IDE

.ary, N.W. Bldg

EC 2 1965 taken from the library.

274

ORRGANIC CHEMISTRY: RADIOACTIVE CARBOHYDRATES, SUGARS

IN SOLUTION, ALDOL CONDENSATIONS, MOLECULAR STRUCTURE,

SYNTHESIS OF SELECTED COMPOUNDS, AIR POLLUTION STUDIES,

REFERENCE MATERIALS (ORGANIC)

JULY 1964 TO JUNE 1965
THE NATIONAL BUR]

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standards; determination of physical constants and properties of materials; development of for testing materials, mechanisms, and structures, and making such tests as may be ne> - lady for government agencies; cooperation in the establishment of standard practices for in< tion in codes and specifications; advisory service to government agencies on scientific and t- problems; invention and development of devices to serve special needs of the Government; a«-: e to industry, business, and consumers in the development and acceptance of commercial standi I* and

simplified trade practice recommendations; administration of programs in cooperation wit! ted States business groups and standards organizations for the development of intec of practice; and maintenance of a clearinghouse for the collection and di>- m of scii h nical, and engineering information. The scope of the Bureau's activities i? - in the f nfj listing of its four Institutes and their organizational units. Institute for Basic Standards. Applied Mathematics. Electricity. M- ^|

Atomic Physics. Physical Chemistry. Laboratory Astrophysics * Radiation Ph\sics I Laboratory:* Radio Standards Physics; Radio Standards Engineering. Office of Standard Refe

Data. Institute for Materials Research. Analytical Chemistry. Polymers. Metallurgy

rials. Reactor Radiations. Cryogenics.* Materials Evaluation Laborato- ' f >ta ence Materials. Institute for Applied Technology. Building Research. Information Technology. Performance

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* Located at Boulder, Colorado 80301. ** Located at 5285 Port Royal Road, Springfield, Virginia 22171.
NATIONAL BUREAU OF STANDARDS

Technical Note 274

ISSUED DECEMBER 3, 1965

ORGANIC CHEMISTRY: RADIOACTIVE CARBOHYDRATES, SUGARS

IN SOLUTION, ALDOL CONDENSATIONS, MOLECULAR STRUCTURE,

SYNTHESIS OF SELECTED COMPOUNDS, AIR POLLUTION STUDIES,

REFERENCE MATERIALS (ORGANIC)

JULY 1964 TO JUNE 1965

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, 1963* and became part of the Institute for

Materials Research in the February I, 19^, reorganization.

It consists at present of seven sections and about 85 technical personnel encompassing some J>0 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 my 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 Technical

Note. In 1965 we plan to issue these summaries for all of

our sections. The following is the first annual report on

progress of the Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

ii
PREFACE

The Organic Chemistry Section originated in a combination, in 1948, of the Polarimetry Section of the Optics

Division and the Organic Chemistry Section of the Chemistry

Division. In the reorganization of i960, the Section was

assigned to the Physical Chemistry Division, which became

part of the Institute of Basic Standards in the reorganization of February 1, 1964. The Section was transferred to the

Analytical Division of the Institute for Materials Research

on February 17, 1965. This report covers the principal

activities of the Section in both Institutes from January 1,

1964 to July 1, 1965. Considerable background material is

presented for exposition of the overall program.

Most of our activities arise from need, on the part of

the scientific public, for assistance on problems within the

scope of the Bureau's mission, within the area assigned to

the Division and Section and within the special competence

of the staff. Certain activities have goals, largely

determined by the needs of the public. Thus, the primary

14 goal of the ' C-labeled carbohydrate project is to make

urgently needed, labeled carbohydrates available to the

scientific public; the goal of the metallo-organic standards

project is to provide suitable standards for determination of

metals In lubricating oils; the goal of our phase of the air

pollution project is to obtain knowledge on the oxidation of

iii
hydrocarbons that occur in polluted air. We conduct a

vigorous research program in order to maintain competence in

the ever-increasing number of specialized techniques of

organic chemistry. By so doing, we are able to support the

over-all Bureau program and render aid to the scientific

public. In selecting projects, preference is given to

problems of wide applicability and to the development of

basic principles. We prefer projects which yield methods of

measurement, or techniques applicable to a variety of problems

We carefully avoid merely extending methods or techniques

that follow known patterns. In the sections that follow,

some of the current and recently completed activities of

the staff are described briefly.

In order to describe experimental procedures adequately,

it has been necessary occasionally 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

IV
TABLE OF CONTENTS

PAGE

1. RADIOACTIVE CARBOHYDRATES 1

Synthesis of Carbon-l4-labeled Carbohydrates . . 1

General Methods «•....„... 1

D-Glucose-3- C 3

Synthesis of Tritium-labeled Carbohydrates ... 6

General Methods 6

Preparation of Intermediates ......... 9

3-t-Labeled Carbohydrates 9

Preparation of Carbon-l4- and Tritium-labeled

Carbohydrates for the Scientific Public ..... 10

Isotope Dilution and Double-label Methods of

Analysis 11

Determination of Kinetic Isotope-effects .... 13

Special Apparatus and Techniques ......... 17

Transfer of Liquids 18

Filtration 20

Removal of Solvents . „ 23

Reduction of Aldonic Lactones by Sodium

Amalgam 25

Chromatography ..... 29

Techniques for the Assay of Carbon-l4 and

Tritium ............... 30

2. SUGARS IN SOLUTION 36

Mutarotation Reactions ........ 37

Enolization Reactions ...... 4l

Successivej Reversible Processes ....... -i-1

v
TABLE OF CONTENTS (cont'd)

PAGE

Measurement of Rates of Primary Enolization . . 4^

Measurement of Rates of Interconversion of

Sugars . 44

Thermodynamic Stability of Isomeric Hexoses . . 46

Reversible Condensation and Cleavage Reactions . 47

Cleavage and Re synthesis of Hexoses ...... 47

Formation of Straight-chain Ketoses and

Branched—chain Aldoses by the Aldol

Condensation .......... 49

Rearrangements of the Saccharinic Acid Type ... 51

3. ALDOL CONDENSATIONS 57

Syntheses via Aldol Condensations

Cyclopentitols 60

4. MOLECULAR STRUCTURE 62

Cyclic Polyhydroxy Ketones ... 62

Phenylhydrazono-phenylazo Compounds

Acetamido-deoxyketoses

5. SYNTHESIS OF SELECTED COMPOUNDS 75

6. AIR POLLUTION STUDIES 76

Introduction

Interaction of Aromatic Hydrocarbons with

Oxygen under Pressure ....

Apparatus 78

Results

Oxidation of Polycyclic,, Aromatic Hydrocarbons

on Particulate Matter .....

VI
TABLE OP CONTENTS (cont'd)

PAGE

Determination of Losses Due to Adsorption.,

During Filtration of Aqueous Solutions of

Polycyclic, Aromatic Hydrocarbons . . 89

Preparation of Reference Compounds; Purification

by Column Chromatography 94

7. REFERENCE MATERIALS (ORGANIC) 96

Rare Organic Compounds ............. 96

14 ~b C- and ^H-Labeled Carbohydrates 96

Sugar Standards ................. 96

Metallo-organic Standards .... 96

Tris(l-phenyl-lj3~butanediono)chromium(lII)

(Standard Reference Material No. 1078) .... 97

Tris ( l-phenyl-l_, 3-but anediono ) ir on( III

)

(Standard Reference Material No. 1079) .... 98

8. PUBLICATIONS, REPORTS AND LECTURES ....... 100

Publications ........ .. 100

Manuscripts Accepted for Publication ...... 101

NBS Reports ........... * 101

Lectures .................... 102

9. PERSONNEL ' 104

10. ACKNOWLEDGEMENTS ................ 105

11. LIST OF REFERENCES 106

Vll
LIST OF FIGURES

FIGURE NO. PAGE

l4

1. Synthesis of a 1- C-labeled sugar 2

14

2. Synthesis of D-glucose~3- C . .

3. Standard-tapered test tube with radioactive

sugar 18

4. Sintered-glass filter pipet . . .» „ 19

5. Apparatus for transfer of liquids 20

6. Filtration apparatus 21

7. Apparatus for filtration in a closed system . 22

8. Distillation apparatus for determining

water-t 23

9. Trap for preventing entrainment losses in

f reeze-drying ....... ....... c . 24

10. Tube for reduction of lactones with sodium

amalgam .......... 26

11. Apparatus for reduction of aldonic lactones . 26

12. Sodium amalgam in the form of pellets .... 2

13. Radioautograph of chromatogram of impure

l^C-labeled sugar

14. Scan of chromatogram of pure tritium-labeled

sugar

15. Prior mechanisms for the formation of "a"-Dglucosaccharinic acid ,~. .

16. Proposed mechanism for the formation of

"a"-p-glucosaccharinic acid ....

17. Mechanisms for the formation of pentaacetoxybenzene and hexaacetoxybenzene ........ 64

18. Proposed structure for a typical 2-oxc-1.3-

bis(phenylhydrazono) compound

vm
LIST OF FIGURES (cont'd)

FIGURE NO. PAGE

19. NMR spectrum of 2-oxo-l,3-bis (phenylhydrazono)cyclohexane (hybrid structure) ... 69

20. Diagram of cell for spectrophotometric

study of the interaction of hydrocarbons

with gases 79

21. Photograph of apparatus for the spectrophotometric study of the interaction of

hydrocarbons with gases 80

22. Spectral changes induced by treatment of

benzene with oxygen 84

LIST OF TABLES

TABLE NO.

1. Completeness of cyanide addition to

2,3-_0-isopropylidene-D-glycerose ....... 4

2. Isotope effects in oxidation of aldoses-1-t

with iodine in alkaline solution at 25 °C . . . 16

3. Determination of isotope effects in the oxidation of aldoses -1-t with iodine in alkaline

solution by the double-label technique .... 17

4. Determination of tritium: carbon-l4 ratios

with a dual-channel , liquid scintillation

counter .............. 35

5. Migration of carbon atoms in the formation

of "a"-D-glucosaccharinic acid 56

6. Changes in concentration of phenanthrene in

filtrate, during filtration of solutions of

phenanthrene in distilled water 91

ix
ORGANIC CHEMISTRY: RADIOACTIVE CARBOHYDRATES , SUGARS

IN SOLUTION, ALDOL CONDENSATIONS , MOLECULAR STRUCTURE,

SYNTHESES OF SELECTED COMPOUNDS, AIR POLLUTION STUDIES,

REFERENCE MATERIALS (ORGANIC)

July 1964 to June 1965

Edited by Horace S. Isbell

ABSTRACT

The report describes work in progress in the Organic

Chemistry Section of the Analytical Chemistry Division at the

National Bureau of Standards. It presents reviews of recent

contributions of the Section on the following subjects:

Synthesis of carbon-14- and tritium-labeled carbohydrates,

isotope dilution and double-label methods of analysis, determination of kinetic isotope effects, use of solvent

isotope-effects for studying pyranose-furanose interconversions, measurements of rates of primary enolization, rates

of rearrangement of one sugar to another by bases, . estimation

of the relative stabilities of isomeric hexoses, reversible

aldol condensations, mechanism for the formation of

saccharinic acids, formation of branched-chain aldoses and

linear ketoses by aldol condensations, stereochemistry of

monoaminotetrahydroxycyclopentane derivatives, cyclic polyhydroxy ketones, phenylhydrazono-phenylazo tautomerism,

acetamido-deoxyketoses, syntheses and properties of selected

organic compounds, interaction of aromatic hydrocarbons with

oxygen, oxidation of polycyclic, aromatic hydrocarbons on

particulate matter, loss on filtration of aqueous solutions

of polycyclic, aromatic hydrocarbons, oxidation products of

pyrene, and preparation of l-phenyl-l,3-butanedione chelates

of chromium and iron for use as newmetallo-organic standards.

x
Key Words

Radioactive carbohydrates

Carbon- 14-labeled carbohydrates, synthesis of

Glucose- 3-C-14

Tritium-labeled carbohydrates, synthesis of

Isotope dilution techniques

Double-label method of analysis

Cyanohydrin synthesis

Reduction of aldonic lactones

Liquid scintillation counting

Hexoses-3-C-14

Pentoses-2-C-14

Tetroses-l-C-14

Apparatus for radiochemical syntheses

Kinetic isotope effects, determination of

Acetobacter suboxydans^ isotope effect in oxidation by

Sugars in solution

Mutarotation reactions

Enolization reactions

Rate of enolization, measurement of

Epimeric sugars, interconversion of

Thermodynamic stability of sugars

Alkaline rearrangement of sugars

Water-t, in measurement of enolization

Saccharinic acids, formation of

Aldol condensations

Branched- chain aldoses, formation of

Ketoses, synthesis by aldol condensation

Cyclopentitols, synthesis by aldol condensation

Molecular structure

Cyclic polyhydroxy ketones

Ketoses, acetamido-deoxyPhenylhydrazones, tautomerism of

2-Oxo-l,3-bis(phenylhydrazones)

Synthesis of organic compounds

Dimethanesulfonate of 2,2 l (phenylimino)diethanol, carcinostatic activity of

Olefinic alditols, method for synthesis of

Acetamido carbohydrates, infrared spectra of

Air pollution studies

Oxidation of polycyclic, aromatic hydrocarbons, bibliography on Interaction of aromatic hydrocarbons with oxygen

Apparatus for spectrophotometric measurements

Oxidation of hydrocarbons on particulate matter

Photochemical changes in thin-layer chromatograms

Adsorption of hydrocarbons on filters

Oxidation products of pyrene

Separation of pyrenediones by column chromatography

Reference materials, organic

Metallo-organic compounds

XI

1. RADIOACTIVE CARBOHYDRATES

( H. L. Frush , N. 3. Holt, H. S. Isbell,

L. T. Sniegoski, and C. W. R. Wade)

A. Synthesis of Carbon- 14-labe led Carbohydrates

1. General Methods

For a number of years, the Organic Chemistry Section has

carried out an extensive program on the development of methods

for synthesizing and utilizing carbohydrates position-labeled

with carbon- 14 or tritium. These urgently needed radioactive

compounds have been supplied to nearly every major biological

research laboratory in the country and to several laboratories abroad. Methods of synthesis and analysis have been

described in approximately 40 published papers, and over 2500

samples have been supplied to other research workers. (Some

of the methods are given in references 1 to 8, inclusive.)

Gradually, as commercial laboratories have become able to

synthesize radioactive carbohydrates (frequently, by our

published methods), the Section has discontinued supplying

them, except under special circumstances (as, for instance,

when commercial supplies have been temporarily depleted)

.

The Section does conduct certain difficult syntheses of

radioactive carbohydrates when (a) there is a demonstrable

need for a compound; (b) the compound, if available, would

have numerous applications in research; and (c) commercial

production of the compound is not feasible.

In order to prepare a radioactive sugar position-labeled
with carbon-l4, it is necessary to begin with a sugar having

one less carbon atom in the chain, and to extend the chain by

a series of reactions, starting with the addition of a

C-labeled reagent. The reaction that we chose for adaptation

to the semimicro scale necessary in the synthesis of a

ik r t 1- C -labeled sugar is the so-called Kiliani synthesis L9J.

It begins with the addition of cyanide (cyanohydrin reaction)

as indicated v 'Tow:

CN

RCHO

(Na*CN)

*CN HOCH + KCOH

I I R R

hydrolysis

*C02 *C0 H

2H HOCH + HCOH

I I R R

separation

lactonization

*i *C=0

I HOCH

I R'O-1

lactonization

* i *C=0

I HCOH

I R'O-1

reduction

(NaHg

x

) reduction

(NaHg

x

)

*CHO

I HOCH

I R

14 1- C -Sugar A

I HCOH

I R

14

1^ C-Sugar B

14 Figure 1. Synthesis of a 1- C -labeled sugar

2
Addition to the carbon chain creates a new asymmetric

center and affords two compounds. At some stage in the subsequent processes, these two epimers (diastereoisomers differing in configuration at C-2) must be separated. This has

usually been accomplished on the semimicro scale by cocrystallization with the non-labeled (carrier) compound.

Addition of the carrier reduces the specific activity of the

14 compound separated. For the synthesis of 1- C -labeled

sugars, the diastereoisomers are usually separated as the

salts of the corresponding aldonic acids.

14 14 For the preparation of 2- C -labeled sugars, a 1- Csugar is allowed to react with non-labeled cyanide in a

second Kiliani synthesis. This synthesis again creates

epimers, thus drastically lowering the yield of the desired

compound. Furthermore, the separation of epimers by cocrystallization with the non-labeled form again lessens the

specific activity. Nevertheless, the Section has developed

14 methods for the preparation of several 2- C -labeled sugars

[10]; some of these are now commercially available.

2. D- Glucose -3 -C

A procedure for the synthesis of this compound has

recently been developed [11]. In order to synthesize

14 D-glucose-3- C , it was necessary to start with the threecarbon compound D-glycerose (used as its 2,5-0-isopropylidene

derivative) and extend the carbon chain to six carbon atoms,

by three successive, addition reactions. Carbon- l4-labeled

3
cyanide was used only in the first of these additions. The

use of carriers in each step would have resulted in such

dilution of radioactivity as to have made the final product

useless. Consequently, we employed large-scale paperchromatography for separations at the tetronic and pentonic

levels. In order to ascertain optimal conditions for the

first cyanohydrin reaction, the rate and completeness of

addition of cyanide in various buffers were determined. The

results, given below, show that the most rapid and complete

reaction with cyanide occurred in the presence of sodium

bicarbonate and sodium carbonate.

Table 1. Completeness of cyanide addition to

2,3-0-isopropylidene-B-glycerose.

Time (days)

Buffer 3 5 7 10 12

% % % % %

NaHSO^

3 67.3 69.6 70.7 74.1 75.0

NaHCO, 78.6 88.7 9^.6 97.7 98.9

Nao C0,

2 3 72.7 82.7 88.5 93.7 95.6

NaOAc ) HOAc /

67.3 73.2 75.5 77.9 79.1

Because of the absence of the stabilizing pyranoid ring

in the tetroses, tetronic lactones cannot be reduced to

tetroses by the methods successfully employed for reducing

pentonic and higher lactones. Thus, both sodium amalgam

reduction and catalytic hydrogenation of an erythronic

lactone lead to the fully reduced product, erythritol [12,13],

4
However, a modified Rosenmund reduction [14],, as first

used by Cook and Major on fully acetylated aldonyl chlorides

[15]* appeared to provide a possible semimicro method for

14 preparing D-erythrose-1- C . The method had already been

applied by Glattfeld and Kribben [13] to the macro-reduction

of DL-erythronic acid (to give an over-all yield of

DL-erythrose of less than 4$). Subsequently, Ladenburg et al .

[lo] improved the yields of acetylated aldonic acids by use

of the cadmium salt of the acid for the acetylation. For the

semimicro preparation of tri-O-acetyl-D-erythronyl chloride,

it seemed probable that phosphorus pentaehloride, used by

Cook and Major, would be more convenient than thionyl chloride,

employed by Glattfeld and Kribben. In pilot experiments on

non-labeled material, one-millimole quantities of cadmium

D-erythronate were f reeze-dried, acetylated with a mixture of

acetic anhydride and hydrogen chloride, converted into triO-acetyl-D-erythronyl chloride with phosphorus pentachloride,

and reduced by the Rosenmund procedure to tri-O-acetylaldehydo-D-erythrose . The over-all yield, as determined by

titration, was 90%.

The reactions are as follows:

Cd(OH) p

Ac

p

O

H0H2 C(CH0H) 2C02H

^

[H0H2C(CH0H) 2C02

] 2Cd - *- (HC1)

AcOH

2

C ( CHOAc

)

2 C02H ^L- AcOHgC ( CHOAc

)

2C0C1

PCI,- H

2

(Pd)

AcOH2 C( CHOAc

)

2CH0
The chromatographic separation of epimers at the

4-carbon stage proved to be complicated by the formation of

condensation products. Consequently, the tetronic acid

14 mixture was converted into the two D-tetroses -1- C , and

14 thence into a mixture of the four D-pentonic- 2- C acids.

After lactonization and paper chromatography, this gave, as

14

one fraction, a mixture of 2- C-labeled D-arabinonic and

D-xylonic lactones. This mixture of lactones was reduced to

14

a mixture of the corresponding sugars, and D-arabinose- 2- C

was separated chromatographically; this sugar was then used

for the third cyanohydrin synthesis. The entire reaction

scheme, given in Figure 2 yielded not only the desired

14 D-glucose-3>- C but also numerous by-products of great value

for the study of reaction mechanisms, for the determination

of isotope effects, and for use as starting compounds in the

preparation of other labeled carbohydrates.

B. Synthesis of Tritium- la.be led Carbohydrates

1. General Methods

14 The versatility of a C-labeled compound in chemical

and biological research can be greatly extended by the simultaneous use of the tritium-labeled compound; various doublelabel techniques are then possible. Implemented by liquid

scintillation spectrometry, double-label methods have wide

application in biological studies, in evaluation of isotope

effects, and in determination of reaction mechanisms. For

these reasons, the Section has developed (a) methods for the

6
2,3-O-Isopropylidene-D-glycerose

14, cyanohydrin synthesis (Na CN) . Epimerlc D-tetronic cyanohydrins

I hydrolysis

I neutralization of acids with Cd(OH)„

* 14

2

14 Cadmium D-erythronate-1- C and D-threonate-1- C . Tri-O-acetyl-D-erythronyl- 1- C and

D-threonyl-l-14c chlorides

I Rosenmund reduction

* 14 14 Tri-O-acetyl-aldehydo -D-erythrose- 1- C and D-threose- 1- C

cyanohydrin synthesis (NaCN)

hydrolysis

Four D-pentonic acids,

Aliquot Taken for Remaining Steps

|lactonization of acids

I chromatographic separation

14

A. D-Lyxono-l,4-lactone-2- C (held on paper)

Radioactivity

(millicuries)

132

116

95

68

(71%)

B.

C.

14 D-Ribono-l,4-lactone-2- C (held on paper)

14 D-Arabinono-l^-lactone-g- C

|-Xylono-l y 4-lactone-2-l7?C

\ reduction of band C (NaHg ) X

chromatographic separation

19.0

14 D-Arabinose-2- C

^ 14 D-Xylose-2-C (held on paper)

cyanohydrin synthesis (NaCN)

Epimeric D-hexonic cyanohydrins

hydrolysis, and separation of acids by use of carriers

7.0

14 Potassium D~gluconate- 3- C

i " —14*

D— -Glucono- 1 ,

7 5 -lac tone--3-C

reduction (NaHg )

' 14

X

D-Glucose-3- C

1/ 14

D-Mannono-1.4-lactone-3- C 3.0

3.6

2.8

14

Figure 2. Synthesis of D-glucose- 3- C .

7
synthesis of tritium-labeled as well as carbon-l4-labeled

carbohydrates, and (b) techniques for using double-labeled

compounds

Carbohydrates position-labeled with tritium must be

synthesized by methods entirely different from those already

described. In general, reduction processes (carried out with

tritiated reagents, such as the tritiated metal hydrides)

have proved to be the most convenient and practicable for

position-labeling. Thus, reduction of an aldehyde group then

yields a primary alcohol having a carbon-bound tritium atom.

Reduction of a keto group likewise introduces tritium, and

simultaneously creates a new asymmetric center; the two

tritium-labeled diastereoisomers resulting can usually be

separated by chromatography. Reduction of aldonic lactones

produces aldoses- 1-t and alditols- 1-t ; the proportions depend

on the experimental conditions.

We have also used other reduction methods, such as (a)

catalytic tritiation and- (b) reduction of lactones with

sodium amalgam in tritiated water. Method (a) requires a closed system for circulating tritium gas, but yields highactivity products; method (b) necessitates the use of excessive amounts of radioactivity in the form of tritiated

water, because the tritium introduced comes from the solvent.

However, method (b) is convenient for preparing low-activi'

aldoses- 1-t . Some synthetic procedures developed in this

laboratory are given in references 17 to 20, inclusive.

8
2. Preparation of Intermediates

v m

Because reduction of a/C=0 group to a^C^ „ group is an

effective method for introducing carbon-bound tritium into a

carbohydrate molecule, we investigated carbohydrate intermediates possessing the former group. Some of these intermediates are commercially available [e.g., 2-keto- and

5-keto-D-gluconic acid (D- arabino-2-hexulosonic acid and

D-xylo-5-hexulosonic acid), and the common uronic acids

(D-glucuronic, D-mannuronic, and D-galacturonic acids)].

Other needed carbonyl compounds can be obtained only by laborious chemical syntheses, involving oxidation of a carbohydrate derivative in which all but one>CHOH group is protected by O-substitution. A third group of carbonyl intermediates may be obtained by certain stereospecific, bacterial

oxidations. Thus, Acetobacter suboxydans accomplishes the

following oxidation:

R R

I I HCOH HCOH

I I HCOH C=0

CH2 0H CH20H

Agrobacterium tumefaciens oxidizes C-3 of many D-glucopyranosyl

and D-galactopyranosyl derivatives.

3. 3-t-Labeled Carbohydrates

We have applied all of the described methods (for the

preparation of intermediates and reduction thereof) in the
synthesis of a number of aldoses and alditols labeled at C-l,

C-2, and C-6. Work is now in progress on the preparation of

certain important 3-t -labeled carbohydrates.

Through the kindness of Professor M. Bernaerts, Brussels,

we have received samples of 3-keto-lactose and 3-ketomaltobionic acid. By means of a culture of Agrobacterium

tumefaciens , we have oxidized several disaccharides and

aldob ionic acids, and, by chromatography, have shown the

presence of the corresponding 3-keto compounds. By use of

various tritiated reducing reagents, these 3-keto compounds

are being reduced either differentially (keto group only) or

completely. The products will be chromatographically

separated either before or after hydrolysis of the disaccharide

linkages. The resulting 5-t -labeled carbohydrates, particularly

sucrose, lactose, D-glucose, and D-galactose will have many

important applications. There are innumerable analytical and

technological possibilities for the use of the tritium-labeled

carbohydrates ; these will undoubtedly be developed when the

materials become available.

C. Preparation of Carbon- 14- and Tritium-labeled Carbohydrates for the Scientific Public

After development, the described synthetic methods have

been used for preparing sufficient supplies of radioactive

sugars to furnish needed quantities to other laboratories.

Each preparation requires careful handling through a complex

series of processes, and final proof of a high degree of

purity, usually by chromatographic methods.

10
As radioassay methods have grown more sophisticated,

research workers have become concerned with smaller and

smaller amounts of "radiochemical impurities. Since, on

storage, decomposition of radioactive organic compounds can

occur by self-radiation, we periodically monitor our supplies

by chromatography and, when it is needed, supply information

to other workers concerning purity and methods of synthesis

and analysis. This project fills an urgent need on the part

of medical and biological research workers. Although the

simpler radioactive carbohydrates are now made commercially,

the Bureau remains an important source in this country for

difficultly synthesized radioactive carbohydrates.

D. Isotope Dilution and Double-label Methods of Analysis

The analysis of organic materials is greatly facilitated

by the use of carbon- l4- and tritium-labeled compounds, of

which the Organic Chemistry Section has a unique and valuable

collection. The usefulness of radioisotopes arises in large

measure from the ease and sensitivity of measurement of radioactivity. One of the most useful analytical techniques,

isotope dilution, permits the analysis of mixtures that are

otherwise extremely difficult to evaluate. Thus, an aliquot

of a mixture of labeled products can be added to a known

quantity of a (non-labeled) suspected constituent, and the

total activity of the carrier (purified by recrystallization

or chromatography) indicates the amount of the labeled constituent in the aliquot. The method can be used whenever

pure compounds are available.

11
The technique is even more effective in the so-called

double-label method. In this, an aliquot of an unknown

mixture, labeled with radioisotope A, is added to a suspected

constituent having a known activity of radioiosotope B.

Amounts of the doubly labeled compound too small for gravimetric procedures can then be isolated by chromatography or

by addition of non-labeled carrier (because such addition

does not change the ratio of A to B) . The activity of A in

the aliquot (in the form of the given compound) is then

calculated from this ratio and the amount of radioisotope B

that was added.

We have developed a combination of the two techniques

just described which permits the kinetic study of isotope

effects, or differences in the rates of reaction of labeled

and non-labeled molecules. In this analytical technique,

both isotopes are present in the compound before reaction ; one is situated at or near the reaction center, and the other

is remote from it. When an isotope is directly involved in a rate-determining step of a reaction, a relatively large

(primary) isotope effect occurs. An isotopic atom remote

from the reaction center has little or no effect on the

reaction rate. If an isotope effect exists in the reaction

of a doubly labeled compound, the ratio of the two isotopes

in both the residual reactant and the product changes progressively as the reaction proceeds, and the isotope effect

can be determined from this change. At various stages of the

reaction, non-labeled carriers of either the reactant or the

12
product are added to aliquots of the reaction mixture, and

the ratio of isotopes is determined in the doubly labeled

compound. The method is particularly suitable for use with

carbon-l4 and tritium.

E. Determination of Kinetic Isotope-effects

The isotope effect is usually expressed as k*/k, in which

k* and k are, respectively, the rate constants for the labeled

and non-labeled forms of the reactant molecule. For a reaction

that is first order (or pseudo first-order) with respect to

the reactant, the value of k*/k can be calculated by the

equations of Stevens and Attree [21] as modified by Ropp [22]:

k*/k = 1 + [log r'/log (1 - f)] (1)

k*/k = log(l - rf )/log(l - f

)

(2)

In these equations, f_ is the fraction of starting material that has reacted. "When the equations are applied to

radioactive compounds, r_ is the ratio of the molar specific

activity of the accumulated product to that of the initial

reactant; and r_' is the ratio of the molar specific activity

of the residual reactant to that of the initial reactant.

(The value for the molar specific activity of the accumulated

product of 100$ reaction may be used instead of that for the

initial reactant.)

Determination of _f , £, and r_' by conventional methods is

difficult, because the product and the residual reactant must

be separated and rigorously purified. However, the task has

been greatly simplified by the simultaneous use of two radioisotopes [23], If p° is the ratio of the functional to the

13
reference isotope in the initial reactant, p_ the ratio in the

residual reactant, and p' the ratio in the product., then r_ =

E'^R ^' = JP/P°* and equations 1 and 2 become, respectively:

k*/k = 1 + [log(p/p°)log(l - f)] O)

and k*A = log(l - fp'/p_°)/log(l - f ) (4)

When the reaction is complete, p_' becomes equal to p (provided that the functional isotope is retained in the product

and that there are no side reactions).

By means of the analytical method outlined, values for

k*/k for carbon-l4- and tritium- labeled compounds involved in

the following reactions and processes were measured and reported in the publications cited: Oxidation of labeled

D-mannitols and D-glucitols with Acetobacter suboxydans [24,

25]; recrystallization of g-glucose- 1-t (single label only)

[26]; oxidation of g-glucose- 1-t and g-glucose -1- C with

iodine [23]; recrystallization of D-mannose- 1-t phenylhydrazone [27]; oxidation of aldoses- 1-t with bromine [28]; oxidation of aldehydes and aldoses- 1-t with sodium chlorite [29].

On the basis of the isotope effects determined, a unique

mechanism was proposed for the oxidation of aldehydes and

aldoses with chlorite ion. The mechanism accounts, for the

first time, for the unusual stoichiometric relationships in

this reaction.

The work on the oxidation of aldoses-1-t with bromine (a

currently controversial subject) showed clearly that various

amounts of the slowly oxidized a anomers mutarotate to the p

14
anomers before oxidation, and that the relative amounts of

the a anomers oxidized directly and indirectly depend on the

conformational stability of the pyranose form.

An analytical method was also developed for kinetically

determining a tritium isotope-effect, without the use of

double labels, for reactions in which water-jt is formed from

a nonvolatile reactant [30]. In this method, the equation of

Ropp is employed, and f is estimated from the amount of reagent consumed or by other convenient means. The solution

containing the products of partial reaction and the residual

reactant is diluted with water to a known volume. A portion

of this solution is freeze-dried, and the total activity of

the water-t (the product of partial reaction) is calculated.

The ratio of the activity of the water-t to the activity of

the initial reactant equals rf_. Substituting the values of

f and rf in equation (2) gives k*/k. The precision of the

method is illustrated in Table 2. Results obtained by the

double-label method, given in Table 3* show that the agreement is good, considering that the values from the water-t

method are based on the early part of the reaction period,

whereas those from the double-label method are based on the

latter part.

15
Table 2. Isotope effects in oxidation of aldoses-1-t

with iodine in alkaline solution at 25 ^c—

Aldose-1-t Fraction of

aldose oxidized

Radioactivity in

original

reactant

Radioactivity of

water-t

formed

Isotc

effec;

L-Arabinose

f

0.100

.200

.300

|JLC

6.05

6.30

5.64

0.092

.194

.262

Avg.

k*/k

0.15

.14

.13

0.14

D-Xylose .165

.200

.303

15.90

13.78

13.83

.347

.511

.756

Avg.

.12

.17

.16

0.15

D-G-lucose .100

.200

.300

7.60

7.43

7.56

.120

.252

.409

Avg.

.15

.15

.16

0.15

D-Mannose .107

.198

.313

9.82

7.55

7.47

.143

.214

.3.:-

Avg.

.13

.13

.12

0.13

D-Galactose .101

.199

.317

6.26

6.35

6.42

.082

.186

.340

Avg.

.12

.13

.1

.:•

D-Talose .100

.200

.300

10.86

11.26

10.09

.194

.340

.515

Ay .

.1*

.14

.1:

0.15

L-Rhamnose

monohydrate

.100

.200

.300

16.71

11.28

' 12.40

.239

.330

.504

Ay

.1-

.13

O.lj

16
Table 3. Determination of isotope effects in the oxidation

of aldose s-JL-t with iodine in alkaline solution

by the double-label technique.

Fraction of Ratio of functional Isotope

aldose oxidized to reference isotope effect

in carrier mixture

k*/k

.- 14 D-Gluco se-6- C-l-t

0.0 9.,12 , , a^-r .761 30,,89 0.15

.785 30,,66 .21

.839 43,.05 .15

.882 52.,17

14 .-^C-l-t

Avg.

•18

0.17

p--Mannoi3e-2-

0.0 7..72 _—__

.726 23,M .14

.756 26,.39 .13

Avg. 0.14

F. Special Apparatus and Techniques

In the foregoing sections, we have omitted description

of the techniques employed in the preparation and use of

radioactive carbohydrates. In this section, we describe

general procedures, apparatus, and techniques which have been

developed (or modified) for the synthesis or analysis of

carbon-l4 or tritium-labeled compounds. The techniques are

chiefly modifications of procedures developed in many

laboratories [31]. Much of the synthetic work on labeled carbohydrates is carried out on a semimicro scale in test tubes or

round-bottomed flasks having standard-tapered joints (19/38 or

24/40). The tubes are conveniently made from blank, standardtapered, outer joints; hollow stoppers sufficiently long to

accomodate a spatula are made from the inner joints (Figure 3).

17
Figure 5. Standard-tapered test tube with

radioactive sugar.

1. Transfer of Liquids

Several methods are used for the transfer or removal of

radioactive solutions. Formerly., disposable pipets were drawn

from Pyrex-glass tubing (O.D. 7 mm) and fitted with rubber

bulbs of 1- or 2 -ml capacity; this type of pipet is now

commercially available.

A special filtration pipet., made in the Section, is

often used for removing mother liquor from crystals in a test tube (Figure 4). To make the pipets., high-melting glass

(Vycor) is crushed in a mortar, and successively sieved

through screens of 40., 60, and 100 mesh per inch. Pyrexglass tubing (O.D. 8 mm) is drawn out, and so cut that the

18
;>

Figure 4. Sintered-glass filter pipet.

narrow opening is about 1 or 2 mm in diameter. The narrow

end of the tube is then tamped in the sieved glass collected

by either the 60-mesh or the 100-mesh sieve , so that about

2 to 5 nun of the end of the tube is filled with glass

particles of the desired size; the tamped end is then

slightly sintered in a flame. From a series of pipets so

prepared, only those that have a satisfactory flow-rate

under vacuum are selected; in use, the sintered-glass pipet,

equipped with a rubber bulb, serves as a small, frittedglass filter. Usually, radioactive compounds are not

removed from the test tube in which they are crystallized;

instead, the mother liquor is separated by means of a

sintered-glass pipet, the crystals are appropriately washed,

and the washings are similarly removed.

It is often convenient to transfer a liquid from one

flask or test tube to another; the simple apparatus of

Figure 5 is effective for such transfers. The flask is

rinsed in the same manner in which it is drained. Formerly,

narrow, glass tubing was used for connecting the two

containers; however, narrow, flexible-plastic tubing, which

is now available, obviates breakage and the need for several

sizes of glass siphons.

19
opening

to control

vacuum^

to trap

and vacuum

line

narrow

plastic

tubing

Figure 5. Apparatus for transfer of liquids.

2. Filtration

For filtering radioactive solutions, we devised the

glass funnel of Figure 6, which is used in a variety of

sizes, the larger and wider ones containing a perforated

porcelain or fritted-glass plate. The opening of a small

funnel is usually closed with a plug of glass wool or a

glass "nail" made by flattening the end of a narrow glass

rod; this is covered with a layer of paper pulp (tamped

down) and, if decolorization is necessary, with a small

amount of a decolorizing carbon (provided that the solution

to be filtered does not have a high specific radioactivity),

If more than a few milliliters of solution are to be

filtered, a transfer siphon (shown in the sketch) is fitted

to the funnel, permitting the transfer of a radioactive

solution without loss.

20
rr

to trap <-^,

and vacuum

line

fitted to' % flask

or test

tube

$ 19/38

or 24/40

inner joint

=^\

narrow glass

or plastic tubing

Figure 6. Filtration apparatus.

Figure 7 shows apparatus designed for filtering

solutions of compounds in a closed system. At the start of

the process, flask C contains the solvent , and flask A

(initially in the lower position) contains the solid to be

dissolved. Under vacuum, solvent is distilled from flask C

to flask A, and the solid is dissolved, Flasks A and B are

then rotated 180 degrees, flask B is cooled, and the solution

filters through the fritted-glass disk into flask B. By

cooling the top of flask A (with powdered, solid carbon

dioxide supported on the flask by a cork ring) and slightly

warming flask B, solvent condenses on the upper wall of

flask A, thereby rinsing it. This solvent returns to flask

B when flask B is again cooled.

21
Figure 7. Apparatus for filtration in a closed

system.

The solution in flask B may be freeze-dried (see

following section) j or the compound may be brought to

crystallization by evaporating part of the solvent on a rotary vacuum still or by adding a poor solvent.

We designed the apparatus of Figure 8 for use in

evaluating tritium isotope-effects in reactions in which

water-t_ is evolved, as, for example, in the water-t_ method

described on page 15 of this report.

22
Figure 8. Distillation apparatus for determining water-t.

vacuum line.

5. Removal of Solvents

Solvent removal has been one of the most serious

problems in the development of methods for synthesizing

radioactive carbohydrates. For concentration of the

relatively large volumes of solution often obtained as the

eluant from columns of ion-exchange resins, we have used a

rotary vacuum still of the conventional type; however, each

distillate is tested for radioactivity (that occasionally

may be mechanically carried into the distillate as spray).

If radioactive, the distillate is separately re-evaporated,

and, when reduced to a small volume, is transferred to the

principal radioactive residue.

Water, acetic acid, and j)-dioxane are frequently

23
removed from solutions of radioactive materials by freezedrying. A solution is frozen in a round-bottomed flask,

and the solvent is removed by sublimation. Figure 9 shows

the use of an "activity trap" (A) so designed that solid

to

vacuum

line

Figure 9. Trap for preventing entrainment losses

in f reeze-drying.

radioactive residues (which, if fluffy, may tend to puff

out of the flask) may be readily reclaimed. The contents

of the solvent trap (which is cooled in a mixture of solid

carbon dioxide and an organic solvent) are tested for

radioactivity before they are discarded.

24
To concentrate liquids in test tubes , we have used a

commercially available, multiple-outlet, vacuum still

(modified for use with standard-tapered test tubes) that

rapidly swirls the contents of the tubes in a bath at

constant temperature. The apparatus is used with a cold

trap and a vacuum pump.

4. Reduction of Aldonic Lactones by Sodium Amalgam

In slightly acid solutions, lactones of aldonic acids

are reduced by sodium amalgam to the corresponding sugars.

Because the reaction of the amalgam with water produces

sodium hydroxide, it has been customary to add acid,

usually sulfuric, from time to time as the solution

approaches neutrality. However, for the reduction of

radioactive aldonic lactones with sodium amalgam on a

semimicro scale and in a closed system, it was necessary

to devise new procedures and apparatus. Instead of the

step-wise addition of sulfuric acid, we use an excess of

(slightly soluble) sodium hydrogen oxalate. The reductions

are conducted in a heavy-walled, standard-tapered glass

tube that has a side arm for adding amalgam (Figure 10)

;

the tube is fitted with a stainless-steel stopper having a

greaseless bearing that accommodates a stainless-steel

stirrer. In use, the reduction tube is kept in an ice

bath (Figure 11). The sodium amalgam used in the reduction

is prepared in the form of pellets (Figure 12) by pouring

molten amalgam into a column of mineral oil [32].

25
1/4 STAINLESS STEEL STIRRER

STAINLESS STEEL STOPPER J 24/40, FITTED WITH GREASELESS

BEARINGS

« HEAVY -WALLEO

GLASS TUBE

Figure 10. Sodium amalgam reduction apparatus

Figure 11. Apparatus for reduction of aide
Figure 12. Sodium amalgam in the form of pellets.

Prior to the reduction of a

' C-labeled lactone, the

amount of sodium amalgam required for the maximal yield of

the corresponding sugar is determined by reducing onemillimole samples of the non-labeled lactone with various

quantities of sodium amalgam in the presence of excess

(solid) sodium hydrogen oxalate. In a typical preparation

14 of a radioactive sugar, one millimole of the C-labeled

aldonic lactone and 2 g of sodium hydrogen oxalate are

placed in the reduction tube (Figure 11). Twenty

milliliters of ice water is then added, and, after the

stirrer has been started, 4,6 g of (5%) sodium amalgam

pellets are introduced into the tube through the side arm.

27
The mixture is vigorously stirred in the ice bath for about

an hour. The reduction tube is then removed from the ice

bath, mercury is pipetted from the mixture, and the solution

is neutralized by the addition of sodium hydroxide solution

(sufficient to produce a faint, permanent pink color with

phenolphthalein indicator); the color is then discharged by

the dropwise addition of a solution of oxalic acid. Five

volumes of methanol are added, and the crystalline salts

are removed by filtration, washed with methanol, and

discarded. The combined filtrate and washings are

concentrated under reduced pressure to a thin sirup, to

which four volumes of methanol are added; the small crop

of salts thus precipitated is removed and washed as before.

The filtrate is freed from methanol under reduced pressure,

and an aqueous solution of the product is passed through a column of mixed cation- and anion-exchange resins (about

20 ml). The resin is washed with water until the radioactivity of the last part of the effluent is negligible.

The combined eluant is tested for ionic impurities by use

of a commercial conductivity-meter of the type used for

testing the purity of distilled water. The salt-free

solution is concentrated under reduced pressure, and freezedried (Figure 9). The residue, dissolved in a few drops of

water, is transferred to a tared test-tube (Figure 3).

The solution is diluted with two volumes of methanol,

nucleated with the crystalline sugar, ana treated with

28
sufficient 2-propanol to produce incipient turbidity.

Additional 2-propanol is added as crystallization proceeds

Finally, the mother liquor is removed by means of a

capillary filter-pipet (Figure 4), and the crystals are

washed with a mixture of methanol and 2-propanol. The

compound is recrystallized, usually with filtration of

the solution (Figure 6), until it is chromatographically

pure.

5. Chromat ogr aphy . We have used paper chromatography extensively in

synthesizing and testing both carbon-l4- and tritiumlabeled compounds. Radioautographs of chromatograms of

carbon-l4-labeled compounds are useful for detecting

impurities (Figure 13) , or following the course of a

reaction. For certain difficult separations or purifications, large-scale paper chromatography has been used for

preparative purposes. Bands containing carbon-l4-labeled

Figure 1J> . Radioautograph of chromatogram of

impure *- C-labeled sugar.

29
compounds, precisely located by radioautographs, are cut

out of the paper , and eluted.

Chromatograms of tritium-labeled compounds , because

of the weaker radiation, do not produce satisfactory

radioautographs. However, by use of a commercial

chromatogram- scanner, it is possible to ascertain the

purity of tritium-labeled compounds (Figure 14), and even

—

1

*- 1

.7

1-

2 1

; H

Figure 14. Scan of chromatogram of a pu]

tritium- labeled suge

to determine the location of bands of tritium-labeled

compounds on a large-scale, preparative chromatogram.

These techniques will be discussed in more detail in a

forthcoming paper.

6- Techniques for the Assay of Carbon-14 and Tritium

Utilization of carbon-14 and tritium as research tools

requires simple and precise methods of analysis. The method
of- choice depends on the equipment at hand and the

substance to be analyzed. In prior publications from this

Section, techniques have been described for determining

carbon-l4 and tritium by assaying samples in films [33, 34]

and in solution [4] with a 2-tt, windowless, gas-flow,

proportional counter. The methods require relatively

simple equipment , but they are laborious.

At present, most of our measurements are made with a

dual-channel, liquid scintillation counter which greatly

simplifies measurements of radioactivity. With this

instrument, assays of carbon-l4 and tritium may be made

separately, or simultaneously with double-labeled compounds,

without changing the setting of the instrument. This

flexibility in the use of the dual-channel instrument is

achieved in the conventional manner [35]* by critically

setting the pulse-height discriminators of channel 1 for

monitoring tritium, those of channel 2 for monitoring

carbon-l4, and the voltage of the photomultiplier tubes

for optimum, over-all monitoring.

* For the simultaneous assay of carbon-l4 and tritium in a

liquid-scintillation counter, Okita and co-workers [36]

described three procedures, which they designated as (a) the

screening method, (b) the simultaneous-equation method, and

(c) the discriminator-ratio method. The procedure outlined

here is essentially their method (b). Under the conditions

described, the method has the advantage that, without

changes in setting, it permits counting of either isotope

alone, or of the two in the same sample, with fairly high

efficiency.

31
In order to obtain the best settings for the

pulse-height discriminators, plots are made of the counting

efficiencies, at various pulse-heights, of both carbon-Inland tritium in the scintillator solution of choice. The

optimal setting is taken as that point on the graph at

which the highest contrast-ratio (ratio of the counts

occurring in one channel to those occurring in the other)

is obtained for both isotopes with minimal loss of counting

efficiency for channel 1 (tritium). For selecting the

voltage of the photomultiplier tubes, integral,

discrimination-bias curves (counting efficiency versus high

voltage) are plotted for each isotope. From these curves,

obtained with discriminator settings of 40 to 500 v (for

channel l) and 300 to 1000 v (for channel 2), for the

instrument at hand, an optimal setting of 1225 v was found

for counting carbon-l4 and tritium in the p_-dioxanecontaining scintillator solution described below. The value

for the high voltage is the optimal neither for carbon-14

nor for tritium, but is a compromise that permits (a)

satisfactory counting of either isotope separately, and

(b) simultaneous counting of the two isotopes in a mixture.

We have found scintillator solutions prepared from

p_-dioxane or toluene as described by Hayes [37] to be

highly satisfactory; because of considerations of solubility

we ordinarily use the former solvent. The p-dioxane

32
* scintillator solution contains 7 g of PPO , 0.3 g of

dimethyl-POPOP , and 100 g of naphthalene per liter; the

* toluene scintillator solution contains 5 g of PPO and 0.3

g of dimethy1-P0P0P per liter. By use of 0.1-ml aliquots

of aqueous j radioactive solutions in 10-ml quantities of

the p_-dioxane scintillator solution, and with the settings

given above, we have found the following counting

efficiencies: in channel 1, about 26$ for tritium and 25$

for carbon-l4; and in channel 2, about 3.7$ for tritium and

72$ for carbon-l4.

The radioactivity of each isotope in a mixture

containing both carbon-14 and tritium (T) may be expressed

by the following equations [26]:

14 _ Nlh

£ - M

2h — l , and (5)

dps c-,h - c 1"2 "2"1 Qhn N^ - NgCl

—dps h

i

c 2

" h

2

C l

where N., = net counts per second of channel 1, N

2 = net

counts per second of channel 2, h^ = T efficiency of channel

14

1 3 h

2 = T efficiency of channel 2, c1 = C efficiency of

channel 1, Cp = C efficiency of channel 2, and dps =

disintegrations per second. After the four counting

* 2,5-dipnenyloxazole.

** 2,2 -p_-phenylenebis(4-methyl-5-phenyloxazole)

33
efficiencies have been determined, equations 5 and 6 can be

simplified (by combining constants) to the following

convenient forms

^dps = M2 - BN1 (7)

T, = CNn - DNC (8) —dps 12 v ' —dps

The values of the combined constants A, B, C, and D apply

to a given set of conditions only.

This method of counting double-labeled compounds

requires careful determination of counting efficiencies.

14 The best results are obtained when the T/ C ratio lies

between 20:1 and 1:1.

In order to check the accuracy and precision of the

double-label counting procedure, we prepared and assayed

a series of solutions containing (in each solution) known

14 amounts of D-glucose-1- C and D-glucose-1-t . In each

analysis, 0.100 ml of an aqueous solution was counted in

10 ml of the p_-dioxane scintillator solution. Each sample

was counted five times, for 100 seconds each time. The

results are reported in Table 4.

34
o|

x:

4J •H •

13

u

cu

CO 4-> o c

•H 3

4-1

O

Cfl o

i-l c

<f o

p-4

H 1 4.) C CO o r-4

JO. T—1 n H

cfl 4-1 u c

.. •r-4 e o a w H 1 4-1 •a

•i-l •r-l u 3

4-1 cr H

14-4 r—I O

r— c (U o c

•H c

4-J CO

cfl J3 C a

•pJ i e .—< ^ crj 0) 3

4-) TJ

CD Q CO

<t

cu

i-4 -a

CO H

CDO3

CD

!-l CD

M-t

M-)

•r-4 Q

"0c3ocOO

CD

CO

CO 4JBoOe

cOco

14-1 oco

•I-l u

•>-l cooaEou

<J-ol

H|U

H|O

H|

CN

CDCC

cO Xio

CD3C

cO uu

H|U

HI

aa

•a

CNl

on

ON

CN

onO

m ON On

r-l

O + +

co

m o+ oi oI

co

CMi oI

ONo

VO oI o+

00

ca • • a 00 •vJ- -a <f m

mm

vO

CN

CN

0000

ON ao

oo co <j- in in

CMmmm

CON iO

a, cn on

T3

O CM CO CM

mmm

.—i ON

CO

inom

CNm

co

oo o

CM

CM+Oo

00 vO m o m r~-

CMini mo

vOi

CO o 1—1

<* 1—1 r-4 CN on vO 00

•H i—4 vom ON 00 ON ON 1—1

4-t CO o <t- CM ON r^ <t o o U CM t—l i—i

r» r^ co O co r^. co r-~ vo

co vo co

co o CN m r*- i—i

CO

CNO

r—1

CN 00

ON vO

mmCMon

r-4 CN

m

on

r-4

ON

l^»

r-4

COvO

r—1

CNm

r-4 CM

VOmm00

00

on r-4 00 ONi—4 m

in

T—I

i—t o

00

in

co

-Ct- i—l 00

CNvO

O

COm

vO

CN

m

co vOCO

ON

r-1

co o 00 r-^ CO CN m CN oo on

•r-4 r-~ r-- co 00 r-»

O on i—4 4J • o CO on <t- CM ON r^ m O O ^ r-4 r—1

i—1

<!

i—l vO

cn r^ P- vO 3G

•r4

4-1 CON

•r(U

cO -ac

co 4JCO

m

ON r—1

VO • • CM

CO rII II CN CM x: O . r. .^ O 00 O vO • • vO <*- CN CN

CO

in

CM

i~>. co

00

ON

CO

CN

CN

vO

<1- o

r-4 co

ON I CO I -d- I r-4 I oo

IDco

14-4

4-1 c

CDou

CD

P-I

35
2. SUGARS IN SOLUTION

( H, S. Isbell , H. L. Frush, R. Schaffer,

C. ¥. R. Wade and R. A. Peterson)

For over 100 years, the complex and manifold reactions

of the sugars in alkaline solution have fascinated organic

chemists and engaged their attention. Notwithstanding painstaking and extensive investigations, knowledge of the interconversion and degradation reactions of sugars in alkaline

solution is still incomplete.

In a monumental work, Nef and his students spent manyyears in separating and identifying the products of the alkaline degradation of sugars, but obtained very few quantitative

data [38]. Modern, tracer methods of analysis are far more

powerful than the classical methods, and the reaction mixtures

can now be readily analyzed. However, the problem is complicated by the existence of several competing reactions, some

of which are reversible and some not. Determination of the

equilibrium proportions of the products of the reversible

reactions, and the relative rates for all of the reactions,

would be of great value to workers in theoretical organic

chemistry.

Our Section is in a particularly favorable position to

obtain much of the desired information because we have high

competence in the preparation, purification, and analysis of

carbohydrates, as well as an extensive collection of

36
carbon-l4- and tritium- labeled sugars that are useful for

following the course of the reactions by tracer techniques.

In developing the program, we have studied the main

processes involved in the transformations; these include (a)

mutarotation reactions, (b) enolizations, (c) reversible

condensation and cleavage reactions, and (d) rearrangements

of the saccharinic acid type.

A. Mutarotation Reactions

"When dissolved in water, reducing sugars undergo certain

reversible, isomerization reactions which result in changes

in optical rotation known as mutarotation. The rationalization of mutarotation reactions is an intriguing and important

problem in carbohydrate chemistry.

The mutarotation process is known to involve the

reversible interconversion of several modifications of the

sugar [39]. For each sugar, this results in an equilibrium

between two pyranose forms, two furanose forms, an open-chain

form, and solvated and ionic modifications of these. The

mutarotations of D-galactose, D-talose, D-ribose, g-glycerog- ido-heptose, and certain other sugars give clear evidence

for two types of reaction, namely, a-p pyranose interconversions and pyranose-furanose interconversions [4o]. The

mutarotations of D-glucose, D-xylose, lactose, and certain

other sugars arise almost entirely from reversible a-p

pyranose interconversions. The mutarotation of D-fructose,

31
however , arises in large measure from a pyranose-furanose

interconversion [4l],

The a-p pyranose interconversions (reactions of type I),

have been investigated thoroughly,, but relatively little work

has been done on pyranose-furanose interconversions (reactions

of type II). Isbell and Pigman [4l] showed that the two types

of reaction differ markedly in their heats of reaction, activation energies, rates of reaction, and sensitivity to acidbase catalysts. It has been tacitly assumed that a-P

pyranose and pyranose-furanose interconversions take place

through a common, open-chain intermediate. Other mechanisms

are possible, especially a change in configuration without

ring cleavage, or a change in the size of the ring by an

intramolecular, opposite-face mechanism. In view of these

possibilities, it seemed desirable to study both types of

mutarotation, in water and in deuterium oxide. Prior to the

present work, others had studied the mutarotation of D-glucose

and certain other sugars by isotope effects [42], and had obtained important information on the reaction mechanisms, but

no pyranose-furanose interconversion had been so investigated.

Therefore, we undertook a study of isotope effects in the

mutarotation of D-glucose, a typical a-f3 pyranose interconversion, and the mutarotation of D-fructose, a typical

pyranose-furanose interconversion. The work has now been

reported in a Ph.D. thesis by a member of the staff (C.W.R. Wade)

[43].

38
The ultimate objective of the work was to elucidate the

mechanisms for pyranose-furanose interconversions and to show

how these reactions differ from a-|3 pyranose interconversions.

The research plan- included the following tasks: (a) Development of convenient methods for preparation and study of

crystalline deuterated sugars; (b) determination of highly

accurate data on the rates of mutarotation for certain sugars

in HpO, in the presence of acid and base catalysts } and for

the same sugars in DpO_, in the presence of deuterated acids

and bases; (c) determination of the Arrhenius heats of activation for the two types of reaction in HpO and in DpO; (d)

comparison of the catalytic effects of acid and base catalysts

on the two types of reaction in HpO and in DpO; (e) determination of the relative rates of mutarotation in HpO and in DpO

(isotope effects) for the acid-, base-, and water- catalyzed

reactions of types I and I_l; and (f ) formulation of satisfactory mechanisms for the two types of mutarotation reaction.

In accordance with this plan, methods were successfully

developed for the preparation and purification of the

jD-deuterium analogs of ct-D-glucopyranose, a-D-xylopyranose,

and a-D-fructopyranose. The rates of mutarotation of the

O-hydrogenated sugars in HpO and of the JD-deuterated sugars

in DpO, were determined in the presence of sulfuric acid

(0.015 M> 0.0025 M, and 0.00125 M) > potassium acid phthalate

(0.001 M), and o-nitrophenol (0.01 M) , at 3.9° and 20.0°C.

39
The rates of mutarotation of a-D-glucopyranose in H

2 and V^O

in the presence of 0.0006 M sodium hydroxide were also

determined. The rate constants were used to calculate the

activation energies, acid-base catalytic effects, and isotope

effects (k/k*). Catalytic-rate equations were calculated to

show the relative effectiveness of the catalytic pairs

H-5

+/D^0+

5 OH"/OD", and H

2 0/D2

0. In all cases, for a given

pair of catalysts and at a given temperature, "normal" isotope

effects (k/k*>l) were found for the rates of mutarotation of

each sugar. The results were examined and interpreted as

functions of a- (3 pyranose or type I interconversions (for

D-glucose) and of pyranose-furanose or type II interconversions

(for D-fructose).

The activation energies, catalytic effects, and rate

constants clearly show parallel differences in type I and

type II reactions in both solvent systems. However, both the

measured and the calculated (catalytic) isotope effects show

that three distinct but similar mechanisms are concurrently

operative for both types of reaction. In strongly acid

solutions, both a-f3 pyranose and pyranose-furanose interconversions occur by rapid, pre-equilibrium, proton transfers,

followed by the rate-determining step. In slightly acid

solutions, proton transfers occur in the rate-determining

step. In basic solutions, pH measurements and isotope effects

show that the sugar loses a proton to the base in a rapid step

kQ
and that the formation of the sugar anion precedes the ratedetermining step. Thus, in highly acid solution, the general

acid-catalyzed mechanism predominates; in slightly acid

solution, the water-catalyzed mechanism prevails; and, in

alkaline solution, the base-catalyzed mechanism is most

important.

The results support reaction mechanisms advanced by

Isbell and Frush in an earlier publication [44]. In each

instance, the open-chain form of the sugar is the intermediate

in the mutarotation reaction. Although present in small proportions, the open-chain intermediates give rise to reactions

characteristic of aldehydic and ketonic compounds. Some of

these will be considered next.

B. Enolization Reactions

1. Successive, Reversible Processes

In alkaline solution, the open-chain form of the sugar

in the equilibrium mixture undergoes enolization. By this

process, an aldose yields a 1,2-enediol, and a ketose yields

both a 1,2- and a 2,3-enediol. According to Nef [58]*, reversion of a 1,2-enediol gives two epimeric aldoses plus the

corresponding ketose, and reversion of the 2,3-enediol gives

two ketoses having the carbonyl group at C-2 plus two ketoses

having the carbonyl group at C-3. Enolization of the resulting two 3-ketoses yields a 2,3-enediol plus two j5,4-enediols.

Reversal of the process, beginning with each of the

41
3,4-enediols, gives (hypothetically) two ketoses having the

carbonyl group at C-j5.j.plus two having the carbonyl group at

C-4. Presumably, by this type of reaction, all of the

aldohexoses and ketohexoses possible are formed from a single

hexose.

The early work of Lobry de Bruyn and Alberda van

Ekenstein [45] provided clear evidence for the formation of

the 1,2- and 2,3-enediols; however, the existence of the 3*4-

and 4,5-enediols of hexoses remained uncertain. Formation of

D, L-sorbose by alkaline rearrangement of D-glucose and of

D-fructose indicated possible extensive isomerization [46],

but the authors suggested that the L-sorbose may arise by

fragmentation and recombination rather than by successive

14 enolization and de-enolization. Later, by use of D-glucose -1- ( Sowden and Thompson [47] established that D, L-sorbose is

formed almost exclusively by the enolization—de-enolization

mechanism (which must include formation of the 3,4- and

4,5-enediols), and that fragmentation and recombination play

only a minor role in the isomerization. The existence of

3,4-enediols was recently confirmed [48] by R. Schaffer of our

staff, who prepared crystalline ft -D-manno

3-hep tulo s e monohydrate

and found that it rearranges in alkaline solution, via a 2,3-enediol, to D-gluco -heptulose, and, via a 3,4-enediol, to

a transitory, epimeric 3-ketose. This intermediate yields a 2,3-enediol and, ultimately, D-altro-heptulose plus D-allo42
heptulose. Thus, Nef ' s idea of the existence of all possible

enediols is confirmed.

Ordinarily., an equilibrium state is not reached in these

interconversion reactions. Hence, the proportions of the

isomeric sugars in the reaction mixtures depend on the extent

of the reaction. In the early stages of the reaction, the

composition of the reaction mixture depends on the rates of

formation of the various sugars. In the final stage of the

reaction (pseudo-equilibrium state) the proportions of the

sugars present in the mixture depend on their relative thermodynamic stability. To understand the behavior of sugars in

alkaline solutions, we need to know, for each sugar, the rate

of enolization, the relative rates of conversion of the

enediols into the several sugars, and the relative thermodynamic stability of each sugar in the pseudo-equilibrium

mixtures.

2. Measurement of Rates of Primary Enolization

Heretofore, quantitative measurements of the rates of

enolization have not been attempted because the system is so

complicated. However, we .have recently developed a method

for measuring the rates of enolization of the sugars under a

variety of conditions. The method involves treating the sugar

under any desired condition with a base catalyst in the

presence of tritiated water. After a short reaction time,

the mixture is acidified. This introduces carbon-bonded

43
tritium into the molecule (see equation 9). Assay of the

Ri>g i£2^ RC-ig iliOrL R

«*_H (9)

IK (slow) o h (fast) o n H y HO" HO

reaction mixture for tritium, after removal of excess

tritiated water and oxygen-bonded tritium, provides a measure

of the enediol formed. By acidifying the reaction mixture

when only a small fraction of the sugar has been converted into

enediol, one obtains a measure of the rate of the primary

enolization. By this method, we have measured the rate of

enolization of D-glucose in the presence of various catalysts.

The results are now being prepared for publication, and

similar measurements are being made for other sugars.

3. Measurement of Rates -of Interconversion of Sugars

Prior to the present work, Sowden and Schaffer [49 ] had

applied tracer methods to the study of the alkaline rearrangement of D-glucose, D-mannose, and D-fructose in water and in

deuterium oxide. They fully confirmed the mechanism for the

interconversion of the three sugars through the 1,2-enediol,

and showed the practicability of tracer methods for obtaining

detailed information on the reactions.

The following method has now been developed for studying

the interconversion reactions: A sample of the C- labeled

sugar (usually 0.1 millimole) is treated with a base catalyst

under controlled conditions. After the desired reaction

44
period has elapsed, the reaction mixture is treated with a

cat ion- exchange resin to remove the base. The solution is

then passed through an anion-exchange resin to remove the

saccharinic acids. The carbon- 14 retained on the resin is a

measure of the saccharinic acids formed. The deionized

solution is divided into aliquots, and the labeled products

are measured by the isotope-dilution technique, using nonlabeled carriers.

We have now completed measurements on the rearrangements

of D-glucose, D-fructose, and D-mannose at various temperatures, in 0.036-N calcium hydroxide, in 0.04-N sodium

hydroxide, in dry pyridine, and in aqueous pyridine. Interconversion of the sugars at 5°C is surprisingly slow. After

nine months, 85 to 90$ of the starting sugar remained intact.

Under the conditions used, there is no appreciable difference

in the action of calcium hydroxide and sodium hydroxide. We

found that, in the early stages of the reactions, (1)

D-glucose gives D-mannose and D-fructose in nearly equal proportions; (2) D-mannose gives much more D-fructose than does

D-glucose; and (3) D-fructose gives more D-mannose than does

D-glucose.

A study of the inter conversion reactions of D-galactose,

D-talose, D-sorbose (D-xylo-hexulose ) , and D-tagatose (D- lyxohexulose) is also in progress. The work entails very careful

purification of the labeled sugars used as tracers and of the

sugars used as carriers. We plan to study, ultimately, the

45
interconversions of all of the hexoses and of as many pentoses

and heptoses as possible. Measurements made in the early

stages of the reactions give information on the relative rates

of formation of the various sugars one from another. Tl

measurements supplement xhe measurements of the rates of

enolization made by use of tritiated water (see Section E.2).

Measurements of the composition of mixtures obtained after

long reaction-periods are being used to estimate the relative

stabilities of the isomeric hexoses. This subject will be

considered next.

4. Thermodynamic Stability of Isomeric Hexoses

In an equilibrium system, Gibbs free-energy differences

can be calculated from the equation G = -RTlnK. Because

some of the reactions of sugars in alkaline solution are not

reversible, accurate equilibrium constants cannot be obtained.

Approximate values can be estimated from the proportions of

the various sugars in pseudo-equilibrium states. By approaching the pseudo-equilibrium state from each of the constituent

sugars, it may be shown that the equilibrium constants lie between certain values. We have estimated equilibrium constants

from the ratios of D-mannose to D-glucose and D-fructose to

P-glucose in two reaction systems — one, aqueous 0.04-N

sodium hydroxide at 35°C and the other, 9-percent aqueous

pyridine at 120°C . With aqueous pyridine at 120°C, the freeenergy difference in calories for conversion of D-glucose into

D-mannose was found to be of the order of +1000, and, for

46
D-glucose into D-fructose, of the order of +300 calories per

mole. The study is being continued in order to obtain moreaccurate values.

C. Reversible Condensation and Cleavage Reactions

In alkaline solution, sugars establish equilibrium states

consisting of furanose and pyranose modifications, together

with small proportions of carbonyl and enolic forms. We have

already considered sugar transformations, in alkaline solution,

that do not involve a change in the carbon skeleton of the

sugar. However, certain rearrangements occur with alteration

of the carbon chain. Some of these take place by cleavage of

the carbon chain followed by resynthesis by aldol condensation.

1. Cleavage and Resynthesis of Hexoses

Tracer methods provide a means for evaluating the alteration in the carbon chain. In a rearrangement starting with

a sugar that is position-labeled with carbon-l4, the extent

of cleavage and resynthesis can be estimated from changes in

the distribution of carbon-l4 in the substrate and in the

14 product. Thus, cleavage of a hexose- 1- C yields a 1-labeled

triose-enediol and non-labeled D-glycer aldehyde as shown in

equation 10.

icOH CH 2 0H CH 2 0H

COH COH C=0

H0C>H HOC-H H0C>H § Hfr-lOyiH

—*

HC=0 HC-^-iH (10)

HCOH HCOH HCOH

CH 2 0H CH 2 0H CH 2 0H

I

*

47
The cleavage may start with either the 1,2-enedicl (I) or

the free ketose (II), and may proceed by the electron shifts

depicted in the formulas. Presumably, the keto path is the

more important [50]. The nascent, labeled triose-enediol

equilibrates with dihydroxyacetone (l,3-dihydroxy-2-propanone)

and becomes labeled at both C-l and C-3. Except for the label,

the enediol is the same as the enediol of D-glyceraldehyde.

Hence, both fragments become labeled at C-l and C-J>, and the

hexulose formed by aldol condensation of the labeled fragments

would be labeled at carbon atoms 1,3,4, and 6. To provide

information on possible cleavage and resynthesis reactions,

we have degraded some of the sugars obtained by rearrangement

of the labeled sugars in the tracer studies already described.

Our results show that the cleavage and resynthesis reaction

14 l4 in the preparation of D-fructose- 1- C from D-glucose- 1- C

by the action of 0.04-N alkali does not exceed 2$ of the total

reaction.

Presumably, under highly alkaline conditions, the

cleavage and resynthesis reactions reach pseudo-equilibrium

states. It might be expected that repetitive aldol condensations would give compounds having long carbon-chains. But,

surprisingly, the pseudo-equilibrium mixtures obtained by

alkaline rearrangement of lower sugars do not contain appreciable proportions of sugars having more than 3ix carbon atoms.

Inasmuch as the composition of the mixture at equilibrium is

determined by thermodynamic stability, it may be concluded

48
that sugars having more than six carbon atoms are less stable

than the lower sugars.

2. Formation of Straight-chain Ketoses and Branched- chain Aldoses by the Aldol Condensation

In an aldol condensation, one reactant must be in the

carbonyl form and the other in the enolic form, as shown in

equation 11.

H H R' . _ H H R' c-c-c

II H H

R-C + -C=C > R-C-C-C (11)

IK

^ '^ II K J <y o o)

H H

The two components of the addition reaction may be (a)

tautomeric forms of the same compound, or (b) different

compounds. Depending on the nature of the reactants, it is

possible to obtain either linear ketoses or branched-chain

aldoses. Our results show that, when the hydroxyl group at

C-2 of the enediol is unsubstituted, linear ketoses are formed.

In the unsubstituted enediol, hyp ere onjugat ion of the R 1

group at C-2 enhances the nucleophilic properties of C-l, and

hence promotes substitution at this point.

Thus, Schaffer and Isbell found that 2,4-0-ethylideneg-erythrose (III) and dihydroxyacetone (presumably in the enolic

form) yield a straight-chain 5,7-O-ethylideneheptulose (IV)

[51]; and Schaffer and Cohen found that, by self -condensation,

2-erythrose, in alkaline solution, yields a straight-chain

3-octulose (V) [52]. Presumably, the reactions take place as

49
shown in equations 12 and 13.

f8-H + HCH' 0H -* t8-H-b-»-

CH20H (12)

i^ 8

8> 1^4

H0

m IE

H H H / 1x n HHH HO HHOHO (13)

HOH.C-C-C-C + <C=C-C-CH2 OH > H0H 2 C-C-C-C-C -C-C-C

2 OH

O II ^+^ k H H O » H HHO 65 HHHO

H H 2-

When the hydroxyl group at C-2 of the enediol is substituted, an electron shift occurs from the hydroxyl group at

C-l to carbon atom 2. This shift increases the nucleophilic

properties of C-2, with the result that the addition takes

place between this carbon atom (instead of C-l) and C-l of

the aldehyde, to yield a branched-chain aldose. The mechanism

is depicted in equation 14.

R' H/-K H» H R' H , fih\ C=^ + C-R » C-C-C-R* K 1^)

r\ IK M Co R O* R H

H

As discussed in Section 3. A of this report, Schaffer and

Isbell [53*5*1-] found that branched-chain aldoses are formed in

high yield by condensation of sugar derivatives in which the

hydroxyl of C-2 is blocked by substituent groups. Formation

of the branched-chain compounds is unusual, and has not heretofore been explained.

50
). Rearrangements of the Saccharinic Acid Type

• In addition to the reversible aldol condensations just

iiscussed, essentially non-reversible, saccharinic acid rearrangements take place in alkaline solutions of sugars. As

Dointed out by Nef [35], saccharinic acid formation takes

place through isomerization of the sugar to an a-dicarbonyl

compound, followed by a benzilic acid type of rearrangement

Df the latter, with hydration. However, Nef ' s concept of

the mode of isomerization of the original sugar to the

Intermediate a-carbonyl compound did not prove sound. But,

Ln 19^9 an acceptable course for the initial isomerization

fjas developed by Isbell [55]. In "the revised mechanism, the

i-dicarbonyl intermediates for production of the various

types of saccharinic acid arise from enolization followed

oy ^-elimination of a hydroxyl group.

rhus, formation of metasaccharinic acid was depicted as in

equation 15

.

H-C*xT

(C-OH

H-C i -OH

I

1 CHOH

I CHOH

H-C = C^-H

"5

H-C

CHOH

CHOH

H-C=0

H-C-H H

+

OH

CHOH

I CHOH

C0

2

H

CHOH

CHp

I 2

CHOH

I CHOH

(15

CH

2 0H CH

2

0H CH2 0H CH

2

0H

1,2-Enediol Metasaccharinic

acid

51
Similar mechanisms were formulated for the production of

saccharinic acids from 2,3- and 3,.^-enediols, and these have

been largely confirmed [50].

However, tracer studies reveal that, in some instances,

saccharinic acids may be formed by two or more paths. Thus,

in the formation of "a"-D-glucosaccharinic acid (VT) from

D-mannose-1- C , Sowden, Blair, and Kuenne [56] found 39$ of

the carbon- 14 at the methyl carbon atom (the expected position)

and 57$ at the tertiary carbon atom. As pointed out by Kenner

and Richardson [57]* carbon- Ik at the tertiary carbon atom

could arise from fragmentation and resynthesis of the sugar,

followed by rearrangement of the 1,3-labeled hexose. However,

Sowden, Blair, and Kuenne noted that this process, if operating alone, could account for only 50$ of the isotope at the

tertiary carbon atom; this proportion would be lessened by

14 direct isomerization of the hexose -1- C . Lemieux suggested [58] that the unexpected result might

arise from two concurrent reactions, one by way of the NefIsbell mechanism, and the other by way of an internal-return

mechanism in which D-mannose-1- C (XI) is converted into

D-glucose-2- C (XII), which then rearranges, through

D_-fructose-2- C (XIII) and the conventional mechanism, to

"a"-D-glucosaccharinic acid (See Figure 15). According to th

proposal, Sowden, Blair, and Kuenne ' s result would require,

as an intermediate, B-fructose labeled at C-l and C-2 in the

ratio of 39 to 57, respectively.

52
C0o H

t^C-C-OH

HCOH

I HCOH

CH2 OH

"a"-D-Glucosaccharlnic Acid

(2-C-Methyl-D-ribo-pentonic Acid)

si

Nef-Isbell Mechanism

HC-OH

c=o

U

HH-C-OH

C

3

H7°3

H* HC-OH

,(•OH

C-*OH

C

3

H7°3

37JD

HC

5

C^OH

C=0

C

3

H7°3

IK

*

C H,

C=0

C=^0.

OH

C

3

H7°3

CO.H

J

2

H.C-COH

3

I C

3

H7°3

3ZI

Lemieux Mechanism

.Hv

0'' N

I - I*

H-Cs jC-H

0=^C-H

HCOH

I HCOH

I C^OH

XT

H-C=0

I*

HC-OH

I HOCH

HCOH

I HCOH

I C^OH

SI

CH-OH

I*2

c=o

I HOCH

HCOH

I HCOH

I C^OH

TTTT

CH

II*2

C-OH

I c=o

I F

HCOH

I HCOH

I CI^OH

xnz:

CH„

i* 3

,C=0

lc=o

v

l HCOH

OH

HCOH

I C^OH

xe:

co2 h

* i *

v H.C-C-OH - 3

I HCOH

I HCOH

I C^OH

3zr

Figure 15. Prior mechanisms for the formation of

"a"-D~s:lucosaccharinie acid.

53
We have now separated and analyzed the labeled-D-fructose

14 formed from D-mannose-1- C under conditions similar to those

used for preparing "a n -D-glucosaccharinic acid. Alkaline

degradation of 'the labeled D-fructose to the next lower

aldonic acid (D-arabinonic acid) showed 98% of the carbon-l4

to be at C-l. Hence, the rearrangement cannot take place by

the mechanism suggested by Lemieux. The lack of carbon-l4 in

the labeled D-fructose at positions other than C-l also shows

that Kenner and Richard's proposed fragmentation of D-mannose

and recombination cannot play a major role in the rearrangement.

Because none of the proposed mechanisms fully account

for the experimental results, we have formulated a new

mechanism (see Figure 16). it differs from others in that

the "a"-glucosaccharinic acid labeled at the tertiary carbon

atom is derived from C-3 of the parent sugar, instead of from

C-2, as in the Lemieux mechanism. The mechanism effects an

interchange of C-l and C-3 of the metasaccharinic acid. We

believe that the new mechanism, in conjunction with the NefIsbell mechanism, will account for the supposedly anomalous

results

The Nef-Isbell mechanism, the Lemieux mechanism, and the

newly proposed mechanism involve a benzylic acid rearrangement

of the same a-diketone. The diketones for the three mechanisms

are produced by different paths and are labeled in different

positions. Presumably, in each instance, the benzylic acid

rearrangement may occur in either of two ways, depending on

54
HC^O

h HOC-H

I HOCH

HCOH

I HCOH

I C^OH

HC-^OH

HOC ) HO-CH

:

I HCOH

I HCOH

I CHoOH :H2

*HC=0

I HOC

II CH

I HCOH

I HCOH

I C^OH

*HC=0

I 0=C

I HCH

I HCOH

I HCOH

CHoOH :H2

H ^H

HCH

H C

2

H

5 2

H

C?

c*

HOC<\

r o HCH / VYl X

C

2

H

5 2

HOC

S

C*

I

1

'

1! -! + HCH

\

^

'

i //i /

^C / \

H C

2

H

5 2

xm XEE xsrnr

H

c?

i©*C

/

I c*

«2

h

c H

7 C

2

H

5 2

CH_

( c=o

HCOH

C

2

H

5 2

OH

C09 H

'*

-* Ho C-C-0H

3

I HCOH

C

2

H

5 2

XIX XX 3ZE

Figure 16. Proposed mechanism for the formation

of "a"-D-slucosaccharinic acid.

55
which of the two carbonyl groups is attacked by the hydroxyl

ion. To account for the presence of carbon-l4 at the tertiary

14 carbon atom of the product from D-mannose-1- C , with the new

mechanism the attack of the hydroxyl ion must be at C-2,

causing migration of the methyl group to C-3; with the

Lemieux mechanism,, the attack must be at C-J> 3 causing

migration of the CUH7CU group to C-2. Table 5 shows the

positions of C-l, C-2, and C-3 of the parent D-mannose in the

"a"-D-glucosaccharinic acid that would be formed by the three

mechanisms. To test the proposed mechanism experiment ally

,

14

we have rearranged samples of D-mannose-1- C and

14 D-mannose -3- C , and are now determining the distribution of

carbon- 14 in the resulting "a"-D-glucosaccharinic acid.

Table 5. Migration of carbon atoms in the formation

of "a"-D-glucosaccharinic acid.

If CH^ migrates If C^H^O^ migrates

C-l in CH^ C-l in CH^

Nef-Isbell Mechanism C-2 in COgH C-2 in R^COH

C-3 in R^COH C-3 in COgH

C-l in C0 2

H C-l in R^COH

Lemieux Mechanism C-2 in CH^ C-2 in CH^

C-5 in R^COH C-3 in COgH

C-l in R^COH C-l in COgH

Proposed Mechanism C-2 in COgH C-2 in R^COH

C-5 in CH

3

C-2 in CH^

56
3. ALDOL CONDENSATIONS

(R. Schaffer )

A. Syntheses via Aldol Condensations

The sustained interest that carbohydrates have commanded

because of their immense economic value and the ever-widening

recognition of their biochemical importance and variety has

resulted in the characterization of complete families of these

substances. This unique assemblage of linear and cyclic

stereoisomers has been put to use in modern organic chemistry

as model compounds in studies of reaction mechanisms involving

stereochemical transformations and the subtleties of molecular

shape (conformation). However, some of these compounds are

not easily obtained from the syntheses through which they are

known; also, there are some, as yet unknown, that should be

made, for they would favorably complement those currently

available. The possibilities of devising easier access to

some of the known compounds and to desirable new ones are

twin objectives of the stereochemistry project. We have

chosen to accomplish these objectives by use of seemingly

complex syntheses which have been neglected in the past because they presented the troublesone prospects that their use

would lead to a multiplicity of products and to severely increased stereochemical problems; however, such obstacles may

today be overcome by employing the effective separation

techniques and powerful analytical instrumentation that have

been recently developed.

57
Our interest in these complex syntheses began while undertaking a detailed investigation of the structure of a dialdopentose derivative. In studying it, we were able to attribute

the unusual properties of the compound to the presence of a

hemiacetal form of a 1,3-dioxane ring that joined two molecules together [59]. However, this stabilizing feature

vanished in the presence of alkali, which, we found, catalyzed

an unexpected aldol condensation between two molecules of the

dialdose, yielding a 4-C-formyldialdo-nonose, probably the most

complex monosaccharide known. Its discovery indicated that

aldol condensations, although not typical reactions of carbohydrates, might usefully be secured with other suitably

selected derivatives [53* 5^1. This was proved possible with

the synthesis of a 2-C-(l,2-dihydroxyethyl)-D-glucose [60] from

D-ethylidene-D-erythrose. (The method of preparation of

D-erythrose presented in this paper has been recommended above

any other proposed [6l].) In addition, the potentiality of

aldol reactions of aldose derivatives was further demonstrated

by utilizing two different reactants for the preparation (in

excellent yield) of a 4-C-(hydroxymethyl) pentose [62]. Although of importance in themselves as branched-chain sugars

(related, for example, to streptose, a carbohydrate component

of streptomycin), the new substances proved to be useful

source-materials from which exceedingly valuable compounds

could be derived; thus, from the 4-C-(hydroxymethyl)pentose,

58
the enantiomorph of the rare, natural sugar D-apiose was

readily prepared [62]; and D-manno-3-heptulose , the first

known example of a 3>-ketose, was obtained from the

2-(l,2-dihydroxyethyl)-D-glucose [60 ] . This extraordinary

ketose had a structure thought to have only transitory

existence as an intermediate in isomeriz,ation reactions. Its

properties gave convincing evidence for previously proposed

enediol mechanisms [48], and settled a controversial attempt

to ascribe a 3-heptulose structure to a carbohydrate isolated

from a biological source [63]. In addition to the 3-heptulose,

we were able to make available a second crystalline, unsubstituted 3-ketose that was discovered to be an aldol-condensation

product of D-erythrose [52], The readiness with which

D-erythrose undergoes transformation to D- gluco~L- glycero-3-

octulose makes probable to us that it will be observed in

biological systems.

With the aid of a former summer employee, Mr. Eugene

Barron, evidence of the formation of a third 3-ketose has

been obtained in an aldol condensation of D-threose. It is

hoped that work with this new substance can soon be resumed.

We have learned, by private communication, that our experiments are stimulating interest in other laboratories in the

chemistry of these compounds.

We have been able, also, to devise a simple aldol

synthesis for higher ketoses having their keto group at

59
carbon atom 2. The problem of separating isomeric products

was encountered in this process , but was simply overcome

through the use of chromatography, as these products were

previously known compounds [51]. Their nature and compositior

were found to be altered when the same reactants in unsubstituted form were allowed to enter the reaction. In this way,

the previously difficulty obtainable D-allo-heptulose , which

was not detected in the aldol synthesis with substituted

reactants, could be isolated in a useful yield [64].

B. Cyclopentitols

At present, we are concerned with the synthesis and

characterization of cyclopentane derivatives having a

functional group on each carbon atom. Surprisingly little is

known of pentasubstituted cyclopentanes . Such products would

be the 5-carbon analogs of the cyclitols, which function as

the model compounds for stereochemical studies on cyclohexanes. The cyclopentane derivatives may be expected to

serve similarly for cyclopentane chemistry, and they should

be particularly valuable in shedding light on cyclopentane

conformations. Our cyclopentane derivatives are being prepared from a new compound which is unusual in having two

hemiacetal groups in one ring of its structure. In a series

of reactions, it is converted into certain novel monoaminotetrahydroxycyclopentane derivatives. Assignment of configuration to each of the asymmetric carbon atoms of the

50
products is now under investigation.

Our success in elucidating structural features of the

new compounds with which we are concerned is related to a

combination of reliance on the classical approach of organic

chemistry and utilization of the remarkable information that

analytical instrumentation can provide.

61
- 4. MOLECULAR STRUCTURE

(H. S". Isbell , A. J. Fatiadi, E. J. McDonald)

A. Cyclic Polyhydroxy Ketones

On oxidation, cyclohexanehexols (inositols) yield keto

derivatives which, by successive enolization and elimination

reactions, can be transformed into polyhydroxybenzenes [65].

In progressing from cyclohexane derivatives through cyclic

olefinic intermediates to aromatic compounds, reactions and

intermediate compounds are encountered which are eminently

suited for the study of reaction mechanisms and basic concepts

of theoretical organic chemistry.

In a recent publication [66], we reported the preparation

and properties of D,L-xylo-trihydroxycyclohexenediolic acid

(XXI) from the products of oxidation of myo- inositol with

nitric acid. The structure of compound XXI was established,

and certain unique and highly interesting reactions were

found. The new compound reduces Tillmans reagent, reacts

with iodine in neutral and acid solution, produces a blue

color with a solution of ferric chloride, and exhibits other

properties characteristic of the enediolic acid structure

R—C=C—C—R'. The compound is readily oxidized to xylo- HO HO

4,5,6-trihydroxy-l,2,5-cyclohexanetrione, an important new

ketone. Compounds XXI and XXII are related to ascorbic acid

and dehydroascorbic acid, respectively; like ascorbic acid

62
and dehydroascorbic acid, they are readily interconverted by

oxidation and reduction.

XK. m

Acetylation of XXI and of XXII under basic conditions

proceeds with simultaneous aromatization, resulting in the

formation of pentaacetoxybenzene and hexaacetoxybenzene,

respectively. On hydrolysis , the latter compounds yield

pentahydroxybenzene (benzenepentol) (XXIII) and hexahydroxybenzene (benzenehexol) (XXIv)^ respectively. Oxidation of

XXIV, by air in alkaline solution, yields tetrahydroxy-pbenzoquinone (XXV); further oxidation yields the anion of

rhodizonic acid (XXVI) and cyclohexanehexone (XXVTI) [65].

°1/ \f°

0^ ^0

XXVII

Synthesis of pentahydroxybenzene from myo-inositol

provides a means for obtaining this highly interesting but

little known polyphenol. Freshly prepared methanolic

solutions of pentahydroxybenzene are slightly yellow; they

65
turn deep red on exposure to air. Ferric chloride solution

acquires pink color that rapidly deepens to magenta. At

room temperature, the compound reduces Benedict solution and,

also, silver nitrate, the latter almost instantly. The

compound may have useful medical applications.

Formation of pentaacetoxybenzene and hexaacetoxybenzene

from XXI and XXII, respectively, was found to be in accordance

with a mechanism previously advanced by Isbell [55] for the

production of aromatic compounds from keto-inositols (see

Figure 17). The mechanism involves successive enolization

reactions, with cleavage of acyloxy groups beta to the enolic

hydroxyl groups.

2S

AcO £0

AC2 (bQSe)

Ac0

^AC /

H OAC

-OAc

AcO £0'

OAc

H OAc

AcO OH

*+/ \ OAc

AcO OAc

AcO OAc

-> H- / \ OAc

AcO OAc

xxn

Ac2 (base)

AcO OAc AcO OAc

-> AcO-f

AcO OAc

Figure 17. Mechanisms for the formation of pentaacetoxybenzene and hexaacetoxybenzene.

o4
B. Fhenylhydrazono-phenylazo Compounds

The structure of phenylhydrazono compounds in general is

a subject of high current interest because, by use of spectrograph^ and resonance methods , some of the conventional

structures have been shown to be incorrect. Thus, Mester [67]

and others [68] have found that crystalline phenylosazones of

sugars normally have a cyclic, chelate structure, first suggested by the Fiesers [69 ], instead of the conventional

structure. Furthermore, it has been shown that certain

phenylhydrazono compounds undergo complex tautomeric changes

[70].

We have found that xylo-4,5,6-trihydroxy-l,2,3-cyclohexanetrione (XXII) (described in the preceding section of

this report) forms a crystalline bis (phenylhydrazone) having

some unusual properties. The bis (phenylhydrazone) obtained

under mild conditions gives a yellow solution in toluene which

turns red on standing. Evaporation of the toluene solution

gives a dark-red, crystalline product. The change in color

suggests a change from a phenylhydrazono to a phenylazo

structure.

From the method of synthesis, the bis (phenylhydrazono)

compound could be either a l-oxo-2,3-bis (phenylhydrazone) or

a 2-oxo-l,3-bis (phenylhydrazone) (XXVTII). Either compound

should be capable of existing in tautomeric modifications.

Hence, it was of interest to investigate the structures of

XXVIII and related compounds in considerable detail.

65
To determine the positions of the phenylhydrazono groups

of XXVIII, we oxidized the red bis(phenylhydrazone) with

sodium metaperiodate and separated the reaction product. It

would be expected that the 2-oxo-l, 3-bis (phenylhydrazone)

would yield, by oxidation, the dialdehyde, XXIX. The initial

oxidation product was found to rearrange, with cleavage of

water, to a pyridazine derivative, XXX; this compound was obtained in good yield.

H HC =

OH N-N-Ph | H HC=0

f-<

c=N" NPh °VAn

(oh >° > °=c > I l +p. H0Vh PhN-N=C PhN=NAX^N" Ph

' N-NPh H I H

H HC=0

xyvin xkik xxx

Formation of XXX established that the bis (phenylhydrazone)

of xylo-4,5j6-trihydroxy-l,2,3-cyclohexanetrione is, indeed,

2-oxo-l, 5-bis (phenylhydrazono ) -xylo-4, 5 , 6-tr ihydroxycyclohexane (XXVIII). The work also provided a route to new

pyridazine derivatives having unusual structural features.

The reactions and properties of these compounds are being

studied.

After establishing the positions of the substituent

groups in our 1, 3-b is (phenylhydrazone ) , we turned our attention to the structures of the two forms of the substance. To

ascertain whether other 2-oxo-l, 3-bis(phenylhydrazones) likewise exist in tautomeric forms, we prepared and studied the

following previously known compounds: 2-oxo-l, 3-b is (phenyl

66
hydrazono)propane (XXXI); 2-oxo-l,3-bis (phenylhydrazono)cyclohexane (XXXII); 3-oxo-2,4-bis(phenylhydrazono)cyclohexane

carboxylic acid (XXXIII); 2-oxo-l,3-bis (phenylhydrazono)cyclopentane (XXXIV); and 2-oxo-l,3-bis (phenylhydrazono)indan (XXXV)

H H HC=N-NPh N-NPh HOC N-NPh

C=0

I HC=N-NPh

H

2XXT ZXM KYm

H H N-NPh N-NPh

N-NPh

H

zxxnz: mr

In each instance the product obtained by reaction of a

trione with phenylhydrazine under mild conditions crystallized

as a crude , yellow-orange mixture which turned red when heated

in toluene (or other anhydrous solvent ), and formed dark-red

crystals on removal of the solvent.

The initial product from the reaction of 1,2,3-cyclohexanetrione and phenylhydrazine under mild conditions

crystallized from aqueous ethanol as a yellow monohydrate.

A freshly prepared, ice-cold, aqueous ethanol solution of the

hydrate shows an absorption band at about 400 mu. which shifts

rapidly to a longer wavelength (about 480 mu.). After equilibration and evaporation of the solvent, the red, anhydrous

form of the compound is obtained. The IR, UV, and NME spectra

67
of the hydrate indicate that it has a conventional phenylhydrazono structure. However, the spectra of the red modification of the same compound and the red forms of the other

2-oxo-l,3-bis(phenylhydrazones) listed above show that they

are not true oxo-phenylhydrazones; they do not show infrared

absorption in the regions characteristic of either the free

carbonyl group or the imino hydrogen atom. All of the

compounds give three characteristic absorption bands with

Aw 243 to 259, 283 to 290 and 468 to 488 m^i, respectively, max

The similarity of the spectra support the conclusion that all

of the red compounds have the same type of structure. The

spectra are in accordance with the enolic, hybrid, phenylhydrazono-phenylazo structure depicted in Figure 18 for 2-oxol,3-bis(phenylhydrazono)cyclohexane (XXXII). The structure

Q

Ph

=£&

n t"

Ph

+/

/ \

Ph

\

H

0-

N —

Ph

.Ph N—

//

\

-

\ /

N =N

H

Ph

N=N

Ph

/ —

I

\N—

H

Ph

Ph

N —

/ V

/

\ —

N=N

Ph

Figure 18.

xxxii

Proposed structure for a typical 2-oxo

l,3~bis(phenylhydrazono) compound.

68
of XXXII was further confirmed by its NMR spectrum in deuterated

chloroform (see Figure 19). The observed low-field absorption,

at about l4 ppm, is indicative of intramolecular hydrogenbonding (presumably between the imino hydrogen atom and the

c arb onyl gr oup )

.

500 400 300

-^-V'VNJi'l.^. . •****,

80 7.0 6.0 5.0 4.0 3.0 2.0

TMS

PPM (8)

Figure 19. NMR spectrum of 2-oxo-l,3-bis (phenylhydrazono)cyclohexane (hybrid structure).

Confirmation of the enolic hybrid, phenylhydrazonophenylazo structure for the red forms of compounds XXVIII

,

and XXXI - XXXV, inclusive, has been obtained from a comparison of the spectra of the compounds with those of model

compounds having similar structural features. This work is

now being prepared for publication.

The trihydroxycyclohexanetrione derivative XXVIII gives

rise to many unusual reactions. By acetylation with a basic

catalyst, it give l,4-diacetox-2,6-bis (phenylazo)benzene

69
(XXXVI) in almost quantitative yield (see equation 16)

H

1 HO N-NPh

H

AcO N-NPh

J, r Vo AC2°>

hoY_/ ( H N-NPh

NOAc r° AcOV- /

H NTNPh

I H

i H

XXVIII

H N ="-NPh

\

hA#>

AcO yi =/

H N==NPh

AcO OAc ( 16

)

H N=NPh

XXXVI

Compound XXXVI presumably arises from removal of the imino

hydrogen of XXVIII by the action of the base catalyst followed

by the changes depicted. The mechanism is based on the concept

of successive, electron displacements [55] advanced by

Isbell and used subsequently for the rationalization of many

reactions in the carbohydrate field.

C . Ace tamido-deoxyket oses

By oxidation of 1-acetamido-l-deoxy-D- glycero -D- galacto- heptitol with Acetobacter suboxydans , a new ketose (XXXVTI),

closely related to D-fructose (XXXVIII) and perseulose

(XXXIX) _, was obtained [71]. The new sugar, 7-acetamido-7-

1 HOC-CH20H

1

1 HOC-CH,OH

1 HOC-CH.OH

HOCH

I HOCH HOCH

HCOH

I HCOH HCOH

HCOH HCOH

1 HCOH1 OCH

1

1

1 r^w "T

UClla tJL.ll H2 C-NH-C-CH3

II H2COH

XXXVII XXXVIII XXXIX

70
deoxy-L-galacto -heptulose , was shown to be a pyranose by comparison of its optical rotation with that of p-D-fructopyranose

Some of the properties of the new 7-acetamido-7-deoxy-Lgalacto-heptulopyranose were determined for characterization.

It melts at 139 to l4o°C. With 1-butanol: pyridine : water

(3:2:1) as an ascending, developing solvent , the mobility of

the compound on Whatman No. 1 paper is 0.88 that of fructose.

Color formation with staining reagents sensitive to ketoses

is as follows: phloroglucinol, violet; urea phosphate, yellow

(bright yellow fluorescence); resorcinol, tan (violet

fluorescence); orcinol, purple (red fluorescence). The

compound reacts with Somogyi copper-reducing reagent at approximately the same rate as does fructose.

The following equations represent the optical rotation

of 7-acetamido-7-deoxy-q-L- galacto-heptulopyranose in water

at 20 C and 3.5 C, respectively:

t a] 20

D

- -l5 .5e-°?96t _97 . l3

[a] 3 - 5

D

= -ll.ge-- 0101^ -105.8,

where t_ is the time (in minutes) after dissolution of the

sample.

In conjunction with the synthesis of XXXVII, methods for

the preparation of 1-amino-l-deoxyalditols were examined.

Catalytic reduction of glycosylamines proved to be unsatisfactory, because of the instability of the glycosylamines in

neutral or slightly acid solution [72]. Reduction of sugar

oximes [73 ] was satisfactory, and this method is recommended

71
for small-scale preparations of 1-amino-l-deoxyalditols from

readily available aldoses. Reduction of 1-nitro-l-deoxyalditols, prepared from the lower sugars by condensation with

nitromethane [7^-], proved to be the most satisfactory method;

reduction takes place readily, and gives the amines in high

yield.

72
5. SYNTHESIS OF SELECTED COMPOUNDS

( R. S. Tipson and A. Cohen)

The purpose of this project is to devise chemical

procedures for the synthesis of new organic compounds] to

study mechanisms and kinetics of reactions involved in the

preparation of these materials and of required intermediate

compounds; and to determine the physical and chemical properties

of the compounds, for use as reference materials. The work

not only affords such materials for physical, chemical, and

biological research, but serves in developing fundamental

principles involved in organic reactions.

In one phase of this project, N,N-bis(2-chloroethyl)- and

N, N-b is (2-iodoethyl) aniline and their hydrochlorides were prepared. Methods were devised for synthesis, in high yield, of

the dibenzenesulfonate, dime thane sulfonate, and di-p-toluenesulfonate of 2,2 ' -(phenylimino)diethanol. Each compound was

converted into the corresponding p_-aldehyde . The interrelationships and structures of the compounds were proved, and

their properties were determined. An article describing this

work was published [75]. In an informal arrangement with the

National Cancer Institute, each compound synthesized was submitted for evaluation of possible carcinostatic activity. The

above-mentioned dimethanesulfonate was found to "have substantial activity in at least one test system."

We discovered [75] that, in the reaction of methane73
sulfonates with sodium iodide to give the corresponding iodo

derivatives, the salt that is precipitated is not CH-,SCUNa 3 but the double salt 4(CH,S0-,Na) -Nal; this salt had been

described by Collmann in 1868 [76]. Our discovery has since

been confirmed [77]. The significance of the finding is that,

in the past, various workers have used the weight of the salt

formed as a measure of the extent of the reaction, and they

had assumed that the salt was CH^SO^Na. Our work shows that

an apparent yield of 152$ represents an actual yield of 100$

of CH-^SO-^Na, and that other apparent yields must be adjusted

in the same ratio to provide the actual yield of CH-^SO-^Na.

Another phase of this project is concerned with derivatives of alditols as reference materials. Typical sulfonic

esters of alditols have been prepared, and their physical and

chemical properties have been determined. In particular, work

has been done on the mechanisms and kinetics of nucleophilic

substitution reactions and of elimination reactions in

aprotic, dipolar solvents.

The initial work has been performed with sulfonic esters

of D-mannitol, and a variety of derivatives of this alditol

have been crystallized for the first time, including 3,4-diO-(methylsulfonyl) -D-mannitol (XL) and 3,4-di-0-p-tolylsulfonyl-D-mannitol (XLI), and the 1,2,5,6-tetraacetates of esters

XL and XLI. After purification of these products, their

physical properties were determined; these properties included

74
melting point, specific optical rotation, and infrared

absorption spectrum.

The rates of hydrolysis, by 80$ acetic acid, of the

isopropylidene groups from l,2:5 a 6-di-0-isopropylidene-Oj^—

di-0-(methylsulfonyl)-D-mannitol (XLII) and 1,2 :5,6-di-0-

isopropylidene-3 5 ^l— di-_0-p-tolylsulfonyl-D-mannitol (XLIII)

were determined by methods involving optical rotatory power,

and optimal preparatory methods for XL and XLI were developed.

A simple method was devised for elimination of the sulfonyloxy

groups in XLII and XLIII, thereby converting them into the

corresponding acetal of trans -^-hexene-D- threo-1,2,5 ,6-tetrol,

which was hydrolyzed by 80$ acetic acid to the free, unsaturated tetrol, a. compound of considerable interest. This tetrol

and its di-O-isopropylidene acetal have been characterized for

use as reference materials. The work will be extended to

D-glucitol and other alditols.

Earlier work of the Section on acetamido derivatives has

been extended. In collaboration with Professor Venancio

Deulofeu, Buenos Aires, Argentina, we have examined the infrared spectra of some kO 1-acetamido derivatives of alditols and

related compounds. Correlations have been made between absorption bands in the spectra and the structures of the

various compounds. These results will be valuable for identification and in checking for purity of these compounds.

75
6. AIR POLLUTION STUDIES

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

J. H. Gould, M. N. Inscoe, and H. S. Isbell)

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

Pyrolysis or incomplete combustion of carbon-containing

materials at high temperatures gives rise to polycyclic,

aromatic compounds. Consequently, many compounds of this

nature have been detected as pollutants in the air of large

cities, particularly where dispersal is retarded.

Such air pollutants include many polycyclic, aromatic

hydrocarbons and certain of their oxidation products. Some

of these hydrocarbons are carcinogenic to animals and, possibly,

to man. Eleven carcinogenic hydrocarbons have thus far been

detected in automobile exhaust fumes; five are also present in

practically all airborne particles (such as dust and soot) [78].

From particulate matter, obtained from polluted air,

quinones of such hydrocarbons as pyrene, benzo [a]pyrene, and

dibenzo

[

def , mno ]chrysene, as well as other oxidized organic

compounds (largely unidentified as yet), have been isolated

and identified [79]. This suggests that, after release of

reactive hydrocarbons Into the atmosphere, some oxidation may

normally occur in the air. Indeed, over thirty years ago, it

was discovered [80 ] that, on exposure to air containing ozone,
the carcinogenic benzo[a,]pyrene is transformed into less

carcinogenic or noncarcinogenic material. Such oxidation is

to be expected, as polluted air contains a number of oxidants:

the nitrogen oxides, oxygen, and ozone of unpolluted air,

together with such compounds as sulfur dioxide, produced by

combustion.

The oxidation of polycyclic, aromatic hydrocarbons is

also of interest because, in evolving ways for reducing air

pollution, attention is currently being given to the development of chemical devices for oxidizing atmospheric pollutants

produced by incomplete combustion and pyrolysis in automobile

and Diesel exhaust fumes.

In considering the fate of these hydrocarbons and methods

for minimizing their effects as air pollutants, it thus became

of interest to assemble all of the available information on

(1) the oxidants effective in the oxidation of polycyclic,

aromatic hydrocarbons, (2) the relative reactivity of the

hydrocarbons, (3) the conditions under which oxidation proceeds,

(4) the chemical mechanisms involved when such oxidations occur, and (5) the products formed.

Therefore, a critical review entitled "Oxidation of

Polycyclic, Aromatic Hydrocarbons" was written; this is to be

published as NBS Monograph 87 [8l], As part of our study on the

problem of air pollution, an attempt will be made to maintain

a current bibliography of pertinent scientific literature on

77
this subject j with the object of serving our group and others.

B. Interaction of Aromatic Hydrocarbons with Oxygen under

Pressure

To gain an understanding of the oxidation of polycyclic,

aromatic hydrocarbons , information regarding their behavior

with oxygen under pressure is a useful first step. Consequently, we have observed the spectral changes produced by the

interaction of a number of aromatic hydrocarbons with oxygen

at a number of pressures and temperatures , relative to a

reference spectrum of the sample treated with nitrogen.

1. Apparatus

In order to be able to investigate the effect of temperature and pressure on the reaction of oxygen with the aromatic

hydrocarbons , we devised and constructed a pressurizing system

in an absorption-cell thermostat that we had previously constructed for use with the Cary Model-l4 Spectrophotometer.

In its working range of +60 to -100°C, this sample-cell thermostat maintains the temperature of the sample within a few

degrees C. Simultaneously, the solution in the absorption

cell is subjected to a fixed, moderate pressure of the chosen

gas (to date, oxygen; but such gases as nitric oxide and sulfur

dioxide could be used). The upper limit of the gas pressure

used has been thirty pounds per square inch (gauge), a limit

set by the strength of the ordinary absorption cells employed.

The apparatus is shown in Figures 20 and 21. It employs

78
Figure 20. Diagram of cell for spectrophotometer ie study

of the interaction of hydrocarbons with gases.

a V-bottomed, metal cell-container (A) which aligns the

absorption cell in the sample-beam of the spectrophotometer.

Container A is held inside a metal box (B) by means of rigid,

Styrofoam insulation (C). Both A and B are fitted with quartz

windows (D), which are insulated from the walls by cork

gaskets. Both sets of windows are in contact with electric

heaters which prevent the formation of fog or frost at the

lower temperatures employed. Moreover, these heaters are the

primary source of heat when the system is operated above room

temperature.

79
Figure 21. Photograph of apparatus for the spectrophotometry study of the interaction of

hydrocarbons with gases.

The sample compartment is attached below a replica (E)

of the sample-compartment cover by four studs and is secured

by four wingnuts. To the top of E is fastened a one-liter

Dewar flask (F); this contains the liquid nitrogen used as the

refrigerant, and is stoppered. The fluid for transfer of heat

is dry air at low pressure or tank nitrogen, admitted by the

hose connection (K). This gas stream is split in two, and

passes through two identical needle-valves (I) which are

coupled, back to back, by a. knob (J). This arrangement ensures

80
that (1) the valves will operate in an inverse manner, and (2)

the total flow through the apparatus will remain about the

same, regardless of the distribution of the heat-transfer gas.

It also provides a crude means of setting the response of the

system to the action of the temperature-control system.

The first stream of gas is conducted directly to the

sample-compartment via the mixing chamber (H) and tube (G),

both of which are insulated with Styrofoam. After the gas

has bathed the absorption cell, it is vented to the atmosphere

through a tube (not shown)

.

The second stream of gas is admitted into the top of the

Dewar flask (F) past an electrically operated valve (L) which

is either open to the atmosphere or closed, depending on the

degree of cooling to be applied to the sample in order to

maintain the low temperature selected. When cooling is required, valve L closes and the liquid nitrogen is forced from

F, through the control valve (M), and into the mixing chamber

(H), where it evaporates, thereby cooling the primary stream

and, subsequently, the absorption cell.

The open or closed condition of valve L is determined by

the temperature-control system; this consists of two copperconstantan thermocouples (one for the sample and one as a

reference), a potentiometer, and an amplifier whose output

operates valve L. The thermocouple for the sample is pressed

against the top of the absorption cell by means of a spring.

The reference junction is kept in an ice bath. The opposed

81
output from the thermocouples is applied, through a reversing

switch j to the input of the potentiometer. This potentiometer,

which is the primary temperature-measuring device, also acts

as the balance unit for the control system, because the input

to the amplifier is obtained from the galvanometer of the

potentiometer. The amplifier is a Brown Recorder amplifier,

so modified as to provide a single-ended output of reversible

phase. The AC output of the amplifier is rectified, and then

applied to the control valve L, so as to inject or withold

refrigerant as determined by the direction of the current

through the galvanometer.

After the proper setting of the phasing switches has been

achieved, the temperature of the absorption cell can be set,

and maintained, merely by turning the potentiometer dial to

the millivolt reading required for production of the desired

temperature

By means of a second system, the solution under study is

treated with a gas under pressure. In operation, the pressureregulated gas is admitted, from a tank, into the absorption

cell, by means of a spring-loaded, injector assembly (N). The

gas is conveyed from the tubing connections (P) by capilla:

tubing. The upstream and downstream pressures are recorded

by gauges (R) and (S), respectively.

The seal at the neck of the absorption cell is provided

by a stopper that had. been cast, on a paraffin block, from a silicone rubber vulcanizing at room temperature.

82
2. Results

Figure 22 shows the changes observed in the spectrum

of a solution of benzene in 2-methylheptane on treatment

with oxygen, relative to the spectrum of the same

solution on treatment with nitrogen. The fine structure

present is attributed to singlet-triplet transitions [82], and

the general absorption underlying it is the result of charge

transfer between the benzene (donor) and the oxygen (acceptor)

.

To date, we have made observations on the spectral

changes produced by the interaction of oxygen with solutions

of naphthalene, anthracene, naphthacene, phenanthrene,

b en z [a] anthracene, and pyrene in inert solvents, relative to

the spectra of the same solutions treated with nitrogen. The

magnitude of the changes produced by oxygen is a function of

the oxygen pressure and the concentration of the aromatic

hydrocarbon.

Although the spectral changes observed for the polycyclic

hydrocarbons mentioned are not so spectacular as for benzene,

either in magnitude or complexity, they are readily observed.

With the exception of naphthacene and benz [aj anthracene, the

oxygen-induced absorption produced in the spectrum of each of

these hydrocarbons is (a) completely removed on displacing

the oxygen by treatment with nitrogen, and (B), in intensity,

directly proportional to the absolute pressure of oxygen

applied.

83
0.9

0.8

0.7 -

0.6 —

B 0.5

z

<

CO<ro

</> CD 0.4

<

0.3

0.2

0.1

0.0

1

- 1

i

—

i

\

1 \

—

I i

—

1 > fi

i •t

—

\

i

—

\ i

* - \ 1 —

- u —

\ \

V

—

-» V

ij —

1 1 1 1 1 1 1 1 1 300 350

WAVELENGTH

Benzene in 2-Methylheptone

(1:1 by volume)

———under Nitrogen

10 psig Oxygen —— 30 psig Oxygen

400 mp

Figure 22. Spectral changes induced by treatment of

benzene with oxygen.

84
However, for naphthacene, the absorption does not appear

to "be proportional either to the pressure or the square of the

pressure (over the whole range of available pressures). In

addition, naphthacene gives a large response to oxygen treatment relative to its concentration, as compared with the behavior of the other hydrocarbons studied.

For naphthacene and benz [a, ] anthracene, we observed nonreversibility on treating the oxygenated solutions with

nitrogen. (Such nonreversibility was not observed for the

spectra of the other hydrocarbons studied. ) It indicates the

formation of a permanent chemical compound of the hydrocarbon

with oxygen under pressure. For naphthacene, formation of

this compound was promoted by irradiation with light from a

tungsten source or from the near-infrared source of the Cary

Model-l4 spectrophotometer. Whether such promotion can be

effected for the other hydrocarbons has not yet been studied.

C. Oxidation of Polycyclic, Aromatic Hydrocarbons on

Particulate Matter

Observations made during the application of thin-layer

chromatography to characterization and determination of purity

of various polycyclic, aromatic hydrocarbons led to our discovering that alterations which take place in the appearance

of the spots are often the result of photochemical reactions

on the thin-layer chromatogrsms [83 ]

.

Observations were made on spots of 15 representative

hydrocarbons following exposure to ultraviolet light. The

85
adsorbents used were silica gel G, aluminum oxide G, cellulose

powder 5 and acetylated cellulose (21$). Four of the hydrocarbons (phenanthrene, chrysene, triphenylene, and picene)

show no changes after such exposure, other than a slight

fading of fluorescence when allowed to stand in room light

for several days. On silica gel G and aluminum oxide G, pronounced changes occur, both in the appearance and in the

fluorescence of the 11 other hydrocarbons. Spots of the hydrocarbons having an anthracenic structure (anthracene, naphthacene, benz [a, ] anthracene, dibenz [a,, cj anthracene, and dibenz-

[a,h]anthracene) turn yellow or a yellow-tan; spots of condensed hydrocarbons (pyrene, benzo[a,]pyrene, benzo[_e ]pyrene,

perylene, benzo[ ghi jperylene, and coronene) turn darker tan

or brown. At the same time, the appearance of the spots under

ultraviolet illumination also changes. The fluorescent color

characteristic of each hydrocarbon (usually a shade of blue

or green) becomes dull and is gradually lost, while the spots

take on an orange or red coloration, usually changing to such

a deep red that they appear completely dark. A halo (blue,

green, orange, or red, depending upon the hydrocarbon involved)

is often seen at the edge of the dark spot. On powdered

cellulose or acetylated cellulose, the behavior of these 11

hydrocarbons is somewhat similar, although the changes in

appearance and fluorescence are much less extensive and develop

much more slowly.

86
After the initial exposure to ultraviolet light, these

changes take place even when the plates are kept in the dark.

Similar, but slower, changes also occur on plates kept in

ordinary room-light, without exposure to other ultraviolet

illumination. The changes are accelerated by continuous

irradiation, either by long-wave length ultraviolet light or

by light of 253.7 mu.. The nature of the developing solvent appears to have

little effect on the colors observed. However, although it

has sometimes been recommended that the chromatopiates be

read while they are still wet [84,85] we have found that the

presence of solvent often accelerates the changes in the spots.

This effect is particularly noticeable with chlorinated solvents,

For example, on a plate coated with silica gel G and developed

with carbon tetrachloride, a pyrene spot, examined under ultraviolet light while still wet, turns dark and loses much of its

fluorescence within 5 minutes. However, if the plate is allowed to dry for 10 to 15 minutes in the dark before being

exposed to ultraviolet light, the spot is more stable and the

blue-green fluorescence is still strong after several hours,

although by this time some darkening of the spot can be observed

in visible light.

In some instances, this darkening can be utilized in the

visualization and identification of hydrocarbon spots. Benzene

solutions of pyrene, benzo[a.]pyrene, benzo[e_]pyrene, and

benzo

[

ghi jperylene were separately spotted on a silica gel G

87
plate (about 10 u.g of hydrocarbon per spot), developed with

heptane , dried in the dark for 15 minutes , and exposed to

continuous, long-wavelength ultraviolet illumination. The

pyrene and benzo [ajpyrene spots turned brown within 5 minutes,

and all four spots were easily visible in room light after 15

minutes of ultraviolet irradiation. In another experiment,

the gradual appearance of a brown spot on a silica gel G

chromatogram aided in demonstrating the presence of pyrene as

an impurity in a sample of chrysene.

The reactions which give rise to these changes in the

hydrocarbon spots are undoubtedly quite complex. Presumably,

such photo-oxidations as have been reported for anthracene on

alumina or silica gel [86] are involved. This is supported

by our identification of 1,6-pyrenedione and 1,8-pyrenedione

among the numerous products of the reaction of pyrene on

silica gel G.

The work is being extended to oxidation, on a larger

scale, on particulate matter in the presence of air and ultraviolet light. For separation, identification, purification,

and quantitative determination of the products formed, column

chromatography and gas —liquid chromatography are being used,

in conjunction with ultraviolet, infrared, and nuclear magnetic

resonance spectroscopy.

88
D. Determination of Losses Due to Adsorption, During

Filtration of Aqueous Solutions of Polycyclic, Aromatic

Hydrocarbons

In the course of some preliminary experiments on

solubilization, undertaken by us two years ago, erratic results

were obtained for the solubilities of pyrene and benzo[a]pyrene

in aqueous solutions of polyvinylpyrrolidone (PVP) or

deoxyribonucleic acid (DNA); these were traced to adsorption

of the hydrocarbons by the filter media.

Although it has been pointed out that some adsorption of

a solute may occur during filtrations [87], there is apparently

no general awareness of the possible magnitude of such losses

with dilute solutions of certain solutes. Loss by adsorption

of polycyclic, aromatic hydrocarbons during filtration [88]

has been mentioned briefly, and such adsorption has been

shown [89] to be the cause of a major discrepancy in some

recent attempts [90] to determine the solubilities of such

hydrocarbons in aqueous solutions of DNA. However, there has

been no detailed report on the extent of losses due to adsorption.

To evaluate the extent of this adsorption, studies were

initially made on solutions of phenanthrene in distilled water,

because phenanthrene is sufficiently soluble in water to permit

changes in concentration to be observed spectrophotometrically

by means of the strong absorption band at 251 mo.. The solutions

used had been pre-f iltered, usually through a membrane filter

89
(Polypore 27A; see Table 6, footnote c), and, on visual examination in ultraviolet light [91]* showed no particles of

undissolved phenanthrene . All spectrophotometric measurements

were made on a Cary Model-1^ spectrophotometer, using matched,

fused quartz, absorption cells with the appropriate solvent

or solution as a reference. To reduce errors arising from

the background absorption caused by (a) ultraviolet-absorbing

materials leached from the filters and (b) light-scattering by

suspended particles, the base-line technique [92 J was used for

determinations of concentration [Note 1].

The results are shown in Table 6, from which it may be

seen that, during filtration, phenanthrene is adsorbed from

aqueous solutions by various filter media. Losses are

particularly pronounced when membrane filters are used. The

amount of hydrocarbon removed from solution by adsorption during

filtration depends to some extent on the composition of the

filter, but the pore size of the filter also has a very marked

effect, indicating the influence of changes in surface area

as well as in the rate of filtration. The pronounced increase

in the amount of hydrocarbon removed from solution when two

filters are used instead of one (see the data obtained with

Polypore 27A) is also a result of both increased surface area

and decreased rate of flow. The effect of changes in the rate

of filtration alone may be seen particularly well in the data

for Polypore AM-1 and for Polypore GM-6, where a decrease in

the rate of filtration resulted in a sharp decrease in the

90
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91
concentration of phenanthrene in the filtrate.

Losses were also noted when aqueous solutions of naphthalene, pyrene, or anthracene were filtered, the relative losses

being greater with the less-soluble hydrocarbons. Thus, when

an aqueous solution of pyrene was filtered through a sinteredglass filter (fine frit), the concentration of pyrene in the

first 50-ml portion of filtrate was hk% of the original concentration. Also, no anthracene could be detected spectrophotometrically in the final 20-ml portion of filtrate after

filtration of 270 ml of an aqueous suspension of anthracene

through Polypore AM-7.

When solutions of pyrene, benzo [ajpyrene, benz [a.] anthracene

dibenz [ a, h] anthracene, or similar hydrocarbons in aqueous

solutions of PVP or DNA were filtered, there was a marked loss

of hydrocarbon in the filtrate. No adsorption of hydrocarbon

was observed from solutions in aqueous ethanol (20 or 50$) or

in other organic solvents.

Such polymeric materials as PVP and DNA were also adsorbed

from aqueous solution, but the filters were quickly saturated

by these materials and, after the first 20 to 50 ml of filtrate

had been collected, further changes in concentration of the

filtrate were not usually observed. In contrast to this rapid

saturation, and the saturation of membrane filters by protein

[93], the filters are not readily saturated by the hydrocarbons.

Occasionally, in addition to the errors introduced by this adsorption [Note 2] during filtration, particles of some hydro92
carbons (seen as bright, fluorescent specks when the filtrates

are viewed in ultraviolet light) pass through paper or sinteredglass filters. However , the centrifugation procedure of

Kofahl and Lucas [95 ] was found to give fairly reproducible

solutions of hydrocarbons even in viscous solutions containing

highly polymerized DNA. Two centrifugations, with Pyrex-wool

plugs centrifuged down onto the residue, were sufficient to

ensure removal of suspended hydrocarbon. Comparison of the

spectra of the resulting solutions and of the original suspensions, using the base-line technique, showed the change in

concentration of the dissolved hydrocarbons, due to centrifugation, to be less than 10$. By this method, considerably less

variation in concentration is observed than when filtration

is used to remove undissolved hydrocarbon. The loss may arise

from adsorption on the centrifuge tubes and on the glass-wool

plugs.

Note 1. It is especially important to use this method in

solubilization experiments if the solvent is a

solution of a polymeric material. The hydrocarbon

often appears to bring about changes in the lightscattering and sedimentation properties of the

polymer, with the result that the background absorption of the reference blank and the experimental

solution are no longer exactly comparable. On applying the base-line technique to the spectral curves

shown in Fig. 3 of reference [90] and estimating the

93
concentration from readings at jQ5, 395-* and ^°5 mji-j

we have found that the concentration of benzo [a.]pyrene

remains quite constant (approximately 1 x 10" M)

during the centrifugation. The spectral changes

observed appear to be the result of the more rapid

sedimentation of the DNA in the hydrocarbon-containing solution than in the solution of DNA only (cf,

[88]). We have observed similar effects with PVP

solutions of aromatic hydrocarbons.

Note 2. Munck [9^] used silicone-treated glassware to lessen

the adsorption of steroids from aqueous solutions

,

but in the present work, similar treatment of frittedglass filters with a silicone preparation (Desicote,

Beckman Instrument Co. ) did not produce any clearcut effect on the amount of phenanthrene adsorbed

during filtration. The reduced affinity of the

treated surface for the hydrocarbon appears to be

counteracted by the effect of the reduction in the

rate of filtration brought about by the hydrophobic

surface of the filter.

E-. Preparation of Reference Compounds; Purification b}

Column Chromatography

For use in identification of materials by thin-layer

chromatography and gas —liquid chromatography, samples of pure

reference compounds are needed, so that their behavior can be

established.

9^
For each hydrocarbon whose oxidation is studied, a number

of oxidation products that might possibly be formed under the

particular conditions of oxidation are required. Thus, in

connection with studies on the oxidation of pyrene, the following reference compounds were prepared: 1-, 5-, and 4-pyrenol,

1)6- 3 1,8-, and 4,5-pyrenediol, and l,3:>6,8-pyrenetetrol.

In addition, methods have been developed for separation

of the pyrenediones by column chromatography [96]. The 1,6-

pyrenedione was separated from 1,8-pyrenedione on a column of

silica gel, with glacial acetic acid as the eluant. The 1,6-

pyrenedione was then finally purified on a column of activated

alumina, with benzene as the eluant. The properties (including

ultraviolet and infrared spectra) of purified 1,6- 1,8-, and

4,5-pyrenedione and of l-oxo-6,7-phenalenedicarboxylic

anhydride were determined.

95
7. REFERENCE MATERIALS (ORGANIC)

( H. S. Isbell and Staff)

A. Rare Organic Compounds

The development of numerous instrumental methods of

analysis whereby substances are determined both qualitatively

and quantitatively by comparison of an unknown with a standard

has created a strong demand for a wide variety of reference

materials. During the past year, our Section has fulfilled

21 requests from research workers elsewhere for small samples

of rare compounds prepared in the course of our research program and not available elsewhere. The compounds have been of

great value to these research workers, as shown by letters

expressing appreciation for the service. During the coming

year, we plan to improve and extend this service.

14 "5 B. C and ^H- Labeled Carbohydrates

Under our labeled carbohydrate project, we have maintained

a supply of the labeled carbohydrates and have furnished

research workers in other institutions with kj> samples (5250 (ic)

during the current year.

C. Sugar Standards

Production and distribution of standard samples of dextrose

and sucrose are being continued. During the year, 458 samples

of the sugar standards were sold.

D. Metallo-organic Standards

Preparation and distribution of metallo-organic standards

9
is being continued. During the year, 1332 samples of metalloorganic standards were sold. Our supplies of ferric cyclohexanebutyrate (Standard Reference Material No. IO58) and of

tris ( 2' -hydroxyacetophenono) chromium (III) (Standard Reference

Material No. 1072) were exhausted. Inasmuch as the compounds

used as the earlier standards are slightly hygroscopic , we

reinvestigated the derivatives of l-phenyl-l,3-butanedione

for use as new standards. Measurements of the changes in

weight of the materials used as the earlier standards and of

the new l-phenyl-l,3-butanedione chelates showed that the

latter are less susceptible to change in weight on exposure

to air under laboratory conditions. Hence, l-phenyl-1,3-

butanedione chelates were used for new standards for iron and

chromium. Procedures for preparation of the new standards,

and for preparing stable solutions of the compounds, are given

in the sections that follow.

1. Tris (l-phenyl-l,3-butanediono)chromium( III) ( Standard

*Reference Material No. 1078) — » '— »..^.» iii^m m..+ »- 1 ! .i » —— ' Chromium(lll) chloride hexahydrate (293.1 g, 1.1 moles)

is dissolved in 1200 ml of water in a 5-liter flask equipped

with a stirrer and heated in an oil bath. The mixture is

vigorously stirred while a solution of 487 g (3 moles) of

l-phenyl-l,3-butanedione in 2000 ml of ethanol is slowly

added. The resulting solution is heated and stirred while a

solution of 200 g of urea in 350 ml of water is added during

97
30 minutes. The mixture is heated and stirred overnight , after

which the precipitate is collected and washed with hot water

until the aqueous washings are colorless. Finally, the solid

is washed with ethanol and air-dried; weight, 456 g (85$).

About sixty g of unreacted diketone may be recovered from the

filtrate.

The dry, crude product is dissolved in 2000 ml of chloroform and stirred while 1000 ml of ethanol is added. The mixture

is stored overnight in a refrigerator. The crystalline product

is separated by filtration, recrystallized from chloroform as

before, dried, and powdered (100 mesh).

Anal. Calcd. for C^H^CrOg: Cr, 9-71. Found: Cr, 9.58.

2. Tr is ( 1-phenyl-l , 3-butanediono ) ir on( III ) . (Standard

Reference Material No. 1079)

l-Phenyl-l,3-butanedione (502.7 g, 3 moles) is dissolved

in 1500 ml of absolute ethanol in a 3-liter flask, and

vigorously stirred while 1200 ml of concentrated ammonium

hydroxide is slowly added. The resulting solution is added,

with stirring, to a hot solution of ferric chloride hexahydrate

(270.2 g, 1 mole) in 400 ml of water. The dark-red compound

begins to separate immediately. The mixture is stored overnight in a refrigerator, and the resulting crystals are

separated by suction filtration, and washed, first with hot

water, and then with ethanol. The air-dried, crude compound

weighs about 500 g (92$).

98
The compound is recrystallized from 1500 ml of hot

chloroform by addition of 900 ml of warm ethanol. Crystallization begins after about 10 minutes , and is allowed to continue

overnight. The crystals are collected on a filter, air-dried,

and recrystallized as before from chloroform. The yield of

the pure product is about 444 g (82$).

Anal. Calcd. for C^ H

27Fe0g: Fe, 10. 35 . Found: Fe, 10.26; . The new standard reference materials are crystalline, and

are easily prepared in high yield. They are relatively stable

in the presence of moisture. After exposure of a 0.5-g quantity

of each compound to 75$ humidity for 120 hours, the iron

derivative gained 0J mg and the chromium derivative gained

0.2 mg. Both samples readily lost this moisture on being

heated for 1 hour at 110°C.

The solubilities of these new standards in lubricating

oils are increased by the use of 6-methyl-2,4-heptanedione

and 2-ethylhexanoic acid, the reagents used previously for

Standard Reference Materials IO58 and 1072. The solutions

prepared with these reagents are stable, and compatible with

the other NBS metallo-organic standards.

99
8. PUBLICATIONS , REPORTS , AND LECTURES

A. Publications

Tritium-labeled Compounds. X. Isotope Effects in the

Oxidation of Aldoses-1-t with Bromine.

H. S. Isbell and L. T. Sniegoski, J. Res. NBS 68A , (Phys.

and Chem.) No. 2, 145 (1964).

Cyclic Polyhydroxy Ketones. II. xylo-Trihydroxycyclohexenediolic Acid and Keto-inositols

A. J. Fatiadi and H. S. Isbell, J. Res NBS 68A , (Phys. and

Chem.) No. 3, 287 (1964).

Tritium-labeled Compounds. XI. Mechanism for the Oxidation

of Aldehydes and Aldoses- 1-t with Sodium Chlorite.

H. S Isbell and L. T. Sniegoski, J. Res. NBS 68A , (Phys.

and Chem.) No. J>, 301 (1964).

Synthesis of Higher Ketoses by Aldol Reactions. II.

Unsubstituted Heptuloses.

R. Schaffer, J. Org. Chem. 29, 1471 (1964).

The Isomerization of D-manno-3-Heptulose by Alkali.

R. Schaffer, J. Org? Chem. 29, 1473 (1964).

Report on Sugars and Sugar Products - October 1963.

E. J. McDonald, J.A.O.A.C. 47, 69 (1964).

Photochemical Changes in Thin-layer Chromatograms of

Polycyclic, Aromatic Hydrocarbons.

M. N. Inscoe, Anal. Chem. 36, 2505 (1964).

Methods in Carbohydrate Chemistry, Vol. 5, Academic Press,

(I965). (64) Determination of Reducing End-groups by

Reaction with C ^"-Labeled Cyanide.

H e S. Isbell.

Synthesis and Ring Structure of 7-Acetamido-7-deoxy-L-galacto - heptulose.

E. J. McDonald, J. Res. NBS 69A, (Phys. and Chem.) No. 3,

291 (1965).

Deuterium Isotope Effects in the Mutarotations of a-D-Glucose

and P-D-Fructose.

Ph.D. Thesis by Clarence W„ R. Wade, submitted to Georgetown

University, May 14, 1965.

100
B. Manuscripts Accepted for Publication

Oxidation of Polycyclic, Aromatic Hydrocarbons.

R. S. Tipson. Accepted for publication as NBS Monograph 87

Separation of Pyrenediones by Column Chromatography.

A. J. Fatiadi. Accepted for publication in Journal of

Chromatography

.

14 Synthesis of D-glucose-3- C and Related Compounds.

H. L. Frush, L. T. Sniegoski, N. B. Holt, and H. S. Isbell.

Accepted for publication in Journal of Research, NBS.

C. NBS Reports

(Quarterly Reports on Air Pollution Program prepared jointly

with certain Sections of the Physical Chemistry Division)

NBS 836O - Quarterly Report AIR POLLUTION PROGRAM - 1/1/64-3/31/64. P. J. Ausloos, H. S. Isbell, R. Klein,

R. Stair, R. S. Tipson.

NBS 8363 - Special Report AIR POLLUTION PROGRAM - 4/27/64 -

R. S. Tipson.

NBS 8512 - Quarterly Report AIR POLLUTION PROGRAM - 4/1/64-6/50/64. H. S. Isbell, R. Klein, R. E. Rebbert,

C. A. Samson, R. Stair, R. S. Tipson.

NBS 8582 - Quarterly Report AIR POLLUTION PROGRAM - 7/1/64-9/50/64. H. S. Isbell, R. Klein, R. E. Rebbert,

R. Stair, R. S. Tipson.

NBS 8634 - Quarterly Report AIR POLLUTION PROGRAM T

10/1/64-12/51/64. H. S, Isbell, R. Klein, R. E. Rebbert,

R„ Stair, R. S. Tipson.

NBS 8683 - Quarterly Report AIR POLLUTION PROGRAM - I/I/65-3/3I/65. H. S. Isbell, R. Klein, R. E. Rebbert,

R. Stair, R. S. Tipson.

NBS 8930 - Quarterly Report AIR POLLUTION PROGRAM - 4/I/65-6/3O/65. R. Klein, R. E. Rebbert, R. Stair,

R. S. Tipson.

101
D. Lectures

1/6/64 - Nomenclature of Sugar Conformers . Chemistry Seminar - Georgetown University,, Washington, D. C. - R. S. Tipson

1/13/64 - Higher Sugar Synthesis by Aldol Reactions. Chemistry

Seminar - Georgetown University., Washington., D. C. - R. Schaffer

1/20/64 - Isotope Effects in Chemical and Biological Reactions.

Chemistry Seminar - Georgetown University, Washington.,

D. C. - H. S. Isbell

5/1/64 - Phenylhydrazono-phenylazo Structures for 2-0xo-l,3-

Bis (phenylhydrazono) Derivatives. Bi-sectional ACS

regional meeting, University of Maryland - A. J.

Fatiadi and H. S. Isbell

5/28/64 - Branched-chain Monosaccharides. Fifth Annual

Medicinal Chemistry Symposium., University of New

York at Buffalo - R. Schaffer

7/9/64 - Application of Isotope Methods of Analysis. Gordon

Research Conference, Tilton, N. H. - R, Schaffer

7/13/64 - Interpretation of Reactions in the Carbohydrate

Field in Terms of Consecutive Electron Displacements.

Internationale Symposium uber die Chemie der

Kohlenhydrate, Miinster, Germany - H. S. Isbell

9/1/64 - Isomerization of Reducing Sugars in Alkaline

Solution. ACS - Division of Carbohydrate Chemistry,

Chicago, 111. - H, S. Isbell , R. A/ Peterson, H. L.

Frush, and N. B. Holt

9/1/64 - Oxidation of Polycyclic, Aromatic Hydrocarbons

Pertinent to Air Pollution. ACS - Division of Water,

Air, and Waste Chemistry - Symposium on Air Pollution,

Chicago, 111. - H„ S. Isbell , R. S. Tipson, and

A. J. Fatiadi

9/1/64 - Oxidation of Polycyclic, Aromatic Hydrocarbons on

Particulate Matter. ACS - Division of Water, Air,

and Waste Chemistry - Symposium on Air Pollution,

Chicago, 111. - M„ N, Inscoe

3/12/65 - Program of Organic Chemistry Section. Analytical

Chemistry Division Staff Meeting, Chem. Bldg.

Lecture Room. - H. S. Isbell

102
5/7/^5 - Phenylhydrazono-Phenylazo Taut omerism. Bi-sectional

ACS regional meeting , University of Maryland - A, J. Fatiadi and H. S. Isbell

5/7/65 - Use of Solvent Isotope Effects in the Study of

Pyranose—Furanose Interconversions . Bi-sectional

regional ACS meeting 5 University of Maryland - C. W. R. Wade

105
9 . PERSONNEL

Organic Chemistry, H. S. Isbell, Section Chief

R. S. Tipson

H. L. Frush

R. Schaffer

C. W„ R. Wade

N. B. Holt

A. J. Fatiadi

A. Cohen

E„ J. McDonald, retired

L. T. Sniegoski, resigned

R. A. Peterson, transferred

J. H. Gould " M. N. Inscoe " E. Barron, Summer Student, terminated

T. Andrews " " LWOP

104
10 . ACKNOWLEDGEMENTS

The staff of the Organic Chemistry Section wish to thank

Mrs. R. S. Mad'dock for her assistance in preparing this report

We also express our thanks to Dr. J. K. Taylor and staff of

the Analysis and Purification Section for consultation,, for

use of equipment j and for many analyses.

The Editor acknowledges with thanks the cooperation of

all members of the staff of the Organic Chemistry Section. He

is particularly indebted to Dr. H. L. Frush who critically

reviewed all parts of the report and made many improvements,

and to Dr. R. S. Tipson who rendered editorial assistance and

advice on nomenclature. Special thanks are expressed to Mrs.

Y. S. Sladen, Section secretary, whose cooperation and zealous

work made expeditious publication possible.

105
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Ill


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