ANALYTICALBIOCHEMISTRY64, 45-52 (1975)

Mass Spectrometry of Uronic Acid Derivatives Part V. Fragmentation of Methyl Derivatives of Methyl 4-Deoxy-fl-L-Threo-Hex-4-Enopyra nosiduronic-Acid VLADIMIR KOV~IK AND PAVOL KovX~ Institute of Chemistry, SIovak Academy of Sciences, 809 33 Bratislava, Czechoslovakia

Received May 31, 1974: accepted August 19, 1974 The basic fi-agmentation mechanism of methyl(methyl 4-deoxy-2,3-di-Omethyl-/?-L-threo-hex-4-enopyranosid)uronate has been deduced by deuteromethylation analysis, metastable transition measmements, and by interpreting the spectra of weakly excited foregoing molecules. The differences in fi-agmentation of partially methylated derivatives of methyl 4-deoxy-/3-1:threo-hex-4enopyranosiduronic acid compared to that of the fully methylated substance are discussed in detail and the criteria are proposed for identification of the compounds concerned by mass spectrometly.

The fission of acidic heteropolysaccharides and polyuronates by enzyme-catalyzed (2-4)/3-elimination resulting in the formation of 4,5unsaturated 4-deoxyhexuronides appears to be a general means of degradation of these substances in nature. In addition, poly(hexopyranosid)uronates also undergo depolymerization by base-catalyzed fl-elimination reaction (5-7). The B-ehmination process, which may occur also under neutral conditions (8) was previously studied by treatment of esterified uronic acid derivatives with a number of basic agents using mesyloxy (9-1 1), acetoxy (1 1,12), methoxy (1 1,13), glycosyl (14), hydroxyl (15), and isopropylidene (16) as leaving groups. The information accumulated supports the assumption (17,18) that B-elimination products may be found among the products of methylation of uronic acid derivatives. With this in mind we have attempted to elaborate a method for the determination of the structure of methyl derivatives of 4,5-unsaturated 4-deoxyhexuronic acids and have studied the mass spectroscopic fragmentation of methyl derivatives of methyl 4-deoxy-/3-k-threo-hex-4enopyranosiduronic acid. 45 Copyright © 1975 by Academic Press, Inc. Printed in the United States. All rights of reproduction in any form reserved.

46

KOVACIK AND KOV~C

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47

URONIC ACID DERIVATIVES

MATERIAL A N D METHODS

Compounds investigated R1

R2

R~

1 2 3 4 5

CH3 CH3 CD3 CH3 CH~

CH3 CD3 CD3 H D

CH3 CH3 CHa CHa CH3

6

H

H

CH:~

R1

R2

R3

7 8 9 10 11

D CH3 CH3 CHz CH~

D CHa CH3 H D

CH3 H D H D

12

H

H

H

Compounds 1, 4, and 6 were prepared as described (16,19). Cornpounds 2 and 3 were made by trideuteriomethylation of 4 and 6 with CD3I and AgzO. Saponification (20) of 1, 4, and 6 afforded compounds 8, 10, and 12, respectively. O-Deuteration of substances was accomplished by evaporation of solutions of 4, 6, 8, and 10 in DzO directly in the mass spectrometer. COOR,

~o~

\ //OMe r OR]

Mass Spectrometry The mass spectra were obtained with a MCh 1306 mass spectrometer (USSR) at an ionizing potential of 70 eV. The inlet temperature was 20-40°C, and that in the ionizing chamber 120-130°C. Compound 1 was also studied using electrons of 10, 11, 12, and 13 eV. The peak intensities are given in percentage of the total ionization % E45. Exact massmeasurements of ambiguous peaks were done with the accuracy of 10 ppm using Varian MAT, SM-1B spectrometer. The latter instrument was used also for finding the metastable transition by defocusation technique (Scheme 1). The metastable peaks found in the spectra are in the text represented by their calculated values. To avoid introduction of new nomenclature of ions use was made of the formal resemblance of the ions discussed with those produced in the fragmentation of permethylated methyl hexopyranosides (21) and the symbols introduced by Kochetkov are used throughout. RESULTS A N D DISCUSSION The mass spectra of methyl(methyl 4-deoxy-2,3-di-O-methyl-/3-Lthreo-hex-4-enopyranosid)uronate 1, its 3-O-trideuteriomethyl-, and 2,3di-O-trideuteriomethyl analog 2, and 3 are given in Table 1. In addition

48

KOVACIK AND KOVAC TABLE 1 OF Methyl(Methyl4-Deoxy-2,3-di-O-Methyl-B-L-Threo-Hex-4Enopyranosid)Uronate AND ITS TRIDEUTERIOMETHYL ANALOGS

MAss SPECT~

m/e 238 235 232 207 204 201 172 169 147 144 141 137 134 131 130 127 129 116 113 114 111

I

2

3

m/e

0.080

107 104 101 98 92 91 89 88 85 81 78 75 76 73 71 62 59 57 55 48 45

0.058 0.089 0.086

0.094 0.034

0.199 1,003

0.304 0.301

0,884

0.424 0.169

0.634 0.049 0.223 0.403 0.111

0.111 0.179 0.051 0.248 0.149

0.055 0.101

0.397

0.237

0.874

0.437

0.026 0_189 0.189 0 240

1

2

0.735 0.785

0.240 1.045 0.523

2.086 59.595 5.761

2.398 64.500 0.892

7.350

5.042 1.998

9.734 0.397 1.788 0.298 0.795

0.039 0.461 0.430 1.691 0.369 0.953

6.655

8.116

3 0.129 0.171 0.394 0.274 2.228 65.078 4.628 0.543 1.286 4.542 4.799 4.228 0.150 0.514 1.086 0.143 0.743 3.657 3.086

to these data, the proposed fragmentation scheme (Scheme 1) is based on the metastable transition measurements and the spectra obtained with electrons of 10-13 eV. Scheme 1 shows the disintegration series and also the relative contributions of the produced isomeric ions. The main course of the fission of 1 and 3 is retro-Diels-Adler fragmentation, where the charged ions are mainly those of the structure M e O h C H - - C H - - O M e (H1) which, together with the products of their disintegration, form about 80% of the total ion intensity. H; ions containing the atoms C(3)-C(6) are visible only in the spectra taken with electrons of 11-13 eV. After an impact with electrons of 70 eV the ions Ha readily disintegrate and after splitting off of the carbomethoxy radical the ions at m/e 85 are formed, the accurate measurement of which (in the fragmentation of 1) confirmed the elemental composition C4H50~,. The ions J1, composed of two isomers and making about 8% of the total ion intensity, are formed even after impact with electrons of 10 eV. The cleavage of a methoxyl group from the molecular ions in the form of a radical occurs simultaneously at the anomeric center and at C-3 in the ratio 4 : 1. After elimination of methanol the ions A.~ are produced. The least abundant ions are those of the Series C. The ions C1, formed after +

1

z

URONIC

49

ACID DERIVATIVES

a molecule of H C O O C H a had been eliminated, could be seen only in the spectra obtained with low energy electrons ( 1 1 - 1 3 eV). These ions are precursors to the ions Cz formed after the cleavage of a methoxyl radical from either C-2 or from the carbomethoxyl group (7:3). Scheme 1 has wider applicability than to that of the fragmentation of methyl derivatives of the title acid. In an analogous manner the structure of methyl(methyl 2-O-benzyl-4-deoxy-3-O-methyl-fl-L-threo-hex-4enopyranosid)uronate (22) and methyl(4-deoxy-l,2-O-isopropylidene-flL-threo-hex-4-enopyranosid)uronate (16) could be elucidated. In the fragmentation of methyl(methyl 4-O-deoxy-2-O-methyl-B-Lthreo-hex-4-enopyranosid)uronate 4 and methyl (methyl 4-deoxy-Cl-ethreo-hex-4-enopyranosid)uronate 6 (Table 2) the course of all four Series, shown in Scheme 1, can be seen. Compared to the permethylated derivative 1, the values m/e of the corresponding ions containing hydroxyl groups are shifted to lower values by 14 and 28 units. In Series A the elimination of methanol is accompanied by liberation of water (187 ~ 169 in the fragmentation of 4, 173 --* 155, m* = 138.9 in that of 6). From the ions H1 of the fragmentation of 6 at role 74 a methyl radical is cleaved offto give rise to the ions H 2 a t Ill/t2' 5 9 ( m * = 47_0). The ions H; are at role 71 and as found by the accurate mass measurement, have the composition C:~H.~O,. The poor abundance of the ions J1 at role 61 is TABLE 2 MASS SPECTRA OF METHYL DERIVATIVES OF Methyl 4-Deoxy-fi-L-Threo-Hex-4-Enopyranosiduronic Acid

mle 218 204 187 186 173 172 169 158 155 144 141 131 129 127 113 111 101

4 (2.6)

6 (61

8 (2.31

10 (2)

0.053 0.102 I).155

0.106 0.483 0.638

0.186 I),029 0.043 0.172 0.114 0.815 0.915 0.086

0.102

0.153 0.024 0.071

0.828 0.629

0.143 0.273 0.630

0.284 0.261 0.376 1.050 0.590

0.297 0,285 0,333 0,368

2.145 0.600 0.110 0.279 0.166

0.758 0.615 0.200 0.157

m/e 99 98 89 88 87 85 75 74 73 71 69 61 59 57 55 45

4 (2.6)

6 (6)

8 (2.3)

10 (2)

0.431 1.526 2.247 55.274

0.286

0.590 0.958 2.430 51.553

0.368 1.699 2.245 44.316

5.605 6.149 0.797 14.498 1.098 1.150

1.948 0.974 2.435 14.788 4.335 1.390 1.615 1.592 0.725 2.043 17.104

2.100 1.537 1.975 10.646 4.607 1.099 0.910 2.545 0.653 1.745 9.910

0.400 2.503 3.332 60,603 10.982 0.400 1.087 8.723 0.644 1.573 3.232

1.664 1.303 2.890 5.145

50

KOVACIK AND KOVAC

caused by their disintegration, after elimination of methanol, to the ions at m/e 29 (23). In the spectra of the methylated acids 8 and 10 the peaks of the molecular ions are not present, which is a significant difference compared to the spectra of the methyl esters 1-7. The fragmentation proceeds essentially according to Scheme 1. Due to its thermal instability a clear spectrum of the title acid 12 could not be obtained. In addition to the fragmentation described above the compounds containing one and two hydroxyl groups 4, 6, and 10 undergo an additional mode of fission not occurring when the carbon atoms C-2 and C-3 bear methoxyl groups (as in 1-3 and 8). From the molecular ions of 4 and 10 having one hydroxyl group a molecule of methanol is eliminated to give rise to the ions (M-32) + (m* = 158.7 for 218 ~ 186 in 4). It could be proved by analyzing the spectra of the deuteroanalogs 5 and 11 that there is an interaction of the methoxyl group with the adjacent hydroxyl group resulting in the formation of an epoxide. The spectrum of 7, the deuteroanalog of 6, showed the interaction of the two hydroxyl groups producing the ions (M-18) + ( m * = 169.6 for 204--~ 186). The ions (M-32) + and (M-18) + disintegrate after elimination of CO from the epoxide ring to give rise to the ions at m/e 158 (in the case of 4 and 6) and m/e 144 (in the case of 10) (m* = 134.2 for 186 --~ 158, found in the spectra of 4 and 6). The obtained theoretical information about the fragmentation of methyl derivatives of methyl 4-deoxy43-L-threo-hex-4-enopyranosiduronic acid can be used in practice for the structure determination of its fully and partially methylated derivatives which may arise in methylation analysis of uronic acid-containing substances. Table 3 contains the features characteristic of the fragmentation of the possible methylated derivatives of the title acid. The proposed features characteristic of the fragmentation of methyl (methyl 4-deoxy-3-O-methyl-/3-e-threohex-4-enopyranosid) uronate and of the corresponding acid are based on the fragmentation scheme of the permethylated derivative 1 and the differences between its fragmentation and those of the partially methylated derivatives and free acids. Using Table 3 the number and the location of the methyl groups in methyl derivatives of the title acid can be assigned according to the following criteria: 1. The presence of the peak of the molecular ions confirms the esterification of the carboxyl group. 2. The value of m/e of M, A1 (A2) or (M-32) + ions gives the number of the methyl groups present in the molecule. 3. The m/e value of Ha ions (the base peak of the spectrum) and H2 ions shows the substitution at C-2. 4. The location of intense signals of the ions H.~ at m/e 85 or 71 (4-8%

51

URONIC ACID DERIVATIVES TABLE 3 OF O-Methyl DERIVATIVES OF Methyl 4 - D e o x y - f l - E - T h r e o - H e x - 4 - E n o p y r a n o s i d u r o n i c A c i d

CHARACTERISTIC

FEATURES

Symbol of ions

m/e

M

232 218 204 201 187 173 186 172 169 155 88 74 73 59

A1

M-32 (or M-18) Az H1 H2

H" J~ Intensity ratio

OF

FRAGMENTATION

(2.3.6)

(2.6)

(2.3)

XXX

XXX

XXX

(3.6)

(2)

(61

XXX XXX

XXX

XX

85 71

X

75 61 85/71

X

XX

X

14.5

XX

0.5

X

• • •

X

X

- •

5.

5-10

0.4

- "

Peak intensities: • ~< 0 . 5 : 0 . 5 < • • ,a 1.5:1.5 < • 4 < x ~< 8; 8 < xx