April 5 , 1960
CARBOXYL-REDUCED CHONDROITIN AND CHONDROSINE
1G73
benzene and chromatographed over alumina (60 g.). The v”,: 1740 (ketone in five-membered ring), 1695, 1668 (a$following fractions were collected: (1) benzene, 44 mg.; (2) unsaturated ketone) and 1598 ern.-’. benzene-ether 3:1 , 180 mg.; (3) benzeneether 1: 1, 226 ~ ~ Calcd. ~ forl c .~ ~ H 77.93; ~ ~H,~8.53. ~ :~ , - ~ ~ ~ ~ i : mg.; (4)ether, 86 mg.; (5)chloroform, 340 mg., Fraction i7.46; H , 8,79. 3 only crystallized; repeated crystallizations from ether, ISRAEL m.p. 19&197”, [ a ] D f 48” (C 0.61, ,A, 240 mp ( E 9.000); REHOVOTH,
c,
c,
[ C O X T R I B U T I O X FROM TtIB D E P A R T M E N T O F C H E M I S T R Y O F
THEO H I O
STATE CNIVERSITY]
Chondroitin Sulfate Modifications. I. Carboxyl-reduced Chondroitin and Chondrosine BY
11.L. WOLFROM AND BIENVENIDO 0 . JULIANO’ RECEIVED AUGUST13,1969
Carboxyl-reduced chondroitin (11) was prepared by exhaustive sodium borohydride reduction of chondroitin methyl ester (I) in borate buffer. Partial acid hydrolysis of 11, N-acetylation and carbon column fractionation of the hydrolyzate facilitated the isolation and characterization of D-glucose, 2-acetarnido-2-deoxy-~-~-galactose monohydrate and the sole disaccharide, 3-~-~-~-glucopyranosyl-2-acetamido-2-deoxy-a-~-galactopyranose dihydrate (111). Compound I11 was readily degraded in alkaline solution to n-glucose and a Morgan-Elson reactive sugar, “anhydro-N-acetyl-D-galactosamine.” Sodium borohydride treatment of I11 gave the alditol I V , chromatographically identical t o the product derived from chondrosine methyl ester hydrochloride. N-ilcetylhexosaminols were shown to give positive Morgan-Elson reactions. Formula VI11 is proposed as the correct structure for the periodate oxidation product of N-acetylcl~ondrosinol(VI).
The acid instability of hexuronic acids has been frequently noted.2 Owing to the difficulty of attaining complete hydrolysis without destruction of the component monosaccharides, an alternative approach used for studying mucopolysaccharide structure has been to convert the hexuronic acid residues to hexose units by reduction of their methyl esters with sodium b ~ r o h y d r i d e . ~ -Since ~ no configurational changes were involved, deductions from the hydrolysis of reduced material have been applicable to the original polymer. We report herein the preparation of 96% carboxyl-reduced polymeric chondroitin (desulfated chondroitin sulfate A) (11) and also carboxylreduced chondrosine, the sole disaccharide from the acid hydrolysis of 11, isolated as the crystalline N-acetyl dihydrate derivative 111, m.p. 155-157’, [(u]D 4-47 + +1g0 (water). Substance 11, [a]D 11’ (dimethyl sulfoxide), was derived, in 71% yield, from chondroitin metbyl ester6 (I) by exhaustive sodium borohydride reduction’ ; the first reduction was 6G% efficient, the second increased the reduction to 86y0 and the third to 96%. These results are in striking similarity to the reduction of the methyl ester of desulfated chondroitin sulfate B5 with sodium borohydride, where 66 and 85% reductions of the L-iduronic acid to L-idose on the first and second reductions of the polymer were attained. Partial acid hydrolysis of 11, N-acetylation and carbon column fractionation of the hydrolyzate, facilitated the isolation of D-glucose, 2-acetamido-2-
+
(1) National Science Foundation Predoctoral Fellow, 1957-1958, under Grant NSF-G4494 to The Ohio State University: C . F. Kettering Research Foundation Fellow, 1958-1959. (2) R. L. Whistler, A. R. Martin and M. Harris, J . Research N u l l . Bur. Sfandurds, 24, 13 (1940); E . Stutz and H. Deuel, Helv. Chim. Acta, 41, 1722 (1958). (3) B. Weissmann and K. Meyer, THISJ O U R N A L , 74, 4729 (1952); 76, 1753 (1954). ( 4 ) E. A. Davidson and K. hleyer. i b i d . , 76, 5686 (1954). ( 5 ) R. W. Jeanloz and P. J. Stoffyn, Federation Puor., 17, 1078 (1958). (6) T.G. Kantor and If.Schubert, THISJOURNAL, 79, 162 (1957). (7) Harriet L. Frush and H. S. Isbell, i b i d , , 7 8 , 2844 (1950).
deoxy - a - D - galactose (N - acetyl - D - galactosamine) monohydrate and the sole disaccharide, 3-O-~-~-glucopyranosyl-2-acetamido-2-deoxya-Dgalactose dihydrate (111). D-Glucose was characterized as its crystalline 6-pentaacetate and the crystalline 2 - acetamido - 2 - deoxy - a - D - galactose monohydrate was further characterized as the crystalline P-pentaacetate.8 Carboxyl-reduced chondrosine gave positive ninhydrin and Elson-Morgang reactions. Its crystalline N-acetyl derivative I11 gave a positive Morgan-Elsonlo reaction. On acid hydrolysis of 111, D-glucose and 2-amino-2-deoxyD-galactose (D-galactosamine) were detected by paper chromatography. The infrared spectra of I1 and I11 were very similar. The disaccharide I11 was degraded readily in dilute alkali solution to D-glUCOSe and a MorganElsonlo reactive sugar (Rglucose129, “anhydroN-acetyl-D-galactosamine,” different from 2-acetamido-2-deoxy-~-galactose (Rglucose 1.2). This is by analogy to the reported” alkaline degradation of 3-0-~-~-galactopyranosyl-2-acetamido-2-deoxyD-glucose to D-galactose and “anhydro-N-acety1-1glucosamine” (Rglucose 1.70), different from 2-acetamido-2-deoxy-~-g~ucose (N-acetyl-D-glucosamine) (Rglucose 1.24). The fact1’ that the 4-O-P-D-galaCtOpyranosyl analog did not yield “anhydro-N-acetylD-glucosamine” and the 6-O-P-~-galactopyranosyl compound gave, instead, 6-O-P-~-galactopyranosyl- “anhydro-N-acetyl-D-glucosamine”with Rglucose 1.04, should make alkaline degradation a valuable tool for determining 0-substitution of N-acetylhexosamines. 2-Acetamido-2-deoxy3-O-methyl-~-glucose also provided “anhydro-Nacetyl-D-glucosamine,” which is of unknown structure. 4-0- and 6-0-substitution for I1 can be discounted from the results of the alkaline degradation. I t s positive Morgan-Elsonlo test eliminates ( 8 ) M. Stacey, J. Chem. Soc., 272 (1944). (9) L. A. Elson and W. T.J. Morgan, Biochem. J., 27, 1824 (1933). (10) W. T. J. Morgan and L. A. Elson, ibid., 28, 988 (1934). (11) R. Kuhn, Adeline Gauhe and H. H. Baer, Chem. Ber., 87. 289 1138 (1954).
AI. L. U’OLFROM AND RIENVENIDO0 . j v L I m o
1674
k
A
OH
I
NHCOCH~
H
m
ITOl. s2
I
I
OH
H
I
NHCOGH3
m
the (1 + 4)-linkage since 4-0-substituted N- Levene, l8 is hereby reinterpreted. The diamide acetylhexosamines were unreactive to the rea- glycitol (VI) from the 0-deacetylation of the Ngent,l l--13 acetylchondrosinol heptaacetate methyl ester (V) Sodium borohydride reduction of I11 readily of Levene, l8 on periodate oxidation, underwent gave 3-O-/3-~-g~ucopyranosy1-2-acetamido-2-deoxyformaldehyde and formic acid scission in the reD-galactit01 (IV), as the sirupy main product, duced portion to yield initially the intermediate which was chromatographically identical (Rglucose VII. Formula VIII, as suggested by Toro-Feli0.76), in three developer systems, to IV4 derived ciano, l9 is proposed for the crystalline oxidation from chondrosine methyl ester hydrochloride. The product is01ated.l~ The formation of such a glycitol IV was still Morgan-ElsonLo reactive and substituted dioxane ring (VIII) should be favored gave (by paper chromatography) D-glucose as the and such a ring has been well substantiated in the only reducing sugar on acid hydrolysis. Com- difructose dianhydrides.2o Its presence is in pounds I11 and IV have similar Rglucose values. harmony with t h e reportedL7one mole further up2-Acetamido-2-deoxy-~-galactose and the corre- take [IX] of oxidant. Scaled molecular models sponding alditol have been shown to also have show that VI11 could be formed readily. Color similar Rfvalues,14 whereas similar Rfvalues have tests showed that the hexuronic acid was intact in been reported for 2-acetamido-2-deoxy-~-galactose V, thus supporting the established4 component in various solvent systems.16 Substance IV was sequence for chondrosine. resistant to alkaline degradation under conditions That the /3-D-glucopyranosidic linkage in I1 is which degraded 111. Its trace impurities of D- more stable than the 2-acetamido-2-deoxy-/3-~glucitol and “anhydro-iV-acetyl-D-galactosaminol” galactopyranosidic linkage is consistent with cornmay be accounted for by the degradation of 111 in parative kinetic data21 showing that the rate of the alkaline borohydride medium prior to reduction glucosidic hydrolysis of methyl 2-acetaniido-2to IV, and subsequent reduction of these products. deoxy-/3-D-glucopyranoside was 9-18 times that of The isolation of only one disaccharide, 111, from methyl /3-D-glucopyranoside.22 Since desulfation I1 implies that the stability of the 0-D-glucuronidic of chondroitin sulfuric acid proceeds faster than linkagel6 alone cannot account for the selective glycosidic cleavage in acid solution,6the absence of nature of the acid hydrolysis of chondroitin sulfate sulfate groups in 11 does not affect the problem. A to chondrosine4 since conversion of this to a /3- HydroIytic studies of I1 showed the presence of D-glucosidic linkage also gave the related disac- N-acetylated sugars, I11 and 2-acetamido-2-decharide I11 and none for the alternative sequence. OXy-D-gakKtOSe, during the first two hours of reOn the basis of the established component sequence action, denoting that glycosidic cleavage was faster for chondrosine, after Davidson and M e ~ e r , ~ (17) M. L. Wolfrom, R. K. Madison and M. J. Cron, ibid., 74, 1491 the periodate oxidation data of Wolfrom and co- (1952). (18) P. A. Levene, J. Biol. Chem., 140, 267 (1941); P. A. Levene workers,’? based upon the sequence utilized by (12) R. W. Jeanloz and Monique Tr&mlkge,Federation Proc., 16, 282 (1956). (13) S. A. Barker, A. B. Foster, M. Stacey and J. M. Webber, J . Chem. Soc., 2218 (1958). (14) W. R. C. Crimmin, ibid., 2838 (1957). (15) S. Roseman and J . Ludowieg, T H I S JOURNAL, 76, 301 (1954). (16) R. L. Whistler and G. N. Richards, THISJOURNAL, 80, 4880 (1958).
and F. B. LaForge, ibid., 16, 69 (1913). (19) E. D. Toro-Feliciano, M.Sc. Thesis, The Ohio State University, 1957.
(20) Emma J. McDonald, Aduances in Carbohydrate Ckem., 2 , 253 (1946). (21) A. B. Foster, D. Horton and M. Stacey, J . Chcm. SOC.,81 (1957); see also R . C. G. Moggridge and A. Neuberger, ibid., 745 (1938). (22) E. A. Moelwyn-Hughes, Trans. Faraday SOL,26,603 (1929).
CQ2CH3
AcO Hf-O$OAc H
- study showed hours at 5 " . Recrystallization was effected in the samc that a hydrolysis time of 2.25 to 3 hr. (IOO', 1r5 s o h . in AT manner; L-ield 0.18 g. of white crystals, m.p. 115-120' sulfuric acid) was required for an optimum yield of uinliy- (preliminary softening), [ ~ ] ' S D $84' (c 1.04, water, final, drin and Elson-Morgan reactive carboxyl-reduced chondro- downward mutarotation); X-ray powder diffraction data: sine from I , by paper chromatographic analysis on \\'hatman 10.530s4D(l,l), 7.80tn, 7.lSw, 5.16m(3), 4.64n1, 4.40n1, 4.19sSo. 1 filter paper with 1-butanol, pyridine aiid water ( l , l ) ,S.YOw, 3 . 6 3 ;j.lgvw, ~ ~ ~ 2 . 1 7 ~ ~Stacey8 . cites 120( 8 : 2 :1.5 by vol.) developer and both (separately) the El122' and +SO0 for this substance. soii-Murgan9~32 and alkaline silver nitrate33 indicators. A n d . Calcd. for C8H16N08.H20:C, 40.16; IF, 7.16; Substance I1 dissolved in iV sulfuric acid after 1 hr. of reN, 5.86. Found: (2,4030; H , 7.37; S,5.81. fluxing. Morgan-Elsonlo reactive zones, 2-acetamido-2deoXy-D-galaCtOSe ( N-acetyl-D-galactosamine) ( RRluOOSe 1.2) 2-Acetam~do-tetra-O-acetyl-2-deoxy-~-~-galactopyranoSe. and 111 (X,I,,,,,, 0.74) were rioted during the first 2 hr. of -The procedure was essentially that of Stacey.s An hydrolysis. -In amount of 4.00 g. of I1 was refluxed for amount of 90 mg. of 2-acctamidc1-2-deoxy-cy-~-galactose 2.25 lir. in 125 nil. (c 3.2) of N sulfuric acid and the conled monohydrate described :ibove was suspended in 1.2 nil. of Iiydrolyznte was neutralized with solid barium carbonate. acetic aiihyclride a n d shaken with powdered, fused zinc .\fter tlic filtr,itioii of inorganic residue, tlie filtrate was made cliloride (30 mg.) f i r 24 iir. The reaction mixture was acitlic nith 10 nil. of iv hydrochlixic acid prior t o ,V-acetylapoured into 4 vol. ~f ice-water and the suspension was care. solution of the concentrated yellow liydrolyzatc tio11.l~ % fully neutralizcd by the addition of solid sodium carbonate. in water (75 ml.) was treated a t 0" with 7.5 nil. of methancl, The mixture was made slightly alkaline with dilute sodiuni 90 nil. (settled vol.) of Dowex 1 3 ' (carbonate form) and 2 hydroxide and extracted 6 times with chloroform (10-ml. 1111. of acetic anhydride and stirred for 90 min. a t 0-5". portions). The combined extracts were dried over anhpThe reaction mixture was filtered and the filtrate and wash- drous sodium sulfate overnight, filtered and the filtrate and ings were passed through a column (180 X 12 mm., diam.) washings concentrated under reduced pressure until cryso f Dowex 5034 ( H + form) to remove any non-acetylated tallization ensued. Then the mixture was diluted with amino sugar. Paper chromatographic analysis at this ethanol and kept at 5'; yield 40 mg. (37'4) of white stage sliowed the absence of 2-amino-2-deoxy-~-galactosc, crystals, ri1.p. ZED,[ c u ] * ~ D+8' ( c 0.4, chloroform); X-ray D-glucuronic acid and cetylchondrosine and the presence powder diffraction data: 8.1039w407.44~(2,2),6.28111, 5.10only of distinct zones for D-glucose, %-acetamido-l'-deox?--D- vw, 4 .00s( l ) , 3.84m, 3 . 6 5 v ~ ,3.31s( 2,2), 3.03vw, 2.34111. galactose and 111. The X-acetylated hydrolyzate was frac- Stacey8 cites 235' anti f 7 ' f i r this substance. (Kucliar C u i ~ g r o u n d ~column ~) tiirnated 011 a cnrbo11~~ < 4 n d . Calcd. fur C,eH,iKOlo: C , 4'3.35; 1-1, 5.9;; S, (210 x 44 mm.,diam.) previously washed with 2 liters of water. After placing the saniple UII the column, the 3.ti0. Found: C, 49.25; H, 5.85; X, 3.70. cliroinatograni was tlcveloped with water (9 liters), 3-O-~-~-Glucopyranosyl-Z-acetaniido-2-deoxy-~-~-galacethanol (4.: liters), 3:; ethanol (2.8 liters), 5% ethanol ( 3 . 2 tose Dihydrate (III).-Crl-stallization of the pure I11 fracliters) and 6'jo ethanol ( 4 liters). By paper chromatot i m s ( 1.O g.) involved the addition of it small volunle of graphic analysis it was shown that D-glucose only was pres- ethanol t o t h e drj- sirup and keeping a t 5'. Kecrystallizaent in the first liter of the water eflluent but was mixed with tion w a s effected in the same manner; yield 0.40 g. (lO',4) 2-acetamido-2-deoxy-~-galactose in the rest of the water from 11) of white tnicroncedles, m.p. 155-157' (preliminary effluent; pure 2-acetamido-2-deoxy-~-galactose was eluted softening), [ o ] ' ~ D +ai (extrapolated) + + l Y 0 (final, c with 2% ethanol, but was contaminated with I1 in the 3Yo 1.07, water); X-ray powder diffraction data: 1 4 . 3 3 8 ~ , 4 " ethanol eluate; and the 5 7 , and 670 eluates contained pure 13.0vs(l), 1 0 . 4 ~ ( 2 , 2 ) , 7.36vw, G.56vw, 4.91vw, 4.39s, 11. The fractions corresponding to the above pure hydrol- 1.36m, 4.14~(2,.'), 4.(1tin, 3.88in, 3.60111, 3.33w, 2.S9w, yzates were concentrated, turbidity was renioved by filtra- 2.79vw, 2.7 1vtv, 2.31 v w , 2.28vw, 2.1 ivvlv, 2.1~ V V W , tion through a fritted-glass filter arid trace impurities were 1. Y O v w , 1 . 8 7 ~ ~ ~ . removed by passing through a column (60 X 13 mm., .lnuZ. Calcd. for C l , l l 1 2 ~ S 0 , ~ ~ 2 1C~, ,40.09; 0: H, 6.97; tliam.) of mixed-bed resin (Amberlite MU-337) and the N,3.34; H20, 8.59. 1~;ound:C , 40.09; H, 7.09; S , 3.27; H 2 0 , 8.32; Morgu-Elsoil'" tcst, ( + j .
SPRUCE LIGNINWITH &BUTYLHYPOCHLORITE
April 5 , 1960
1677
matographically identical, with three dcvelopcrs, to sirupy Its infrared spectrum was strikingly similar to that of 11, except for an 807 em.-’ peak in 111, absent in 11. The 3-0-P-D-glucopyranosyl-2-acetamido -2- deoxy - D - galactit01 886 cm.+ band may be due to the C1-H absorption of 13-D- (IV) prepared from authentic chondrosine according to Davidson and Meyer .4 The principal and non-reducing glucopyranose.31 Hydrolysis of I11 ( 5 mg.) in 3 ml. of IV sulfuric acid and subsequent paper chromatography of the spot ( Rgluoose 0.76) was unreactive to aniline hydrogen hydrolyzate showed D-glucose and 2-amino-2-deoxy-~-ga1- phthalate,41but was reactive to the alkaline silver nitrate3a and (purple) to the E l s ~ n - M o r g a n g *indicators. ~~ Trac?: of actose (D-galactosamine). D-glucitol ( Rgluoose 1.O) and Morgan-Elsonlo reactive anTo determine the alkali stability of 111, an amount of 5 hydro-iv-acetyl-D-galactosaminol,” ( Rglueose1.8) also were mg. of I11 was treated with 1 ml. of 0.04 N sodium carbonate for 2 hr. a t room temperature and the mixture was detected, with alkaline silver nitrate,S3 in both preparations. Besides I V and “anhydro-N-acetyl-D-galactosaminol,”“ analyzed paper chromatographically. Aside from 111, D( ,~-acetyl-D-glucosaminol) glucose and a Morgan-Elsonlo reactive sugar ( Rgluoose 1.8), 2-acetamido-2-deoxy-~-glucitol~~ “anhydro-N-acetyl-D-galactosamine,”lidifferent from 2- also gave the characteristic purple color of the Morganacetamido-2-deoxy-~-galactose( RBiuoose 1.2), were detected. Elsonlo reaction with the reagent.3z Characterization of 3-0-(Methyl Tri-0-acetyl-P-D-glucoAn amount of 100 mg. of 111 in 5 ml. of 50% methanol pyranosy1uronate)-2-acetamido-tetra - 0 -acetyl 2 - deoxy - Dwas added in portions, with stirring, to a solution of 40 mg. of sodium borohydride in 5 ml. of 0.1 hl borate buffer (PH 8) galactitol (V) .4Z-Substance V17J*gave a positive uronic acid reaction. at 0’. The mixture was stirred a t 0’ for 2 hr., an additional assayzQand a negative Elson-Morgang~~~ hr. a t room temperature and acidified to p H 5 with acetic (41) S. M. Partridge, Nature, 164, 443 (1949). acid and passed through a column (100 X 13 mm., diam.) (42) Experimental work by Mr. J. pi. Schumacher. of mixed-bed resin (Amberlite MB-337). A hygroscopic W. Palmer, Elizabeth M. Smyth and K. Meyer, J . B i d . sirup was obtained after carbon” (Nuchar C ~ n g r o u n d ~ ~Chein., ) (43) J. 119, 491 (1937). column purification and solvent removal under reduced pressure; yield 60 mg. This product was found to be chro- COLUMBUS 10. OHIO
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