ORGANIC MASS SPECTROMETRY, VOL. 27, 799-806 (1992)

Characterization of Glucosamine and N-Acetylglucosamine Oligomers by Fast Atom Bombardment Mass Spectrometry

C. Bosso

A. Domard* Centre de Recherches sur les Macromolicules Vbgbtales (CERMAV-CNRS), BP 53X, 38041 Grenoble Cedex, France

Laboratorie &Etudes des Materiaux Plastiques et des Biomatbriaux (URA CNRS No. 507), Universitb Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918,69622 Villeurbanne Cedex, France

The oligomers of N-acetylglucosamine [degree of polymerization (d.p.) 2-61 and of glucosamine (d.p. 3-9) were systematically studied by the fast atom bombardment technique. Positive- and negative-ion detection were used successively in each case. The latter provided more information in respect of the number of fragmentation path- ways. In addition to the classical fragmentations usually obtained with other oligosaccharidic chains, new species specific to these two series were observed. They depend mainly on the nature of the substituents located on the C(2) positions of the glucosidic units constituting the chain.

INTRODUCTION

Since the first results on monosaccharides were reported by Barber et al.' in 1982, there have been numerous publications on applications of the fast atom bombard- ment (FAB) technique for the characterization of oligo-

Various mechanisms for the cleavages of glycosidic links have been proposed7** and the rules governing the fragmentation of the oligosaccharide chains refined by tandem mass spectrometric studies on model compounds subjected to negative-ion FAB.'-"

This work was focused on the study of the oligomers of N-acetylglucosamine with degrees of polymerization (d.p.) varying from 2 to 6 and those of glucosamine with d.p. 3-9. The structures of these two series are shown in Fig. 1. These oligomers were obtained by acidic hydrolysis of chitin and poly(glucosamine), respectively, followed by separation by steric exclusion chromatog- r a p h ~ . ' ~ , ' ~ The study of the various products by both positive- and negative-ion FAB [FAB( +) and FAB( -)]

showed that the extent of fragmentation and the frag- mentation pathways are a function of the nature of the substituent on the C(2) position of the glucosidic repeat- ing units. A comparison was made with previously published results obtained with other oligosaccharide series.'

EXPERIMENTAL

The FAB mass spectra were recorded on a Nermag R lOlOC mass spectrometer with 2000 u as the upper limit of detection. The instrument was equipped with an M-Scan Wallis-type gun.

For the two families of oligomers studied, a glycerol matrix acidified with 0.1 p1 of 0.1 M hydrochloric acid was used. The oligomers were obtained as described previously.' The glucosamine oligomer series were all isolated in the hydrochloride form.

(B) n=0to7; d.p.=3 to9

Figure 1 . Structures of the products studied. (A) N-acetylglucosarnine oligorners; (B) glucosarnine oligorners.

0030-493X/92/070799-08 $09.00 0 1992 by John Wiley & Sons, Ltd.

Received 29 January 1992 Revised manuscript received 21 March 1992

Accepted 22 March 1992

800 C. BOSS0 AND A. DOMARD

RESULTS AND DISCUSSION

Study of oligomers of N-acetylglucosamine

The results of a preliminary mass spectrometric study of N-acetylglucosamine oligomers prepared from chitin by a different method of depolymerization has been report- ed previ~usly. '~*'~ The purpose of that work was essen- tially to characterize the purity of the products and not to investigate systematically the various fragmentation mechanisms. This paper will discuss the behaviour of the oligomers when they are subjected to FAB(+) and FAB( -) and in particular the types of cleavage that are observed.

Study using positive-ion FAB. Analysis of the results obtained with the oligomers of d.p. 2-6, subjected to FAB(+), reveals three types of cleavage which are depicted in Fig. 2. In addition to the classical ions [M + H]+ characterizing the relative molecular mass of the products, a new fragmentation appears giving

[ MH-CHI=C=O I+/ CHqOH

H O Y w o \ r

[M + H - CH,=C=O]'. Reputure of the glycosidic links leads to a series of ions starting at m/z 204 then followed by species separated by Am = 203, the number of species depending on the particular oligomer (Table 1). Examples of the mass spectra for the d.p. 3 and 5 oligomers are shown in Fig. 3. The relative molecular mass (RMM) characterization of these oligomers is made relatively easy by the presence of the [M + HI+ ions, although fragmentation becomes more and more difficult as the d.p. increases, and is particularly so for the d.p. 6 oligomer whose RMM is 1236. In this series of products, the ions from pathway B or C give more intense peaks than in the case of the series of cello- and malto-oligosaccharides analysed previously.' This can be related to the strong inter- or intra-molecular hydro- gen bonds which are established as the d.p. increases and which reduce considerably the solubility in water.

Study using negative-ion FAB. Comparison of the spectra obtained by the two methods of analysis shows FAB( -) as giving more information in respect of the number of fragmentation pathways. In addition to the classical deprotonated ions, the spectra show an intense peak corresponding to [M - H - H,O]-, which was vir-

[ M+H]+

1 B o r C

-O* n-1 tondl NHAc

+H+

OH 4

A

Figure 2. Main pathways of fragmentation of the oligomers of N-acetylglucosamine obtained by positive-ion FAB in the glycerol matrix.

Table 1. Main ions observed by FAB(+) and FAB(-) with the N-acetylglucosamine oligomers in a glycerol matrix

Ions observed by FAB(+) Ions observed by FAB( -)

D.p. of Pathway Pathway Pathway B Pathway oligomer RMM [M + HI+ [M + H - CH,-C-O]+ Pathway A B or C [M - H I - B or C or C - H,O Pathway E Pathway G G - H,O

2 424 425 383 222 423 220 262 322 304 204 407 204 222 220 202,405 262 322 304

3 627 628 586 407 425 626 423 608 465 525 507 61 0

204,407 222 220 202,405 262 322 304 425 829 423 608, 811 465 525 507 628 626 668 728 71 0

202 405

4 830 831 789 610, 813

204, 407 222, 425 220, 423 202,405 262,465 322, 525 304, 507 5 1033 1034 992 610, 813 628, 831 1032 626,829 608, 81 1 668, 871 728, 931 71 0, 91 3

204,407 222,425 220, 423 202,405 202,465 322, 525 304, 507 6 1236 1237 1195 61 0. 81 3 628, 831 1235 626,829 608. 81 1 668,871 728, 931 71 0,913

1016 1014

1016,1219 1034 1032 1014.1217 1074 1134 1116

CHARACTERIZATION OF GLUCOSAMINE AND N-ACETYLGLUCOSAMINE OLIGOMERS 80 1

'""1 1461 G 100 1277

I d p.=3 d.P.=5

m'z mz

Figure 3. Positive-ion FAB mass spectra of d.p. 3 and 5 oligomers of N-acetylglucosamine in a glycerol matrix (G).

tually absent in the results obtained with cello- and malto-oligosaccharides.5 Moreover, other ions produc- ed by the cleavage of the glycosidic linkages according to pathway B or C are observed (Fig. 4). These give a peak at m/z 220, then a series of ions, regularly separat- ed by Am 203, up to the molecular ion (Table 1). This last pathway of cleavage is also accompanied by the loss of H,O giving an ion at m/z 202 and a series of others separated by Am 203, the number depending on the particular oligomer.

The main pathway is represented by the E series which is characterized by a first signal at m/z 262 and, as above, a series of others separated by Am 203. A final series of fragments constitutes pathway G.16 It gives a signal at m/z 322 and various others separated by Am 203. This latter pathway of cleavage is also accompa- nied by a loss of H 2 0 (Table 1).

To the authors' knowledge, this series of oligosac- charides is the first which gives so much information on

spectra obtained in the negative-ion mode (Fig. 5). Although the decomposition pathways B/C or E give signals of similar intensity to those of the series of cello- or malto-oligosaccharides, this is not the case for the G and G - H 2 0 fragmentations, which give considerably enhanced signals in the case of the oligomers of N - acetylglucosamine.

Study of the oligomers of glucosamine

As represented in Fig. 1, oligomers of glucosamine were isolated in the hydrochloride form. Nevertheless, under the conditions used in our experiments, only the oligo- mer of d.p. 3, having an RMM of 501 for the free amine form, gives a FAB mass spectrum where the amine func- tion on C(2) appears in the hydrochloride form. The other oligomers of d.p. 4-9 give spectra where this sub- stituent is in the NH, form.

. .- I M-H I /

[M-H-H~O] -------

NHAc n to n=O

NHAc n to n=O

E B or C

Figure 4. Main pathways of fragmentation of the oligorners of N-acetylglucosamine obtained by negative-ion FAB in a glycerol matrix.

802 C. BOSS0 AND A. DOMARD

d.p.=3 d p = 5

200 300 600

nYz mZ

Figure 5. Negative-ion FAB mass spectra of d.p. 3 and 5 oligomers of N-acetylglucosamine in a glycerol matrix (G)

100

____I r:

323

02

i36

i

9p" Figure 6. Positive- and negative-ion FAB mass spectra of a d.p. 3 glucosamine oligomer in the hydrochloride form in a glycerol matrix.

CHARACTERIZATION OF GLUCOSAMINE AND N-ACETYLGLUCOSAMINE OLIGOMERS

100

803

369

d.p.-4

I A

B o r C Figure 7. Main pathways of fragmentation of the oligorners of glucosarnine obtained by positive-ion FAB in a glycerol matrix.

Case of the oligomer of d.p. 3. The oligomer of d.p. 3 is the only unit which, under the conditions used, allows con- firmation of the B structure represented in Fig. 1. The spectrum obtained by FAB( +) (Fig. 6) shows the pres- ence of an ion at m/z 574 corresponding to [C18H,,013N,Cl,]+ and one at m/z 395 corresponding to [C,,H,,O,N,Cl,]+, which is due to cleavage of the glycosidic link. However, it is important to note the very low intensity of these signals compared with the series of intense signals due to species in which the C(2) amine group is present, not as the hydrochloride, but as the free NH, form. This form is the only one observed in the FAB(+) spectra of the higher homologues (d.p. 4-9) under the experimental conditions used. The d.p. 3 oligomer also shows a peak for the [M + H]+ ion at m/z 502, corresponding to [CI8H,,Ol3N3]+, in addi- tion to a peak for the [M + H - H,0] ' ion at m/z 484 and for the fragments at m/z 323 ([C12H2,08NZ]+) and

341 ([Cl2HtSO9N2]+). The FAB( -) spectrum confirms these observations. It shows the presence of an [M - HI- ion at m/z 608 corresponding to [C,8H,,01,N,Cl,]-, with M corresponding to [C,,H,80,,N,Cl,], and a new form, [M + Cl]-, with M = [C,,H3,0,,N,], which gives the most intense signal at m/z 536 and which corresponds to [C,,H,,O,,N,Cl]-. This latter form will be the main species observed in the FAB(-) spectra of the oligo- mers of higher d.p. It is accompanied by fragments which have retained a chloride ion in their structure but the spectrum does not show the [M - HI- form corre- sponding to the structure having the free NH, form on all the glucosidic rings of the oligomer.

Study using positive-ion FAB. Analysis of the mass spectra obtained with the oligomers of d.p. 3-9 leads to the identification of three pathways of fragmentation.

lo/ d.p.=6

x 5 I I I I I I i

8?2 - 7 900

985

9671 1 .

mz mz

Figure 8. Positive-ion FAB mass spectra of d.p. 4 and 6 oligomers of glucosamine in a glycerol matrix (G).

804 C. BOSS0 AND A. DOMARD

Under the experimental conditions, except for the case mentioned above, only the NH, form of the amine groups on the C(2) is detected.

The first degradation pathway (A) represents the cleavage of the glycosidic linkages leading to an ion giving a signal at m/z 323 and other peaks separated by Am 161, the number depending on the particular oligo- mer (Fig. 7). A second decomposition pathway also cor- responds to cleavage of the glycosidic linkages but, in this case, with migration of protons leading to the B or C fragmentation mode. It gives a peak at m/z 341 and signals separated by Am 161 varying with the d.p. of the oligomers. These cleavage pathways A and B or C give very intense peaks, contrary to those obtained in the case of cello- and malto-oligosa~charides,~ where pathway A was the most important. A last mode of fragmentation consists in the cleavage of the glucosidic ring giving the pathway D represented in Fig. 8. These spectra show a series of peaks starting at m/z 369 and with the others each separated by Am 161. This is in contrast to the case of cello- and malto-oligosaccharides where, for similar experimental conditions, it was not observed.

Study using negative-ion FAB. As mentioned above, except for the case of the oligomer of d.p. 3, the -NH3+ C1- form of the amine groups on the C(2) position of each glucosidic unit of these oligomers was not directly visible. Nevertheless, the presence of chlorine, mainly in the [M + C11- form, can be observed for all the com- pounds studied (Table 2). This behaviour is very differ- ent from that shown by most of the carbohydrates studied in a glycerol matrix, acidified with HC1. In these cases, the presence of an [M + Cll- ion of low abun- dance is often found simultaneously with an [M - HI- ion of high abundance.

Two fragmentation pathways allow the structure of an oligomer to be established (Fig. 9). The first mode of cleavage corresponds to the rupture of the glycosidic links with migration of protons and attachment of a C1- ion. It generates a signal at m/z 375, which corre- sponds to an ion obtained by paths B or C + C1-, and a series of peaks increasing by Am 161 at each step. These ions may lose a molecule of water, thus providing another series of ions starting at m/z 357 (Fig. 10). A second fragmentation pathway [D + C1-] corresponds to cleavage of the glucosidic ring and leads to a series of ions with C1- attached, starting at m/z 403 and increas- ing by Am 161 each step. This behaviour is different from the case of N-acetylglucosamine oligomers where the E and G pathways are well defined but where the D mode of decomposition is not observed. Similarly, the behaviour of the glucosamine oligomers differs from that of the cello- and malto-oligosaccharide series in which fragmentation pathway G is observed whereas pathway E is not.

The failure to detect the -NH,, HC1 structure despite the fact that the species were isolated in the hydrochloride form may be due to several factors. First, as a general rule, the hydrochloride form, whatever, the product, is rarely observed by FAB. This may be related to some instability of this form when subjected to this technique. Second, the dielectric constant of glycerol is close to 42, which is much lower than that of water and favours the -NH, , HCl form. Third, the hydrophilicity and the pK, values of these oligomers increase with decrease in the d.p. Finally, FAB does not differentiate between the forms -NH,, HCL and -NH3+ t C1-. Hence the small amount of hydrochloride salt structure observed with the oligomer of d.p. 3 could be attributed to both the presence of water and to the high pK, value of this oligomer, leading to the formation of some of the

Table 2. Main ions observed by FAB( +) and FAB( -) with the glucosamine oligomers in glycerol matrix

Ions observed by FAB(+) Ions observed by FAB(-)

D.p of RMM Pathway Pathway B Pathway B oligomer (NH, form) [M + H] + Pathway A B or C Pathway D [M + CI] - or C + CI- or C + CI- - H,O

501 3 4 662

5 823

6 984

7 1145

8 1306

9 1467

502 323 484

663 323 484 645

323,484 824 645, 806

985 323,484 645, 806

967 323, 484

1146 645, 806 967, 11 28 323, 484

1307 645,806 967, 11 28

1289 323, 484

1468 645, 806 967, 11 28

1289, 1450

341

341 502

341 502 663

341, 502 663, 824

341, 502 663, 824

985 341, 502 663, 824 985, 11 46

502, 663 824, 985

1 146, 1307

369

369 530

369 530 691

369, 530 691, 852

369, 530 691, 852

1013 368, 530 691, 852

1013, 1174

530, 691 852, 101 3 11 74,1335

536 375

375 536 697

375 858 536

697 1019 375, 536

697. 858

375, 536 1180 697, 858

1019 375, 536

1341 697,858 1019, 1180

375, 536 1502 697, 858

1019,1180 1341

357

357 51 8

357 51 8 679

357, 518 679, 840

357, 518 679, 840

1001 357, 518 678,840

1001, 1162

357, 518 679, 840

1001,1162 1323

Pathway D + CI-

403

403 564

403 564

725 403, 564 725,886

403, 564 725,886

403, 564 725, 886

1047

403, 564 725, 886

1047, 1208

CHARACTERIZATION OF GLUCOSAMINE AND N-ACETYLGLUCOSAMINE OLIGOMERS 805

Figure 9. Main pathways of fragmentation of the oligomers of glucosamine obtained by negative-ion FAB in a glycerol matrix.

536

518

d.p.=6

697

! 679 I

1 1

101

CONCLUSION

375

d.p.=8

I

Figure 10. Negative-ion FAB mass spectra of some oligomers of glucosamine in a glycerol matrix.

-NH3+ + C1- structures which are more stable than the -NH, , HCl structure during FAB.

The study of these two families of compounds shows the variety of cleavage pathways which can be observed with them, either by FAB(+) or by FAB(-). The results depend much more on the substituent on the C(2) position of the glucosidic ring than on the glyco- sidic linkage. This conclusion, is easily deduced from a comparison of the results obtained with these two series

1341

with those previously observed for the series of cello- and malto-oligomers. Whereas the study in the positive- ion mode shows a more or less classical behaviour, the study with negative-ion FAB seems to give a better rep- resentation of the structures of these two families of compounds, as was previously reported by some workers using tandem mass spectrometry.'-' '

Acknowledgements

We thank Dr G. A. F. Roberts for correcting the English and L. Patron for technical assistance.

REFERENCES

1. M. Barber, R. S. Bordoli, R. D. Sedgwick and J. C. Vickerman,

2. A. Dell, H. R. Morris, H. Egge, H. Von Nicola'i and G. Strecker,

3. A. Dell, W. York, M. McNeil, A. G. Darvill and P. Albersheim,

4. C. Bosso. J. Defaye, A. Heyraud and J. Ulrich. Carbohydr. J. Chern. SOC., Faraday Trans. 1 78, 1291 (1 982).

Carbohydr. Res. 11 5, 41 (1 983).

Carbohydr. Res. 1 17,185 (1 983).

Res. 125,309 (1 984).

806 C. BOSS0 A N D A. DOMARD

5. C. Bosso, A. Heyraud and L. Patron, Org. Mass Spectrom. 26,

6. D. Fraisse, J. C. Tabet, M. Becchi and J. Raynaud, Biomed.

7. A. Dell, Adv. Carbohydr. Chem. Biochem. 45, 19 (1 987). 8. J. C. Prome, H. Aurelle and D. Prome, p. 10 in Abstracts ofthe

Third European Symposium on Carbohydrates Chemistry. Bio- chemistry, Technology, 16-20 September 1985, ed. by J. Defaye.

9. A. Ballistreri, G. Montaudo, D. Garozzo, M. Giuffrida and G. Impallomeni, Rapid Commun. Mass Spectrom. 3,302 (1 989).

10. D. Garozzo, M. Giuffrida, G. Impallomeni, A. Ballistreri and G. Montaudo, Anal. Chem. 62, 279 (1 990).

321 (1991).

Environ. Mass Spectrom. 13, 1 (1 986).

11. J. W. Dallinga and W. Heerma, Biol. Mass Spectrom. 20, 21 5 (1 991 ).

12. A. Domard and N. Cartier, in Chitin, Chitosan Sources, Chem- istry, Biochemistry, Physical Properties and Applcations, ed. by G. Skjak-Braek, T. Anthonsen and P. Sandford, pp. 71-86. Elsevier, (1 989).

13. A. Domard and N. Cartier, Int. J. Biol. Macromol. 11, 297 (1 989).

14. C. Bosso, J. Defaye, A. Domard and A. Gadelle, Carbohydr. Res. 196, 57 (1986).

15. W.-T. Wang, R.-F. Yao and W.-G. Chai, Chin. Biochem. J. 2, No. 3 (1 986).

16. A. Dell and C. E. Ballou, Carbohydr. Res. 120, 95 (1983).