Application of mass spectrometry to methylated monosaccharides identification

134

SHORT COMMUNICATIONS

glucose and acetylglucosamine could be linked in some manner to a second polymer, a glycerol phosphate polymer (i.e. a teichoic acid) resembling that of Lactobacilhts casei 9 (but without esterified D-alanine residues). Further work will be needed to decide between these possibilities. We are indebted to Dr. H. J. FROMM for a sample of ribitol dehydrogenase of

A. aerogenes. Bacteriological Service, University of Liege, Li@e (Belgium) and Pharmacology Department, Washington University, St. Louis, Mo. (U.S.A.)

j . M. (~-HUYSEN

1 j. M. GHUYSEN, Biochim. Biophys. Acta, 5 ° (1901) 4z3 . 2 j. M. GHUYSEN, M. LEYH-BOUILL~ AND L. DIERICKX, Biochim. Biophys. Acta, 63 (I962) 28(). a j. M. GHUYSEN, M. LEYH-BOUILLE AND 1~. DIERICKX, Biochim. Biophys..4cta, ()3 (1962) 297. a M. R. J. SALTON AND G. MILHAUD, Biochim. Biophys. Acta, 35 (1959) 254. 5 j. M. GHUYSEN ANn J. L. STROMINGER, in the press. s j. T. PARK AND M. J. JOHNSON, J . Biol. Chem., 18i (I949) ~49. 7 H. J. FROMM, Biochim. Biophys. Acta, 57 (1962) 369. 8 S. ESTRADA-PARRA, P. A. REBERS AND M. HEIDELBERGER, Biochemistry, () ([962) [~ 75. 9 M. V. XELEMAN AND J. BADDILEY, Biochem. J., 8o (1961) 246.

Received June 2oth, 1963 Biochim. Biophys. Acla, 83 (1964) 132 134

~c 83oo2

Application of mass spectrometry to methylated monosaccharides identification The standard procedure for the structural analysis of carbohydrate biopolymers, ba~ed on methylation, still remains tedious and wasteful, due to the lack of appropriate methods for the identification of methylated monosaccharides. This is despite the fact that the methylation procedure 1,2, and the methods for the separation of methylated sugars which are produced by hydrolysis 3~, have been considerably improved during the last few years. We have recently investigated the pathways of fragmentation of a-methyl2,3,4,6-tetra-0-methyl-D-glucoside (I) after electron impactL The structures of the majority of the fragments produced were established on the basis of the mass spectra of a-methyl[2-0-Me-2Ha]-2,3,4,6-tetra-0-methyl-D-glucoside (II), a-methyl[2,3-di-0Me_2Ha]-2,3,4,6-tetra-O-methyl-D-glucoside (III), ~-methylE4,6-di-O-Me-2H3~-2,3,4,6 tetra-0-methyl-D-glucoside (IV) and ~-methyl[6-O-Me-2H3]-2,3,4,6-tetra-O-methy 1D-glucoside (V). The data obtained make it possible to predict the positions of the major peaks in the mass spectra of all the other possible methyl [Me-2Hal-tetra 0-methyl- and [di-Me-2H~-tetra-O-methyl-glu cosides*. The spectra predicted, together with the spectral data measured for I-V are shown in Table I. These data reveal that each of the compounds exhibits a unique mass spectrum, which can thus establish, unequivocally, the position of the -C2H3 group. * Obviously the f r a g m e n t a t i o n p a t t e r n of :~-nlethyt[tri-O-Me-2Ha]-tetra-O-methyl-D-g luc°sides m u s t be essentially similar to t h a t of methylEO-Me-~Hs!-tetra-O-methyl-n-g lucosides-

Biochim. Biophys..4cla, 83 (1964) 134-136

L

*"

oo t,a

x

¢o

4~,~

~"

173 162

152

145 131

III

149

145 131

III

48 (5 ° )

45

45 (5 °)

88

88

IOI (90)

IO 4 (IO)

179

176

176

173 159

IOI

19o (84) 187 (I6)

19 °

187

221

149 148

152 148 134

149 148

134 (6o) 131 (4 ° ) 114 (50) I I I (50) lO 4 (3 o) IOI (7 o) 91 (95) 88 (5) 48 (15) 45 (85)

lO4 (7 o) IOI (3 o)

91 (16) 88 (84)

48 (5)

45 (95)

II 4 (tO) I I I (90)

159 (5 ° ) 162 (5 o)

159 (5 ° ) 162 (5 o)

45 (7 ° )

48 (30)

91 (84) 88 (16)

lO4 (90) I0I (IO)

I I 4 (4 o) I I I (60)

134

179 176

176 162

173

19 ° (9 ° ) 187 (IO)

221 2o8

2

19o (25) 187 (75)

221 208

3

179

208

221

205

4

219

6

205

(D

45 (60)

48 (4 o)

94 (79) 91 (21)

lO 7 (3 o) IO 4 (60) IOI (I0)

114 (50) I I I (50)

137

152 151

162

176

182

193 (16) 19o (84)

224 211

2,3

45 (7 ° )

48 (30)

91

45 (20)

48 (8o)

9I (84) 88 (I6)

lO 4

II 4 (60) I I I (4 o)

lO 7 (60) lO 4 (4 o)

134

137 (60)

152 148

165 (5 ° ) 162 (5 o)

176

179

193 (9 ° ) 19o (IO)

224 2o8

2,6

45 (85)

48 (15)

88 (5)

94 (16) 91 (79)

lO4 (95) I0I (5)

134 (4 ° ) 114 (4 o) I I I (60)

137 (60)

152 151

165 (5 ° ) 162 (50)

176

182

193 (io) 19 ° (9 ° )

224 211

3,4

45 (4 ° )

48 (60)

91 (95) 88 (5)

lO4 (35) IOI (65)

114 (I0) I I I (90)

134

155 148

165 (5 ° ) 162 (5 o)

173

179

193 (25) 19o (75)

a24 2o8

3, 6

( I ) A N D DEUTERATED ANALOGUES

134 (4 ° ) 114 (90) I I I (IO)

149 151

165 (5 ° ) 162 (5 o)

179

I82

193 (75) 19 ° (25)

224 211

2,4

Position o f - C 2 H n groups

MASS SPECTRA (m/e VALUES)OF METHYL-2,3,4,6-TETRA-O-METHYL-~,D-GLUCOSIDE

TABLE I

45 (5 ° )

48 (5 o)

91 (16) 88 (84)

lO 7 (I0) lO 4 (60) I0I (30)

I I I (50)

114 (5 0)

131 (4 ° )

134 (60)

I5"> 148

165

176

179

193 (84) I9 o (16)

224 2o8

4, 6

)....t

o

o ,-4

I3t)

SHORT COMMUNICATIONS

Hence, a c o m p l e t e l y new a p p r o a c h to the identification of aldohexoses is provided. The m e t h y l a t e d m o n o s a c c h a r i d e is c o n v e r t e d to the c o r r e s p o n d i n g m e t h y l glycoside a n d s u b s e q u e n t l y d e u t e r o m e t h y l a t e d w i t h C ' H a I . The c o n s t a n t s of the substance o b t a i n e d will indicate the p a r e n t monosaccharide, whereas comparison of its mass s p e c t r u m with the d a t a p r e s e n t e d in Table I will enable assigning the positions of -C~Ha groups, i.e., positions of u n p r o t e c t e d h y d r o x y l s in the m e t h y l a t e d monosaccharide p r o d u c e d during h y d r o l y s i s of the polymer. The p r o p o s e d m e t h o d has the a d v a n t a g e over existing m e t h o d s in t h a t it makes possible the s t a n d a r d i z a t i o n of p r o c e d u r e for all type.~ of m o n o s a c c h a r i d e s as well as the a d v a n t a g e of requiring no a u t h e n t i c samples besides r e a d i l y available p e r m e t h y l monosaccharides. This m e t h o d of m e a s u r e m e n t of the mass s p e c t r u m needs only minor q u a n t i t i e s of c o m p o u n d , a n d in c o m b i n a t i o n with gas -liquid, or t h i n - l a y e r ~ c h r o m a t o g r a p h y , it should simplify c o n s i d e r a b l y the s t r u c t u r a l analysis of c a r b o h y d r a t e polymers. The a p p r o a c h m u s t r e m a i n valid on going to m e t h y l ethers of a n y t y p e of monos a c c h a r i d e - aldopentoses, 6-deoxyaldohexoses, ketoses, deoxv- a n d d e o x y a m i n o sugars, etc. The e s t a b ! i s h m e n t of the necessary e x p e r i m e n t a l d a t a fl)r these f u r t h e r a p p l i c a t i o n s is now in progress. Institute f o r Chemistry of N a t u r a l Products, A c a d e m y of Sciences, .lloscow (U.S.S.R.)

N . K . Kocm,:rKov O. ,~. CHIZHOV

t 1~. I(UHN, t-1. TRISCHMANNAND [. 1.6w, ..lngew. Chem., t, 7 (1955) 32. z 1¢.. tQUHN, H. ]3AI.;RAND A. SEI,'.LIGI,;I~,,.=lll~Z. Chem., ()l t (i958) 23(2. a G. N. 1,2OWKAI~aNY,Advan. Carbohydrate Chem., 9 (1954) 3o4. ~lW. \V. BINKLEY, Advan. Carbohydrate Chem., 1o (I955) 55. 5 C. T. BISHOp AND F. 1'. CooPeR, Ca~..[. Chem., 38 (I9()O) 38S. 6 I t . \V. KIRCHER, Anal. Chem., 32 (1960) 1103. v N. K. I(OCHETKOV, (). ,~. (~HIZHOV, N. S. \~7ULFSON AND |~. ~I. ZOLOTARI:\:,

Tetrahedro~z, in

the

press. s K. ]-IEYNSAND 11. F. (;Ri"TZMACHI~;R,:tHqeW. Chenz., 74 (I9()2) 387. Received N o v e m b e r 4th, I9() 3 Biochim. Biophys..4 cta, 83 (i c)t)4) ~34-130