Identification of Sialic Acid and Related Acids as Acetylated Lactones by Gas Chromatography/Mass Spectrometry

JOURNAL OF MASS SPECTROMETRY, VOL. 31, 267-274 (1996)

Identification of Sialic Acid and Related Acids as Acetylated Lactones by Gas Chromatography/Mass Spectrometry

Yvonne Nygren, Sten-Ake Fredriksson and Bo Nilsson* National Defense Research Establishment, Department of NBC Defense, S-901 82 Umea, Sweden

A procedure for characterization of sialic acid-like monosaccharides, i.e. 3deoxy-2-ulosonic acids, by gas chromatography/mass spectrometry is described. The free monosaccharides were reduced with NaBH, and the resulting 3-deoxyaldonic acids were acetylated. Five-membered ring lactones were obtained as the major products. The keto-deoxy compounds N-acetylneuraminic acid, N-glycolylneuraminic acid, 3 d e o x y - D - m a n n ~ 2 - o c t u l ~ o ~ acid and 3-deOXy-D-glyCerO-D-galaCtO-2-nOnU~OSO~C acid were investigated. A detailed study of the electron impact ionization fragmentation pathways of the acetylated lactones were conducted using tandem mass spectrometry. The mass spectra allowed unambiguous structural identification of the acidic monosaccharides.

KEYWORDS: gas chromatography/mass spectrometry; tandem mass spectrometry; sialic acid; lactones; acetylation

INTRODUCTION

Carboxylic acid-containing monosaccharides are constituents of several glycoconjugates. The uronic acids glucuronic and iduronic acid make up, together with N- acetylglucosamine and N-acetylgalactosamine, the repeating disaccharide unit in the polysaccharide portion of proteoglycans. Galacturonic acid (GalA) can be found as polygalacturonic acid in pectin from cell walls of plants. The biological function of uronic acid- containing polymers is often that of structural elements. The ketodeoxy acid 3-deoxy-~-manno-2-octulosonic acid (Kdo) is an integral part in the inner core of lipo- polysaccharides and connects the carbohydrate chain via lipid A to the outer cell membranes of Gram- negative bacteria. Other commonly found ketodeoxy acids are the sialic acids N-acetylneuraminic acid (NeuSAc) and N-glycolylneuraminic acid (NeuSGc) and their numerous O-acetylated derivatives. Kdo is mostly found in internal positions whereas Neu5Ac and Neu5Gc are in non-reducing terminal positions.

The sialic acids are common constituents in glycopro- teins and gangliosides. NeuSGc has not been found in human glycoconjugates. About 10 years ago another sialic acid-like monosaccharide, 3-deoxy-~-glycero-~- galacto-2-nonulosonic acid (Kdn) was discovered in gly- coproteins and gangliosides from rainbow trout eggs.’*2 This structure differs from sialic acid by an OH group at C-5 instead of an acetamido group. NeuSAc, NeuSGc and Kdn also exist as homopolymers where the mono- mers are joined by a2-8 or a2-9 ketosidic linkage^.^.^

Author to whom correspondence should be addressed.

Several biological functions have been ascribed to the sialic acids, of which one is more prominent, i.e. Neu5Ac is the receptor for influenza virus and part of the receptor for several bacteria. A common chemical feature of the ketodeoxy acids is their susceptibility to acid hydrolysis. Treatment with mild acid releases these sugars specifically without cleavage of most other glyco- sidic linkage^.^ Hydrolysis of O-acetylated derivatives of Neu5Ac with preservation of the O-acetyl groups can be carried but by using propionic acid.6 Liberation of sialic acids from glycoconjugates can also be achieved by enzymatic methods. There are a number of sialidases available from various sources with different specificities for substituents and binding positions.’-’

Determination of sialic acids has traditionally been carried out by colorimetric method^.'^-'^ Identification of O-acetyl derivatives of NeuSAc and Neu5Gc can be performed by thin-layer chromatography (TLC)14 and high-performance liquid chromatography (HPLC).’ Analysis of acidic monosaccharides by gas chromatography (GC) requires derivatization of both the carboxylic acid and the hydroxyl groups. Analysis of sialic acids by electron impact mass spectrometry (EIMS), as peracetylated methyl ester methyl glycosides, and eluci- dation of the fragmentation patterns has been used for structural studies of modified sialic acids.16 Per-O-tri- methylsilyl ether methyl ester derivatives are commonly used in the GC analysis of sialic acids.” Recently, a method based on periodate oxidation, reduction, per- methylation and analysis by fast atom bombardment (FAB) MS was published for the identification of Neu5Ac and the determination of its linkage position in glycoprotein oligosaccharides and gangliosides.’ * Later the periodate oxidation technique and further derivatization were applied to the analysis of various sialic acid derivatives by FABMS.l’ Periodate oxidation and

CCC 1076-5 174/96/030267-08 0 1996 by John Wiley & Sons, Ltd.

Received 13 September 1995 Accepted 5 December 1995

268 Y. NYGREN, S.-A. FREDRIKSSON AND B. NILSSON

borohydride reduction have also been used to charac- terize a2-8 sialic acid polymers.'' This paper describes a GC/MS method for the analysis of some carboxylic acid-containing 3-deoxy-2-ketomonosaccharides as acetylated lactones.

EXPERIMENTAL

Materials

The monosaccharides, D-galacturonic acid, N- acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-~-manno-2-octulosonic acid were purchased from Sigma Chemicals. 3-Deoxy-D-glycero-D-galacto-2- nonulosonic acid was a generous gift from Professor Yu-Teh Li, Tulane University. All reagents used were of analytical grade.

Reduction and acetylation

Monosaccharides (0.1-0.2 mg) were reduced with an equal amount of NaBH, or NaBD, at room temperature for 2 h. Acetic acid (1 ml) was added and the sample was concentrated to dryness. Boric acid was volatilized by evaporation with 3 x 0.5 ml of methanol. Samples were desalted using Dowex 50W-X8 ion- exchange resin, 100-200 mesh (H+-form). Acetylation was carried out under alkaline conditions in 0.75 ml of acetic anhydride-pyridine (2 : 1, v/v) at room temperature overnight and at 100°C for 30 min. Acety- lation under acidic conditions was performed in 0.5 ml of trifluoroacetic anhydride-acetic acid (2 : 1, v/v) at room temperature for 15 min." Ethanol (1 ml) was added an after 30 min the sample was evaporated to dryness. The acetylated product was extracted with 0.5 ml of chloroform after addition of 1 ml of water. The chloroform phase was washed three times with water (3 x 1 ml) and concentrated to dryness. Residual pyridine was removed by evaporation with 0.1 ml of toluene.

GC and MS

GC was performed on a Hewlett-Packard Model 5890 instrument equipped with a flame ionization detector. Separations were accomplished on a DB5-HT (J & W Scientific) capillary column (30 m x 0.32 mm id., film thickness 0.1 pm) using a temperature programme from 40-300 "C at 20 "C min-l. GC/MS in the EI mode was carried out using a Hewlett-Packard Model 5970 mass- selective detector. Mass spectra were recorded at 70 eV with an ion source temperature of 200 "C.

A VG 70-SQ hybrid sector-quadrupole tandem mass spectrometer (EBqQ geometry) was used for FABMS and MS/MS measurements in the EI mode. The resolution of the sector part of the instrument was

adjusted to 1000 (10% valley definition) and unit resolution of the quadrupole mass filter. FAB mass spectra were obtained using xenon atoms at 8 keV kinetic energy and a current of 1 pA. A 2 p1 volume of the sample solution was mixed with 2 pl of the matrix dithioerythritoldithiothreitol (9 : 1, v/v) on the probe tip.

For measurements in the EI mode, the ion source temperature was 200"C, trap current 200 PA and electron energy 70 eV. Nitrogen was used as target gas for collision-induced dissociation. The precursor ion was isolated by MS1 and transmitted into the quadrupole collision cell. The target gas pressure was adjusted to attenuate the precursor ion to approximately 50% at 40 eV collision energy (laboratory frame of reference), which gave a pressure reading of 3 x mbar on the ion gauge close to the collision cell. Product ion spectra were obtained by scanning the quadrupole mass analyzer from m/z 10 to 300 using a 1 s scan time. Pre- cursor ion spectra were recorded by scanning the magnet from m/z 300 down to m/z 100 while monitoring the selected product ion using MS2. The mass scale of MSl was calibrated using perfluorokerosene. Mass calibration of MS2 was accomplished by admitting the m/z 614 ion, generated from perfluorotributylamine, into the collision quadrupole and using low- and high-mass ions of the product ion spectrum for calibration.

RESULTS

Reduction and acetylation

Since Kdo, Kdn, Neu5Ac and Neu5Gc are keto sugars, after NaBH, reduction they give two products, rep- resenting the 2-epimers of the reduced form. As expected, no reduction of the carboxylic acid was seen. It was noted that acetylation under alkaline conditions (acetic anhydride-pyridine) gave less by-products when the derivatization was performed at room temperature overnight than at 100°C for 30 min. Comparison of acetylation under alkaline and acidic conditions (trifluoroacetic anhydride-acetic acid) showed that alkaline conditions gave the highest yield in quantification by GC with flame ionization detection. The reduced and acetylated products were analysed by GC and structurally characterized by EIMS and FABMS.

Analysis by GC and GC/MS

The reduced and acetylated monosaccharides were analysed by GC and GC/MS and the total ion chromatogram o6tained is shown in Fig. 1. The reduced hexuronic acid GalA is known to form a lactone and was therefore included for studies of the fragmentation by MS. Two well separated epimers in about equal amounts were detected from each of the keto acids Kdo, Kdn, Neu5Ac and NeuSGc. Quantification by GC relative to an internal standard (glucitol hexaacetate)

ANALYSIS OF ACETYLATED LACTONES BY GC/MS 269

8 9 10 11 12 13 14 15 16 17 Retention Time (min)

Figure 1. Total ion chromatogram of carboxylic acid-containing monosaccharides after reduction and acetylation. Peaks: 1 - GalA; 2 - Kdo; 3 = Kdn; 4 - Neu5Ac; 5 - NeuSGc. TIC =total ion current.

showed that these products were quantitatively dominant and that no other products were detected by GC, when the acetylation was carried out under alkaline conditions as described above. The nitrogen-containing acids NeuSAc and NeuSGc gave a lower response in the GC analysis using a flame ionization detector. This was also observed in the analysis of acetylated 2-acetamido- 2-deoxyhexitols by GC.

Characterization of EIMS

The derivatives obtained from these acidic mono- saccarides show a EI-induced fragmentation similar to that in the EI mass spectra of alditol acetate^.^'-'^ Briefly, El mass spectra of alditol acetates without a nitrogen atom and prepared after NaBH, reduction are characterized by primary odd mass numbers, due to

%

'i'

homolytic cleavage processes of carbon-carbon bonds in the alditol to produce oxonium ions. Secondary fragments are formed from the primary fragments by elimination of acetic acid (60 u), ketene (42 u) and acetic anhydride (102 u). The molecular ion (M') is not observed in EIMS of alditol acetates; however, secondary even-mass fragments are derived from the putative Mi' ion by eliminations of the above species. When a nitrogen atom is present, e.g. in 2-acetamido-2-deoxyhexitols, the most abundant ions are those with the positive charge located on the nitrogen in the acetamido group and recognized by their even mass numbers. The base peak in spectra of alditol acetates, including the spectra presented here, is the acetylium ion, H3CCEO+, of m/z 43. The abundance of this ion is high and dominates the mass spectrum. Therefore, in this paper spectra are displayed from m/z 50. Other ions present in alditol acetates are the diacetyloxonium ion,

145 7 73 ec 201

273

60 80 100 120 140 160 180 200 220 240 260 280 300 fnk

Figure 2. El mass spectrum of GalA after NaBH, reduction and acetylation.

270 Y. NYGREN, S.-A. FREDRIKSSON A N D B. NILSSON

100 -

50 -

A 115

'i"

0 ii

% B

100 -

143 157

145 139 /

0

1117

217 257

1259

Figure 3. El mass spectra of (A) Kdo and (B) Kdn after NaBH, reduction and acetylation.

[(CH,CO),OH] +, of m/z 103, and the triacetyloxonium ion, [(CH,CO),O]+, of m/z 145. The heaviest ion in alditol acetates is usually the [M - 591' ion (M - CH,COO'), which then goes through a series of

eliminations (acetic acid, ketene and acetic anhydride) giving odd-mass secondary ions (no nitrogen) or even- mass ions (one nitrogen).

The mass spectrum obtained from reduced and acetylated GalA is shown in Fig. 2. An abundant ion of

mfz 145 is seen together with m/z 73, a two-carbon frag- ment containing two O-acetyl groups. Upon reduction with NaBD, , the relative abundances of these primary fragments decreased, whereas those of m/z 74 and 146 increased. No other ions indicating longer segments of the alditol chain were observed. The ion at m/z 201 is consistent with a five-membered lactone ring involving the hydroxyl group at C-3 and the carboxylic acid group. Supporting evidence was furnished by the


subsequently m/z 99 by elimination of ketene. A fragmentation pathway leading to m/z 115 and 157 is proposed by starting from the molecular ion of m/z 346, which is not seen, by opening of the lactone forming an acylium radical ion. Loss of an acetoxy radical (59 u), elimination of carbon monoxide (28 u) and acetic anhydride gives m/z 157, which decomposes to m/z 115 by elimination of ketene.

A primary ion at mJz 273 formed by cleavage of the C-1-C-2 bond and containing the lactone gives rise secondary ions at m/z 171 and 231 by elimination of acetic anhydride and ketene, respectively. A series of even mass fragments, m/z 100, 142, 184, 226, 244 and 304, originate from the molecular ion by eliminations of acetic acid and ketene. Analysis by FABMS gave an

0 I1 0

I1

- 60

mh143 m/z 83

Figure 4. Fragmentation pathway of the five-membered lactone ring of the ketodeoxy acids.

NaBD,-reduced sample where, as expected, no mass increase was observed for this ion. Surprisingly weak secondary fragments containing the lactone ring are formed by elimination of acetic acid, giving m/z 141 and

0 II 100 -

50-

3 -< c\ p I

21?

143

21 4 360 {r

=YJ AcOCH

1

i I I

HCOAc

HCOAc 'i !R 11s

360 J 200 50 100 150 250 300 350 400 d 2

% 100- 1 0

II B

73 I

A c S r ( ' \ ,

418 l3

AcOCH,CHNCH

AcOCH -IJ 272

1

! 2 :

I I

HCOAc 50.

HCOAc

73-

172

418

/

150 200 250 300 350 400 50 100

Figure 5. El mass spectra of (A) Neu5Ac and (B) Neu5Gc after NaBH, reduction and acetylation

272 Y. NYGREN, S . - k FREDRIKSSON AND B. NILSSON

HC=NHAC I

A C O ~ H HCOAc

I

HCOAC I CH,OAc

d z 360

+ i l HC= NHCCH,OAc

HYOAc HYOAc

AcOFH

CH,OAc

m/z4ia I

AcO ACO

AcHN S : H 2 0 A c ? AcOCH,CHN

Ac m/z 360

Ac m/z 41 a

0 (AcOCH,CNHAc)

- l O l \

OAc (AcNHAc) AcO

\+ 0 > CH, \+ /CH,OAC 0

m/z 157 m/z 139

- 4 2 1

m/z 139 m/z 157

\42 -6f

0

b 0 CH,

m/z 97 m/z 97

Figure 6. Fragmentation pathways for the ions of m/z 360 and 41 8 present in the spectra of NeuBAc and Neu5Gc. respectively, in Fig. 5

[M + 231 + ion at m/z 369, providing supporting evidence for the structure. The FABMS analysis showed, in addition, other less abundant signals, presumably due to quasi-molecular ions of degradation products not detected by GC/MS.

Reduction of keto sugars gives rise to epimers and the mass spectrum of one of the epimers, obtained from Kdo, is shown in Fig. 3 (spectrum A). From the ion at m/z 143 it can be concluded that lactonization involving the carboxylic acid and the hydroxyl group at C-4 of reduced Kdo has taken place. Support for this five- membered ring closure with inclusion of C-2 was obtained from the NaBD,-reduced sample, which

showed a shift to m/z 144. Further fragmentation by elimination of acetic acid from m/z 143 gives m/z 83 (Fig. 4). A series of primary cleavage products at m/z 73, 145, 217 and 289 shows a linear sequence of four 0- acetylated carbons not contained in the ring structure. Mechanisms leading to secondary fragments from these ions have previously been proposed for alditol acetates, e.g. the base peak at m/z 115, which originates from m/z 217 by elimination of acetic acid followed by elimination of ketene.23,24 Further support for the ring size of the lactone is provided by the primary ions at m/z 215, 287 and 359. Secondary ions are formed from m/z 287 and 359 by elimination of ketene to give m/z 245


and 317, respectively. Usually elimination of ketene is preceded by elimination of acetic acid, except when an acetamido group is present. Some secondary ions containing the lactone seem to be a result of direct elimination of ketene without prior elimination of acetic acid. Analysis of the lactone was also carried out with FABMS and gave an [M + 23)' ion at m/z 455 consistent with the above-proposed structure. Also in this case other species, not detected by GCIMS, were observed. The other peak in the chromatogram of Kdo gave a mass spectrum similar to the above. However, some differences in the relative abundances were observed. These differences are not easily rationalized and were not investigated further.

One of the spectra obtained from Kdn after reduction and acetylation is shown in Fig. 3 (spectrum B). The primary cleavage ions of m/z 73, 145, 217, 289 and 361 show a linear stretch of five 0-acetylated carbons. A series of secondary fragments formed by eliminations of acetic acid and ketene are seen, analogous to the Kdo spectrum and as in alditol acetates. As in the previous spectrum, the five-membered lactone ring is determined by the ions of m/z 143 and 83. Supporting evidence for the presence of the lactone is provided by the primary cleavage ions of m/z 215, 287, 359 and 431. Also in this case some lactone-containing ions give secondary ions by direct elimination of ketene, e.g. m/z 245 and 317. The molecular mass was confirmed by an [M + 23)' ion of m/z 527.

Two products were also obtained from Neu5Ac due to epimerization, as above (Fig. 1). The EI mass spectrum of one of these products is shown in Fig. 5 (spectrum A). As in the EI mass spectra of alditol acetates of 2-acetamido-2-deoxyhexitols, abundant frag- ment ions containing the acetamido group are prevalent. The nitrogen-free ions are of low abundance, which is particularly relevant for the lactone-containing ion at m/z 143 as compared with the same ion in the Kdo and Kdn spectra (Fig. 3). Reduction with NaBD, gave the expected mass shift to m/z 144. An abundant ion at m/z 360 indicates a linear sequence containing the acetamido group not involved in the ring formation. Other ions formed by cleavages in the linear alditol chain are of low abundance or even absent. The ion at m/z 360 gives rise to secondary fragments giving m/z 318 and 300 by elimination of ketene and acetic acid, respectively. Further discussions of the secondary fragments derived from m/z 360 are presented below. Another abundant primary ion at m/z 214 containing the lactone moiety is formed by cleavage on the other side of the acetamido group. Secondary ions formed by elimination of ketene and acetic acid are observed at m/z 172 and 154, respectively. The base peak at m/z 139 is a result of eliminations from m/z 360, which was further investigated by MS/MS and is discussed below: An abundant ion at m/z 215 can be traced back to the molecular ion by alternating eliminations of acetic acid and ketene. FABMS of this compound showed an [M + 231' ion at m/z 526, confirming the lactone structure.

One of the two spectra obtained from Neu5Gc after reduction and acetylation is shown in Fig. 5 (spectrum B). As expected, the nitrogen-containing ions are dominant and the lactone-determining ions at m/z 143

and 83 are weak as in the Neu5Ac spectrum. The base peak in the spectrum is the acetoxyacetylium ion CH,C(O)OCH,C=O' of m/z 101. The primary cleavage ion of m/z 418 indicates a linear sequence including the acetylated N-glycolyl group and four 0-acetyl groups. Secondary ions from m/z 418 give m/z 376 by elimination of ketene, m/z 358 by elimination of acetic acid and subsequently m/z 316 by elimination of ketene.

A primary cleavage ion at m/z 272, combined with m/z 230 formed by elimination of ketene, supports the presence of the lactone ring. Further discussions of fragmentation routes of the ions produced from m/z 418 are presented below. FABMS of the compound gave an [M + 23]+ ion at m/z 584.

Analysis by MS/MS

In order to study the fragmentation pathways and espe- cially fragments containing the N-acetyl and N-glycolyl groups in Neu5Ac and NeuSGc, respectively, MS/MS analysis was employed.

Both Neu5Ac and Neu5Gc give rise to a series of secondary ions at m/z 97, 139, 157 and 259. The product ion and precursor ion spectra of m/z 259 showed that these ions represent a major fragmentation pathway originating from the primary precursor ions at m/z 360 for Neu5Ac and m/z 418 for Neu5Gc (Fig. 5). These secondary ions have odd mass numbers and therefore should not contain the nitrogen atom. A mechanism for this fragmentation pathway is proposed in Fig. 6. After a primary cleavage followed by cyclization, eliminations take place to give m/z 259. For Neu5Ac the nitrogen is eliminated as N-acetylacetamide (101 u) and for Neu5Gc as N-(acetoxyacety1)acetamide (159 u). From the ion at m/z 259 acetic acid can be eliminated from three positions to form m/z 199, which then can eliminate ketene giving m/z 157, or eliminate another acetic acid forming m/z 139. Finally, further eliminations of acetic acid from mjz 157 and of ketene from m/z 139 give two possible structures for the m/z 97 ion. The MS/MS analysis also revealed that the ion at m/z 170 found in the spectra of Kdo, Neu5Ac and Neu5Gc is formed from the respective molecular ions. Further fragmentation of m/z 170 by two consecutive eliminations of ketene gives first m/z 128 and then m/z 86.

DISCUSSION

All monosaccharides investigated could be identified after reduction and acetylation as five-membered ring lactones. None of the sugars were able to form six- membered rings. The ability to form lactones depends on the orientation of the ring substituents. In the GalA case the 0-acetyl groups at C-4 and C-5 together with the ethylene glycol residue (C-1 and (2-2) all have a trans orientation, whereas in the GlcA case they would be cis oriented, which make the lactone unstable As expected, after NaBH, reduction the keto group gave two products, in about equal proportions, differing in the orientation of the OH group at C-2. The fact that

214 Y. NYGREN, S.-A. FREDRIKSSON AND B. NILSSON

C-3 is a deoxy function in all these keto acids makes the orientation of the 0-acetyl group at C-2 less important for the stability of the lactones. The GC behaviour of these compounds is excellent. However, when a nitrogen atom is present the response is lower when using a flame ionization detector. The same observation is made for acetylated 2-acetamido-2-deoxy sugars, which makes quantification by GC less accurate. Usually a response factor has to be calculated from a standard solution. The only products detectable by GC and GC/MS were the lactones. Analysis by FABMS revealed in addition, however, other products in minor proportions. As other products were also formed, for quantification by GC a standard solution has to be prepared. The objective of this study was to develop a method for the MS identification of ketodeoxy acids rather than quantification. The reason why GalA was included is mainy for studies of acetylated lactones by GC/MS.

It was also noted that the conditions for acetylation were of vital importance for reducing the number of side reactions. Alkaline acetylation carried out at room temperature instead of 100 "C significantly reduced the formation of by-products. The EI fragmentation shows abundant ions characteristic of the lactone ring for the nitrogen-free compounds, i.e. m/z 201 for GalA and m/z 143 for Kdo and Kdn (Figs 2 and 3). When a nitrogen atom is present (NeuSAc and NeuSGc), fragmentation in the vicinity of the amide group dominates.

The mass spectra of the 2-epimers for each ketodeoxy sugar were similar but not identical. The differences were related to the relative abundances. A question that could be asked is whether it is possible to relate the differences in the relative abundances of some ions to the orientation of the ring substituents. It has previously been shown that methyl ester methyl glycosides of 0- methylated glucuronic and galacturonic acids showed differences in the relative abundances of some ions, making it possible to distinguish between these two acids.25 Furfhermore, in premethylated disaccharide alditols differences in the abundances of ions derived from the hexosyl residue can be used to differentiate between glucosyl, galactosyl and mannosyl residues.26

NeuSAc and NeuSGc have a common fragmentation route giving rise to the odd mass numbered secondary ions of m/z 259, 199, 157, 139 and 97, all originating from m/z 360 and 418, respectively. A mechanism involving elimination of the nitrogen function is proposed for this pathway (Fig. 6). The same ions are also observed for acetylated 2-acetamido-2-deoxyhexitols. No mechanism explaining the formation of these secondary ions has been presented previously.

Earlier methods based on GC have included derivatization of the carboxylic acid to an ester followed by acetylation or other derivatizations of the hydroxyl groups. The advantage of using the method presented here is that derivatization is carried out in one step and stable derivatives are obtained.

REFERENCES

1. D. Nadano, M. Iwasaki, S. Endo, K. Kitajima, S. lnoue and Y.

2. Y. Song, K. Kitajima, S. lnoue and Y. Inoue, Trends Glycosci.

3. T. E. Rohr and F. A. Troy, J. B i d . Chem. 255,2332 (1 980). 4. S. Kitazume, K. Kitajima, S. lnoue and Y. Inoue, Anal.

Biochem. 202, 25 (1 992). 5. G. Reuter, R. Pfeil, S. Stoll, R . Schauer, J. P. Kamerling, C.

Versluis and J. F. G. Vliegenthart, Eur. J. Biochem. 134. 139 (1 983).

6. T. P. Mawhinney and D. L. Chance,Anal. Biochem. 223, 164

7. C. Fischer, S. Kelm, B. Ruch and R. Schauer. Carbohydr. Res.

8. A. Kluge, G. Reuter, H. Lee, B. Ruch-Heeger and R. Schauer.

9. E. Miiller. C. Schroder, R. Schauer and N. Sharon, Hoppe- SeyIerS 2. Physiol. Chem. 364, 141 9 (1 992).

10. P. Bohm, S. Dauber and L. Baumeister, Klin. Wochenschr. 32, 289 (1 954).

11. L. Warren, J. Biol. Chem. 234,1971 (1 959). 12. D. Aminoff, Virology 7, 355 (1 959). 13. G. W. Jourdian. L. Dean and S. Roseman. J. B i d . Chem. 246,

Inoue, J . Biol. Chem. 261,11550 (1 986).

Glycotechnol. 4,216 (1 992).

(1 994).

213,263 (1991).

EUf. J . cel l B i d . 59. 12 (1 992).

430 (1971).

14. H-P. Buscher, J. Casals-Stenzel and R. Schauer, Eur. J . Biochem. 50,71 (1 974).

15. A. K. Shukla and R. Schauer. J. Chrornatogr. 244, 81 (1 982). 16. N. K. Kochetkov, 0. S. Chizhov, V. I. Kadentsev, G. P. Smir-

nova and I . G. Zhukova, Carbohydr. Res. 27, 5 (1973). 17. G. Reuter and R. Schauer,Ana/. Biochem. 157,39 (1986). 18. A . 4 . Angel, F. Lindh and B. Nilsson, Carbohydr. Res. 168, 15

19. A. E. Manzi, A. Dell, P. Azadi and A. Varki, J. Bid. Chem. 265,

20. G. Ashwell, W. K. Berlin and 0. Gabriel, Anal. Biochem. 222,

21. E. J. Bourne, M. Stacey, J. C. Tatlow and J. M. Tedder, J. chem. soc. 2976 (1949).

22. K. Biemann, D. C. DeJongh and H. K. Schnoes,J.Am. Chem. Soc. 85,1763 (1 963).

23. L. S. Golovkina, 0. S. Chizhov and F. V. Wulfson, In. Akad. NaukSSSR,Ser.Khim.11,1915 (1966).

24. J. Lonngren and S. Svensson, Adv. Carbohydr. Chem. 29,41 (1 974).

25. V. Kovacik, S. Bauer, J. Rosik and P. Kovac, Carbohydr. Res. 8, 282 (1 968).

26. J. Lundsten, F. Lindh, S. Svensson and B. Lindberg, Carbo- hydr. Res. 254, 15 (1 994).

(1987).

8094 (1 990).

495 (1994).

Documents

Identification of Sialic Acid and Related Acids as Acetylated Lactones by Gas Chromatography/Mass Spectrometry