XZIay 20, l%X
S P E C I F I C POLYS.\CCIIhRIDE OF T Y P E
VI
2-41
PNEUMOCOCCUS
recrystallized from ethyl acetate. A sample prepared from no hydroxyl absorption. The distilled methylated sugar (517 mg.) had b.p. 170-180" (bath temp.) a t 0.01 mm. and authentic 4-0-methyl-~-glucosehad m.p. 156-158" and a OMe, 55.7%; calcd. ClsHaaOlo; OMe, 52.9%. We are inmixed melting point of 156-159'. In an attempt to make other derivatives of 3- and 4-0- debted to Mr. Y. Tanaka for the infrared spectra. Hydrolysis of Methylated Disaccharide .-The methylated methyl-D-glucose for comparison, the dibenzyl mercaptals were made by the usual procedure from authentic specimens. disaccharide (437 mg.) was dissolved in sulfuric acid (25 T h a t from 3-Chnethyl-~-glucosewas recrystallized with dif- ml., 1 N)and hydrolyzed on the steam-bath for 11 hours, [a] 84.8", constant. The sirup (396 mg.) obtained after ficulty from ethanol containing a few drops of water and had The mercaptal from 4-~-methyl-~-glucose neutralization (BaC09) showed two components of Rf 0.68 m.p. 66-69'. and 0.83 in solvent A . T h e mixture was separated on prereadily crystallized from absolute ethanol, and had m.p. washed Whatman 3 M M paper using the same solvent. The 158-159 '. faster component was extracted with ether and the slower Methyl 2-0-( 2,3,4,6-Tetra-0-methyl-a-~-glucopyranosyl)-3,4-di-0-methyl-~-xylopyranoside .-To a portion (864 one with ethanol. Identification of 2,3,4,6-Tetra-O-methyl-~-glucose .-Caremg.) of the neutral disaccharide methyl glycoside was added sodium hydroxide (6 ml., 40%) and dimethyl sulfate (0.5 ful chromatographic examination of the fraction having R f 0.83 showed the presence of a small amount of a faster comml.). The mixture was stirred a t high speed a t a temperature of 4j-55' and 0.5-1111. portions of dimethyl sulfate ponent (tri-0-methyl-L-rhamnose ? ) and because of this it added every 15 minutes for 1.5 hours. This treatment was was not possible to obtain the tetra-0-methyl-D-glucose repeated twice and after 4-5 hours the bath temperature was crystalline. A small amount of A7-phenyl-2,3,4,6-tetra-0raised to 80" for 0.5 hours. This cycle was repeated twice methyl-D-glucosylamine was obtained with m.p. 122-132' and finally after neutralization to PH 8.9 with sulfuric acid and when mixed with an authentic sample 130-136". Identification of 3,4-Di-O-methyl-~-xylose .-The cotnthe product was isolated by continuous extraction with chloroform overnight. The partly methylated sirup (6'75 mg.), ponent with Rr 0.68 was chromatographically pure and was oxidized with bromine in the ordinary way for 48 hours. which showed only weak infrared absorption in the hydroxyl distilled a t region, was dissolved in methyl iodide (20 ml.) and Drierite The derived 3,4-di-O-methyl-D-sylono-6-lactone 140-160" (bath temp., 0.01 mm.), crystnllizcd spontane( c a . 1 g.) added. Silver oxide ( 5 g.) was added in small portions over 9 hours to the refluxing and stirred solution. ously and had m.p. and mixed in .p . 64-66'. After three such methylations the infrared spectrum showed VANCOUVER, B. C., CANADA
[CONTRIBUTION FROM
THE
IXSTITUTI2
OF
MICROBIOLOGY, RUTGERS,THESTATE UNIVERSITY]
The Specific Polysaccharide of Type VI Pneumococcus. and Reactions BY
PAUL
A . REBERS .4ND
LfICHAEL
I. Preparation, Properties
HEIDELBERGER
RECEIVED NOVEMBER 24, 1958
+
Purified specific polysaccharide of type VI pneumococcus, S VI, [a]D 129", contains D-galactose, D-glucose, L-rhamnose, a poly01 definitely identified as ribitol and phosphorus in approximately equimolar amounts. The P occurs as a labile diester, and alkaline hydrolysis yields a stable, serologically inactive monoester, [ O ] D 106". Periodate oxidation of S VI produces formaldehyde but no formic acid; the galactose is attacked, but the glucose and rhamnose are resistant. After alkaline hydrolysis of S VI, glucose and rhamnose are still attached to the phosphorus-containing portion in periodateresistant linkages in the original ratio, but now, besides formaldehyde, formic acid is liberated, apparently from the galactose.
+
3)-O-~-glucop~ranc,s~.l-( 1 ~)-O-LS V I seems to be a linear polymer of the unit - -4 or 2-O-~-galactopyranosyl-( 1 -+
rhamnopyranosyl(1
-+
I
1 or 3)-ribitol-3 or 1-0-P-0--
ll
Pneumococci of the serological Types 11, V and VI form a triad, of which Types V and VI were originally considered sub-types of I1.z Hecause the serological relationships reflect differences and similarities in the capsular specific polysaccharides which, among pneumococci, are the principal determinants of immunological type specificity, a study of the fine structures of these three carbohydrates4 is considered likely to clarify the as yet obscure relation of chemical constitution to the immunological specificities and cross reactivities (1) This study was carried o u t under a grant from t h e National Science Foundation. ( 2 ) 0. T. Avery, J . E x p e r . M e d . , 22, 804 (1915); G. Cooper, M. Edwards and C. Rosenstein, ibid., 49, 461 (1929). (3) M. Heidelherger and 0. T. Avery, i b i d . , 38, 73 (1923); 40, 301 (1924); 0.T.A w r y and M. Heidelberger, i b i d . , 38, 81 (1923); 42, 367 (1925); M. Heidelberger, W. F. Goebel a n d 0. T. Avery, ibid., 42, 727 (1926). (4) Hereinafter referred t o a s S 11, S V and S VI.
involved and to serve as a model for similar investigations. The chemistry of S I1 and S V is being unravelled by Prof. Maurice Stacey and Dr. S. A. Barker, respectively, of the University of Birmingham, England, and some progress with S I1 has already been r e ~ o r d e d . ~The present paper is concerned with initial stages of the chemical study of S VI. Experimental Materials and Methods.-+ VI, isolated by the phenol process,B was generously supplied by E. R. Squibb and Sons, through the kindness of Mr. T. D . Gerlough. Type V I anti-pneumococcal horse serum was furnished by the Division of Laboratories, S e w York State Department of Health. Thanks are also due to Dr. N. K. Richtrnyer for many of the samples of polyols and to Dr. F. Smith for the sample of Lthreitol. (5) K.Butler and M . Stacey, J . Chem. Soc.. 1537 (l9;b). (6) T. D. Gerlough, U. S. P a t . 2,340,318(Feb. 9, 1944); J. W.Palmer and T. D. Gerlough, Science, 92, 155 (1940).
Nitrogen in all polysaccharide samples was determined by a micro-Kjeldahl method.7 Inorganic phosphorus was determined by t h e method of Fiske and Subbarow8 or by a modificationg in which p-semidine replaces sodium l-naphthol-2-ami1io-4-sulfonate. Organic phosphorus was estimated after digestion with sulfuric acid and 30Yc hydrogen peroxideg~'Owhich had been absorbed with kaolin in order to reduce its P content." All centrifugations were carried out at 0" unless otherwise noted. Measurements of optical activity were made a t room temperature, 25-30', in a Rudolph Model 80 polarimeter with a sodium lamp. Unless otherwise designated, water was used as solvent and the concentration was 1-270. Oxidations by periodate were carried out as suggested by Periodate consumption was determined according t o Fleury and Lange.I3 i l n y formic acid liberated was estimated by addition of ethylene glycol and titration under N s with 0.01 iV Ba(OH)*t o the initial p H . Formaldehyde was identified and determined by precipitation with dimedone"9'4 or by t h e use of chromotropic acid.'j Dialyses were carried o u t in Visking cellophane tubiiig, p/l" diameter, t h a t had been pretested for leaks. pH was determined with a Model G Beckman pH meter. Paper chromatograms were run with Whatman KO. 1 filter paper b y a descending technique a t room ternperatures (25-30'). T h e solvent mixtures used were: ( A ) BuOII: pyridine: H20, 6: 4 : 3lB; ( B ) BuOH: E t O H : €120: NH3, 40: 10:49: 1'7; (C) ~-collidine-€I~O.~7 Sugars were located on chromatograms with reagents: ( D ) aniline-trichloroacetic acid1*; (E) aniline hh-drogen phthalate.'g (E') Polyols20 as well as sugars'' were detected witli ainmoni:~cnl silver nitrate; (G) phosphate esters irere located by a niodification of t h e Hanes-Isherwood procedure.*' Paper electrophoresis was carried out in borate buffer (0.05 AI, pH 10) on Whatman S o . 3?*or glass paper.23 Purification of S VI.-Of the methods tried, precipitatior, by alcohol in t h e presence of calcium acetate yielded the best results. A 7% aqueous solution was centrifuged at 105,000 times gravity for 1 h r . in a Spinco ultracentrifuge. After addition of calciuni acetate a t pH 5 t o t h e clear supernatant u p t o 5% ( ~ v . / v . ) ,957c ethanol was added to 0.75 vol. T h e small precipitate of high N content was removed by centrifugation and the main fraction was precipitated h y an additional 0.15 of the new volume of alcoho1. After four such precipitations, it showed [orlo 1 1 2 1 t o 127O, 0.06 to 0.2y0N and 4.0 t o 4.2y0 P, ascompared with 1-113'; 0.3 t o 0.4, and 4.0 t o 4.2, respectively, reported by Brown.24 Fusion with sodium failed t o show sulfur or halogens. Three g. of purified material was obtained from 8 g . of starting product. T h e N content could be reduced to 0.06% with(7) R . Markham, Biochem. J . , 36,i90 (1942); I < . A . Kabat and 31. h l a y e r , "Experimental Iminiiii~~rlieiniitrJ.,"C. C Thuinas, Slxiug field. Ill., 1948. IbbaRow,J . Bioi. c/lrm.,81, 629 (192CI). (9) R . I,. Dryer, 4.R . Tammes and J. I . Roiith, preprint of piihlication, "Determination of Phosphorus in Huily I'liiids," I a s t i n a n Organic Chemicals, Rochester, AT. Y . (10) W.W. Umbreit, R. 11. Burris and J. F.Stauffer, "Xanometric 'Techniques a n d Tissue Rletabolism," 2nd Ed., Burgess Publishing Co., hfinneapolis, Minn., 1051, p . 190 (11) S . C. Chang and Rf. L. Jackson, S c i e n c r , 124, 1209 (1956). (12) J. R. Dyer, "Methods of Biochemical Analysis," Vol. 111, edited by D. Click. Interscience Publishers, Inc., S e w York, iY.Y . , 1%5. (13) P. F. F l e u r y a n d J. Lange, J . phurin. c h i m , 17, 107, 19G (1933). (14) D . Vorlander, 2 . a n d . Chern.. 77, 241 (1929); J. H. Yoe and I.. C. Reid, Ind. Eng. C h e m . , A m l . E d , 13, 238 (1941). (15) M. Lambert and A. C. S e i s h , Can. J . Reseovch, 28B, 83 (1950). (16) E. Chargaff, C. I,, Levine arid C. Green, J . B i d . Lhi2m., 175, 67 (1948). (17) S . hi. Partridge, N u l u r e , 158, 270 (1940); B i o c h e m J . , 42, 238 (1948). (18) R. M. McCready and 13, A. RIcComb, A n d . C h r m . . 26, LGL,? (1954). (19) S. M. Partridge, S n l n r e , 164, 413 (1949). (20) L. Hough, ihid.. 165,400 (1950). (21) R . S. Bandurski and B Axelrod, .I. B i d . Cht'm., 193, 403 ( I 951). ( 2 2 ) A . H . Vocter, J . C h ~ i i i . $ m . , Q X ? (195:i). (?,
