NBS

Organic Chemistry: Air Pollution Studies;

Kinetic Behavior of Sugars in Solution

Carbon-14- and Tritium-Labeled Carbohydrates;

Characterization of Chemical Structures,

Phenylhydrozono-Phenylazo Tautomerism; Synthesis

of Research Materials, Cyclopentitols and

Related Substances; Novel Research Materials;

Standard Reference Materials (Organic)

July 1965 through June 1966

405

Edited by Horace S. Isbell

***eA u of *

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

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THE NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards1 provides measurement and technical information services

essential to the efficiency and effectiveness of the work of the Nation's scientists and engineers. The

Bureau serves also as a focal point in the Federal Government for assuring maximum application of the physical and engineering sciences to the advancement of technology in industry and commerce. To

accomplish this mission, the Bureau is organized into three institutes covering broad program areas of research and services: THE INSTITUTE FOR BASIC STANDARDS . . . provides the central basis within the United

States for a complete and consistent system of physical measurements, coordinates that system with the measurement systems of other nations, and furnishes essential services leading to accurate and uniform

physical measurements throughout the Nation's scientific community, industry, and commerce. This

Institute comprises a series of divisions, each serving a classical subject matter area: —Applied Mathematics—Electricity—Metrology—Mechanics—Heat—Atomic Physics—Physical

Chemistry—Radiation Physics—Laboratory Astrophysics2—Radio Standards Laboratory,2 which

includes Radio Standards Physics and Radio Standards Engineering—Office of Standard Refer- ence Data. THE INSTITUTE FOR MATERIALS RESEARCH . . . conducts materials research and provides

associated materials services including mainly reference materials and data on the properties of materials. Beyond its direct interest to the Nation's scientists and engineers, this Institute yields services which are essential to the advancement of technology in industry and commerce. This Institute is or- ganized primarily by technical fields: —Analytical Chemistry—-Metallurgy—Reactor Radiations—Polymers—Inorganic Materials—Cryogenics2—Materials Evaluation Laboratory—Office of Standard Reference Materials.

THE INSTITUTE FOR APPLIED TECHNOLOGY . . . provides technical services to promote the use of available technology and to facilitate technological innovation in industry and government. The

principal elements of this Institute are: —Building Research—Electronic Instrumentation—Textile and Apparel Technology Center

Technical Analysis—Center for Computer Sciences and Technology—Office of Weights and Measures—Office of Engineering Standards Services—Office of Invention and Innovation—Clearinghouse for Federal Scientific and Technical Information.3

1 Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D. C,

20234.

2 Located at Boulder, Colorado. 80302.

3 Located at 5285 Port Royal Road, Springfield, Virginia, 22151.
UNITED STATES DEPARTMENT OF COMMERCE • John T. Connor, Secreta.y

NATIONAL BUREAU OF STANDARDS • A. V. Astin, Director

NBS TECHNICAL NOTE 405

ISSUED SEPTEMBER 30, 1966

Organic Chemistry: Air Pollution Studies;

Kinetic Behavior of Sugars in Solution; Carbon-14- and Tritium-Labeled Carbohydrates;

Characterization of Chemical Structures,

Phenylhydrazono-Phenylazo Tautomerism; Synthesis

of Research Materials, Cyclopentitols and

Related Substances; Novel Research Materials; Standard Reference Materials (Organic)

July 1965 through June 1966

Edited by Horace S. Isbell

Organic Chemistry Section

Analytical Chemistry Division

Institute for Materials Research

NBS Technical Notes are designed to supplement the Bureau's regular publications program. They provide a means for making available scientific data that are of transient or limited interest. Technical Notes may be

listed or referred to in the open literature.

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C., 20402 - Price 60 cents
FOREWORD

The Analytical Chemistry Division was established as a separate Division at the National Bureau of Standards on

September 1, 19^5^ and became part of the Institute for

Materials Research in the February 1, 1964 reorganization.

It consists at present of nine sections and about 100 technical personnel encompassing some 45 different analytical competences from activation analysis and atomic absorption to

vacuum fusion and X-ray spectroscopy. These competences

,

and in turn the sections which they comprise, are charged

with research at the forefront of analysis as well as awareness of the practical sample, be it standard reference

material or service analysis. In addition it is their

responsibility to inform others of their efforts.

Formal publication in scientific periodicals is highly

important. In addition, however, it has been our experience

that informal, annual summaries of progress describing efforts

of the past year can be very valuable in disseminating information. At the National Bureau of Standards such publications

fit logically into the category of a Technical Note. In 1966

we plan to issue these summaries for all of our sections.

The following is the second annual report on progress of the

Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

Ii
PREFACE

This Is the second in a series of annual progress

reports of the Organic Chemistry Section of the Analytical

Chemistry Division. The report, in the form of an NBS

Technical Note, covers the principal activities of the Section

for the period July 1, 1965 to June 30, 1966.

We have been greatly pleased with the interest accorded

our last Technical Note (No. 274). It appears to have filled

a real need for providing the public with desired information

on work in progress. In addition, we have found the report

to be a convenient means for answering inquiries made to the

Bureau concerning certain problems of current interest. The

report provides an overall view of the work of the Section,

and furnishes more information than can be given by letter.

In the present report, we have sought to convey not only

information as to what we are doing but as to why we are doing

it. In some instances, we have presented a historical resume

of our work to give perspective regarding our present activities.

We trust that this treatment will provide the scientific public

with a better understanding of our overall research goals. At

various points in the Note, reference is made to unpublished

infrared and ultraviolet spectrograms. We shall be pleased to

furnish copies of these on specific request.

In order to describe experimental procedures adequately,

it has occasionally been necessary to identify commercial

materials and equipment in this report. In no case does such

identification imply recommendation or endorsement by the

National Bureau of Standards, nor does it imply that the material

or equipment identified is necessarily the best available for

the purpose.

Horace S. Isbell, Chief

Organic Chemistry Section

Hi
TABLE OF CONTENTS

PAGE

1. AIR POLLUTION STUDIES

Reference Compounds; Preparation, Purification,

and Characterization 1

Techniques 1

Polycyclic, Aromatic Hydrocarbons 3

Oxidation Products of Polycyclic, Aromatic

Hydrocarbons 4

Photo-oxidation of Polycyclic, Aromatic Hydrocarbons on Solids 16

Anthracene on Silica Gel 16

Pyrene on Silica Gel 20

Pyrene on Other Solids 24

Perylene on Silica Gel 25

Other Hydrocarbons on Silica Gel G or Alumina. 26

Anthracene, and Pyrene, on Air-borne,

Particulate Matter from Cincinnati, Ohio . . 27

Pyrene on Maryland Soil 28

Pyrene on a Silica Gel —Alumina Mixture ... 28

2. KINETIC BEHAVIOR OP SUGARS IN SOLUTION 29

Conformational Analysis 29

Pioneer Work at NBS 29

Current Work at NBS 35

Mutarotation Reactions 38

Enolization Reactions 40

Measurement of Rates of Primary Enolization . 40

Pseudo-equilibrium States 42

Reversible Condensation and Cleavage Reactions . 42

Saccharinic Acid Rearrangements 42

iv
TABLE OF CONTENTS (CONT'D)

PAGE

3. CARBON-14- AND TRITIUM-LABELED CARBOHYDRATES . . 46

l4 Synthesis of a-L-Rhamnose- 1- C (6-Deoxy-a>-

L-mannose- 1- ^cT . 46

Discussion of Method 46

Experimental Details 48

Synthesis of ct-L-Fucose- l- C (6-Deoxy-aL-galactose- l-^C ) 51

Synthesis of D-Glucose-4-t s D-Galactose-4-t , and.

D-Glucitol-4-t 7 51

Liquid Scintillation Counting; Study of Oxygen

Quenching 56

Large-scale Paper Chromatography 6l

Procedures 62

4. CHARACTERIZATION OF CHEMICAL STRUCTURES; PHENYLHYDRAZONO-PHENYLAZO TAUTOMERISM 69

xylo-4 , 5 » 6-Trihydroxy-2-oxo-l , 3-his (phenylhydrazono )-

cyclohexane and 4-Oxo-l-phenyl-5-phenylazo-3~

pyridazine Derivatives 69

Structures of 2-0xo-l, 3-bis (phenylhydrazono

)

Compounds 69

Inosose Derivatives 71

5. SYNTHESIS OF RESEARCH MATERIALS; CYCLOPENTITOLS

AND RELATED SUBSTANCES 72
TABLE OF CONTENTS (CONT'D)

PAGE

Nit rome thane Synthesis 73

Structure of Sodium Salt 73

Conversion into Nitrocyclopentane Derivatives.

The Aminocyclopentane Derivatives and Their

Structures 76

6. NOVEL RESEARCH MATERIALS 80

Unsaturated Alditols 80

1-Acylamido Derivatives of Aldoses (Furanoid,

Pyranoid, and Acyclic) 83

7. STANDARD REFERENCE MATERIALS 91

New Program on Standard Reference Carbohydrates . 91

Rare Hexoses 91

1,2-0-Isopropylidene-L-idose 92

Metallo-organic Standards 99

Cadmium and Calcium Standards 100

8. PERSONNEL AND ACTIVITIES 104

Personnel Listing ..«.„..... 104

Publications . . . . . ». 104

Manuscripts in Process of Publication 105

NBS Reports . . 105

Lectures •... . . 105

9. ACKNOWLEDGMENTS . . „ . . . . 106

10. REFERENCES . . . . . . . . . . . 107

vi
LIST OF FIGURES

FIGURE NO. PAGE

1. Portion of equipment used in measurement of

ultraviolet, visible, and infrared spectra . .

2. Column separation of oxidation products of

pyrene 11

3. Influence of OH group at C-3j direction of

dipole moments 38

4. Apparatus used in the determination of

enolization by radiochemical techniques ... 4l

5. Liquid scintillation counter used for

determining carbon-l4 and tritium 60

6. Simplified preparation of stirrup and wick for

heavy-paper chromatogram 63

7. Addition of auxiliary wick to heavy-paper

chromatogram . . .

• 64

8. Method for nondestructively locating bands on

developed, heavy-paper chromatogram 64

9. Use of tracing and chromatogram scanning to

locate bands of high-activity, tritiated

compounds 66

10. Paper chromatogram scanner used for assay of

carbon-l4- and tritium-labeled compounds . . . 66

11. Illustration of capacity of heavy (seed-test)

paper; dependence on relative R values . . . 67

12. Illustration of capacity of fast, medium-weight

paper, prepared with thinner wick 68

13. NMR spectrometer used for determining

geometric arrangement of protons in cyclopentane derivatives .... 74

vil
LIST OF FIGURES (CONT'D)

FIGURE NO .

14. NMR spectra of V, III, and III-diacetate . . .

LIST OF TABLES

TABLE NO . 1. Photochemlcally induced changes In the

absorption spectra of pyrenediones in methanol

2. Ultraviolet and visible spectra of some

perylene derivatives . . .

3. Products from the photo-oxidation of anthracene

(0.25 g) on silica gel G ........... .

4. Ultraviolet absorption bands of anthracene and

compounds A and B

5. Some products of the oxidation of pyrene on

silica gel G ........... . . „ . .

6. Enolization of D-glucose

7. Effect of oxygen on counting efficiency for a tritium source . ........

8. Composition of scintillation solution employed

9. 1-Acetamido compounds measured, and index to

spectrograms thereof .............

PAGE

75

13

15

18

19

22

86

viii
ORGANIC CHEMISTRY SECTION: SUMMARY OF ACTIVITIES

JULY 1965 TO JUNE 1966

Edited by Horace S. Isbell

ABSTRACT

This report describes work in progress in the Organic

Chemistry Section of the Analytical Chemistry Division of the

NBS Institute for Materials Research. It includes certain

historical material, presented to give perspective to the overall program. The following research areas are discussed:

Air-Pollution Studies . Work in this field is directed toward

the study of the oxidation of polycyclic aromatic hydrocarbons.

Results are reported for the oxidation of naphthalene,

anthracene, phenanthrene, pyrene, and perylene with a variety

of oxidants. The results obtained with periodic acid as an

oxidant are particularly noteworthy. Studies of photooxidation of the polycyclic hydrocarbons on silica gel, alumina,

soil, and air-borne particulate matter have revealed, for each

hydrocarbon, products that may arise in contaminated air under

smog conditions

.

Kinetic Behavior of Sugars in Solution . Pioneer work at NBS

Is reviewed with respect to recent interest in the relationship

between conformation and chemical reactivity. In some instances,

reactions take place either with a change in conformation or

with a change in configuration. It has been shown that oxidation

of beta anomers of aldopyranoses by bromine takes place in large

measure directly, but oxidation of the alpha anomers takes place

(a) by direct oxidation with change In conformation and (b) by

way of anomerization. For an alpha (axial) anomer, the extent

of the reaction that takes place by way of anomerization

parallels the conformational stability of the anomer. Another

important contribution in this area is an explanation, in terms

of dipole moments, for the (previously observed) large Influence

of the hydroxyl group of C-3 on the conformation of the pentopyranose ring. The ring tends to assume that conformation in

which this hydroxyl group is equatorially oriented, because the

dipole —dipole interaction with the ring oxygen atom is then at

the minimum. Other work reported on this project includes

results from the study of mutarotation reactions in H2O and D2O,

measurement of rates of enolization of sugars by hydrogen

tritium exchange, and a chromatographic method for ascertaining

the distribution of carbon-l4 in l^C-labeled "a"-D-glucosaccharinic acid and in l^C-labeled acetic acid.

ix
Carbon-l4- and Tritium-labeled Carbohydrates , Syntheses are

reported for a-L-rhamnose- l--i-4c , a-L-fucose- 1-l^C , p-glucose-4-t , D-galactose-4-tJ and D-glucitol-4-t T New and improved techniques

are described for heavy-paper chromatography and for the nondestructive location of bands on developed chromatograms

.

A unique application of a liquid scintillation counter was

made in a study of the interaction of fluorescent hydrocarbons

with oxygen. It was found that the scintillation properties of

pyrene and 1,1' -bipyrene on exposure to a carbon-14 or a tritium

source are extremely sensitive to the presence of oxygen.

Mixtures of pyrene and 1,1' -bipyrene in toluene were found to be

effective media for study of scintillation counting.

Characterization of Chemical Structure; Phenylhydrazono- phenylazo Tautomerism . The positions of the phenylhydrazono

groups in xylo-4,5.?^-''trlhydroxy-2-oxo-l 3 3-'bis (phenylhydrazono)

cyclohexane were established by degradation. A series of new

4-oxo-l-phenyl-5-(phenylhydrazo)-3-pyridazlne derivatives were

prepared. A simple procedure was devised for preparing inososes?

from their phenylhydrazones by means of ion-exchange resins.

Synthesis of Research Materials? Cyclopentitols and Related

Substances . Application of the nitromethane synthesis to a dlaldehyde (previously prepared in this laboratory from

2,3-0--isopropylldene~I)-lyxose) gave two nitrocyclopentitols

which, on reduction, gave two unique aminocyclopentitol

derivatives. The structures of these compounds were determined

by chemical and physical methods.

Novel Research Materials . A highly efficient procedure was

developed for introduction of nonterminal unsaturation into

alditol derivatives. The infrared spectra of 60 carbohydrate

acetamido derivatives were measured and critically evaluated,

and no bands were found that could be correlated with the absence

or presence of the pyranoid or furanoid ring.

Standard Reference Materials . Preparation of a series of

standard reference carbohydrates was begun, and results obtained

in the preparation of 1,2-0-isopropylidene-L-idose are reported.

Certain oil-soluble calcium and cadmium chelates were

investigated for use as new metallo-organic standards. The

solubilization technique previously developed is discussed.

Key words:

Air-pollution studies, oxidation of polycyclic aromatic

hydrocarbons, sugars in solution, conformational analysis,

carbon-l4- and tritium-labeled carbohydrates, large-scale

paper chromatography, phenylhydrazono-phenylazo tautomerism,

cyclopentitols and related substances, unsaturated alditols,

infrared spectra of acylamido derivatives, carbohydrate

reference materials, metallo-organic standard reference

materials.
1. AIR POLLUTION STUDIES

( R. S. Tipson , A. Cohen, and A. J. Fatiadi)

This work is supported, in part, "by the Division of Air

Pollution, Public Health Service, U. S. Department of Health,

Education, and Welfare.

A. Introduction

Because of the presence of many polycyclic, aromatic

hydrocarbons and certain of their oxidation products in

polluted air, information was needed on such hydrocarbons.

In addition, studies are being conducted on the oxidation of

polycyclic, aromatic hydrocarbons found in polluted air.

Reactions with a variety of oxidants are being examined, and

the oxidation products (peroxides, aldehydic acids, quinones,

etc.) are being identified.

B. Reference Compounds; Preparation, Purification, and

Characterization

Before proceeding to study the oxidation of a hydrocarbon,

a very pure sample of the hydrocarbon is required. Furthermore,

a pure sample of each of the anticipated oxidation products is

needed .

1 . Techniques

In addition to the customary techniques of purification

and characterization, the following have proved useful.

a. Adsorption Chromatography on a Dry Column [l].

The dry column is useful for preparative work, and its

separative efficiency is reported to approach that of thinlayer plates. Use of pressure (nitrogen) with micro-columns

facilitates development. Before use in dry columns, silica

gel was treated with glacial acetic acid, washed with water
until neutral, and activated at 115 C. In the course of the

work, we found that silica gel G contains an impurity (or

impurities), dissolved by methyl alcohol, that absorbs at 228

and 231 nm (nanometer), with a shoulder at 26l nm (in

dichloromethane )

.

b. Visualization of Spots on Thin-layer Chromatograms . In visualization of the spots on thin-layer chromatograms, a spray of concentrated sulfuric acid (alone or in methyl alcohol),

followed by heating, has been found useful; it reveals products

not visible under ultraviolet light prior to spraying. Such

spots are then further characterized by their appearance under

ultraviolet light.

c. Characterization of Compounds. This was

accomplished with the aid of visible, ultraviolet, infrared,

and nuclear magnetic resonance spectrometry, and by gasliquid chromatography (see figure 1).

Figure 1. Portion of equipment used in measurement of

ultraviolet, visible, and infrared spectra.
2. Polycyclic, Aromatic Hydrocarbons

Commercial sources for most of the polynuclear hydrocarbons

that have been detected as air pollutants were located, and 22

of these were purchased. They are being examined for purity by

ultraviolet, infrared, and chromatographic techniques.

a. Anthracene

.

Two commercial samples of anthracene

were examined (l, reagent grade; 2, highly purified by zone

melting). By use of petroleum ether saturated with 97$ methyl

alcohol (aqueous), and thin-layer chromatograms on commercial

silica gel sheets (activated at 115 C for 0.5 hr), sample 1

showed two impurities, but sample 2 showed no impurities. A

sample of 2 was now applied, as a spot, to silica gel G and

developed with benzene. Under ultraviolet light, only

anthracene was detected. However, when the plate was sprayed

with concentrated sulfuric acid and heated on a hot plate, a bluish-purple spot appeared that was not anthracene (which

gives a grey spot); neither was it anthraquinone (which gives

a yellow spot). This is under further study.

b. Naphthacene

.

This compound was found, by thinlayer chromatography on silica gel (with benzene as the solvent),

to contain four impurities, and two of these reacted with

iodine vapor. Recrystallization from (a) concentrated sulfuric

acid and water, and (b) boiling xylene failed to remove these

impurities. Attempted purification by vacuum sublimation

I o eliminated two of the impurities, giving a product of mp 34l C

(in an open capillary tube, with darkening); lit. mp, 3^-1 C.

This is under further study.

c. Pyrene

.

Commercial pyrene is slightly yellow;

ordinarily it contains tetracene [2]. It has been found that

the impurities can be removed easily by treatment, at room

temperature, of an acetone solution of crude pyrene with solid

3
potassium permanganate (with stirring), followed by filtration

using a small amount of decolorizing carbon. Concentration of

the solution gave a white product which was washed successively

with water saturated with NaHSO and then with water, and airdried in the dark. Crystallization of the material from glacial

acetic acid gave a highly pure, colorless product (mp 152-154 C),

which showed one spot on thin-layer chromatograms

.

d. Perylene

.

This compound, was purified by

chromatography on a column of silica gel (with glacial acetic

acid as the solvent) giving product of mp 276-278 C (lit. mp,

273-274 °C).

3. Oxidation Products of Polycyclic, Aromatic Hydrocarbons

a. Commercial Compounds. Oxidation products (needed

for reference) that are commercially available were purchased

and tested for purity. To aid in the identification of possible

products, the infrared absorption spectra were recorded for

commercial samples of: I, anthracene (l, reagent grade; 2, zone

refined ); II, anthraquinone (sublimed); III, 1 ,2-dihydroxyanthraquinone; IV, 1 ,4-dihydroxyanthraquinone; V, 1,5-dihydroxyanthraquinone; VI, 1 ,8-dihydroxyanthraquinone; VII, 2,6-dihydroxyanthraquinone ; VIII, 1,2,3-trihydroxyanthraquinone; IX, 1,2,4-

trihydroxyanthraquinone; X, 1,2,7-trihydroxyanthraquinone; XI,

1,2,10-anthratriol; XII, 1 ,4,9,10-anthratetrol ; and XIII, anthrone.

To aid in subsequent identification, thin-layer chromatograms

were run on commercial silica gel sheets (activated for 0.5 hr

at 115 C) for I, II, and IV, using benzene as the solvent. A

mixture of these 3 compounds was readily resolved by use of this

solvent

.

Pure samples of certain hydroxy derivatives of anthraquinone

were needed for use in identifying the products from the photooxidation of anthracene on particulate matter.

4
A sample of commercial 1,2-dihydroxyanthraquinone (ill)

was sublimed at 193 °C/0.27 mm; it then had mp 290-291 °C

(lit. mp, 291-292 C); after recrystallization from absolute

ethyl alcohol, mp 288-291 °C, (4° per min); 290-292 °C (12°

per min). Thin-layer chromatography of III on silica gel G

with (l) petroleum eth^r saturated with 97$ methyl alcohol

gave a purple spot at the origin; (2) glacial acetic acid

gave slight migration with tailings (all purple); and (3)

75:24:1 benzene —ethyl acetate —glacial acetic acid gave a yellow spot with a violet tail. When sprayed with magnesium

acetate in methyl alcohol, the yellow soot became purple.

When the acetic acid in the solvent mixture was replaced by

formic acid, the separation was greater, but the materials

had similar colors and overlapped. Thin-layer chromatography

of III on kieselguhr G with benzene as the solvent gave a

yellow front with a pale-violet tail; when sprayed with magnesium

acetate in methyl alcohol, this became completely purple.

Partial separation of III was attempted on a dry column

(6 mm i.d. x 22 in.) of silica gel, using 0.050 g of the

2 material and a nitrogen pressure of less than 2 lb/in. ; the

developer was 75*24:1 benzene —ethyl acetate —formic acid.

Pale-green and pale-violet impurities remained on the column.

The material from the eluate was re-examined by thin-layer

chromatography on kieselguhr G with benzene; the violet tail

was no longer present. However, with silica gel G and

75:24:1 benzene —ethyl acetate —formic acid, it was found that

complete separation had not yet been attained. This is under

further study.

A sample of 1,8-dihydroxyanthraquinone (Vl) was

recrystallized from hot glacial acetic acid; mp 191-193 C

(lit. mp 193-197 C; 196 C). Thin-layer chromatography of

5
VI on silica gel G, with benzene as the solvent, showed one

spot. However, chromatography on Whatman No. 1 paper, with

97$ aqueous methyl alcohol and detection with methanolic

magnesium acetate, gave a yellow and an orange spot which

overlapped. This is under further study.

Work on the other compounds is in progress.

b. Miscellaneous Synthetic Compounds. Exploratory

experiments were conducted on the oxidation of naphthalene,

anthracene, phenanthrene, and pyrene with a variety of oxidants.

The oxidation studies were directed to a study of (a) the

reactions of polycyclic, aromatic hydrocarbons with oxidants

heretofore not adequately investigated, and (b) reactions

pertinent to the basic chemistry of the oxidation reactions,

and the intermediates and oxidation products formed from

polycyclic, aromatic hydrocarbons. : -";•-.

Significant results were obtained with periodic acid as

the oxidant. This reagent has only once before been employed

for oxidation of a compound (cholesterol) in this field. By

oxidation of the hydrocarbons with periodic acid in a 1:4

ratio, the following gave the products in the yields cited

in parentheses:

1. Naphthalene in glacial acetic acid gives

1,4-naphthoquinone (69 to 75$).

00

2. Anthracene in N,N-dimethylformamide gives

anthraquinone (92 to 95$).
II

il

3. Naphthacene in NjN-dimethylformamide gives

naphthacenequinone (80$).

II

4. Phenanthrene in _p_-dioxane gives phenanthrenequinone

(45 to 55#).

°*_*°

5. Benz[a}anthracene in glacial acetic acid gives

benz[ a ]anthracene-7,12-dione (20$)

.

7
c. Pyrene Derivatives. Pyrene in glacial acetic

acid gave, with periodic acid, an unexpected condensation

product (yield, nearly quantitative). The compound

crystallizes in light-yellow plates that melt at 334 to

336 C. The substance has a strong blue fluorescence; it has

been identified as 1,1 ' -bipyrene, formed by hydrogen

abstraction and dimerization.

This compound had been synthesized earlier from

1-bromopyrene [3]. The prior synthesis was expensive because

it required preparation of the 1-bromo derivative. The newly

discovered preparation begins with readily available rawmaterials and is very simple. Hence, the compound can now be

readily made in any desired quantity. A manuscript describing

the method is being prepared for publication.

The residue from the preparation of 1 ,1 ' -bipyrene gave a small yield of 1,6-pyrenedione, 1,8-pyrenedione, and an iodo

derivative of unknown structure.

Oxidation of 1,1 ' -bipyrene with chromic acid in glacial

acetic acid did not yield the anticipated quinones.

Nitration gave a mixture, from which two crystalline nitro

compounds have been separated. The fluorescence spectrum of

1,1 ' -bipyrene was found to be 35 times as strong as that of

pyrene

.

8
Pyrene with sodium periodate (catalyzed with ruthenium

dioxide) in aqueous acetone is reported [4] to give an 11$

yield of 4,5-pyrenedione in 16 days.

4,5-Pyrenedione

We have found that, if this oxidation is conducted In aqueous

N,N-dimethylformamide containing 20 to 25 percent of glacial

acetic acid, a similar yield (10$) of 4,5-pyrenedione is

obtained after 36 to 48 hr. (if the oxidation is permitted

to proceed for 72 hr, ring-opening occurs.) Sodium

periodate may be replaced by sodium chlorate, which is cheaper.

Studies in progress have revealed that pyrene reacts

readily with Ce(SC>K) , K S Op, active manganese dioxide,

oxygen on platinum, benzoyl peroxide, and m-chloroperoxybenzoic

acid, whereas it appears inert to KBO , Bi , and HCIOk.

The following pyrene derivatives were prepared by known

methods, and their visible, ultraviolet, and Infrared absorption

spectra were recorded:

(1) 1,1 ' -bipyrene

j

(2) 1-bromopyrene, 1-iodopyrene, l-nltropyrene, and

1,6- and 1,8-dinitropyrene;

(3) 1,6-pyrenediol, and 1,6-pyrenedIol diacetate;

1,8-pyrenediol , and 1 ,8-pyrenediol diacetate;

and 4, 5-pyrenediol;

(4) 1,6-pyrenedione, 1,8-pyrenedione, and

4,5-pyrenedione; and
(5) 1-pyrenol, 3-pyrenol, 4-pyrenol, and

1,3.,6,8-pyrenetetrol

.

Arnold and Larson [5] reported that pyrene In "boiling glacial

acetic acid is oxidized with 30$ H

P°p to S ive a mixture of

1,6- and 1,8-pyrenedione. This preparation was repeated, but

close examination of the reaction product revealed that it was

a complex mixture which could not be separated by chromatography

on silica gel, deactivated Al , or Florisil. The IR spectrum

of the product showed peaks at 5.8 u.m (indicative of the presence

of opened-ring components) and 6.14 u.m (conjugated, aromatic

quinones )

.

An Improved method for preparing the 1,6- and

1,8-pyrenediones was devised in which pyrene in glacial acetic

acid is oxidized at 85 °C with 30$ H_0n , catalyzed by Vo c . The

oxidation proceeds vigorously, and temperature control is

required for mild oxidation. The method gave a mixture of

1 ,6-pyrenedione and 1,8-pyrenedione in 60$ yield. It was

found that the formation of 1 ,8-pvrenedione is favored by low

II

II

Pyrene 1,6-Pyrenedione 1,8— Pyrenedione

temperatures. The reaction mixture, which was separated by

column chromatography, also contained unreacted pyrene,

"pyrenic anhydride," and hydroxylated pyrene derivatives.

10
I-Oxo- IH-phenalene-6,7-dicarboxylic

anhydride ("Pyrenic anhydride")

A direct method for the separation of 1,6- from

1,8-pyrenedione has been developed and published [6]. The

method consists of chromatography on a column of silica gel,

using glacial acetic acid as the eluant. The 1,6-pyrenedione

is then purified by chromatography on a column of activated

alumina, with benzene as the solvent (see figure 2).

Figure 2. Column separation of

oxidation products of

pyrene

.

11
work: was begun on the oxidation of pyrene with lead

tetraacetate, a reagent not heretofore used with pyrene. The

hydrocarbon, in glacial acetic acid, was treated for 92 hr

at room temperature, with 2.4 moles of Pt^OAcK oer mole.

The major product, identified as 1,6-diacetoxypyrene, was

obtained in 40-50$ yield.

The oxidation of pyrene with active MnO was also studied.

Active MnO is known to oxidize labile primary alcohols to

aldehydes, but heretofore no oxidation of hydrocarbons with

MnO had been reported. Reaction of pyrene with active MnO

in benzene gave 5$ of 1,6-pyrenedione; reaction in glacial

acetic acid gave 2Fffo of 1,6-diacetoxypyrene and 7$ of

1,6-pyrenedione

.

In connection with our studies on the preparation and

separation of pyrenediones, we have observed some unusual

photochemical changes. When a solution of a pyrenedione in methyl

alcohol is exposed to daylight, chemical changes occur which

can be followed with the ultraviolet spectrophotometer. The

changes in the absorption spectra in the visible and

ultraviolet regions are summarized in table 1.

From table 1, it may be seen that the changes for

4, 5 -pyrenedione are greater than for 1,6-pyrenedione and

1,8-pyrenedione ; there is comolete disappearance of the

conjugated quinoid structure, indicating extensive degradation

and fragmentation of the molecule. For 1,6- and 1,8-pyrenedione , the appearance of strong bands at 243-248 nm possibly indicates

change from the quinoid to a benzenoid structure (e.g., by

formation of the corresponding dimethoxypyrenes ) . For the

solution of 1,6-pyrenedione, strong bands appear at 352-355 nm,

379-383 nm, and 401-404 nm after only 3 to 4 days of exposure

to daylight. For pyrenic anhydride , the rate of photo-induced

12
change was lower than for the pyrenediones; the appearance of

strong bands at 230 and 328 nm possibly indicates change from

the benzenoid to a conjugated quinonoid structure. The

presence of new products was verified by evaporation of each

of the solutions and thin-layer chromatography of the residue

Table 1. Photochemical ly induced changes in the absorption

spectra of pyrenediones in methanol.

Pyrene- Initial

dione

1,6-

cone. (10

mole/ liter)

2.75

Observed bands (nm)

30 min after dissolution 90 days of exposure

to daylight

239 (infl.), 242,

272, 282, 355, 383,

404, 447 (infl.)

237, 265, 277, 400, ',28,

448

1,8- 2.47 238, 282, 351, 366, 395,

458

243, 366, 425 (infl.)

4,5- 3.01 238, 254, 280, 291, 308,

322, 420

No bands; infl. at

239, 257, 304, 372,

415

"Pyrenic

anhydride"

3.09 256, 367, 396 (infl.),

408 (infl.)

230, 251 (infl.),

328, 410 (infl.)

infl. = inflection.

d. Perylene Derivatives . Reference compounds

obtained by the chemical oxidation of perylene were needed

for comparison with oxidation products resulting from its

photo-oxidation.

Oxidation of commercial perylene by published procedures

[7,8] gave impure 3,10-perylenedione that could not be purified

by many recrystallizations; thus, after successive

recrystallization from pyridine —N,N-dimethylformamide,

13
N,N-dimethylformamide —_o-dichlorobenzene , and nitrobenzene,

the material still contained impurity showing bands at 5.65

and 5.80umin the infrared spectrum.

3.,10-Perylenedione has now been obtained pure by

adsorption chromatography using two different systems: (a)

silica gel and glacial acetic acid, and (b) Darco adsorption

carbon and nitrobenzene. The purified dione had mp 390-392 C

(decomp.); the bands in its ultraviolet and visible spectra

were somewhat different from those recorded in the literature

[8] (see table 2).

The bands observed for purified 4-oxo-4H-benz[ de ] - anthracene-7,8-dicarboxylic anhydride and 1,5-anthraquinonedicarboxylic acid are listed in table 2, as these compounds

are also formed in the oxidation.

The preparation and purification of other perylene

derivatives are in progress.

COOH

Perylene 3,10-Perylenedione

HOOC

4_Oxo-4H-benz[deJ - 1 ,5-Anthraquinoneanthracene-7,8-dicarboxylic dicarboxylic acid

anhydride

e. Benz[ a] anthracene Derivatives . A survey of the

literature and of chemical catalogs revealed that none of the

anticipated products of the oxidation of benz [a] anthracene are

commercially available. Hence, we have started the preparation

of such benz[ a] anthracene derivatives for use as reference

compounds

.

14
Table 2. Ultraviolet and visible spectra of some perylene derivatives.

3, 10-Perylenedione

Xmax ( nm)

MeOH Cone . Cone

.

H

2

S0

4

H

2

S0

4

[8]

4-0xo-4H-benz[de] anthracene7,8-dicarboxylic anhydride

Xmax (nm)

MeOH Cone. Cone.

H

2

S0

4

H

2

S0

4

[8]

215

230sh 230

238 241 238

245sh 246sh 246.5

258 256.5 254sh

266 265sh 271

272sh 270sh

285 285 278-283

293sh 297sh 290 290

307 307

314 315 311 312sh 311

319 325

335 334sh 335

343sh 347 341

350 352 354 352

362 362

375

363

392sh 403

411 412sh

415sh 422sh 432

488 489 488 485sh 470

548 543

569

580 584

1, 5-Anthraquinonedicarboxylic acid

Xmax (nm)

MeOH

250

272sh

328

390

sh = shoulder.

15
The first comDound of interest, benz [a]anthracene-7*12-diol,

was prepared as the diacetate, by the method of Badger and Lynn

[9]. This entailed treating benz[ a.]anthracene-7,12-dione

(previously prepared from benzfa. ]anthracene and Na_Cr_0 • 2H_0

in acetic acid) with zinc and boiling acetic anhydride for

1.5 hr, and hydrolyzing; a yield of more than 85$ of crude

product was obtained in two crops.

C. Photo-oxidation of Polycyclic, Aromatic Hydrocarbons on

Solids

1 . Introduction

In a preliminary communication [10] from this laboratory,

it was shown that, for certain polycyclic, aromatic hydrocarbons,

when a sample is adsorbed on a suitable solid ( e.g. , silica gel

G) and then irradiated with ultraviolet light in the presence

of air or oxygen, there may result a complex mixture of

oxidation products. For example, in the mixture of products

from pyrene, 1 ,6-pyrenedione and 1,8-pyrenedione were identified.

Presumably, some of the reactions of atmospheric pollutants

involve oxidation of polycyclic hydrocarbons adsorbed on

particulate matter (soot, dust, etc.) in the presence of

oxygen (and other oxidants) and light. Consequently, the

study has been extended, in order to permit us to identify

other products and to determine the proportions of the products.

A simple and convenient procedure for studying this type

of reaction has been devised. The procedure includes exposure

of the hydrocarbon, adsorbed on a solid, to air and ultraviolet

light, extraction of the products from the solid, and column or

thin-layer chromatography of the extract.

2. Anthracene on Silica Gel

To permit us to evaluate and then select the techniques

to be used in extending our study of the oxidation of polycyclic,

16
aromatic hydrocarbons on particulate matter, an investigation

was started on the photo-oxidation of anthracene (as a model

compound) on silica gel G.

A sample (10 g) of a 2.5$ dispersion of zone-refined

anthracene adsorbed on silica gel G was prepared by adding a chloroform solution of the hydrocarbon to the adsorbent and

evaporating off the chloroform on a rotary evaporator. The

resulting white powder was irradiated with ultraviolet light

(366 nm) during 24 hr while being continuously stirred

(egg-shaped, magnetic stirring-bar). The powder was then

placed in a cellulose Soxhlet thimble (pre-extracted with

boiling absolute methyl alcohol) and successively extracted with a variety of solvents; each extract was evaporated to dryness.

For each fraction, the infrared spectrum was recorded, and the

material was examined by thin-layer chromatography with benzene

as the eluant. The results are given in table 3.

Thus, photo-oxidation of anthracene under the conditions

described gives anthraquinone together with material which

does not migrate when benzene is the eluant. This material

gave a negative test with methanolic magnesium acetate,

indicating the absence of phenolic derivatives of anthracene.

No evidence for the formation of 1 ,4-dihydroxyanthraquinone

was found

.

The first two extracts (table 3) were chromatographed on

a dry column [l] of silica gel, with benzene as the developer,

and fractions were collected. Approximately 19.6 mg of

"anthracene" was recovered from the photo-oxidation of 250 mg

of anthracene, indicating that 92$ had been oxidized in 24 hr.

However, although the shape of the ultraviolet spectrum of this

recovered "anthracene" was identical with that of pure

anthracene (the molecular extinction coefficients were not

17
Table 3. Products from the photo-oxidation

of anthracene (0.25 g) on silica gel.

Extract Extractant

No.

1 cold

hexane

2 boiling

hexane

3 boiling

ethyl

acetate

4 boiling

methyl

alcohol

5

d cold

glacial

acetic acid

Wt(g)

Material extracted

Color TLC and IR show

0.130 pale yellow only anthracene (I) +

anthraquinone (II)

0.085 light yellow mainly II, plus some i'

0.138 yellowish

brown

0.033 yellowish

brown

0.027

mainly II, and a trace of I

II, plus hydroxylated material

inorganic acetate plus other

materiale

And an unidentified IR band at 13.08 ^m.

And an unidentified IR band at 12.68 urn. c

" Did not migrate in benzene; pale yallow in the visible, and greyish

blue under ultraviolet light (253.7 nm) . At this stage, the silica gel had a red tingt.

On washing with water, this gave 0.0032 g of water- insoluble, reddish

imaterial which reacted with aqueous permanganate. It was insoluble in

chloroform, but dissolved in hot, dilute, aqueous hydrochloric acid.

determined), thin-layer chromatography of it, with visualization

under ultraviolet light, revealed the presence of a constituent

which had a greenish-yellow fluorescence and which moved

slightly faster than anthracene. Further visualization, by

use of concentrated sulfuric acid aided by ultraviolet light,

showed another constituent (possibljr in trace amounts).

Further fractionation, with 1:9 benzene —hexane, separated

the faster-moving component (compound A) and a component

(compound B) that moved slightly slower than anthracene.

Compound B had a blue fluorescence; its ultraviolet

spectrum was very similar to that of anthracene, but the bands

at the higher wavelengths had all shifted to higher wavelengths

18
"by approximately 11 ran and three new bands appeared, at 273

(shoulder), 285, and 292 ran. A band at 255 ran (for anthracene)

had shifted to 259 nm.

In the spectrum of compound A (as compared with that of

anthracene), the band at 378 nm had shifted to 388 nm, and

other bands had shifted to higher wavelengths. Also, the

relative intensities of the bands at higher wavelengths were

altered, so that the spectrum bears less resemblance to that

of anthracene than does that of compound B. In addition,

there was a great increase in the intensity of the bands at

287 and 291 nm. The bands in this region may be related to

the "quinoid" bands [11] . In table 4, the bands are listed

for anthracene and compounds A and B.

The infrared spectra of A and B do not match those of

any anthracene oxidation products that we have yet recorded.

Consequently, it will be necessary for us to record the

ultraviolet and infrared spectra of many more hydroxylated

anthracene and anthraquinone derivatives.

Table 4. Ultraviolet absorption bands of anthracene

and compounds A and B°.

Anthracene (nm) Compound A (nm)

378

358

341

325

311

292

254

248 (sh)

233 (sh)

388

377 (sh)

368

348

337

327

ca. 317 (infl.)

291

287 (sh)

255

240 (sh)

235 (sh)

Compound B (nm)

391

370

352

334

321

ca. 309

292

285

273 (sh)

259

252 (sh)

233

a

. In dichloromethane, using a Cary Model 14 spectrophotometer,

sh = shoulder; infl. = inflection.

19
3. Pyrene on Silica Gel G

Pyrene adsorbed on silica gel G is colorless, "but has a strong, pale-greenish fluorescence when examined in ultraviolet

light. When the material is exposed in a thin layer to room

light for some time, the pyrene mixture becomes tan and

gradually darkens, losing all fluorescence. The rate at which

the brown color develops depends in part on the presence of

solvents and on the amount of exposure to ultraviolet light.

When the material is exposed to ultraviolet light while moist

with carbon tetrachloride, the tan color appears almost at

once; but, when moist with ethyl alcohol, or after drying before

being examined under ultraviolet light, development of a visible

color-change requires several days. Although ultraviolet light

accelerates the reaction, samples of the material kept in

darkness show a gradual change in color, also.

The reaction appears to be a surface reaction, and its

course is influenced by the adsorbent used. No visible change

was observed with pyrene adsorbed on paper or on a plate coated

with kieselguhr G, and no colored product has been obtained

from pyrene on activated charcoal. On membrane filters

prepared from cellulose esters (Millipore and Polypore filters)

or from polyethylene (Mipor solvent-resistant filters), pyrene

turns tan rather slowly. On chromatographic alumina, the

reaction of pyrene and air gives a reddish color (instead of

the yellowish-tan observed on silica gel G.

)

By elution of the brown material formed from pyrene

adsorbed on silica gel G , and chromatography of the eluate,

five products were obtained in sufficient quantities to observe

their UV spectra. Three of these are yellow, with yellow or

red fluorescence on silica gel G, one is reddish, and one is

20
purple. (Prom pyrene on alumina , the proportions of the yellowproducts obtained are relatively small and the purple product

seems to preponderate.) Work is progressing on (a) the

separation of the reaction products from the large excess of

unreacted pyrene and (b) the identification of these products.

Studies are also in progress on the role of the adsorbent,

the effect of light, the effect of concentration, and other

variables

.

Two of the products described in table 5 have been

identified as 1,6-pyrenedione and 1,8-pyrenedione by comparison

with authentic samples prepared by methods reported in the

literature [6]. Spectral studies suggest the presence of at least

twelve other substances in the reaction mixture. Of these,

five have been obtained in sufficiently pure form to be

characterized by their spectra, but these have not yet been

identified. In light of the reports of the production of

jihydroxyanthraquinones by the photochemical oxidation of

anthracene on alumina [12], it is possible that the polar,

purple and gray materials obtained from pyrene may be

hydroxylated quinones.

In an attempt to identify certain dark-purple compounds

separated by thin-layer chromatography, preparations of

1,6-pyrenediol , 1 ,8-pyrenediol, and 1,4-pyrenedione were

undertaken for use as reference compounds. The diols were

separated in the form of the crystalline acetates. The

4,5-dione was separated by column chromatography.

21
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22
For small-scale work, the proportion of hydrocarbon to

particulate matter was 300 mg of pyrene adsorbed on 5 g of

silica gel G; this was spread in a thickness of ca_. 300 urn on

glass plates (20 x 20 cm), giving an approximate distribution

2

of 0.75 mg of pyrene per cm . The coated plates were

irradiated directly with a UV lamp (>270 nm) by double

exposure; namely, 10 to 12 hr on one side and then 8 to 10 hr

on the other. By double exposure, the yield of oxidation

products was raised by 25 to 35$.

For work on a larger scale, a solution of 3 g of purified

pyrene in hexane was mixed with 50 g of a solid support (e.g.,

silicic acid), the hexane was evaporated off, and the solid

was dried and powdered. The powder was stirred magnetically

(egg-shaped stirrer) in a stoppered Pyrex flask under

irradiation (>270 nm) for 100 to 120 hr (or 200-240 hr),

and then extracted with warm glacial acetic acid. The extract

was evaporated to dryness, weighed, and resolved by column

chromatography with a suitable solvent. The resulting fractions

were checked by thin-layer chromatography, identified,

evaporated to dryness, and weighed.

By treatment of 3 g of pyrene on 50 g of silicic acid

for 120 hr , 2.95 g of material was recovered. Thin-layer

chromatography revealed at least 8 spots; three were

fluorescent, two were purple, one was orange, one was yellow,

and there was a dark-brown spot which did not migrate with the

solvent. By column chromatography of this mixture (2.95 g)

on silica gel with glacial acetic acid as the solvent, there

was obtained 2.8l g of unreacted pyrene and _ca.0.l4 g (ca.

4.5$) of oxidation products. Further column chromatography

of this mixture (0.l4 g) gave 0.042 g of 1,6-pyrenedione

23
{l.hfo oxidation), O.O36 g of 1 ,8-pyrenedione (l.2$), 0.008 g

of 1,6-pyrenediol (0.3$), 0.012 g of 1 ,8-pyrenediol (0.4$),

and a small residue that probably contained other hydroxylated

pyrenes

.

From the preliminary results on the photo-oxidation of

pyrene on silicic acid and on silica gel, the following

conclusions were drawn.

(1) The ratio of 1,6- to 1 ,8-pyrenedione is

approximately 1:1.

(2) The proportions of products may depend on the nature

of the solid support; for example, the proportion of

1,8-pyrenedione produced with silica gel differed from that

obtained with silicic acid.

(3) More of the hydroxylated pyrene material was

produced by 220-265 nm radiation than by the radiation >270 nm.

4 . Pyrene on Other Solids

The oxidation of pyrene on alumina was found to yield

relatively small proportions of the pyrenediones and

considerably more of the darker-colored materials. A purple

substance isolated from this reaction has an absorption band

at a longer wavelength (58O nm) than was observed with any of

the products obtained on silica gel. The isolation of other

products of this reaction is continuing.

Preliminary studies have shown that pretreatment of the

silica gel or alumina with acid or base has a considerable

effect on the character of the reaction products.

Pretreatment with a solution of potassium hydroxide appears

to increase the proportion of darker-colored materials produced,

whereas, after treatment with hydrochloric acid, a browner

color is observed.

24
Pyrene shows considerable change in color on colloidal

silica also, and some changes on zinc oxide or magnesium oxide.

Other hydrocarbons which have been found to undergo color

changes on silica gel or alumina include anthracene,

phenanthrene, benz[_a]anthracene, triphenylene, and perylene.

Investigations on the nature of these reactions are

continuing. The formation of cation radicals, such as is

observed on some silica —alumina catalysts [13] may be

involved in the mechanism of the reaction. The products of

oxidation on zinc oxide, asbestos, or acetylated cellulose

(21$) were found to be quite different from those previously

found for oxidation on silica gel or alumina. In particular,

very little of the 1,6- and 1,8-pyrenediones (which are among

the major products obtained on silica gel or alumina) were

found. It was discovered that pre-treatment of alumina or

silica gel with acid increases the proportion of diones

obtained in the reaction, whereas pre-treatment with a base

decreases the proportion of diones (especially the 1,6-dione),

and increases the proportion of anionic purple and pink

materials related to the diones.

5. Perylene on Silica Gel G

A sample (200 mg ) of purified perylene (mp 278-280 C) was

dissolved in 15 ml of hot toluene (reagent grade) and mixed

with 6 g of silica gel G. The slurry was spread on a glass

plate (20 x 20 cm) which was then heated at 90 C for 10 min.

The layer was now illuminated by ultraviolet radiation

(>270 nm) at 30 to 35 C by the double-exposure procedure for

72 hours. By this time, the original bluish-yellow

fluorescence had lost its intensity, and the white background

had become tan in color. The mixture was extracted with

25
acetone, the extract was evaporated to dryness, and the

residue was examined by thin-layer chromatography on silica

gel G., using 18:1:1 benzene —N,N-dimethylformamide —glacial

acetic acid. From the top to the bottom, at least five spots

were distinguishable: (l) yellow (strong; light-blue

fluorescence) unreacted perylene; (2) orange-brown (weak)

unknown; (3) light brown (strong; light-green fluorescence);

(4) yellow (medium strong; not fluorescent); and (5) dark

brown (strong; not fluorescent) unknown.

Spot 3 was tentatively identified as consisting of

4-oxo-4H-benz[ de] anthracene -7 , 8-dicarboxylic acid or its

anhydride. Spot 4 was positively identified as 3,10-perylenedione by comparison of R values and of ultraviolet and visible

spectra with those of an authentic specimen.

6. Other Hydrocarbons on Silica Gel G or Alumina

Work was begun on the photochemical oxidation of

fourteen other hydrocarbons on particulate matter. Phenanthrene,

triphenylene, chrysene, and picene did not show rapid

photochemical oxidation when adsorbed on either silica gel or

aluminum oxide. These four hydrocarbons are structurally

related and possess no highly active positions. Many of the

other hydrocarbons were quite similar in behavior to pyrene

.

Benz [a.] anthracene and benzo[ ajpyrene reacted more rapidly than

did pyrene.

A more quantitative study was made of the effects of

experimental variables on the oxidation of pyrene adsorbed on

alumina and silica gel and in addition, the behavior of pyrene,

benz[ ajanthracene, and similar hydrocarbons on a variety of

other particulate materials was investigated.

Studies were continued on the identification and

26
quantitative determination of the products formed by treatment

of benztaj anthracene (adsorbed on silica gel and aluminum

oxide) with light and oxygen.

7. Anthracene, and Pyrene, on Air-borne, Particulate

Matter from Cincinnati, Ohio

A sample of air-borne, particulate matter, collected in

Cincinnati, Ohio by the Taft Sanitary Engineering Center and

freed of polycyclic aromatic hydrocarbons by exhaustive

extraction with benzene and then with acetone —methanol, was

presented to us by Elbert C. Tabor.

A sample of anthracene adsorbed on this particulate matter

was photo-oxidized for 24 hr under ultraviolet light (366 nm).

The resulting mixture was successively extracted with benzene,

acetone, and methyl alcohol. The product was examined

by thin-layer chromatography on silica-gel sheets (Eastman

Chromagram), with the following developers: hexane, benzene,

chloroform, ethyl acetate, acetone, and glacial acetic acid.

The spots were visualized under ultraviolet lamps at 366 and

353.7 nm. Most of the material was unchanged anthracene; a small proportion was anthraquinone; and some polar material

j (which may have come from the silica gel itself) remained at

the origin.

Pyrene (0.5 g) adsorbed on this particulate matter (5 g)

was photo-oxidized for 240 hr at 32 +3 C with ultraviolet

light filtered through Pyrex glass (A>270 nm). Successive

extraction of the reaction mixture with acetone and toluene

yielded a trace of oxidation products. Examination of this

on a thin-layer chromatogram (silica gel G and ethyl acetate)

revealed a very weak, yellow spot of 1,6-pyrenedione

MeOH

(Tvmax 429 and 449 nm); no other components could be identified.

27
8. Pyrene on Maryland Soil

A sample of Maryland soil was freed of polycycllc

aromatic hydrocarbons by successive, exhaustive extraction

with water, acetone, toluene, and ethyl alcohol, and was dried

at kO C to constant weight.

Pyrene (2.5 g) was adsorbed on 50 g of this Maryland

soil, and irradiated for 240 hr at 3^+3 C in the presence

of air, as for pyrene on Cincinnati dust. Extraction of the

reaction mixture with acetone and then with toluene gave a 1.2 to 1.8$ yield of reaction products (the balance being

unreacted pyrene). Thin-layer chromatography (silica gel,

ethyl acetate) revealed at least 8 spots. On spraying the

chromatogram with 10$ sulfuric acid in methyl alcohol, and

heating for 10 min at 9° C, fluorescent spots No. 1 and 7

disappeared, indicating that these spots consisted of

hydrocarbons. Spot No. 7 is slow moving, and has a strong

blue fluorescence. Spot No. 1 is unreacted pyrene. The work

is being continued.

9. Pyrene on a Silica Gel —Alumina Mixture

Alumina was pretreated by washing with acid and then with

water; it was dried to constant weight. A 1:1 mixture of

silica gel and this alumina was prepared, and pyrene was

adsorbed on it. The mixture was irradiated, as in the previous

experiment, at 32 + 3 for 240 hr. On extraction, pyrene was

revocered, together with a 3.5 to 3.8$ yield of oxidation

products. By thin-layer chromatography, the major components

were identified as 1,6-pyrenedione and 1,8-pyrenedione . Trace

amounts of diols were detected.

28
2. KINETIC BEHAVIOR OP SUGARS IN SOLUTION

( H. S. Isbell , H. L. Frush, and C. W. R. Wade)

A

.

Introduction

Differences in rates of reaction have been widely used

by chemists for determination of structure and study of

reaction mechanisms, but techniques based on reaction kinetics

have not been generally recognized as an important and distinct

area of analytical chemistry. In many instances, kinetic

methods open up exciting new avenues of research, and reveal

previously unknown phenomena.

In our last report, we pointed out that we are studying,

by means of radiochemical and isotopic methods, the main

processes involved in the transformations of sugars in

alkaline solution. These Include (a) conformational changes,

(b) mutarotation reactions, (c) enolization reactions, (d)

condensation and cleavage reactions, and (e) rearrangements

of the saccharinic acid type. Knowledge of the manifold

reactions that the sugars undergo in solution is essential for

adequate understanding of biological chemistry, because all

areas of biological chemistry touch in some manner on

carbohydrate chemistry. For this reason, some of our earlier

work on the composition of sugar solutions will be reviewed,

and then some of our work in progress will be described briefly.

B. Conformational Analysis

1. Pioneer Work at NBS

As pointed out on page 46 in the Westheimer Report [l4],

in recent texts, and in review articles [15 to 17]* consideration

of the conformation, or shape, of molecules in solution has

become extremely useful for understanding chemical reactions

and for directing them to desired products. In view of the

extremely important position that conformational analysis has

29
assumed, it is of interest to review in historical perspective

our work on this subject.

Our laboratory was the first to attempt to correlate the

effect of conformation on reaction rates and properties of

organic compounds. In 1929, Haworth suggested that certain

sugar derivatives have a strainless ring structure, and that

several forms or "conformations" are possible. In 1930, the

ring structure of the sugars was the subject of a bitter

controversy. Hudson contended from comparisons of optical

rotatory power, that certain sugars and glycosides which

Haworth had shown to possess 1,5 rings do not have this

structure, but have 1,3 and 1,4 ring structures. Studies of

the oxidation of the sugars with bromine did not support

Hudson's hypothesis, and Isbell sought to ascertain how the

sugars that Hudson considered exceptional differ one from

another. Thus, in 1930, Isbell [ 18] wrote, "The parallelism

between the optical rotations of the a- and p-forms of

4-glucosido-mannose with the corresponding forms of mannose

indicates that neither a- nor p-mannose has a 1,4 ring

structure. We cannot definitely state at this time which

form of mannose has a structure similar to the normal form of

glucose. It is entirely possible that Hudson's classification

of ring structures is in reality a classification of a new

type of isomerism. It seems reasonable to presume that there

is a difference in the structures of a- and (3-mannose, the

exact nature of which must remain for the future to determine."

In the next few years, numerous sugars and sugar derivatives

were prepared and studied, to reveal unknown structural

differences and to ascertain the effect of configuration on

these differences. The following quotation from the Journal

of Research NBS In 1937 [19] is noteworthy, because it was the

30
first attempt to correlate the effect of conformation on reaction

rates and to classify sugar derivatives according to what we

now call axial and equatorial substituents

.

"CONFORMATION OF THE PYRANOSE RING

It was pointed out by Haworth that various geometric

forms are possible fbr the pyranose ring. Thus if the

carbon and oxygen atoms comprising the ring were coplanar

the valence angles would be larger than 109°, required to

give a strainless ring; if the ring is strainless the

valence angles for the carbon atoms will be about 109°

and the carbon and the oxygen atoms cannot lie in a single

plane. As measurements of dipole moments for simple

substances containing the pyranose ring reveal that the

oxygen valence bonds form an angle considerably less than

109°, it seems probable that the pyranose ring is bent or

puckered. Although ten or more strainless ring structures

are possible they can be reduced to three types, as

represented in fig. 4: (i) a trans structure in which four

atoms lie in one plane , and the two remaining atoms lie on

opposite sides of this plane; (il) a cis or boat-shaped

structure in which 2 atoms lie on the same side of the

plane formed by the four remaining atoms; (ill) and (IV)

structures in which the carbon atoms are coplanar and the

ring-oxygen is not in the same plane. The monoplanar

strained model is represented by (V).

The X-ray investigations of Cox and others indicate

that the carbon atoms forming the ring in many sugars are

coplanar, while the ring-oxygen lies outside the plane of

the carbon atoms. Two structures of this character are

possible for each hexose; in one of these the sixth carbon

lies approximately in the plane of the carbon ring, as in

(ill); in the other it lies opposite the ring-oxygen,

directed towards the first carbon as in (IV). At this

time there is not a satisfactory chemical method for

deciding which models correspond to the free sugars.

Several sugar derivatives exist which for stereomeric

reasons are limited to certain models. Thus trimethyl

1 ,4-anhydroglucopyranose requires the conformation of a boat-shaped ring in which carbons 1 and 4 lie in the

trough. The model which will serve for this compound,

however, will not serve for 2,3-4 ,6-diacetone a-methyl

mannopyranoside because in order to form a 1,4-anhydro

derivative the hydroxyl on carbon 4 (which is the same

31
for mannose and glucose) must be directed towards

carbon 1 which is in opposition to carbon 6. It is thus

apparent that the conformation of the pyranose ring is

not the same for all sugar derivatives. It is possible

that the various modifications exist in dynamic equilibrium. Consequently the particular modification which

results after condensation with a second group is not

necessarily the same modification as that of the original

Figubb 4.

Models for the pyranose ring.

sugar. It seems probable, however, that structurally

related substances such as a-_£-arabinose, a-d-galactose,

a-d_-fucose, a- 1_- fructose, a-ji-a-mannoheptose, etc.,

have similar ring conformations. It can be observed from

models (ill) and (IV), illustrated in figure 4, that the

ring-oxygen might be directed to either side of the plane

of the carbon atoms. But since no more than two isomers

of any one ring type are known, it seems probable that

32
each free sugar occurs in only one ring conformation.

The separate sugars, however, may have different conformations . If the sugars had the coplanar ring, the alpha and

beta positions would be symmetrically located with

respect to the plane of the carbon-oxygen ring, and

there would be no fundamental difference between the

alpha and beta sugars. But if the oxygen and carbon

atoms forming the ring did not lie in one plane, the

alpha and beta positions would not be symmetrically

located with respect to the carbon-oxygen skeleton, and

would be influenced to different degrees by the oxygen

of the ring. Presumably this unequal influence would

result in differences in the reactivity of the alpha and

beta modifications. The positions of the hydrogen and

hydroxyl groups of carbons 2,3, and 4 might also affect

the reaction rates of the alpha and beta sugars, either

directly or by causing alterations in the conformation

of the pyranose ring to give various strainless ring

isomers. Thus a comparison of the reaction rates of the

alpha and beta pyranoses should provide information about

the conformation of the pyranose ring."

In the same paper, it was pointed out that, "in every

case the beta aldose is oxidized by bromine water more rapidly

than the alpha, and the equilibrium solution contains two or

more substances which differ in reaction rates," and that

"The marked difference in the reactivity of the alpha and beta

sugars supports the hypothesis that the pyranose ring is

dissymmetric and contains a strainless rather than a monoplanar

strained ring. Attention is called to 1,4-anhydroglucopyranose

and to 2,3,^,6-diacetone a-methyl mannooyranoside which for

stereomeric reasons cannot have like strainless ring structures.

Thus the conformation of the ring may vary from sugar to

sugar . . . Consequently, rotational differences may not

correspond to the rotations of the separate asymmetric carbons

but rather to the difference in the rotations of the molecules

as a whole." As a result of these ideas, Isbell sought to

classify the alpha and beta sugars according to what we now

33
call the axial and equatorial positions of the anomeric

hydroxy1 group. At this early date, the concept of conformation

was just beginning to emerge, and there was no basis for actual

assignment of a specific conformation.

Attempts were, however, made to correlate optical rotation,

mutarotation, and bromine oxidation measurements for reducing

sugars having like arrangements for the five carbon atoms

comprising the pyranose ring. The beginning of conformational

analysis was made by Isbell and Frush in 19^0, in a paper [20]

entitled, "Alpha and Beta Methyl Lyxosides, Mannosides,

Gulosides, and Heptosides of Like Configuration." At this time,

an unexpectedly large influence of the configuration at carbon

atom 3 on the properties of the glycosides was noted, and the

close resemblance of each pentose to the corresponding hexose

which has the trans configuration for carbon atoms 3 and 5 was

pointed out. After 26 years, we can now understand and

rationalize this important observation (see page 57 ). Interest

in conformation continued at NBS, and many sugars and sugar

derivatives were prepared to study this subject in the period

1937 to 19^7.

Papers by Hassell and Ottar [21] in 19^7 and by Reeves

[22] in 19^9 established the conformations which are now

accepted for the more important sugars. Modern interest in the

relationship between conformation and chemical reactivity was

awakened by Barton [23] in 1950, when he directed attention

to differences in the chemical behavior of equatorial and

axial substituents of cyclohexanes (see page 71 of [15]).

Even before that time, the Section had in progress a project supported by the Office of Naval Research entitled,

"The Investigation of the Structure, Configuration, and Ring

Conformations of the Sugars and Their Derivatives by Infrared

34
Absorption Measurements."

Work initiated under this project led to the publications

cited in reference [24]

.

2. Current Work at NBS

The present intense interest in conformational analysis

arises from recognition that consideration of conformation

greatly enhances the abilities of chemists to direct chemical

reactions to desired products. Knowledge of conformational

free-energy differences has enabled chemists to predict the

favored conformation, and new techniques , particularly NMR5 have provided the experimental means for definitely establishing the particular conformation adopted by an organic compound.

a. Oxidation of Aldoses by Bromine. Conformation

is a dynamic property; non-bonding interactions determine the

stability or instability of conformers, but a change in

conformation may occur during the reaction. In some instances,

reactions may take place either with a change in conformation

or with a change in configuration.

In development of this concept, Isbell [25] pointed out

that the oxidation of beta anomers of aldoses by bromine takes

place in large measure directly, but oxidation of the alpha

anomers takes place (a) by direct oxidation with change in

conformation and (b) by way of anomerization. Recently, it

was found [26] that "in each Instance, the extent of the

reaction that takes place by way of anomerization parallels

the conformational stability of the anomer. Aldoses which

have an axial hydroxyl group at CI in a highly stable

conformation (e.g., a-D-glucopyranose-CA* and a-D-galactopyranose-CA), resist the change in conformation necessary for

The system for indicating conformation is described in [27].

35
direct oxidation, and react largely through anomerization

(change in configuration). Aldoses of lower conformational

stability (e.g., a-D-lyxopyranose-CA, a-D-talopyranose-CA,

and a-D-mannopyranose-CA) react chiefly by a change in

conformation without a change in configuration.

The rate of the direct reaction depends on the difference

in free energy between the reactants in the ground state and

in a transition state in which the anomeric oxygen atom lies

in the same plane as CI, C2, C5, and the ring oxygen atom.

The free energy required in order to reach this transition

state depends on the structure and the configuration. With

molecules having an axial hydroxyl group at CI in a highly

stable conformation, much energy is required for so changing

the conformation as to bring the hydroxyl group into an

equatorial position. a-D-Glucopyranose and a-D-galactopyranose

have high stability in that chair conformation having an axial

hydroxyl group at CI; this fact accounts for the low rates of

oxidation and the tendency of these sugars to react through a

change in configuration rather than conformation."

It should be noted that the rate of oxidation does not'

depend on the free energy of the sugar, but rather on a freeenergy difference. In fact, some of the most stable sugars

are oxidized at the highest rate. Differences in the rates

for the equatorial anomers may be ascribed to steric hindrance,

but such hindrance may also be considered to be a factor in

the energy of activation. In the oxidation of aldoses with

bromine, the relative importance of the two reaction-paths

depends on the reaction conditions; and hence, the relative

rates, as such, are of little consequence. It is highly

desirable, however, to understand the basic phenomena involved.

Many reactions involve concurrent changes in conformation and

36
configuration, and development of methods for measuring the

two types of change is highly important.

We have recently devised a new method for determining the

two types of reaction in the oxidation of aldoses with bromine.

The method is based on a difference in the isotope effect for

anomerization of the aldose in HO and DO, and the isotope

effect for the direct oxidation of the aldose. There are wide

differences in the isotope effects, and the relative importance

of the paths may be estimated from these. If, as claimed by

Barker, Overend, and Rees [28], the rate-determining step in

the oxidation of a- aldoses consists very largely of anomerizationof the a-aldose to the B-aldose, the rates of oxidation of the

a-aldoses in DO should parallel the rates of anomerization.

Measurements in progress will ultimately answer this question.

b. Influence of the Configuration of Carbon Atom 3

on the Conformation of Aldopentopyranoses . Each pentose is

configurationally related to two hexoses, which differ in the

configuration of C-5. Isbell and Frush [20 ] pointed out that

each pentopyranose closely resembles, in properties, the

corresponding hexopyranose in which C-3 and C-5 have trans

configurations. Thus, L-arabinose, D-xylose, D-lyxose, and

L-ribose respectively resemble D-galactose, D-glucose,

D-mannose, and D-talose more closely than they resemble

L-altrose, L-idose, L-gulose, and L-allose. It was also noted

that "carbon 3 lies opposite the oxygen of the ring and its

attached groups appear to be in a particularly favorable

position to influence the conformation of the ring" (page 133

of [20] ). This influence may now be explained by conformational

analysis

.

In the hexopyranose series, when the - configurations of C-5

and C-3 are opposite, both the CH OH group of C-5 and the OH

37
group of C-3 are equatorial in the stable, CA conformation.

In the pentopyranose series, the large Influence of the OH group

of C-3 may be attributed to a dipole —dipole interaction with

the ring oxygen atom. The molecule assumes that conformation

in which this dipole —dipole repulsive force is at a minimum,

i.e., in which the OH group of C-3 is equatorially oriented

(see figure 3). The conformation then resembles the CA

Equatorial OH at C-3 Axial OH at C-3

Figure 3. Influence of OH group at C-3; direction

of dipole moments.

conformation of the hexopyranose having opposite configurations

for C-3 and C-5. This concept accounts for the previously

noted influence of the configuration of C-3 on the conformation

of the pentopyranoses. The explanation somewhat resembles" that

developed by Lemieux and Chu for the "anomeric effect" (see

page 375 of [15] and page 738 of [17].

C. Mutarotation Reactions

Recent progress in NMR and gas chromatography of silyl

ethers has resulted in a renaissance of interest in the

mutarotation reaction and in the study of the composition of

sugar solutions. In continuation of our study of anomerizations

and pyranose-furanose interconversions, we have made numerous

38
new measurements of the mutarotations of D-fructose and D-glucose

In DO and H

p

0. The work, part of which was described in our

last report, has been incorporated in a manuscript entitled:

"Deuterium Isotope Effects in a-p-Pyranose and Pyranose-furanose

Interconversions . " An abstract of the paper is as follows:

"Rates of mutarotation, activation energies, catalytic

coefficients, and isotope effects are reported for the

mutarotations of a-D-xylose, a-D-glucose, and p-D- fructose in

HO and in DO at 20° and at 3.9 °C. The isotope effects

(k^/k ) for the mutarotation of p-D-fructose (pyranose-furanose —n —JJ — interconversion) parallel in striking manner the 'isotope effects

for the mutarotation of a-D-glucose (a-p pyranose anomerization)

.

For both sugars, the isotope effect is highest in neutral, and

lowest in strongly acid, solution; the parallelism of the values

obtained for the isotope effects for the two reactions under a variety of conditions shows that the rate-determining steps in

the two reactions are much alike. Presumably, in both instances,

the overall mutarotation arises from concurrent reactions

operating on different species of the sugar and showing

substantially different isotope effects.

The following isotope effects were found for the

mutarotation of a-D-glucose at 20 C:

^H

3

0+^D

3

0+

= X - 39; V,cA>2

=

3 - 87; and W^OD " 3 - 03 >

where k - and k - represent the effects of base catalysts in

H_0 and DO, respectively.

The following isotope effects were found for the

mutarotation of p-D-fructose at 20 °C:

*H

3

oV%

3

o+ " lri9 > \(A)2

o

3 - 87; and W±oh - 2 - 88 -

Mechanisms are presented for the several concurrent acid- and

base-catalyzed mutarotation reactions."

39
D. Enolizatlon Reactions

1 . Measurement of Rates of Primary Enolizatlon

With reference to our study of the behavior of sugars in

alkaline solutions, the method which we developed for measuring

the primary enolizatlon of sugars mentioned in our last report

r 29] has been applied to D-glucose under a variety of conditions,

and to several other sugars under limited conditions. The

method involves treating the sugar with a base catalyst in the

presence of tritiated water, whereby the sugar molecule is

converted into an enediol which, upon treatment with acid,

reverts to an isomerized sugar mixture containing one tritiumlabeled atom for each enediol structure formed. All labile

tritium is removed by freeze-drying, and the sample is assayed

for the amount of tritium fixed. The percent enolizatlon is

calculated by assuming that one tritium-labeled atom is fixed

for each enediol molecule formed. The following example

illustrates the technique: Eighteen mg (0.1 mmole ) of D-glucose

is placed in a 4-ml vial, fitted with an air-tight rubber cap

through which hypodermic needles may be inserted. Two needles

are inserted in the stopper, nitrogen (free from carbon

dioxide) is passed through the vial for five min at a rate of

50 ml/min to displace the air. Next, 0.5 ml of 0.035 N NaOH

(2 e 212 mCi of tritium/ml) is injected into the vial and, after

removal of the needles, the sealed vial is placed in a test

tube containing enough water to provide for heat transfer but

not enough to reach the top of the vial. The tube is placed

in the constant-temperature bath illustrated in figure 4.

At the end of the reaction time, the vial is removed from the

bath; two hypodermic needles are inserted, and carbon dioxide

is passed into the vial for 5 min. The solution is left in an

40
Figure 4. Apparatus used in the determination of

enolization by radiochemical techniques.

atmosphere of carbon dioxide for 30 min, after which 3 drops

of glacial acetic acid are added.

The acidified solution is quantitatively transferred with

15 ml of water to a 125-ml, long-necked, round-bottomed flask,

and freeze-dried . After three additional freeze-dryings, by

use of 15 ml of water each time, the residue in the flask is

dissolved in exactly 5 ml of water, and three 0.1-ml aliquots

are removed and assayed for tritium.

All assays were carried out in a liquid scintillation

spectrometer, by use of a p_-dioxane scintillation solution and

the technique described in our last report (page 30 of [29]).

The ratio of the counts per second per mmole of sample to

the counts per second per milliequivalent of the tritiated

water used (as reagent and solvent) times 100 gives the percent

41
enolization. Values for the pseudo first-order rate-constant

were calculated from the relationship

k = 1/t log A/( A-X)

where A is 100 and X is the percent enolization at time t (hr).

Some results for D-glucose are given in table 6. As

expected, the rate of enolization increases with the temperature

and with the concentration of the base. As the reactions

proceed, the solutions turn slightly yellow, and the overall

rate-constant changes, presumably from a variety of reactions.

The initial reaction-rate is characteristic of the primary

enolization. The subsequent changes give rise to pseudoequilibrium mixtures which are being studied by isotopedilution techniques.

2. Pseudo-Equilibrium States

Work was continued on the quantitative determination of

the products of rearrangement of pentoses, hexoses, and

heptoses under conditions approaching equilibrium. Considerable

progress was made on the preparation of carbon-l4 labeled

D-altrose, D-allose, D-talose, D-gulose, and certain heptoses.

These materials are needed for study of the rearrangements by

means of the radiochemical methods which we have developed.

E. Reversible Condensation and Cleavage Reactions

Although our tracer methods provide an exciting new method

for study of the condensation and cleavage reactions, it was

not possible to pursue this subject during the oast year, but

we hope that we shall be able to return to it shortly.

F. Saccharinic Acid Rearrangements

To test the mechanism proposed in the last report (page 55

of [29]) for rationalization of the distribution of carbon-l4

in the "a/'-D-glucosaccharinic acid prepared by Sowden and

co-workers [30} from D-mannose, we rearranged samples of

42
0)

coOo3

!-)60I OH

4-1oco

cON

•H

i-lOcw

0) r-l

cO H

enO £

5-1 43

J-> <t C

CO

COoo

rl 43

en

01 S-i +J 43

CO pi CM£

.N'O

l-l 43

u XI

3> «n

4-1O

J-i 43

O6EH

COeou<

Oua

CU H

CO PioCO

CO i-H o e o ->^ 3 cu T-A r-l O Q on i

J3

43U

43O

J-i CO00

CO 33O

cO

CNin

On

<t

CN oo

00

CM CM m in

co co m

r^ CN • O CO CN

r^

O • • <r CN iH

<t vO i-l • 9 • <t CO CM

CM CM

CM r-l

r^« vo r^ ct> vo • • • • • i—i

r-> cm <f <—i

CM fH r-l

00

O CN CM

O • • • • • ON CO ON CM i—I

in

<tmm

<N<t

00m

rom o

<t

CM cm co

ON

<t

CN in cmom

<t in cooCO

<tON

<to

CM

<N

mm co com in r-» i^ oo

r-l

O

mmCOo mcoo

00 *£>

i-l CO CM

r-l CO 00 <r • • • • r-l r-l V0 CO

i-l

CM <t 00 <t • • • • i—I i—I -<!" i—I

i-l

i-l <f r-l CN • • • • I-l !- <t OI-l

r-~ -<t on co o

1—1 I-l CO ON CMCM

CO vo ro r~» r^ u * • p

I-l <t no

r-l r8

r^ <t• ON

O• c

l-H <t u3 CO

r-l

•H

cuN

CM <t 00 •H

r-lOc

<u

00• I-l CO• <*

i-l CM o ctn r-t r-J

CN <t <o ^O

u

COSO CO vO co• CM• i—1

r-l in CM

r-l 3

CO

<t CM <± <t CD 43 o 4-1 CO • • r-l i—1

CO 00 o

<t

r-l <tON vO

X' oCN rlo u • * 9 • • i—1 o CM v£> rH

0)U

S-i <t vO o o (U r^ ON m a. 00 • • • • O I-l ro r^ 0) 43

4J o CO vO <t o m ON CO •H • • • o r-l r-l CN X|c m O cO • O m

r-l CM vO CO CO

CM oo

in

mm in in

r^ r- r^ r^ r^.

oo oo 00 ON oo

r-l i-l i-l r-l r-l d o o d o

<\

CDu

43

1X1

00o

II

43
D-mannose- 1- C and D-mannose-3^ C, and separated the labeled

saccharinic acid. Prior to degradation of our extremely scarce

labeled material, it was necessary to carry out the procedure

with non-labeled compounds to provide carriers and to perfect

our techniques. All of the necessary intermediates have now

been prepared,, but the final analysis has not been attempted.

To ascertain the distribution of carbon-l4 in the

saccharinic acid, it is oxidized with periodate, and the

resulting acetic acid and formic acid are assayed for carbon-l4.

In order to determine the position of the carbon-l4 in the

acetic acid, It is degraded by the series of reactions given

below:

CH 3 C0 2 H - CH 3 C

S N^f^^N C

6 H 5 CHO

N

! H

U C6 H 5 CH = CH-C

II

HC

N ~~T^^1

KMnO,

N^k^

IV III Intermediates I through IV should show the same radioactivity if the carbon atom of the carboxyl group contains

carbon-l4, but should show a change If carbon-l4 is distributed

between the CH and COOH groups. However, if the carbon-l4 is

in the CH group only, all of the radioactivity should be

eliminated in passing to compound IV. It is imperative,

therefore, that the purity of each intermediate be known before

assays for carbon-l4 are made.

44
Using the technique of thin-layer chromatography, we

found that nanogram quantities of each intermediate can be

rapidly analyzed for purity. One to five nanograms of the

benzimidazole derivative I, dissolved in methyl alcohol, is

spotted on silica gel, and the chromatograph is developed with

2-butanone . After the plates have been dried, the substances

are detected with ultraviolet light, with iodine, or with

sulfuric acid. The spots are sharply separated, and R values

can be used to identify the impurities.

Because of the very small quantities of intermediates

needed, the speed of the method, and the sensitivity of the

reagents, the TLC technique will be invaluable in processing

products, not only from saccharinic rearrangements, but for

radiochemical assay of acetic acid in general

.

45
3. CARBON-14- AND TRITIUM-LABELED CARBOHYDRATES

( H. L. Frush , H. S. Isbell, and N. B. Holt)

A. Introduction

Under our program on carbon-l4- and tritium-labeled

carbohydrates, services to the scientific public have been

continued by supplying research workers with labeled compounds

that were needed. Because of the competence of the staff in

the field of isotopically labeled carbohydrates, requests are

frequently received for additional kinds of labeled materials

that have become important for biological research. However,

developmental work of this nature has been limited in the past

year, because of curtailment of staff through resignation and

retirement. Nevertheless, improvements have been made in the

14 synthesis of two radioactive sugars, L-fucose- 1- C and

14 L-rhamnose- 1- C . The syntheses are described in this report,

together with an improved synthesis yielding D-glucose- 4-t , D-galactose-4-t , and D-glucitol-4-t . The last-named synthesis

was carried out earlier by Dr. L. T. Sniegoski, as part of a

study of isotope effects in bacterial oxidations.

Work has been continued on analytical and purification

techniques. Notable progress has been made in large-scale

paper chromatography, and in the utilization of carbon-l4

and tritium in double-label techniques. Preliminary studies

of oxygen quenching in liquid-scintillation counting emphasize

the need for further work in this area.

B. Synthesis of q-L-Rhamnose -1- C ( 6-Deoxy-q-L-Mannose -l- C)

1 . Discussion of Method

L-Rhamnose occurs free in a few plants, and as a constituent of many glycosides, certain gums, and various

46
bacterial polysaccharides. It is readily prepared from "lemon

flavin," a commercially available dyestuff obtained from blackoak bark (p. 460 of [31]). We have now synthesized

14 g-L-rhamnose- 1- C and have supplied the pure, radioactive

sugar to numerous workers engaged in biological research. The

method developed for the preparation is outlined below.

L-Rhamnose

I electrolytic oxidation

jCa(0H)

2

Basic calcium L-fuconate

co

2

Calcium L-fuconate

2+ degradation (H

p

+Fe ) 5-Deoxy-L-arabinose

Icyanohydrin reaction (Na CN)

14 Epimeric cyanohydrins- 1- C

hydrolysis, and separation

as lactones

J" 14 1

L-Rhamnono-1 ,4-lactone- l- C 6-Deoxy-L-talonoI j I.- Uj it \ 1,4-lactone- l- C

I reduction (NaHg ) 14

X

L - Rhamno s e

1- C

The L-rhamnose was oxidized electrolytically in the

presence of calcium carbonate, by the method of Isbell and

Prush [32], and the resulting calcium L-rhamnonate was

purified through the intermediate separation of basic calcium

L-rhamnonate [33] . The calcium salt was degraded to 5-deoxyL-arabinose [34,35 3* and this was allowed to react with sodium

14 cyanide- C. After hydrolysis of the cyanohydrins, the

epimeric acids were separated as their lactones. L-Rhamnono47
14 1,4-lactone- l- C crystallizes readily, whereas the epimeric

lactone does not. By reduction of L-rhamnono-1 ,4-lactone- l- C

with sodium amalgam,, and use of the nonradioactive sugar as

14

the carrier, a-L-rhamnose- 1- C hydrate was synthesized in an

overall yield of 20.0 %.

2. Experimental Details

a. Calcium L-Rhamnonate . A mixture consisting of

22.8 g of a-L-rhamnose monohydrate (0.125 mole), 4 g of

calcium "bromide, and 6.5 g of calcium carbonate in 500 ml of

water was placed in a 2-liter, 3-necked flask fitted with two

graphite electrodes (l cm diameter) and an efficient stirrer.

The flask was cooled in ice water, and a direct current of

0.2 ampere was passed through the stirred solution. After

34 hr (6.8 ampere-hr) a test for reducing sugar with Benedict

solution [36] was faint, and the electrolysis was stopped.

The electrolyzed solution was filtered, and the calcium

L-rhamnonate was precipitated as the basic calcium salt by the

addition of 20 g of calcium hydroxide. The precipitate was

collected on a Biichner funnel and washed with lime-water until

free from bromide ion. It was then suspended in water, and

•carbon dioxide was bubbled through the (vigorously stirred)

suspension until the supernatant liquid was almost neutral

(colorless to phenolphthalein) . The calcium carbonate was

removed by filtration, and the filtrate was concentrated under

reduced pressure to a sirup from which amorphous calcium

L-rhamnonate was precipitated by the addition of ethyl alcohol.

The crude product weighed 18 g.

b. 5-Deoxy-L-arabinose . A solution of 5 g 112.5

mmoles) of calcium L-rhamnonate and 0,245 g of barium acetate

in 20 ml of water was mixed with a solution of 0.25 g of ferrous

48
sulfate heptahydrate in 20 ml of water. The mixture was diluted

to a volume of 75 ml, placed in a water bath at 50 C, and

treated with 3 ml of 30$ hydrogen peroxide. When the solution

turned dark brown (about one hr), a second quantity (3 ml) of

30$ hydrogen peroxide was added. After an additional hr, the

mixture was diluted with 25 ml of methyl alcohol and 50 ml of

ethyl alcohol, and filtered. The filtrate was concentrated

under reduced pressure to about 25 ml. Titration of an

aliquot with iodine in alkaline solution [37] showed the

presence of 12.4 mmoles of reducing sugar (49.3$ of the

theoretical yield).

14

c. L- Rhamnono-1 ,4-lactone -l- C. A solution

14 / x containing 2.0 mmoles of sodium cyanide- C (30 mCi ; and 10

mmoles of sodium hydroxide in 55 ml was frozen in a 200-ml,

round-bottomed flask. To the flask was added a solution of

1.13 g (12 mmoles) of ammonium carbonate and 2.1 mmoles of

5-deoxy-L-arabinose in 17 ml of water. The mixture was kept

in a refrigerator for one day, and at room temperature for

four days. Sodium carbonate (1.06 g, 10 mmoles) was added,

and the mixture was heated at 100 C, with the addition of

water from time to time, until the evolution of ammonia ceased.

The solution was passed through a column containing 75 ml of

a cation-exchange resin, and the effluent was concentrated

under reduced pressure to a sirup. The sirup was dissolved

in methanol, and the solution was again concentrated; the

process was repeated a third time. Finally, the residue was

dissolved in a few drops of glacial acetic acid, and the

solution was heated at 70 C for one hour, and then

concentrated in a stream of dry air. Heating and concentrating

14

were repeated, and L-rhamnono-1 ,4-lactone-l- C crystallized

49
on nucleation. The crystals were separated, and a second crop

was obtained from the mother liquor after dilution with 200 mg

of nonradioactive L-rhamnono-l,4-lactone . The two crops were

combined, and re-crystallized from methyl Cellosolve by the

addition of ether; yield, 110 mg, 6.737 mCi. By treating the

mother liquor with additional nonradioactive L-rhamnono-1,4-

lactone, several crops were separated; these were combined and

recrystallized; yield, 2l6 mg, 1.674 mCi, and 428 mg, 2.405 mCi.

The total radiochemical yield (10.8 mCi) was 36. percent. The

mother liquor containing 18.0 mCi (chiefly in the form of

L-isorhamnono-l,4-lactone- l- C ), was held for further work.

14

d. q-L- Rhamnose -1- C Monohydrate . The various crops

14

of L-rhamno-1 ,4-lactone- l- C were reduced in quantities of

about 100 mg per reduction tube, as previously described [38] , by use of 15 ml of water in each tube, and, per mmole of lactone,

9.2 g of 5$ sodium amalgam in the form of pellets [39] and 6.4 g

of sodium hydrogen oxalate. Half of the amalgam and oxalate

were added, and, after these had reacted, the remainder was

added. Finally, the reduction products in the tubes were combined,

and the residual acids were neutralized with sodium hydroxide.

Five volumes of methyl alcohol were added, and the mercury and

precipitated salts were separated on a filter, washed with

methyl alcohol, and discarded. The filtrate was concentrated

under reduced pressure to about 10 ml and diluted with five

volumes of methyl alcohol. The salts that precipitated were

removed by filtration, and the filtrate was concentrated, under

reduced pressure, to remove the alcohol. The sirup was

dissolved in water, and the solution was passed through a column

containing 25 ml of a 1:1 mixture of cation- and anion-exchange

resins. The effluent (which was shown to be salt free by

50
testing with a commercial conductivity meter) was concentrated

to a sirup, and brought to crystallization by the addition of

isopropyl alcohol.

By use of nonradioactive a-L-rhamnose as the carrier,

three additional crops of crystals were removed from the mother

liquor. The four crops, when combined and recrystallized,

yielded 617 mg of a-L-rhamnose

1- C having 5.124 mci of

activity. Further use of carrier, and recrystallization of the

product, yielded a croo of 453 nig, having 874 ^iCi of activity.

The radiochemical yield (6.0 mCi) of chromatograpiically pure

l4 l4 a-L-rhamnose -1- C monohydrate was 20.0$ of the sodium cyanide- C

employed in the synthesis.

C. Synthesis of q-L- Fucose -1- C ( 6-Deoxy-q-L- Galactose -l- C)

Polysaccharides containing L-fucose are found in the cell

walls of marine algae, in a few gums, and in certain sub^t.p- -es

of animal origin, such as red blood-cells, blood serum, gastric

mucin, ovarian cyst fluid, and many other materials of biological

importance. Because of the wide-spread occurrence of L-fucose,

14

the _C- labeled, sugar is a valuable research tool. We have

14 carried out the synthesis of a-L-fucose- 1- C , by a method

14 similar to that described for a-L-rhamnose- 1- C monohydrate.

The overall radiochemical yield was 30.2$. A manuscript

describing this synthesis in detail has been prepared for

publication in the NBS Journal of Research.

D. Synthesis of D-Glucose -4-t , D-Galactose -4-t , and D-Glucitol -4-t

( H. S. Isbell and L. T. Sniegoski)

D-Glucitol- 4-t was needed for the determination of isotope

effects in certain bacterial oxidations, and both D-glucose-4-t

and g-galactose- 4-t have numerous anplications in biochemical

and biological research. All three compounds result from the

synthesis depicted below. Briefly, this synthesis involves the

51
preparation, from D-glucose, of a compound having only one

free hydroxyl group (at C-4). This >CHOH is oxidized to >C=0,

and the latter is reduced with a tritiated reagent to >CHOH

in both configurations ( i.e. , the D- gluco and D- galacto )

.

Because the labeled D-galactose compound is formed only by the

tritiated reagent, whereas the labeled D-glucose compound is

diluted with that which was unoxidized at C-4, the resulting

D-galactose has greater specific activity than the D-glucose.

The synthesis employed is a simplification of that of

Kohn and Kohn [40], which, in turn, derives from the work of

Wolfrom et al. [4l,42] on the aldehydo derivatives of the

sugars. In our work, a radiochemical yield (85$) of D-glucose-4-t

and D-galactose-4-t (considerably better than that of Kohn and

Kohn) was obtained by the use of sodium borohydride-_t in methyl

alcohol (instead of lithium borohydride-t_ in ether) as the

reducing agent, and by the use of ion-exchange resins for

purifying the labeled compounds. D-Glucitol-4-t was obtained

in nearly quantitative yield by the reduction of D-glucose-4-t

with sodium borohydride

.

The synthesis of the labeled compounds from D-glucose (i)

involved the preparation, in sequence, of D-glucose diethyl

dithioacetal (il), 2,3:5^6-di-0_-isopropylidene-D-glucose diethyl

dithioacetal (ill), 2,3:5j6-di-0-isopropylidene-D-glucose

dimethyl acetal (IV), purification of IV through crystalline

2,3'«5j6-di-_0-isopropylidene-4-0_-(p_-nitrobenzoyl )-D-glucose

dimethyl acetal (v), saponification of V to IV, oxidation of

IV to 2,3:5j6-di-_0-isopropylidene-D-xylo -hexos-4-ulose dimethyl

acetal (VI), reduction of VI with sodium borohydride-_t

,

hydrolysis to a mixture of D-glucose-4-t (VIl) and D-galactose

4-

t

(VIII), chromatographic separation of VII and VIII, and reduction

of VII to D-glucitol-4-t.

52
I C

2 H

5 SH

CH3OH

JT Acetone TTT CdC03 jy P 2 NC6 H

4 CO.C! HgCI

2 (crude)

(H

3 C)

2 C.

HC(OCH

3 ),

I HCONo

*OCH

I HCOCO C H,N0 o 6 4 2 HCO^

I >(CH 3

) 2

CH30H

(H

3 C)

2 C

H

2 CO

/*

HC(OCH 3

) 2

HCO

I >(CH 3

) H 2 CO

3'2

CrOi

V IV

(H

3 C)

2 C:

HC(OCH 3

) 2

I JHCO^

OCH

I c = o

I HCO^

NaBT4

H

2 CO

C(CH

3

) 2

CHO

I HCOH

I HOCH

I TCOH

I HCOH

1CH 2 OH

CHO

I HCOH

I HOCH

I HOCT

I HCOH

ICH 2 OH

VI VII VIII
Compound II was prepared from 119 g of anhydrous I, 101

ml of concentrated hydrochloric acid, and 100 g of ethanethiol

according to the procedure in [4l], After one recrystallization, the yield was 102 g (54$), mp 127.5 °C (lit. value 127 °C).

Compound III was prepared in crude condition from 101 g

of II, one liter of acetone, and 200 g of anhydrous cupric

sulfate. The yield of sirupy III was 120.8 g. A total of

40 g of III was converted into IV by treatment with mercuric

chloride and cadmium carbonate by the method of Wolfrom e_fc al . [42] as modified by Kohn and Kohn [40] in the use of absolute

methyl alcohol instead of ethyl alcohol, and a lower temperature

(65-70 °C instead of 70-80 °C). The sirupy IV (20.2 g) was

purified by conversion into V. The yield of re-crystallized

product was 8.27 g (27.6$ based on IV), mpl04.5-106 °C (lit.

value, 106-107 °C).

A 4-g sample (8.8 mmoles)of V was saponified with sodium

methoxide in methyl alcohol to sirupy IV (2.89 g), which was

oxidized with 2.89 g (28. 9 mmoles) of chromium trioxide in dry

pyridine. The sirupy product (2.22 g) containing VI reduced

Somogyi solution, and showed a carbonyl band in its infrared

spectrum.

From Kohn and Kohn's data [4o], we estimated that about

a quarter of the 2.22 g of sirupy product, or 555 mg (l.8 mmoles),

was the 4-ketose, compound VI. Without Isolation of this, the

sirup was reduced with 19 mg (0.52 mmole, 1.8l mCi) of sodium

borohydrlde-jt in anhydrous methyl alcohol at °C. Half of

the sodium borohydride-_t was added four hr after the first

half, in order to minimize any isotope effects which might

occur during reduction. After 24 hr, 20.4 mg of ordinary sodium

54
borohydride was added to ensure complete reduction. After

an additional 24 hr, no reducing sugar was present.

The solution was concentrated several times, with addition

of methyl alcohol; cation-exchange resin was added to decompose

excess borohydride and remove sodium ions, and concentration

and addition of methyl alcohol were repeated several times.

After removal of the resin by filtration, and concentration

of the solution to a sirup, the product was hydrolyzed by

adding 25 ml of 0.3 N hydrochloric acid and heating at 98-100 C

for 90 min. A test chromatogram using water-saturated tert - pentyl alcohol showed the presence of only D-glucose and

D-galactose. The D-glucose- 4-t and D-galactose4-

t

spots,

cut out and assayed with a liquid scintillation counter, gave

248 and 332 cps, respectively. Hence, the D-galactose

configuration is favored in the reduction.

The solution was passed through a column of mixed (l:l)

cation- and anion-exchange resins and concentrated, affording

2.05 g of sirupy D-glucose- 4-t (VIl) and D-galactose- 4-t (VIIl).

Radioanalysis of the product showed 1.54 mCi (85.1$ overall

radiochemical yield). A crop of 52 mg of D-galactose-4-t

(3.06 Ci/mg, 159 M-Ci) was obtained by recrystallization from

water—methyl alcohol-isopropyl alcohol. The remaining

material, containing D-glucose-4-t and D-galactose-4-t , was

separated by large-scale paper chromatography on Whatman seedtest paper, with a wick of Whatman No. 3 MM paper (.see page 61).

A triple development was made, with 1-butanol —pyridine —water

(45:25:40) as the solvent. The D-glucose-4-t and D-galactose- 4-t

were eluted from tne paper, and eacn solution was deionized by

passage through a column containing 30 ml of mixed (l:l)

cation- and anion-exchange resins; yield, 551 mg of D-glucose-4-t

55
(0.72 |iCi/mg, 397 u.Ci), 200 mg of D-galactose-4-t (3.24 u.Ci/mg,

648 p,CI), and 143 u.Ci of a mixture of the two from an intermediate

strip.

A sample of D-glucose-4-t (l4o mg, 0.78 mmole) was

reduced to D-glucitol-4-t with a seven-fold excess (50 mg) of

sodium borohydride in aqueous solution [43]. After removal

of cations with a cation-exchange resin and of borate by repeated

concentration of the sirup in the presence of methyl alcohol,,

143 mg of D-glucitol-4-t was obtained. Chromatography in

2-butanone —acetic acid —saturated aqueous boric acid solution

(9:1*1) [44] and scanning of the chromatogram for radioactivity

showed that the D-glucitol-4-t contained no D-glucose.

E. Liquid Scintillation Counting; Study of Oxygen Quenching

( H. S. Isbell , A Cohen)

It is known that oxygen has a reversible quenching effect,

not only on the fluorescence of polycyclic, aromatic hydrocarbons

,

but also on the scintillation from mixtures commonly used for

radiochemical assays. These facts suggested the possibility of

studying the interaction of polycyclic, aromatic hydrocarbons

with oxygen by means of a liquid scintillation counter. It

seemed probable that fluorescent hydrocarbons would act as

scintillators in the counter, and that the reactions with oxygen

might be more or less characteristic of the hydrocarbons.

Knowledge of the quenching effects of oxygen for various

scintillators is desirable, because variations in the oxygen

content of the solutions used in analyses cause substantial

errors^ the more sensitive the scintillator to oxygen-quenching,

the higher the error.

In development of these ideas, a study of the scintillation

properties of pyrene and 1,1' -bipyrene was made. The convenient

method for preparation of 1,1' -bipyrene developed by

56
Mr. A. J. Fatiadi (see page 8 ) makes this heretofore scarce

compound available for research and wide scientific use. The

substance is highly fluorescent; this property offers many

possible applications, as, for instance, a water-insoluble,

invisible, ultraviolet marker, for activating electronic

circuits. It seemed possible that it might have applications

in scintillation counting of radioactivity, because the

fluorescence is at long wavelengths.

For study of the scintillation properties of the hydrocarbons and the effect of oxygen on the scintillation, we

prepared two hydrocarbon-soluble radioactive standards, hexa14 O-acetyl-D-mannitol-1-t and hexa-O-acetyl-D-mannitol-1- C . These provide sources of beta radiation of widely different

energy levels.

A variety of measurements was conducted, but only a few

will be described here, as a more comprehensive study is being

made. In experiments 1 to 6 of table 7, an accurately weighed

quantity of the tritium source (2 to 4 mg) was placed in each

of 6 vials containing a Teflon-coated stirring bar, followed

by 10 ml of the solution to be tested for scintillation (see

table 8) . The vials were sealed with caps containing two

holes and a rubber liner. The solutions were cooled in an ice

bath, and a stream of nitrogen gas (1.5 "to 2.5 liters) was

passed in, through hypodermic needles inserted in the holes of

each cap. Then, the solutions were counted in the liquid

scintillation counter shown in figure 5. The samples were

further successively treated (by the method described) with

air and oxygen; after each treatment, the samples were

counted

.

Experiments 1 and 2 were conducted with conventional

scintillation solutions used for assay of carbon-l4 and tritium.

In the conventional toluene and p-dioxane solutions, tritium

was counted in air with efficiencies of 39 and 30%, respectively.

The count in the p-dioxane solution is less sensitive to oxygen

57
Table 7. Effect of oxygen on counting

efficiency for a tritium source.

Solution3 Flushed Count s/sec/mg of Relative Counting

employed with tritium source count efficiency

%

1 N 506.56 1 45.4

C- Air 435.55 0.86 38.9

°2 246.29 0.49 22.1

2 N 367.84 1 33.0

C— Air 3^4.40 0.91 30.0

°2 225.02 0.61 20.2

3 N

2 35.38 1 0.32

Air 2.53 0.07 0.02

°2 0.17 0.005 0.002

4 N 165.40 1 4.8

Air 40. 61 0.25 0.4

°2 4.37 0.03 0.04

5 N

2

22.10 1 0.20

Air 6.4l 0.29 0.06

°2 0.91 0.04 0.008

6 N

2

186.95 1 16.8

Air 49.36 0.26 0.4

°2 3.42 0.02 0.03

For composition., see table 8.

58
CDa

CDU

>>PH

pqi

hf]

COH o o o H

t3

CD>>oHP

CDO

•H

-PHO

cqo

•H

-P

CO . HH

•HP

i=! •HO

CQ

ChOO

•p•H

CQOPaoo

oo

0)

,0

CO Eh

CD

Ph

0)CICD H

CO ^1P

.Cj P.

CO

PhO

PhO

Ph

CM

CD sO

Ph

Ph

bO M

bfl

bfl

CD

CO -P

H

CD w

CDX

Ha

pi p4

CD

CD rHO

Eh

H

CM

OO ooH « O O

LO !>- o O

O o o o o o

LH LT\ in

m oooH oooH o o o o o o o o m in m m oH

HPHO

CO

tHo

CD P HO

CO

W K\ 4 ITi ID

£h

CD Ph

CO

59
Figure 5. Liquid scintillation counter used for

determining carbon-l4 and tritium.

than that in the toluene solution, but both are greatly affected

by the presence of oxygen. Experiments 4,5., and 6 were conducted

with pyrene and with extremely small quantities of 1 ,1 ' -bipyrene

as scintillators. (The amount of the latter was limited by its

insolubility. ) Both hydrocarbons proved to be effective

scintillators; a mixture of pyrene and 1,1 '-bipyrene is more

effective than either alone. The sensitivity of the counting

efficiency of pyrene and 1,1 ' -bipyrene to the presence of

oxygen is higher than that of the conventional scintillators

used in experiments 1 and 2. The results are of an exploratory

nature, and the investigation is being continued.

60
F. Large-scale Paper Chromatography

( H. L. Frush )

1 . Introduction

Despite the multiplicity of available chromatographic

techniques, paper chromatography continues to be one of the

most versatile techniques for the separation, identification,

purification, and analysis of carbohydrates. With respect to

range in capacity, it has advantages over either thin-layer or

column chromatography. In combination with radiochemical

techniques, it affords sensitive quantitative analyses by nonquantitative isolation methods.

In the syntheses of position-labeled, radioactive

compounds carried out in our laboratories, we have used Daper

chromatography not only for determining purity and for following

the course of reactions, but also for accomDlishing numerous

large-scale separations and purifications in the actual syntheses

[451.

Most large-scale preparative methods for paper chromatography

depend on an increase in bulk achieved by the use of many layers

of thin paper, pressed or rolled together. Some of these methods

require the tedious loading of each sheet, and others suffer

from a lack of uniformity of movement throughout the bulk.

In 1957* Brownell and co-workers [46] described a large-scale,

heavy-paper technique for preparative chromatography in which the

paper (equipped with a supporting stirrup and a wick of thinner

paper) is used, after preliminary purification, in essentially

the manner customary for single sheets. The authors stated

that the dense paper (Whatman seed-test paper, 1 . 5 mm thick) is

capable of separating 0.5 to 3.0 g of material per large sheet

(l8 x 22 in.), depending on the spread in R values. They also

described a method for nondestructively locating the separated

61
bands by staining a "print" obtained by pressing a sheet of

thin paper against the wet chromatog ram.

However, despite the unusually high capacity of the heavy

paper, and the inherent advantages of paper over column

chromatography, the techniques described by Brownell e_t al . have not been widely adopted, presumably because of the

inconvenience of preparation and the fact that movement of

bands through the paper is not so uniform as that in other papers.

Despite these disadvantages, heavy-paper chromatography

has found extensive use in our laboratories for separating both

radioactive and nonradioactive mixtures. Certain simplified

techniques, described below, have facilitated application of

the paper to problems of separation. The techniques are equally

applicable to Whatman No. 17 paper, and, on this paper, afford

separations of 0.2 to 0.6 g per large sheet. The No. 17, like

the seed-test, is a "fast" paper whose properties are improved

by use of a thinner wick, which serves to retard the absorption

of solvent.

2. Procedures

a. Preparation of the Heavy Paper . Figure 6 shows

a simplified method for applying the wick and supporting stirrup.

A strip of Whatman No. 3 MM paper (7 x 18 in.) is loosely

machine-stitched along its short sides, about 2 in. from the

edge, and then folded as shown. The l8-in. edge of the seedtest paper is softened by momentarily dipping it in water, and

blotting the excess. The dampened edge is inserted into the

folded strip of thin paper, and the two are twice machine-stitched

together. To facilitate dripping of the solvent, the lower edge

is conveniently serrated by dampening it and tearing off small

This stitching prevents tearing of the stirrup when the

chromatogram is wet.

62
CONTINUOUS PIECE (7"XI8 ) OF WHATMAN NO. 3 PAPER

GLASS SUPPORT-ROD

MACHINE STITCHING

Figure 6. Simplified preparation of stirrup and wick for

heavy-paper chromatogram.

pieces to form an irregular edge.

•The papers are chromatographically washed with 0.01 N

hydrochloric acid and then with water (two days in each solvent).

The sheets are air-dried, and strips (l in. or more in width)

are trimmed from each long edge; stirrups and wicks are trimmed

to match. A second wick (useful when the paper is washed, or

is developed in a slow-moving solvent) need not be stitched^

but can be added as shown in figure 7

.

Aqueous or alcoholic solutions of mixtures to be

chromatographed (as much as 2 ml ) are applied as a narrow band,

2 cm below the stitching, by means of a pipet having a 1-ml bulb

and a short tip of capillary tubing. The band is loaded from

both sides of the paper, which is then air-dried for' several hr.

The No. 17 paper may be used without purification or trimming,

but with the same stirrup and wick.

b. Nondestructive Location of Bands on the Developed

Chromatogram . Figure 8 illustrates a convenient method for

locating bands of material on the chromatogram after development.

The wet sheet is laid on a glass plate in a well-ventilated hood,

65
ADDITIONAL SHEET 4" X 18" (INSERTED NOT ATTACHED)

DOUBLE WICK

IN USE

V

Figure 7. Addition of auxiliary wick to heavy-paper

chromatogram.

THIN PAPER LAID OVER THICK WET CHROMATOGRAM

STIRRUP AND WICK ^ORIGIN

DIRECTION OF FLOW

Figure 8. Method for non-destructively locating hands on

developed, heavy-paper chromatogram.

and over-laid with a sheet of thin paper (such as Whatman No. 1)

carefully positioned at the origin. A small plastic wheel* is

drawn down the paper in a series of lines, in which good

absorption from the wet chromatogram is caused by the pressure

of the wheel. The tracing thus prepared is sprayed with a

This is a pastry wheel available in stores selling kitchenware.

64
suitable color reagent. Because the color is intense in those

portions of the lines that cross the bands., the limits of the

bands are more clearly discerned than in a "print" [46] of the

entire chromatogram. If it is desired only to ascertain the

progress of a development , a single line, drawn by the wheel

on a strip of paper placed lengthwise down the middle of the

wet chromatogram, will suffice. When the separation of bands

has become satisfactory, and a tracing has clearly defined

them, the chromatogram is air-dried. The outlines of the bands

sketched on the tracing are readily transferred to the

chromatogram by means of a stylus.

14

In the chromatography of C-labeled compounds, a radioautograph most accurately locates the separated bands. By

outlining the bands on the film with series of punctures,

positioning the film on the chromatogram, and dusting it with

carbon, the outlines are transferred to the chromatogram, and

the intact film may be saved for reference.

For tritium-labeled compounds, the tracing technique

previously described has a further advantage. Tritium does

not produce a satisfactory radioautograph from a chromatogram

because of the weakness of its radiation. If insufficient

compound is present to enable the bands to be located by

staining the tracing, the technique illustrated in figure 9

is useful. Individual lines on the tracing are scanned in a paper-chromatogram scanner having a 1:1 correspondence of the

paper and chart (figure 10). This technique has been used in the

chromatography of high-activity, tritium-labeled compounds on

the lower-capacity papers such as Whatman No. 3 MM.

c . Removal and Treatment of Bands of the Developed

Chromatogram . The bands, located by one of the methods

described, are conveniently removed by means of tinners' shears.

65
DIRECTION OF DEVELOPMENT

A

B

C

D

E

F

G

H

TT

• I T

I

I

1

1

* r

*r

-*—' i /"

ORIGIN

* (ON HEAVY PAPER)

SCAN OF H

Figure 9. Use of tracing and chromatogram scanning to

locate bands of high-activity, tritiated compounds

figure lu. J^aper cnroma-cogram scanner used for assay of

carbon-l4- and tritium-labeled compounds.

(Seed-test paper is difficult to cut with scissors and tends

to crumble when cut with a sharp knife.) For elution, the

stirrup and wick are attached to one end of the excised band

by means of several stainless-steel staples, vertically placed

The auxiliary wick is added as shown in figure 7 ? the lower

66
end of the band is trimmed to a point, and the strip is eluted,

usually with 50$ methyl alcohol. Subsequent treatment of the

eluate may include the use of a small amount of a decolorizing

carbon, and, in some instances, of ion-exchange resins.

d. Capacity of the Heavy Papers . Figures 11 and 12

illustrate the capacity of the heavy papers. Obviously, the

ease of separation (and, therefore, the capacity) depends on

2.0 g

SHEET

1.5 g

SHEET

i g

SHEET

0.5 g

SHEET

J I

WHATMAN SEED-TEST PAPER, NO. 3 STIRRUP

AND WICK

d- GLUCOSE

D- FRUCTOSE

L-RHAMNOSE

0.5 TO 2.0 g PER SHEET

D- GLUCOSE

-D -FRUCTOSE

l-RHAMNOSE

Figure 11. Illustration of capacity of heavy (seed-test)

paper; dependence on relative R„ values.

67
600 mq 400 mg 300 mg 200 mg j SHEET SHEET SHEET SHEET

WHATMAN NO. 17 PAPER, NO. 3 STIRRUP AND

WICK

d-GLUCOSE

d-FRUCTOSE

l-RHAMNOSE

200-600 mg/SHEET

d-GLUCOSE

-d-FRUCTOSE

-l-RHAMNOSE

Figure 12. Illustration of capacity of fast, medium-weight

paper, prepared with thinner wick.

the difference in R values. At most indicated levels of

loading, L-rhamnose was separated from D-glucose and D-fructose

Further development, or a second development, would separate

the last two from each other at the one-gram level on the seedtest paper, and at the 400-mg level on the No. 17 paper. The

capacity of the heavy papers can he increased somewhat (if the

decreased length is unimportant) by attaching the stirrup and

wick to the long (22 in.) edge. The chromatographic properties

of the seed-test paper are the same in the two directions. The

R„ values for the seed-test paper are about 0.7 of those for

Whatman paper No. 1.

68
4. CHARACTERIZATION OF CHEMICAL STRUCTURE;

PHENYLHYDRAZONO —PHENYLAZO TAUTOMERISM

( H. S. Isbell and A. J. Fatiadi)

A

.

xylo-4.,5 j6-Trihydroxy-2-oxo-l 3 3-bis (phenylhydrazono )cyclo - hexane and 4-Oxo-l-phenyl-5-phenyIazo-3-pyridazine Derivatives

The positions of the phenylhydrazono groups in xylo -4.,5,6-

trihydroxy-2-oxo-lj3-tiis(phenylhydrazono)cyclohexane were

established by degradation of the compound with sodium metaperiodate, and identification of the reaction product. The

dialdehyde initally formed by periodate oxidation (but not

isolated) cyclized to 4-oxo-l-phenyl-5-phenylazo-5-pyridazinecarboxaldehyde^ which yielded a crystalline methyl hemiacetal,

an oxime_, a semicarbazone, and, by oxidation , the corresponding

monocarboxylic acid and sodium salt. Upon treatment with

phenylhydrazine at 50 C, the carboxaldehyde gave a red.,

crystalline phenylhydrazone which., with phenylhydrazine at

130 C } afforded a product that appears to be 4-oxo-l-phenyl-5-

(phenylhydrazo)-5-pyridazinecarboxaldehyde phenylhydrazone

.

The reactions provide a route to a series of new pyridazine

derivatives which may prove valuable for the synthesis of

unusual compounds for biological and medical research. This

work has been incorporated in a paper which has been accepted

for publication in Carbohydrate Research . B. Structures of 2-0xo-l.,;?-bis (phenylhydrazono) Compounds

A manuscript entitled "Phenylhydrazono —phenylazo

Tautomerism, Part II. Structures of 2-0xo-1^3-bis (phenylhydrazono) Compounds" was prepared, and will be submitted for

publication in the near future. The paper presents measurements

of NMR, infrared., ultraviolet., and visible spectra for twelve

phenylhydrazono compounds , prepared to show that 2-0X0-1,3-

b is (phenylhydrazono) derivatives have a novel phenylhydrazono

phenylazo structure and that they exist in tautomeric forms.

69
In an invited paper presented at an A.C.S. symposium on

"New Reactions in Carbohydrate Chemistry, " the structures of

carboxylic acids, enediolic acids, diphenylf ormazans, and the

enolic tautomers of 2-oxo-l,3-bis (phenylhydrazono) compounds

were compared From the comparisons of the structures, and

consideration of the properties of the compounds, the concept

was advanced that the enediolic acids are analogs of carboxylic

acids in that the =0 and -OH groups are attached to carbon

atoms joined through a -C=C- group. Similarly, it was proposed

OH

that the enolic tautomers of the 2-oxo-l,3-bis (phenylhydrazono)

compounds are analogs of the diphenylformazans in that the

phenylazo and phenylhydrazono groups are also attached to

carbon atoms joined through a -C=C- group. Certain novel

OH

reactions of the 2-oxo-l,^-bis (phenylhydrazono) compounds and

the diphenylformazans were discussed. A reaction with strong

acids, which we discovered and interpreted, is noteworthy.

Thus, a deep-blue color is obtained when a 2-oxo-l,3-bis (phenylhydrazono) compound or a diphenylf ormaz an is dissolved in a strong acid, such as phosphoric acid. Phenylosazones and other

1,2-bis (phenylhydrazono) compounds examined do not give this

intense blue color. The color presumably arises from a cation

of the following type

:

H OH H

I I I »=N-N=C-C=C-N=NR R ©

The reaction is being investigated further,

70
C . Inosose Derivatives

Work was continued on the reactions and properties of

inositol derivatives, especially with reference to aromatization

by cleavage of acyloxy groups beta to enolic hydroxyl groups

[47].

Procedures were developed and published [48] for preparing

inososes from their phenylhydrazones and for separating an

inosose phenylhydrazone from an inosose phenylosazone by means

of ion-exchange resins. In the latter process., the phenylhydrazone —phenylosazone mixture is treated with a cationexchange resin. The phenylhydrazone is hydrolyzed, and the

inosose is obtained in the aqueous extract. The insoluble

phenylosazone is separated with the resin, and extracted from

it with N_,N-dimethylformamide

.

71
5. SYNTHESIS OF RESEARCH MATERIALS:

CYCLOPENTITOLS AND RELATED SUBSTANCES

(R. Schaffer)

A. Introduction

In a study in which a long-known _0-isopropylidene-Dlyxose (i) was finally characterized [49] , one reaction afforded

a new dialdehyde (il) that was found to crystallize as III by

incorporating a water molecule in its structure. (The two

hemiacetal functional groups in a single tetrahydrofuran ring

make this an unusual ring structure for a carbohydrate.) A

more economical source of III is the Isopropylidene derivative

(IV) of naturally occurring D-rlbose, which unexpectedly undergoes oxidation by Deriodate at a much higher rate than I.

rCH2

H 3C CH3 P<

I HO^O^OH NHCO:o HCO hco \< >l ^ r 7 \ / H2

> \ /

H3C CH3 H3C CH3

H0H2 CI/O.oh n m

H3C 3CH3

m

The availability of compound III offered the prospect of

synthesizing pentasubstituted cyclopentanes , 5-carbon-atom

ring analogs of the hexasubstituted cyclohexanes, important

72
biochemicals that serve as model compounds of fundamental

importance for studying the stereochemistry, conformations,

and reaction mechanisms of substances having 6- carbon- atom

rings. The pentasubstituted cyclopentanes hold promise of

equal usefulness for this widespread ring-type.

B. Nitromethane Synthesis

Conversion of the novel dialdose (il) into cyclopentanes

was achieved by treating it in alkaline solution with nitromethane. The reaction may be formulated as involving nitromethane (as the aci -nitrome thane anion) adding to one of the

aldehyde groups of II to form an open-chain, 5-carbon-atom

intermediate having the unreacted aldehyde group at one end and

an aci -nitro functional group at the other, followed by addition

of the latter terminal group to the former to yield the sodium

aci -nitrocyclopentane salt (v) that precipitates from the

reaction mixture.

NO2 Na + .OH

MeN02 NaOMe>

The reaction products possible from this synthesis are the

one with the meso structure depicted as V, another me so salt

(like structure V, except that the two hydroxyl groups are up

and the hydrogen atoms are down), and a DL pair (like V,

except that only one hydroxyl group and one hydrogen atom are

up). However, only compound V was isolated.

1 . Structure of the Sodium Salt

The elementary composition (CqH CvNNa ) according to

chemical analysis of the isolated salt, the presence in its

73
infrared spectrum of peaks that indicate the dioxolane ring,

the nitro group, and hydroxyl groups, and the absence of peaks

for a carbonyl group are consistent with structure V. Its

configuration of asymmetric carbon atoms was established by

use of a proton magnetic resonance spectrometer (figure 13)* a

Figure 13. NMR spectrometer used for determining

geometric arrangement of protons in cyclopentane derivatives.

powerful instrument capable of exhibiting many fine details

of molecular structure. The magnetic resonance spectrum of

the salt in deuterium oxide solution (figure 14) s,hows a pair

of well-spaced singlets, each corresponding in intensity to two

protons. By correlation with published information,, the downfield 2-proton singlet is assigned to the protons on the carbon

atoms to which the hydroxyl groups are bonded. Similarly,, the

upfield singlet is assigned to the two protons on the carbon

atoms to which the dioxolane ring is bridged. Analysis shows

74
NMR in DoO

3 2 10 ppm

N02"Na + HO^OH

HiC CHs

NMR in

Me2 SO

H 1 1- NMR in

acidified

Me2 SO

I I H

HO^Ck OH

98765432 I ppm

IE

NMR in CDCI

i i

i i

AcO ^0^ OAc

98765432 I

H3C CH 3 ppm n-DIACETATE

Figure 14. NMR spectra of V, III, and III-diacetate

that this simple pattern can only be associated with the

symmetrical trans - c is - trans geometrical arrangement of protons,

which is the galacto configuration shown in structure V. This

simple pattern was observed also in the NMR spectra of III and

its diacetate. Conformational analysis would have predicted

the galacto configuration for each of these structures] NMR

provided the proof.

75
2. Conversion Into Nltrocyclopentane Derivatives

The aci -nitro compound tautomerizes to the non-ionized

nitro form when the sodium is replaced by hydrogen. This

transformation generates VI and VII, whose structures differ

only in their configurations at the new asymmetric center.

N02" Na + NOz

H9^\9H H<vkf>H HO^KOH

H3 C ^CH3 ^ 3ZI W

The nltrocyclopentane derivatives VI and VII crystallize as

monohydrates from aqueous solution. The isolated crystals

readily lose the water of hydration on drying in vacuo, but

the dried products do not show characteristic melting points.

Consequently, infrared spectra were employed to characterize

the fractions of hydrated crystalline material isolated. The

spectra were found to be of two distinctive patterns that were

presumed to signify the separation of the individual nitro

compounds. However, the reactions of samples of the "product

type that was obtained in greater abundance resulted in pairs

of isomeric derivatives, and it was found that this product

type was, in fact, a mixture resulting from co-crystallization

of the two hydrated isomers. Separation into pure VI and VII

was effected by fractional recrystallization of the anhydrous

materials from chloroform and then from ether plus pentane

.

3. The Aminocyclopentane Derivatives and Their Structures

a„ Reactions with Sodium Metaperiodate . The amino

compounds VIH and IX obtained by catalytic hydrogenation of VI

and VII, respectively, were particularly useful for determining

76
configurations at the asymmetric carbon atom attached to the

nitro group. The amino derivatives were treated with sodium

metaperiodate, which cleaves bonds between vicinal carbon

atoms attached to hydroxyl and amino groups (and hydroxyl and

aldehyde groups). Rates of this reaction are dependent on the

cis (fast) or trans (slow) relationship of the hydroxyl and

amino groups on the 5-membered ring. Open-chain a-hydroxy

aldehydes are oxidized at intermediate rates. Although amino

compounds VIII and IX could react with two mole-equivalents

of sodium metaperiodate each, at completion the products of

these reactions would be identical] but, on allowing the

oxidation of each amino compound to occur with only one moleequivalent of oxidant, the effects of their different structures

on the course of the reaction (and, hence, their individual

structures) are revealed by the nature of the reaction products

that each gives.

Thus, one of the amino compounds, on equimolecular

oxidation by periodate, gave only the 4-carbon-atom oxidation

product ( II ) , a result which implies that each oxidized

molecule was oxidized a second time and that half of the

original amount of the amino compound was not oxidized at all.

Consequently, this amino compound is VIII, because both

oxidizable groupings on its cyclopentane ring must be trans

HO^KOH HOJ}C0 /°\0H

r NH>< r HCO A 7 TM- HCO HCO \—(

IQ4

> \ y m hco\ / IQ4) \ y oo oo oo oo

H3 C CH3 H3C CH3 H3 C CH3 H3 C CH3

iznr x n

77
for the open-chain 5-carbon-atom oxidation product (X), as it

is formed, to undergo oxidation of its a-hydroxy aldehyde

grouping more readily than either hydroxylamino grouping on

the parent cyclopentane ring. The other amino compound, under

NH2 HO^\QH HO HCO

H3C CH3

IX

the same conditions, gave only the 5-carbon-atom open-chain

product (x), and compound X has the arabino configuration.

Hence, the cyclopentane ring in the present compound has only

highly reactive cis hydroxyl and amino groupings. The parent

is therefore IX. Cleavage of the cyclopentane ring yields the

same open-chain 5-carbon-atom product (x) as would be obtained

from VIII, but X accumulates here, since its cis -substituted.

cyclopentane ring precursor (IX) is a much more reactive

reductant of periodate.

b. Reactions with Nitrous Acid . The configurations

of the compounds assigned structures VIII and IX are supported

by their behavior on treatment with nitrous acid. This reagent

converts an amino into a diazo group, and the latter is

eliminated from its attached carbon atom (nitrogen is. evolved)

with particular effectiveness if the diazo group and a vicinal

trans hydroxyl group are substituents on a 5-membered ring.

The result is the formation of a product having an epoxide

ring. Compound VIII gave such a reaction with rapidity, thereby

affording epoxide XI in high yield. On the other hand, with

compounds where the amino group and the vicinal hydroxyl group

78
are cis to each other, reaction is sluggish and a number of

products usually result. It was found that compound IX, which

is assigned only cis configurations, showed little evidence

of reaction with nitrous acid and no crystalline product could

be isolated.

HO^KOH HO/

S*y MONO, <

/ \

} / 1

H3C CH3 H3C CH 3

T7TTT ~XL

79
6. NOVEL RESEARCH MATERIALS

( R. S. Tlpson and A. Cohen)

Advances in organic chemistry lead to new products for

which improved methods of synthesis are needed; in addition,

suitable methods of purification must be devised, and

appropriate characterization must be developed.

A. Unsaturated Alditols

During the past few years, alditols and related compounds

containing unsaturation have assumed considerable importance.

Although, by the use of elimination reactions in aprotic,

dipolar solvents, a method had been developed for the production

of terminal unsaturation, a simple way of introducing

nonterminal unsaturation was not available. We have now

developed such a method, and a description has been published

[50].

Unsaturation is readily introduced [-51*52] at the two

terminal carbon atoms of an alditol (or an acyclic polyhydroxyalkyl appendage of a cyclic sugar) by the action of sodium

iodide (in a suitable solvent) on a derivative in which a primary and a secondary sulfonyloxy group are contiguous.

(f H0Ts

+ 2 Nal . ?

H

+ 2 NaOTs + Ic CH

2OTs CH

2

2

For sugar derivatives that are acyclic, there is only

one example that has been recorded of a similar reaction

involving two contiguous, secondary sulfonyloxy groups; it

was found that methyl 4-deoxy-2,3-di-0_-(methylsulfonyl )-DLerythronate and -threonate react with sodium iodide [53] to
give the corresponding 2,3-unsaturated esters, but it is known

that the carboxylic ester group has an activating effect [52].

"Unsuccessful attempts were made to prepare an unsaturated

compound" [5^ J from the 3^^-dimethanesulfonate (ila) and

3,4-di-p_-toluenesulfonate (lib) of 1, 2: 5,6-di-_0-isopropylideneD-mannitol (i). Consequently, we decided to re-investigate

this reaction; for convenience, we avoided the use of an autoclave or of sealed tubes. We found that Ila is unaffected on

treatment with a 10$ solution of sodium iodide In acetone

during eleven days at room temperature or eight hr at the

boiling point. However, reaction occurred when various highboiling solvents (for example, 2,5-hexanedione ) were used at

the respective boiling point; free iodine and the sodium

methanesulfonate-iodide salt [53*55] were formed. Unfortunately,

iodination took place, and this made isolation of III difficult.

Hence, we made a search for (a.) a solvent which was capable of

dissolving sufficient sodium iodide and Il(a or b ) to be

practical and which had a boiling point sufficiently high for

the reaction to occur at a reasonable speed, and (b_) a

substance which, when present throughout the reaction, would

combine with the iodine as fast as it was formed (thereby

precluding iodination and promoting the reaction) and which

would prevent the development of acidity.

We found that N,N-dimethylformamide provides a boiling

point high enough for a reasonable rate of reaction and that

zinc dust affords a rapid means for removing iodine. Treatment

of Ila or lib with an excess of sodium iodide (20$ solution

in dry N,N-dimethylformamide )

, in the presence of an excess

of zinc dust, with efficient stirring and with vigorous boiling

under reflux (drying tube) during 5 hr , followed by cooling,

8i
H

H

3<\ /OCH 2 C 1

3 C OCH

1

1 ROCH

1

1 HCOR

HCOv CH, |V1 / \

H

2 CO CH 3

I (R = H)

Ila (R = Ms)

lib (R = Ts)

H,C OCH, 3 \ / , 2 C

/ \ ' H 3 C OCH

CH

II HC

HO) CH, |V

H 2 CO

X X

CH 3

III

Ila + 2.5 Nal

lib + 2 Nal

III + 2 NaOMs • 0.25 Nal + I

III + 2NaOTs+l

filtering j extracting (heptane), evaporating the extract,

retreating the residue with heptane, and evaporating, gave

crystalline 1 ,2:5, 6-di-0-isopropylidene- trans -3-hexeneD- threo -1 ,2,5,6-tetrol (ill) (64$). This was sublimed at

.o o, 45 /0.02 mm, and had mp 80-82 C from aqueous ethanol;

[a]_ + 57.5 (c 1.02, chloroform); J_ ,. 15.5 c.p.s.j it readily

decolorized aqueous permanganate solution. These properties

agree with those recorded [56,57] for compound III prepared

by other methods. Compound III showed v 1307 and 971 cm

max

(trans olefinic; not shown [58] by i) and 1156, 1134, and

1053 cm" (l,3-dioxolane rings; I shows [58] bands at ll60,

-1

-1 1126, and 1044 cm

1

).

82
When compound III was hydrolyzed with &0% acetic acid

(5 hr at 25 C), and the solution was evaporated to dryness

,

trans -3-hexene-D-threo-l,2,5,6-tetrol (IV) was obtained mp

64-65 °C (from ethanol— ethyl acetate); [a]^5 - 13.8° (c 2.00,

water); J^ ^ 15.5 c.p.s.; v max

1653, 1325, and 976 cm"

1

. Anet [57] gave mp 114 °C; [a] D

- 2° (water); J^ ^ I5.5 c.p.s.

For IV, Dr. Anet now finds mp 64-65 °C, [ a ^

5 - 14.6° (c 0.5,

water) , and IR spectrum identical with ours (personal

communication, Sept. 24th, 1965). Moreover, Dr. Haines has

now prepared compound IV, and finds mp 60-62 °C, [a ]j, - 15.3°

(c_ 4.3, water), J-, j. 15.9 c.p.s., and IR spectrum identical

with ours (personal communication, Sept. 28th, 1965)*

Condensation of compound IV with acetone, in the presence of

anhydrous cupric sulfate, regenerated III.

The above procedure for introducing nonterminal

unsaturation should be applicable to a variety of alditol

derivatives, and also to sulfonic esters of suitable derivatives

of certain mono-, oligo-, and poly-saccharides, and cyclitols.

Moreover, it should provide an improvement In the introducing

of terminal unsaturation into alditol and other sugar

derivatives by use of the appropriate sulfonic esters.

Upon conclusion of this phase of the project, work on it

was suspended until that on the topic described in the next

section could be completed.

B. 1-Acylamido Derivatives of Aldoses (Furanoid, Pyranoid , and Acyclic ) This work was conducted, in part, with compounds provided

by Professor Venancio Deulofeu and Dr. Alberto S. Cerezo,

Departamento de Qulmica Organica, Facultad de Ciencias Exactas

y Naturales, Universidad de Buenos Aires, Peru 222, Buenos

Aires, Argentina, and, in part, with compounds prepared at

NBS on a project sponsored by the Office of Naval Research

and described in NBS Report 1358 "Infrared Spectra of Sugar

Acetates and Related Compounds" by H. S. Isbell, F. A. Smith,

C. Creitz, J. D. Moyer, and H. L. Frush, December 1951.

83
The main object of the present project was to record the

infrared absorption spectra of a variety of 1-acylamido

derivatives of aldoses (having furanoid, pyranoid, or acyclic

structures),, in order to find whether previously reported

correlations between the structure and specific bands in the

infrared spectra of certain derivatives of sugars are also

applicable to this group of compounds. In essence, the study

consisted of answering two questions: (a) Do 1-acylamido

compounds having certain structural features show, in their

infrared spectra, the bands previously described as

characteristic of such features, and (b) do 1-acylamido

compounds that lack these structural features fail to show

these bands in their infrared spectra? No infrared band can

be regarded as unique for any feature, of course; and interpretation must be substantiated by other evidence.

For a paper now in process of publication, the spectra

have been recorded for two glycofuranosylacetamides and their

perbenzoates; eleven glycopyranosylacylamides and eight esters

thereof; and thirteen 1,1-bis (acylamido)-l-deoxyalditols and

eight esters thereof „ In addition, a discussion is given of

the spectra previously published [59] f° r five glycopyranosylacetamides and six acetates thereof, and for a 1, 1-bis (acetamido)-

1-deoxyalditol and its tetraacetate; the serial number given

each of these compounds is that originally assigned [59].? with

the letter A suffixed thereto. Also, the spectra previously

published [6o] for three derivatives of D-ribosylamine have

received consideration; the serial numbers for them are those

originally given [60], with the letter B added.

Table 9 gives a list of the compounds studied, and an

Index to the spectrograms; the serial number of a compound

is the same as the number of its spectrogram. The spectra

were measured in the region of 4000 to 667 cm" for compounds

1 to 44, and arranged according to their code numbers [6l].

In a series of articles [62, 63., 64 ] , Barker and co-workers

mg

84

sought to identify, in the range of 960 to 730 cm , infrared
bands characteristic of several aldopyranoses and their

derivatives. They also noted [65] absorption bands for aldoand keto-furanose derivatives. Verstraeten [66] has made a study of infrared bands characteristic of 2-ketoses, and

Nanasi and co-workers [67] have made some correlations for

glycosylamine derivatives.

To be of general usefulness in structural analysis , the

correlations made by Barker and co-workers should be applicable

to the furanoid and pyranoid forms , respectively,, of sugar

derivatives of all kinds, provided that allowance is made for

bands attributable to such other features as benzene rings or

acetate ester groups present in the molecule. Similarly, the

other correlations should be applicable for the features

mentioned.

We have made a careful study of the infrared spectra of

the 60 compounds listed in table 9. w"e found that the useful

correlations between structure and infrared absorption bands

made by Barker and co-workers for certain carbohydrates, and

by Nanasi and co-workers for some N-arylglycosylamines,

cannot be extended to the 1-acylamido compounds we have

studied. This conclusion confirms our previous reports that,

for cyclic acetals of sugars, "distinctive absorption bands

are not apparent for pyranose or furanose rings" [58], and

that, for 1-acetamido derivatives of sugars, "no bands were

noted which could be correlated with the absence or presence

of the pyranoid ring" [68], However, we found that certain

of Verstraeten' s correlations may have some diagnostic value.

This work has been brought to completion, and an article

describing the results is being written.

85
1O /% /-\ •-v /-s

4-1 i-( CN CO <t 21 CM

m <CO vO ^ r.

O v—>

\s >w> v-*' 0) CO a, u

CO 60 i m

m in r>» 00

u ^ \ •> •* CU Q)

U-4 o

i-( CN r-l co <t <r vD <f vO

m vo

CU ti Pi <U • /-N /"*\ co CO CO a

/~\ •">. t-l T-(

cO o o O

Q

O o o O

OOO

00

CO

CU pfpT P2

O pT pf1 pf pT nf pT1 pT o •v CU V v-^ N_^ n_^ N_^ N-^ v_^ •+S N-^ >—' N_X

>-l

p ft

O VO CO r-l r^ co o -* l>» r-l 0\

4-1

—i t>0 • • * • o « aj o r>» 1-1 o o CM r>. r>«

m as <f

a) t—i 'O o 00 co CN 1 CN * <f <o VO CSS a. .-1 + + 1 1 i i + + r-l

co + -? + o x)

4-1 X»»* oo r~» r-l m tf o r*» <f i-i •4-

O

X <h CT\ <t LO i-i vO »jO o i-i CM 00

CU /—\ T-\ r-l i-< r-4 CM CN i-i CN CM CN r-l XI a, o 1 1 1 1 1 1 i 1 1 1 1 c a o r*» m OS CO CO O KO CO o CN 0\

•H N^ <f ON CO LO r-4 CO VO

O r-4 cn r^

i-l T-l r-4 i-4 CN CN 1-1 CM CN CM r-4 X)c

co X)

a)u CU

ti c

CO •H

ca

a

CU cO B CU .-1

co •H CO XI a O c CO O 3 r-4 3 o >> i-l CO a CO co 60 CO 4-1 e w O 1 w CO o q i-l nil n u u M 1 ? H

CO

CI • XI H XJ nn i-i i_3 x) Q

•H o\ d w CU i >. J* CO e a 3 o 4-1

i—i o o 4-1

Q cO

CU r-l

O

Oh 2

ti

4-1

a 3

4-1

a r-l

CO 0) XI a >< •H >> CU >-< •H •V co ti ti

cO o CO 4J CU o ,Q CO 4J CO CO

O •H «H

H

u o CO Q) ti co

N 1 O CO CU ti CU ti ti

a

a

p^j

X c •d

n ti o| S X ti •H ti •H

C cO CO 3 3 i-l a cu CU 1 3 3 •H a •H a CO r-4 r-4 & co a co 4J XI CO CO a cO a cO a >. >N s a cO r-< CO 1 M Eh ti CO r-( CO r-4 i co co fe t> r-l >» w o| 4J P-) t3 tH >% r-l >> wJII g o o >. CO i 0) O >. co >. CO 1 G 4J u CO o •H 4J o CO O co o ca •H CJ >< * o o . r4 | EH • O o o a i X cO J r-4 1-4 3 CN 4J VO J i-l t-H 3 X ti >> CO r-l o 1 >. i-l 1 1 •\ o 1 >> r-l >* CO X t-i cO 1 r-4 X 60 1—4 lO U~l 1 r-l X 60 r-l a o cO 60 SI >> 1 l >-. •* •» S| >*. 1 1 1 i CU l 1 4-1 Qll OH 4-i co CO 4J Oil OH OH OH X3 r4ll Oil • CU 1 | 0) «\ *\ • CU I | 1 I 1 1 1 <: i SJ a O CM 1 SI

CM pq o ca <i S|

ca ca ca VO

a

a

86
CO

<J PQ tn m

PQ o\ cm co m <3 <j <i vo <rj vo r>. co r-t &\ r^ o f-i PQ

com

<y> cr> \0

CM CM

in m

vO

in

CM CM

00 CO

in m

ooo O O O o O /~\ O O

sf^r1 sT

1 sT

CM CM sT*

CM

35 aT*

^w^../*—

v-' **s Vw/ S—' \^ ^—Sw'' *—• CO CM °0 S* CM o CM CM CO o^ CO

• • • • • • • • • • CAO N CO CO CM CO CM m CO r> + I-K-I CM CM CM m H + r-( r-4 + + 1 1 1 i l + +

COO rs, <t in i-i co CM r> o

COO cr> as CO i—i co m CO c^

CM CM .-4 H cm CM CM CM CM T-4 CO vOOO

i in co <t cr\

1 I i—

1

I m CO CO COCt\ <r> <j\ CO O CO m CO CO CM CMtH i-t i-i CM OJ Cxi CM cm i-H

CO CO co CO CO CO CO CO c

T-4 1-1 •-4 r-l 1-1 i-4 t-t i-4 i-4 U U ro U o u U y u

PC 3= pp W Pi a « pp pp o o o CJ> o u u o CJ X—f ^s-^ N_/ ^^ %-.

^^ *w" x_s v_^ m St m CM vO vo St r^ co

CO r*. VO <D CT> o r>s st in

CM iH —4 + CO t-> i-4 co CO + + 1 + CM

1

i-4 +

+ +

co st CT\ l"~- CO CO CO o r^ VO CO CO r^ CM ri CO

i

1—1

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l 1

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1 co r-4

CN) CO co m r^ H CnJ —I CO

1*1 vO co ro r--. CM r*. CM

-A 0) e CP

QJ CJ cO •H C fp r-4 CU a H 1-1 >-, 0) ti CtJ a a Cd fp •H 1-1

CO CO O •H a >.

0) 1-1 iH CO a cO CO cu c >> >. c CO iH o rP 1-1 CO X cO i-i >> 4J •H a O c a >. CO CJ a

CO U C 1 co CO CO i-i P> cp hJII o CO i-4 i-4 ^ 1-4 CO 1 rP •H cO >>

03 60 a ca •H -Q M 'Si O 1 i i -o CO 1 O

<U 1-4 Oil OH >x CO u Oil &

C <u >> 1 i ',< M CO 1 •r\ •H ti X ca ca O CO l ?

u

a *H 1 i i CU 1 OH I CO a OH i-4 r-4 13 iJll 1 r-4 OH

f-l cO i ^ >^ l 1 >, i >» i-4 ca 4-1 4-1 vO ?

i 4-1 ca

CU CO CU >> CU i CJ CJ I i-4 CU i C O d en CP i-i 'J o i—i i-l >^ CJ i-i •H O •H CU CU O i-4 >> td CO >^ >> o ri >> 6 <u OJ P) <u a d c (P a w 4-1 i 1 4-J 4J N 1 4J

cd c c r-4 fi cO iH i-4 CP CO u CU oi o| CU CU CO oi CJ' i-i •H •H 00 •H r-4 a a CO r-4 CU CJ 1 1 o o CJ 1 o

>. B a 1 a ?-. CO CO a >> u cO cO cO d CO rO cO CO CO cfl cd on CO CO i-l i-4 i CO co 1 u I-i i 1 1 1-4 1 O i—i i-l i i-4 o >> >s jii o w oi u 4J oi O] oi 4-J oi

4-1 >^ >. ca >•> CI CO CO i 4J i CJ CJ 1 1 CJ U 1 o CO co i CO tA o O ca o i-i 4-1 4J •H tH •H 4J CJ 1-4 CO O O t-4 o ^a O d i CO !-4 1 1 U J-4 M 1 a U

1-1 Xi ,0. >> 1 X cO PJ pi >> 1-1 CM 4-1 VO KO 4-1 4J 4-1 VO o 4-1 cO •H •i-l ta 1-( >> >-4 i-( CO X cO 1 ! V •\ ! 1 1 •V CO 1 oO i-l S-4 o >M-4 CO 00 a o 00 i-( -* St St St St St St CO st

i 1 1 •H o i l 1 I CJ 1 >> •\ *\ •\ -s •v •% «N •V Oil on Oil & N on Oil on on TJ OH -J CO CO CO co co co co ca CO

1 i 1 O ti 1 1 1 i 1 i CJ •\ •s •\ •\ *\ •V •\ •\ en ca S-l

1 SI

CU CQ

1

ca « ca ca vO ca o

1

CM CM CM CM CM CJ CM CM

87
1ou

4-1 o a

CU to 00 o\ o r-4 CM CO

to CO

r-4 i-t CM CM CN CN CN

(X

u

C/} 00 1 cO cd u

CU 0) co co 1 v£> i CO CO

U-l o

<u c

pd CU s~\ /"N /-\ ^-N ^-\ /»~\ /"> co CO CO CO CO CO c

r-l r-4 r-4 r-l T-l r-4 T-l

co CJ o O 9 o U o

cu « W 33

K

W 33 33

•\ CU o O o O o UO

Q

n

i—i 00

v-^ s-x *—• s_x ^—

N—

v-/ CO r^» r-4 o> CM o a\ • ON oo • o • r-i «* • CO • CM

r-l vO «tf + r-» CN CM + + 1 r-4i + +

<t (N CO

m

m

,—S, o m VO r^. r-4

a c_> a o

>—

|

r-i• 1

r-4i t-I en T-( CM CO <J- t CO <J- o m vO r^ r-4 l-( 00

4-1 coo

ON

a)

r-l XIH

co

CM

a)

fl •v •k t4 a) CU 0)

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CI) i-l

ti &

i-l

a CUa

a

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>n cO

a

•H cO cO

to r-l di

a r-l t-4 o >N a Pi cO >N >N o w CO H r-l co CO 3 o i-l S >^ O o

t-i o >n cO co o CJ 00 3 to i-l' o 3 3 1 T-l o >n c r-l r-i OH 001

too ,0

001 001 cai oili CO t-l>N

cO OHi ONi TJ .-< ca u X cO CO. ca c >> i CO 1 1 1 i 3 a r-< 1 on OH r-l r-l O o >N OH 1 I >N >*

g* t4

a. o I 8

CO.1 9 oN o o o G i r-1 r-l PI P! u u CU r-l >> >n CO CO 1 .Q >>

4J o ON 1 1 ?' ?! 0)o

Pi

ca

d

CJ OI ?

CO cO CO ,a rO cO CO

S-i

U 1 i 1 u

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4-1 4-1 o| o] oi

u 4J

0) CU i i 1 0) cu 1 4J 4-1 -r4 •H •H 4-1 4-> T-l I 1 u U u 1 1 >>vO VO 1 4-1 4J 4J VO VO

C3 •V •v t-4 1 1 1 •v •v O «d" «tf >»o- <t- <* •tf St

•H •\ •v o •\ •\ •V •\ •v ceo CO N CO CO CO CO CO O •V *» c •v •» •» •k •v U CM CM 0) CM CM CM CM CM

fU PQ SI SI

COoH

r-l OsowoI o <m

<t r-lm VO r>^ 00 CT\

O CM V CM CM CM CM CM CO

CO r-l m

un co

CM CM o

4-> •H I u O -U -a >> a cu

cO I •U OH i-5ll CU I I o >.

cO

X o

4-1 •H

P! -H

cO

J-i COI O

4-1 •HO3

r-l00I OHO

XI

•rla

cO o

co cj

pq i I r-l

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4J•H

CJ3

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COi

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OO

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4J

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aO

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". 1 o

r-l r-4 0) o1

•vO vO o o O o O O ^-\ /"\

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v_^ v—/

v_/ ^-X v—/

v_/ v-y v-.' r^ 00 o r~- «* .VO m CO

00 ON t

CM 00 VO COm + + CM + + r-l i + +

r-l r-l r^

O VO vO ON vO

r-4 ON VO <!• ON in r-4 ON

CM r-l rH CM r-l i-l CM r-4 O ON 1 00 1 1 1 1

r-l 00 VO co ON

m r-4 ON

CMo

4-1 •H 1 5-i .rC

•8 >N a cu

CO I I N iJII h4ll

Pi I t

cOM

CO

CU >> X X

>-/ o

co cu •r4 TJ

pq i I r-4

>NXo

CU ID1
co

vO

<VO

CM

co CO

CO m VO r-l r<» •13 o> o 1-4 CM CO <! CO CO co w' CO CO CO <T <r <t <r <r

rO r^ O I r-l

CU Oh Cl> O-, O., CO cr.

PL, CO

co

i

r-< +CM

r-» +

CM+ r-l +

VO

CM

CTi

.-1

r-l OCM Or-l CM

CM

CM

CM

CM

CM

COO

CM

1 o

r-l

CT»

r-l

COO

CM

CM

CM

CM O

CM

CM oCM

CMm

CO CO

co <y\ <j\ vo vo vo o

CM

CO CO CO CO CO u a /—

X

CJ o U O o o

X w >> W W 3 w tn w

o u Oh CJ O CJ o u CJ

-—' %_»• v^ ^—

>w»* s ' v_^ Nw' V

m CO CM r-l r-i o CM CM <f

CM CM m m co CM er> O O 1*-.

1 CM+

+ CO+

VO+VO+ + r-l

1 CO

1

r—

S

CO /"> on O o\ M3 CO <X> en VO r-l CJ o CO r-l CT> o CO CT\ CO CO r^ C^ r-l o CJ CM

1 1 CM

I

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1

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1

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cd CO CO CO <t CM <r r-( m OA CJ CTl r-l r-l CO o CO c^ (TO 00 r^ CO ti a\ a. CM T-l CM r-l r-l r-l r-l r-l r-l ^s r-l ^s

r-lO

4J

r-l •H o Pi 3

H

rQ X CO r-| r-l r-l r»l I-l O r-l O O r-l cd 4J o

4J 4J 1 t r4 4-1 1-4 •H OH OH C r-l •H

Pi O I •H r-l o O

•H 3 r-l r-l ^ o 4J 3

r-lou

rO r-l >. >^ cd 4-1 •H r-l CO 00 O o I-l »H X M

u i N N cO U rl i nj OH a Pi 1 rG r4 Oil X

0) rC

I

1 i a) CJ Oil 4-1 1 1

1 rJH r-l 1 rO XI I >^ OH r-1 >> 1 >> >1 1 1 r-l u i >.

r-lo

X rH J-> X r-l o] o| >, r-l 4-1 OJ

•H

O >. CJ O O i o 1 >^ CJ r-lOU

•H

4JO

CO r-l

CO00

t

OJ 4J o 0) 4-1 m in N r^H 4-1 CJ n3 <U CO TJ •H I i Pi 1 CJ cO

r-lO

4J

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O

3

rO

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CO

I-l a)

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1 o

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3

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1

r-l

r*« 4J

GJO

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r*.

4-1 CJa

0) rO

r-4

4J

CJO

o

Ol

l

i o|

cO

4-1 rQ i 3

COa

rJII

CO TJ CO rl 5-1 c

CU OH

1

C3

i

cduCJ

1

cd PiCU U

r-i601X

o

CO TJ

tH

Ir>» X

o

cu

T-1

W co a) a.

i a

cO

r-l

1 Ol

i 4J

CJ oi 4J

CJ ex

1 Oil OH OH OH

| , P 4J

CJ 1 vO

•V

Csl Pi o •H U

4-1

1 •HS-i

4J

1

o

cu 'O

tX

o

co 13

I>,X

o

<u •a

X

o

(D

CM o in

CO »v

CO •»

in

•> rO

to rO

1

4-1

1 <!•

4J

1

4-1

1 m m

i CM

t vO •H CO CO •H ol to CO en CO en co

r-l

| W •% •» w •> •V «\ •s -\ •%

i r-t

1 r-l r-l

i —J r-l 1 CM —I

CM 1 —1

vO CM CM CM CM CM CM

u

cdu

u

CJ H-lCJ Q

C

cd •H

TJu

cdcdoM

4J

to

cd s

. M

>>

XI

TD

tu TJ

•r-l >

o

r-l a

>»

I-l 13

Pi

•r-l • r* N TJ

CJ c

r4 3

CU O u PL, e

• o

CO o

. 03 <J •HX

>, 4-1 XI

LH

TJ O

CJU CJ

cd -r-4 Cu CL

CJ e M cd P^ CO

CO

89
References for Table 9

1. Cerezo, A. S., Deulofeu, V., Carbohydrate Res. 2 f 35 (1966).

2. Hockett, R. C, Chandler, L. B., J. Am. Chem. Soc. £6, 957 (1944).

3. Cerezo, A. S., Delpy, S., Deulofeu, V., paper in preparation.

4. Isbell, H. S., Frush, H. L. , J. Org. Chem. 2J3, 1309 (1958).

5. Tipson, R. S., Isbell, H. S., J. Res. NBS 65A , 31 (1961).

6. Delpy, S., Ph.D. thesis, Buenos Aires, Argentina, 1962.

7. Isbell, H. S., Frush, H. L., J. Res. NBS 46, 132 (1951) RP2186.

8. Frush, H. L., Isbell, H. S., J. Res. NBS 47, 239 (1951) RP2248.

9. Tipson, R. S., J. Org. Chem. 26, 2462 (1961).

10. Brigl, P., Miihlschlegel, H. , Schinle, R. , Ber. 64, 2921 (1931).

11. Deferrari, J. 0., Deulofeu, V., J. Org. Chem. 22, 802 (1957).

12. Isbell, H. S., Smith, F. A., Creitz, E. C, Moyer, J. D., Frush,

H. L., "Infrared Spectra of Sugar Acetates and Related Compounds,"

NBS Report 1358, December 1951.

13. Deulofeu, V., Deferrari, J. 0., Anales Asoc. Quim. Arg. _38, 241 (1950)

14. Isbell, H. S., Frush, H. L., J. Am. Chem. Soc. 71, 1579 (1949).

15o Deferrari, J. 0., Deulofeu, V., J. Org. Chem. _17, 1097 (1952).

16. Deferrari, J. 0., Ondetti, M. A., Deulofeu, V., J. Org. Chem 24,

183 (1959).

17. Deferrari, J. 0., Deulofeu, V., J. Org. Chem. _17, 1093 (1952).

18. Gros, E., Lezerovich, A., Recondo, E. F., Deulofeu, V., Deferrari,

J. 0., Anales Asoc. Quim. Arg. _50, 185 (1962).

19o de Labriola, E. R. , Deulofeu, V., J. Org. Chem. JL2, 726 (1947).

20. Deulofeu, V., Deferrari, J. 0., J. Org. Chem. L7, 1087 (1952).

90
7. STANDARD REFERENCE MATERIALS

( H. S. Isbell , H. L. Frush, and R. Schaffer)

A. New Program on Standard Reference Carbohydrates

1. Rare Hexoses

The development of highly sensitive, modern methods of

instrumental analysis , and the heightened interest in

carbohydrates because of their immense economic value and

their many roles in biological processes, have resulted in a real and growing need for a large group of unavailable

carbohydrates of high purity as standard reference materials.

These are in demand for the identification of unknown compounds,

for quantitative measurements by instrumental techniques, for

studying and testing biological systems and processes, and as

nucleating crystals for the preparation of sugars in other

laboratories

.

For many years, the Organic Chemistry Section has maintained

a position of leadership in the development of methods for the

synthesis of rare sugars and sugar derivatives. In a program

covering a number of years, the Section has developed methods

for synthesis of sugars position-labeled with carbon-l4 and

tritium, and supplied these radioactive sugars on a fee basis.

The use of this service is world-wide. As the result of basic

research and numerous publications, the scientific public looks

to our Section for both information and materials in the

carbohydrate field.

In a new project now underway, it is planned to prepare

a series of standard reference carbohydrates, and to supply

these on a fee basis. Certain commercially unavailable hexoses

(D-allose, D-altrose, L-galactose, D-gulose, L-idose, D-sorbose,

D-tagatose, and D-talose) will be prepared, in the highest

purity feasible, in quantities of 10 to 100 g (depending on the

Individual characteristic of the sugar) . Because the preparation

of these materials is arduous and time-consuming, samples will

be issued to the public in amounts of only 10 to 100 mg.

91
2. 1, 2-0- Isopropylldene -L- Idose ( R. Schaffer ) At present , there is no evidence for the natural

occurrence of Idose., but both mirror-image forms of synthetic

idose have been prepared although neither has been obtained

as a crystalline material [69]. This sirupy aldohexose is

unstable. On storage,, it is reported to transform spontaneously

into the ketohexose sorbose [70], thus undergoing a reaction

ordinarily catalyzed by alkali. Furthermore ^ in aqueous acid.,

the sugar tends to form an equilibrium mixture with its

1,6-anhydride, a product that results from loss of a molecule

of water between the hydroxyl groups at C-l and C-6 of the

sugar [71]. Fortunately_, derivatives of idose do not suffer

such instability. Consequently , a derivative from which the

free aldohexose can be readily liberated is of particular value

for preserving the sugar and providing idose of optimum purity.

Crystalline 1,2-0-isopropylidene-L-idose [72], which can be

synthesized from D-glucose by a series of reactions, serves

this purpose.

The transformation of D-glucose into 1,2-0-isopropylideneL-idose involves the following steps: D-glucose (I) —* 1^2?5,6-

di-O-isopropylidene-D-glucose (II) —* 1,2-0-isopropylideneD-glucose (III) —- 1, 2-0-isopropylidene-L-xylo-pentodlaldose

(IV) —« "dimer" (V) —- 5-cyano-l 5 2-0-isopropylidene-D-glucopentose (VI) + 5-cyano-l,2-0-isopropylidene-L-ido_-pentose

(VII) —- barium 1,,2-0-isopropylidene-D-glucuronate (VIII ) +

barium 1^2-0-isopropylidene-L-iduronate (IX) —- calcium 1,2-

O-isopropylidene-L-iduronate (X) —- 1,2-0-isopropylideneL-idurono-6,3-lactone (XI) —- 1,2-0-isopropylidene-L-idose (XII),

Details of the conversion of D-glucose into its acetone

diacetal (II) and of the latter into monoacetone-D-glucose (III)

have been presented in many places [73] and are therefore not

discussed here. Periodate oxidation of III to the monoacetonexylo-dialdose (IV), with crystallization of the "dimeric"

product (V), was reported by Schaffer and Isbell [7^-].

92
a. Dlmerlc 1, 2-0- Isopropylidene -;[)-xylo -pentodialdose

Hemlhydrate (V) . A stirred solution of 50 g of sodium metaperiodate in 400 ml of water in a 1-liter flask, surrounded by

an ice bath, was treated during 30 min with 50 g of 1,2-0-

Isopropylidene-D-glucofuranose, added in small portions. After

the solution had been stirred an additional 20 min, the excess

periodate was decomposed by the addition of ethylene glycol.

The solution was concentrated by freeze-drying, and the residue

was extracted with four 100-ml portions of chloroform. The

extracts were combined, clarified by filtration through carbon,

and concentrated under reduced pressure to a heavy sirup. By

dissolution of the sirup in 100 ml of water and reconcentration

under vacuum, the trace of chloroform was removed. The

concentrate was dissolved in 50 ml of water, filtered, and

stored in the refrigerator for several weeks. The crystals

that formed were separated, washed with water, and then

recrystallized from water to give 29.9 g of a molecular dimer

of 1,2-0-isopropylidene-D-xylo -pentodialdofuranose which

crystallizes with 0.5 molecule of water of hydration. Additional

hydrate crystallized after lyophilization of the mother liquors,

dissolution of the residue in an equal weight of water, and

further storage at low temperature. Alternatively, this

concentrate can be fractionally distilled at high vacuum, as

described by Iwadare [75] in his original preparation of sirupy

IV. The distilled sirup is not as pure as the crystalline

product, but is nevertheless adequate as a starting material

for the cyanohydrin synthesis. Extraction procedures provide

a less pure sirupy product [76].

Conditions for adding cyanide to the free aldehyde group

of IV were carefully studied [77 1 when the reaction was employed

in this laboratory with radioactive cyanide for the synthesis

of D-glucose -6- C . Since the cyanide addition reaction adds

an asymmetric center to the L- xylo reactant, one product (VI)

has the D-gluco and the other (VTI) the L- ido -configuration

.

95
The relative proportions of the two products (VT and VII) are

affected by the buffer present in the reaction medium. We

have found that, with an acetic acid-sodium acetate buffer,

the reaction gives a maximum of the D-gluco epimer (VT) and

that less acidic buffers lead to a diminished yield of VT but

an Increased yield of the L- ido epimer (VII). However,

distinctly alkaline buffers were found to catalyze the aldol

reaction of the dialdose IV [78], which resulted in lower

yields of both VT and VII. The optimum yield of VII was obtained

by use of a bisulfite-buffered reaction. Although our analytical

details and an optimum-yield synthesis of the L-ido -epimer were

published [79 1 > a recently published procedure for preparing

the L-ido product utilized a bicarbonate-buffered cyanohydrin

reaction. In our procedure, the cyanohydrin addition products

VI and VTI are saponified by dilute alkali, and by use of

cation-exchange resin are then converted into the salts VIII

and IX. Advantage Is taken of the marked difference in the

solubilities of these barium salts to separate the epimers.

Compound VTII crystallizes on concentrating an aqueous solution

of VTII and IX, and replacement of the remaining water by methyl

alcohol completes the separation, as compound IX remains in

solution. The L- ido epimer is then purified by converting IX

into the calcium salt X, which crystallizes readily. Other

workers [76,80] have transformed the salt mixture obtained on

saponification of VI and VTI into their corresponding lactones,

and separated the latter products by column chromatography.

However, the salt-crystallization process we use is no less

effective for the separation, and has the advantages of being

much more rapid than the chromatographic method and readily

utilizable for separating any quantity of material.

b . Calcium 1 , 2-0-Isopropylidene -L- idofuranuronate

Dihydrate (X) . An aqueous solution of 12.54 g of 1,2-0-isopropylidene-L-xylo-pentodialdofuranose (IV), 12. 65 g of sodium

pyrosulfite, and 3.4 g of sodium cyanide was prepared at

and allowed to warm to room temperature. After three days,

94
the solution was heated at 90° for one hr, and then treated

with 7 g of sodium carbonate,, and refluxed for three hr. The

solution was cooled in an ice-bath., and passed through a column

containing 350 ml of cation-exchange resin at ice temperature.

The effluent, including washings, was delivered into a flask

containing 41.6 g of barium hydroxide octahydrate, immersed in

an ice-bath. After removal of excess barium hydroxide by

carbonation and filtration, the filtrate was concentrated under

vacuum to a sirup that yielded 8.5 g of crystalline barium

1,2-0-isopropylidene-D-glucofuranuronate monohydratej addition

of methyl alcohol gave a further 0.5 g. The mother liquor was

passed through a column containing 75 ml of cation-exchange

resin at ice temperature. The effluent, including washings,

was delivered into a flask containing 5 g of calcium carbonate.

The mixture was filtered, and the filtrate concentrated under

vacuum. Calcium 1,2-0-isopropylidene-L-idofuranuronate dihydrate

separated. The product, 4.7 g, was recrystallized from water

with the addition of methyl alcohol.

c . l,2-0-Isopropylidene -L- idurono-6,3-lactone (XI) . Lactone XI is prepared from X by use of a cation-exchange resin

to replace the calcium by hydrogen. The acid thus obtained

loses water on refluxing in toluene solution. An almost

quantitative conversion into XI is effected. An ice-cold,

aqueous solution containing 20 g of X was passed through icecold cation-exchange resin, and the effluent and washings were

concentrated under reduced pressure. After several additions

of ethyl alcohol and reconcentrations, the concentrate was

dissolved in acetone, toluene was added, and the solution was

heated gently for 2 hr to allow the acetone to escape and the

toluene to reflux. Filtration and concentration gave large

crystals of XI.

95
HCOH

I HCOH

HOCH

I HCOH

I HCOCH 2 OH

Me 2 CO

1 HCO.

| "^C

^Me

HCO

I ^Me

1 HOCH

1 iinLUfr\

HCO.

l>

H„CO

^Me

X Me

HCO^ .Me

HCO

I X

Me

I HOCH

I HCO

HCOH

CH 2 OH

11 III

V HCN

(Of IV) HS0 3~

HCO. Me

HCO^ x

Me

HOCH

HCO

HCOH

I C=N

VI

+

HCO Me

1 /

C

HCO^ \xMe

HOCH

HCO

OH

HCO. .Me

I C

HCO Me

HOCH

HCO

HCOH

COO Ba

VIII •> +

HCO. Me

HCO i*le

HOCH

HCO

->

HOCH

C=N

HOCH

COO \

VII

96

IX
HCO ,Me

HCO Me

HOCH

» HCOHC=0

IV

HCO. ^Me

C

HCO ^Me

HO —C— C —C —C

V

HCO. .Me

HOT ^Me

HOCH

HCO

HOCH

COO £2-

2

X

HCO.

HCO

1 >

1

.Me

XMe

1 OCH

nr-n

HOCH

C =

XI

>

HCO- -Me

HCO xMe

HOCH

HCO

HOCH

CH 2 OH

XII 97
d. 1,2-0- Isopropylidene -L- idose (XII) > Lithium

aluminum hydride effectively reduces the lactone to XII. A

stirred solution of 10 g of XI in anhydrous ether was treated

slowly with a clear solution of lithium aluminum hydride in

ether, and then refluxed for 15 min. Addition of 100 ml of

ethyl alcohol (slow at first) led to the dissolution of . precipitated solids,, and, after a solution of 10 ml of

concentrated acetic acid in 150 ml of water had been added,

the ether was evaporated under a jet of nitrogen, and the

aqueous solution was treated with cation-exchange resin at

. Concentration of the effluent, and reconcentrations with

ethyl alcohol, gave a sirup that dissolved readily in ethyl

acetate. 1,2-0-Isopropylidene-L-idose crystallized on

concentration of the ethyl acetate solution. The product was

recrystallized from ethyl alcohol plus pentane.

98
B. Metallo-organic Standards

1. Introduction ( H. S. Isbell ) At the time of the initiation of the metallo-organic

standards program at NBS, metal naphthenates were used for

spectrographic measurements of metals in lubricating oils.

Because of variations in the composition of naphthenic acid

and lack of oil-solubility of some metal naphthenates, we

sought other metallo-organic oil-soluble compounds as standard

reference materials.

In development of this project, we prepared numerous salts

of fatty acids; some of these had certain desirable properties,

but certain salts formed gels with petroleum products. These

gels are similar to Napalm (a coprecipitated aluminum soap from

napthenic acid and the fatty acids of coconut oil, used for

incendiary munitions). Presumably, gel formation arises from

cross-linked metal complexes. To avoid formation of gels, we

devised the technique of adding a highly fat-soluble beta- diketone to the mixture containing the standard. Presumably,

the beta- dike tone unites with some of the co-ordination centers

of the metal, and thereby prevents cross-linking and formation

of a gel. Use of this solubilizing technique permits considerable

latitude in the compounds used in preparing solutions of the

metals in lubricating oils. At the time we developed the

solubilizing technique^ we had already prepared supplies of

some of the standards we now issue. With depletion of the stock

of materials, we are now attempting to obtain improved products.

Exploratory work has revealed that many of the metals of interest

to this project form crystalline derivatives with 1-phenyl-l ,3-

butanedione. With the solubilizing technique, some of these

compounds are suitable for use as standards. Procedures for

99
preparation of the chromium and iron chelates of 1-phenyl-l , 3-

butanedione were described in our last Technical Note (No. 274),

and the preparation of the cadmium and calcium chelates is

de s c ribe d he re .

1 . Cadmium and Calcium Standards ( C . W . R.. ' Wade ) Cadmium 2-ethylhexanoate and the cadmium derivative of

1-phenyl-l, 3-butanedione were prepared, and studied for use as

metallo-organic standards. Both compounds dissolve in the

solubilizing mixture previously developed by us, and give

stable solutions of cadmium in petroleum oils. Cadmium

2-ethylhexanoate is, however, liquid at room temperature and

is therefore not suitable for use as a metallo-organic standard.

The cadmium chelate of 1-phenyl-l ,3-butanedione, however, was

found to have the desired properties, and was selected for use

as the standard material. Procedures for the preparation of

both compounds are given below, as well as for the corresponding

calcium compounds.

a. Cadmium 2-Ethylhexanoate » A solution of 7.98 g

(0.03 mole) of cadmium acetate dihydrate in 50 ml of water

was heated to 70 C and stirred, while a hot, aqueous, 20$

solution of sodium 2-ethylhexanoate (prepared from 4. 32 g,

0.03 mole, of 2-ethylhexanoic acid) was added. Cadmium

2-ethylhexanoate separated as a viscous, amber sirup which

was washed with two 5 -ml portions of hot water and dissolved

in 15 ml of ethyl alcohol. The solution was filtered and

evaporated. Crystallization of the sample was effected by

dissolving the viscous mass in 5 nil of pentane and refrigerating the solution* After about 24 hr, long thin needles

separated, but on being warmed to room temperature, the

crystals melted*

100
The compound dissolves readily in benzene, xylene,

chloroform, methyl alcohol, toluene, heptane, or carbon

tetrachloride; but, because of its low melting point, it is

not suitable for use as a standard sample.

b. Cadmium Bis(l-phenyl-l ,3-butanedione ) Chelate . Cadmium acetate dihydrate (2.66 g, 0.01 mole) was dissolved

in 5 ml of water and mixed with 5 ml of concentrated ammonium

hydroxide (sp gr 0.90). The solution was heated to 80 C and

vigorously stirred, while 3.2 g (0.02 mole) of 1-phenyl-l ,3-

butanedione (benzoylacetone ) in 5 ml of hot aqueous ethyl

alcohol (50$) was added slowly.

The cadmium bis(l-phenyl-l ,3-butanedione ) chelate was

precipitated immediately. After the mixture had been stirred

for a further 30 min, the material was washed on the funnel

with two 5-ml portions of hot water. The crude sample, after

recrystallization from 30 ml of ethyl alcohol, weighed 3*9 g

(89.6$) and melted at 210-211 C with decomposition.

The solubility of the compound in oil, using 6-methyl-2,4-

heptanedione and 2-ethylhexanoic acid as solubilizing agents,

is equivalent to, or better than, that of cadmium cyclohexanebutyrate used as NBS Standard Sample 1053. Additional tests

showed that the compound may be readily recrystallized from

N,N-dimethylformamide, or from ethyl alcohol.

The preparation was repeated with 4 kg of cadmium acetate

and 4.8 kg of 1-phenyl-l, 3-butanedione . The crude crystalline

product weighed 6.1 kg. After recrystallization from N,N-dimethylformamide, the compound will be used for a new cadmium organometallic standard.

c. Calcium 2-Ethylhexanoate - NBS Standard Sample 1074A . 2-Ethylhexanoic acid. (1.8 kg, 12.5 moles) was neutralized with

20$ aqueous sodium hydroxide, using phenolphthalein as the

101
Indicator (faint pink). The resulting solution was heated on

a water bath to about 90 C and vigorously stirred, while a hot, aqueous solution of 763 g (6.88 moles) of calcium chloride

in 2 1 of water was slowly added. The calcium salt began to

separate immediately as a sticky product which, after continued

heating and stirring, became crystalline. The solid product

was collected by filtration and washed three times with 500-ml

portions of hot water. The crystalline material (1980 g) was

recrystallized from 3 1 of ethyl alcohol (water being added to

incipient turbidity), and washed on the funnel with cold

acetone—ethyl alcohol (1:4). The recrystallization was

repeated, and the white compound was ground (agate mortar and

pestle), sieved (60-mesh), and dried in a rotary vacuum drier

(95 C/5mm), with continuous mixing.

After the dried material had been resieved, the product

was again thoroughly mixed, and submitted to Sections 310.05

and 310.02 for analyses. . Anal . Calcd. for C ,H CaO,: Ca, 12.3. Found: Ca, 12.5.

Spectrochemical analysis: Silicon and strontium were each

found to be less than 0.01 percent, and all other metallic

impurities were each found to be less than 0.001 percent.

Results of the drying tests suggested that this standard

should be dried in a desiccator over phosphorus pentaoxide

for at least 48 hr.

d. Calcium Bis(l-phenyl-l ,3-butanedione ) Chelate . Calcium hydroxide (7.4 g, 0.1 mole) was mixed with 100 ml of

water and heated to boiling, and the mixture vigorously stirred

while 32.4 g of l-phenyl-l,3-butanedione in 100 ml of ethyl

alcohol was added. Reaction occurred immediately, with rapid

dissolution of the calcium hydroxide and precipitation of the

' 102
calcium bis(l-phenyl-l,3-butanedione ) chelate. The off-white

precipitate was thoroughly washed with two 5-ml portions of

hot water and air-dried; weight, 36.8 g (100$).

The dry, crude product was dissolved in 150 ml of hot

ethyl alcohol, and the solution was kept at room temperature

overnight. The resulting crystals were collected on a filter

and recrystallized; weight 32 g, mp above 260 C. The compound

dissolves readily in the solubilizing mixture previously

developed for use in spectrographic analyses, and will give a stable solution of 500 ppm of calcium in oil.

The use of this compound as a replacement for the present,

oil-soluble, calcium standard is contemplated.

103
8. PERSONNEL AND ACTIVITIES

A

.

Personnel Listing

H. S. Isbell, Section Chief

R. S. Tipson

H. L. Frush

R. Schaffer

C. W. R. Wade

N. B. Holt - Retired Nov. 1965

A. J. Fatiadi

A. Cohen

B

.

Publications

Oxidation of Polycyclic, Aromatic Hydrocarbons.

R. S. Tipson j NBS Monograph 87 (Sept. 1965).

14 Synthesis of D-Glucose-3- C and Related Compounds.

H. L. FrushJ L. T. Sniegoskl, N. B. Holt, and H a S. Isbell,

J. Res. NBS 69A , 535 (I965).

Separation of Pyrenediones by Column Chromatography.

A. J. Fatiadi, J. Chromatog. 20, 319 (1965).

NBS Technical Note 274. Organic Chemistry Section - 1/1/64 - 6/30/65.

Edited by H. S. Isbell. (October 1965)

Action of Zinc Dust and Sodium Iodide in N,N-Dimethylf ormamide

on Contiguous, Secondary Sulfonyloxy Groups: A Simple Method

for Introducing Nonterminal Unsaturation.

R. S. Tipson and A. Cohen, Carbohydrate Res. 1, 338 (1965).

Preparation of Inososes From Their Phenylhydrazones by Use of

a Cation-exchange Resin; Separation of Certain

Phenylhydrazones From Phenylosazones

.

A. J. Fatiadi, Carbohydrate Res. 1_, 489 (I966).

Phenylhydrazono-phenylazo Tautomerism. Part I. xylo-4,5 ,6-

Trihydroxy-2-oxo-l,3-bis (phenylhydrazono)cyclohexane and

4-0xo-l-phenyl-5-phenylazo-3-pyridazine Derivatives

.

H S. Isbell and A. J. Fatiadi, Carbohydrate Res. 2, 204

(1966).

104
C

. Manuscripts in„ Process of Publication

Large-scale, Preparative Paper Chromatography.

H. L. Frush. . (Approved for publication.)

Deuterium Isotope Effects in a-p Pyranose and in Pyranose

Furanose Interconversions

.

H. S. Isbell and C. W. R Wade. (Manuscript completed for

J. Res, NBS)

Infrared Absorption Spectra of Some Aldofuranoid, Aldopyranoid,

and Acyclic 1-Acylamldo Derivatives of Sugars.

R. S. Tipson, A. S. Cerezo, V. Deulofeu, and A. Cohen.

(Manuscript completed for J. Res. NBS)

D. NBS Reports

(Quarterly Reports on Air Pollution Program prepared jointly

with certain Sections of the Division of Metrology and the

Division of Physical Chemistry)

NBS 8985 - Quarterly Report AIR POLLUTION PROGRAM - 7/1/65-9/30/65. R. Klein, R. E. Rebbert, R. Stair,

R. S. Tipson.

NBS 9031 - Quarterly Report AIR POLLUTION PROGRAM - IO/I/65-I2/31/65. R. Klein, R. E. Rebbert, R. Stair,

R. S. Tipson.

NBS 9092 - Quarterly Report AIR POLLUTION PROGRAM - 1/1/66-3/31/66. R. Klein, R. E. Rebbert, R. Stair,

R. S. Tipson

NBS 9373 - Quarterly Report AIR POLLUTION PROGRAM - 4/1/66-6/30/66. R. Klein, R. E. Rebbert, R. Stair,

R. S. Tipson.

E

.

Lectures

9/15/65 - Reactions of 2-0xo-l, j5-"bis (phenylhydrazones ) and

Diphenylformazans . 150th A C.S. Meeting, Atlantic

City, N. J. Symposium on New Reactions in

Carbohydrate Chemistry. H. S. Isbell . 1/17/66 - The Use of Paper Chromatography in the Separation,

Purification, and Analysis of Labeled Carbohydrates.

A.C.S. Winter Meeting, Phoenix, Arizona.

H. L. Frush

1/19/66 - 2,3-0-Isopropylidene- erythro -tetrodialdose and its

Conversion into Nitro- (and Amino- ) cyclopentanetetrols . A.C.S. Winter Meeting, Phoenix, Arizone. R. Schaffer

105
9. ACKNOWLEDGMENT

The Organic Chemistry Section wishes to thank Dr. J. K.

Taylor and staff of the Analysis and Purification Section

for assistance and use of equipment during the past year.

We also wish to express our appreciation to Mrs. R. S. Maddock,

Administrative Assistant for the Division of Analytical

Chemistry in Charge of Publications, who assisted in preparing

the report for publication.

The editor deeply appreciates the cooperation and help

of all members of the staff of the Organic Chemistry Section

in writing the report. He is particularly indebted to Dr.

H. L. Frush who critically reviewed all parts of the report

and made many improvements. Special thanks are expressed to

Mrs. Y. S. Sladen, Section secretary, whose tireless efforts

made expeditious publication possible and whose cooperation

and patience made a difficult task pleasant.

106
10 . REFERENCES

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.

[2] Clar, E., Ber. 65, 1427 (1932).

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[5] Arnold, R. T., Larson, R„, J. Org. Chem. 5., 250 (1940).

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[16] Angyal, S. J., Australian J. Sci. 28, 173 (1965).

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107
[19] Isbell, H. S., J. Res. NBS 18, 505 (1937) RP990.

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RP1274.

[21] Hassel, 0., Ottar, B., Acta Chem. Scand. 1, 929 (1947).

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D., Smith, F. A., J. Res. NBS 57, 179 (1956) RP2708;

Isbell, H. S., Smith, F. A., Creitz, E. C, Frush,

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(1957) RP2772; Tipson, R. S., Isbell ,~HT~S . , Stewart,

J. E., ibid . 62 , 257 (1959) RP2960; Tipson, R. S.,

Isbell, H. S., ibid . 64A , 239 (i960); 64A , 405 (i960);

65A , 31 (196l))~63A, W9 (1961); 66A , 31 (1962).

[25] Isbell, H. S. 3 J. Res. NBS 66A , 233 (1962).

[26] Isbell, H. S., Sniegoski, L. T., J, Res. NBS 68A, 145

(1964).

[27] Isbell, H. S., Tipson, R. S., Science 150 , 793 (1959);

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.

[28] Barker, I. R. L., Overend, W. G., Rees, C. ¥., Chem.

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.

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[34] Ruff, 0., Ber. deut . chem. Ges. 32, 550 (1899)5 35,

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108
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[57] Kline. G. M., Acree, S. P., BS J. Res. 5, 1065 (1950)

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ibid . 64A, 135 (i960).

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.

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(1956).

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H. S., J. Res. NBS 69A, 535 (1965).

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(1965).

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109
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Soc. 1951, 1218.

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R. S., J. Med. Chem. 6, 822 (1963)5 Richardson, A. C,

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.

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H., J. Chem. Soc. 1954 , 171.

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(1946). ~

110
[75 1 Ref . 513 PP. ^85 and 484. &

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5864 (1957).

[75] Iwadare, K., Bull. Chem. Soc. Japan, 16, 40 (1941).

[76] Wolfrom, M. L , Thomas, G. H. S., Methods

Carbohydrate Chem. 2, 52 (1965).

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(1958)5 81, 2178 (1959).

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[80] Shafizadeh, P., Wolfrom, M. L., J. Am. Chem. Soc. 77,

2568 (1955).

Ill
NOTES

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