of the present invention 
are useful either alone, or as a unit within a larger peptide. 
The term "isostere" is defined in Korolkovas, A., Essentials of Molecular 
Pharmacology: Background for Drug Design, Wiley-Interscience, New York, 
1970, p. 50. As used herein, a peptide isostere is intended to apply 
broadly to compounds or groups that are similar, according to the 
generally accepted view in the art, in their external electronic shells; 
the term also applies in a more restricted fashion to compounds or groups 
with similar localization of regions of high or low electron density in 
molecules of similar size or shape. According to Korokovas (supra) the 
term "classical isostere" refers to systems wherein peripheral electron 
layers are the same (for example, Cl, Br, I), or systems related by 
Grimm's hydrogen displacement law (for example, O, NH and CH.sub.2) are 
proposed to have some similar properties. The term "nonclassical 
isosteres" refers to molecules whose steric arrangement and electronic 
configuration are similar, for example, --CO-- and --SO.sub.2 --. As used 
herein, both classes of isosteres are intended. Thus, a peptide isostere 
as used herein is a peptide which may include natural amino acids, 
derivatives of amino acids, or structures which are generally not 
considered to be part of an amino acid or a peptide structure, but which 
have similar steric properties and electronic configuration. Examples of 
peptide isosteres known in the art are the hydroxyethylene dipeptide 
isosteres that act as non-hydrolyzable renin inhibitors (Evans. B. F. et 
al., In: Peptides: Structure and Function, Proc. 9th Amer. Peptide 
Symposium, C. M. Deber et al., eds, Pierce Chemical Co., 1985, p. 743). 
By the term conformationally restricted is intended any peptide or isostere 
having a covalent bond which is either not present in the native peptide 
which it is intended to mimic, or is more stable than the bond which may 
already be present in the native peptide. This term may be exemplified by 
the placement of a single bond between the carbon atoms which in the 
native peptide are part of two separate side chain carboxylic acid 
functional groups, such as the omega-carboxylic acid groups of glutamic 
acid or aspartic acid. A further example is the substitution of Cys 
residues with other residues that may be bonded according to the present 
invention, thus replacing a more labile disulfide bond with a more stable 
C--C bond. 
These conformationally restricted peptide units are designed to correspond 
to particular kinds of secondary structure or tertiary structure, 
including the .alpha.-helix, .beta.-turn, and reverse turn. For a detailed 
description of secondary structure, see: G. E. Schulz et al., Principles 
of Protein Structure, Springer Verlag, New York 1985, in particular 
Chapter 6, pp. 108 et seq.; Creighton, T. E., Proteins: Structure and 
Molecular Properties, W. H. Freeman & Co., New York, 1983, in particular 
Chapter 6, pages 199 et seq. and Table 6-5. These references are hereby 
incorporated by reference in their entirety. Preferably, for maintaining a 
structure more stable than the native structure, for example, an 
.alpha.-helix, .beta.-turn or .beta.-meander, a covalent bond is 
introduced in place of the naturally occurring hydrogen bond, using the 
methods of the present invention. One of ordinary skill in the art will 
readily appreciate how to determine the sites in a peptide for introducing 
a covalent linkage in order to achieve the desired structural 
stabilization, without undue experimentation. It will also be apparent to 
one of ordinary skill in the art which amino acid substitution to make, 
and how to achieve protection and subsequent deprotection of the 
non-reacting sites, in order to practice the methods of the present 
invention. 
Conformationally restricted peptides of the present invention can be 
peptides corresponding to an intact biologically active peptide, or to a 
fragment thereof. They may also be random peptide sequences created solely 
for the purpose of producing a particular secondary or tertiary structure 
for use in studying the effects of the conformation of larger 
polypeptides. For example, the peptides may be short sequences, such as 4 
amino acids to create a .beta.-turn or 5 amino acids to create an 
.alpha.-helical structure or an .alpha.-helix-promoting structure, which 
are later incorporated into larger peptides or protein analogues. In such 
short sequences, two particular residues, typically the terminal residues, 
are selected to permit the desired electrochemical reaction. However, the 
intervening amino acids can be more broadly and nearly randomly selected. 
Such selection will involve knowledge of the attributes of the specific 
amino acids, such as charge, size, known proclivity to enter into 
particular secondary structures, etc. However, such a short sequence need 
not be identical in sequence to a fragment of a known, biologically active 
peptide. 
According to the present invention, the conformationally restricted peptide 
is produced by an electrochemical method, either oxidative or reductive. A 
preferred oxidative coupling reaction useful in the present invention is 
the Kolbe coupling reaction, which can couple omega-carboxylic acid 
moieties present in glutamic and aspartic residues within the peptide. 
Thus, as an initial strategy, the present inventor utilized intramolecular 
Kolbe coupling to couple two specifically positioned residues in a short 
peptide (see Examples below). 
The methods of the present invention can be used for any peptide or 
isostere having at least two amino acid residues. Particular functional 
groups to be linked may be present naturally in the peptide, for example 
the carboxylic acid groups on the Glu or Asp side chain, or be introduced 
in the form of an "artificial" amino acid or an amino acid derivative, 
such as diaminosuberic acid, as discussed below. 
For example, the secondary structure of vasopressin and somatostatin are 
each defined in part by a Cys-Cys disulfide bond. Replacement of this 
disulfide bond by a dimethylene bridge represents a conservative 
modification, and preserves biological activity. 
In one embodiment of the present invention, the naturally occurring amino 
acids of the native peptide sequence are used to prepare precursors for 
electrochemical modification. This feature makes the approach of the 
present invention both convenient and inexpensive. 
The economy of the methods of the present invention compared to prior art 
methods for the formation of a dimethylene bridge in a peptide can be 
readily appreciated by the following analysis. In the method of Hase et 
al. (supra) for preparation of a dicarba-Arg.sup.8 -vasopressin, the 
artificial amino acid Z-L-aminosuberic acid is required. This compound is 
sold by Peninsula Labs (1990-1991 price list) for $105 per gram. The 
actual reagent used in the prior art synthesis is Z-L-aminosuberic acid 
gamma-t-butyl ester, which must be protected by an additional four 
reaction steps and costs $145 per gram. In comparison, the reagent needed 
for the Kolbe method is Z-L-glutamic acid gamma-t-butyl ester, which is 
commercially available at $9 per gram (Bachem Bioscience, Inc.). Thus, the 
reagent alone as used in the method of the present invention represents a 
&gt;95% savings in cost over the reagent used in the method of the prior art. 
In other embodiments of the present invention, artificial amino acids are 
synthesized, using methods well-known in the art (see below), wherein 
these amino acids are specialized to exploit unique or useful reactivity 
properties of particular substituents such as halides and tosylates. Such 
modified amino acids may then be incorporated into a desired polypeptide, 
for example, during peptide synthesis, to create a starting material for 
an electrochemical reaction in accordance with the present invention. 
For example, in oligopeptides, conformationally locked secondary structure 
units may be prepared. By appropriate positioning of two latently reactive 
carboxylic acid side chains, Kolbe electrooxidative coupling would allow 
the formation of cross-linked .alpha.-helix (FIG. 9) or reverse turn 
structures (FIG. 1) which may then be manipulated as a building block in 
constructing larger polypeptide hormone analogues. 
The strategy of electrochemical coupling of peptide fragments can be 
extended to encompass a large variety of types of coupling reactions in 
addition to the Kolbe synthesis. Among the possibilities of alternative 
coupling methods are such reactions as the coupling of an aliphatic amine 
with another amine to generate a diazo species (Fuchigami, T. et al., 
Bull. Chem. Soc. Japan 53:2040 (1980)), and the oxidative coupling of two 
aromatic rings (Bechgard, K. et. al., Tetrahedron Lett. 13:2271 (1972)). 
In addition to oxidative electrochemical coupling, as described herein, 
methods based on reductive electrochemical coupling reactions may be used. 
For example, the electrochemical reductive coupling of 
alpha,omega-dihalides has generated nearly quantitative yields of strained 
rings (Fry, A. J. et al., J. Org. Chem. 38:2620 (1973)). Serine and 
threonine residues protected by acid labile t-butyl groups can be 
selectively deprotected during the acid cleavage from the 
4-[2',4'-dimethoxyphenyl(aminomethyl)]phenoxy resin. The hydroxyl group 
can be readily converted to chloride or bromide using P(C.sub.6 
H.sub.6).sub.3 /CX.sub.4 or P(C.sub.6 H.sub.6).sub.3 Br.sub.2 without 
disturbing the normal functionalities present in peptides. In an 
alternative strategy, the hydroxyls are tosylated with TsCl/pyridine, and 
then followed by the electrochemical reduction to install an ether 
cross-linked bridge. These reactions are illustrated in more detail in the 
Examples, below. 
The methods of the present invention are useful in the study of all 
peptides having biological activity. Thus, the scope of this invention is 
unlimited. Specific examples of peptides on which work has already been 
conducted in accordance with the present invention, or which are 
candidates for such work, include: the developmentally relevant peptides 
nerve growth factor, insulin-like growth factors I and II, epidermal 
growth factor, human growth hormone, insulin and oncogene-encoded 
proteins. Other useful peptide targets for the electrochemical 
cross-linking methods of the present invention include vasopressin, which 
may be involved in brain development (D. De Wied, Prog. Brain Res. 60:155 
(1983)), .alpha.-melanocyte stimulating hormone, which has been linked to 
fetal development (Sawyer et al., supra), somatostatin, which is known to 
inhibit the release of growth hormone (A. Gomez-Pan et al., Clin. 
Endocrinol. Metab. 12:469 (1983); P. Brazeau et al., Science 179:77 
(1973)); and cholecystokinin peptides (Charpentier, B. et al., 1988, 
supra). Theoretically, however, the present invention is useful in the 
production of conformationally restricted peptides corresponding to every 
biologically active peptide. 
Spectroscopic methods well-known in the art, including nuclear magnetic 
resonance, infrared spectroscopy, and circular dichroism, are used to 
reveal the detailed nature of the actual secondary structure of the 
modified peptide. 
Nuclear magnetic resonance spectrometry is a powerful tool for peptide 
structure analysis. In the absence of diffraction-quality crystals, NMR 
offers the most precise method available for determining peptide 
structure, and will provide information on the nature of peptide structure 
most relevant to a dissolved state. NMR thus helps establish the detailed 
conformational structure in the neighborhood of the electrochemically 
coupled moieties. Two-dimensional NMR techniques have been successfully 
applied to peptides with molecular weight up to about 15 kDa, much larger 
than the peptides described in the Examples below, using a variety of 
pulse sequences (Westler et al,, supra; C. Griesinger et al., Amer. Chem. 
Soc. 110:7870 (1988)). Techniques which have been exploited extensively to 
determine details about peptide structure utilize nuclear Overhauser 
enhancement effects which can provide information about interatomic 
distances and through-bond coupling parameters which can reveal dihedral 
angles between coupled atoms. Vicinal spin-spin coupling constants .sup.3 
J.sub.NH.alpha. provide a reliable basis for confirming secondary 
structures suggested by interproton distance maps (K. Wuthrich, NMR of 
Proteins and Nucleic Acids, Wiley-Interscience, New York, Chap. 9, 1986). 
Direct evidence for the formation of a hydrogen-bonded network necessary 
for forming an .alpha.-helix or .beta.-sheet is accessed from an 
examination of the backbone amide-proton exchange rates, as was achieved 
in studies of bovine pancreatic trypsin inhibitor (BPTI) (G. Wagner et 
al., J. Mol. Biol. 160:343-61 (1982)). A segment of .alpha.-helical 
secondary structure is expected to exhibit slow exchange rates for all 
residues in the sequence. Spin-lattice relaxation lifetime measurements 
describe the regions of the oligopeptide which are more rigid or more 
flexible. 
Circular dichroism spectropolarimetric studies provide important 
information on the secondary structure of the oligopeptides of the present 
invention (A. Wollmer et al., In: Modern Methods in Protein Chemistry, 
Walter de Gruyter & Co., Berlin (1983)). Analysis of CD spectra by 
comparison with reference spectra of regular polypeptides or globular 
proteins which have been previously characterized by X-ray 
crystallographic methods allows the calculation, with varying degrees of 
success, of the observed spectra in terms of idealized .alpha.-helix, 
.beta.-sheet, and random coil structures. The CD spectra of an idealized 
.alpha.-helix shows minima at 208 nm and 222 nm and a maximum at 195 nm, 
while the .beta.-structure exhibits a minimum at 217 nm and the random 
coil has a minimum below 200 nm. Studies which examine the effect of 
concentration on CD spectra are used to determine any role of 
self-aggregation in the formation of secondary structure. CD spectra are 
measured in a variety of solvent conditions ranging from buffered aqueous 
systems to mixtures of water and miscible organic solvents such as 
trifluoroethanol (TFE). Hydrogen-bond disrupting solvent systems should 
produce mainly unordered structures, while the increasingly hydrophobic 
solvents, which have been suggested to mimic the dielectric environment 
within a globular protein, should progressively accentuate the formation 
of helical structure (J. W. Nelson et al., Proteins 1:211 (1988)). Several 
computer programs are available which permit straightforward analysis of 
CD data by such methods (S. W. Provencher et al., Biochemistry 20:33 
(1981); J. P. Hennessey et al., Anal. Biochem. 125:177 (1982); N. 
Greenfield et al., Biochemistry 8:4108 (1969)), affording 
semi-quantitative determinations of the proportions of secondary 
structural types. 
Induction of the biologically active conformation is thought to result from 
interaction with the membrane-bound receptor surface (E. T. Kaiser et al., 
Science 223:249 (1984)). The air-water interface has been extensively 
studied as a convenient model for simulation of the physical interactions 
of biological interfaces (B. R. Malcolm, Progress in Surface and Membrane 
Science, Academic Press, New York, pp 189-229 (1973)). The behavior of 
peptides at the air-water interface characterizes the specific type of 
secondary structure hypothetically induced (or otherwise present) at the 
receptor surface. Peptides containing amphiphilic .alpha.-helical and 
.beta.-strand structures form stable monolayers on buffered Langmuir 
troughs, which on compression produce clearly distinguishable 
pressure/area (.pi.-A) isotherms (Malcolm, supra). Fitting the isotherm 
data to the two-dimensional equation of state .pi.[A-A.sub.o 
(1-k.pi.)]=nRT, where A.sub.o is the limiting area at .pi.=0, and k is the 
compressibility constant. Helices tend to be more compressible and less 
aggregated than .beta.-strands. Film balance data are invaluable 
especially for comparing the interactions of the hormone analogs of the 
present invention with native peptide hormones. 
There have been few studies of the .beta.-turn secondary structure or 
models of .beta.-turns at the air-water interface, and therefore the 
peptides of the present invention incorporating such turns will be useful 
for obtaining such information. A theoretical model for vasopressin 
interaction with its receptor incorporates both a hydrophobic and 
hydrophilic surface (Smith, G. W., Dev. Endocrinol. 13:23 (1981)), leading 
to the expectation that the structure is amphiphilic. 
The biological activity of each internally cross-linked peptide hormone 
analogue may be examined in a variety of assays. Novel cross-linked 
peptide hormone analogues will be examined for either agonist and 
antagonist properties. 
The activity of peptide hormones which act by stimulating the second 
messenger cyclic adenosine-5'-monophosphate (cAMP) is determined by their 
ability to stimulate cAMP formation in vitro using preparations of 
membrane-bound adenylyl cyclase using the standard .sup.3 H/.sup.31 P 
double-label protocol (Y. Salomon et al., Anal. Biochem. 58:541 (1974)). 
The activity and specificity of vasopressin analogues is evaluated at V1 
and V2 receptors. 
The effects of growth factor analogues on embryogenesis is preferably 
assessed using rat embryonal carcinoma (EC) cell lines, which mimic the 
early morphological and biochemical stages of mammalian development in 
vitro (G. R. Martin, Science 209:768 (1980)). Analogues of IGF-I, IGF-II 
or MSH analogues are examined for activity in this way. 
Somatostatin analogues are assayed for inhibition of the release of growth 
hormone (P. Brazeau, et al., Science 179, 77 (1973)). 
Active compounds are tested for their susceptibility to enzymatic 
proteolytic degradation processes using methods well-known in the art. For 
example, see: Veber, D. F., et al., Nature 280:512 (1980)). 
The preclinical and clinical therapeutic use of the present invention in 
the treatment of disease or disorders will be best accomplished by those 
of skill, employing accepted principles of diagnosis and treatment. Such 
principles are known in the art, and are set forth, for example, in 
Braunwald, E. et al., eds., Harrison's Principles of Internal Medicine, 
11th Ed., McGraw-Hill, publisher, New York, N.Y. (1987). 
The peptides and compositions of the present invention are well suited for 
the preparation of pharmaceutical compositions. The pharmaceutical 
compositions of the invention may be administered to any animal which may 
experience the beneficial effects of the compositions of the invention. 
Foremost among such animals are humans, although the invention is not 
intended to be so limited. 
Thus, the present invention provides a method for treating a subject in 
need of treatment with a peptide hormone agonist or antagonist. Using 
methods described herein, or other methods well-known in the art for 
establishing biological activity of a peptide hormone, one or ordinary 
skill in the art will be able to determine without undue experimentation 
the agonist or antagonist activity of a peptide hormone analogue according 
to the present invention. Such a peptide hormone analogue may then be 
administered to a subject having a deficiency or dysregulation in the 
activity of the peptide hormone, in order to treat such a deficiency or 
dysregulation. 
By the term "treating" is intended the administering to subjects of peptide 
according to the present invention for purposes which may include 
prevention, amelioration, or cure of disease associated with a deficiency 
in a peptide hormone or dysregulation in the activity of the hormone. 
According to the present invention, a subject, preferably a mammalian 
subject, more preferably a human, is treated with a peptide according to 
the present invention. Such treatment may be performed alone or in 
conjunction with other therapies. 
The present invention thus includes pharmaceutical compositions containing 
the peptide of the present invention along with a pharmaceutically 
acceptable excipient. Also included is a pharmaceutical composition 
comprising the peptide, in combination with an additional therapeutic 
agent plus a pharmaceutically acceptable excipient. 
In addition to the pharmacologically active compounds, the pharmaceutical 
compositions of the present invention may contain suitable 
pharmaceutically acceptable carriers comprising excipients and auxiliaries 
which facilitate processing of the active compounds into preparations 
which can be used pharmaceutically. Preferably, the preparations, 
particularly those preparations which can be administered orally and which 
can be used for the preferred type of administration, such as tablets, 
dragees, and capsules, and also preparations which can be administered 
rectally, such as suppositories, as well as suitable solutions for 
administration by injection or orally, contain from about 0.01 to 99 
percent, preferably from about 20 to 75 percent of active compound(s), 
together with the excipient. 
To enhance delivery or bioactivity, the peptides can be incorporated into 
liposomes using methods and compounds known in the art. 
The peptides are formulated using conventional pharmaceutically acceptable 
parenteral vehicles for administration by injection. These vehicles are 
nontoxic and therapeutic, and a number of formulations are set forth in 
Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., 
Easton, Pa. (1980). Nonlimiting examples of excipients are water, saline, 
Ringer's solution, dextrose solution and Hank's balanced salt solution. 
Formulations according to the invention may also contain minor amounts of 
additives such as substances that maintain isotonicity, physiological pH, 
and stability. In addition, suspensions of the active compounds as 
appropriate oily injection suspensions may be administered. Suitable 
lipophilic solvents or vehicles include fatty oils, for example, sesame 
oil, or synthetic fatty acid esters, for example, ethyl oleate or 
triglycerides. Aqueous injection suspensions that may contain substances 
which increase the viscosity of the suspension include, for example, 
sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the 
suspension may also contain stabilizers. 
The peptides of the invention are preferably formulated in purified form 
substantially free of aggregates and other protein materials, preferably 
at concentrations of about 1.0 ng/ml to 100 mg/ml. 
A typical regimen for treating a peptide hormone deficiency, for example, 
comprises administration of an effective amount of the appropriate 
conformationally-restricted peptide agonist administered over a period of 
one or several weeks and including between about one and six months. 
The peptide of the present invention may be administered by any means that 
achieve its intended purpose. For example, administration may be by 
various parenteral routes including subcutaneous, intravenous, 
intramuscular, intraperitoneal, intradermal, transdermal, 
intracerebroventricular, intrathecal or buccal routes. Alternatively, or 
concurrently, administration may be by the oral route. Parenteral 
administration can be by bolus injection or by gradual perfusion over 
time. 
It is understood that the dosage of peptide administered will be dependent 
upon the age, sex, health, and weight of the recipient, kind of concurrent 
treatment, if any, frequency of treatment, and the nature of the effect 
desired. The ranges of effective doses provided below are not intended to 
limit the inventor and represent preferred dose ranges. However, the most 
preferred dosage will be tailored to the individual subject, as is 
understood and determinable by one of skill in the art. 
The total dose required for each treatment may be administered by multiple 
doses or in a single dose. The peptide may be administered alone or in 
conjunction with other therapeutics directed to the treatment of the 
deficiency or dysregulation. 
Effective amounts of the peptide are from about 0.01 .mu.g to about 100 
mg/kg body weight, and preferably from about 10 .mu.g to about 50 mg/kg 
body weight. 
Having now generally described the invention, the same will be more readily 
understood through reference to the following examples which are provided 
by way of illustration, and are not intended to be limiting of the present 
invention, unless specified. 
EXAMPLE I 
Intramolecular Electrochemical Coupling of Precursor Peptide Hormones and 
Structure Fragments 
The general synthetic strategy will include: 
(1) preparation of an oligopeptide sequence containing the latent 
electrochemically reactive moieties in a form protected for the duration 
of the chemical steps leading up to the coupling event; 
(2) inducing the electrochemical coupling under structure promoting 
conditions such as solvents which stabilize hydrogen bonds, for example, 
trifluoroethanol; and 
(3) deprotecting all orthogonally protected functional groups. 
Larger peptide hormones are likely to have many different labile side 
groups not compatible with either peptide coupling reactions or 
electrochemistry. A sufficient variety of protecting groups are now 
available which are stable both to the FMOC protocols and to the 
electrooxidation and electroreduction conditions. 
An example which will demonstrate the ease and utility of the approach is 
the nonapeptide hormone [.sup.8 Lys]-vasopressin, which contains a 
disulfide bridge linking cysteines at positions 1 and 6 (FIG. 2). Exchange 
of the cysteine residues with gamma-t-butyl glutamyl residues, followed by 
the standard electrochemical coupling protocol, will result in the 
convenient replacement of the sulfur atoms of the cystine bridge with 
isosteric methylenes. The crude cross-linked peptide will be purified by 
size-exclusion chromatography (to eliminate intermolecularly coupled 
dimeric products) and by high performance liquid chromatography (HPLC). 
The conformationally restricted peptide units, as prepared, will contain 
protecting groups at the N-terminus and all reactive substituents, and 
will thus be in a form suitable for incorporation into an elongating 
polypeptide chain using standard methods of solid-phase fragment 
condensation (Kaiser, E. T., et al., Science 243:187 (1989)). Progress in 
fragment coupling is analyzed by amino acid analysis. After the 
conformation-restricting fragment is incorporated, the remaining 
individual residues of the sequence are incorporated using Merrifield or 
Sheppard methods (E. Atherton et al., J. Chem. Soc., Chem. Commun. 537 
(1978)). 
The identity of each peptide analogue prepared by the new cross-linking 
method of the present invention will be confirmed by amino acid analysis 
and by electrospray ionization mass spectrometry or fast atom bombardment 
mass spectrometry. Prior to incorporation into the synthesis of peptide 
analogues, the fragments will be examined by a variety of spectroscopic 
techniques including circular dichroism and nuclear magnetic resonance 
spectroscopy in order to determine the conformational structure which was 
stabilized as a result of the electrochemical process. By an iterative 
procedure, it will be possible to fine tune the nature of the 
conformational restriction by conducting the electrochemical process under 
altered conditions, or by selecting different positions for the 
electroactive residues. 
EXAMPLE II 
Preparation of a Tetrapeptide With a .beta.-Turn 
Tetrapeptides having a .beta.-turn was prepared according to the following 
scheme (see FIG. 1): 
A. Z-Glu-(gamma-O-t-Bu)-Gly-Gly-Glu-(gamma-O-t-Bu) (SEQ ID NO:1) 
(1) synthesis of an N-protected sequence 
-Glu-(gamma-O-t-Bu)-Gly-Giy-Glu-(gamma-O-t-Bu) (SEQ ID NO:1) on the 
2',4'-dimethoxy-4-benzhydrylamine resin using 9-fluorenylmethoxycarbonyl 
(FMOC) protocols (Fields, G. B., Int. J. Peptide Prot. Res. 35:161 
(1990)). 
(2) cleavage with trifluoroacetic acid to give 
Z-NH-Glu-Gly-Gly-Glu-NH.sub.2 (SEQ ID NO:1); and 
(3) electrochemical oxidative coupling at a platinum gauze electrode at 
0.degree.-5.degree. C. in methanol/pyridine. 
The reaction proceeded cleanly to give the desired product, with only minor 
side products, as indicated by thin layer chromatography. 
The cross-linked tetrapeptide showed an interesting concentration 
dependence, indicating an increasing extent of self-association as the 
concentration increased. This suggests the possible formation of a more 
extended .beta. structure. 
Circular dichroism spectra were measured in aqueous KF and phosphate 
buffers, and exhibited minima varying between 209 nm and 218 nm, and 
between 185 and 195 nm, at concentrations between 0.1 and 35 .mu.M. These 
spectral features resemble characteristics found in previous model type I 
and II' .beta.-turns (R. W. Woody, In: The Peptides: Analysis, Synthesis, 
Biology, vol. 7, V. Hruby (ed), Academic Press, New York, 1985, Chapter 
2). 
[Leu]Enkephalin, the X-ray crystal structure of which demonstrates the 
presence of a type III' .beta.-turn (G. D. Smith et al., Science 199:1214 
(1978)), shows similar signs of association by CD and NMR at millimolar 
concentrations in DMSO and TFE (M. A. Khaled et al., Biochem. Biophys. 
Res. Comm. 76:224 (1977)). 
The cross-linked tetrapeptide containing Gly.sup.2 -Gly.sup.3, the most 
conformationally flexible residue combination, generates a structure with 
moderately restricted conformation according to CD. A negative band at 191 
nm did not change with concentration, while a second negative band shifted 
position from 216 nm at low concentration to 208 nm at high 
concentrations, suggesting the contribution of mobile or distorted 
.beta.-turns susceptible to polar solvent disruption. 
B. Z-Dsu-Pro-Gly-Dsu (SEQ ID NO:11) 
A second tetrapeptide having a well-defined .beta.-turn due to the 
replacement of Gly.sup.2 with Pro, a residue with high propensity to form 
turns in native globular proteins was also synthesized. The 
Z-Dsu-Pro-Gly-Dsu (SEQ ID NO:11) cross-linked between the Dsu residues was 
synthesized as follows (FIG. 8). 
Twenty mg of the linear peptide was dissolved in 10 ml methanol (freshly 
distilled) in a 50 mL glass beaker. To this was added 76 .mu.l of 
triethylamine and 0.9 mg of metallic sodium. The beaker was charged with 
two wire mesh platinum electrodes and a calomel reference electrode. The 
reaction mixture was placed in an ice water bath and charged at constant 
voltage +2.8 V, using a Model 363 EG&G Potentiostat/Galvanostat for a 
period of 60 minutes. The initial current which was at 0.8 A fell to 0.02 
A in that period. The current was monitored for another 1 hour and showed 
no detectable decrease. Thin layer chromatography (TLC) of the reaction 
mixture on silica gel (plastic, coated with UV 254+ material) gave one PMA 
positive spot with an R.sub.f of 0.32 (chloroform/ethyl acetate, 95/5). 
The starting material appears on the baseline in this solvent system. The 
starting material was earlier tested for purity by TLC in n-butanol/acetic 
acid/water 4/1/1 and gave one spot with an R.sub.f of 0.45. 
The reaction mixture was then poured into a round bottom flask and solvent 
was removed under reduced pressure to give 12 mg of a dark brown viscous 
oil. HPLC analysis of an aliquot of this material (in acetonitrile), using 
a C18 (Vydac, 300 .ANG.) reverse phase column showed presence of the 
starting material r.sub.t 20 min. and two other peaks at r.sub.t 24 min. 
(analyzed to be two materials eluting very closely) and r.sub.t 32.5 min. 
The fraction eluting at 32.5 min was collected an analyzed for amino acid 
content and gave Gly (1.00) to Pro (1.00) showing no presence of glutamic 
acid. The crude product from the electrochemical step was taken up in 
chloroform/ethyl acetate (95/5) and washed with 10% sodium carbonate 
solution and 0.5M citric acid solution. The organic layer was washed with 
saturated sodium chloride solution and dried over anhydrous magnesium 
sulfate and filtered. Upon removal of the solvent, a colorless filmy 
material (10 mg) was obtained. This material was taken up in 
acetonitrile/water 50/50 and analyzed using a C18 reverse phase column, 
using isocratic conditions (50% acetonitrile/water/0.1% TFA) over 30 
minutes, showing that much of the starting material and by-products were 
removed by the workup procedure. The crude product was taken up in the 
minimal amount of acetonitrile/water (1:1) and purified using a C18 10.mu. 
pore-size reverse phase column using isocratic conditions 50% 
acetonitrile/water. The fraction eluting at r.sub.t 34 min was collected 
and, after removal of volatile solvents, was lyophilized to give 4 mg of a 
white flaky solid that was submitted for FAB mass spectroscopy: M+ 473 
(calculated, 473). Amino acid analysis by reverse phase HPLC after 
hydrolysis of an aliquot of the peptide in propionic acid/HCl (1:1) at 
150.degree. C. for 45 min showed the absence of any Glu in the product, 
though the analysis gave responses for the PTC derivatives of Pro and Gly. 
Upon analysis by circular dichroism (as above), the Pro-Gly tetrapeptide 
showed a stable negative band at 193 nm, unaltered by concentration 
(3.7-346 .mu.M, 0.01M KF). When dissolved in trifluoroethanol, the 
cross-linked Pro-Gly system develops a stronger negative band at 190 nm 
and small positive bands at 202 nm and 220 nm. This spectrum agrees well 
with theoretical spectra for .beta.-turns (Chang et al., Anal Biochem. 
91:13 (1978)). 
C. Reverse Turn Subunit 
A coupling sequence as shown in FIG. 1 will be used to establish a reverse 
turn subunit. This reaction involves 
(1) preparation, on a 4-[2',4'-dimethoxyphenyl(aminomethyl)]phenoxy resin 
(e.g., see: H. Rink, Tetrahedron Lett. 28:3787 (1987)) of a tetrapeptide 
consisting of two benzyl ester-protected gamma-glutamates separated by two 
"spectator" residues (glycines); 
(2) cleavage from the resin with trifluoroacetic acid; and 
(3) subsequent electrochemical cross-linking in pyridine-methanol at pH 
7.0. 
A tetrapeptide such as the ones described above may contain any amino acid 
in position 2 and 3 that is consistent with the secondary structure 
desired. Preferably, the amino acid residues in these positions are 
Gly-Gly, Pro-Gly or Gly-Pro, consistent with achieving a stable beta turn. 
Such a structure can be utilized according to the present invention as a 
building block for a conformationally stable analogue of any peptide known 
or proposed to have a beta turn as a structural element which is important 
for its biological activity. Many peptides with beta turns are known in 
the art, for example the antibiotic gramicidin and the hormone vasopressin 
(see below). 
EXAMPLE III 
Synthesis of [Dsu.sup.1,6,Leu.sup.8 ]Vasopressin 
A. Z-Glu-Tyr(OBz)-Phe-Gln-Asn-Glu-Pro-Leu-Gly-NH2 (SEQ ID NO:2) 
The linear peptide [Glu.sup.1,6,Tyr(OBz).sup.2,Leu.sup.8 ]vasopressin was 
synthesized on a 4-[2',4'-dimethoxyphenyl(methylamino)]phenoxy resin using 
FMOC amino acid active esters. The first amino acid was introduced on the 
solid-phase support by coupling with dicyclohexylcarbodiimide (DCC) and 
diisopropylethyl amine (DIEA) in methylene chloride/dimethyl formamide 
(1:1). The reaction was monitored till no more free amine was detectable 
by the Kaiser test. The determination of the substitution level was 
carried out by cleaving the FMOC group from an aliquot of the resin-amino 
acid and determining the optical density of the liberated fluorene the 
substitution level of the first amino acid loaded was 0.23 mmol/g. The 
remaining residues of the peptide were added using five equivalents of the 
FMOC-amino acid active ester, prepared separately by reacting the 
FMOC-amino acid with diisopropyl carbodiimide and hydroxybenzotriazole in 
methylene chloride/dimethyl formamide (1:1) for one hour at 0.degree. C. 
The FMOC group was cleaved using piperidine (50% in dimethyl formamide). 
The glutamic acid residues were introduced with side chain acid functions 
protected with t-butyl groups in order to facilitate simultaneous 
deprotection with the cleavage of the peptide from the resin. Tyrosine was 
introduced with the phenyl O-benzyl protected. The N-terminal amino acid 
(glutamic acid) was introduced as the N.sup..alpha. -benzyloxycarbonyl (Z) 
derivative. The finished peptide was cleaved from the resin using 
trifluoroacetic acid (50% in methylene chloride). The resin was 
subsequently washed with TFA (20% in methylene chloride). The peptide 
solution in TFA and methylene chloride was filtered, and the volatile 
solvents were removed under reduced pressure to give a dark viscous oil 
which was taken up in 25 mL 50% acetonitrile/water. After removal of 
acetonitrile under reduced pressure, the water suspension of the peptide 
was lyophilized to give 800 mg of crude peptide, a flaky yellow powder. 
Four hundred milligrams of the linear diacid crude peptide were dissolved 
in 20 mL of 50% acetonitrile/water and analyzed by HPLC (Vydac, C18 
reverse phase, 300 A, 4.6.times.250 mm, A: 0.1% TFA/water, B: 0.1% 
TFA/acetonitrile, 0-50% B linear gradient over 30 min). The fraction 
eluting at 16.12 minutes possessed an amino acid analysis corresponding to 
the expected product. The bulk of the sample was purified on a 
semi-preparative HPLC column (Vydac, C18 reverse phase, 300 A, 
10.times.250 mm, A: 0.1% TFA/water, B: 0.1% TFA/acetonitrile, 0-50% B 
linear gradient over 30 min). Fractions were pooled together, and after 
removal of volatile organic solvents, the water suspension of the peptide 
in water was lyophilized to give 126 mg of a flaky white solid. 
B. [Dsu .sup.1,6,Leu.sup.8 ] Vasopressin 
Five milligrams of the linear diacid peptide amide were dissolved in 10 mL 
methanol/pyridine (3:1). To this was added 4.3 mg of sodium methoxide and 
0.1 mg sodium metal in a 50 mL glass beaker. The glass beaker was fitted 
with two cylindrical wire mesh platinum electrodes and placed in an ice 
water bath. The cell was charged at +2.8 V (relative to a standard calomel 
reference electrode) for one hour. TLC on silica gel showed the presence 
of a substance faster moving than the starting material in 
n-butanol/acetic acid/water (3:1:1; Rf=0.63), and stains PMA positively. 
The initial current was 750 mA, which had fallen to 25 mA in this time. 
The reaction was allowed to go for another one hour at which point the 
current fell to 20 mA. After 4.4 hours the current reached a saturation 
value of 20 mA, and did not decrease further. The reaction mixture was 
poured into a tared round bottom flask and the volatile solvents were 
removed under reduced pressure. The viscous oil that was obtained was 
taken up in 20 mL chloroform/ethyl acetate (95:5), filtered, and washed 
with 10% sodium carbonate solution (2.times.20 mL) and 0.5M citric acid 
solution (2.times.20 mL). The organic layer was then washed with saturated 
sodium chloride solution (2.times.10 mL) and dried over anhydrous sodium 
sulfate. After removal of solvent, a brown powdery solid was obtained. 
This solid was taken up in 10 mL acetonitrile and water (1:1) and analyzed 
by HPLC (Vydac C18, 300 A column, 4.6.times.250 mm, 50% 
acetonitrile/water, isocratic, 1.2 mL/min) which showed the presence of 
one major component at 34.5 min. The crude peptide was purified by HPLC 
(Vydac C18, 300A, 10.times.250 mm, 50% acetonitrile/water, isocratic, 3 
mL/min). After removal of volatile solvents from the pooled fractions the 
aqueous suspensions were lyophilized to give 2.3 mg of a flaky white 
solid. The fraction eluting at 34.81 min was collected and analyzed by 
fast atom bombardment was mass spectroscopy demonstrating the presence of 
M+ 1213.5 (calculated, 1213.5). A small amount of this sample was 
dissolved in 100 .mu.L of acetonitrile and analyzed on a Vydac C18 
analytical column under isocratic conditions (50% acetonitrile in water, 
40 minutes), and was found to be homogenous. 
EXAMPLE IV 
Active Analogues of Somatostatin (FIG. 3) 
Electrochemical coupling techniques may be applied both in fragments from 
which model peptide hormones may be constructed by fragment condensation 
and also in whole peptide hormones. For the latter case, somatostatin 
analogues provide a second application of the cross-linking method with 
potential therapeutic significance. Somatostatin, a cyclic 
tetradecapeptide containing a Cys-Cys disulfide bridge, is a 
multifunctional regulatory hormone isolated from the hypothalamus. A 
previous cyclic somatostatin agonist which binds tightly to receptors in 
the anterior pituitary and inhibits growth hormone secretion (A. Gomez-Pan 
et al., Clin. Endocrinol. Metab. 12:469 (1983); P. Brazeau et al., Science 
179:77 (1973)) has been observed to greatly slow the proliferation of 
solid tumors in human small cell lung carcinoma (J. E. Taylor et al., 
Biochem. Biophys, Res, Commun. 153:81 (1988)). 
Redesign of agonists, such as those above, can be implemented using the 
methodology according to the present invention and will result in the 
development of a pharmacophore of greater activity and stability. 
A dimethylene-bridged somatostatin-(2-15) analogue is prepared on a 
2',4'-dimethoxy-4-benzhydrylamine resin using FMOC protocols (Fields, G. 
B. et al., supra) (see FIG. 3). The Lys residues will be protected with 
benzyloxycarbonyl (CBZ) groups, and Ser and Thr with benzyl groups. The 
Glu(gamma-t-Bu) side chain will be solvolyzed during the resin cleavage 
step, thereby preparing the electroactive moieties for the coupling step. 
The Trp-8 residue, when protected by the 2,4-dichlorobenzyl-oxycarbonyl 
group (DCZ), will be stable to TFA, mild amine base, and electrooxidative 
conditions (Y. S. Klausner et al., J. Chem. Soc., Perkin Trans., I: 627 
(1977)). After cleavage from the resin with TFA, the protected peptide 
amide will be oxidatively coupled, and deprotected under mild 
hydrogenolysis conditions (Pd-C, 4% HCOOH, MeOH). The bridged peptide will 
be purified by gel filtration on a Bio-Rad P-2 column and C-4 reverse 
phase HPLC. 
NMR evidence (E. M. van den Berg et al., In: Peptides: Structure and 
Function, C. M. Deber et al., (eds), Pierce Chemical Co., Rockford, Ill., 
pp. 619ff (1987)) for .beta.-turn formation stabilized by intramolecular 
hydrogen bonding between Thr-10(NH) and Phe-7(C.dbd.O) suggests a second 
model involving the insertion of Glu residues instead of Phe-6 and Phe-11. 
The hydrophobic dimethylene bridge will disrupt to a lesser extent the 
native hydrophobic interactions at these positions than will polar 
bridges. 
EXAMPLE V 
Active Analogs of Insulin and IGFs (FIG. 4) 
First, in order to determine the functional domain(s) of the peptide, 20-30 
residue fragments of the 67-residue IGF-II are prepared wherein each pair 
of Cys residues is replaced by Glu (G. B. Fields et al., Int. J. Peptide 
Protein Res. 35:161 (1990)). A second design includes partial replacement 
of Cys residues with Glu, making some bridges dimethylenes while others 
remain as disulfides. The Cys residues will be protected by standard 
methods (Fields et al., supra), such that they may be deprotected 
sequentially after the electrochemical coupling step. The reduction in 
molecular freedom due to the dimethylene bridges is expected to stabilize 
the overall conformational structure of the region. An insulin analogue 
can be similarly prepared, and is indeed a simpler procedure because of 
the presence of only two pairs of Cys residues. 
A synthetic target involves a convergent double coupling of a 
differentially protected fragment which preserves portions of both A and B 
domains. The N-terminal tripeptide is deleted since des(1-30)IGF-I 
exhibits enhanced potency in promoting organ growth (C. Gillespie et al., 
J. Endocrinol. 127:401 (1990)). The Lys residues will be protected with 
CBZ groups, Ser and Thr with benzyl groups, and Tyr with didborobenzyl 
(DCB). t-Butyl esters of Glu will be cleaved during resin cleavage giving 
the C-terminal amide diacid, ready for the first electrochemical step. Asp 
and Glu residues uninvolved in bridge formation will be protected as 
benzyl esters. Phenacyl glutamic esters can then be cleaved selectively 
with thiophenol (C. C. Yang et al., J. Org. Chem. 41:1032 (1976)), making 
available the reacting moieties for the second electrochemical step. 
Finally, all remaining benzyl protecting groups can be removed at once 
under catalytic transfer hydrogenolysis conditions (FIG. 4). 
The second strategy is based on crystallographic data on insulin which 
demonstrates the presence of helical structure in the A and B chain (T. L. 
Blundell et al., Adv. Prot. Chem. 26:279 (1972)), and on an examination of 
helical net diagrams of IGF which support the contention that several 
stretches have amphiphilic structure. Thus putative .alpha.-helical 
stretches can be stabilized in the peptides by bridging [i,i+4] residues 
located on the presumed hydrophobic face of portions of the hormone 
predicted to be amphiphilic. Maintaining the secondary structures of 
synthetic fragments of IGF will allow a comparison of their relative 
activities and therefore provide information on function for each region. 
EXAMPLE VI 
MSH Analogues (FIGS. 5-7) 
Fragment studies have suggested that the region .alpha.[.sup.7-9 ]MSH 
containing Phe-Arg-Trp constitutes the minimal necessary structural 
element for hormone activity. The bridging method used herein provides a 
simple strategy for restricting the conformation of this region. 
Initially, the conformation restricting linkage will be installed around 
this "active site" tripeptide. Three structures are shown in FIGS. 5-7. 
Each design entails a larger region about a putative active site, intended 
to access incrementally higher degrees of molecular flexibility. 
EXAMPLE VII 
Electrochemical Reductive Coupling of alpha,omega-dihalides 
The electrochemical reductive coupling of alpha,omega-dihalides has 
generated nearly quantitative yields of strained rings. Serines and 
threonine residues protected by acid labile t-butyl groups can be 
selectively deprotected during the acid cleavage from the 
2,4-dimethoxy(aminomethyl)-phenoxy resin. The hydroxyl group can be 
readily converted to chloride or bromide using P(C.sub.6 H.sub.6).sub.3 
/CX.sub.4 or P(C.sub.6 H.sub.6).sub.3 Br.sub.2 without disturbing the 
normal functionalities present in peptides. 
##STR1## 
Reductive conditions of the otherwise protected dihalopeptide will lead to 
the corresponding cross-linked peptide. 
For preparation of dihalopeptides, a solution of triphenylphosphine in 
acetonitrile is cooled in an ice bath and treated with slightly less than 
an equivalent amount of bromine (or other halogen) while stirring 
vigorously. The bis(serine) peptide (dialcohol) is dissolved in minimal 
dimethylformamide and added to the phosphine mixture over 5 minutes. 
Solvents are evaporated. The residue is taken up in ether, washed with 
sodium carbonate solution, and dried over magnesium sulfate. After 
solvents are removed, the residue is used directly in the coupling step. 
A solution of dihalopeptide in acetonitrile/0.3M tetramethylammonium 
bromide is electrolyzed with the potential set at -0.9 to -1.4V vs. 
Ag/AgCl (KCl) reference, and the temperature is kept at 
20.degree.-25.degree. C. The electrolysis is terminated when the current 
falls below 10% of the initial value. After most of the acetonitrile is 
removed under vacuum, the residue is taken up into ether, washed with 
water, and purified by gel filtration and HPLC using conventional 
techniques. 
In an alternative strategy, the hydroxyls are tosylated with TsCl/pyridine, 
and then followed by the electrochemical reduction to install an ether 
cross-linked bridge. 
##STR2## 
The oxyanion generated in situ is a convenient intermediate for increasing 
the length of the bridging tether by one bond. Amides, ethers, and esters 
are highly resistant to further electrochemical reduction. 
The references cited above are all incorporated by reference herein, 
whether specifically incorporated or not. 
Having now fully described this invention, it will be appreciated by those 
skilled in the art that the same can be performed within a wide range of 
equivalent parameters, concentrations, and conditions without departing 
from the spirit and scope of the invention and without undue 
experimentation. 
While this invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications. This application is intended to cover any variations, uses, 
or adaptations of the inventions following, in general, the principles of 
the invention and including such departures from the present disclosure as 
come within known or customary practice within the art to which the 
invention pertains and as may be applied to the essential features 
hereinbefore set forth as follows in the scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acid 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GluGlyGlyGlu 
1 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acid 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GluTyrPheGlnAsnGluProLysGly 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AlaGlyGluLysAsnPhePheTrpLysThrPheThrSerGlu 
1510 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ProSerGluThrLeuGluGlyGlyGluLeuValAspThrLeuGlnPheValGlu 
15 1015 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
SerTyrSerLeuGluGluPheArgTrpGluLysProVal 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(D) OTHER INFORMATION: Leu-4 is Nle 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
SerTyrSerLeuG luHisPheArgTrpGluLysProVal 
1510 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(D) OTHER INFORMATION: Glu-4 and Glu-10 are Dsu 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
SerTyrSerGluGluHisPheArgTrpGluLysProVal 
1510 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(D) OTHER INFORMATION: Glu-1 and Glu-4 are modified at 
their side- chains 
(ix) FEATURE: 
(D) OTHER INFORMATION: Glu-1 is modified at the amino 
end to add a Z group 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GluProGlyGlu 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GluGluAlaAlaGlu 
15 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GluPheArgSerAlaAspLeuAlaLeuLeuGluThrTyrGlu 
1510 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(D) OTHER INFORMATION: Glu-1 and Glu-4 are Dsu 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GluProGlyGlu 
1