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DEC 9 1970

NBS TECHNICAL NOTE 547

Organic Chemistry

Section:

Summary of Activities

July 1969 to June 1970

NATIONAL BUREAU QF STANBAtW!

UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

NATIONAL BUREAU OF STANDARDS • Lewis M. Branscomb, Director

Ls-47 u

>• NBS

-i.

TECHNICAL NOTE 547

ISSUED NOVEMBER 1970

Nat. Bur. Stand. (U.S.), Tech. Note 547, 134 pages (Nov. 1970) CODEN:: NBTNA

Organic Chemistry Section:

Summary of Activities

July 1969 to June 1970

Robert Schaffer, Editor

Organic Chemistry Section

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

i? w j y*.

V,, ov *

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.

(Order by SD Catalog No. C 13.46:547), Price $1.25.
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 1, 196*1, reorganization.

It consists at present of nine sections and about 100 technical personnel encompassing some 60 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 a highly

important output of our laboratories. In addition, however,

it has been our experience that informal, annual summaries of

progress describing efforts of the past year can be very valuable in disseminating information about our programs. A word

is perhaps in order about the philosophy of these yearly

progress reports . In any research program a large amount of

information is obtained and techniques developed which never

find their way into the literature. This includes the

"negative results" which are so disappointing and unspectacular but which can often save others considerable work. Of

importance also are the numerous small items which are often

explored in a few days and which are not important enough to

warrant publication—yet can be of great interest and use to

specialists in a given area. Finally there are the experimental techniques and procedures, the designs and modifications

of equipment, etc., which often require months to perfect and

yet all too often must be covered in only a line or two of a journal article.

ii
Thus our progress reports endeavor to present this information which we have struggled to obtain and which we feel

might be of some help to others . Certain areas which it

appears will not be treated fully in regular publications are

considered in some detail here. Other results which are

being written up for publication in the journal literature

are covered in a much more abbreviated form.

At the National Bureau of Standards publications such as

these fit logically into the category of a Technical Note.

In 1970 we plan to issue these summaries for all of our

sections . The following is the sixth annual report on progress of the Organic Chemistry Section.

W. Wayne Meinke, Chief

Analytical Chemistry Division

111
PREFACE

This is the sixth annual progress report of the Organic

Chemistry Section of the Analytical Chemistry Division, and

it describes the activities of the Section from July 1, 1969

to June 30, 1970. Unlike the formal, journal publications

that emanate from the Section from time to time, this report

summarizes the activities of the entire Organic Chemistry

Section during that period.

Following the established pattern within this Division;

the programs of the Section have both a research and a sample

aspect: about half of the total effort of the Section has

been applied to research, and the remainder to Standard

Reference Materials (SRMs). The Section must concern itself

with many subjects in organic chemistry in supporting the

National Bureau of Standards' overall mission, which is to

contribute, where possible, to the solution of nationwide

problems through the enhancement of meaningful measurement.

To that end, in the past year, much of the Section's effort

was directed to developing SRMs needed in clinical chemistry

laboratories. This has entailed working with organic compounds that fall into a number of classical areas of organic

chemistry. To maintain overall unity in the Section's program,

considerable attention is given to one major area of organic

chemistry, the carbohydrates, developing therein the Section's

competences in organic analysis, characterization, and

synthesis. These competences are utilized to the maximum

extent for the development of SRMs. Our efforts in serving

clinical chemistry have been fostered by the National Institute

of General Medical Services of the National Institutes of

Health.

IV
In this report, in order to describe procedures

adequately, it has occasionally been necessary to identify

commercial equipment. In no case does such identification

imply NBS recommendation or endorsement, nor does it imply

that the equipment identified is necessarily the best

available for the purpose.

Robert Schaffer, Chief

Organic Chemistry Section
TABLE OF CONTENTS

PAGE

CLINICAL STANDARDS 2

Cholesterol. Post-issuance Studies 3

Thin-layer Chromatography of Cholesterol

Samples 3

Liebermann—Burchard Analysis H Differential Scanning Calorimetry (DSC)

and Phase-solubility Analysis (PSA) 4 Urea (Clinical). Post-issuance Studies 5

Determination of Purity of Urea by

Differential Scanning Calorimetry 5

Determination of Moisture Content of Urea

by Karl Fischer Titration 5

Determination of Purity of Urea by Phasesolubility Analysis 6

Bilirubin 6

Standard Solutions of Bilirubin 6

Some Estimates of the Millimolar

Absorptivities of Bilirubin in

Non-aqueous Solvents 10

Millimolar Absorptivities in Methyl Sulfoxide

for Bilirubin Diazo-coupled by an Evelyn

Malloy Procedure 12

Millimolar Absorptivities for Bilirubin

Diazo-coupled by the Jendrassik—Grof

Procedure 14

D-Glucose (Clinical) 19

Analysis of D-Glucose by Gas —liquid

Chromatography 19"

Apparent Purity of D-Glucose by

Differential Scanning Calorimetry 22

Determination of Moisture Content of

D-Glucose Samples 2

4

VI
TABLE OP CONTENTS (CONT'D)

PAGE

Cortisol 24

Physical Properties of Some Cortisol

Samples 25

Determination of Purity by Differential

Scanning Calorimetry 26

Spectrometric Properties. 27

Thin-layer Chromatography of Cortisol ... 28

4-Hydroxy-3-methoxy-DL-mandelic Acid (VMA) ... 30

Melting Point of Commercial Samples of

VMA. 30

Thin-layer Chromatograohy of Commercial

Samples of VMA . . ,. . . 31

Spectrometric Properties 32

Examination by Gas —liquid Chromatography

(G.l.c.) of the Per ( trimethylsilyl

)

Derivatives of Samples of VMA 37

Determination of Purity of VMA by

Differential Scanning Calorimetry 37

D-Mannitol (Clinical) 38

Thin-layer Chromatography (T.l.c.) of

D-Mannitol 38

Paper Chromatography of D-Mannitol 39

Optical Rotation of Samples of

D-Mannitol 40

3-Nicotinamide Adenine Dinucleotide (3-NAD) and

its 1,4-Reduced Form (3-NADH) 42

Loss of Weight on Drying 45

Elemental Analysis 45

Paper Chromatography of 3-NAD 46

Gas —liquid Chromatography of Commercial

3-NAD after Per ( trimethylsilyl )ation ... 51

Spectrometric Methods 52

Enzymic Assay of 3-NAD 53

CARBOHYDRATE SYNTHESIS, STRUCTURE, AND

CHARACTERIZATION: NOVEL RESEARCH MATERIALS

AND MODEL COMPOUNDS 55

Synthesis of the Isomeric Di-O-isopropylidene-3-

D-fructopyranoses 56

VII
TABLE OF CONTENTS (CONT'D)

PAGE

Synthesis of g-Psicose 58

Model Parameters for the Analysis of Skew

Conformations by P.m.r. Spectroscony 66

The Conformations of 1 ,2 : 4 ,6-Di-(3-benzylidenea-D-glucopyranose Derivatives 73

Iterative Analysis of the Proton Magnetic

Resonance Spectra of 1 ,2 : 3 ,5-Di-C-isopropylidenea-D-glucofuranose Derivatives 77

Cyclic Polyhydroxy Compounds 86

Determination of Inososes with an Alkaline

Solution of Copper(II) Oxalate —Tartrate

Complex (the Somogyi Reagent); Structure

of the Reagent (by E.s.r. Studies), and

Reaction Mechanisms Involved 86

E.s.r. Evidence for a Dimer Structure of

the Alkaline Copper (II) Tartrate Complex

in the Fehling Reagent at 77 K 91

Preparation of Inositol Hexasulfate 94

3. GENERAL ORGANIC CHEMISTRY 95

Novel Iodination of the a-Methylene Group in

1 , 3-DIphenyl-l , 3-propanedione and Related

B-Diketones with Periodic Acid 95

Degradation of Bilirubin with Chromic Acid .... 99

Degradation of Hemin and Related Bile Pigments

with Periodic Acid 101

Isolation of a Biliverdine Type of Reaction

Intermediate on Oxidation of Bilirubin with

Periodic Acid 103

4. DETECTION AND IDENTIFICATION OF TRACES OF

ORGANIC COMPOUNDS 105

2 ,4 ,6-Trinitrotoluene 105

vm
TABLE OF CONTENTS (CONT'D)

PAGE

Hexahydro-1,3 , 5-trinitro-s-triazine (2) 106

Detection of 2 by Diazonium Coupling 106

Detection of 2 by Formation of

Formaldehyde 106

Detection of 2 by Formation of Hydrazine. . . 107

Detection of 2 by the Franchimont Test. . . . 110

Spectrodensitometric Means of Detecting

Low Levels of 2 Ill

5. PERSONNEL AND ACTIVITIES 11

3

Personnel Listing 113

Publications 113

Manuscripts in Process of Publication .... 114

Lectures H°

6. ACKNOWLEDGMENTS H7

7. REFERENCES ll8

LIST OF FIGURES

FIGURE NO .

1. Determination of the purity of a volatile

compound by gas —liquid chromatography. ... 20

2. Recording an optical rotatory

dispersion spectrum 4l

3. P.m.r. spectra of 1 at 100 MHz; the signals of

the aromatic protons are not shown, (a)

Single resonance spectrum, (b ) theoretical

spectrum from iterative analysis, (a)

irradiation of H-l , and (d) irradiation of

H-2 and H-3 68

4. Adjustment of the strength of the magnetic

field, at the console of a nuclear magnetic

resonance spectrometer 70

5. Positioning a sample tube, prior to insertion

into the probe head of a nuclear magnetic

resonance spectrometer 71

ix
LIST OP FIGURES (CONT'D)

FIGURE NO . PAGE

6. Partial p.m.r. spectra of 4. (a) Observed

spectrum of solution in pyridine at 100 MHz,

and (b) computed spectrum after iterative

analysis as a seven-spin system 75

7. Partial p.m.r. spectra of 1. (a) Observed

spectrum at 220 MHz, and \b) computed

spectrum after Iterative analysis as a seven-spin system 79

8. Partial p.m.r. spectra of 1- N. (a)

Observed spectrum of solution in benzene-*^

at 220 MHz, and (b) computed spectrum after

iterative analyses of two- and six-spin

systems 83

9. P.m.r. spectra of 2. (a) Computed spectrum

after iterative analysis by magneticequivalence factoring, and (b) observed

spectrum of solution in 8:1 (v/v) acetone-^,-

benzene at 90 MHz 85

10. E.s.r. spectra of frozen (77 K) aqueous

alkaline solutions of Cu(II) complexes:

spectrum A, tartrate —oxalate (the

Somogyi reagent); B, tartrate (the Fehling

reagent); C, citrate (the Benedict reagent);

and D, tetraamine hydrate (cuprammonium, the

Reeves reagent). (All four low-field

e.s.r. spectra on the left side of the dotted

line were recorded at somewhat greater

field-modulation amplitude than the spectra

for the g ^2 region. ) 92

LIST OF TABLES

TABLE NO . PAGE

1. Dependence of absorption on pH, for

"azobilirubin" in methyl sulfoxide 13

2. Millimolar absorptivities of bilirubin and

"diazobilirubin" in human serum 17

3. Millimolar absorptivity of bilirubin

in human-serum albumin 18
LIST OP TABLES (CONT'D)

TABLE NO. PAGE

4. Isomerization of D-glucose during melting

to T .... 7 21

max

5. Effect of duration of heating at 0.625 K/min

on the apparent purity of D-glucose (clinical)

as measured by differential scanning

calorimetry 23

6. Percent of water in samples of D-glucose .... 24

7. Some physical properties of samples of

Cortisol 26

8. T.l.c. data on commercial samples of

Cortisol 29

9. Melting points of samples of VMA 31

10. T.l.c. data on commercial samples of VMA .... 33

11. Approximate percent of impurity in commercial

samples of VMA as indicated by mass

spectrometry 34

12. Re-estimation of purity of commercial samples

of VMA by mass spectrometry 35

13. Relative retention times and percentages of

components of samples of per ( trimethylsilyl )ated

VMA and related compounds 36

14. Analysis of purity of VMA by differential

scanning calorimetry 37

15. Thin-layer chromatography of D-mannitol .... 39

16. Specific rotations, at various wavelengths, of

solutions of commercial samples of D-mannitol

in acidified ammonium molybdate solution ... 4l

17. Ultraviolet absorption peaks of 3-NADH at

pH 10 43

18. Analysis of a commercial sample (lot no. 2331)

of 3-NAD . . .-. 46

XI
LIST OF TABLES (CONT'D)

TABLE NO. PAGE

19. Paper chromatography of samples of commercial

3-NAD. Detection by fluorescence-quenching

or KOH-spraying, or both 48

20. Paper chromatography of 3-NAD (lot no. 2331) • 50

21. Retention times of products obtained by

per ( trimethy lsilyl)ation of commercial

3-NAD (lot no. 2331) 51

22. Ultraviolet spectral data for 3-NAD (lot no.

233D 52

23. Enzymic assay of 3-NAD (lot no. 233D by

reduction with ethanol in the presence of

horse-liver alcohol dehydrogenase 54

24. Coupling constants (Hz) of 6-deoxy-l ,2 : 3 ,5-diC-isopropylidene-a-rj-glucofuranose

derivatives 8l

25. Chemical shifts (t, p. p.m.) of 6-deoxy-l, 2:

3 , 5 -di-O-isopropylidene-a-D-gluc ofuranose

derivatives 8l

26. Determination of inososes with the Somogyi

reagent 87

27. Magnetic parameters for the Cu(II) complexes

of the Somogyi (A), Fehling (B), Reeves (C),

and Benedict (D) reagents at 77 K 93

28. Reagents used to detect hydrazine after

generation from 2 on a t.l.c. plate 109

Xll
ORGANIC CHEMISTRY SECTION: SUMMARY OP ACTIVITIES

JULY 1969 TO JUNE 1970

Edited by Robert Schaffer

This report of the Organic Chemistry Section of the

National Bureau of Standard's Institute for Materials

Research provides a summary of a year's scientific activity;

as such, it covers both work that was completed and some

that is still in progress. The development of Standard

Reference Materials (SRMs) and their certification

constitute a significant output that we term the sample

aspect of our programs. The research component appears In

the form of journal publications.

About one-half of the report concerns SRMs.

Spectrophotometric measurements of bilirubin and of

"azobilirubin" in simple solvents and in serum or proteins

are given much attention. D-Glucose for use as a clinical

standard Is examined by gas---liquid chromatography and

differential scanning calorimetry to ascertain changes in

the proportions of the anomeric forms of the sugar during

these measurements. Additional compounds for development

as SRMs are Cortisol, 4-hydroxy-3-methoxy-DL-mandelic acid

(VMA), g-mannnitol, and the reduced form of 6-NAD. Some

properties thus far studied on commercially available

specimens are reported.

In carbohydrate research programs, the purity of

1, 2 : 4 , 5-di-C-Isopropylidene-B-fructose and the importance

of using It pure for conversion into pure g-psicose are

described. The proton magnetic parameters of the

unquestioned j skew conformation of 3-0-benzoyl-l, 2

,

H-0-

benzylidyne-a-g-ribopyranose are analyzed. These parameters

have major utility as a model for conformational analysis.

Iterative analysis of p.m.r. spectra and the conformations

of some g-glucose derivatives are described. Also, work is

reported on cyclic polyhydroxy compounds, including

1
oxidation, electron spin resonance, and new derivatives.

Finally, a number of other topics are described:

iodination of 3-diketones with periodic acid, and reactions

of bilirubin, hemin, and related bile pigments; also, some

work on the detection of polynitro aromatic compounds is

given.

Key words: "Azobilirubin" ; bilirubin; 3-NAD; 6-NADH;

cholesterol; Cortisol; g-mannitol; electron spin resonance;

proton magnetic resonance; skew conformations; Standard

Reference Materials; uric acid; VMA

.

1. CLINICAL STANDARDS

In 1967, the Section undertook to develop the first

of the Bureau's SRMs specifically directed to filling a need in laboratories doing clinical chemical analysis.

Cholesterol (SRM 911) was issued that year, and since then

the Section was responsible for the Urea (SRM 912), Uric

Acid (SRM 913), and Creatinine (SRM 91.4) that have been

issued. A few months ago, a tally of laboratories served

with these SRMs numbered in excess of 700; many were

outside the United States.

Two additional organic SRMs are about to be issued.

These are SRM Bilirubin and SRM g-Glucose. Considerable

effort was required to develop criteria for the Bilirubin

SRM because of the difficulty of working with a material

that is not stable at its melting point, nonvolatile,

poorly soluble, and unstable. At this writing, the SRM

itself is scheduled to be evaluated in July 1970, and

therefore, data on it are not included in this report. We

have been very much concerned with the methods by which the

SRM bilirubin can be dissolved, and combined with serum

proteins; for analytical use, it is required in that matrix
We report some of the results we have obtained by using

such procedures

.

Clinical laboratories perform D-glucose analyses by

many methods chemical as well as biochemical (i.e., with

enzymes). To ensure that the reference material is

compatible with all clinical techniques, the D-glucose used

for the clinical SRM has been more thoroughly characterized

than the Dextrose (SRM 4la) currently issued by NBS for the

chemical reducing methods used widely in industrial

carbohydrate technology.

Development of four new SRMs for clinical analysis

has begun. These are Cortisol, 4-Hydroxy-3-methoxy-gL- mandelic Acid (VMA) , g-Mannitol, and the reduced form of

3-Nicotinamide Adenine Dinucleotide (3-NADH).

Some data collected on two of the SRMs previously

issued, namely, Cholesterol and Uric acid, are reported.

We have been reexamining the former of these from time to

time since it was issued, in order to ascertain evidence

of any change in its purity. The latter had not been

reexamined by us since it was certified.

A. Cholesterol. Post-issuance Studies

In Technical Note 457 3 we reported analyses performed

on the cholesterol SRM six months after issuance under a provisional certificate, and, in Technical Note 507,

further studies at two additional six-month intervals. The

objective of this continuing study is the evaluation of

the shelf-life of the standard, particularly as it is known

that cholesterol may undergo decomposition. The material

has now been tested again.

1. Thin-layer Chromatography of Cholesterol Samples

(A. Cohen)

Chromatography was performed on activated Silica Gel G

plates with 1:1 (v/v) ethyl acetate —heptane as described

3
in Technical Note 457. Use of an atmosphere (in the

chamber) that was not saturated with vapors of the solvents

gave better resolution.

Samples of the bulk supply of cholesterol that had been

stored in a freezer for 30 months showed no significant

evidence of change, but samples that were sealed in amber

bottles kept at room temperature exposed to laboratory

fluorescent lighting for 22 months, and samples in amber

bottles that were loosely capped to ensure exposure of the

cholesterol to air for 22 months, were found to be

increasingly less pure, respectively, than the freezer-stored

supply. No attempt was made to assess these changes

quantitatively

.

2

.

Liebermann—Burchard Analysis

(A. Cohen)

Measurements made by our modification of the

Liebermann—Burchard method for cholesterol (see Technical

Note 457) showed no significant difference between samples

of the freezer-stored cholesterol and those left exposed

to air and light for nearly 2 years.

3

.

Differential Scanning Calorimetry (DSC) and Phase- solubility Analysis (PSA)

(R. F. Brady, Jr.)

DSC and PSA were applied to cholesterol samples as

described in Technical Note 457 [1]. The purity values

for the freezer-stored cholesterol were found to be 99-7

mole-percent by DSC, and 99.7 weight-percent by PSA.

Samples kept exposed to air in amber containers at room

temperature for six months were found to be 99.4 mole-percent

pure by DSC and 99.4 weight-percent pure by PSA.
B . Urea (Clinical). Post-issuance Studies

1. Determination of Purity of Urea by Differential

Scanning Calorimetry

(B. Coxon)

By use of ultra-pure indium (2.5620 mg), a calibration

factor of 0.02360 meal/unit area was determined at the time

of this study.

Portions of urea (0.9485 mg of sample 1, and 1.0840 mg of

sample 4) were heated in sealed sample-pans from 401 K to

406 K at 0.625 K/min. The thermograms were divided into

seven sections between ^402.5 K and 405-5 K, and the area

of each section was determined in duplicate by planimetry.

The values of purity obtained were 99.82 mole-percent

for sample 1, and 99-81 mole-percent for sample 4; these

results are essentially unchanged from those determined

on the same samples two years earlier.

2

.

Determination of Moisture Content of Urea by

Karl Fischer Titration

(J. H. Thomas)

Portions of urea (4.8388 g and 4.7266 g of sample 1 and

4.7196 g and 4.7252 g of sample 4) were dissolved in absolute

methanol, and titrated with Karl Fischer reagent to an end

point of 45 yA on a Waco titration apparatus. The Karl

Fischer apparatus was first calibrated with milligram

quantities of water in the range of the moisture content

of the urea sample. In this calibration, the weights of

water used divided by the corresponding volumes (V) of

Karl Fischer reagent required give a factor (F). The

expression

F x V x 100

weight of urea
gives the % of moisture in the sample. The percentages of

moisture in the two samples were 0.050 and 0.047; and 0.048

and 0.050, respectively.

3. Determination of Purity of Urea by Phase- solubility Analysis

(R. F. Brady, Jr.)

An analysis of the purity of a sample of urea was

performed by using phase-solubility analysis. The solvent

chosen was isopropyl alcohol, in which urea is soluble to

the extent of 29.0 mg/g of solvent. For details of the

technique, see reference [2]. The sample of urea was found

to be 99.9% pure; the accuracy of the technique is considered

to be ±0.1%. Within experimental error, this analysis is in

excellent agreement with the analysis of purity by

differential scanning calorimetry.

C . Bilirubin

(B. F. West and B. A. Johnson)

1. Standard Solutions of Bilirubin

Bilirubin in serum exists (a) as the free compound

adsorbed on serum albumin, as, otherwise, free bilirubin

would exhibit its high insolubility in aqueous media, and

(b) in a soluble, conjugated form, e.g., as the derivative

in which each of its carboxyl groups is esterified by OH-1

of g-glucuronic acid.

Although the development of standards for bilirubin

conjugates is not now feasible because of difficulties in

isolating and purifying such conjugates from natural

sources or, alternatively, difficulties in their synthesis,

bilirubin itself is available, and it is used for

standardizing clinical assays. By modification of the

conditions of coupling with diazotized sulfanilic acid,

clinical chemical procedures afford the means for obtaining a

6
separate estimate of (a) the proportion of the conjugated

bilirubin, and (b) the sum of the conjugated and free

bilirubin.

In 1962, a committee with representatives from the

American Academy of Pediatrics, the College of American

Pathologists, the American Association of Clinical Chemists,

and the National Institutes of Health recommended a procedure for preparing solutions of bilirubin in serum

for use in standardizing clinical assays [3]. In their

words, "an accurately weighed quantity of an 'acceptable

bilirubin' Is dissolved completely and as quickly as

possible (less than 5 rain) in subdued light at room

temperature in 0.1 M sodium carbonate solution, the

quantity of the latter being selected to constitute 2

percent of the final volume of the prepared standard.

[Occasionally, bilirubin preparations are encountered

which are difficult to dissolve in the carbonate D When

this occurs, 0.05 M sodium hydroxide may replace the 0.1 M

sodium carbonate, provided the resultant solution is used

within 5 min. ] (These two bracketed sentences appear as a footnote.) The clear red solution is immediately diluted

with an 'acceptable serum diluent' to a final volume

selected to give a desired final concentration of not less

than 5 mg/100 ml. An 'acceptable serum diluent,' to be

used both in the preparation of this standard and in any

subsequent dilutions for calibration purposes, is defined

tentatively as pooled serum having an absorbance of less

than 0.100 at 4l4 my and 0.040 at 460 my at a dilution of

1:25 in 0.85% NaCl . " The recommended procedure (and the

Committee's accompanying criteria for the bilirubin and the

serum to be used for standards preparation) proved to be a milestone in upgrading clinical analysis for bilirubin.

As a careful reading of these direction might suggest,

bilirubin is not readily dissolved within the recommended

7
conditions, nor is it easy in subdued light to see whether

all of a specimen has dissolved in the deep-red solution

that is produced. Furthermore, once dissolved in alkali,

bilirubin undergoes decomposition. The net effect is a

great likelihood of a less-than-optimal preparation of the

standard solution, particularly if the procedure is carried

out only occasionally or by an analyst unaware of the many

pitfalls

.

Some further salient facets needing attention were

added by Gambino [4]: "Several precautions must be

observed when applying the Committee recommendations on a uniform bilirubin standard. The standard should be made

with human serum or plasma that is less than 4 hours old and

that has been protected from light and heat. Many samples

of older, heated, or irradiated sera caused slight to severe

suppression of azobilirubin color when tested by the

Submitter in four variations of the Malloy—Evelyn method,

the Powell—O'Hagan method, and the Jendrassik—Grof method.

Michaelsson and Schellong also recommended fresh serum.

The standard must be protected from light. The pH of the

standard should be brought to 7.3 —7-5. When bilirubin

crystals are dissolved in sodium carbonate, there should

not be any brown tint, and, when the same crystals are

dissolved in chloroform, there should not be any brown tint.

If 5 vol. of chloroform—bilirubin solution are washed with

1 vol. of 5% (w/v) sodium bicarbonate, the bicarbonate wash

should be free of dark brown pigment."

When our work on developing a bilirubin standard

reference material led us to the preparation of bilirubinenriched sera, we recognized the desirability of finding

an alternative means for effecting dissolution of the

crystalline material one that would avoid the use of

aqueous alkali. We attempted to do this with methyl

sulfoxide (Me„SO) as solvent, and only later learned that
work with this solvent had already been published by Gadd

[5]. Billing, Halsam, and Wald [6] have further studied the

use of this solvent. In their work, proportions of Me ? SO

in the serum are utilized that far exceed the 2 percent

(v/v) restriction employed in the American Committee's

method; also, they used frozen, pooled serum (not fresh

serum) as the diluent. We describe some work along these

lines . A further modification of the dissolution procedure has

been described by Dybkaer and Hertz [7], who used a 0.1 M

solution of potassium cyanide In formamide to dissolve the

bilirubin. In it, bilirubin is stable for several hours,

and a remarkably high concentration of the pigment can be

obtained. The solution, which they denoted as B-CN-F

(bilirubin in potassium cyanide —formamide) , is readily

used to enrich fresh, pooled serum to as high as 25 mg/100 ml.

Most interestingly, on fractionation by size-chromatography

,

the enriched serum (made to pH 9-5 before adding the B-CN-F)

was found to have the bilirubin distributed only in the

albumin fraction, as found in hyperbilirubinemic serum.

Hence, their technique employs serum made alkaline to that

pH prior to enrichment, and the enriched preparation is

neutralized afterwards for storage. The authors reported

the utility of such enriched sera as standards for the assay,

by direct spectrophotometric measurement, of the free

bilirubin of hyperbilirubinemic sera.

Without having in hand the bilirubin that would be

certified as the Bureau's SRM, we undertook to examine

bilirubin samples by utilizing the clinical-chemical

techniques of bilirubin diazo-coupling, in the absence or

presence of protein, and bilirubin spectrophotometry. By

assuming that each bilirubin sample was pure, millimolar

absorptivities were calculated, to provide a basis for

comparison for each test and technique. As the bilirubin

content of the sample examined is not accounted for in the

9
calculation, these millimolar absorptivities have only

relativistic value.

a . Some Estimates of the Millimolar Absorptivities

of Bilirubin in Non-aqueous Solvents

Our customary procedure was to weigh (by microbalance)

3.5 to 7 mg of bilirubin on a pan, and then wash the sample

into a 0.5- to 1-liter volumetric flask by use of solvent.

Thus, dilution was not normally employed to bring the

solution to a concentration that would provide a reading of

about 0.7 absorbance unit on our Cary 14 spectrophotometer.

Dissolution in chloroform usually required brief warming at

^70°C in a water bath, and this was performed under subdued,

incandescent light. Volumes were adjusted at 20 °C, out

solutions were warmed to 25 °C prior to recording of their

spectra. Unless otherwise specified, the chloroform used

was reagent grade that contains 0.75% of ethanol.

i . In Chloroform

Two portions of a bilirubin sample were weighed. One

was heated in the dark at 117 °C/0.3 torr in an Abderhalden

apparatus for 10 h. Then the two samples were dissolved

in reagent chloroform. The heated sample showed e 61.7;

the unheated sample showed e „ 61.5. The similarity of

values showed that the heat-treatment had little effect on

the result; alternatively, the content of volatile material

in this sample seemed to be low, and heating did not appear

to "harm" the bilirubin.

Another sample of bilirubin was simultaneously examined

in hydrocarbon-stabilized chloroform and in (our usual)

ethanol-stabilized chloroform. The e .. values obtained were mM

61.0 and 60.6, respectively. The difference, although

small, appears to be significant; we have obtained other,

similar data.

10
In our extraction experiments, we had observed that,

after extraction with water, chloroform solutions of

bilirubin invariably show lower absorbances, and, to learn

whether this was related to the selective removal of the

ethanol from the chloroform, we measured the absorbance of

bilirubin dissolved in chloroform that had been freed of

ethanol by extracting it twice with water and then drying

(NapSCK) and distilling. In the ethanol-free chloroform,

we obtained an e ,, 58.7 On addition of ethanol to this mM

solution to a volume fraction of 0.002, e „ increased to

59-3 » and e „„ remained at 59.3 when the ethanol content of ^ ' mM

the solvent was raised to 0.4 and then to 1 percent. Hence,

the composition of the chloroform used in obtaining the

millimolar absorptivity of bilirubin must be carefully

specified. The X of these chloroform solutions was

max

453 nm.

ii. In Methyl Sulfoxide

Bilirubin dissolves very readily in this solvent. A

minute or so of immersion of the mixture in the water of

an ultrasonic-cleaning bath ensures completion. Because

of the costliness of the solvent, only 2 to 3 mg of bilirubin

was employed and the solutions were made to 250 ml. The

values of e „ are ^64, distinctly higher than found in mM ' ° ° chloroform; the X is like that found in chloroform. IllctX

iii. In Formamide Made 0.1 M in Potassium

Cyanide

This solvent is prepared by dissolving an appropriate

weight of the salt in formamide. Bilirubin solutions in

this solvent were made to ^7 mg/1 . As in methyl sulfoxide,

dissolution is facile and is ensured by a brief ultrasonic

treatment. The values of e .. in this solvent are ^64, and mM ' the X is 453, as in methyl sulfoxide and in chloroform,

max '-" J

11
2 . Millimolar Absorptivitles in Methyl Sulfoxide for

Bilirubin Diazo-coupled by an Evelyn —Malloy Procedure

The Evelyn—Malloy procedure for total bilirubin in

serum [8] was adapted to the examination of bilirubin

in methyl sulfoxide alone (i.e., without any serum or

protein present). We used a concentrated solution of

bilirubin in Me„SO and, at first, diluted this with methanol,

the diluent normally employed in the procedure with serum.

However, there was wide scatter in replicate tests, and this

seemed to be due to the turbidity that resulted from

admixture with the methanol. The latter was therefore

replaced by Me

?

SO as the diluent, and a great improvement

in precision resulted.

In this modification, ^10 mg of bilirubin (carefully

weighed) was dissolved in 25 ml of MepSO, and 1.0-ml

aliquots were diluted to 50 ml with Me

? S0. For testing,

aliquots (5 ml) of the latter solution were mixed with

equal volumes of the diazo reagent. Three replicates on two

dilutions of the same bilirubin—MepSO solution gave an

average e of 50.1 (range 49.3—50.7)-

Experiments were then conducted to determine whether

there might be an optimal pH for the reaction in MepSO.

The concentration of hydrochloric acid in the diazo

reagent was therefore varied, and it was found that an

increase in e resulted from use of a lower proportion

of hydrochloric acid. This change in e „ was accompanied

by a wavelength shift toward the red; the visible color

of the diazotized mixture was pink. As use of less than

0.1 ml of the concentrated acid, the condition giving the

highest value of e ... was deemed undesirable, further

investigations of effects of pH were not pursued. Hence,

the optimal pH was not ascertained. Data are given in

table 1.

12
Table 1. Dependence of absorption on pH , for "azobilirubin"

in methyl sulfoxide

Concentrated

hydrochloric

acid (ml) a mM

0.1 3-31 528 61.9

0.25 2.93 529 60.6

0.5 2.92 532 59.6

1.0 2.73 537 58.6

1.25 2.35 538 58.4

1.5 2.27 5^2 57.9

2.0 2.13 544 56.4

2.5 2.09 547 55-5

3.0 1.98 550 54.7

3.5 1.93 552 53-5

4.0 1.80 554 52.4

4.5 1.92 555 52.1

aPer 100 ml of sulfanilic acid solution.

When two different stock solutions of bilirubin in

Me„S0 were diluted with additional Me ? S0 to provide

concentrations from 14.5 to 3 ug/ml, and these were

tested with the diazo reagent of "optimal composition,"

a linear relationship was found for the absorbances

measured at 528 nm at the concentrations tested.

Prom time to time over a period of two months, two

commercial bilirubins were remeasured by this diazo

procedure, each test involving weighing, dissolution, and

diazo-coupling . One material showed, in 5 tests, an

average& e .. of 62.1. with results ranging from 61.2 to mM 3 & &

62.9; the other material showed, in 4 tests, an average

e ,. of 62.5 and a range of 61.8 to 62.9. mM &

13
A sample of a commercial, lyophilized, bilirubincontrol material (bilirubin on bovine albumin) was examined

by the Me„SO-modified Mall oy-- Evelyn procedure. The sample

was reconstituted according to the manufacturer's directions,

and then further diluted with Me

p

SO to a suitable

concentration. The A for the diazo-coupled product was the

max ^ ^ same as we had found for bilirubin diazo-coupled in the absence

of the protein (i.e., 528 nm); however, the e was found to

be 50.6 based on the concentration stated for the samples.

An £ lower than ^62 was to be expected, in keeping with

the well-known effect of protein on the millimolar

absorptivity of "azobilirubin .

3 . Millimolar Absorptivities for Bilirubin

Diazo-coupled by the Jendrassik— -Grof Procedure

In this method, the coupling reaction of bilirubin with

diazotized sulfanilic acid at pH ^5-5 produces a red

solution. Addition of alkali before the absorbance is

measured brings the pH to ^13.4 and changes the color to

green. Variations of the basic method that enhance its

usefulness have been reported in the literature.

As our Interest was the utilization of bilirubin in

preparing standard solutions for use by clinical laboratories

to calibrate their analyses, •> we determined values of e ,. for » mM

the bilirubin in these solutions, as well as for the

diazo product obtained.

The excellent, bilirubin-dissolving capacity of formamide

made 0.1 M in potassium cyanide was used by Dybkaer and

Hertz [7] for enriching serum pools prepared from freshly

collected blood. We attempted, at first, to perform the

diazo-coupling of the B-CN-F (see p. 9) solution in

the absence of added protein, by the Jendrassik—Grof

procedure as described by Gambino [4], with the aim, again,

of obtaining values of e M uncomplicated by the presence

14
of protein; however, this procedure did not seem to be

usable without protein present, and further effort along

this line was abandoned. In Its place, we worked with

B-CN-F and its enrichment of human sera.

A number of separate preparations of B-CN-F at a bilirubin concentration of ^7 mg/1 were scanned by

spectrophotometry, and were found, at A 453 nm, to give max

e M ^64. These absorption spectra were otherwise very

similar in appearance to that of solutions of bilirubin In

chloroform.

Dybkaer and Hertz specified that fresh, pooled, human

serum be used as the matrix for enrichments. As adequate

supplies of fresh, human serum are not always available,

it was of interest to us to examine the applicability of

frozen, pooled serum, especially as this was available

to us for use. A rather cloudy serum was used in the first

of our enrichments with B-CN-F. The serum had been made

alkaline (to pH ^9-5) > and was then enriched. We followed

Dybkaer and Hertz's use of tetraborate buffer as diluent

to afford direct spectrophotometric measurement of the

bilirubin, and found, with non-enriched serum (diluted with

buffer) as the blank, that e .. of the bilirubin corresponded

to ^50 for eight entirely separate enrichments. But, on

diazo-coupling, the e values obtained ranged widely, from

77 to 90, values that individually are much higher than

might have been expected. With use of the same serum (not

made basic beforehand) 5, the values of e ,. for "diazobilirubin" mM

were still in the 80's. We think that these very high

e „ values may be due as much to turbidity as to the use of

"old" serum.

Further work with B-CN-F enrichments was next conducted

on fresh serum that we obtained (by way of the Medical

Office at NBS) 4 —5 h after the blood had been drawn. After

adjustment of the pH to ^9-5j the serum was treated with

15
B-CN-F, to provide a concentration of bilirubin of ^20

mg/100 ml of serum; then, the serum was neutralized with an

amount of acid equivalent to the alkali initially used.

Aliquots of this serum were separately frozen at -28°C;

aliquots of non-enriched serum were frozen as well. Thawed

aliquots were diluted (1:10) with 0.85% saline for (a) direct

spectrophotometry , with tetraborate buffer used to effect a further 1:10 or 2:5 dilution, and water used as the

blank; and (b) diazo-coupling following Dybkaer and Hertz,

employing the Michaelsson modification of the Jendrassik—

•

Grof procedure.

Four different serum-pools were used. Pool 1 was

enriched with one preparation of B-CN-F, and aliquots

were studied over a period of several days. Pool 2 was

enriched with 3 different B-CN-F solutions; similarly,

pools 3 and 4 were each divided into three separate

portions, and each was enriched with a new B-CN-F

concentrate. Each enrichment with B-CN-F provided a concentration of bilirubin of ^20 mg/100 ml of serum.

The results obtained by study of each aliquot for

bilirubin and "diazobilirubin" millimolar absorptivities

are shown in table 2.

We were also interested in knowing how purified, humanserum albumin might behave as a matrix for enrichment with

B-CN-F. For this purpose, commercial, lyophilized,

human-serum albumin was dissolved in 0.85% (w/v) saline,

to provide a 2.5% (w/v) solution of the protein. Portions

of this solution were enriched with B-CN-F, eibher (a) with

no special pretreatment , (b) after being brought to pH 9*5

and then neutralized, or (c) enriched as in (b), but not

subsequently neutralized. However, it was found that,

independent of their mode of preparation, aliquots obtained

from these enrichments were very unstable. As an alternative,

the albumin solution was made up in a phosphate buffer at

16
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pH 7-4 (following a suggestion by Dr. A. Hainline) , and

enriched as in (a) utilizing B-CN-F and, for comparison,

with solutions of bilirubin in 0.05 M sodium hydroxide.

Table 3 provides an overall summary of the results

from these enrichments. The values of e .. given for each

mAf to enrichment are averages obtained from the denoted number of

replicates by each procedure. Enrichments 1 —5 with B-CN-F

provided values of e ,. that were much less reproducible than mM ^ those of 6---10 (by sodium hydroxide) . The ranges of e do not reveal the distinct trend to diminish with time that

was found in the B-CN-F enrichments.

Table 3. Millimolar absorptivity of bilirubin in human-serum

albumin

Enrichmen t Base Time

studie

(days

;d

)

Nc

re

at

. of

plices

e mA

Direct

Av. Range

r Diazo

Av. Range

1 A 11 8 55.4 1.2 71.2 1.2

2 A 10 6 50.9 1.7 68.4 2.0

3 A 6 10 53.4 1.3

4 A 6 6 52.4 0.9 66.9 2.1

5 A 12 8 52.2 1.8 67.3 2.1

6 B 8 10 55.5 0.4

7 B 12 8 55.6 0.9 70.6 4.2

8 B 8 8 55.2 1.2 68.9 1.0

9 B 8 4 57.9 1.4 68.6 2.0

10 B 8

, 0.05

nm.

M

4

sodium hyd

55.4

roxide

0.8

*. b

At

67.8

A max

1.2

a

A, 0.

c nm.

1 M

'At

KCN-F; B

A 600

max

^467

18
D . g- Glucose (Clinical)

Analyses for g-glucose in blood constitute an important

diagnostic test, and thus, in the near future, NBS will

issue standard samples of B-glucose for clinical use.

Dextrose, issued by NBS as Standard Reference Material 4la,

is the standard for chemical reducing methods of sugar

analysis. The clinical standard, for which more extensive

investigations of the physical properties of the D-glucose,

its quantitative purity, and impurities have been made, is

intended for enzymic and other clinical methods. Preliminary

analyses were described in a previous report [9]-

1 . Analysis of g-Glucose by Gas —liquid Chromatography

(B. Coxon and J. H. Thomas)

a. Crystalline Material

In order that a minimum proportion of the a-p_-glucose

would be anomerized to 3-D-glucose, portions (5 nig) of

samples 2B and 4B were per ( trimethylsilyl)ated by use of

1 ml of Tri Sil Z at 0°C for 10 min. The anomers were

separated on a glass column (6 ft. x 0.25 in. O.D.); the

solid support was Chromosorb WAWS (80 —100 mesh) with a liquid phase of 3% of SE-30. The column was operated

isothermally at 180 °C, and the separated components were

detected with a flame-ionization detector operated at 220 °C.

The sample size was 1 yl.

The areas of the peaks due to a and 3 anomers were

obtained by triangulation. The percentages of a and 3

anomers were then calculated from the total areas, with the

assumption that anomeric trimethylsilyl derivatives have the

same response factor.

The percentages of 3-D-glucose found, for four

determinations on each sample, were: for 2B, 0.43; 0.40;

0.42; and 0.50; for 4B, 0.53; 0.60; 0.52; and 0.53.

19
Figure 1. Determination of the purity of a volatile

compound by gas —liquid chromatography.

b . Melted Samples of g- Glucose

In order to assess the significance of the determination

of the purity of B-glucose by differential scanning

calorimetry, samples of g-glucose were heated under nitrogen

in the calorimeter and were analyzed by g.l.c. of the

trimethylsilyl ethers at various stages of the melting

process .

20
Samples of g-glucose 03 mg) in open aluminum pans were

flushed six times with nitrogen, and were heated from 380 K

to 4l4 K at 80 K/min. Heating of each sample was then

continued; one sample was heated to a maximum temperature

(T ) of 4l8 K, corresponding to a small amount of

max

premelting, a second sample was heated to 420 K 3 corresponding to the maximum rate of heat input on the DSC

curve (melting ^60% complete) , and a third sample was melted

completely (T 421 K)

max

At T , the aluminum sample-pan was rapidly removed

max

from the calorimeter, and was allowed to cool to room

temperature. The melted g-glucose was then trimethylsilylated

by stirring the sample pan and contents with Tri Sil Z (1 ml)

at 0°C for 1 h. The resulting solutions were then analyzed

by g.l.c. as described in 1 (a). The results are shown in

table 4.

Table 4. Isomerization of D-glucose during melting to T = to & & max

T

max

(K)

a anoraer 3 anomer

(%)

29 8

b

99.5 0.5

418 97.8 2.2

420 55.2 43.5

421 45.3 52.3

Other components

1.3

2.4

Probably furanoid or acyclic forms.

Unmelted.

21
The fact that isomerization reactions proceed during the

differential scanning calorimetry of g-glucose shows that

the results for purity obtained by this technique should be

treated with some caution. However, in spite of this,

surprisingly good agreement was obtained between the

proportions of a-D-glucose measured by DSC and by g.l.c.

It is easily shown that heats of isomerization of the

order of 278— -500 cal/mole* have little effect on the mole-%

purity measured, as these heats of Isomerization are small

compared with the heat of fusion of D-glucose (see table 5).

2. Apparent Purity of g- Glucose by Differential

Scanning Calorimetry

(B. Coxon and R. F. Brady, Jr.)

By slight variations in procedure with DSC, different

values can be obtained for the mole-percent purity of the

D-glucose

.

a . Initial Procedure

Calibration of a Perkin—Elmer Model DSC-1B instrument

against ultrapure indium (2.562 mg) gave a calibration

factor of 0.02658 meal/unit area of recording chart.

Sample weights were determined on a Cahn electrobalance

which was calibrated against a standard 5-mg weight before

each weighing. Portions of g-glucose (2.8895 mg of sample

4B, and 3.3720 mg of sample 2B) in open sample-pans were

evacuated and flushed with nitrogen six times and were

heated from 380 K to 4l4 K at 80 K/min, and then from 4l4 K

^Literature values [10] of 278 and 299 cal/mole for a-g-glucose

in aqueous solution at 25 and 35 °C, respectively. If it

is assumed that the heat of isomerization is proportional

to temperature, a value of 536 cal/mole can be estimated

for 421 K (the mp of a-D-glucose)

.

22
at 0.625 K/min. The segment of each DSC thermogram between

4l8 and 424 K was analyzed by planimetry, duplicate

measurements being made on each of eight sectors under the

curve to assure reproducibility. Purity values thus

obtained were 98. 61 and 98.58 mole-percent with portions

of g-glucose samples 4B and 2B,* respectively. However,

the presence of a proportion of 3 anomer (see section 1) and

the moisture content must be borne in mind.

b . Effect of Duration of Heating on the Apparent

Purity

The calculated purity of D_-glucose was found to be

dependent on the duration of the slower heating prior

to melting. Portions of 2-glucose were heated in open

sample-pans under nitrogen from 298 K to temperature T at

80 K/min, and then from T at 0.625 K/min up to the melting

point (T ) . Higher calculated values of mole-percent

purity were obtained when the duration of heating before

melting was lessened, presumably because of decrease in the

extent of anomerization or dehydration (or both) that would

lower the purity of the sample. The results are summarized

in table 5

.

Table 5. Effect of duration of heating at 0.625 K/min on

the apparent purity of g-glucose (clinical) as

measured by differential scanning calorimetry

T

(K)

T

m

(K)

Duration of

slow heating M

p Purity

(min)

( cal/mol) (J/mol) (mole-#)

411 420.7 15.5 8280 34,700 98.75

414 422. 7 13.9 7860 32,900 99.03

417 422.5 8.8 8300 34,700 99.40

417 422.0 8.0 8210 34,400 99.41

418 422. 7 7.5 8100 33,900 99.47

420 423. 8 6.1 8100 33,900 99.43

*Determined on material as received from the manufacturer

23
3 . Determination of Moisture Content of D-Glucose

Samples

(J. H. Thomas)

Karl Fischer (K.F.) titrations of samples 4B and 2B

of p_-glucose were made by the standard method, except that

Karl Fischer diluent was used instead of methanol. The

moisture content was then cross-checked by another direct

method, namely, the near-infrared (NIR) spectrophotometric

method that is specific for water [11], for which, in the

present analysis, a cell having a 5-cm path-length was used,

because of the low content of water. The percentages of

water found are given in table 6

.

Table 6. Percent of water in samples of D-glucose

Sample number

4B

2B

Water (%)

by K.F. titration

0.055

0.055

0.051

0.056

0.052

0.053

by NIR

0.057

0.055

0.060

0.055

Cortisol

(R. F. Brady, Jr.)

Cortisol (1) is one of several hormones secreted by the

CH 2 OH

C =o

OH

1

24
adrenal cortex. It is essential to the normal functioning

of the human body, and many pathological conditions result

from abnormalities in the amount of Cortisol produced. Such

malfunctions as Addison's disease are engendered by Cortisol

deficiency, whereas excessive amounts of this hormone may

result in such equally serious disorders as Cushing's syndrome

The evaluation of adrenocortical activity is a critical

step in the diagnosis of these diseases, but, because of

the variety of adrenocorticosteroids and their various

reduction and degradation products, many difficulties are

encountered in the determination of these compounds in

plasma and urine.

A pure, crystalline Cortisol is being developed as a Standard Reference Material for use as a clinical standard

for assaying Cortisol In body fluids.

1 . Physical Properties of Some Cortisol Samples

a . Melting Point

Melting-point determinations in open Pyrex capillary

tubes were made on four commercial samples of Cortisol.

Each tube was immersed in a bath preheated to 200 °C, and was

then heated at a rate of 0.5 °C/min until melting was

complete. Their melting ranges are recorded in table 7-

The melting point of Cortisol is given in the literature

[12] as "217—-220° with some decomposition."

b . Optical Rotation

Approximately 60 mg of Cortisol from each of the

commercial sources was accurately weighed (to 10 yg) into

a 10.00-ml volumetric flask, and dissolved in sufficient

absolute ethanol to make 10.00 ml of solution. The samples

appeared to differ In ease of dissolution. The optical

rotation at 589 nm of each solution in a 1-dm tube was

measured by using a Perkin—Elmer Model l4l photoelectric

25
polarimeter, and the rotations are given in table 7. The

optical rotation of Cortisol is given in the literature

| 2; 'D c . Elemental Analysis

[12] as [a] 22 +167° (c 1, absolute ethanol)

Manufacturer'1 s m p

lot number (°C)

52580 219-222

58B-2240 212-216

2167 216-220

U4174 220-224

a^- -1 b

Results of elemental analyses of the four commercial

samples of Cortisol are given in table 7. The values

calculated for C H are: C, 69.6; H 3 8.3$.

Table 7- Some physical properties of samples of Cortisol

22 [a]~ Analysis T

D m

(degrees) %C %R (°C)

+167.

l

a 69.5 8.4 221

+166.

8

b 69.6 8.3 211

+l66.4 a 69.8 8.4 223

+164. 9° 69.3 8.3 218

"Dissolved rapidly. "Dissolved slowly. Dissolved very

slowly

.

2 . Determination of Purity by Differential Scanning

Calorimetry

Each sample of Cortisol was examined by using a Perkin

Elmer Model DSC-1B Differential Scanning Calorimeter. A

weighed quantity of Cortisol in an open aluminum dish was

placed in the sample cell of the calorimeter, and the dish

was covered with a hemispherical aluminum dome. A

similarly covered, empty aluminum dish was placed in the

reference cell. With the calorimeter cover locked in place,

the sample chamber was flushed with a gentle stream of

dry nitrogen for 3 min in order to remove oxygen. The

calorimeter temperature was then raised from 298 K to

about 5 K below the melting point, at a rate of 80 K/min,

26
and then at a rate of 0.625 K/min, until melting was complete

The procedure of operating under a nitrogen atmosphere was

used with the Cortisol because the decomposition that attends

the melting of cholesterol while it is exposed to air had

thereby been prevented, and by employing this technique,

satisfactory DSC analyses of the purity of cholesterol had,

in fact, been obtained [1]. However, with Cortisol,

"decomposition" apparently continues to take place during

melting, even under the foregoing conditions. Thus,

the endotherms obtained continued to show extremely broad

"melting" ranges and low peak-amplitudes; also, the

temperature of maximum peak-amplitude (T ) was different

for each sample. Hence, differential scanning calorimetry

is not applicable for determining the purity of Cortisol

because of the instability of this compound on heating.

3 . Spectrometric Properties

a. Infrared Spectrum

Commercial samples of Cortisol in a potassium bromide

pellet show a well-defined i.r. spectrum having major

bands at 3440 (OH stretch), 3015, 2980, 2950, 2925, 2895

(all CH stretches), 1720 (C-20 carbonyl stretch), 1645

(C-3 carbonyl stretch), 1610 (C=C stretch), 1455, 1435,

1420, 1395, 1350, 1325, 1290, 1280, 1240, 1210, 1140, 1125

1105, 1070, 1060, 1040, 1015, 950, 930, 910, 875, 790, 755,

and 710 cm

-

. Infrared spectra were recorded with a Perkin—Elmer

Model 257 grating infrared spectrophotometer. The spectra

of the four commercial samples were identical with the

published spectrum [13]; no impurities were evident from

inspection of any of these spectra.

27
b . Nuclear Magnetic Resonance Spectrum

Each sample of Cortisol (^60 mg) was dissolved in 0.4

ml of methyl sulfoxide-dv- containing 2% of tetramethylsilane

as the internal standard. Spectra were obtained by using

a Varian A-60 spectrometer operating at 37 °C. The n.m.r.

spectrum of Cortisol contains a "methylene envelope" from

60.6 to 2.8, upon which are superimposed two singlets

for the protons on C-18 (60.66) and C-19 (61.40). A

singlet at 63-38 may be assigned to the proton of the

hydroxyl group on C-17, and a broad peak (having some

fine structure) at 54.1—5.0 may be assigned to the

protons of the two other hydroxyl groups, on C-ll and C-21.

Two additional singlets, at 65.12 and 5.57, may be assigned

to the protons on C-21 and C-4, respectively. The spectra

of the four commercial samples were superposable , and none

showed any evidence of impurity.

4 . Thin-layer Chromatography of Cortisol

Thin-layer chromatography was performed with Silica Gel

G, activated at 110 °C, as the adsorbent, coated on glass

plates (10 x 20 cm); the depth of the adsorbent layer was

250 ym. Chloroform—methanol (97:3, v/v) , hexane —ethyl

acetate (1:1, v/v), and benzene—isopropyl alcohol (4:1,

v/v) were the developing solvents . Samples in acetone

(2 yg/yl) were applied in small spots by using a Hamilton

syringe. After development, plates were examined under

ultraviolet light, and in no instance was any zone made

visible. Plates were then sprayed with methanol —concentrated

sulfuric acid (5:1, v/v) and heated at 110 °C for 10 —15

min to develop the spots. The results are summarized in

table 8.

28
Table 8. T.l.c . data on commercial sampl es of cort

Manufacturer '

s

lot number

Developing

solvent

R

F

Id entificati

52580 A 0.09

0.19

Cortisol

cortisone

58B-2240 0.09 Cortisol

2167 0.03

0.09

0.19

unknown

Cortisol

cortisone

U4174 0.09

0.20

Cortisol

cortisone

52580 B 0.08

0.11

Cortisol

cortisone

58B-2240 0.08 Cortisol

2167 0.08 Cortisol

U4174 0.08 Cortisol

52580 C 0.59 Cortisol

58B-2240 0.59 Cortisol

2167 0.59 Cortisol

U4174 0.59 Cortisol

A, Chloroform—methanol (97:3 v/v) ; B, hexane —ethyl acetate

(1:1, v/v); C, benzene —isopropyl alcohol (4:1, v/v).

The identification of one of the minor spots as cortisone

(2) was made by comparison with authentic cortisone in the

OH

2

29
three developing solvents employed. The R values were: in

b chloroform—methanol (97:3, v/v) , 0.19; in hexane —ethyl

acetate (1:1, v/v), 0.11; and in benzene —isopropyl alcohol

(4:1, v/v), 0.59-

F. 4-Hydroxy-3-methoxy -g^-mandelic Acid (VMA)

( R. S. Tipson , A. J. Patiadi, and R. F. Brady, Jr.)

4-Hydroxy-3-methoxy-2-mandelic acid (1) (vanil-,

COOH

I HCOH ^

OCH.

OH

vanillyl-, or vanillin-mandelic acid, VMA) is a constituent

of normal and pathological human urine, as it is the major

urinary metabolite of epinephrine and norepinephrine.

Consequently, it is important that a well-characterized VMA

reference standard be made available for use in clinical

laboratories. For the assays used by clinical chemists, the

g^ form gives the same responses as the D form, and so we

have studied samples of the racemate.

1 . Melting Point of Commercial Samples of VMA

(A. J. Fatiadi)

Melting-point determinations were made, on samples

[seven commercial, and one that we had purified by

successive recrystallization from 1:1 (v/v) benzene

30
.acetonitrile and then from acetonitrile] of giL-VMA, in Pyrex

capillary tubes with the contents "exposed to the air. These

tubes were placed in a preheated bath (at 125 °C) and heated

at a rate of 1 °C/min to the temperature of melting (or

decomposition) . The melting range was read from a mercury

thermometer and is reported, without correction, in table 9.

Table 9- Melting points a of samples of VMA

b Sample lot no. Melting point

(°C)

recrystallized 134-135

86085 133-13^

none 132-134

100-0220 131.5-132.5

5113 131-132

V-1451 130-132

1449 130-131

900796 129-131

Literature [14] mp , 134 —135 °C (with decomposition).

With decomposition; uncorrected.

2 . Thin-layer Chromatography of Commercial Samples

of VMA

(A. J. Fatiadi)

Thin-layer chromatography was performed on layers

(^250 ym thick) of adsorbent coated on glass plates

(5 x 20 or 10 x 20 cm). The adsorbents tested were

(1) cellulose powder activated at 105 °C for 10 min, and

(2) Silica Gel G, not activated. The developing solvents

employed with adsorbent 1 were: butyl alcohol saturated

with 3 M hydrochloric acid (solvent A), 20:20:1 (v/v) amyl

alcohol—formic acid—water (solvent B) , and 13:7 (v/v)

propyl alcohol—water (solvent C). For adsorbent 2, the

solvent used was 4:1:1 (v/v) chloroform—acetic acid—water

31
(solvent D) . For detection of spots with adsorbents 1 or

2, a spray of diazonium solution was used; this consisted

of a 1:1:2 (v/v) mixture of (a) a solution of 0.1 g of

p-nitroaniline in 2 ml of 3 M hydrochloric acid, (b) a

solution of 0.2 g of sodium nitrite in 100 ml of water,

and (c) a solution of 10 g of potassium carbonate in 100 ml

of water. The plate was developed, sprayed, and dried at

60 °C for 30 min; fluorescent spots were located under u.v.

light (254 nm). This spray detected ^2.5 Pg of VMA

.

For plates of adsorbent 2, a spray consisting of 3 M

sulfuric acid in methanol could also be used. After the

plate had been sprayed, it was heated at 110 °C for 3 min,

giving nonf luorescent , dark-brown spots that changed to

purple. By use of this spray, ^4 ug of VMA could be detected

The following approximate R values were observed for

t VMA: adsorbent 1, 0.90 (solvent A), 0.71 (B) , 0.61 (C);

adsorbent 2, . 80 (A) and 0.23 (D) . The results obtained

for commercial samples of VMA examined with adsorbent 1,

solvent A, and the diazonium spray are given in table 10.

3 . Spectrometric Properties

(A. J. Fatiadi)

a. Infrared Spectrum

Infrared spectra were recorded with a Perkin—Elmer

Model 257 grating infrared spectrophotometer. The purified

sample of VMA (in a potassium bromide pellet) gave a well-defined i.r. spectrum having major bands at 3350 (OH),

2980 and 2940 (C—H stretch), 1870, 1748 and 1715 (C=0

stretch), 1610, 1520, 1440, 1385, 1330, 1275 and 1240

(=C-0-C, aromatic ether), 1200, 1160, 1140, 1070, 1040,

870, 835, 785, 740, and 710 cm

-1

(benzene ring vibration).

The spectra of the seven commercial samples were superposable

b

.

Nuclear Magnetic Resonance Spectra

A sample (^70 mg) of purified VMA was dissolved in

32
Table 10. T.l.c. data on commercial samples of VMA

Manufacturer's r _ _ T , .... L . a

lot number F values Identification

86085 0.90 VMA

0.04 (trace) U

None 0.92 VMA

0.31 U

100-0220 0.89 VMA

0.3^ / 0.24 U

0.06 ) 5113 0.91 VMA

0.62 ) 0.28 (u

0.03 1 900796 0.91 VMA

0.62 ) 0.35 U

0.02 > V-1451 0.88 VMA

0. 32 ) 0.18 |u

0.12 J 1449 0.90 VMA

0.59 ) 0.32 U

0.05 ) U = unidentified.

0.5 ml of methyl sulfoxide-d,- containing 2% of

tetramethylsilane as the internal standard. Spectra were

obtained by using a Varian A-60 spectrometer operating at

32 —35 °C. The n.m.r. spectrum showed signals for a singlet

at 6 3-70 (-0CH~ group); a singlet at 6 4.95 (presumably

x -J due to -C-H, not exchangeable with D

p 0) ; a multiplet

centered at <5 6.8 (aromatic protons), and a broad peak at

6 8.65 that, on treatment with a drop of pyridine-d changed

to a sharp singlet at 6 8.73. The spectrum does not show

clearly the phenolic hydroxyl proton, and the chemical shifts

of other protons in the spectra depend very much on the

nature of the solvent used. 00
c . Mass-spectrometric Examination of Commercial

Samples of VMA

(A. J. Patiadi)

Mass spectra of seven commercial samples of VMA were

analyzed for purity at a sample temperature of ^140 °C with

a Bell and Howell Consolidated Double-Focusing Mass

Spectrometer Model 21-491. The spectra were measured, and

were first examined for gross differences

.

A calculation of the approximate purity was made by

summing the ion current obviously arising from compounds

other than the VMA. Table 11 lists the samples in order

of decreasing purity. The numerical value is a rough

approximation of the impurity relative to the content of the

major component.

Table 11. Approximate percent of impurity in commercial

samples of VMA as indicated by mass spectrometry

Sample lot no. Total impurity, %

none ^0.01

100-0220 ^0.01

5113 ^0.01

86085 ^0.02

900796 ^0.08

V-1451 ^0.20

1449 % 0.25

On the basis of these results, the last 3 samples were

not examined further. More-exact calculations of the

contribution of impurities to the spectra of the first

4 samples and a recalculation of the order of purity were

made. The numerical values shown in table 12 are the sums

of ion currents due to readily identified ions not probable

in the spectrum of VMA. The values may be compared to a

value of unity for the molecule ion current of VMA;

34
consequently, the recalculation showed the purity, in

decreasing order, as given in table 12.

Table 12. Re-estimation of purity of commercial samples

of VMA by mass spectrometry

Sample lot no. Total impurity, %

86085 0.028

none 0.028

100-0220 Q.O34

5113 0.059

In addition, two commercial samples of VMA were heated

for 4 h at 110 or 14.0 °C, and their mass spectra were

compared with that of unheated material. The spectra of

the first two samples, heated at 140 °C, were found to contain

a considerable proportion of a compound tentatively identified

as vanillin. The proportion of the molecule ion of vanillin

(from an authentic sample) was found to be 6 percent.

The spectrum of sample 100-0220 (heated at 100 °C/1 torr

for 4 h) was compared with that of unheated sample. Again,

a large proportion of vanillin was found. The proportion

of the molecule ion peak of VMA to the molecule ion peak of

vanillin was found to be 25%.

In the spectra of both mixtures, in addition to vanillin,

small proportions of heavier ions were found in the vicinity

of mass 300. The proportion of the mixture represented by

these heavier ions (occurrence of decarboxylation of VMA at

these temperatures is obvious) is probably less than 1% of

the total. The mass spectra were measured by E. E. Hughes

of the Microchemical Analysis Section, most of whose comments

are provided.

35
Table 13,

Sample

lot no.

Relative retention times and percentages of

components of samples of per ( trimethylsilyl) ated

VMA and related compounds

Major component Impurity

Approximate Approximate Approximate Approximate

retention ^ retention * time (min) time (min)

VMA

re cry stallized

(benzene)

900796

86085

100-0220

None

5113

V-1451

1449

Vanillin

Vanillic acid

16.6

16.9

16.8

17.1

17.3

17.5

17.5

17.3

7.0

10.0

99.7

99.3

99.2

99.1

99.0

99.0

98.8

98.5

98.0

99.0

10.0

23.2

6.6

10.4

23.9

32.0

6.4

9.7

23.1

6.7

8.1

10.5

24.5

6.8

10.6

32.0

7.0

10.0

11.2

24.0

7.1

9.8

24.2

33.0

6.8

10.8

23.6

33.6

3.4

6.7

0.1 ( A)

0.2 ( B)

0.2 ( c)

0.1 ( A)

0.3 ( 'B)

0.2 ( ;b)

0.2 ( 'c)

0.1 ( ;a)

0.3 < 'B)

0.2 ( ;c)

0.2 ( :a)

0.2 ( ;b)

0.3 < ;b)

0.3 :c)

0.3 [A)

0.3 :b)

0.4 [c)

0.1 [A)

0.1 [B)

0.3 ;b)

0.3 :c)

0.3 [A)

0.4 ;b)

0.1 :b)

0.4 [c)

0.2 [A)

0.4 'B)

0.3 'B)

0.5 'D)

0.3 [c)

a A, vanillic acid; B, unknown; C, vanillin; and D, isovanillin

36
4

. Examination by Gas— liquid Chromatography (G.l.c.)

of the Per (trimethylsilyl) Derivatives of Samples

of VMA

(A. J. Fatiadi)

Samples of VMA (seven commercial and one that we had

purified) were individually examined by

per( trimethylsilyl)ation, followed by g.l.c. Separation

was achieved on a glass column (6 ft. x 2 mm i.d.) of

Chromosorb Q (60 —80 mesh) with a liquid phase of 3% of

OV-17. The column was operated isothermally at 150 °C,

and the separated components were detected with a flameionization detector operated at 200 °C; the temperature

at the injection port was 180 °C. The sample size was ^10 \i\ The samples were per ( trimethylsilyl)ated by use of

Tri Sil Z. Vanillin and vanillic acid were used as

reference compounds. The results are summarized in table 13

5

.

Determination of Purity of VMA by Differential

Scanning Calorimetry

(R. P. Brady, Jr.)

Seven commercial samples of VMA were analyzed for

purity by differential scanning calorimetry. The technique

employed was similar to that described earlier [1] for the

analysis of cholesterol by DSC. The results are summarized

in table 14.

Table 14. Analysis of purity of VMA by

differential scanning calorimetry

A#„

Purity (mol %)

99.2

99.1

99.0

98.9

98.6

97.7

Manufact urer s m p F

lot numb er (K) cal/mol J/mol

86085 399.1 8270 34,620

None 399.2 8180 34,240

5H3 399.7 8000 33,490

100-0220 400.2 8470 35,460

V-1451 398.6 8270 34,620

1449 399.8 7605 31,830

900798 a No distinct melting point

37
G. Q-Mannitol (Clinical)

(B. Coxon and J. H. Thomas)

Fats, which occur as part of the lipid content of

serum, are composed of a variety of fatty acid residues

that are esterified to the glycerol moiety common to

these compounds. Saponification liberates the glycerol, and

it can be assayed by periodate cleavage of its vicinal

carbon atoms, with the liberation of formaldehyde. The

latter is estimated from the color produced by reaction

with chromotropic acid. Only the primary carbon atoms

are converted into formaldehyde; the secondary carbon

atom provides formic acid, and chromotropic acid is

unaffected by this acid. As a standard for fats by use of

this test, glycerol would be very unsatisfactory. On the

other hand, 0-mannitol is a non-hygroscopic solid that

can be purified to a high degree and can serve in place

of the glycerol, because it affords the same number of

formaldehyde molecules per molecule of it oxidized. Its

higher molecular weight is advantageous for use as the

standard

.

Samples of D-mannitol were obtained from four different

suppliers and are designated as samples 1, 2, 3 a and 4.

1 . Thin-layer Chromatography (T.l.c.) of g-Mannitol

T.l.c. was conducted on 20-cm glass plates coated with

Silica Gel G. The samples were applied to the plates as

0.5- or 1-ul aliquots of 10$ solutions in water,

corresponding to applied amounts of 0.05 and 0.1 mg,

respectively. It was found that separation of g-mannitol

from g-glucitol* is difficult to achieve, and so the use

*D-Glucitol is a likely impurity in preparations of D-mannitol

that have been produced commercially by electrolytic

reduction of D-fructose.

38
of plates of Silica Gel G impregnated with various inorganic

complexing reagents was investigated. In these studies,

the unactivated layer of silica gel was sprayed with the

solution of complexing reagent and then activated at 125 °C

for 10 min, before application of the sample, except that,

for acidified ammonium molybdate, ammonium molybdate

solution was sprayed on the plate, which was then activated,

cooled, and sprayed with 5% sulfuric acid. After air-drying

of the plate, the samples were applied, and t.l.c. was

performed immediately. The results are given in table 15-

Although small differences (<0.1) of the i?„ values

h

of D-mannitol and D-glucitol were noted, these compounds

have not been separated completely by any of the systems

thus far investigated. However, several of the methods

afforded separations of impurities in the D-glucitol sample.

Table 15. Thin-layer chromatography of D-mannitol

Impregnation Solvent R value

reagent

None A 0.4

Saturated boric A 0.3

acid solution B 0.3

Saturated

sodium tetraborate

solution C 0.1

None C 0.5

Ammonium

molybdate

sulfuric acid C 0.3

A, 4:1 (v/v) methanol—chloroform; B, methanol; C, 4:1 (v/v)

methanol —water.

2 . Paper Chromatography of D-Mannitol

Aliquots (5 yl) of 10$ solutions of samples 1 —4 of

0-mannitol (and of g-glucitol) in water were applied to

Whatman filter paper No. 1, and paper chromatography was

39
performed by the descending method with 8:2:1 (v/v) ethyl

acetate—pyridine —water (0.5 mg of each alditol). The

chromatograms were sprayed with sodium metaperiodate

solution (20 mM) , followed after 2 min by silver nitrate

solution (5%)- All of the samples of g-mannitol (Man) showed

a trace of impurity having i? M 0.14. Under these conditions,

D-glucitol has R.„ 1.12, but this value is not sufficiently = b Man ' J different from that of g-mannitol (1.0) to permit the

detection of small amounts of D-glucitol in large applications

of 0-mannitol. The sample of D-glucitol was found to contain

significant proportions of two impurities having Z? M 0.22

and 0.03.

3 . Optical Rotation of Samples of D-Mannitol

Ammonium molybdate solution was prepared by dilution

3 of 50.000 g of the crystalline tetrahydrate to 1 dm with

distilled water. Each sample of g-mannitol (^0.08 or ^0.04 g,

accurately weighed) was dissolved in an aliquot (20.0 ml)

of ammonium molybdate solution. The resulting solution

was then diluted to 25.0 ml with 0.5 M sulfuric acid.

Measurements were made either at 20 °C, by use of a

thermostated, 1-dm cell in a Perkin—Elmer Model l4l

polarimeter, or at ^24 °C in a non-thermostated, 0.1-dm

cell in a Cary Model 60 spectropolarimeter . With either

of these instruments, measurements at wavelengths below

370 nm were not possible, owing to the very strong

absorption of the acidified ammonium molybdate solution.

Indeed, an ultraviolet absorption spectrum (recorded by

A. Cohen) of a solution prepared by 1,000-fold dilution of

acidified, stock, ammonium molybdate solution with water

showed an absorption maximum at 214 nm (absorbance ^1.45).

Solutions of 2-mannitol in acidified ammonium molybdate

solution are unstable, and, during storage at room

temperature for 16 h, deposit colorless crystals. On

further storage, the supernatant liquid becomes bright blue.

40
values of the specific rotation, mainly for the concentration

range of 1.60 —1.65, are given in table 16.

Figure 2.

Table 16.

Recording an optical rotatory dispersion spectrum

Specific rotations, at various wavelengths, of

solutions of commercial samples of D-mannitol in

acidified ammonium molybdate solution

Waveler

(A), ir

igth

1 nm

589(Na^D)

578

546

436

r 1 20 degrees, for samples 1 —

4

1

+139.0 a (l4l.9) b

b

b

145.1(144.4)

166.1(167.3)

297.3(299.8)

2 + 138.

6

; 145-5

166.0

297.3

+138.9 (141)

145.2(147)°

166.1(169)°

296.7(303)°

20 'Literature value [15], [a]f, +139.2° (e 1.60) b

D

+138.9

145.4

166.0

297.2

The values

in parentheses were measured on a less concentrated solution

(a 0.32); literature [15] values, [a]^° +140 . 3 and +141° . c The values in parentheses were obtained from studies of

optical rotatory dispersion at ^24 °C, and are based on

measurements of recorder chart-paper (<7 cm) with a precision

of ±0.2 mm.

41
H. 3-Nicotinamide Adenine Dinucleotide (3-NAD) and Its 1,4 - Reduced Form (3-NADH)

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

Among the organic compounds now being studied for

development as Standard Reference Materials for use in

clinical chemical analysis is 3-NADH (2), the 1,4-reduced

form of 3-NAD (1). Although 3-NAD was discovered in 1906, and

O — CH, ov N© )) O —CH2 ,CX

H 2 NCO

HO OH

OH

/

O

H2 NCO

where R =

HO

isolated and identified in 1936, it was not crystallized as

the free acid until 1964. As far as we are aware, physical

criteria for highly purified 3-NAD have not been reported in

the literature.

42
Similarly, although purification of 3-NADH was vastly

improved in 1961 by use of column chromatography on DEAFcellulose, physical criteria for the highly purified compound

have apparently not been published. For example, as may be

seen from table 17, the most recent value for e ,. at 340 nm

is 6.25 (determined in 1957), but the value of 6.22 (determined

in 1948) is usually cited.

Table 17. Ultraviolet absorption peaks of 3-NADH at pH 10

e ,. at 260 nm e ,. at 340 nm 4 ,- A /A-,. iri References mM mM 260 340

1

2

3

4

5

6

7

15.9 2.54 a

8

5.93-6.31 (av.

6.18) but they

gave 6.22 9

6.3 10

16.9 2.72-2.87b

11

6.25 12

16 2.56 c 13

14.4 2.32 b 14

4.,78

5,,90

5.,12

27 (NBS cal

6.30)

cs

5.,47

5..50

6,.27

2.4 b

15

14.0 (or

slightly lower) 2.24 or less 16

2.32 17

2.32 18

14.4 6.2 2.32 19

p Y) o Assumed £oim = 6.27. Assumed e,^ = 6.22. Assumed

Zohn = 6.25 (average of values in Refs. 4, 9, and 12).

^3
References to table 17.

1. Warburg, 0., Christian, W., Biochem. Z. 28J, 291 (1936).

2. Haas, E., Biochem. Z. 291, 79 (1937).

3. Negelein, E., Wulff, H. J., Biochem. Z. 293., 351 (1937).

4. Ohlmeyer, P., Biochem. Z. 2JT7, 66 (1938).

5. Negelein, E., Bromel, H., Biochem. Z. 301, 135 (1939);

303 , 132 (1939)

•

6. Schlenk, F., J. Biol. Chem. 146, 619 (1942).

7. Kubowitz, P., Ott, P., Biochem. Z. 314., 94 (1943).

8. Drabkin, D. L. , J. Biol. Chem. 157, 563 (1945).

9. Horecker, B. L., Romberg, A., J. Biol. Chem. 175 , 385 (1948).

10. Colowick, S. P., footnote 4 on p . 389 of Ref. 9-

11. Lehninger, A. L., Biochem. Prepn. 2, 92 (1952).

12. Wallenfels, R., Christian, W., Methods Enzymol. 3.,

882 (1957).

13. Rafter, G. W., Colowick, S. P., Methods Enzymol. 3.,

887 (1957).

14. Siegel, J. M., Montgomery, G. A., Bock. R. M., Arch.

Biochem. Biophys. 82, 288 (1959).

15. Dalziel, R., Biochem. J. 84_, 240 (1962).

16. Silverstein, E., Anal. Biochem. 12, 199 (1965).

17. McComb, R. B., Gay, R. J., Clin. Chem. 14, 754 (1968).

18. Hartel, A., Helger, R., Lang, H., Z. Rlin. Chem. Rlin.

Biochem. 6, 259 (1968)

.

19. Specifications and Criteria for Biochemical Compounds,

Tipson, R. S., (Ed.) Publication 1344, National Academy

of Sciences —National Research Council, Washington, D. C.,

1967, CoE-7, p. 207.

44
Our initial studies have been made on the oxidized

form (3-NAD), because it has been reported to be more

stable than the reduced form. Moreover, owing to its high

hygroscopicity , studies were made with a single sample before

the techniques developed were applied to other samples of

3-NAD and to samples of 3-NADH (which is reported to be

highly unstable to both moisture and light). The following

preliminary results have been obtained with commercial

samples of 3-nicotinamide adenine dinucleotide

.

1

.

Loss of Weight on Drying

(A. Cohen and A. J. Patiadi)

On being dried at 25 °C/0.1 torr, a sample (299.875 nig)

of 3-NAD (lot no. 2331) lost the following decrements in

weight (in mg) after various total time-intervals (given,

in hours, in parentheses): 1.7 (1), 0.4 (2), 1.1 (20), 0.3

(24), 0.15 (26), 0.06 (28), and 0.02 (30). Total loss in

weight in 30 h, 3. 73 nig (1.24%). Further drying was not

carried out.

After 2 days of drying under the same conditions, lot

2459 (2 samples of ^350 mg, accurately weighed) lost 4.9

and 4.8$; lot V3730 (3 samples of ^100 mg) lost 2.3, 2.3,

and 2.3$.

The theoretical values for Cp-,Hp 7 N

7

0-. lP

? are as follows:

anhydrous, 0.0; 1 HpO, 2.6; 2 HpO, 5.2; 2.5 HpO, 6.4; 3 HpO,

7.5; and 4 HpO, 9.8$.

2

.

Elemental Analysis

The results of analysis of a commercial sample of 3-NAD

are given in table 18.

45
Table 18. Analysis of a commercial sample (lot no. 2331) of

3-NAD

Calculated for

Q H N p Ash

C 21H

27 7 14P

2

anhydrous 38.0 4.1 14.8 9.3 0.0

1 H

2 37.0 4.3 14.4 9.1 0.0

2 H

2

36.1 4.5 14.0 8.9 0.0

2.5 H

2 35.6 4.6 13.8 8.7 0.0

3 H

2

35.2 4.6 13-7 8.6 0.0

4 H

2

3^.3 4.8 13.3 8.4 0.0

Found

not dried 36.3 4.7 13.6 8.1 2.5

By emission spectrographic analysis of a sample (lot no

2331) 3 the following semiquantitative estimates were

obtained: Al, 50; B, 5; Ca, 100; Cr, 30; Cu, 40; Pe 3 70;

Mg, 20; Na, 2000; Ni , 25; and Si, 50 ppm of the sample.

3 . Paper Chromatography of 3-NAD

(A. Cohen)

Chromatograms of 3 -NAD on Whatman No. 1 chromatography

paper were obtained by use of the following developers [16]

solvent A, 66:1:33 (v/v) isobutyric acid—cone. ammonium

hydroxide —water, pH 3-7; B, 7:3 (v/v) 95$ ethanol—1.0

M ammonium acetate, pH 7-5; C, 2:1 (v/v) pyridine—-water

[17]; and D, 3:1 (v/v) acetone —water [18]. 3-NAD and each

of the reference compounds were dissolved in 1:3 (v/v)

ethanol—water, and the solutions were individually spotted

in the following amounts: "adenosine diphosphoribose"

sodium salt (ADPR) , 2.4—4.8 yg; a-NAD, 1.35—2.70 yg;

nicotinamide, 7-5 yg; 3-NADH, 4 —8 yg; and 3-NAD, 27, 135,

and 270 yg, by use of Drummond, disposable micro-pipets

.

46
After development of the chromato grams , the compounds

were detected by their fluorescence-quenching effect when

irradiated with 254-nm light; 366-nm light failed to reveal

any of these compounds, except 3-NADH. A spot corresponding

to nicotinamide, used as reference compound, was evident with

190- or 270-yg applications of 3-NAD run in solvent A, as

well as in solvent B. The R„ values of the various compounds

t in each of these solvents are listed in table 19 . Whether

any of the reference components are impurities initially

present in the 3-NAD or may be present as artifacts formed at

the pH employed for the paper chromatography is still under

study

.

To maximize the possibility of finding impurities that

might not be visibilized by quenching under 25^-nm light,

an alternative detection method was sought. A very useful

procedure employed a spray consisting of methanolic potassium

hydroxide (15 g in 100 ml of methanol). Our exploration of

this spray formulation was based on the observation of

Kaplan et at. [20] that aqueous 5 M sodium hydroxide induces

in 3-NAD a blue fluorescence that is forty times as intense

as that caused by M potassium cyanide. The alkali-induced

fluorescence of 3-NAD had been discovered earlier [21] . We

tried potassium hydroxide (5 M) as a chromatographic

detection reagent but found that it causes some curling of

the paper chromatograms . Our methanolic spray, when applied

at 25 °C, gives a highly fluorescent product with 3-NAD under

366-nm irradiation. The R„ values, and fluorescence with

this spray (in parentheses), were as follows: Solvent A,

nicotinamide, O.87 (none); a-NAD, 0.34 (blue); ADPR-Na

salt, 0,20 (none); solvent B, nicotinamide, 0.7^ (none);

ADPR-Na salt, 0,17 (none); a-NAD, 0.10 (blue); and 3-NADH

(which has an inherent blue fluorescence), 0.15 (blue).

^7
Table 19 . Paper chromatography of samples of commercial

3-NAD. Detection by fluorescence-quenching or

KOH-spraying, or both

Sol- Manufac:turer's lot no. Fl uor- Identification

vent 2331 V3730 2459 escence

with KOH

A

b 0.87 0.86 0.85 none nicotinamide

(?)

0.71? 0.71? none trace, unknown

0.45 0.44 c 0.45 none unknown

0.37 0.37 0.38 blue 3-NADd

0.21 0.21 0.22 none ADPR

[0.21] e [0.21] e [0.22] e blue unknown

0.13 0.13 0.14 blue unknown

0.07? 0.07? 0.08? blue trace, unknown

B

f 0.71 vw g 0.71 vw g 0.71 vw g none nicotinamide

0.29 vw s 0.2 8 vw s 0.28? vw s none unknown

0.18 0.17 0.18 none ADPR , 0.11 0.10 , 3 -NAD

[0.10] n 0.12 blue

none none unknown

0.02 0.02 0.10 blue unknown

,Application of 240 ug.

Descending.

,Blue fluorescence with 366-nm light.

Because of its R value, close to that of 3-NAD, the

possibility of presence of a-NAD cannot be excluded.

Visibilized only by spraying with KOH; blue fluorescence in

interior portions of ADPR spot; tailing (blue fluorescence)

f to slower spot.

Ascending.

gVery weak (by quenching)

.

A fluorescent impurity, having the same Rp

value as 3-NAD,

seen (only in this sample) under 366-nm light; impure 3-NAD

can contain [19] a fluorescent impurity that is not 3-NADH.

48
We have found our detection spray to be very sensitive

and helpful in differentiating impurities associated with

3-NAD and 3-NADH . The blue fluorescence given on spraying

does not appear immediately on the chromatogram, but only

begins to be visible after ^2 min and has increased

substantially after ^10 —15 min. The limit of sensitivity

of the spray has not yet been determined; however, in one

test, k yg of a commercial sample of 3-NADH, developed

with solvent C and apparently pure by inspection under

366-nm light, was found with the spray reagent to exhibit

another fluorescent spot, possibly 3-NAD.

In solvent B, ADPR is not well separated from 3-NAD, but

may overlap it partially; however, their discrimination

presents no difficulty when the detection spray is used.

In all four solvent systems, a -NAD cannot be resolved

from 3-NAD if the proportion of the latter far exceeds that

of the former, and so, if the a- were present as an impurity

in 3-NAD, the a -NAD would not be detected.

With solvent A, lot V3730 of 3-NAD under 366-nm light

showed a fluorescent impurity that was not found in other

commercial samples examined. Lots 2331 and 2459 3 on the

other hand, contained an impurity with this same R value,

t but it was nonfluorescent . With solvent B, this fluorescent

impurity in lot V3730 was not resolved from the 3-NAD.

Descending paper chromatography [17] of the reference

compounds for 24 h with solvent C showed R values (relative

to R = 1 for 3-NADH) and fluorescence with the spray (in

parentheses) as follows: ADPR-Na salt, 0.93 (none); a -NAD,

0.74 (blue); 3-NADH has an inherent blue fluorescence.

Nicotinamide was also used as a reference compound, but was

not detected because, under these conditions, it was eluted

from the chromatogram. The results of descending paper

chromatography on commercial 3-NAD (lot 2331) in solvent C

are given in table 20. Results with solvent D were similar

to those in C.

49
Table 20. Paper chromatography a of 3-NAD (lot no. 2331)

Solvent R

b

c

c 0.97

0.93

0.76

0.44

D

C 1.28

1.14

0.73

0.37

Fluorescence Ident if icat.

with KOH

none unknown

none ADPR

3-NADa blue

blue unknown

none unknown

none unknown

blue 3-NADd

blue unknown

.Application of 268 yg.

Relative to R = 1 for 3-NADH.

Descending (24 h)

.

Or a-NAD, or both.

For descending paper- chromatography in solvent D, the

R values (relative to if = 1 for 3-NADH), and fluorescence

with the spray (in parentheses), were: ADPR'Na salt, 1.02

(none); a -NAD, 0.75 (blue); 3-NADH has an inherent blue

fluorescence. A commercial sample (lot 2331) of 3-NAD

showed three impurities in solvent D (see table 20). Here

ADPR-Na was not evident, but a very fast-moving impurity

(R = 1.14) was evident by its fluorescence-quenching.

Surprisingly, the 3-NAD spot showed an unexpected blue

fluorescence under 366-nm light. In some way, this may be

related to the complex with acetone that 3-NAD has been

reported [21] to form in alkaline medium.

Attempts to resolve a-NAD from 3-NADH by t.l.c. on PEIcellulose with solvent B or with 0.2 M Tris buffer (pH 9.0)

were unsuccessful.

50
3-NADH examined by descending chromatography on paper

with solvent C appeared to be pure under 254- and 366-nm

irradiation, but exhibited a fluorescent impurity at R 0.50

h when sprayed with the methanolic KOH . 3-NAD run as a reference standard at a nearby position showed R„ 0.51-

h

4 . Gas —liquid Chromatography of Commercial 3-NAD

after Per ( trimethylsilyl)ation

(A. J. Patiadi)

A sample (5 mg) of commercial 3-NAD (lot no. 2331) was

per ( trimethylsilyl) ated by use of 0.5 ml of Tri Sil Z at

25 °C for 60 min and 24 h. The products were injected into

a glass column (6 ft. x 2 mm I.D.) having Chromosorb Q

(60 —80 mesh) as the solid support, with a liquid phase of

3% of OV-17 . The column was operated isothermally at 150 °C 3 and the components were detected with a f lame-ionization

detector operated at 200 °C. The sample size was 10 yg. The

retention times observed for the components detected are

given in table 21. Work is still in progress on the

identification of the components

.

Table 21 . Retention times of products obtained by

per ( trimethylsilyl )at ion of commercial 3-NAD

(lot no. 233D

Approximate retention time (minutes) of components observed

after

1 hour

3-

4.

6

.4

7.

9.

2

.6

15. 2

24.,0

24 hours

3.,6

7.

9.

,0

,4

14,.8

23,.8

38,,4

Time elapsed after mixing 3-NAD with Tri Sil Z.

51
5 . Spectrometric Methods

(A. Cohen)

Approximately 3 —4 nig (carefully weighed) of commercial

3-NAD was rapidly dissolved in water or in tris (hydroxymethy l)aminomethane (Tris) buffer, adjusted to pH 8 with

glacial acetic acid; total volume 100 ml. A sample, dried

under high vacuum without heat, was similarly dissolved.

Ultraviolet spectra of the solutions were recorded from

600 to 220 nm by use of a Cary Model 14 u.v. spectrophotometer. (The spectra showed no absorption from 600 to

350 nm.) The solutions were then stored in the dark at

20 —25 °C, and re-examined periodically. No change was

apparent in 3 days. The data are given in table 22.

Table 22. Ultraviolet spectral data for 3-NAD (lot no. 233D

Solvent Time at mM . . . 25 °C (days) At At A 250 7/1 260 A 280 //1 260

231.5 260

nm nm

Not dried

Water 8.01 17.5 0.83 0.24

3 8.15 17.5 0.82 0.23

51 decomposed

7.86 17.4 0.82 0.21

3 7.89 17.4 0.83 0.21

Tris buffer

(pH 8)

Dried

7.85 17.5 0.82 0.21

18 7.67 17.6 0.80 0.20

41 7.29 17.35 0.79 0.20

pH 7 Manuf. data — 17.6 0.82 0.21

pH 7 NRC [22] — 18.0 0.83 0.22

0,.83

0,.82

0,.82

0,.83

0,.82

0,,80

0..79

0,,82

0,.83

52
6. Enzymic Assay of 3-NAD

(A. Cohen)

When 3-NAD is reduced, a peak characteristic of 3-NADH

appears at 3^0 nm, and the initial absorbance at 260 nm is

diminished in intensity. The NRC values of e „. for the

reduced compound are 14.4 at 260 nm and 6.2 at 3^0 nm [22]

(see table 17). It is commonly accepted that lower values

of A

?(

- C] /A^ lin are indicative of purer 3-NADH; consequently,

it would also be indicative of a purer 3-NAD as the

reactant

.

Horse-liver alcohol dehydrogenase (HLAD), 2.3 U/mg [23],

was used. Commercial 3-NAD (lot no. 2331; 4.811 mg), 40 ml

of 0.5 M ethanol in Tris buffer (0.1 M unadjusted; pH ^9.8),

and 2 mg of HLAD in 2 ml of Tris buffer were mixed, and

diluted to 100 ml with Tris buffer. The blank was a comparable solution without 3-NAD. The reaction reached

completion in 38 —50 min, and was read at 75 min.

Absorbances were measured at 3^0 nm, at 260 nm, and at two

minima. The solution was then left exposed to laboratory

light at room temperature, and was re-examined after 4, 8,

and 18 days. The results are summarized in table 23-

53
Table 23. Enzymic assay of 3-NAD (lot no. 2331) by

reduction with ethanol in the presence of

horse-liver alcohol dehydrogenase

e mM at

a NRC Time ke:pt at 25 °C (days)

ill* •* (nm) constants

[22] for

3-NADH

i1 8 18

^235 6. 74 6. 82 6.80 6.76

260 14.4 V1.6 ll1.7 14. 7 15.0

290 l. 39 1. 51 1.49 1.92

340 6.2 b

5. 85 5. 88 5.71 5.61

Absorb

ance

ratio

A 25Q /A 260 0.82 0.,80 0. 80 0.79 0.79

A 2QQ /A 260 0.23 0.,25 0. 25 0.26 0.27

A 26q /A 340

2.32 2.. 49 2. 50 2.58 2.68

,For resulting 3-NADH.

Manufacturer's data for this lot, 5-9 ±0.2

54
2. CARBOHYDRATE SYNTHESIS, STRUCTURE, AND CHARACTERIZATION:

NOVEL RESEARCH MATERIALS AND MODEL COMPOUNDS

The Section's area of research specialization in

organic chemistry continues to be the carbohydrates. Two

ketoses were examined: Q-fructose and D-psicose, the former

because Its 1, 2 : 4 , 5-di-0-isopropylidene derivative lacked

sufficient purity to be used unequivically in subsequent,

synthetic experimentation. The nature of the problem

with its purity was resolved and, as an outgrowth, a higheryield process for making the pure derivative was developed.

Work with g-psicose was related to this . Only by use of

that pure g-fructose derivative (i.e., free of the

contaminating 2 , 3 :

4

, 5-di-0-isopropylidene isomer) was a pure g-psicose obtainable; by utilizing the pure "diacetonefructose" as the starting material, confounding aspects in

publications from several laboratories on the synthesis of

2-psicose were clarified.

Proton magnetic resonance studies of interesting

carbohydrates were actively pursued during the year.

Although there is considerable interest in understanding the

relative importance of chair and nonchair conformations of

molecules that contain six-membered rings, there is a paucity of proton resonance data for unequivocal examples

of nonchair conformations that can serve as a reference for

conformational equilibria. Certain rigid carbohydrate

molecules can be most useful in this regard. Our p.m.r.

study of 3-0-benzoyl-l, 2 ,4-0-benzy lidyne-a-D-ribopyranose

has now defined the degree of applicability of previously

developed relationships between coupling constants and

dihedral angles with the locked, skew conformation of this

compound

.

55
A p.m.r. study was also conducted on 1 , 2-0-alkylideneD-glucopyranoses to settle any controversy concerning their

adopting a possible skew conformation. The resonance

parameters found by our study showed clearly that the

compounds possess chair conformations

.

Computer programs have been of considerable importance

for the analysis of complex p.m.r. spectra. Now, too, the

Section possesses a high-resolution spectrometer having

heteronuclear resonance capability, and its use is extending

the range of our work.

Inosose oxidation by the Somogyi reagent was examined,

and led to the examination of the reactant in this and other

copper-complex reagents by electron-spin resonance. The

reaction with the Somogyi reagent appears to involve radical

or one-electron mechanisms, depending on the reaction

temperature. A facile synthesis of inositol hexasulfate has

been developed. This compound is of interest, since it is

the sulfuric acid analog of phytic acid, a naturally

occurring myo -inositol derivative.

A. Synthesis of the Isomeric Di -Q- isopropylidene -g-gfructopyranoses

(R. F. Brady, Jr.)

1 . Introduction

Large amounts of 1, 2 : 4 3 5-di-0-isopropy lidene-3-Dfructopyranose (2) were required as the starting material

for a synthetic program leading to crystalline derivatives

of rare sugars. Samples of this diacetal were therefore

purchased from commercial sources, and, for comparison,

samples were synthesized by published procedures. By

per( trimethylsilyl) ation followed by g.l.c. analysis, it

was found that most of these samples were impure, the

contaminant of 2 being its isomer, namely, 2 , 3 :

, 5-di-Cisopropylidene-3-D-fructopyranose (3). A procedure was

56
therefore sought for freeing the 2 from this impurity,

with the highest possible recovery of pure 2.

In addition, as the literature on the synthesis of 2

records yields lying only in the range of 50 [24] to 66%

[25], it was desired to attempt to devise a method that would

give pure 2 in higher yield.

2. Results and Discussion

A suspension of crystalline g-fructose (1) in acetone

containing sulfuric acid (as catalyst) was stirred, and the

reaction was monitored after various time-intervals by g.l.c

analysis of per ( trimethylsilyl)ated, neutralized aliquots

;

the diacetal observed first was compound 2.

It was found that, at high concentrations of catalyst

(>5%) 3 2 isomerizes rapidly to 3. The concentration of 3

is maximal at equilibrium; indeed, 3 is Isolable in

satisfactory yield if the reaction is allowed to reach this

stage

.

CH 2 OH

£

HO J> <k

Jj/Ui Me

Me-C—

O

Me

CH2 OH

57
In contrast, at low concentrations of catalyst ($0.5%) , compound 2 slowly isomerizes to 3- By arresting the reaction

at the point at which the concentration of 2 is maximal,

compound 2 was isolated in 80% yield.

Use of intermediate concentrations of catalyst, or

premature processing of the reaction mixture, affords

mixtures of diacetals 2 and 3, such as those reported

by Ohle and Roller [26] and Bell [25]. Such mixtures

are more difficult to separate, and isolation of either

isomer in pure form is attended by great diminution in

yield

.

Apparently, 2 is the product of the kinetically

controlled condensation of g-fructose with acetone; that

it is observed first is, presumably, a consequence of the

higher reactivity of the primary hydroxyl group on C-l,

as compared with that of the secondary hydroxyl group

on C-3. However, in 2, the spiro- fusion of the 1,3-

dioxolane ring to the pyranoid ring creates strain [27]

at C-2. This strain is relieved when 2 rearranges to

3, because the 1, 3-dioxolane ring becomes ois-fused. to the

pyranoid ring, and diacetal 3 greatly preponderates in the

mixture at equilibrium. Thus, in acetone containing 5%

of sulfuric acid, each pure isomer is converted, within 5

min at room temperature, into an equilibrium mixture

consisting of 9^% of 3 and 6% of 2; hence, 3 is

thermodynamically more stable than 2.

These results have been incorporated in an article

which has been accepted for publication [28].

B . Synthesis of g- Psicose

( R. S. Tipson , R. F. Brady, Jr., and B. F. West)

1 . Introduction

In Technical Note 457, we described preliminary studies

on the synthesis of g-psicose from D-fructose, undertaken

58
because a crystalline derivative of g-psicose was desired

for issuance as a Standard Reference Material. We have,

in particular, re-investigated the preparation of its

1, 2 : 4, 5-di-0-isopropylidene derivative, which is crystalline.

D-Psicose (D-ribo-hexulose , 5a) is a rare sugar that

occurs in the antibiotic psicofuranine [29] (9-3-Bpsicofuranosyladenine) and in Itea plants [30]; it has

also been found in the nonf ermentable fraction of cane

molasses [31] s where it may have been formed by the action

of the lime used in sugar manufacture. g-Psicose was

first synthesized [32] in 1936, by the pyridine-cataly zed

isomerization of g-allose; it has also been prepared by a 6-step synthesis [33] from g-ribose, by a 4-step synthesis

[34] from D-fructose, by mild isomerization of Q-glucose

with alkali [35] , and by isomerization of g-fructose by

N3 N

1 -dicyclohexylcarbodiimide in hot methanol [36] to

D-psicose, g-glucose, and p_-mannose, followed by

fermentation with bakers' yeast. However, some of these

methods require expensive starting-materials, and all

of them are tedious and give low overall yields

.

In 1967, McDonald [37] described a new synthesis of the

sugar, starting from g-fructose (la), that involved the

following steps. Compound la was converted into l,2:4,5-diC-isopropylidene-$-g-fructopyranose [28] (2a*), and this

was oxidized with methyl sulfoxide —acetic anhydride

(oxidant I) to 1, 2 : 4, 5-di-0-isopropylidene-3-D-eri/ thvo-2 , 3-

hexodiulo-2 ,6-pyranose (3); it was stated that the 3-acetate

(2c) of compound 2a was simultaneously formed, but that

recrystallization of the crude product afforded pure 3.

Reduction of 3 with sodium borohydride in aqueous methanol

then gave 1, 2 : 4 , 5-di-0-isopropylidene-3-D-piZ?o-hexulopyranose

*Takagi and Rosenstein [38] have found that, in crystalline

2a, the conformation of the molecules is intermediate

between 1C (U) and H* (g)

.

59
OR

RO

O

CH,OR

ip

Me2 SO

Ac 2 ->

Ip—

O

+ (2c, R = Ac)

2d,R =CH 2 SMe

NaBH4

cAp

°\/ OR

IP

H +

fC ^| 0R. CH20R

RO T~T

RO OR

4

a. R= H

b, R= SiMe 3

5

60
(4a) contaminated with "only a trace" of the corresponding

D-fructose diacetal (2a), presumably removed on

recrystallization of crude 4a; and acid hydrolysis of

compound 4a afforded D-psicose (5a).

Because this synthesis provides a direct means for

preparing g-psicose, it was decided to repeat this procedure

to produce a supply of the crystalline 1 . 2 : 4, 5-di-0-

isopropylidene-3-D-psicopyranose (4a) for use as a Standard

Reference Material. Three months after publication of

McDonald's Note [37], a more detailed article describing

similar work was published independently [39]; certain of

the conclusions described in the latter article had already

been arrived at by us [40]. Finally, in 1968, another

article was published on the same subject [4l]. The present

work was done in order to explain certain discrepancies

between the results of the three sets of workers [37,39,41].

2 . Results and Discussion

In our study, McDonald's procedure [37] for preparing

3 was first repeated in accordance with her instructions.

Compound 2a was oxidized during 24 h at 25 °C with a solution of acetic anhydride (1 molar proportion) in

methyl sulfoxide (70 molar proportions), and crude compound

3 was isolated in a yield of ^50% of the theoretical, as

compared with the 70% yield of crude product reported by

McDonald [37]. Because of this result, a series of

experiments was performed in which the molar proportions

of 2a to acetic anhydride (A) to methyl sulfoxide (B) were

1:2:42, 1:16:42, and 1:21:42, but the yield of 3

(recrystallized) was only raised to 52 —54% of the

theoretical. As the oxidation of 1 mole of 2a with McDonald's

proportions would require the use of 5 liters of methyl

sulfoxide, the molar proportions of 2a:A:B were changed to

1:10:10, in contrast to the ratios of 1:1:70 [37], 1:23:46

61
[39], and 1:43:85 C^l] reported in the literature. The

reaction was monitored by t.l.c. on Silica Gel G, and we

found that (a) the time necessary for completion of the

reaction with the 1:10:10 ratios was 4 days, as compared

with 2 days [37] and 1 day [39,41]; and (b) only two products

are formed. James and co-workers [39] reported that three

products are formed, whereas the other workers [37, 4l] found

only two.

In all of these experiments, we found that, accompanying

compound 3, there was a large proportion of a different

crystalline compound (X) that, solely on the basis of R„

t values in t.l.c., had been regarded by McDonald [37] as

being the 3-acetate 2c. (The rather close R„ values of

X and 2c in her solvent system explain the misidentification.) However, we found that the second product (X) shows

no carbonyl absorption in its i.r. spectrum, and therefore

could not be the acetate 2c. To verify this conclusion, we

prepared an authentic specimen of acetate 2c by the method

of Fischer and Noth [42], and found that the i.r. spectrum

of 2c, which shows bands at 1735 (C=0) and 1240 cm

-1

(C-0),

is entirely different from that of compound X. A mixture of

compound X (m p 82.5—83 °C) with compound 2c (m p 76 —77 °C)

melted at 59 °C. Compound X was identified [39] as 2d, the

3-(methylthio)methyl ether of compound 2a.

To effect separation of the diulose acetal 3 from X (2d),

McDonald [37] recrystallized the crude product from methanol,

to afford 3; compound 2d was not recovered. James and

co-workers [39] isolated 3 by recrystallization of the crude

product from petroleum ether; the mother liquor was

evaporated to dryness and the resulting syrup was crystallized

from petroleum ether to give a mixture of crystalline 3

(as needles) and 2d (as rectangular plates). These were

separated "mechanically," and recrystallization of the

platelets from petroleum ether gave 2d. We have found that

62
the two compounds are readily separated by dissolution of

the mixture in absolute methanol (5 ml/g) and addition of

water (2.5 ml/g); the ether 2d crystallizes out almost

quantitatively and is filtered off, the mother liquor is

evaporated to dryness, and this residue, crystallized from

hexane, gives 3.

Whereas the diulose acetal 3 has been reported to have

[a] -100.4°(in acetone) [37] , -126. 4° (in chloroform) [39] , and

-106°(in ethanol) [41], we found [a]^ 5 -104.7°(in acetone),

-119. 2° (in chloroform), and -113- 5° (in ethanol) . Because of

these discrepancies, we decided to prepare compound 3 by

oxidation of 2a with ruthenium tetraoxide [4l] (oxidant II)

by an Improved procedure [43]. Compound 3.- obtained in

this way, was homogeneous by t.l.c. and g.l.c. analysis;

it had the same specific rotations as those we had found for

3 prepared by use of oxidant I.

The reduction of pure compound 3 was effected with sodium

borohydride by a modification of the procedure of James and

co-workers [39]. G.l.c. analysis of the 3-( trimethylsilyl)

ether of the "crude" crystalline product, under conditions

that would have detected the presence of 0.1$ of compound

2b, revealed that the reduction product consisted solely

of 4a. Hence, the reduction is completely stereospecific

.

This result is not in agreement with that of the earlier

workers. By t.l.c., McDonald [37] found "a trace" of 2a in

the product obtained on sodium borohydride reduction of 3

(prepared with oxidant I). Cree and Perlin [4l] reported

that, on lithium aluminum hydride reduction of 3 (prepared

with oxidant II), "only a trace of the pj-fructose derivative

was formed during the reduction, and sodium borohydride in

aqueous ethanol was almost as exclusively selective in

favoring the production" of 4a. In contrast, on reduction

of 3 prepared with oxidant I, James et at. [39] found, by

g.l.c. analysis, 10$ of 2a in the crude 4a. obtained by

63
reduction with lithium aluminum hydride, and 1.8$ of 2a

when 3 was reduced with sodium borohydride.

We have sought an explanation for these discrepancies.

The possibility that 2a, as a contaminant of 4a, arose from

2d as a contaminant of 3 was excluded, as we found that 2d

is unaffected by sodium borohydride under the conditions

used for reducing 3 to 4a. Hence, the only explanation

possible is that a small proportion of 2a remained unchanged

on oxidation of 2a, and that it was carried as a contaminant

of 3 (and, thence, of 4a); the presence of a small

proportion of 2a in 3 would not be readily revealed by the

t.l.c. procedures that had been used [37,39,41].

In addition to 3 and 2d, James et al . [39] found, in

the mixture obtained by treating 2a with oxidant I for 1

day, a third component that was not unoxidized 2a. It has

been established [28] that many commercial and laboratory

samples of 2a are contaminated with 2 , 3 : 4 , 5-di-0-

isopropylidene-3-D-fructopyranose (6a).* We have now

found that compound 6a is converted into a mixture of

2,3:4, 5-di-(9-isopropylidene-aZ(ie?2i/<i(9-3-D-araZ?ino-hexosuloIp-O

CH 2 OR

[O]

[H]

Ip-O

+ 6c

+ 6d

a, R=H

b, R=SiMe3 cy R = Ac

d, R=CH2 SMe

*The S* conformation depicted was found by Maeda and

co-workers [44]

.

64
2, 6-pyranose (7) and the 1-acetate (6c) and

l-(methylthio) methyl ether (6d) of 2, 3 : 4, 5-di-0-

isopropylidene-3-D-fructopyranose on treatment with oxidant I

under the conditions that we had used for oxidation of

compound 2a. In the t.l.c. developer used by James et at.

[39 3, compound 7 has i?„ 0.39, and compounds 6c and 6d have

S_ 0,72; the latter is the 2?_ value reported [39] for the

"third product" detected in the oxidation of 2a by oxidant I,

showing that their 2a might, indeed, have been contaminated

with 6a. The presence of a small proportion of 6a in 2a

would not be readily revealed by the t.l.c. procedures that

had been used [37,39,41].

Reduction of compound 7, under the conditions that we

had used for the reduction of compound 3, afforded compound

6a. Thus, if 6a is a contaminant of 2a, it will give its

oxidation product (7) as a contaminant of 3; on reduction'

of this mixture, 6a will be a contaminant of 4a, and thence,

D-fructose (la) will be a contaminant of the desired D-psicose

(5a). We therefore emphasize that, for preparation of pure

4a, the diacetal 2a used as the starting material must

be free from diacetal 6a, and, before reduction, the diulose

acetal 3 must be free from 2a.

Such contamination of 4a by 2a or 6a is most readily

detected by hydrolysis of 4a to 5a, because 5a has [a ]„ +4.7°

(in water) [33], whereas la (from any 2a or 6a present as a contaminant) has [a ]_ -92° (In water) [45]. On hydrolysis

of our samples of 4a with 0.1 M oxalic acid at 65 °C, the

resulting solution of 5a had [a 1 D +4.7° (In water) and the

sugar was homogeneous by t.l.c. [27] and by g.l.c. of its

per( trimethylsilyl) derivative (5b). The previous workers

[37,39,^1] did not report having applied these tests to

their samples of 4a.

65
25 Freshly recrystallized 4a had m p 68 —69 °C and [a] D

-116.4° (in acetone), but, after it had been kept for several

months at room temperature, the m p was 56 —58 °C. This

change has been observed before [39] , and cited as evidence

for a supposed rearrangement of crystalline 4a to crystalline

1, 2 :

3

, 4-di-0-isopropylidene-3-D-psicofuranose (8); however,

we found that the change in m p of 4a was unaccompanied

by any change in R„ value, i.r. spectrum (in solution), or

b optical rotation whereas compound 8 has [32] m p 57 —58.5 °C

20 and [a]

n -98.2° (in acetone). Hence, the change in m p

of 4a. must be ascribable to physical change from an unstable

to a stable crystal modification, and not to a chemical

change in structure.

Consequently, for the preparation of pure D-psicose (5a)

via oxidation of 1, 2 : 4 , 5-di-0-isopropylidene-3-B - fructopyranose (2a), the latter must be free from its

2,3:4,5 isomer (6a), which is oxidized to the corresponding

aldosulose acetal. Pure 1, 2 : 4 , 5-di-0-isopropy lidene-3-

^-erythro-2

,

3-hexodiulo-2 , 6-pyranose (3) undergoes

stereospecific reduction with sodium borohydride to give

only 1, 2 : 4, 5-di-C-isopropylidene-3-D-^^^c>-hexulopyranose (4a),

which exists as two different crystal modifications.

Compounds 3 and 4a. have been characterized, and discrepancies

in the literature have been explained.

These results have been incorporated in an article that

has been accepted for publication [46].

C . Model Parameters for the Analysis of Skew Conformations

by P.m.r. Spectroscopy

(B. Coxon)

1 . Introduction

Although the relative importance of chair and nonchair

conformations is of considerable interest in the

conformational analysis of carbohydrate molecules, there

is a scarcity of p.m.r. data for genuine, or widely

66
accepted, examples of nonchair conformations [47], especially

as several of the earlier assignments of a skew conformation

to carbohydrates have been questioned or withdrawn. In

order to determine the magnitudes of some of the protonresonance parameters that may be expected to be

characteristic of the skew conformations of pyranose

compounds, the p.m.r. spectrum (at 100 MHz) of 3-C-benzoy 1-

1,2, 4-0-benzylidyne-a -g-ribopyranose (1) has been analyzed

by iterative, computer methods. This rigid system provides

a suitable model for the testing of theoretical and

empirical equations that relate various types of coupling

constant with dihedral angle.

2 . Discussion

Inspection of molecular models of 1 reveals that the

molecule is rigidly locked In the conformation la by the

three-point attachment of the benzylidyne group.

H-3

H-5'

H-5

1a

The assignments of the signals of the ring protons In

the spectrum (see fig. 3a) of 1 in chloroform-d were

confirmed by noting solvent shifts, and by double-resonance

experiments. Some of the double-resonance spectra are

shown in fig. 3 (c and d)

.

67
H-3

H-3 H-2

5.05 5.10 5.59 5.64 5.87

(b)

J U I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I —> <— 5Hz

i-i *

H-3

H-2'

(d)

Figure 3. P.m.r.

-^M^u^

spectra of 1 at 100 MHz; the signals of the aromatic

protons are not shown. (a) Single resonance spectrum, (b)

theoretical spectrum from iterative analysis, (a) irradiation

of H-l, and (d) irradiation of H-2 and H-3.

68
Initial parameters for the iterative analysis of the

spectra were derived by ABX analysis of the H-3, H-2, and

H-4 nuclei with H-l decoupled (fig. 3c), and by ABX analysis

of the H-5, H-5', and H-4 signals in the single-resonance

spectrum (fig. 3a); H-5 resonates at lower field than H-5'.

With H-l decoupled, H-2 still resonated as a quartet

(fig. 3c) whose spacings suggested long-range coupling with

H-4. This situation was confirmed by irradiation of H-4.

Good agreement was obtained between the observed spectrum

(fig. 3a) and the theoretical spectrum (fig. 3b) calculated

from the parameters obtained after 13 iterations

.

For the particular values of proton—proton dihedral

angles (<J>) present in la, the corresponding values of the

vicinal coupling-constants may be used to test the

applicability of the Karplus equation,

<j> = cos~

1

r 0.5 ±[0.25 + 36(J + 0.281 1/2

1 , (1)

1 IB i

to carbohydrate systems. The form [48] of this equation is

convenient, in that solution of the quadratic equation in

cost}) provides two roots, one of which corresponds to

0° S <f> < 88°, and the other, to 88° <:

<J> ^ 180°, with the

use of the same constants for each type of angle.

The vicinal-proton, dihedral angles obtained by

measurement of Dreiding molecular models of la, and the two

angles that were calculated from each vicinal-coupling

constant by using equation (1), have been correlated with

the angles that would be expected for an idealized,

symmetrical skew form [49] of the pyranoid ring.

In a molecular model of la, the conformation is somewhat

distorted from the idealized skew form, as only the

corresponding values of <{>„ k were in close agreement

therewith. The values of <j>„ „, <j>„ k, and cK _ calculated

from equation (1) agree quite well with the values estimated

69
Figure 4. Adjustment of the strength of the magnetic field,

at the console of a nuclear magnetic resonance

spectrometer

.

by study of molecular models, but the agreement is less good

for 6-. n and cb ,. ._ . . 1,2 4,5'

The value of the geminal coupling (JV _ , 11.7 Hz) of 1

is intermediate between those (10.8 —10.9 and 13.4—14.0 Hz)

found [50] for a anomers or 3 anomers of D-ribopyranose

tetrabenzoate or of the related 1-halides, for which a dependence on the orientation of the vicinal, electronegative

substituent (benzoyloxy) on C-4 has been indicated [50].

Although the value of 11.7 Hz for J n ,_ , is closer to those

5,5

of the a anomers, the orientation of 0-4 with respect to H-5 ? and H-5 more nearly resembles the trans - gauche arrangements

found [50] in the 3 anomers.

70
SB .;

J

Figure 5. Positioning a sample tube, prior to insertion

into the probe head of a nuclear magnetic

resonance spectrometer.

More-detailed considerations suggest that these results

can be interpreted in terms of the proximity of a and p

orbitals of the H —C-5 bonds and the ring oxygen-atom,

respectively, which can lead to a p -* a electron transfer

that results in a positive increment to the geminal

coupling-constant , in addition to that due to a-electron

withdrawal by a suitably disposed, electronegative substituent

The long-range coupling J^ j, of 1, amounting to 2.45 Hz,

is probably the largest thus far observed over four

saturated bonds of a carbohydrate system. In the skew

conformation la, the dihedral angles <J>

' (of H-2 and C-4,

71
about the C-2 —C-3 bond) and <J> ' ' ' (of H-4 and C-2, about the

C-3 —C-4 bond) are ^170° and ^171°, respectively, and,

therefore, the orientation of H-2, C-2, C-3 3 C-4, and H-4

is very close to the planar W-arrangement that is considered

[51j52] to be optimal for coupling ( J) over four saturated

bonds. In la, H-2 and H-4 have quasi and equatorial

orientations with respect to the plane of the pyranoid ring.

The positive sign of J" ?

u is in agreement with the

indirect, through-bond mechanism proposed [53] by Barfield.

However, Barfield's mechanism [53] does not satisfactorily

account for the reported [54] variation of the sign of J

with the equatorial —equatorial or equatorial —axial

orientations of ring protons of the chair conformations of

pyranoses. Barfield's theory [53] has been interpreted [55]

in terms of the equation

J = 0.7(cos 2

<|)' + cos

2

cf>''') - 0.3- (2)

Substitution in this equation of the values of <j> ' and <f> '

'

' for la gives for J a value of 1.06 Hz that does not agree

with the value of J p

u observed, namely, 2.45 Hz. Such

^ > 4

ij disagreement has been noted [55] for J" in a series of

tetrachlorobicyclo[2, 2, l]heptane derivatives, and it has

been suggested [55] that better agreement with experiment

could be obtained by use of the equation

V = Acos^'cos 2

^

' ' ' - B, (3)

where B = 0.35, and A = 0.31 for 0° $ <J> ' , (J)'" ^ 90°, 1.07

for 0° < <j> ' < 90° <: <f>' !1 <: 180°, or 3. 61 for 90° <: $

' , <p

' '

'

<< 180° . For la, equation (3) yields J 3.07 Hz, which agrees

more closely with the value observed for J„ u

.

These results were incorporated in an article that has

been published [56].

72
D. The Conformations of 1 , 2 : 4 , 6-di -6>- benzylidene -g-gglucopyranose Derivatives

(B. Coxon)

1. Introduction

There is little doubt that the favored conformation of

methyl 4 , 6-<9-benzylidene-a-g-glucopyranoside and of other

similar derivatives having the same configuration at C-4

is the CI (0) form. However, there has been controversy

concerning the possible chair or skew conformations of

1, 2-0-alkylidene-a-rj-glucopyranose derivatives. The

investigation of derivatives containing both 1,2- and

4,6-cyclic acetal substituents was, therefore, of interest,

and 1 , 2 : 4 , 6-di-<9-benzy lidene-a-2-glucopyranose (1) and the

3-acetate (2), 3-benzoate (3), and 3-methanesulfonate (4)

prepared by Fletcher and co-workers [57] have been found to

be suitable for study by p.m.r. spectroscopy at 100 MHz.

O CH

2 . Discussion

P.m.r. spectra (100 MHz) of compounds 1 —4 were

recorded for their solutions in chloroform-d or pyridine

73
or in mixtures thereof. Although parts of the spectra

were complex, because of overlap of multiplets, the signal

multiplicities observed for the protons on the sugar ring

were only those to be expected from coupling over two, or

three, bonds. The spectral assignments for these protons

were confirmed by spin-decoupling and spin-tickling, and by

solvent shifts. Comparison of the chemical shifts of the

benzylic protons with those of the O-benzy lidene-a-Dglucopyranoses and 4 , 6-0-benzylidene-a-rj-glucopyranosides

indicated that, for each compound, the singlet at low field

could be assigned to the 1,2 benzylic proton, and that at

higher field, to the 4,6 benzylic proton.

The chemical shifts of the protons attached to the sugar

ring of 1 —4 were sufficiently different that their spectra

could readily be analyzed by first-order methods. However,

an estimate of the accuracy of the parameters so derived, and

of the validity of the analysis, was obtained by iterative

analysis [58] of the spectrum (see fig. 6a) of compound 4

a.s a seven-spin system (H-l —H-5 , H-6e, and H-6a). Prom

spin-tickling experiments on 2, it was established that the

sign of J r r is the opposite of that of JV c and J c c„> be ,6a 5 3 6e D 3 va

and, therefore, J c c was made negative in the iterative ' be ,ba

analysis of the spectrum of 4. Support for the validity of

the assignments was provided by the good agreement between

the theoretical spectrum (fig. 6b) that had been computed

from the refined parameters and the observed spectrum

(fig. 6a), and by the small error-parameters obtained. From

the coupling-constant data for 4, it was calculated that the

maximum differences between the first-order value and the

refined value of each coupling constant is about 13% of the

latter, and that the corresponding mean difference for seven

coupling-constants is 6%.

74
5.7 5.9 6.1 6.3 T

Figure 6. Partial p.m.r. spectra of 4. (a) Observed

spectrum of solution in pyridine at 100 MHz,

and lb) computed spectrum after iterative analysis

as a seven-spin system.

75
The values of j. u , Ju t- 3 and jy r shown by 1 —4 are

appropriate for pairs of protons having a trans -diaxial

orientation,9 and the medium-sized values of J r c observed 5,oe

are typical of protons having a gauche-axial —equatorial

orientation. However, the values of j 1 „ are consistently

^1 Hz larger, and the values of J _ , ^3-6 Hz smaller, than

those of methyl 4 ,6-0-benzylidene-a -g-glucopyranoside

,

indicating that the proton-— proton dihedral angles <£-, ? and

(J) 9

_ of 1 —4 are smaller than those of the a -Q-glucopyranoside

.

d , J

Application of the refined Karplus equation [59] to compounds

1 —4 gave the approximate values cf>, 9 38 —39° and <j>_ _ 142 — 145° , which compare favorably with the values of 25° and

135° > respectively, measured on Dreiding molecular models.

The results are consistent with the conformation depicted

in formula 5> in which the /77-dioxane ring adopts a chair

shape, and in which the pyranoid ring has a flattenedchair shape (because of attachment of the 1, 2-C-benzy lidene

group). As compound 1 had been prepared [57] under acidic

conditions, it is assumed that the phenyl group of the

4 , 6-0-benzylidene substituent is equatorially attached to

the chair shape of the m-dioxane ring [60, 61].

76
Although study of molecular models revealed that the

cyclic acetal substituents would not sterically prohibit

the adoption of a skew shape by the pyranoid ring of the

dibenzylidene derivatives (were this energetically feasible),

only a small value of J„ j. (0.03 Hz) was obtained from the

iterative analysis of the spectrum of compound 4. Hence,

there is no evidence for any conformational equilibrium

involving that skew shape of the pyranoid ring in which H-2

and H-4 and their intervening carbon atoms have an almost

planar W-arrangement [52].

In conclusion, the favored conformation of l,2:4,6-diC-benzylidene-a-D-glucopyranose derivatives is that in which

the pyranoid ring has a f lattened-chair shape. Nevertheless,

as the coupling constants J „ _ and J~ u of these derivatives

are, for all of the compounds studied, significantly larger

than those of 1, 2-0-alkylidene- and -arylidene-a-gglucopyranoses , there is evidently less conformational

distortion of the di- than of the mono-benzylidene acetals

.

This situation may be ascribed to the tendency of the

4, 6-0-benzylidene substituent to increase the rigidity of the

pyranoid ring.

Numerical data are given in an article that has been

accepted for publication [62].

E . Iterative Analysis of the Proton Magnetic Resonance

Spectra of 1, 2 : 3 , 5-Di -O- isopropylidene -a-Q-glucofuranose

Derivatives

(B. Coxon)

1 . Introduction

In continuation of previous work [48,63,64] on the

conformations of 1, 2 : 3 , 5-di-0-arylidene- and -alkylidene-aB-glucofuranose derivatives, an investigation by p.m.r.

spectroscopy of the conformations of 1, 2 : 3, 5-di-0-

isopropylidene-a-D-glucofuranose derivatives has been started

A necessary preliminary to the assignment of conformation

77
by this technique is analysis of the p.m.r. spectra, which,

depending on the relative magnitudes of the chemical shifts

and coupling constants may be simple or complex. Analysis

of complex spectra is assisted considerably by the

availability of spectrometers that operate at a high

strength of magnetic field (>20 T) , and by use of computer

programs that (a) calculate a trial, theoretical spectrum

and then (b) refine, with the input of suitably assigned,

experimental transition-frequencies, the initial parameters

until the theoretical spectrum and the experimental

transitions converge to a "best" fit according to a criterion

of least squares

.

In the present report are described the chemical shifts

and coupling constants obtained by iterative analysis of (a)

6-deoxy-l ,2:3, 5-di-0-isopropylidene-6-phthalimido-a-Dglucofuranose (1), (b) 6-deoxy-l, 2 : 3, 5-di-<3-isopropylidene-6-

15 15 phthalimido-a-D-glucofuranose-6- N (1- N) , and (c) 6-deoxy1, 2 : 3 } 5-di-(3-isopropylidene-a-2-glucofuranose-d-,

?

( 2-<i,

?

) .

2 . Results and Discussion

15 The synthesis of compounds 1 and 1- N has been described

previously [65,66]. At a radiofrequency of 100 MHz, their

p.m.r. spectra in several different solvents had resisted

analysis, because of overlapping of multiplets . However,

at a frequency of 220 MHz (see figs. 7a and 7b), these

multiplets are sufficiently well separated for a set of

trial parameters to be obtainable by simple, first-order

analysis

.

The p.m.r. spectrum of compound 1 was analyzed by means

of the LA0CN3 program [67] as a seven-spin system consisting

of H-l, H-2, H-3, H-4, H-5, H-6, and H-6 T

. In the iterative

part of the calculation, 66 theoretical transitions were

assigned to 21 experimental line-positions, and variation

of seven chemical shifts and of the coupling constants

78
H-l

(a)

4.23 5.49 5.60 5.72 5.79 6.09 6.14 T

(b)

A

10 Hz

Figure 7. Partial p.m.r. spectra of 1. (a) Observed

spectrum at 220 MHz, and (b) computed spectrum

after iterative analysis as a seven-spin system

79
^1 2' J

2 3' J

3 k>

J

k 5' J

<5 6> J<5 6" and J

6 6' was P ermltted

A good fit of experimental and theoretical spectra (see

figs. 7a and 7b) was obtained after only one iteration. The

refined coupling-constants and chemical shifts are shown in

tables 2k and 25, respectively, together with the

15 corresponding parameters of compounds 1- N and 2.

N CH:

Jl OCH _ o |^o

MeMe

O

O—C-Me

Me

1 (n= un)

1- 15 N (N= 15 N)

CH- CH3

Me-C

I Me.Q

OCH

(CD3 ) 2 CO

O

O-C-Me

I Me

D3 C-CD,C

OO

O-C—CD: CD,

2-d 12

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15 15

For compound 1- N, the introduction of a coupled N

nucleus gave eight nonequivalent nuclei, each with spin

1/2, thus exceeding the capabilities of the LA0CN3 program.

Fortunately, however, H-2 and H-3 are only very weakly

coupled, and so the eight-spin system was divided into (a)

15

a six-spin sub-system consisting of N, H-3, H-4, H-5, H-6,

and H-6', and {b) a two-spin system consisting of H-l and H-2.

Each of these sub-systems was then analyzed separately. For

the iterative analysis of the six-spin sub-system, 60

theoretical transitions were assigned to 26 experimental lines,

and variation of the proton chemical-shifts and the coupling

constants j^^, j^ 5

, j^^, J^,, Jg^,, J

6 15N

, and J

6

, 15

B

15

was allowed, but not of the N chemical-shift, for which

no experimental data are as yet available (for the purposes

of the calculation, this shift was assigned an arbitrary,

fixed value). Iteration proceeded for four cycles, with r.m.s.

errors of the line positions of 0.775, 0.183, 0.157, and 0.157.

For the iterative analysis of the trivial, two-spin sub-system,

the four theoretical transitions were assigned to the four

observed lines of the H-l and H-2 doublets, and variation

both of chemical shifts and of the coupling constant was

permitted. Iteration proceeded for three cycles, with r.m.s.

errors of 0.070, 0.001, and 0.001. The refined parameters

are shown in tables 24 and 25, and the agreement between the

theoretical spectrum calculated from the refined parameters

and the observed spectrum at 220 MHz is depicted in fig. 8.

This theoretical spectrum is a composite of those of the twoand six-spin sub-systems.

Compound 2-cL „ was synthesized by direct exchange of the

isopropy lidene groups in 6-deoxy-l , 2 : 3 , 5-di-C-isopropylidenea-0-glucofuranose (2) with acetone-d/- in the presence of

concentrated sulfuric acid, anhydrous copper(II) sulfate, and

Drierite, The dodeca-deuterated derivative was used for

these studies because, in the p.m.r. spectrum of the non82
(a)

II l5 ^ N- CH-, iro1 ' -CH Me ¥>

Me \L CH—Me

Me

\T 220 MHz

H-4 H-2 H-5 H-3 H-6 H-6

4.22 5.49 5.60 5.71 5.78 6.08 6.13 7"

(b)

Jl

10 Hz \J ill Ml "U.

Figure 8 Partial p.m.r. spectra of 1- y N. (a) Observed

spectrum of solution in benzene-<ig at 220 MHz,

and {b) computed spectrum after iterative analyses

of two- and six-spin systems.

83
deuterated compound 2 at 90 MHz, the H^C-6 signal of interest

was obscured by the signals of the isopropylidene methyl

groups. The experimental line-positions obtained from 2-d, „ were average values of four spectra at 90 MHz. These were

recorded by sweeping twice from low to high frequency, and

twice from high to low frequency.

In the spectrum of 2-<i

1 „, the H-C-6 signal showed as a

sharp doublet due to coupling with H-5- This spectrum was

analyzed as an A^BCDEF system consisting of H_C-6, H-5, H-4,

H-3, H-2, and H-l by using a magnetic-equivalence factoringprogram that has recently become available [68] in iterative

form (UEAITR) . The purpose of this program, for which

compound 2-d-.

?

was designed as a test case, is to use any

magnetic equivalence present in the system (such as that for

the methyl protons) to factorize the Hamiltonian n.m.r.

function, and thereby decrease the size of the corresponding

Hamiltonian matrix and, also, the computer time required.

In previous analyses [63] of seven-spin systems by use of the

LA0CN3 program, it had been found necessary to limit

differentiation of the largest Hamiltonian matrixes to the

diagonal terms, in order that the iterative calculations

should not take an excessive time on the computer.

By using the UEAITR program, computation of a trial,

theoretical spectrum for 2-d-.

? took 25 sec. Initial inputparameters for the calculation were obtained by simple,

first-order analysis of the spectrum, and, indeed, H-2 was

not observably coupled to H-3, as in the 6-deoxy-6-

15 phthalimido derivatives 1 and 1- N, and in other a-Dglucofuranose derivatives [48,63,64]. In the iterative

computation, 118 theoretical transitions were assigned to

17 experimental lines, and variation of the six chemicalshifts and coupling constants J-. „, J~ ~

? J~ k, Ju ,-, and

JV r was permitted. The experimental and theoretical spectra

were in excellent agreement after four Iterations (figs. 9b

and 9a, respectively). The r.m.s. errors of these iterations

84
(a)

H-l H-2 H-3 H-4 H-5

10 Hz

4.06 5.46 5.83 5.92 6.39

l i

i ~n r"> v • •'•"

D,CCH3 0—CH„ Alt?

D 3 C n

6-j-cD3 CD3

AT 90 MHz

Figure 9. P.m.r. spectra of 2. (a) Computed spectrum after

iterative analysis~by magnetic-equivalence

factoring, and (b) observed spectrum of solution

in 8:l(v/v) acetone-^—benzene at 90 MHz.

85
were 0.240, 0.083, 0.080, and 0.080, and the final probable

errors of parameters (see tables 24 and 25) were <:0.028.

The values of J ^ ^ shown in table 24 are considerably

larger than those of the 1 , 2 : 3 , 5-di-0-arylidene and

-alkylidene derivatives studied previously [48,63,64]; this

indicates that the chair shapes of the m-dioxane rings of

15

1. 1- N, and 2-<i, p have either been modified in some way,

or are involved in conformational equilibria different from

those of the derivatives previously studied. This difference

may be attributable to the presence of 1 : 3- syn-diaxial

CH^ —CH„N or CD~ —CH- interactions in the anticipated chair

forms of 1, 1-15/1/, and 2-cLp.

Further work is planned, using variable-temperature

techniques

F . Cyclic Polyhydroxy Compounds

(A. J. Fatiadi)

1. Determination of Inososes with an Alkaline Solution

of Copp er(II) Oxa.latib —Tartrate Complex (the

Somogyi Reagerit) ; St:ructure of the Reagent (by

E.s .rStudies ), and Reaction Mechanisms Involved

Four pentahydroxycyclohexanones (inososes) have been

analyzed with the Somogyi reagent, and empirical equations

for their quantitative determination have been derived. It

was found that reaction of the Somogyi reagent with inososes

affords nonstoichiometric quantities of cuprous oxide, and

that the reducing power varies with the Inosose.

The following inososes were analyzed and equations for

each were determined (see table 26): a racemic mixture of

2^-2 , 3, 4, 6/5-pentahydroxycyclohexanone (D-epi-inosose-2)

(1) and 22-2, 4 , 5 , 6/3-pentahydroxycyclohexanone (2-epiinosose-2) (2), the 2 enantiomorph 2, 2,4,6/3,5-

pentahydroxycyclohexanone (wi/o-inosose-2) (3), and

22-2,3 j, 5/4, 6-pentahydroxycyclohexanone (D -my o-lnosose-1) (4).
G o

T3 CD ^"N

CD > C\J CO O rH

cdO

H C\J

m VD

.D CD II =r VD LP, VD Q V5.

CO

m

0^on

rHCMOCO00

-Q

G + + + + O

-p •H

XXX

X c P CM CM CO

CD cd rHH rH

bO

G

H rH rH rH

cd

a1 • • • • CD H O O O O G

II II II II . CD •H £ • _£

>> >H >H >H >H

•H ro-P

hO 6 O Oe

CM CO

O CO •H m cm

CO cd cd s CO CD G S G O T3 £ O ^ :§: G

-P ^ 1 •H i cd O G P VD LP, VD VD t3

,£ CD LP cd rH rH rH H c\ P • P G P • G

•H

O CD •H cd CM

O £ s T3 e CD •HP OQ cd

CD • «"» •H

CO rH r-{ > E 6 CD CO 13 T3 £ LP II G G \ T3 •H O • CM I MX G

Oh 6 OG + CM ? ro? ^r ? cd O

rH i CO rH r- S G • cd CO

GO P G

rH LT\ COO

•H 1 c

•H P p P cd in -h G cd G on CD G

•H • CD

O -H 6 O CO G 6 G CM CD G

CD 1 CM rH •> CO CX CD P CD CD 1 1 G O P CD CO CO CD CM CD O C CD CD Q CO 1 CO •H -H ,£ T3

CO CO CD

O

p P CO CO CO Cd Oh Cm • G G O G O CO O

VD

HH

G COG

p •H

CM 1 H1 OG

H1 G hO

CD 6 G • CD CD fX, •^ H O Q X3

rH 0) a. 1 Si

G 11 s

X2 1 00

O £ O . G

cd Gill 1 ^ 1 O >H CO

G

Eh Oil

J s Qll cd

D
OH

A hvOH

OH HO

HO

OH

An e.s.r. study of the Somogyi reagent (from aqueous

solution at 77 K), described later, showed that the Cu(II)

complex present in the reagent has mainly a monomeric

structure. By comparison, the e.s.r. spectrum of Cu(II)

complexes at 77 K of the deep-blue Fehling reagent revealed

a monomer-dimer structure, that of the Benedict reagent

showed the presence of a relatively low concentration of

dimer as compared to monomer, and that of the Reeves

cuprammonium reagent showed primarily the monomeric structure

Spectrophotometric and e.s.r. studies of the mechanisms

of oxidation of inososes with the Somogyi reagent at moderate

temperature are in agreement with a one-electron transfer

process; however, at higher temperature, an extensive

degradation of the inosose by the Somogyi reagent is

doubtless caused by generation of transient radicals during

the oxidation, as evidenced by results of a radical-trapping

experiment

The mechanisms suggested for the oxidation of inososes

with the Somogyi reagent at high temperature (involving a transient radical intermediate) and at moderate temperature

(a one-electron, transfer process) are shown in schemes 1

and 2, respectively.

These results have been incorporated in an article that

has been submitted for publication.

88
OH

Cu 2+

O2

OH

OH

OH

+ Cu + + o:

-^ decomposition

R = H,OH

R' = OH

Scheme 1
OH

Cu

2+ OH"

->

R = H, OH

10

11

Scheme 2

90
2 . E.s.r. Evidence for a Dlmer Structure of the Alkaline

Copper (II) Tartrate Complex in the Fehling Reagent

at 77 K

Application of low- field e.s.r. spectroscopy to structural

study of certain frozen Cu(II) complexes [69] and vanadyl

complexes [70] prompted our work. The e.s.r. spectra of

poly cry stalline, frozen (77 K) aqueous solutions of four

reagents commonly used in the carbohydrate field are shown

in fig. 10. The low-field lines of the e.s.r. spectrum of

frozen Fehling reagent (spectrum B) are evidence of some

partially resolved structure; this low-field portion

(1.0 —2.0 kgauss) of the spectrum can be attributed to

forbidden transitions associated with AM = 2 components

2+ 2 + (dimer structure) [70,71], and can arise from Cu Cu

ion-pairs [69]. The AM = 2 transitions occurring as the

2+ 2 + result of Cu Cu dipole interaction should appear at

approximately half the field (about 1.6 kgauss, g ^4) of

the g ^2 (3.3 kgauss) [69-71], and the actual spectrum

obtained at high gain (for spectrum B) shows a structure at

about 1.5 kgauss which is characteristic of a dimer.

A possible structure for the dimer that exists in Fehling

solution at 77 K is one in which two tartrate hydroxylcarboxylate ligands (C^HpOg) bridge the Cu

2+

(0H) Cu

2+

(0H)

ion-pair to give two f ive-membered, chelate rings; a polynuclear, complex structure for this reagent had been

suggested without proof [72].

The e.s.r. spectrum of the Benedict reagent at 77 K

(spectrum D) shows a broad, rather weak, low-field signal

that is somewhat shifted to higher field (1.5 to 2.0 kgauss);

its presence can be ascribed to a relatively low concentration

of dimer, as compared to monomer, although the dimer structure

91
Figure 10. E.s.r. spectra of frozen (77 K) aqueous alkaline

solutions of Cu(II) complexes: spectrum A,

tartrate —oxalate (the Somogyi reagent); B,

tartrate (the Pehling reagent); C, citrate (the

Benedict reagent); and D, tetraamine hydrate

( cuprammonium, the Reeves reagent). All four

low-field e.s.r. spectra on the left side of the

dotted line were recorded at somewhat greater

field-modulation amplitude than the spectra for

the g ^2 region.

92
was found [73] for Cu(II) citrate complex at different pH

values. The very weak (or absent) low-field lines in the

e.s.r. spectra of the Somogyi (spectrum A) and Reeves reagent

(spectrum C) at 77 K are indicative of a preponderantly

monomeric structure for these two Cu(II) complexes.

The spectrum of the monomeric species (AM = 1) shows a characteristic, single, intense, perpendicular component,

and less-intense, four-line, parallel components in the

g ^2 region. Some overlap is observed in all four e.s.r.

spectra, but it is particularly severe in spectrum C. The

magnetic parameters for the monomeric species are given in

table 27-

Table 27. Magnetic parameters for the Cu(II) complexes of

the Somogyi (A), Pehling (B), Reeves (C), and

Benedict (D) reagents at 77 K

-1 -4

Reagent gi gi A i

i , cm x 10

A 2.058 ±0.003 2.302 ±0.005 164 ±10

B 2.048 ±0.004 2.294 ±0.003 188 ±10

c 2.072 ±0.001

D 2.065 ±0.003 2.291 ±0.002 165 ±5

Indicated limits of error for the g and A values are

maximum estimated errors.

The presence of extra features in the high-field portion

(AM = 1) of the e.s.r. spectra for A, B, and D (see fig. 10,

indicated by arrows) could readily be confused with the

perpendicular components from 63 Cu and 6

5

Cu nuclei; however,

it is known [74,75] that separation of lines due to 6 3 Cu and

6

5

Cu is a difficult task that requires a special technique.

However, the extra peak in the e.s.r. spectrum of the

Fehling reagent (77 K) was also observed for two samples

of the reagent prepared from salts of pure

53 Cu and 6

5

Cu

isotopes, respectively.

93
When, for either the Somogyi or the Fehling reagent,

the frozen solution is warmed to room temperature, an extra

signal is produced (at 3-245 kgauss for A), in addition to

the usual, four-line, e.s.r. spectrum of the Cu(II) complex.

The origin of this and other extra signals may be associated

with the presence of paramagnetic superoxide, ion-ligand . The author thanks T. Murphy for specimens of salts of

pure 6 3 Cu and 65 Cu, and N. Adams for the e.s.r. measurements

3 . Preparation of Inositol Hexasulfate

Inositol hexasulfate (1) is conveniently prepared by

direct interaction of my o-lnosltol either with fuming

sulfuric or chlorosulfonic acid. Hexasulfate 1 was isolated

1 R = S03 H

2 R = SO3K

from the reaction mixture as its hexapotassium salt (2) in

55 —62% yield by (a) an exchange reaction with potassium

chromate, or (b) direct neutralization with potassium

hydroxide

Fuming sulfuric acid containing 30% of sulfur trioxide

was placed in an Erlenmeyer flask provided with a magnetic

stirring-bar, a thermometer extending to the bottom of the

flask, and a loosely stoppered drying-tube containing

calcium chloride. The acid was heated to 60 °C (oil bath),

94
and finely powdered mz/o -inositol was introduced in small

increments during 2.75 —3 h. The reaction is exothermic,

and the temperature of the reaction mixture should be kept

at 60 —65°C by cooling. If the temperature exceeds 80 °C,

some decomposition usually ensues, as shown by darkening

of the solution. Sometimes, near the end of the reaction,

the temperature must be raised to 70 —75 °C for 10 to 15 min,

in order to dissolve any unreacted material. The light-yellow

to brownish yellow solution containing my o-lnosltol

hexasulfate was then cooled to about 35 °C, and was carefully

poured into a stirred slurry of a solution of potassium

chromate (10$ excess) in water with crushed ice. The

greenish yellow suspension was then diluted with cold

methanol, stirred for 30 min at —3 °C, and filtered. The

precipitate was washed successively with ice-cold water and

ice-cold 1:1 water—methanol. The wet, somewhat colored

product was then recrystallized from hot water; cooling and

washing produced snow-white needles of 2 (as the dihydrate

when dried in vacuo at room temperature); yield 53 —55%; the

2- 2 + test for S0|, by addition of Ba was negative. This

procedure has been published [76].

3. GENERAL ORGANIC CHEMISTRY

(A. J. Fatiadi)

A . Novel Iodination of the q-Methylene Group in 1,3-

Diphenyl-1, 3-propanedione and Related g-Diketones

with Periodic Acid

In continuation of a study [77] of new applications of

periodic acid in organic chemistry [78], uses of this

oxidant have now been extended to the specific iodination

of the a-methylene group of certain B-diketones

.

95
A solution of 1, 3-dipheny 1-1, 3-propanedione (la, enol

content In chloroform-d, ^92%, by p.m.r. spectroscopy) in

acetic acid was stirred with an aqueous solution of

paraperiodic acid at 95 °C for 15 min, and then at room

temperature for 60 min. The mixture was next treated

with water at room temperature to yield snow-white needles

of the 2-iodo derivative lb.

R H O^-H H O^H

Ph—C—C —C —Ph Ph—C— C—C —Ph Ph —C—C—C— Ph

II \ + A 1 \ + A

O R' O jj o jJ

OH

u, R = R'= H

b, R = H, R'= I

c, R = R'= I

d, R = R'= Br

The mass spectrum (probe-temperature, 30 °C) of lb did

not show the molecular ion (m/e 350), but did exhibit an

ion peak at m/e 223 due to elimination from lb of iodine,

2+

~

observed as a molecular ion at m/e 25 4 (Ip ) and as an ion

peak at m/e 127 (I + HI). The molecular ion (m/e 350)

was observed at higher probe-temperature (40 °C). Compound

lb showed AMe0H 250 (e M 18.5) and 330 nm (e M 1); A KBr 5-90

max mAf mM ' max

and 6.00 (C=0, non-hydrogen-bonded), 15-30 and 16.68 ym

i (-C-I); p.m.r. data in CDC1 , 67-30 —8.20 (m 10 H, aromatic)

x -J and 66.97 (s, 1 H,-CH=C ). The monoiodo derivative lb was

also obtained (in 50^ yield) on treatment of la in acetic

acid with an aqueous solution of iodic acid at 95 °C for

20 min and at room temperature for 24 h.

96
Treatment of an acetic acid solution of la with an

aqueous solution of paraperiodic acid at 95 °C for 30 min and

at room temperature for 30 min gave yellow crystals of the

2,2-diiodo derivative (lc) (19 —21$), m.p. 152—153 °C (95$

EtOH). Compound lc exhibits moderate stability (loss of

iodine occurs, to give lb) on storage as the solid, or in

solution; it shows moderate loss of iodine on

recrystallization from ethanol. The mass spectrum (LKB-9000,

probe-temperature, 60 °C) exhibited a molecular-ion peak at

MpOH

m/e 476; other spectral data: a 248 (e ,, 3.1), 290(sh) maXK"R'R (e „ 4.9), and 440 nm (e ,. 0.2); X 6.03 (C-0, nonmM mM max , hydrogen-bonded), 15-28, 16.28, and 17-65 ym (-C-I); p.m.r.

data in CDC1-, 67-20 —8.10 (m, 10 H, aromatic).

When iodic acid was used instead of periodic acid,

little or no formation of the diiodo derivative lc was

observed. The use of a larger proportion of the reagents

(iodic or periodic acid) did not improve the yields of

the products lb or lc

.

Compounds lb and lc were readily converted into the

known dibromo derivative Id, m p 94 —96 °C, on treatment of

their solution in carbon tetrachloride with bromine at room

temperature. Compounds lb and lc were converted into the

starting material la on extraction of their chloroform

solutions with an aqeuous solution (5%) of sodium hydrogen

sulfite

.

On treatment of solutions of the foregoing compounds in

methanol with a solution of cupric acetate in methanol, the

bis-copper chelate complex was precipitated, immediately for

la, after three min for lb, and not at all after 24 h (a

deep-green solution) for compounds lc and Id; this test serves

to distinguish between these compounds. The progress of

formation of the product was monitored by p.m.r. spectroscopy

(CDClO as follows: for disappearance of the enolic

hydroxyl groups at 517.16 for la, a shift of the peak at

97
66.82 (-CH=C\ ) for la to 66.97 (-CH=cO for lb, and

disappearance of the latter for lc.

Formation of the mono-iodo derivative from other acyclic

(enolic) 8-diketones has been observed; for example, from

1 , 3-diphenyl-l, 3-butanedione, 2, 4-pentanedione (which gives

a vigorous reaction), 2,4-hexanedione, hexafluoro-2 ,4-

pentanedione, and others.

Application of /l/-iodosuccinimide, used in iodination

of the methine group of certain enol acetates [79] s or of

other iodinating reagents [80], failed to iodinate the

methylene group in la. When sodium metaperiodate was used

instead of periodic acid, or the acetic acid was replaced by

Nj W-dimethylformamide or p-dioxane, none of the reactions

mentioned were observed.

E.s.r. monitoring of the reaction of periodic acid with

la showed no radical participation, indicating the ionic

character of the iodination process. The behavior of

periodic (or iodic) acid towards la indicates structural

lability of the I — bond in the reagents. This high

lability of the I — bond may be associated with a relatively weak, double-bond character in the I — bond

[8l]. Iodination of la can be envisaged as involving

addition of the iodonium ion to the double bond, to give

the intermediate 2, although addition of a molecule of

hypoiodous acid, or its anion (an iodoso ion 10 ), with

subsequent elimination of a molecule of water to give lb,

as shown in intermediate 3> is not excluded. The relatively

modest yield of the iodo compounds may be due to an

association of the enolic diketone with the reagent [82]

that could impede formation of intermediates 2 and 3-

The results were incorporated in an article that has

been published [83].

98
Degradation of Bilirubin with Chromic Acid

bilirubin (1) was suspended in acetone, cooled to about

Bilirubin

1, R = -CH= CH 2

P = -CH2CH2 C02 H

10 °C, and treated with chromic acid [84 —86] (1% CrQ in 2 M

sulfuric acid). The suspension was stirred for two hours;

by this time, all solids had dissolved to give a deep-brown

solution. The latter was extracted with ether, dried

(sodium sulfate), concentrated to a thin syrup, neutralized

with aqueous sodium carbonate, and filtered. The clear

filtrate was extracted with 4:1 (v/v) ether—dichloromethane,

and the extract was washed with water, dried (sodium sulfate),

and set aside (solution A) for isolation of 3.

The alkaline extract was neutralized (6 M hydrochloric

acid) to about pH 2 and extracted with ether. The extract

was washed, dried, evaporated to a syrup, and left to

crystallize in an open dish at room temperature for 72 h,

to give crystalline, crude hematinic acid (2), m p 96 —98 °C.

The product was dissolved in ether, the suspension was

filtered (carbon) , and the filtrate chromatographed on a column (1 x 20 cm) of silica gel, and eluted with 1:5 (v/v)

ether-—pentane; a middle fraction was collected, evaporated

to dryness, and the crystals recry stallized from ether

pentane (carbon); white crystals of 2, m p 110 —112 °C; lit.

[87] m p 112—113 °C.

99
H 3 C

Hematinic acid

Extract A was evaporated to dryness, the product dissolved

in ethyl acetate, and the solution chromatographed on a column (1 x 20 cm) of Florisil by elution with 1:6 (v/v) ethyl

acetate—pentane and then with 1:3 (v/v) ethyl acetate

pentane . A syrupy product was obtained; on crystallization

from ^60% aqueous ethanol (carbon) and cooling (72 h), it

gave microcrystalline needles of 3 S m p 66 —67 °C; lit [87]

m p 68 —69 °C . Compound 3 was homogeneous by t.l.c. [Silica

H 3 C

CH3 CH 2

H

2

- Ethyl-3-methylmaleimide

100
Gel G; 1:5:3 (v/v) cyclohexane—carbon tetrachloride —ethyl

acetate (solvent A); with a spray of 3% solution of benzidine

in methanol]; R„ 0.44 ±0.02, close to that reported [85,86].

It is interesting that the oxidation mixture from 1

(containing five or six components by t.l.c.) included a substantial proportion of an unknown component showing a strong blue fluorescence; the presence of a component with

this property was not reported by Rudiger and co-workers

[84 —86] in their comprehensive study of degradation products

of bilirubin and biliverdine derivatives . The presence of

this component was also observed In degradation products of

bilirubin and hemin with periodic acid (see later).

The unknown fluorescent component has the following

R„ values: solvent A [85], O.76 ±0.03, solvent B [4:1 (v/v)

benzene —ethyl acetate], 0.70 ±0.02. The unknown compound

can be isolated by use of a Florisil column with solvent B.

C . Degradation of Hemin and Related Bile Figments with

Periodic Acid

Bile pigments are usually degraded by chromic acid [85,86];

however, It has been found in this laboratory that aqueous

solutions of periodic acid and even iodic acid can oxidize

porphyrins (hemin, hematin, heme), as well as bile pigments

(bilirubin, biliverdine, urobilin, and others). Usually,

the co-solvent used in these oxidations is acetic acid,

tetrahydrofuran, or acetone. Although acetone in the presence

of periodic acid and oxidizable substrate produces substituted

iodo-acet ones , degradation of hemin (4) in the presence of

this solvent proceeds faster than in the other solvents.

However, for a systematic study of the oxidation of bile

pigments with periodic acid, use of tetrahydrofuran—acetic

acid as co-solvent was found advantageous. The degradation

products are usually identified by t.l.c. (activated Silica

101
H 2 C = CH

H,C

H 3 C

CH = CH.

Hemin (ch loro hemj n)

Gel G, 20 x 20 cm or 5 x 20 cm plates, solvents A [85,86] or

B, and 3% benzidine solution in methanol as the spray [85^86]

to give fluorescent spots), by comparison of their R values

b with those of authentic samples and with those reported in

the literature.

Hemin was suspended in acetone, treated with 4 M aqueous

periodic acid and stirred for 3 h at room temperature. The

white solid precipitated was filtered off, and washed with

acetone, and the filtrate was neutralized with 10% aqueous

sodium hydrogen carbonate. The neutral solution was

extracted with 4:1 (v/v) ether—dichloromethane, and the

extract was thoroughly washed with water, dried (sodium

sulfate), and evaporated to give a very pungent syrup

(neutral fraction)

.

102
T.l.c. analysis of the neutral fraction showed components

having the following R values (solvent A [85,86]): (a)

h before spraying with benzidine spray, R 0.74 (blue

h

fluorescence) and 0.41 (strong blue fluorescence); (b) after

spraying with the benzidine spray, i?_ 0.91 (yellow with

t purple fluorescence), 0.74 (blue fluorescence), 0.44 (yellow),

0.41 (blue fluorescence), 0.37 (red-purple), 0.30 (weak

yellow), 0.24 (strong purple-brown), 0.17 (weak yellow),

0.075 (strong purple), and 0.00 (at origin). The spot having

R„ 0.44 corresponds to compound 3 [85,86], and that of i?„

h ~ h

0.24, to 2-ethylidene-3-methylsuccinimide [85,86].

On acidification of the sodium hydrogen carbonate

solution with hydrochloric acid, extraction with ether, and

evaporation, a small amount of crude 2 was isolated.

D . Isolation of a Biliverdine Type of Reaction Intermediate

on Oxidation of Bilirubin with Periodic Acid

Treatment of a solution of bilirubin (1) in methyl

sulfoxide with an aqueous solution of periodic acid, with

stirring at 15—20 °C for 10 min, gives at first a dark-green

solid which can be filtered off (prolonged treatment of

this solid with periodic acid causes degradation) . The

solid can be recrystallized from methanol —ether, to give a MeOH bluish green, microcrystalline solid 5 (A 226, 272 sh,

~ max

340, 372, and 655 nm) having a biliverdine type of

absorption; the product contains a free iodine atom and is

believed to be a charge-transfer complex.

A sample recrystallized from methanol —pentane (room

temperature) and dried at 110 °C/10 mtorr for 2 h had C = 53.3,

H = 4.61, N = 7.45, and I = 16.1%. However, the proportion

of iodine ( I ) in 5 varies from one preparation to another,

n ~ and this is characteristic of certain iodine charge-transfer

compounds [88]

103
O N

Me

1, Bilirubin

n

H 5 I06 NaBH4

5, Bijiverdine -iodine complex

where P = —CH2 CH2 C02 H

R = -CH=CH2

Treatment of a methanol solution of complex 5 with sodium

borohydride in methanol at room temperature, followed by

acidification with 3 M hydrochloric acid and extraction with

chloroform, with thorough washing of the extract with water

0HC1 (which is imperative),3 gives to bilirubin,3 X 3 ^51 nm

max

(e 59.9), in 80$ yield. This is the first report of a biliverdine derivative being reduced to bilirubin (1) without

either the olefinic or carbonyl groups in 1 being affected.

A note on this study is in preparation for publication.

104
4. DETECTION AND IDENTIFICATION OF TRACES OF ORGANIC

COMPOUNDS

In NBS Technical Note 507, we reported some thin-layer

chromatographic techniques for detecting low levels of two

compounds, namely, 2,4 ,6-trinitrotoluene (1) and hexahydro1 s 3 , 5-trinitro-s-triazine (2). Alternative means for their

H 3 \ /NO;

NOdetection at low level have been investigated, where

there seemed to be potential for lower levels of detection,

or interferences with detection might be minimized or averted

A. 2,4 ,6-Trinitrotoluene (1)

(A. Cohen)

We had reported for compound 1 that, after development

on a silica gel plate with 9:1 (v/v) benzene —pentane, and

spraying of the dried plate with ethylenediamine in methyl

sulfoxide [89], faint pink spots were detectable with as

little as 15 ng of compound. These spots did not fluoresce

under 366-nm light. However, we now find that, when the

sprayed plate is dipped in liquid nitrogen [90], and

immediately examined under 366-nm light, the spots fluoresce

(pink), and 10 ng of 1 is detectable.

105
B. Hexahydro-l,3 a 5-trinitro-s- triazine (2)

1 . Detection of 2 by Diazonlum Coupling

(A. Cohen)

With compound 2, we had reported its detection at low

levels by spraying the developed plate with methanolic

potassium hydroxide and heating at 120 °C for 5 min. This

produces potassium nitrite, which can oxidize a sprayed-on

solution of Benzidine Blue [91] to give weak-blue or

purplish spots (nonfluorescent on cooling in liquid nitrogen).

The production of N0~ offers the potential for using a whole series of reagents, via the diazonium coupling-reaction,

which could provide colored products of fluorescent compounds

that might lead to still lower levels of detection. For

example, after completing the treatment of 2 to produce

potassium nitrite, the plate can be sprayed with 0.5% of

sulfanilic acid in 1:1 water—acetic acid and with 0.3% of

Wj/V-dimethyl-1-naphthylamine in 1:1 water —acetic acid [92];

in that publication, 1-naphthylamine was used. Quantities

comparable to those with the Benzidine Blue spray can be

detected, but" these spots are pink. They fluoresce when

treated with liquid nitrogen and 366-nm light.

2 . Detection of 2 by Formation of Formaldehyde

(A. Cohen)

Formaldehyde is liberated from 2 on treatment with

concentrated sulfuric acid [93] s and J acid (6-amino-lnaphthol-3-sulfonic acid) (0.5$) in sulfuric acid [9^1 can

detect the formaldehyde liberated. After spotting with 30

ng of compound 2, developing as usual, spraying with the J

acid reagent, and warming for 8 min at 120 °C, a very weak

yellow fluorescence appears under 366-nm irradiation. This

fluorescence is enhanced when the plate is immersed in

liquid nitrogen and immediately examined under u.v. light.

106
3 . Detection of 2 by Formation of Hydrazine

(W. J. Rossiter, Jr.)

In conjunction with the preceding studies, additional

experimentation on the detection of trace quantities of 2

was attempted. It was proposed that hydrazine, generated

by reduction of 2 with zinc and acetic acid, might provide

the basis for detection of 2. There is literature precedence

for the production of hydrazine from 2 [95]: hydrazine had

been isolated as aromatic azine derivatives from the

reduction of 2 [95] » as well as of hexahydro-1 ,3, 5-trinitrosos-triazine [96] with zinc and aqueous base. In both cases,

the reaction solutions had been neutralized with acid (and

thus, possibly, acidified) before the addition of the

aromatic aldehyde that trapped the hydrazine.

On exposure to acid vapors, aromatic nitro compounds are

known to be reduced on t.l.c. plates coated with silica gel

in which zinc has been incorporated [97]. This method was

chosen for the reduction of 2. Thus, after spotting with

known quantities of 2, zinc-containing t.l.c. plates were

immersed in an atmosphere of acetic acid vapor for various

lengths of time at various temperature; then the plates were

removed and sprayed with an alcoholic solution of

p-(diethylamino)benzaldehyde . Variations in the reaction

conditions produced some plates on which, after spraying,

orange spots appeared at points where 2 had originally been

spotted. These orange spots were assumed to represent

bis[p- (diethylamino)benzal ]azine formed in the reaction of

the generated hydrazine with p- (diethylamino)benzaldehyde

.

Maximization of the reaction conditions afforded detection

of 2 at a level of 25 ng.

We wished to demonstrate rigorously that hydrazine was,

indeed, being generated in the reduction and then visibilized

as a colored, aromatic azine. This confirmation was

accomplished by the combined techniques of t.l.c. and u.v.

107
visible spectrophotometry. By t.l.c, it was demonstrated

that the colored spot generated on the plate from 2 migrated

in different solvent systems with the same R values as those

t displayed by authentic samples of the aromatic azines

expected. Two azines, bis[p- (diethylamino)benzal ]azine and

salicylalazine, were used for this test. Next, when examined

by u.v. —visible spectrophotometry, an extract of the orange

spot generated on the t.l.c. plate from 2, and assumed to be

bis[p- (diethylamino)benzal]azine, exhibited the same, longwavelength, absorption band as an authentic sample of

bis [p-(diethylamino )benzal]azine . This evidence, coupled

with the literature data [95 5 96] previously cited, leads to

the conclusion that hydrazine is generated on the t.l.c.

plate from reduction of 2 with zinc —acetic acid.

Other known methods [98 —104] for detection of hydrazine

were next investigated, in an effort to improve the detection

of 2. Table 28 lists the various color-spot tests employed;

as the table shows, only salicylaldehyde enhanced the

detection of 2; the lower limit of detection with this

reagent is 10 ng when the t.l.c. plate is examined under a 366-nm lamp immediately after immersion in liquid nitrogen.

Without cooling in liquid nitrogen, the limit of detection

with salicylaldehyde is ^50—60 ng.

As 2 was being detected at the lowest level by the

fluorescence of salicylalazine, it was thought that extraction

of the salicylalazine spot and fluorimetric analysis of the

extract might possibly provide additional, and more conclusive,

evidence for the presence of 2. However, it was subsequently

determined that salicylalazine does not fluoresce in solution

[105]. Accordingly, a study was undertaken to find a substituted aromatic aldehyde or ketone that could be

employed in the detection of trace quantities of 2 through

the formation of an azine that does fluoresce in solution.

Of a number of eligible compounds, 2-hydroxy-l-naphthaldehyde

108
Table 28. Reagents used to detect hydrazine after generation

from 2 on a t.l.c. plate

Reagent

p-Et

2

NC

6

H lj CHO <

p-Me

2

NC

6

H i| CH=CHCHO'

o-HOCgH^CHO"

Picryl chloride'

2-Nitro-l,3-

indandione

Ammoniacal silver

nitrate

References

98

99, 100

0HCCH=CHCH

2

CH0 101

p-Me

2N-o-HOCgH CHO'

102

103

104

Limit of Visibilization on

detection the t.l.c. plate

of 2 (ng)

25

25

10

120

50

<500

<500

<500

a faint yellow spot

on a colorless background

with a small amount,

a dark spot on a yellow background;

in larger quantity,

the spot is red

under the 366-nm

lamp, with liquidnitrogen cooling, a faint pink spot on

a blue background

red spot on a yellow

background

under visible light,

light yellow on a colorless background;

under the 366-nm lamp,

pink on a maroon

background; no color

enhancement on liquidnitrogen cooling

orange spot on a brown background

no distinct color;

light spot on a tan

background

brown spot on a gray

background

Reagent used was 1% , in absolute alcohol solution.

'Reagent formed from 2% aqueous 1- ( 4-pyridyl)pyridinium

chloride solution by mixing with an equal volume of 2M

,NaOH solution immediately before spraying.

'Formed in the usual way for preparing the Tollens reagent.

109
appears the most promising of those tested thus far. By

using this aldehyde, 2 can be detected on a t.l.c. plate at

the 50-ng level. Experimentation is in progress to determine

whether the 2,

T -dihydroxy-l ,1 '-napthalazine can be extracted

from a t.l.c. plate and the fluorescence spectrum of the

extract measured without interference.

4 . Detection of 2 by the Franchimont Test

(W. J. Rossiter, Jr.)

The Franchimont test [106] is an analytical colorreaction specific to the W-nitro group both of /V-nitroamines

and /V-nitroamides . The basis of this test is that the Nnitro compound produces a characteristic color, usualy violet,

when added to a mixture of zinc and acetic acid in the

presence of certain aromatic amines. W-Nitroso compounds

produce no color under identical conditions.

We have adapted this test to the low-level detection of

2. The test, as employed for our purposes, has some

similarity to the preceding method of detecting 2 through

hydrazine formation. Thus, the zinc that is required for

the test is incorporated into the silica gel used for

coating the thin-layer plate, and acetic acid is used as the

vapor. However, the difference between the two methods of

detecting 2 is that, in the Franchimont test, the t.l.c.

plate is sprayed with an alcoholic solution of an aromatic

amine and dried before immersion in the acetic acid vapor.

The amine used was 2-naphthylamine

.

Our adaptation of the Franchimont test is conducted in

the following simple way. A quantity of 2 is spotted on a t.l.c. plate precoated with Silica Gel GF-25^ containing 5%

of zinc dust (by weight). The plate is sprayed, until wet,

with a 1% alcoholic solution of 2-naphthylamine, and, after

partial air-drying, the plate is placed in an atmosphere of

acetic acid vapor, where it is allowed to remain for 30 min

110
before being removed. During this time, if a sufficient

quantity of 2 is present, yellow spots start to appear. At

the end of the 30-min reaction period, spots of 2 in the 2.0-

to 5.0-yg range are yellow with a violet tinge; in the 0.1-

to 1.0-yg range, they are pale yellow; and, below 0.1 yg,

they cannot be observed in ordinary, visible-wavelength light.

However, under the 366-nm lamp, spots representing quantities

of 2 as low as 5 ng can be observed by fluorescence. At the

5-ng level, the spot is a pale, light blue on a deeper blue

background. As the amount of 2 is increased, the intensity

of the fluorescence increases, but there is no apparent

increase in color intensity when the t.l.c. plate is immersed

in liquid nitrogen.

Other aromatic amines have been examined in this t.l.c.

version of the Pranchimont test. Many produce a yellow to

violet-yellow spot on the t.l.c. plate. However, none tested

has produced a fluorescent spot. Thus far, only with

2-naphthylamine in this test has this low level of detection

of 2 been achieved.

Little seems to be known about the mechanism of the

Franchimont test [107]. It is possible that such knowledge

would aid in its further exploitation.

5 . Spectrodensitometric Means of Detecting Low

Levels of 2

(A. Cohen)

Spectrodensitometric methods for detecting 2 at low

levels were examined. This procedure affords the possibility

of detecting the compound without the destruction that occurs

when spray reagents are used, and thus, of recovering the

compound for further identification. Several commercial

densitometers were used in the examination of 250-ym thick,

silica gel plates, fluorescent and nonfluorescent , that had

been spotted with 30 — 60 ng of compound 2, developed, and

dried. The plates were examined by u.v. absorption and by

111
fluorescence quenching- reflectance methods. Usually, a larger amount of the compound had to be spotted also, in

order to determine the approximate region where the soughtfor trace of the compound might be. Its exact location

might still require mm-by-mm examination of the region.

The smallest amount of compound detectable with a spectrodensitometer was at the 30-ng level. To detect this

amount requires high instrument-gain and/or recorderexpansion. Hence, background noise is substantial. A

major part of this high background may result from

imperfections in the adsorbent layer or, possibly, unequal

distribution of the fluorescing material in the adsorbent.

These imperfections may limit the usefulness of this

technique

.

112
5. PERSONNEL AND ACTIVITIES

A. Personnel Listing

R. Schaffer, Section Chief

R. S. Tipson

B. Coxon

A. J. Fatiadi

R. F. Brady, Jr.

W. J. Rossiter, Jr.

B. F. West

A. Cohen

J. H. Thomas

B. A. Johnson

D. W. Vomhof, Research Associate to 9-26-69

D. Niner, Guest Worker (1969)

J. James, Guest Worker (1970)

B. Publications

NBS Technical Note 507. Organic Chemistry Section.

7/1/68 - 6/30/69.

Edited by R. Schaffer (October, 1969).

Synthesis of the Two D_-2-Pentuloses • New Derivatives

of ^-ery thro -Fentulose . R. S. Tipson and R. F. Brady, Jr., Carbohyd. Res. 10 , 5^9 (1969).

15 Studies of N-Labeled Amino Sugars. Synthesis and

Spectroscopy of Derivatives of 6-Amino-6-deoxy-0-

glucose-£-l5ii7.

B. Coxon, Carbohyd. Res. 11, 153 (1969).

Review of Organic Structure Determination, by D. J.

Pasto and C. R. Johnson.

B. Coxon, Capital Chemist 1_9, 143 (1969).

Review of Organic Synthesis, by R. E. Ireland.

R. F. Brady, Jr., Capital Chemist 19. 239 (1969)

.

Phenylhydrazono—phenylazo Tautomerism. Part III.

Reactions of Phenylformazans and Certain

Bis (phenylhydrazones) with Strong Acids.

H. S. Isbell and A. J. Fatiadi, Carbohyd. Res. 11,

303 (1969).

113
A Comparison of the Spectrophotometric and Gaschromatographic Methods with the Vacuurn-oven Procedure

for the Determination of Moisture in Starch Hydrolyzates

D. W. Vomhof and J. H. Thomas, Ann. Proc . Am. Soc

Brewing Chemists, 1969 , 139.

Novel Aromatization of a Trihydroxycyclohexanetrione

(Triket oinositol ) to a Dibenzo-p-dioxin Derivative.

A. J. Fatiadi, Carbohyd. Res. 12, 130 (1970).

Preparation of Inositol Hexasulfate.

A. J. Fatiadi, Carbohyd. Res. 12, 293 (1970).

Novel Iodination of the a-Methylene Group in 1,3-

Diphenylpropane-1, 3-dione and Related 3-Diketones

with Periodic Acid.

A. J. Fatiadi, Chem. Commun. 1970 , 11.

Conversion of Certain Cyclic Phenylhydrazino Derivatives

into Phenylazo Compounds with Periodic Acid.

A. J. Fatiadi, J. Org. Chem. 35, 831 (1970).

The Synthesis, Equilibration, and Conformations of

Diastereoisomeric 1 , 2-0-Isopropylidene-3 ,5-0- (methoxymethylidene )-6-0-p-tolylsulf onyl-a-P_-glucofuranoses • Conformational Evidence from a Nuclear Overhauser Effect.

B. Coxon, Carbohyd. Res. 12, 313 (1970).

Review of The Amino Sugars. The Chemistry and Biology

of Compounds Containing Amino Sugars; Volume IA

,

Chemistry of Amino Sugars, edited by R. W. Jeanloz.

B. Coxon, Carbohyd. Res. 13., 319 (1970).

Model Parameters for the Analysis of Skew Conformations

of Carbohydrates by P.M.R. Spectroscopy.

B. Coxon, Carbohyd. Res. 13, 321 (1970).

Review of Biokhimiya. Uglevody . Uspekhi v Izuchenii

Stroeniya i Metabolisma, by B. N. Stepanenko.

A. J. Fatiadi, Carbohyd. Res. 13., ^72 (1970).

C . Manuscripts in Process of Publication

Isotopic Methods in Carbohydrate Chemistry.

R. Schaffer. (Manuscript accepted for "The

Carbohydrates," edited by W. Pigman and D. Horton,

Chapter JI5, Vol. 2B, in press)

Occurrence, Properties, and Preparation of Naturally

Occurring Monosaccharides, Including 6-Deoxy Sugars.

R. Schaffer. (Manuscript accepted for "The

Carbohydrates," edited by W. Pigman and D. Horton,

Vol. IA , in press)

114
Infrared Spectroscopy of Carbohydrates.

R. S. Tipson and P. S. Parker. (Manuscript accepted

for "The Carbohydrates," edited by W. Pigman and

D. Horton, Vol. IB, In press)

Conformational Analysis via Nuclear Magnetic Resonance

Spectroscopy

.

B. Coxon. (Manuscript accepted for "Methods in

Carbohydrate Chemistry," edited by R. L. Whistler

and J. N. BeMIller, Vol. 6, in press)

The Conformations of 1 , 2: 4 , 6-Di-0-benzylidene-a-r3-

glucopyranose Derivatives.

B. Coxon. (Manuscript accepted for publication in

Carbohydrate Research)

Electron-spin Resonance Study of an Alkaline Solution

of Copper(II) Oxalate—meso-Tartrate Complex (the

Somogyi Reagent) and Related Complexes.

A. J. Patiadi. (Manuscript accepted for publication

in J. Res . NBS

)

Cyclic Acetals of Ketoses. Part III. Re-investigation

of the Synthesis of the Isomeric Di-O-isopropylidene-Dfructopyranoses

.

R. F. Brady, Jr. (Manuscript accepted for publication

in Carbohydrate Research)

Cyclic Acetals of Ketoses. Part IV. Re-investigation

of the Oxidation of 1 , 2: 4 ,5-Di-0-isopropylidene-3-Dfructopyranose with Methyl Sulfoxide—Acetic Anhydride.

R. S. Tipson, R. F. Brady, Jr., and B. F. West.

(Manuscript accepted for publication in Carbohydrate

Research)

Determination of Moisture in Starch Hydrolyzates by

Near-infrared and infrared Spectrophotometry.

D. W. Vomhof and J. H. Thomas. (Manuscript accepted

for publication in Analytical Chemistry)

Determination of Moisture in Syrups by Gas —-Solid

Chromatography

.

D. W. Vomhof and J. H. Thomas. (Manuscript submitted

for publication in J. Ass. Of fie . Anal. Chem.)

Determination of Inososes with an Alkaline Solution of

Copper(II) Oxalate —Tartrate Complex (the Somogyi

Reagent); and Reaction Mechanisms Involved.

A. J. Fatiadi. (Manuscript submitted for publication

in Carbohydrate Research)

Evidence for Adsorption as the First Step in the Solidstate Oxidation of Benzenehexol with Active Manganese

Dioxide

.

A. J. Fatiadi. (Manuscript completed)

115
D . Lectures

15

9/8/69 —Studies of ^N-Labeled Amino Sugars. Synthesis

and Spectroscopy of Derivatives of 6-Amino-6-

15 deoxy-D-glucose-£- N. Carbohydrate Division,

158th American Chemical Society Meeting, New

York, New York. B. Coxon . 12/V69 -Chemistry of Keto Sugars. Student Affiliate

Chapter of the American Chemical Society, Mount

Saint Mary's College, Emmitsburg, Maryland.

R. F. Brady, Jr . 12/17/69— The Aldol Reaction in Carbohydrate Chemistry.

Chemistry Department, American University,

Washington, D. C. R. Schaffer . 3/18/70— Current Status of Clinical Standard Reference

Materials. Northeast Section of the American

Association of Clinical Chemists, Boston,

Massachusetts. R. Schaffer . 3/19/70 — Current Status of Clinical Standard Reference

Materials. Clinical Chemistry Laboratory,

Hartford Hospital, Hartford, Connecticut.

R. Schaffer.

3/23/70 —Bilirubin Standard Reference Material. 5th Annual

Meeting of the Association for the Advancement of

Medical Instrumentation, Boston, Massachusetts.

R. Schaffer . 5/27/70 —Experiences with Computerized, Iterative Analysis

of the N.M.R. Spectra of Carbohydrates. Joint

ACS—CIC Meeting, Toronto, Ontario, Canada.

B. Coxon.

116
6. ACKNOWLEDGMENTS

The editor gratefully acknowledges the cooperation of

his associates in the Organic Chemistry Section in the

preparation of this report. Special thanks are due Dr.

R. S. Tipson for facilitating the editorial effort.

Miss Barbara Hyde, Section Secretary, brought the manuscript

to final form. We wish also to thank Mrs. R. S. Maddock,

Administrative Assistant for the Division of Analytical

Chemistry in Charge of Publications, for assisting in its

publication

117
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77, 5337 (1955).

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