Preparation and properties of several polysaccharide sulfates

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Preparation

$6,

and Properties of Several Polysaccharide Sulfates’

ROY L. WHISTLER From

the

Department

3641 (1961)

of Biochemistry,

AND

W. W. SPENCER

Purdue

Received April

University,

Lafayette,

Indinrla

3, 1961

Amylose, amylopectin, guaran, cellulose, and laminaran are sulfated to different degrees with very little degradation in dimethylformamide with triethylamine-sulfur trioxide complex at 0”. A 1 + 6 linked dextran and alginate are unreactive. Periodate oxidation of the polysaccharide sulfates of degree of substitution of one suggests that half of the sulfuric ester groups may be on position C-6. Amylose, amylopectin, and guaran sulfates precipitate solutions of gelatin, egg albumin, and methylene blue but not human plasma. They form films but absorb large amounts of moisture and readily disperse in water, Unlike carrageenan their solutions do not gel with potassium or ammonium ions. Sulfuric ester groups are readily eliminated from amylopectin sulfate but not from laminaran sulfate.

and by sulfamic acid in an aqueous solution of urea (16). Starch has been sulfated with The earliest reported sulfation of a polyconcentrated sulfuric acid (3, 17-20), chlosaccharide is that of Braconnot (1)) who in rosulfonic acid (21), and sulfur trioxide 1819 dissolved linen in cold concentrated vapors (6). A complex of sulfur trioxide and sulfuric acid and after dilution with water a tertiary amine was first used by Wurzobserved the presence of a new acid which burg, Rutenburg, and Ross (22) to prepare he called “acide vigito sulphurique.” Later low degree of substitution, granular, undeanalysis of this material by de Carolles (2)) graded starch sulfates. Sulfamic acid in an Fehling (3), and Marchand (4) showed the aqueous solution of urea (23) has also been acid to be a sulfuric acid ester of degraded used. Dextran has been sulfated with chlocellulose. Since then several hundred meth- rosulfonic acid (24-27)) sulfur trioxide ods have been described for the sulfation of (24), sulfuric acid (24)) and sulfuryl chlocarbohydrate material. ride (24). Sulfated alginic acid has been Many methods have been evolved with a prepared with chlorosulfonic acid (2%31). view toward production of commercial polyOther polysaccharides which have been sulsaccharide sulfates. Thus, cellulose has been fated by chlorosulfonic acid in pyridine are sulfated bv means of concentrated chloro- chitin (32-34)) pectin (32, 35)) xylan (32, sulfonic a&d (5) I sulfur trioxide vapors (6, 36)) hyaluronic acid (37), and laminaran 7)) chlorosulfomc acid-pyridine mixture (38). Chondroitin sulfate has been sulfated (8-12)) concentrated sulfuric acid in the both with a mixture of sulfur trioxide and presence of an aliphatic alcohol (13-15), sulfur dioxide and also with a mixture of sulfur dioxide and chlorosulfonic acid (39). 1 Journal Paper No. 1736 of the Purdue AgriMost sulfation procedures have either cultural Experiment Station, Purdue University, Lafayette, Indiana. Presented in part before the induced excessive polysaccharide degradation or led to a low, unevenly distributed Carbohydrate Division of the American Chemical ester content with concomitant low soluSociety at the 138th National Meeting in New York, N. Y., September ll-16,1966. bility of the derivative. INTRODUCTION

36

POLYSACCHARIDE

In recent years interest has also developed in the production of polysaccharide sulfat,es with blood anticoagulant activity. The present investigation was undertaken to produce high molecular weight polysaccharide sulfates with different levels of ester content. EXPERIMENTAL POLYSACCHARIDES Amylose was fractionated by the 1-butanol method of Schoch (40). Amylopectin was fractionated from commercial corn starch by the pentasol method of Wilson, Schoch, and Hudson (41). Guaran was prepared from guar flour (42). Commercial grade laminaran, gum arabie, and alginic acid were used in this work. Dextran from Leuconostoc mpsentcroides NRRL B-512 F was kindly furnished by the Northern Utilization Research and Development Division, U. S. Department of Agriculture, Peoria, Illinois. SCLFATI~N Fifteen grams of polysaccharide was introduced into 500 ml. dimethylformamide at 0”. The desired amount of trimethylamine-sulfur trioxide complex (43) Tvas added, and the reaction was allowed to proceed at 0” for 24 hr. with constant stirring. The product was dialyzed against a solution of 10% sodium hydrogen carbonate for 24 hr. and against distilled water for 48 hr. The polysaccharide sulfate sodium salt was then obtained by freeze-drying. SULFATE

ANALYSIS

The polysaccharide sulfate sample (0.5-l g.) was refluxed in 250 ml. of a 10% hydrochloric acid solution overnight, and the sulfate was precipitated by the addition of 25 ml. of a 10% barium chloride solution. The precipitate was transferred to a weighed Gooch crucible, heated 1 hr. at 300”, and ignited 1 hr. at 600”. VISCOSITY Viscosities were determined using a modified Ubbelohde viscometer (44). Necessary dilutions were made in the side arm of the viscometer. National Bureau of Standards standard viscosity oils were used to calibrate the viscometer. FILM

STREXGTH

Films were cast with a doctor blade with a clearance of 0.02 in. from approximately a 10% aqueous solut,ion (w/v) onto a clean glass surface. Films wem allowetl to rquilibratr :it 70°F. and 65%;

3T

SULFATES

relative humidity for at least 48 hr. prior to testing. Stress-st,rain diagrams were obtained for the films on a Scott IP2 incline plane serigraph. Tensile strengths were based upon the original cross SPCt.ion of the film. The tensile strength values given are the average of those from at least five strips. OWOTIC

?V~OLECULAR WEIGHT

Osmotic pressures of solutions of polysaccharide sulfates in 5% sodium chloride solution were determined at 25.0 ‘- 0.02” by use of the StabinImmergut (45) modification of the Zimm-Myerson (46) osmometer. Gel cellophane membranes, kindly furnished by the American Viscose Corporation, Philadelphia, Pa., were aged in the solvent three or more days before use. Redistilled t.oluene was used as the manometric liquid. Static mcasurement,s were obtained and the difference in height of the liquids was measured to 0.01 mm. PERIODATE Periodate the method (47).

OX~D.~TIO~-

oxidations were done according to of Anderson, Greenwood, and Hirst PRECIPITATION

TESTY

To separate l-ml. portions of a 1% solution of the neutral sodium salt of the sulfated polysaccharides at room temperature were added equal volumes of the following solutions: 2% gelatin, 2% egg albumin, 3% calcium chloride, saturated ammonium sulfate, and saturated potassium chloride. Another l-ml. portion, under the same conditions, was treated with 5 drops of an aqueous methylene blue solution. Amylose sulfates were titrated potentiometrically (48). REACTION

WITH ,~LCOHOLIC

Po~hssrun~ HYDROXIDE Two grams of polysaccharide sulfate was treated with 200 ml. of 2.5 N potassium hydroxide in 50% ethanol at 80” for a specified period of time. The mixture was poured over crushed ice, and to this was added 100 ml. of a 4 N hydrochloric acid solution. The solution was dialyzed against distilled water for 48 hr., concentrated to 100 ml., and freeze-dried. Desulfat.ed amplopectin sulfate (100 mg.) was dissolved in 2 ml. of 72% sulfuric acid at O”, diluted to 30 ml., and heated on a steam bath for 12 hr. After cooling, the solution was neutralized with barium carbonatc. Barium salts were rcmovcd b> filtration. The solution was concentrated to 1 ml. and chromatographed on Whatman No. 1 papri with 1-butanol-ethanol-water (2: 1: 1). Sugars rere clrirctr~d with a spray of silver nitrate in ncrtonc.

38

WHISTLER RESULTS

AND

AND

DISCUSSION

Sulfation of amylose, amylopectin, and guaran proceeds swiftly and easily with triethylamine-sulfur trioxide complex. As the TABLE

I

SULFUR CONTENT OF POLYSACCHARIDE PREPARED WITH DIFFERENT MOLAR OF SULFATING Molar ratio of triethylamine-sulfur trioxide complex/ mole of monosaccharide unit

0.6

0

5.7 7.3 13.1

1.5 1.8 2.1 3.0 4.5

-

/ Amylopectin

D. S.

0.9

0

14.8 17.5

0.35 0.48 1.1

Amylose Amylose Guaran Guaran

TABLE VISCOSITY

CHARIDE

Amylose Amylose Amylose

D. S.

0 0.8!

0 0.04

0

0

SULFATES CHLORIDE

0.85 0.45 2.55 0.66 6.0 0.86 9.1 1.4 12.8 1.6 13.5

-

0.04 0.14 0.38 0.65 1.09 1.2

DATA

WEIGHT xl

7,

303,000 376,000 132,000 161,500

1052 1027 815 567

III NUMBERS

POLYSACSODIUM

OF

IN 0.1 N SOLUTION

Limiting viscosity number

Material

D. S.

sulfate sulfate sulfate

1.01 1.42 2.01

150 190 170

0.00 0.03 0.45 0.86 1.40

54 72 80 114 94

0.00 0.04 0.26 0.65 1.20

510 520 490 540 490

Amylopectin Amylopectin Amylopectin Amylopectin Amylopectin Guaran Guaran Guaran Guaran Guaran

%S

3.00 2.01 0.00 1.20

sulfate

LIMITING

D. S. ______

D. S.

triacetate sulfate

Guaran

II

MOLECULAR

Material

(

%S

7.0 9.2 11.1 1.42 14.6 2.0115.6

TABLE OSMOTIC

RATIOS

AGENT

Amylose %S

SULFATES

sulfate sulfate sulfate sulfate

sulfate sulfate sulfate sulfate

SPENCER

molar ratio of the sulfating agent to polysaccharide increases, the degree of substitution (D.S.) increases until a value 1.1-2.0 is reached (Table I). A higher molar ratio of sulfating agent effects little increase in the D.S. At a 4.5 molar ratio of sulfating agent to polysaccharide, chromatographic grade cellulose is sulfated to a D.S. of 1.40, a-cellulose to a D.S. of 1.55, and a highly reactive grade of cellulose (i2vicel I 2 is esterfied to a D.S. of 2.04. Under the same conditions of sulfation dextran from Leuconostoc mesenteroides NRRL B-512F which has approximately 95% 1 + 6 linkages (49) is substituted to a D.S. of 0.06. Similarly, alginic acid with no primary hydroxyl groups undergoes no substitution. The sulfation procedure wit,h triethylamine-sulfur trioxide complex seemsto induce very little polysaccharide depolymerization as indicated by osmotic molecular weights and viscosity measurements (Tables II and III). Amylose sulfated to a D.S. of 2.01 has nearly the same degree of polymerization as a sample of carefully acetylated amylose. Guaran on sulfation undergoes a slightly greater depolymerization. Maintenance of a high molecular weight during various sulfations is substantiated by the determination of limiting viscosity numbers (Table III). Viscosity determinations on polysaccharide sulfates are easily and significantly measured in 0.1 M sodium chloride solutions. Such measurements suggest that very little polysaccharide depolymerization occurs when sulfation is not carried beyond a D.S. of 1. The effect of increasing D.S. on viscosity in water solution for amylose, amylopectin, and guaran sulfates is shown in Table IV. Continued increase in viscosities occurs in amylopectin and guaran sulfates until a D.S. of approximately one is reached, whereas, with amylose sulfate, viscosity increases continuously with D.S. over the range investigated. Since amylose sulfates below a D.S. of 1 swell, but do not form complete solutions in water, measurements below this level of substitution cannot be made. A surprisingly large increase in vis2 “Crystalline tainable from delphia, Pa.

Cellulose” (hydrocellulose) American Viscose Corporation,

obPhila-

POLYSACCHARIDE

co&y is dereloped with amylopectin sulfates and to a lesser extent with guaran sulfates when t’he D.S. is increased to approximately 0.7-0.9. This degree of sulfation appears to give rise to maximum viscosity for these derivatives. The very 10~ degree of substitution obtained with dextran and alginic acid, which have no or very few primary hydroxyl groups, suggeststhat in extensively sulfated polysaccharides t)he major, and perhaps the entire, initial sulfation involves the primary hydroxyls. More concrete information on the distribution of sulfate groups in amylosc, amylopectin, and guaran sulfates is obtained by periodate oxidation (Table V). The data suggest t’hat at a D.S. of one, up t,o 50% of the sulfate groups may be located at carbon C-6. This greater reactivity of primary hytlroxyls is commonly observed in polysaccharides. Ricketts (50) subjected dcxtran sulfate, with an average D.S. of one, to oxidation with sodium inet,aperiodate. Formic acid (0.2 mole) was produced, thus indicating a random distribution of the sulfate ester groups on the secondary hydroxyl groups. Since the free acid form of the polysaccharidc sulfates are st’rongly dissociated, autohydrolysis proceeds rapidly. Even in the freeze-dried condition t,he acid form of the polysaccharide derivatives undergo extensive depolpmerization within a short period of time. Amylose and guaran sulfates in the sodium form produce clear films with moderate tensile strength (Table VI). The films of the sulfated polysaccharides are strongly hydrophilic and have higher moist’ure content than fihns of unsulfated material under the same humidity conditions. The sulfated polysarcharitle films dissolve rapidly in water. Guaran, amylose, and amylopectin sulfates, except those with low D.S., combine with gelatin, egg albumin, and methylene blue to form gels or precipitates (Table VIII. They do not precipitate the proteins of human plasma. Thus, they are similar in action to the naturally occurring polysaccharidc sulfate carrageenan obtained from the red seaweed Chondrus crispus. Unlike carragcenan, howrvcr, they do not have the

39

SULFISTES TABLE IV VISCOSITIES OF O.Z$$ POLYSACCHARIDE SULFATE

COMPARATIVE

SOL~~TIONS Amylopectin

sulfate

charm

__-

sulfate

’

Amylose

sulfate

-

D. s.

cps.

___.

--.-

0.00

1.1 1 1.2 1.4 I .6

0.04 0.30 0.45 0.8G

1.40

1.1

0.26 0.65

5.6

, 2.9

8.8

l.OB

( 7.6

TABLE PERIODATE

1.01 I 1.40 , 2.00

(

V

CONSUMED BY SULBATED POLYAACCHARIDE Ioles periodate

consumed

D. s.

Material

Amylose Amylose sulfate Guaran Guaran sulfate Amylopectin Amylopectin sulfate

0.00 1.10 0.00 1.09 0.00 0.90

TABLE TENSILE

5.5 5.8 6.1

STRENGTH

SULFATED

7 days

l5 days

0.31

0.85

0.00

0.39 1.06

0.85 0.46 1.08 0.47

0.31 0.76

0.46:

0.45

i

0.90 1.00 0.50 I 0.56

i

VI AND

POLYSACCHARIDE I

1 day

ELONGATIOK

OF

FILMS Tensile strength

-

Material

D. S.

Elongation

Amylose Amylose sulfate Guaran Guaran sulfate

0.00 1.10

13 7.5

6.5

13

1.2

20

9 13

1.1

15

4.8

19

0;

( 0.00 1.09

Moisture %

specific propert’y of gclation in solution with potassium or ammonium ions. Amylose sulfates of less t’han D.S. of 2 show in solution a purplish blue color with iodine, but on potentiometric titration show no complex formation with iodine. While the sulfate ester groups of all polysaccharidc sulfates are hydrolyzable by

30

WHISTLER

AND SPENCER

acid, they show a marked difference in their elimination under alkaline conditions. Unless their removal leads to the formation of anhydro rings, elimination with alkali will proceed slowly (51). From analogy with p-toluenesulfonyl esters it might be asTABLE EFFECT

VII

SULFATED

OF

POLYSACCHARIDES

SOLUTIONS

OF PROTEINS

METHYLENE

-

Material

BLUE

- 2% 2% egg -

D. S.

gelatin SOlUtion

ON

AND

-

2rlbu-

min iOlUtion

-

HUman iv P lasm a

%:i;leene

____ f”

+

-

0.00

-

-

-

0.04 0.65 1.20

+ +

-

-

+ +

-

Carrageenan Guaran Guaran Guaran Guaran

sulfate sulfate sulfate

Amylose Amylose

1.10

+

+

-

2.01

+

+

-

0.00 0.03

-

-

-

sul-

-

-

sul-

0.45

+

+

-

sul-

0.85

+

+

-

sulfate sulfate

Amylopectin Amylopectin fate Amylopectin fate Amylopectin fate

-

a + denotes

-+++ ++ +++ +++ +++ ++ +++

-I

-

-

formation

of an insoluble

precipi-

tate. I

I

I

I

TIME, FIG.

1.

Rate

of

laminaran and (B) with a 2.5 N solution 50% ethanol.

sulfate

I

I

16 20 HOURS

24

I

elimination

that for the formation of an ethylene oxide ring the sulfonic acid group must be adjacent, to a free hydroxyl group in a trans configuration (52, 53). Ricketts (50) found that alkali eliminated sulfate groups from dextran sulfate and that the rate was slower the higher the degree of sulfation. By ionophoresis he was able to detect glucose, gulose, altrose, and mannose in the hydrolyzate of the desulfated material. This would indicate that the elimination proceeded by the formation of an ethylene oxide ring with Walden inversion at the carbon atom losing the sulfate ester group. Sulfate ester groups in C-6 position are labile to alkali if the hydroxyl group in position C-3 is free. Under these conditions rapid elimination occurs with the formation of 3,6-anhydro rings (52). Thus, a single sulfate group at any position in a n-glucose unit of amylose or amylopectin sulfate should be labile to alkaline conditions. A single sulfate ester group at any position in a n-glucose unit in laminaran should be stable. No adjacent hydroxyls in the bans configuration occur in laminaran, and its polymeric 1 + 6 linkage prevents 3,6-anhydro ring formation. These observations ‘are verified by examinat’ion of the behavior of laminaran and amylopectin. sulfates of D.S. of 1 in alcoholic potassium hydroxide (Fig. 1). Little elimination of sulfate occurs from laminaran, and even that may arise in large part from sulfuric acid groups situated on the mannitol end units (54). As expected, elimination of sulfuric acid ester groups occurs in amylopectin sulfate. While some n-glucose units may be disubstituted and consequently not lose all of their sulfate, the high loss of sulfate observed suggests that the major portion of n-glucose units are monosubstituted. After alkaline treatment of the amylopectin sulfate, acid hydrolysis and paper chromatography of the hydrolyzate leads to the observation of two components. The ma3or one has a flow rate corresponding to n-glucose and the minor one to authentic 3,6-anhydro-n-glucose. No evidence is observed for the presence of 2,3-anhydro-nglucose.

sunned

26 from

amylopectin sulfates at of potassium hydroxide

(A)

80” in

POLYSACCHARIDE ACKNOWLEDGMENTS The authors gratefully acknowledge grants from t,lie Corn Industries and the Department of Health, Education, and Welfare which helped to support this work. REFERENCES 1. BRACONNOT, H., Ann. chim. et phys. [2], 12, 185 (1819). 2. CAROLLES, B. DE, Ann. 52, 412 (1844). 3. FEHLINC, H., Ann. 53, 135 (1845). 4. MARCI~AND, B., J. pmkt. Chena. 35, 199 (1845). 5. CLAISSO~Y, P., J. 7mkt. Chcm. 69, 1 (1879). 6. TRAUBE, w., French Patent 657,204 (1929). 7. TRAUBE,

W.,

BLASER,

B., AND GRWERT,

C. &r.

61B, 745 (1928). 8. GEBAUER-F?LSE~C,

E., U. S. Patent

36. KAI.LI,A, 37. 38. 39. 40.

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.4sD

41. E.,

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and

Gf'llllhw

Irld.

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41

27. PAYYE, H. G., AXD BAKER, P. J., Am. J. Xed. Tecknol. 19,219 (1953). 28. MONES, H. Cr., Belgian Patent 496,541 (1950). 29. LEE, J., C. S. Patent 2,599,564 (1952). 30. ~~LBURN, H. E., U. S. Patent 2,727,889 (1955). 31. BERGER, L., U. S. Patent 2,694,058 (1954). 32. CUSHIIIG, I. B., AND KRATOVIL, E. J., V. S. Patent 2,755,275 (1956). 33. WOLFRO>I, M. L., SHEN, T. M., AND SU>IIJI~;RS, C. G., J. Am. Chem. Sot. 75, 1519 (1953). 34. British Patent 740,152 (1955) 35. ~OCLER, I<.. Swiss Patent 326,791 (1958).

1,734,291

(1929).

13. K4GAlvA.

SULFATES

K. x.

vos,

.4ND H~SEXANS,

E., Experi-

cnlia 2, 222 (1946) ; C. A., 40, 6165 (1946). H.~DIDL~N, A., G. S. Patent 2,599,172 (1952). O’N~LI,, A. N., Can. J. Chem. 33, 1097 (1955). Mm-em, Ii. H., PIHOU& R. P., AXD ODIER, M. E., liclv. Chim. Acta 35, 574 (1952). SCHOCH, T. J., J. Am. Chenz. Sot. 64, 2957 (1942). WILSOX, 13. J.. JR., S~HO~~I, T. J., AKD Huusos. C. S., J. Am. Churl. Sot. 65, 1380 (1943).

42. HEYNE,

E., AND WHISTLER,

R. I,., J. Am.

Chcm.

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C., dcv

J. Am. C/wrrti~

Ciwm. unrl

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(1954).

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