Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal α2→3 and α2→6 sialogalactosylated linkages at subpicomole levels

Article abstract of DOI:10.1021/ac990674w

Perbenzoylated sialooligosaccharides were found to be stable derivatives, giving intense signals during the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis in the positive-ion mode. Terminal Neu5NAcα2→3 and α2→6Gal units of

Full text of DOI:10.1021/ac990674w

Anal. Chem. 1999, 71, 4969-4973
Mass Spectrometric Analysis of Benzoylated
Sialooligosaccharides and Differentiation of
Terminal r2f3 and r2f6 Sialogalactosylated
Linkages at Subpicomole Levels
Peng Chen, Ulrike Werner-Zwanziger, Donald Wiesler, Marty Pagel, and Milos V. Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
crystal structure of the polyomavirus/ Neu5NAcR2f3lactose com-
plex has recently been solved.14 Additionally, Varki et al.15 found
that CD22â, a glycoprotein present on the surface of B cells,
P erbenzoylated sialooligosaccharides were found to be
stable derivatives, giving intense signals during the matrix-
assisted laser desorption/ ionization (MALDI) mass spec-
trometric analysis in the positive-ion mode. Terminal
Neu5 NAcr2 f3 and r2 f6 Gal units of oligosaccharides
undergo characteristic structural changes during benzoy-
lation, yielding easily recognizable mass spectral patterns.
Subpicomole carbohydrate samples were successfully
benzoylated and analyzed through MALDI mass spec-
trometry.
recognizes only the R-2,6-linked sialic acid residues, whereas Weis
16
and co-workers
had shown that both R2f3- and R2f6-
sialogalactosidical linkages are effective with the influenza virus
hemagglutinin.
Even though the use of specific enzymes (sialidases) can lead
to a selective release of either Neu5NAcR2f3 or Neu5NAcR2f6
residues, direct structural information concerning the nature of
the corresponding sugars and the course of such enzymatic
reactions is difficult to obtain at low sample levels without
assistance from other structural techniques. Traditional analytical
tools, such as nuclear magnetic resonance (NMR) spectroscopy
and X-ray crystallography, require relatively large quantities of
material for structural characterization. The permethylation with
the gas chromatography/ mass spectrometry (GC/ MS) method
suffers from difficulties because of the low volatility caused by
the ionic nature of the sialo residues. The introduction of matrix-
assisted laser desorption/ ionization (MALDI) mass spectrometry17
has revolutionized the analysis of large biological molecules
including carbohydrates. The oligosaccharide molecules are often
desorbed/ ionized as sodiated and potassiated ions, with minimum
backbone fragmentation, which has led to substantially simplified
mass spectra. However, since many antigenic surface sialoglycans
are often small in size (commonly with molecular weights of less
than 1000), their MALDI signals can be overwhelmed by the
matrix background in the low-mass/ charge region. Moreover,
during the MALDI-MS measurements, the charged glycans are
known to present notorious analytical problems, such as the loss
of sialyl residues during the desorption/ ionization process and
the requirements for careful and tedious sample purification. To
overcome these difficulties, methylation of the sialyl carboxy
group18 has been used to stabilize the sialyl residues, while
1-8
With growing interest
in the biological functions of
glycoconjugates, structural information about various glycans is
becoming increasingly important. Many biologically relevant
carbohydrates, such as oligosaccharides in different glycoproteins,
are highly heterogeneous, but frequently present in low abun-
dance. Demands have been increasing for new analytical meth-
odologies that could provide structural details at picomole levels
and below.9,10
N-Acetylneuraminic acid residues, positioned terminally at
the nonreducing sites in glycans, are spatially accessible and
often responsible for many recognition and binding events.11,12
Neu5NAc R2f3Gal and Neu5NAcR2f6Gal linkage units are
commonly found in many O-linked and N-linked glycans. The
necessity of differentiating these two linkage types has been
pointed out by Cahan and Paulson,13 who demonstrated that the
polyomavirus (a tumor-producing virus) requires Neu5NAcR2f
3Gal, but not Neu5NAcR2f6Gal, for hemagglutination; the
* Corresponding author: (tel) 812-855-4532; (fax) 812-855-8300; (e-mail)
novotny@indiana.edu.
(1) Dwek, R. A. Chem. Rev. 1 9 9 6 , 96, 683-720.
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Nature 1 9 8 8 , 333, 426-431.
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(12) Schauer R. Adv. Carbohydr. Chem., Biochem. 1 9 9 2 , 89, 6338-6342.
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10.1021/ac990674w CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/23/1999
Analytical Chemistry, Vol. 71, No. 21, November 1, 1999 4969

amination with a peptide10 has also been developed to enhance a
sample desorption/ ionization. Unfortunately, many recognition
glycans are linked to proteins through oxygen,19 most often
necessitating a release by the alkaline borohydride reduction.20-22
As a result, alditols are obtained, which are no longer amenable
to facile or reductive amination.
In this study, we have found that perbenzoylation23 stabilizes
the sialyl residues for the MALDI-MS measurements and allows
the analysis of charged sialoglycans, along with benzoylated
neutral glycans, in the positive-ion mode. A method of differentiat-
ing Neu5NAcR2f3glycans and Neu5NAcR2f6glycans at subpi-
comole levels has also been established through chemical
derivatization and a MALDI-MS measurement.
aqueous solution, to a 5-mL glass reaction vial and lyophilized
thoroughly to dryness before the addition of an excess amount
of benzoylation bulk solution (at a stoichiometric amount of 500
µL/ per mg of glycan), prepared by dissolving benzoic anhydride
in an equal weight of solvent, 1-methylimidazole. The reaction vial
was sealed with a Teflon-lined cap, and the benzoylation reaction
was carried out at 110 °C for 5 h. After benzoylation, the vial was
cooled to room temperature and the solution was diluted with a
10-fold excess of acetonitrile before a further mixing with an equal
volume of water and elution through a fritted plastic column
packed with 5 mL of Diaion HP-20 resin. The loaded cartridge
was washed with 200 mL of 50% acetonitrile aqueous solution,
while the benzoylated glycans were eluted with 15 mL of pure
acetonitrile and recovered upon evaporation through either
nitrogen flow or in a vacuum. A preparative reversed-phase HPLC
was used for a further purification. 13C NMR (benzene-d6, 400
MHz, δ): region I has 13 peaks (1 lactone carbonyl carbon, 1
amide carbonyl carbon, and 11 benzoyl carbonyl carbons), 172.94,
169.07, 166.61, 166.28, 166.17, 166.12, 165.96, 165.84, 165.47,
165.43, 165.26, 163.21, 159.42; region II has 2 peaks (corresponding
to 2 carbons that have 2 alkoxyl groups attached), 102.05, 97.54;
region III has 16 carbons (each has 1 proton and 1 alkoxyl group
attached), 76.72, 74.29, 73.70, 72.91, 72.71, 71.97, 70.87, 70.50,
69.51, 68.77, 67.59, 67.45, 63.19, 62.93, 62.85, 62.70; region IV has
2 peaks (both belonging to carbons of the sialic acid residue; one
is free of any alkoxyl group while the other bears the acetamido
EXPERIMENTAL SECTION
Materials. Neu5NAcR2f3lactose, Neu5NAcR2f6lactose, a
mixture of both (2f3) and (2f6) saccharides (with 85% of the
2f6 form), Neu5NAcR2f6Galâ1f4GlcNAcâ1f3Galâ1f4Glc,
benzoyl anhydride, and 1-methylimidazole were purchased from
Sigma (St. Louis, MO). 4-(Dimethylamino)pyridine (DMAP) and
2 M (trimethylsilyl)diazomethane (TMSCHN2) in hexane were
products of Aldrich (Milwaukee, WI). 3′-Sialyl-Lewis X was
obtained from Calbiochem Corp. (San Diego, CA) and purified
with the aid of the Dionex anion-exchange chromatographic
system. HPAE-grade sodium hydroxide was purchased as a 50%
(w/ w) aqueous solution from Acros Organics (Pittsburgh, PA),
while anhydrous sodium acetate was received from Fluka (Mil-
waukee, WI). Sep-Pak C18 cartridges (containing 100 mg of
sorbent) were products of Waters Corp. (Milford, MA), and Diaion
HP-20 resin was purchased from Supelco, Inc. (Bellefonte, PA).
The arabinosazone matrix for mass spectrometry was synthesized
in this laboratory, as described previously.24
1
substituent), 48.32 39.15. H NMR, and two-dimensional DEPT
and HETCOR NMR results (benzene-d6, 400 MHz, δ): acetyl CH3,
1.41 (s); 5 pairs of CH2 (all double doublet) 1.80 and 2.63, 4.42
and 4.91, 4.56 and 4.68, 5.00 and 5.26, 5.43 and 6.26; 14 CH protons,
3.11 (double d), 3.91 (d), 3.99 and 4.00 (2 overlapped protons),
4.62 (overlapped), 5.10 (d), 5.66 (double d), 5.70-5.82 (3 over-
lapped protons), 5.88 (double d), 6.02 (m), 6.6-7.2 (2 protons,
overlapped with benzoyl protons). FT-IR (thin film, cm-1): 3400-
3200 (br, w), 3100-2800 (br, m), 3067 (m), 2966 (m), 1969 (w),
1917 (w), 1728 (vs), 1601 (m), 1537 (m), 1452 (m), 1373 (w), 1267
(vs), 1175 (s), 1107 (s), 1026 (m), 897 (w), 849 (w).
P urification of Benzoic Anhydride and 1-Methylimidazole.
A 25-g sample of benzoic anhydride was dissolved in 15 mL of
benzene at 50-60 °C, before addition of 50 mL of cyclohexane.
The solution was cooled to room temperature and allowed to
crystallize at 4 °C for 5 h. The crystals were collected through
filtration and submitted to recrystallization following the same
procedure. The final product was dried in a vacuum. 1-Methylimi-
dazole was distilled under vacuum before its use and stored with
the molecular sieve added.
P reparation of Benzoylated Neu5NAcr2f3lactose Alditol
(1 b) for NMR Study. Neu5NAcR2f3lactose (5 mg) was dis-
solved in 250 µL of 1 N sodium borohydride solution. After a 3-h
reaction under stirring at room temperature, the sodium ions were
removed from the solution through a fritted plastic syringe packed
with 3 mL of Dowex 50WX2-400 cation-exchange resin. The resin
bed was washed with 3 mL of deionized water. The eluents were
combined and lyophilized. The glycan alditols were made free of
borate through repetitive evaporation in methanol. The Neu5-
NAcR2f3lactose alditol product was transferred, as a 20-µL
P reparation of Benzoylated Neu5 NAcr2 f6 lactose Aldi-
tol (3 b) for NMR Study. The Neu5NAcR2f3- and 2f6lactose
mixture (50 mg, 85% of 2f6 content) was reduced and benzoylated
following the above procedures with a 10-fold scale-up in the
chemicals except that a longer benzoylation time of 8 h was used
to increase the yield of 3 b. The product 3 b was well separated
from other benzoylation products in the reversed-phase HPLC
elution due to its higher hydrophobicity. 13C NMR (benzene-d6,
400 MHz, δ): region I has 14 peaks (1 amide carbonyl carbon
and 13 benzoyl carbonyl carbons), 169.15, 166.40, 166.18, 166.01,
165.87, 165.85, 165.81, 165.71, 165.66, 165.60, 165.46, 165.35,
165.26, 165.24; region II has 2 peaks (corresponding to 2 carbons
that have 2 alkoxyl groups attached), 100.63, 97.36; region III has
16 carbons (each has 1 proton and 1 alkoxyl group attached),
77.62, 73.88, 73.52, 72.41, 71.85, 71.56, 71.04, 70.97, 70.42, 69.29,
68.44, 68.32, 63.55 (highest peak; may contain 2 carbon signals),
63.26, 63.19; region IV has 2 peaks (one connected to the amide
and the other without any alkoxyl attached), 51.65, 36.40. 1H NMR,
and two-dimensional DEPT and HETCOR NMR results (benzene-
d6, 400 MHz, δ): 5 pairs of CH2 (all double doublet) 2.61 and
2.79, 4.32 and 4.43, 4.67 and 5.04, 4.77 and 4.86, 4.88 and 5.66; 14
(19) Hounsell, E. F.; Davies, M. J.; Renouf, D. V. Glycoconjugate J. 1 9 9 6 , 13,
19-26.
(20) Carlson, D. M. J. Biol. Chem. 1 9 6 8 , 243, 616-626.
(21) Iyer, R. N.; Carlson, D. M. Arch. Biochem. Biophys. 1 9 7 1 , 142, 101-105.
(22) Chen, P. Doctoral Thesis, Department of Chemistry, Indiana University,
Bloomington, 1998.
(23) Daniel, P. F. Methods Enzymol. 1 9 8 7 , 138, 94-116.
(24) Chen, P.; Baker, A. G.; Novotny, M. V. Anal. Biochem. 1 9 9 7 , 244, 144-
151.
4970 Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

CH protons, 3.96 (t), 4.72 (d), 4.96 (overlapped), 5.02 (d), 5.25
(m), 5.34 (m), 5.63 (double d), 5.94 (m), 6.11 (m), 6.19 (d), 6.37
(double d), 6.53 (d), 6.6-7.2 (2 protons, overlapped with benzoyl
protons). FT-IR (thin film, cm-1): 3400-3200 (br, w), 3065 (w),
2918 (s), 2851 (m), 1968 (w), 1914 (w), 1728 (vs), 1601 (m), 1452
(m), 1316 (m), 1260 (vs), 1177 (m), 1107 (s), 1069 (s), 1026 (m),
860 (w), 845 (w).
1H and 13C spectra for proton and 13C NMR spectra, including
two-dimensional recordings, are provided as Supporting Informa-
tion.
Benzoylation of 5 0 -pmol Amounts of Carbohydrates for
a MALDI-MS Study. A 50-pmol sample of Neu5NAcR2f3lac-
tose, Neu5NAcR2f6lactose, Neu5NAcR2f6Galâ1f4GlcNAcâ1f
3Galâ1f4Glc, or 3′-sialyl-Lewis X was transferred, as a 10-µL
solution, to a 0.1-mL glass reaction vial and lyophilized thoroughly
to dryness before the addition of 40 µL of derivatizing solution
prepared by dissolving benzoyl anhydride in an equal weight of
1-methylimidazole. The benzoylation of neutral glycans was carried
out at 110 °C for 5 h in a tightly capped reaction vial. After
benzoylation, the vial was cooled to room temperature and the
solution was diluted with a 10-fold excess of acetonitrile. The
mixed solution was transferred into an equal volume of water and
mixed well before an elution through a Waters Sep-Pak C18
cartridge. The loaded cartridge was washed with 40 mL of 50%
aqueous acetonitrile, while the benzoylated glycans were eluted
with 2 mL of pure acetonitrile and recovered upon evaporation
through either nitrogen flow or vacuum evaporation.
MALDI Mass Spectrometric Measurements. All experi-
ments were carried out on a Voyager Linear BioSpectrometry
workstation MALDI/ time-of-flight mass spectrometer from Per-
Septive Biosystems (Framingham, MA). The arabinosazone bulk
matrix solution was prepared by dissolving 1 mg of arabinosazone
in 60 µL of ethanol and 40 µL of deionized water. All analyses
used the on-plate mixing procedure, in which, typically, a 0.4-µL
aliquot of the matrix solution was placed on a polished stainless
steel plate, and before drying occurred, 0.2 µL of the acetonitrile
solution, containing 500 fmol-1 pmol of benzoylated glycans was
quickly added and mixed on the plate using a micropipet. The
sample/ matrix solution dries out very fast, and a MALDI mass
spectrometric analysis can normally be performed in several
minutes.
Figure 1. MALDI mass spectrum of benzoylated carbohydrates:
500 fmol amounts of benzoylated Neu5NAcR2f3lactose (A) and
Neu5NAcR2f6lactose (B). Inset: 10 femtomole of benzoylated
Neu5NAcR2f3lactose, detected with 0.05-µL acetonitrile sample
solution and 0.05-µL arabinosazone matrix solution in 60% ethanol.
dazole, such transacylation has been minimized while the ben-
zoylation rate is comparable to that with DMAP/ pyridine. Figure
1 depicts a MALDI-MS analysis of a 500-fmol sample of benzoy-
lated Neu5NAcR2f3lactose and Neu5NAcR2f6lactose from 50-
pmol sample pools using an excess amount of benzoic anhydride
in 1-methylimidazole. The structural differences of these two
sialoglycans result in very different product patterns for benzoy-
lation (due to the different numbers of hydroxy groups accessible
for benzoylation) and a characteristic structural alteration during
the derivatization.
For a fully benzoylated sialosugar with free carboxyl groups,
its mass can be calculated using the following formula:
mass ) (NeuNAc - H2O)d + HexNAce + Hexf + Fucg +
104.11(3d + 2e + 3f + 2g + 1) + H2O + Na ) (291.26 -
18.01)d + 203.19e + 162.14f + 146.14g + 104.11(3d +
2e + 3f + 2g + 2) + 18.01 + 22.99
Methylation of Carboxyls in Sialosugars. Approximately 100
pmol of benzoylated Neu5NAcR2f3lactose or Neu5NAcR2f
6lactose was transferred, as a 65-µL solution in anhydrous ben-
zene, to a 0.1-mL glass reaction before addition of 10 µL of anhy-
drous methanol and 25 µL of 2 M TMSCHN2 in hexane. Sub-
sequently, the vial was sealed with a Teflon-lined cap and the
solution was stirred at room temperature for 8 h. Extra reagents
and solvents were removed through nitrogen flow and the
methylation product was dissolved in 100 µL of acetonitrile for
an MS measurement.
where d, e, f, and g represent the number of the N-acetyl-
neuraminic acid (NeuNAc), N-acetylaminohexose (HexNAc),
hexose (Hex), and fucose (Fuc), respectively.
The benzoylated sialosugars, with free carboxyl groups, have
been found by us to be desorbed/ ionized as dehydrated products,
with masses of 18 Da less per a sialo unit than those of the
expected sodiated molecular ions, as accounted for in the formula
as (NeuNAc - H2O). Therefore, for a fully benzoylated Neu5NAc-
lactose, a peak at m/ z 1784 is to be expected. A MALDI-MS
measurement on 1 a shows a major peak at m/ z 1680 (Figure 1A),
which is 104 m/ z less than m/ z 1784. The difference of 104 Da
represents one benzoyl group addition; hence it is clear that 1 of
the 11 hydroxy groups of the free sugar remained underivatized,
most likely the one that is in just the right position to undergo
spontaneous lactonization25 with the carboxyl group of the
RESULTS AND DISCUSSION
1-Methylimidazole has been found to be a good solvent and
catalyst for benzoylation, replacing pyridine and DMAP used in
the former protocol.23 The use of DMAP facilitates the benzoy-
lation reaction, but it has been found by us to cause a substantial
degree of transacylation (replacement of acetyl groups in the sugar
molecules by the benzoyl groups). With the use of 1-methylimi-
Analytical Chemistry, Vol. 71, No. 21, November 1, 1999 4971

neuraminic moiety to yield structure 1 a. In this case, however,
the loss of one H2O molecule did not occur because of the
dehydration of the free carboxyl group in the MALDI desorption/
ionization process, but due to the ester formation during lacton-
ization. Moreover, an equilibrium between the lactone and the
corresponding hydroxy acid is believed to exist, for after TM-
SCHN2 is added to the benzoylated sugar (represented by the
peak at m/ z 1680), the resulting methyl ester (with a mass/ charge
of 1712) can further be benzoylated to yield a peak at m/ z 1816
in the mass spectrum (data not shown), indicating a complete
benzoylation of all 11 hydroxyl groups. It is worthwhile to note
that methylation of the carboxyl group results in a mass increase
of 32 (as compared to the reaction with a free carboxyl acid group),
of which 14 Da is being contributed by a methylene addition and
18 Da by a recovered water molecule. It appears that such a
lactone/ hydroxy acid equilibrium is too unfavorable for a complete
benzoylation of the sugar, but it may be shifted to accommodate
the methylation process.
The configuration of the Neu5NAcR2f6lactose molecule does
not permit an analogous lactone formation. Here, every hydroxyl
group should be accessible to attack by benzoic anhydride. This
is supported by the MALDI-MS measurement (Figure 1B) that
reveals a peak at m/ z 1784, corresponding to a fully benzoylated
product 2 (as compared to the one at m/ z 1680 that can be
converted completely to m/ z 1784 upon extented benzoylation).
In the 2f6 isomer case, a peak at m/ z 1846 (62 Da higher than
that at m/ z 1784) is also seen, while no such addition was observed
with a 2f3-linked oligosaccharide before methylation. Substitution
of benzoyl (105) for the acetyl (43) group of the amide moiety is
thereby suggested. It is of interest that such mass addition by 62
Da is of insignificant intensity for the 2f3 isomer, whose carboxyl
group is supposedly tied up as a lactone; however, mass spectral
evidence indicates that the substance represented by the m/ z 1846
peak is not converted to a methyl ester upon treatment with
(trimethylsilyl)diazomethane, in contrast with the substances
represented by m/ z 1680 and 1784 peaks in the case of the 2f6
isomer. For this reason, an interaction between the carboxyl group
and the amide nitrogen has been proposed, resulting in ejection
(25) Yamamoto, Y.; Onda, M.; Takahashi, Y.; Inoue, Y.; Chuˆjoˆ, R. Carbohydr.
Res. 1 9 8 8 , 182, 31-40.
4972 Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

of the acetyl group and replacement of it by benzoyl (which is, of
course, present in large excess), leading to structure 3 a.
For further NMR characterization, the reduced versions of
Neu5NAcR2f3- and 2f6lactose were used for benzoylation. The
use of benzoylated alditols in the NMR measurements is necessary
in avoiding the well-known complexity associated with the ano-
meric configurations. The benzoylated alditols were purified
through reversed-phase HPLC to yield 1 b and 3 b, which give
mass spectroscopic patterns similar to those of their counterparts,
1 a and 3 a, except for an upward mass/ charge shift of 106 for
each corresponding peak. This is because reduction has intro-
duced two hydrogen atoms (2 Da) and created a new hydroxyl
group to be benzoylated (104 Da). Although the NMR spectra of
highly benzoylated products were quite complex, they were in
general agreement with the proposed reaction mechanisms and
MS evidence.
Such perbenzoylated glycans can be detected at high sensitivity
by MALDI-MS, as depicted in the inset of Figure 1, where a 10-
fmol amount of benzoylated Neu5NAcR2f6lactose was observed
at a signal-to-noise ratio of 10:1. Differentiation between 2f3 and
2f6 linkages in the manner described was shown to be general
when applied to selectin-binding ligand, sialyl-Lewis X, and a
human milk carbohydrate, Neu5NAcR2f6Galâ1f4GlcNAcâ1f
3Galâ1f4Glc, as exemplified in Figure 2.
The benzoylation strategy enables a direct structural determi-
nation of the sialocarbohydrates. The number of sialic acid
residues can easily be derived from the mass/ charge values and
confirmed by a facile methylation experiment. Arabinosazone
appears to be a suitable MALDI matrix for both the benzoylated
sialo- and asialosugars. It is conceivable that the benzoylation/
MALDI-MS approach, when coupled with a use of selective neur-
aminidases, could also reveal sialyl linkage information on other
multisialylated glycans. Thus, the capability of analyzing both the
neutral and acidic glycans and differentiating their terminal 2f3
and 2f6 sialyl galactosidical linkages appears very valuable for
future correlation studies of carbohydrate structures and their
biological functions.
Figure 2. MALDI mass spectrum of benzoylated carbohydrates: 1
pmol of benzoylated sialyl-Lewis X (A) and Neu5NAcR2f6Galâ1f
4GlcNAcâ1f 3Galâ1f4Glc (B). Arabinosazone in 60% ethanol was
used as the matrix solution.
SUPPORTING INFORMATION AVAILABLE
13C NMR spectrum of benzoylatedNeu5AcR2f3lactose alditol
1
(1 b) in deuterated benzene; H NMR spectrum of benzoylated
Neu5aCR2f3lactose alditol (1b) in deuterated benzene; 1H COSY
diagram of benzoylated Neu5AcR2f3lactose alditol (1 b) in
1
deuterated benzene; H spectrum of benzoylated Neu5AcR2f
6lactose alditol (3 b) in deuterated benzene; 13C and DEPT NMR
spectrum of benzoylated Neu5AcR2f6lactose alditol (3 b) in
deuterated benzene; HETCOR diagram of benzoylated Neu5AcR2f
6lactose alditol (3b) in deuterated benzene; and 1H COSY diagram
of benzoylated Neu5AcR2f6lactose alditol (3 b) in deuterated
benzene are available. This material is available free of charge
via the Internet at http:/ / pubs.acs.org.
ACKNOWLEDGMENT
Received for review June 18, 1999. Accepted August 3,
1999.
This research has been supported by Grant 24349 from the
National Institute of General Medical Sciences, U.S. Department
of Health and Human Services.
AC990674W
Analytical Chemistry, Vol. 71, No. 21, November 1, 1999 4973