analysis of permethylated glucosaminyl-glucosaminitol disaccharides by combined gas-liquid...

Analysis of Permethylated Glucosaminyl- Glucosaminitol Disaccharides bv Combined Gas-liquid Chromatography Miss Spectrometry'

Michael Jensen, Dietmar Borowiak, Hans Paulsenj and Ernst Th. Rietschel Max-Planck-Institut fur Immunbiologie, D-7800 Freiburg and Institut fur Organische Chemie und Biochemie der Universitat Hamburg,$ D-2000 Hamburg-13, W. Germany

The a!-and /?-forms of permethylated 1,3-, 1,4- and 1,6-linked N-acetylglucosaminyl-N-acetylglucosaminitol disaccharides have been analysed directly (without hydrolysis or further modifications) by combined gas-liquid chromatography mass spectrometry. Gas-liquid chromatography facilitated the separation of the a - and /?-isomers of each disaccharide pair. In every case, the respective a-form was slower than the 0-form. While, additionally, the a! - and p-forms of the 1,6-linked glucosamine disaccharide could be separated from those of the 1,3- and 1,4-linked disaccharides, the a- and /?-forms of the latter two could not be resolved from each other with the liquid phases used. All three disaccharides could be readily differentiated and characterized by mass spectrometry, however. Specific fragments for each glucosamine disaccharide could be defined. Therefore, combined gas-liquid chromatography mass spectrometry allowed an unequivocal determination of the anomeric configuration and the position of the glycosidic linkage in the glucosamine disaccharides.

INTRODUCTION EXPERIMENTAL

The lipid A component of lipopolysaccharides (endo- toxins) from various groups of gram-negative bacteria consists of a substituted /?-1,6-linked D-glucosamine disaccharide.'** The anomeric configuration of this disaccharide has been established in previous studies with the aid of ~-N-acetylglucosaminidase.3'4 The position of the glycosidic linkage has been iden$fied by methylation analysis among other methods. However, the interpretation of gas-liquid chromatograms obtained after methylation, acetolysis, reduction and peracetyl- ation of reduced glucosamine disaccharides has been complicated by the fact that not only the expected N-methylacetamido but also N-acetylacetamido derivatives were f ~ r m e d ~ ' ~ from the glucosaminitol residue, depending on the position of the substituent.

In a new approach to the analysis of lipid A, one natural and various synthetic N-acetylglucosamine disaccharides were permethylated and analysed directly after reduction, without hydrolysis, by gas-liquid chromatography mass spectrometry (GLCMS). The results show that reduced permethylated N-acetylglucosamine disaccharides can be separated by GLC according to the anomeric configuration and the position of the glycosidic linkage within the disaccharide. The disaccharides investigated could be fully characterized and identified by their retention time and MS analysis.

t Abbreviations: N-acetylglucosamine = 2-acetamido-2-deoxy-~- glucose; A'-acetylgalactosamine = 2-acetamido-2-deoxy-~-galac- tose.

N- Acetyl-D-glucosamine disaccharides and reference compound

The per-0-acetylated derivatives of N-acetyl- D-glucosaminyl a -1,3-~-acetyl-~-g1ucosamine,~ N - acetyl - D - glucosaminyl - p - 1,3 - N - acetyl - D - gluco - samine,8 N-acetyl-D-glucosaminyl-a- 1 . 4 - ~ - a c e t y l - ~ - gl~cosamine,~ N-acetyl-~-glucosaminyl-a - 1,6,-N- acetyl-~-glucosamine~ and N-acetyl-D-galactosaminyl- a-1,3-N-acetyl-~-galactosamine*~ were synthesized as described. D-Glucosaminyl-a- 1,6-~-glucosamine was prepared from Salmonella minnesotu Re-lipopolysac- charide as described previ~us ly .~ N-ACetyl-D-glUCOS- aminy~-@-1,4-N-acetyl-D-g~ucosamine (chitobiose) as well as maltotriose (internal standard) were from Senn Chemicals (Dielsdorf, Switzerland).

Methylation of glucosamine disaccharides

The peracetylated glucosamine disaccharides (1-2 mg, 0.1 ml) were de-0-acetylated and reduced by treatment with NaBH4 (10 mg, 0.1 ml) or NaB2H4 (10 mg, 0.1 ml). After acidification (acetic acid) and evaporation with methanol, permethylation was performed according to Hakomori" and the products purified by Sephadex LH-20.

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CCC-0306-042X/79/0006-0559$03.50

BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 12, 1979 559

M. JENSEN, D. BOROWIAK, H. PAULSEN AND E. TH. RIETSCHEL

Analysis of permethylated reduced N-acetylglucosamine disaccharides

Gas-liquid chromatography was performed on a Varian Aerograph (1400) equipped with a flame ionization detector. Methylated disaccharides were separated on the following phases: (1) 10% SE-30 on Gas-Chrom Q (100-120 mesh) at 270°C; (2) 3% OV-17 on Gas- Chrom Q (100-120 mesh) at 270 "C; (3) 2,5% QF-1 on Gas-Chrom Q (100-129 mesh) at 260 "C; (4) 3% OV- 225 on Gas-Chrom Q (100-120 mesh) at 260 "C.

In all cases glass columns (3 mm x 200 cm) were use;; nitrogen served as carrier gas (30 ml min- ). Permethylated maltotriitol was used as an internal standard (R , = 1.00).

Mass spectrometry

This was performed using a Finnigan mass spectrometer (model 3200) connected to a gas chromatograph (10% SE-30). Mass spectra were taken at an ionizing potential of 70 eV (2000 V acceleration voltage), ionization cur- rent intensity 0.7 mA, ion source temperature 50 "C, atomic mass units range 35-510, scan time 2ms and sensitivity lo-' AJV.

RESULTS

Gas-liquid chromatography of permethylated reduced N-acetylglucosamine disaccharides

Gas-liquid chromatographic analysis (10% SE-30, 270 "C) of the permethylated N-acetylglucosaminyl-N- acetylglucosaminitol disaccharides showed that the anomeric forms could be well resolved (Table l), the ,B-form eluting from the column faster than the a -form in each case (Fig. 1). In addition, the a- and @-isomers of the 1,6-linked disaccharides separated from those of the 1,3- and 1,4-linked disaccharides which eluted faster

Table 1. Relative retention times of the a- and @-forms of permethylated N-acetylglucosaminyl-N-acetylglucosaminitol disaccharides as obtained on different stationary phases. Retention times are relative to permethylated maltotriitol (Rt = 1.00)

Relative retention time

Stationary phase and temperature rC) Permethylated reduced N-acetyl-0-glucosamine SE-30 OV-17 OV-225 OF-1

disaccharide? linked (270) (270) (260) (2601

(I 1.3 0.86 0.89 2.40 3.62 p 1.3 0.76 0.69 ndb ndb

(I 1.4 0.85 0.88 2.33 3.58 p 1.4 0.77 0.71 1.96 3.26

a 1.6 1.11 1.21 3.48 5.34 (3 1.6 0.93 7.00 2.66 3.93

a Rt of a-l,3-linked N-acetyl-D-galactosamine disaccharide = 0.92 LSE-30).

nd = not determined.

8 16 24

Time (min)

Figure 1. Gas-liquid chromatogram of permethylated N-acetylglucosaminyl-N-acetylglucosaminitol disaccharides (10% SE-30, 270°C). The peaks correspond to p-1,3+p-1,4 (peak 1). (1-1,3+(1-1,4 (peak 21, 0-1.6 (peak 3) and (1-1.6 (peak 4) glucosamine disaccharides. Peak IS corresponds to permethylated maltotriitol (internal standard). The peak eluting from the column after 5 min represents contaminating phthalic acid ester.

(Fig. 1). In contrast, the @-1, 3- and p-1,4-linked disaccharides (R, = 0.76 and 0.77, respectively) as well asthe a-1,3-anda-1,4compounds(Rt=0.86 and0.85, respectively) possessed comparable retention times (Table 1) even when other temperatures (data not shown) or stationary phases (OV-17, OV-225 and Q F-1) were used. Table 1 summarizes relative retention times (R, maltotriitol = 1.00) of the disaccharides investigated as obtained on four liquid phases with differing polarity.

Mass spectrometry of permethylated reduced N-acetylglucosamine disaccharides

The 1,3-, 1,4- and 1,6-linked permethylated N-acetylglucosaminyl-N-acetylglucosaminitol disaccharides were also analysed by combined GLCMS (SE-30). The mass spectra of the a- and @-forms of the disaccharides were qualitatively identical and under the conditions tested quantitative differences not reproducible. There- fore, only results obtained with the a-isomers will be discussed. Fragments at mlz 260, 228, 196 and 154, which were not shifted after reduction with sodium borodeuteride (Table 2, Figs. 2-4 were common to the mass spectra of 1,3-, 1,4- and 1,6-linked disaccharides. These fragments derive from the non-reducing glucosamine residue of the disaccharide and are most probably generated by the fragmentation pathway A (Scheme l).12313 In addition, all spectra exhibited fragments at m/z 378 (410-32), 346 (378 -32) and 276, which were increased by one mass unit on reduction of the respective disaccharide with borodeuteride (incomplete shift in the case of the 1,6-linked disaccharide). These fragments are probably produced by the D series of fragmentation (Scheme l).12v*3 From the D series ions at m / z 336 and 304 (336 - 32) would be expected. They are clearly evident in the spectrum of the 1,6-linked disaccharide (Fig. 4). They are also present in spectra of

560 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 12, 1979 @ Heyden & Son Ltd, 1979

ANALYSIS OF PERMETHYLATED GLUCOSAMINE-DISACCHARIDES

Table 2. Fragments common to mass spectra of 1,3-, 1,4- and 1,6-linked permethylated N-acetylglucosaminyl-N- acetylglucosaminitol disaccharides

Shift by one mass unit after

Fragment NaBZHd reduction Origin of fragment

(mlz) 260

A series fragmentation (GlcN)

- C-1-C-2 cleavage in GlcNol

(1) D series fragmentation

228 (260 - 32) 196 (228-32) 154 (196-42)

507 (M -45)

378 (410-32) 346 (378 - 32)

G I C N O I ~

276 + D and Aseries (GlcNol)

142 (+)" (1) Dseries (2) C-3-C-4 cleavage in GICNOI~ (1 74- 32)

130 C-2-C-3 cleavage in

a (+)=partly shifted. 'in 1,4- and 1,B-linked disaccharides.

88 (130-42) + '} GlcNol

the 1,3- and 1,4-linked molecules, but with low intensi- ties. The prominent fragment at m / z 142 is also generated by the D series fragmentation. With the 1,4- and 1,6-linked disaccharides, however, this fragment is partly shifted to m/z 143 after borodeuteride reduction, indicating that in these cases the ion at m / z 142 is also produced via another fragmentation pathway (see below).

H,C-OCH,

1 p 3

I I I

HC-N=C-CH,

HC-OCH,

HC-OCH,

H,C-OCH,

H,CO 260

H,C-OCH,

I 5%

I I

HC-KC-CH,

HC-OCH,

HC-OCH, I

YC-OCH, 1 HC-OCH,

I 1 HC=6,

CH-0-CH, H,CO'

m/z 110 (378.3161

Scheme 1. A and D series fragmentations of 1.6-linked permethylated N-acetylglucosaminyl-N-acetylglucosaminitol.

Other fragments also observed in the mass spectra of all three disaccharides investigated (Table 2, Figs. 2 4 ) include those at m / z 130 and 88 (130-42), which are increased by one mass unit after reduction with sodium borodeuteride. These ions derive from the glucosamini- to1 residue of the disaccharides and are well known from mass spectroscopic studies on permethylated or partially methylated N-acetylglucosaminitol (cleavage of C-2- C-35,6). None of the spectra contained the molecular ion (m/ z 552); however, a fragment at m / z 507 ([M -45+) was seen (Figs. 2-41. The 1,3-linked disaccharide (Fig. 2 , Table 3). This was characterized by ions at m/z 419 and 463 ([M-89]'), which are formed by cleavage of the C-3-C-4 and C-4-C-5 bonds respectively within the glucosaminitol residue. After reduction with borodeuteride, these ions are shifted to m/z 420 and 464, respectively. The expected characteristic fragment ion at m / z 133 (cleavage of the C-3-C-4 bond of glucosaminitol) is present, although with low intensity. A 1,3-linkage is also indicated by the absence of a peak at m/z 174, which represents a major ion in the mass spectra of 1,4- and 1,6-linked glucosamine disaccharides. In contrast to the 1,4- and 1,6-linked compounds, the fragment at m/r 142 is unchanged after borodeuteride reduction indicating that here it derived exclusively from the non- reducing glucosamine residue (D series). The 1,4-linked glucosamine disaccharide (Fig. 3). The mass spectrum of this compound exhibited characteristic fragments at m/z 422 and 390, the primary ion being produced by cleavage of the C-2-C-3 bond of the glucosaminitol residue (Table 3). These fragments are not shifted on reduction with borodeuteride. The fragments at m/z 174 and 475 (cleavage of the C-3-C-4 and C-1 -C-2 bonds within the alditol portion, respectively), which do not occur in the spectrum of the 1,3-linked disaccharide (Fig. 2) are present, however, in the spectrum of the 1,6-linked disaccharide (Fig. 4,

Table 3. Fragments specific to mass spectra of 1,3-, 1,4- and 1,6-linked permethylated N-acetylglucosaminyl-N- acetylglucosaminitol disaccharides

Fragment Shift by Fragment present in spectra of produced by

one mass unit disaccharides bound cleavage within glucosaminitol after NaB2H,

Fragment reduction 1.3 1.4 1.6' betweenCatoms

( m l z ) 463 + + 41 9 + + 133

422 390

4.5 3.4 3,4

2 3 2 3

(422 - 32)

+ 4,5 21 8 + + + 1 2 47 5

(507 -32) 174 + + + 3.4

336 + ( + I ( + I 304 + (336 - 32)

- -

- - - ( + I - -

- + +

- - - - -

- -

- -

-

( + ) (+ ) + +} fragmentation Dseries

'The part of fragments mlz 378 and 346 (378-32) not shifted after NaB2Hd is indicative for a 1,6-linkage.

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100

- %

In c Q)

- - - HZC-OCH, c

> 380 X 50

HP C-OCH,

300 350 400 450 500

rn /I

Figure 2. Mass spectrum of perrnethylated N-acetylglucosaminyl-a-l,3-N-acetylglucosaminitol.

Table 3). The fragment at m / z 142 is partially derived from the ion m/z 174 by elimination of methanol. Both fragments are shifted by one mass unit after reduction of the 1,4-linked disaccharide with borodeuteride, the fragment at m/z 142 only partially, however, indicating that part of it derives from the glucosamine residue (D series). The 1,6-linked disaccharide. This is primarily characterized by a fragment at m / z 218, which is produced by cleavage of the C-4-C-5 bond within the alditol portion of the disaccharide (Fig. 4, Table 3). This fragment is shifted to m/z 219 after reduction of the disaccharide with borodeuteride. Other characteristic fragments include that at m/z 475 which is unchanged, and those at m / z 174 and 142, which are partly increased by one mass unit after borodeuteride reduction. About 50% of the peaks at m / z 378 and 346 (378 - 32) were shifted after borodeuteride reduction, indicating that they derive from the D series fragmentation, while the other SOYO, being unchanged after borodeuteride reduction, derived from the glucosaminitol residue (cleavage of the C-3-C-4 bond) of the disaccharide. Surprisingly, in the 1,4-linked glucosamine disaccharide the ions at m / z 378 and 346 were shifted completely by one mass unit. Therefore, in the case of the 1,6-molecule, the unchanged part of these fragments appears to be characteristic for a 1,6-linkage.

DISCUSSION

Recently, the direct GLCMS analysis of permethylated reduced disaccharides containing a hexose and a N - acetylhexosamine residue has been reported.14'15 In the present paper, permethylated N-acetyl- D-glucosaminyl-N-acetyl-D-glucosaminitol disaccharides have been investigated by combined GLCMS. Our results show that, using a methyl silicon stationary phase (SE-30), the a-forms of the 1,3-, 1,4- and 1,6-linked disaccharides elute slower from the column than the p -forms. Therefore, the anomers can be readily separated from each other. This was also found with the other liquid phases used (OV-17,OV-225, Q F-1). It is noteworthy that the permethylated a- 1,3- and a-1,6- linked D-galactosyl-N-acetyl-D-glucosaminitol disaccharides, under comparable experimental conditions (2.2% SE-30, 240 "C), exhibit smaller retention times than the p-isomers.15

While the a - (and p-) form(s) of the 1,6-linked glucosamine disaccharide could be separated from those of the 1,3- and 1,4-linked disaccharides, it was not possible to separate the a -1,3 from the a -1,4-linked disaccharide or the corresponding p -anomers with the phases used. It has been shown15 previously that a separation of 1,3- from 1,4-linked p-galactosy1-N-



300 X

100 I50 200 250

20 > 380 X 50 HeC- OCH3

I n

I HzC-OCH,

300 350 400 450 500 rn /I

Figure 3. Mass spectrum of perrnethylated N-acetylglucosarninyl-a-l.4-N-acetylglucosarninitol.

acetylglucosaminitol could be achieved with a more polar phase (Q F-1) which was not possible, however, with the corresponding glucosamine disaccharides (Table 1).

An unequivocal characterization and differentiation of the 1,3-, 1,4- and 1,6-linked glucosamine disaccharides was possible by mass spectrometric analysis. Characteristic fragments for each of the three disaccharides could be defined (Table 3). Thus, fragments characteristic for the 1,3-linked disaccharide appear at m/z 463 and 419, those characteristic for the 1,4-linked disaccharide at m / t 422 and 390 and one characteristic for the 1,6-linked disaccharide at m/z 218. These ions are produced by cleavage of carbon-carbon bonds of the glucosaminitol residue of the disaccharide. The mass spectrum of a permethylated N-acetylgalactosaminyl- a- 1,3 -N-acetylgalactosaminitol disaccharide was closely related (data not shown) to that of the corresponding a - 1,3-linked glucosamine disaccharide, except that an unexplained fragment at m / z 364 present in the latter was absent from the former.

In addition to these specific fragments, a number of peaks were common to the spectra of the three disaccharides investigated (Table 2), the majorit deriving from the A and D series of fragmentati~n.’”~ Prom- inent among these are peaks at m / z 260 and 276, which represent, respectively, the cleaved N-acetylglucosamine and N-acetylglucosaminitol portions of the

permethylated disaccharide. In the case of a 1,6-linked reduced and permethylated glucosamine disaccharide carrying amide bound 3-methoxydodecanoic acid, corresponding fragmentf6 m / z 430 and 446 have been identified previously. Similar findings were made with a 1 &linked glucosamine disaccharide with amide linked 3-methoxydecanoic acid, where corresponding fragments at m/z 402 and 418 were found.17

As noted earlier,15 in the low mass range the fragmentation pattern of permethylated N-acetylglucosaminyl-N-acetylglucosaminitol resembles that observed with methylated N-acetylglucosaminitol derivatives in that ions at m / z 130 and 88 pre-

In the case of 1,6-linked glucosamine disaccharides with amide linked long chain fatty acids the ion produced by cleavage of the C-2-C-3 bond of the glucosaminitol residue were present at m / z 300 ((2-12) and m / z 272 (C-10). In addition, fragments at 268 (300 - 32) and 240 (272 - 32) were detected. As expected, the ion at m / z 88 (130 - ketene) observed in spectra of N-acetylglucosamine disaccharides was absent from those of 1,6-linked N-acylglucosamine disacchar ide~. ’~ , ’~ The peak at mf z 218, which represents a characteristic fragment for the 1,6-linked N-acetylglucosamine disaccharide, was not seen either.

It has been postulated that permethylation of N - acetylglucosaminitol leads to the formation of a methyl acetimidate group.536 The formation of this group after

@ Heyden & Son Ltd, 1979 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 12, 1979 563


0 - B -

-

I00 I

HC- N C-CHI 130 (88)

422 HC-OCHs

174 (1421 I

I 378 I HC-OCHn

218

CH3-C CHI HC-OCH3 II 0

10

methylation of N-acetylglucosamine derivatives (with silver perchlorate as catalyst), has been demonstrated. l8 As noted previously, methylation of the oxygen atom of the N-acetyl residue occurs almost exclusively with 6-0-substituted N-acetylglucosaminitol and part1 ( -50%) with 4-0-substituted N-acetylglucosaminitol. The direct GLC analysis of permethylated reduced 1,4- linked N-acetylglucosamine disaccharides, as shown in the present paper, gave only one peak for both the a - and p-forms. Therefore, if the methyl acetimidate derivative had been formed, it could not be resolved from the N-methylacetamido derivative under the conditions employed. (See note added in proof.)

Spectra of the a - and p -forms of each of the 1,3-, 1,4- and 1,6-linked disaccharides resembled each other, and it was not possible to draw mnclusions as to the nature of the anomeric configuration of the non-reduced glucosamine residue from the mass spectroscopic data.

Y

However, the anomeric configuration of the reduced glucosamine disaccharides can be readily obtained from retention times obtained by GLC analysis.

The demonstration that the position of the glycosidic linkage as well as the anomeric configuration of reduced hexosamine disaccharides can be determined by combined GCMS after permethylation without further hydrolysis and modifications of the disaccharide may facilitate structural analyses on lipid A and other hexosamine disaccharide-containing molecules.

Acknowledgements

We would like to thank Mrs Helga Kuttler for the preparation of this manuscript and Ms Helga Kochanowski and Mrs Ulrike Pflugfelder for preparation of the illustrations. We thank Drs 0. Liideritz and H. Mayer for advice and encouragement.

REFERENCES

1. 0. Luderitz, C. Galanos, V. Lehmann, H. Mayer, E. Th. Riet- schel and J. Weckesser, Naturwissenschaften 65,578 (1978).

2. E. Th. Rietschel, S. Hase, M. King, J. Redmond, and V. Lehmann, in Microbiology-1977, pp. 262-268. American Society for Microbiology, Washington, DC (1977).

3. J. Gmeiner, 0. Liideritz, and 0. Westphal, Eur. J. Biochem. 7 ,

4. S. Hase, and E. Th. Rietschel, Eur. J. Biochem. 63,101 (1976). 5. S. Hase and E. Th. Rietschel, Eur. J. Biochem. 63,93 (1976). 6. J. Finne, and H. Rauvala, Carbohydr. Res. 58, 57 (1977).

370 (1969).



7. H. Paulsen, and W. Stenzel, Chem. Ber. 111,2348 (1978). 8. K. Heyns, R. Harrison and H. Paulsen, Chem. Ber. 100, 271

9. H. Paulsen, W. Stenzel, Chem. Ber. 111, 2334 (1978). (1967).

10. H. Paulsen, C. Kolar and W. Stenzel, Chem. Ber. 111, 2358 (1978).

11. S. Hakomori, J. Biochem. 55,205 (1964). 12. N. K. Kochetkov and 0. S. Chizhov, Adv. Carbohydr. Chem.

21, 39 (1966). 13. J. Lonngren and S. Svensson, Adv. Carbohydr. Chem. 29,89

(1974). 14. J. Karkkainen, Carbohydr. Res. 14,27 (1970).

Note added in proof: Recent results of Caroff and Szab6” indicate that during permethylation of N-acetylglucosaminitol (according to the method of Hakomori”) the N-methylacetamido derivative (as shown in Figs. 2 and 3) and not the methyl acetimidate derivative (as shown in Scheme 1 and Fig. 4) is formed. It is likely, therefore, that in the 1 ,&linked reduced and permethylated N-acetylglucosamine disaccharide the glucosaminitol residue is present as the N-methylacetamido derivative.


15. J. Mononen, J. Finne and J. Karkkainen, Carbohydr. Res. 60,

16. S . Hase and E. Th. Rietschel, Eur. J. Biochem. 75, 23 (1977). 17. R. N. Tharanathan, J. Weckesser and H. Mayer, Eur. J. Bio-

18. U. Kraska, J.4. Pougny and P. Sinay, Carbohydr. Res. 50,181

19. M. Caroff and L. Szabo, Biochem. Biophys. Res. Commun. 89,

Received 10 June 1979


371 (1978).

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(1 976).

410 (1979).

BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 12, 1979 565

analysis of permethylated glucosaminyl-glucosaminitol disaccharides by combined gas-liquid...

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