ORGANIC MASS SPECTROMETRY, VOL. 27, 962-966 (1992)

Analysis of Tetrabenzyl N-Glucosidic, N-Mannosidic and N-Galactosidic Isomers using Fast Atom Bombardment/Mass-analysed Ion Kinetic Energy Spectrometry

~~~~~

Lin Yan* and Jiarou Peng Spectral Analysis Laboratory, Analysis and Computation Centre, Box 219, Beijing Medical University, Beijing 100083, China

Xianyun Jiao and Mengshen Cai School of Pharmaceutical Sciences, Beijing Medical University, Beijing 100083, China

A series of new synthetic tetrabenzyl N-glucosidic, N-mannosidic and N-galactosidic isomers were investigated by fast atom bombardment (FAB)/mass-analysed ion kinetic energy (MIKE) spectrometry. The [ M + H] + ions were obtained with high abundance in the FAB spectra when using 3-nitrobenzyl alcohol as the matrix. The FABWIKE spectra provide characteristic daughter ions fragmented from selected molecular parent ions, allowing these isomers to be differentiated. In addition, an interesting rearrangement was found from the MIKE spectra, indicat- ing that tbe benzyl (Bzl) group on the sugar ring is rearranged on to the N atom of the base (R) group to form [R + Bzl+ HI+ and [R + 2BzlI' ions.

INTRODUCTION ~~ ~~ ~~

EXPERIMENTAL

Recently, a series of new tetrabenzyl N-glycosidic, N- mannosidic and N-galactosidic isomers (listed in Table 1) have been synthesized by Jiao and co-workers' by the reaction of l-trichloroacetoxyl or l-trifluoroacetoxyl perbenzylated sugars and protected bases in the pres- ence of the catalyst SnCl, . Since previous studies of the stereoselective synthesis of nucleosides have seldom considered the use of benzyl groups for the protection of the hydroxyl groups of sugars, these syntheses have widened such studies to some extent.

Under the conventional mass spectrometric condi- tions, e.g. using electron impact (EI) and chemical ion- ization (CI), it is not easy to obtain the molecular ion peaks of synthetic N-glycosides, because it is difficult to vaporize these compounds owing to the polarity of their bases and their C ( 1 h N bonds are unstable in glyco- sides protected by benzyl groups. Fast atom bombard- ment (FAB) mass spectrometry' is an efficient method for analysing these compounds, but it does not give suf- ficient information to differentiate isomers with the same relative molecular mass.

Mass-analysed ion kinetic energy spectrometry (MIKE) can overcome this problem. With its selective detection, MIKES allows the common ions from both the sample and the matrix to be ignored and the behav- iour of the relevant molecular ions and their metastable ions, which are most likely to differentiate similar struc- tures, to be observed in isolation. Previous studies have shown the role of FAB/MIKES in studying subtle dif- ferences in the structures of saccharides and solving some stereochemical problem^.^-^

In this paper, we analysed fifteen tetrabenzyl N- glucoside, N-mannoside and N-galactoside compounds using the FABIMIKES technique.

Compounds 1-15 were synthesized by methods described elsewhere' and were purified by thin-layer chromatography. The anomeric configurations of the samples were verified by ' 3C NMR spectroscopy.

FAB and FAB/MIKES experiments were performed on a VG ZAB-HS mass spectrometer fitted with an Ion Tech saddle field gun. Argon was used as the bom- barding gas with an energy of 8 keV and a gun monitor current of 1.8 mA. The accelerating voltage was 8 kV. MIKE spectra were obtained by selecting the ion with the magnetic sector and then scanning the electric sector voltage at a fixed accelerating voltage and mag- netic field. All spectra were recorded with a Model 11-250 data system.

Each of compounds 1-15 was prepared as a chloro- form solution with an approximate concentration of 10 pg p1-'. A 1 p1 volume of the sample solution, sus- pended in about 1.5 p1 of 3-nitrobenzyl alcohol, was loaded on to the stainless-steel tip. After inserting the probe in the ion source, FAB spectra were taken at a scan rate of 10 s per decade. MIKE spectra were obtained according to the same procedure at a scan rate of 12 s per scan.

RESULTS AND DISCUSSION

All the compounds (Table 1) were investigated by FAB mass spectrometry. A careful comparison with glycerol (GLY) and 3-nitrobenzyl alcohol (NBA) as the matrices showed that the [M + HI+ ions were obtained with high abundance when using NBA as the matrix, but

0030-493X/92/090962-05 $07.50 0 1992 by John Wiley & Sons, Ltd.

Received 30 September 1991 Revised manuscript received 26 May 1992

Accepted 1 June 1992

TETRABENZYL N-GLUCOSIDIC, N-MANNOSIDIC AND N-GALACTOSIDIC ISOMERS 963

Table 1. Compounds investigated

OBll

N- G lucosides (1-6)

Type

N- G lucosides

N-Mannosides

N -Galactosides

N - Mannosides N -Galactosides (7-1 2) (13-15)

Compound No.

1 2 3 4 5 6

7 8 9

10 11 12

13 14 15

Anomeric configuration

8- D a- D

8- D 8- D 8- D 8- D

a- D

8- D 8- D a- D

a. 8 - D B- D 8- D

a-D

a. 8-0

RMM

591 634 634 640 648 71 5

591 634 634 640 648 71 5

591 648 71 5

R Name

1,2,4-Triazole R,

Structure

I

R3 Benzimidazole

R4 Thymine

R5 2-N-Acetylguanine

Systematic name

1 - N - (2,3,4,6-0-Tetrabenzyylglucopyranosyl)- 1,2.4-triazole 1 -N- (2.3.4.6-0-Tetrabenzyylglucopyranosyl)uracil 1 - N - (2,3,4,6 -0 -Tetra benzylgl ucopyranosyl) uracil 1 -N- (2,3,4,6-O-Tetrabenzylglucopyranosyl) benzimidazole 1 -N- (2,3,4,6-0-Tetrabenzylglucopyranosyl)thymine 9-N- (2,3,4,6-0-Tetrabenzylglucopyranosyl)-2-N-acetylguanine

1 -N- (2,3,4,6-0-Tetrabenzylmannopyranosyl) -1,2,4-triazole 1 -N- (2,3,4,6-0-Tetrabenzylmannopyranosyluraci\ 1 -N- (2,3,4,6-0-Tetrabenrylmannopyranosyl)uracil 1 -N- (2,3,4,6-0-Tetrabenzylrnannopyranosyl) benzimidazole 1 -N- (2,3,4,6-0-Tetrabenzylmannopyranosyl)thymine 9-N- (2,3,4,6-0-Tetrabenzylmannopyranosyl) -2-N-acetylguanine

1 - N- (2,3,4,6-O-Tetrabenzylgalactopyranosyl) -1.2,4-triazole 1 -N- (2,3,4,6-0-Tetrabenzylgalactopyranosyl) thymine 9-N- (2,3,4,6-0-Tetrabenzylgalactopyranosyl)-2-N-acetylguanine

when using GLY only weak peaks were observed. This result could be attributed to the fact that these tetra- benzyl glycosides have better solubility in NBA than in GLY. In the matrix-background-removed FAB spectra, some fragment ions can be observed in addition to [M + HI'. However, the subtracted spectra do not give sufficient information to differentiate the isomers (Fig. 1).

To obtain further structural information, MIKE spectra were obtained from the protonated molecular ions of the 15 compounds studied. The possible com- positions and m/z values of the major daughter ions are listed in Table 2. The N-glucosides, N-mannosides and N-galactosides are easily differentiated from the differ- ences in the relative abundances of three daughter ions at m/z 415, 431 and 523 in the MIKE spectra, as given in Table 3. As an example, the MIKE spectra of the isomers 1, 7 and 13 with the same relative molecular mass are shown in Fig. 2. Generally, the ion peak at m/z 415 is the base peak in the FAB/MIKE spectra of N- glucosides (except for 6), which indicates that the ion [M + H - RH - BzlOH]' is the most stable. Con- versely, in the FAB/MIKE spectra of N-mannosides (except for 10 and 12), the ion peak at m/z 523 is the base peak, which indicates that the ion [M + H - RH]+ is the most stable. Although only three N-galactosides were studied, their differences from N-glucosides and N-mannosides are quite obvious. The

possible formation pathway of the ions at m/z 415, 431 and 523 is as follows:

'i9 I Y

.z 50

.- P 209

( a )

6f l

I

m /z

Figure 1. Matrix-subtracted FAB spectra of compounds (a) 4 and (b) 10.

964 L. YAN ET AL

Table 2. Probable compositions and m/z values of the major daughter ions obtained from FAB/MIKE spectra of the compounds analysed

Ion composition

[M + HI+ (precursor ion) [M + H - BzlOH]+ [M + H - 2BzlOH]+ [M + H - HBzl]+ [M + H - (2821 - H)]+ [M + H - RH]+ [M + H - RH - BzlOH]+ [M + H - RH - BzlOH - (BzI - H)] +

[M + H - RH - 2BzlOH]+ [M + H - RH - 2BzlOH - ( 2 B ~ l - H)]' [M + H - RH - 2BzlOH - ( BzI - H)] +

[M + H - RH - HBzI]+ [M+H-RH-2BA]+ [RH + H]+ [R+Br l+H]+ [R + 28211 +

[4Bzl- 3H] +

[4Bzl- 3H - C,H,]+ [3Bzl- 2H] +

[3Bzl- 2H + BzlOH] +

[3Bzl- 2H - C,H,]+ [2Bzl- H I +

[2B21 - H + 2BzlOH]+ [2Bzl- H + BzlOH] +

Bzl+

1. 7, 13 2. 3. 8. 9

592 635 484 527 376 41 9 - 543 - 454

523 523 41 5 41 5 325 325 307 307 235 235 21 7 21 7 43 1 431 341 341 70 113

160 203 250 293 361 361 283 283 27 1 271 379 193 193 181 181 397 289 289 91 91

-

-

4.10

641 533

549

523 41 5 325 307 235 21 7 431 341 119 209 300 361 283 271 379 193 181 397 289

91

-

-

6,11.14 8.12,16

649 71 6 541 608 433 500 557 624 468 523 523 41 5 41 5 325 325 307 307 235 235 21 7 21 7 431 431 341 341 127 1 94 21 7 284 307 374 361 361 283 283 271 271 379 - 193 - 181 181 397 397 289 91 91

-

-

The behaviour of 6,12 and 15 is different from that of the other compounds. The relative abundances of the three ions at m/z 415, 431 and 523 are low, so that the differences between them are not obvious. The most intense ion peaks in their MIKE spectra are those at mlz 284 and 608, and the relative abundances of the former show obvious differences. The results are given in Table 4. We can conjecture that this phenomenon, which is different from that with the other glycosides, is due to the influence of the large base group.

From a survey of the MIKE spectra of the 15 glyco- sides, we found that the spectral differences between the isomers caused by the structural differences in the sugar

1001 41 5 I -

f 50 .- I ( a ' .-

c

150 200 250 300 350 400 450 500 550

9 1 1 484

- 523 .- c

$ o-!- , .A; !. , .I 150 200 250 300 350 400 450 500 550

~~ ~

Table 3. Partial FAB/MIKE spectra of the compounds anal ysed

Compound Relative intensity (%)

W e No m/z 41 5 m/z 431 m p 523

N-Glucosides 1 1 00 5 10 13 2 1 00 - 11 3 1 00

4 100 44 5 100 27 12 6 7

- -

- -

N- Mannosides 7 45 44 100 8 36 19 100 9 29 36 100

10 14 89 18 11 29 24 100 12 6 8 5

N-Galactosides 13 71 33 1 00 14 48 100' 71

2 15 9 - a Only the ion peak at m/z 433 was observed.

2,

m + .-

50 .- 0 .- c - $ 0

150 200 250 300 350 400 450 500 550

,i' 2, L .- 2 C 50

.- !2

$ 0

.-

e - 150 200 250 300 350 400 450 500 550

m/z

Figure 2. FAB/MIKE spectra of compounds (a) 1, (b) 7 and (c) 13.

TETRABENZYL N-GLUCOSIDIC, N-MANNOSIDIC AND N-GALACTOSIDIC ISOMERS 965

Table4. FAB MIKE spectra of compounds 6, 12 and 15

Relative intensity (%)

mlz 6 12 15

284 26 61 97 608 100 100 100

ring are far more obvious than those caused by the dif- ferences in anomeric configuration (a, B), as shown clearly in Figs 3-6. Figures 3, 4 and 5 each gives the MIKE spectra of the isomers with the same anomeric configurations, and Fig. 6 gives those with the same sugar ring.

For the six N-mannoside samples, the differences between the MIKE spectra of the a-type and of the /?-type seem similar. For all a-compounds, the ion peak intensities at m/z 415 are higher than those at m/z 431.

200 250 300 350 400 450 500 550 600

200 250 300 350 400 450 500 550 600 m / z

Figure 3. FAB/MIKE spectra of compounds (a) 2 and (b) 8.

= 0 4 . . . , . . . , . , . . . , , , . . ~

200 250 300 350 400 450 500 550 600

200 250 300 350 400 450 500 550 600 m/z

Figure 4. FAB/MIKE spectra of compounds (a) 4 and (b) 10.

415

1

433 I

E A

v) c ._

50 .- z .- e - d o

200 250 300 350 400 450 500 650 600

m /z

Figure 5. FAB/MIKE spectra of compounds (a) 5 and (b) 14.

- ._ ul c

a2

0 0)

f 50 .- ._ + - a 0

200 250 300 350 400 450 500 550 660

m /I

Figure 6. FAB/MlKE spectra of compounds (a) 8 and (b) 9.

Conversely, for all @compounds, the ion peak inten- sities at m/z 431 higher than those at m/z 415.

During the MIKES study, we found an interesting rearrangement phenomenon. For all the compounds investigated, the Bzl group on the sugar ring is rear- ranged on to the N atom of the base group (R) to form [R + Bzl + H]+ and [R + 2Bzl-J' ions. The daughter- ion spectra of these two ions confirmed their structures.

In the MIKE spectra, a series of ions [4Bzl- 3H]+, [3Bzl- 2H]+, [2Bzl- H]+ and Bzl' appeared with fairly high abundances. Further, with these ions as the parent ions, few fragments were observed from their daughter-ion spectra. It is likely that the ions [4Bzl- 3H]+, [3Bzl- 2H]+ and [2Bzl- HI' possess stable resonance structures.

Acknowledgement

Partial support by the Postdoctoral Science Foundation of China is gratefully acknowledged.

966 L. YAN ET AL.

REFERENCES

1. M.4. Cai, D.-X. Qui. Z.-J. Li, J.-X. Yu, X.-Y. Jiao, L.-N. Cai and C. F. Yu, paper presented at the Thirteenth International Congress of Heterocyclic Chemistry, Corvallis, Oregon, U.S.A.. August 1991. 18, 122 (1989).

Nature (London) 293, 270 (1981).

Biomed. Mass Spectrom. 12, 525 (1 985).

4. G. Puzo, J. C. Promeand J. Fournie, Carbohydr. Res. 140, 131

5. M. Becchi and D. Fraisse, Biomed. Environ. Mass Spectrom.

2. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, 6. V. Kovacik, E. Petrakova, J. Hirsch and V. Mihalov. Biomed.

3. E. E. Kingston, J. H. Beynon, R. P. Newton and J. G. Liehr,

(1 985).

Environ. Mass Spectrom. 17,455 (1 988).