NMR assignments for glucosylated and galactosylated N- acetylhexosaminitols: Oligosaccharide alditols related to O-linked glycans from the protozoan parasite Trypanosoma cruzi

Article abstract of DOI:10.1016/S0008-6215(00)00107-5

We report full ¹H and ¹³C NMR assignments for 13 gluco- or galacto- pyranosylated derivatives of GlcNAc-ol, GalNAc-ol or ManNAc-ol, many of which have been prepared by enzymatic methods. These spectra are reference data to aid the structural analysis by NMR spectroscopy of glycosylated alditols derived from the mucin of the protozoan parasite Trypanosoma cruzi. A series of structural reporter groups for the derivatives from this unusual series of O-glycans are described. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00107-5

www.elsevier.nl/locate/carres
Carbohydrate Research 328 (2000) 321–330
NMR assignments for glucosylated and galactosylated
N-acetylhexosaminitols: oligosaccharide alditols related
to O-linked glycans from the protozoan parasite
Trypanosoma cruzi
Christopher Jones a,*, Jose´ O. Previato b, L. Mendonc¸a-Previato b
a Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane,
South Mimms, Herts EN6 3QG, UK
b Instituto de Microbiologia, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersita´ria,
21944-970 Rio de Janeiro, RJ, Brazil
Received 9 February 2000; accepted 11 April 2000
Abstract
1
We report full H and 13C NMR assignments for 13 gluco- or galacto-pyranosylated derivatives of GlcNAc-ol,
GalNAc-ol or ManNAc-ol, many of which have been prepared by enzymatic methods. These spectra are reference
data to aid the structural analysis by NMR spectroscopy of glycosylated alditols derived from the mucin of the
protozoan parasite Trypanosoma cruzi. A series of structural reporter groups for the derivatives from this unusual
series of O-glycans are described. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Parasite mucins; O-Glycans; N-Acetylhexosaminitols; Enzymatic synthesis; Trypanosoma cruzi
1. Introduction
glycan is linked to Thr residues in the peptide
backbone predominantly through a-linked N-
acetylglucosamine [4]. Secondly, the HexNAc-
ol in oligosaccharides isolated by reductive
cleavage can be substituted with b-Galf at
C-4, b-Galp at C-4 or C-4 and C-6, or b-glu-
cosylated [1–3]. The oligosaccharide alditols
isolated from the more complex parasite gly-
cans contain 4,6-disubstituted HexNAc-ol
(usually GlcNAc-ol), rather than 3,6-disubsti-
tuted GalNAc-ol-containing compounds com-
monly isolated from mammalian systems.
Thirdly, further elaboration of the chain is by
galactopyranosylation of the six-arm in a
strain-dependent manner, and galacto-pyra-
nosylation or -furanosylation of the b-
Galf(1“4)- substituent on HexNAc C-4
[1–4]. In our work on the O-glycans from T.
The O-linked glycan chains of the GPI-an-
chored surface mucin from the protozoan par-
asite Trypanosoma cruzi have unusual
structures ([1–4]; Jones et al., unpublished
data). They differ from other O-linked glyco-
protein glycans in three respects. Firstly, the
Abbre6iations: 1D, one-dimensional; 2D, two-dimensional;
COSY, correlation spectroscopy; CPS, capsular polysaccha-
ride; GPI, glycophosphoinositol; HexNAc-ol, N-acetyl-
hexosaminitol; HSQC, heteronuclear single quantum
coherence; PGC, porous graphitised carbon; PNP, p-nitro-
phenyl; TFA, trifluoroacetic acid; TOCSY, total correlation
spectroscopy.
* Corresponding author. Tel.: +44-1707-654753 ext. 211;
fax: +44-1707-646730.
E-mail address: cjones@nibsc.ac.uk (C. Jones).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00107-5

322
C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
cruzi, after release of the glycan with alkaline
borohydride, oligosaccharide alditols are frac-
tionated by gel permeation chromatography
or HPLC on porous graphitised carbon
(PGC) [5,6], and analysed by one- (1D) and
two-dimensional (2D) NMR, methylation
analysis and, when appropriate, fast atom
bombardment mass spectrometry. The iso-
lated oligosaccharides are heterogeneous from
the presence of different HexNAc-ol residues.
derivatives as donors and N-acetylhexo-
saminitols as acceptors. Initially, the enzy-
matic transglycosylation reaction mixture was
reduced directly and a monosaccharide–aldi-
tol fraction isolated directly by HPLC on
PGC. In later experiments, the reaction mix-
ture was fractionated, a disaccharide fraction
isolated, reduced and desalted. In both cases,
the isolated monosaccharide alditols were
purified by HPLC on a 20 cm PGC column
eluted with a shallow acetonitrile gradient. If
the final product remained impure — the
most common impurity was the relevant b-
Hex-(1“6)-Hexitol — a further chromato-
graphic step on an aminopropyl column was
carried out. This method yielded most of the
required oligosaccharide alditols, except that
formation of the b-Hex-(1“3)-HexNAc
derivatives did not occur and yields of b-Hex-
(1“4)-HexNAc-ol were variable. Two disac-
charide alditols were isolated from the
transglycosidation reactions, b-Galp-(1“6)-b-
Galp-(1“6)-GalNAc-ol and b-Galp-(1“6)-
b-Galp-(1“6)-ManNAc-ol, consistent with
sequential addition of Gal residues to the fa-
voured hydroxymethyl groups.
1
At times only enough material for a 1D H
NMR spectrum was obtained, or components
could not be completely resolved. These frac-
1
tions can be identified from the 1D H NMR
spectrum if appropriate authentic model com-
pounds are available, but no systematic study
1
of the H NMR spectra of these compounds
has been reported.
A series of authentic reference com-
pounds — mono-galactosylated and -gluco-
sylated derivatives of N-acetylhexosam-
initols — have been prepared from PNP
derivatives of b-Glc and b-Gal by the method
of Yoon and Ajisaka [7] using enzymic trans-
glycosylation in an aqueous organic solvent
1
13
mix, and their H and C NMR spectra fully
assigned. These compounds also provide refer-
ence samples for methylation analysis.
Glucosylation reactions.—Whilst glucosyla-
tion of GlcNAc and ManNAc only gave only
6-substituted derivatives, two monosaccharide
alditol fractions were isolated from the Gal-
NAc reaction in an approximate ratio of 4:1.
The minor, earlier-eluting b-Glcp-(1“4)-Gal-
NAc-ol fraction was contaminated with b-
Glc-(1“6)-Glc-ol and required re-chromato-
graphy on an aminopropyl column.
2. Results
Oligosaccharide preparation.—Parent com-
pounds for five of the desired alditols (b-Galp-
(1“3)-GlcNAc,
b-Galp-(1“3)-GalNAc,
Galactosylation reactions.—Galactosylation
of GalNAc with recombinant E. coli enzyme
yielded only b-Galp-(1“6)-GalNAc-ol, whilst
use of the enzyme from Aspergillus oryzae
gave a mixture of which the major component
was the b-Galp-(1“6)-GalNAc-ol, and minor
components were b-Galp-(1“4)-GalNAc-ol
and the disaccharide alditol b-Galp-(1“6)-b-
Galp-(1“6)-GalNAc-ol. Galactosylation of
ManNAc gave the b-Galp-(1“6)-ManNAc-
ol and b-Galp-(1“4)-ManNAc-ol as major
and minor products respectively. The NMR
spectrum of the latter was identical with that
of authentic material. Small amounts of b-
Galp-(1“6)-b-Galp-(1“6)-ManNAc-ol were
also obtained.
b-Galp-(1“4)-GlcNAc, b-Galp-(1“4)-Man-
NAc, and b-Galp-(1“6)-GlcNAc) are avail-
able commercially. They were reduced with
sodium borohydride and purified by HPLC on
a PGC column. b-Glc-(1“3)-GlcNAc was
prepared by acid hydrolysis of the capsular
polysaccharide (CPS) from Streptococcus
pneumoniae Type 20, as described [8], except
that the hydrolysis was performed at 80 °C for
90 min with 0.1 M TFA. The hydrolysis mix-
ture was reduced, desalted and b-Glc-(1“3)-
GlcNAc-ol isolated by HPLC.
Other oligosaccharides were prepared ac-
cording to Yoon and Ajisaka [7], using appro-
priate
glycosidases
in
an
aqueous–
triethylphosphate solvent mix with PNP

C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
323
1
COSY to assign the H NMR spectrum and
heteronuclear correlations to assign the car-
bon spectrum. These spectra were used for
linkage determination of some monosaccha-
ride-alditols made by enzymatic methods.
These data are reported in Tables 1–3. Similar
assignments were made for two disaccharide
alditols isolated from the enzymatic trans-
glycosidations.
Epimerisation of GlcNAc-containing oligo-
saccharides.—Attempts were made to prepare
ManNAc-ol derivatives by controlled alkaline
epimerisation of the appropriate GlcNAc-con-
taining disaccharide. Epimerisation of Glc-
NAc in dilute alkali has been studied [9], and
used as a cheap source of GlcNAc/ManNAc
mixtures in the enzymatic production of N-
acetylneuraminic acid [10,11], and is a side
reaction in heparin degradation [12]. Epimeri-
sation of the aminosugar was a potential
source of additional model compounds: avail-
able GlcNAc-containing disaccharides were
treated with base, trapped by reduction, and
the products analysed by HPLC and NMR
spectroscopy. The equilibrium proportions of
GlcNAc:ManNAc after dilute alkali equilibri-
ation have been reported to be 4:1, but NaOH
treatment of b-Galp-(1“4)-GlcNAc with sub-
sequent borohydride reduction gave a mixture
containing b-Galp-(1“4)-GlcNAc-ol and b-
Galp-(1“4)-ManNAc-ol in an approximate
ratio of 2:1. The NMR spectrum could be
interpreted as a mixture of the two com-
pounds, both components of which were al-
ready available in a pure form. Attempts to
use this approach to prepare b-Galp-(1“3)-
ManNAc-ol from the readily available b-
Galp-(1“3)-GlcNAc failed, as reaction in
either dilute sodium hydroxide or dilute
aqueous ammonia gave rise to a complex mix-
ture of degradation products.
Sufficient material was obtained for 13 of
the 18 possible derivatives to allow full NMR
assignment, and partial assignments could be
obtained for one other, which was a compo-
nent of a mixture. Partial NMR data has
previously been reported for two of the com-
pounds studied here. b-Galp-(1“3)-GalNAc-
ol is the product of reductive cleavage of the
Tn antigen from glycoproteins and several
assignment have been reported [13–15]. b-
Glcp-(1“3)-GlcNAc-ol, identified by methy-
lation analysis, was isolated from the
hydrolysate of the S. pneumoniae Type 20
CPS, but only the chemical shifts of the Glc
H-1 and GlcNAc-ol NAc methyl resonances
were reported [8].
Separation of the monosaccharide alditols on
the PGC column.—The use of PGC for the
separation of oligosaccharide alditols was pio-
neered in Hounsell’s laboratory [5,6]. PGC is
robust and the separation depends on hydro-
phobic patches that interact with planar p-sys-
tems in the graphite: components are eluted
with an acetonitrile gradient. The oligosaccha-
ride alditols liberated from parasite mucins
with alkaline borohydride are well resolved
(Jones et al., unpublished work). It was of
interest to us to establish general correlations
between structure and elution time on these
columns as a further aid to characterisation.
In the present work, HPLC on a 10 cm PGC
column proved valuable to isolate low yields
of monosaccharide alditols or disaccharides
without preliminary work-up, as they were
resolved from excess HexNAc, triethylphos-
phate, PNP-glycosides and PNP.
Structural dependence of the HPLC elution
time.—A knowledge of the relative elution
times of structural variants is an aid to struc-
ture determination. Three sets of chro-
matograms were obtained to assess the
importance of three factors, the presence of a
Glc or Gal substituent, the identity of the
HexNAc-ol and the importance of the linkage
position. During HPLC of the six b-Hexp-
(1“6)-HexNAc-ols, the glucosylated com-
pounds eluted later than the galactosylated
analogues. This was also true when comparing
b-Glc-(1“3)-GlcNAc-ol with b-Galp-(1“3)-
GlcNAc-ol. Secondly, in both series of com-
pounds,
b-Galp-(1“6)-HexNAc-ol
and
b-Glcp-(1“6)-HexNAc-ol, the order of elu-
tion was GalNAc-ol before ManNAc-ol be-
fore GlcNAc-ol. The separation of the
galactosylated series is shown in Fig. 1(a):
essentially the same separation was observed
for b-Glcp-(1“6)-HexNAc-ol (data not
shown). However, b-Gal-(1“4)-GlcNAc-ol
The NMR spectra of the monosaccharide
alditols were assigned at 500 MHz and 30 °C
using conventional methods — TOCSY and

324
C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
derivatives eluted. Elution times are compared
elutes
before
b-Gal-(1“4)-ManNAc-ol,
to the peak from permethyl Gal a-methyl
glycoside, present in all samples, at 1.000. The
order observed was “4)-GlcNAc-ol (2.184),
“4)-ManNAc-ol (2.228), “3)-GalNAc-ol
(2.238), “6)-GlcNAc-ol (2.260), “3)-Glc-
NAc-ol (2.261), “6)-GalNAc-ol (2.377) and
“6)-ManNAc-ol (2.391). No derivatives con-
taining “3)-ManNAc-ol or “4)-GalNAc-ol
were available.
whilst b-Gal-(1“3)-GalNAc-ol elutes before
b-Gal-(1“3)-GlcNAc-ol. Within the series b-
Galp-(1“X)-GlcNAc-ol, where X=3, 4 or 6,
the elution order is b-Galp-(1“3)-GlcNAc-ol
before
b-Galp-(1“4)-GlcNAc-ol,
which
eluted before b-Galp-(1“6)-GlcNAc-ol. This
separation is shown in Fig. 1(b). The same
order was observed for the b-Galp-(1“X)-
GalNAc-ol series (data not shown), and in the
ManNAc-ol series, where compounds are
available. The separation of galactosylated
GlcNAc-ol derivatives b-Galp-(1“X)-Glc-
NAc-ol and b-Galf-(1“X)-GlcNAc-ol (X=
3, 4 or 6) by high-performance anion-
exchange chromatography has recently been
reported [16]. The resolution obtained by
HPLC on PGC is comparable with that ob-
tained by anion exchange, and the use of a
volatile solvent system simplifies sample re-
covery for spectroscopic analysis.
3. Discussion
Synthesis of the components.—Using a com-
bination of approaches the majority of the
required monosaccharide alditols were pre-
pared for NMR analysis. The use of glycosi-
dases in organic solvents proved an efficient
route for the preparation of the 6-substituted
derivatives in the small quantities required for
NMR analysis, whilst production of the 4-
substituted derivatives was more problematic
and no 3-substituted alditols were obtained.
Whilst epimerisation of the more readily avail-
Analysis of the partially methylated alditol
acetates (PMAAs) from the N-acetyl-
hexosaminitols by GC and GC–MS on a
DB-1 column showed the order in which the
Table 1
NMR assignments for GalNAc-ol-containing species
Residue
H-1
C-1
H-1%
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
H-6%
NAc
GalNAc-ol
3.740
62.88
Compound not available
3.559
3.688
4.250
52.86
3.852
69.85
3.396
70.82
3.939
71.18
3.654
64.49
3.683
2.057
23.18
b-Glcp-(1“3)-GalNAc-ol
b-Galp-(1“3)-
4.470
105.14
3.789
61.73
4.521
103.50
3.778
63.54
4.465
104.04
3.702
62.82
4.513
103.81
3.744
62.70
4.449
104.44
3.747
62.75
3.667
73.71
4.056
77.60
3.498
76.91
4.022
68.87
3.647
73.86
4.030
68.88
3.512
76.74
3.871
69.55
3.670
73.80
3.876
69.60
3.896
69.67
3.503
70.29
3.398
70.93
3.720
78.05
3.901
69.66
3.730
77.91
3.392
70.76
3.434
70.70
3.933
69.75
3.449
70.84
3.718
76.20
4.183
70.61
3.350
76.93
4.009
70.83
3.712
76.20
4.009
70.75
3.476
76.99
4.125
69.47
3.712
76.29
4.129
69.29
3.773
62.20
3.669
64.07
3.912
61.90
3.705
62.76
3.78
3.773
3.621
3.730
3.606
3.78
72.31
4.383
52.56
3.315
74.49
4.315
52.03
3.533
72.15
4.329
51.93
3.318
74.28
4.266
52.61
3.559
72.09
4.269
52.64
“3)-GalNAc-ol
b-Glcp-(1“4)-
“4GalNAc-ol
b-Galp-(1“4)-
“4)-GalNAc-ol
b-Glcp-(1“6)-
“6)-GalNAc-ol
b-Galp-(1“6)-
“6)-GalNAc-ol
3.728
3.680
3.66
2.043
23.00
2.071
23.15
62.04
3.81
3.681
3.725
3.793
3.772
3.796
2.071
23.15
63.28
3.928
61.83
3.996
72.87
3.772
62.12
4.006
73.02
3.678
3.692
2.065
23.00
2.066
23.03

C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
325
Table 2
NMR assignments for the GlcNAc-ol-containing species
Residue
H-1
C-1
H-1%
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
H-6%
NAc
GlcNAc-ol
3.761
3.635
4.071
3.957
3.596
3.755
3.817
3.648
3.724
3.650
3.761
3.633
2.044
23.35
62.10
4.576
103.93
3.780
61.62
4.509
104.58
3.768
61.64
54.94
3.362
74.22
4.258
54.46
3.551
71.91
4.244
54.49
69.59
3.524
76.71
4.173
76.71
3.676
73.69
4.166
76.73
72.37
3.409
70.95
3.579
71.89
3.904
69.73
3.559
71.88
72.22
3.479
76.71
3.878
71.89
3.715
76.11
3.903
71.73
64.01
3.944
61.93
3.857
63.80
3.776
62.26
3.866
64.11
b-Glcp-(1“3)-
“3)-GlcNAc-ol
b-Galp-(1“3)-
“3)-GlcNAc-ol
3.70
2.048
23.22
3.681
2.031
23.22
b-Glcp-(1“4)-GlcNAc-ol
b-Galp-(1“4)-
Compound not available
4.512
104.12
3.789
62.05
4.505
103.96
3.7
3.579
3.665
73.73
3.947
69.43 a
3.516
76.74
3.980
69.40
3.670
73.72
3.980
69.37
3.927
69.82
3.872
79.79
3.402
70.83
3.681
71.96
3.935
69.68
3.690
71.86
3.697
76.19
3.946
72.28 a
3.479
77.05
3.916
71.08
3.716
76.21
3.916
71.08
3.806
62.22
3.878
63.13
3.934
61.90
4.146
72.42
3.78
3.761
3.754
3.739
3.819
3.78
72.23
4.304
53.79
3.337
74.41
4.089
54.80
3.575
72.01
4.091
54.74
“4)-GlcNAc-ol
b-Glcp-(1“6)-
“6)-GlcNAc-ol
b-Galp-(1“6)-
“6)-GlcNAc-ol
3.659
3.639
3.640
2.063
23.21
2.055
23.22
62.01
4.440
104.52
3.741
61.90
62.05
4.148
72.32
3.836
2.055
23.16
a Assignment uncertain.
Table 3
NMR assignments for the ManNAc-ol-containing species
Residue
H-1
C-1
H-1%
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
H-6%
NAc
C-Me
ManNAc-ol
3.851
61.94
3.768
4.053
53.08
3.880
69.09
3.527
70.41
3.746
71.58
3.824
64.12
3.645
2.041
b-Glcp-(1“3)-ManNAc-ol
b-Galp-(1“3)-ManNAc-ol
b-Glcp-(1“4)-ManNAc-ol
b-Galp-(1“4)-
Compound not available
Compound not available
Insufficient material for full assignment a
4.507
104.02
3.890
63.42
4.503
104.12
3.858
62.12
4.437
104.70
3.859
62.07
3.568
72.39
4.116
54.42
3.330
74.41
4.058
53.25
3.572
72.03
4.061
53.20
3.661
73.79
4.073
68.81
3.513
76.74
3.888
69.12
3.680
73.74
3.894
69.07
3.933
69.74
3.878
78.45
3.396
70.83
3.604
70.36
3.937
69.85
3.619
70.37
3.703
76.12
3.912
72.39
3.467
77.05
3.893
70.67
3.716
76.23
3.892
70.63
3.782
62.27
3.839
61.65
3.930
61.81
4.159
72.85
3.776
62.07
4.161
72.81
3.782
3.839
3.738
3.819
3.776
3.849
“4)-ManNAc-ol
b-Glcp-(1“6)-
“6)-ManNAc-ol
b-Galp-(1“6)-
“6)-ManNAc-ol
3.750
3.760
3.776
2.031
23.34
2.047
23.18
2.048
23.18
1
a Only sufficient b-Glc-(1“4)-ManNAc-ol was obtained for a 1D H NMR spectrum.

326
C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
able GlcNAc derivatives was effective for the
preparation of 4-substituted ManNAc-ol com-
pounds [12], b-Galp-(1“3)-GlcNAc was
rapidly degraded under these conditions and
the required b-Galp-(1“3)-ManNAc-ol could
not be isolated. No experiments were per-
formed in the 6-substituted GlcNAc series, as
the relevant ManNAc-ol derivatives were al-
ready available.
resonance appears to be characteristic of the
stereochemistry of the HexNAc-ol residue,
and, secondly, the pattern of chemical shifts of
the HexNAc-ol H-2, H-3, H-4, H-5, H-6a,
H-6b% and NAc are diagnostic of the substitu-
tion pattern. Many of these resonances are
resolved.
Peak shape of the HexNAc-ol H-2.—The
peak shape of this resonance was characteristi-
cally different in the three HexNAc-ols, due to
NMR assignments.—Full assignment of the
13
3
1H and C NMR spectra of these compounds
the different values of the JH2,H3 coupling
was achieved by conventional methods and
constant. In GalNAc-ol and GlcNAc-ol, the
1
3
3
without difficulty. The 1D H NMR spectra
two JH1a,H2 and JH1b%,H2 couplings are ap-
are key reference data for our work. The
distinction between glucosylated or galactosy-
lated materials is clear from the presence or
absence of highfield resonances between 3.3
and 3.6 ppm, but the determination of the
identity and substitution pattern of the Hex-
NAc-ol is more subtle. A number of ‘struc-
tural reporter groups’ could be defined.
Firstly, the peak shape of the HexNAc-ol H-2
proximately equal (5 Hz), and so, in GalNAc-
ol, where JH2,H3 is small the H-2 resonance
3
appears as a triplet, with barely resolvable
splitting for each peak. In GlcNAc-ol-contain-
3
ing compounds, JH2,H3 is comparable in size
3
3
to JH1a,H2 and JH1b%,H2 and the H-2 resonance
appears as a quartet. In the ManNAc-ol fam-
3
3
ily, the values of the two JH1a,H2 and JH1b%,H2
coupling constants are different, and both dif-
Fig. 1. HPLC separation on a 20 cm 5 mm HyperCarb column of (a) b-Gal-(1“3)-GlcNAc-ol (1), b-Gal-(1“4)-GlcNAc-ol (2)
and b-Gal-(1“6)-GlcNAc-ol (3), and (b) of b-Gal-(1“6)-GlcNAc-ol (3), b-Gal-(1“6)-GalNAc-ol (4) and b-Gal-(1“6)-Man-
NAc-ol (5). The peak indicated by
arises from TSP-d4 present in samples recovered from NMR spectroscopy.

C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
327
mammalian glycoproteins. These include, for
example, b-Galp-(1“3)-GalNAc-ol [13–15],
b-GlcNAc-(1“3)-GalNAc-ol,
b-GalNAc-
(1“3)-GalNAc-ol,
b-GlcNAc-(1“6)-Gal-
NAc-ol and b- -Fuc-(1“6)-GalNAc-ol [13].
L
The anomeric proton chemical shift was re-
ported for b-Galf-(1“4)-GlcNAc-ol [17], and
full assignments for b-Galf-(1“4)[b-Galp-
(1“6)]GlcNAc-ol [1] and these were con-
firmed by Gallo-Rodriguez et al. [18] with
synthetic material.
ManNAc-ol in the T. cruzi mucin.—Analy-
sis of the oligosaccharide alditols obtained
from the GPI-anchored mucin of the proto-
zoan parasite T. cruzi clearly indicated that,
although the major N-acetylhexosaminitol
was GlcNAc-ol, a second HexNAc-ol was also
present, in amounts ranging from 10 to 30%
between samples. Comparison with model
compounds now indicate that this is Man-
NAc-ol rather than GalNAc-ol. It is not clear
whether this arises from ManNAc in the orig-
inal mucin or from rapid epimerisation during
cleavage of the oligosaccharides under basic
condition: this is the subject of ongoing study
and results will be reported elsewhere. The
availability of suitable model compounds and
the identification of structural reporter groups
also demonstrate that the minor component
isolated from T. cruzi Y-strain, originally
identified [2] as a component of a mixture as
b-Galp-(1“3)-GlcNAc-ol, is in reality b-
Galp-(1“4)-ManNAc-ol. Higher homologues
also present therefore are also in this family.
Fig. 2. Peak shapes of the HexNAc-ol H-2 resonance in the
500 MHz NMR spectra of (a) b-Galp-(1“4)-GlcNAc-ol, (b)
b-Glcp-(1“4)-GalNAc-ol, and (c) b-Galp-(1“6)-ManNAc-
ol. In (c), the ManNAc-ol H-2 peak shape is distorted by
second-order coupling to the H-3: this is more severe in the
spectrum of b-Galp-(1“4)-ManNAc-ol, where the ManNAc-
ol H-2 and H-3 are separated by 0.07 ppm.
3
fer from JH2,H3, so the H-2 resonance has
seven lines, with the central line double the
intensity of the flanking lines. This is illus-
trated in Fig. 2.
Structural reporter groups for these compo-
nents.—The chemical shifts of the H-2, H-3,
H-4, H-6a, H-6b% and NAc appear to be char-
acteristic both of the identity and the substitu-
tion pattern of the HexNAc-ol, independent
of whether the substituent is b-Glcp or b-
Galp. These data are summarised in Table 4.
For example, all the ManNAc-ol derivatives
are characterised by highfield H-2 resonance
when 4-substituted, whilst both GlcNAc-ol
and GalNAc-ol have lowfield H-2s. The H-5
of both 4- and 6-substituted GalNAc-ol is
lowfield, in contrast to GlcNAc-ol- and Man-
NAc-ol-containing oligosaccharides. It seems
these rules can be extrapolated to 4,6-disubsti-
tuted systems, when both substituents are in
the pyranose form, but different chemical
shifts for reporter groups apply when the 4-
substituent is a b-Galf residue ([1] and unpub-
lished data).
4. Experimental
Laboratory chemicals were obtained from
Sigma, Aldrich or Fluka, and HPLC grade
MeCN from Fisher Scientific (Loughborough,
UK). b-Glucosidase from almonds, b-galac-
tosidase from E. coli and b-galactosidase from
A. oryzae were obtained from Sigma. Authen-
tic samples of b-Galp-(1“3)-GalNAc, b-
Galp-(1“3)-GlcNAc, b-Galp-(1“4)-GlcNAc
and b-Galp-(1“4)-ManNAc were obtained
from Dextra Laboratories (Reading) and b-
Galp-(1“6)-GlcNAc from Sigma Chemical
Co. (Poole, UK). Disaccharides (1–5 mg)
were reduced with aq NaBH4 (20 mg in 0.5
mL) at r.t. for 6 h, excess NaBH4 destroyed
NMR data reported for related com-
1
pounds.—Full or partial H NMR data have
been reported for a number of related struc-
ture, particularly those related to the oligosac-
charide alditols derived from the various
GalNAc-containing core structures from

Table 4
Structural reporter groups in mono-substituted b-
D
-Hex-(1“X)-
D
-HexNAc-ols and di-substituted
D
-Galp or b- -Galf-(1“4)-(b-
D
D
-Hexp-(1“6)-
D
-HexNAc-ols a
Hexose
HexNAc-ol HexNAc-ol HexNAc-ol HexNAc-ol HexNAc-ol HexNAc-ol HexNAc-ol HexNAc-ol
H-2 H-3 H-4 H-5 H-6 H-6% NAc
b-Glcp-(1“3)- or b-Galp-(1“3)-
b-Glcp-(1“4)- or b-Galp-(1“4)-
b-Glcp-(1“6)- or b-Galp-(1“6)-
GalNAc
GlcNAc b
ManNAc
GalNAc
GlcNAc c
ManNAc
GalNAc d
GlcNAc
4.38
4.26
No compounds available
4.06
4.18
3.50
3.57
4.18
3.85–3.92
3.67
3.88
3.62
3.65
2.043
2.043
4.32–4.33
4.30
4.02–4.03
3.95
3.72–3.73
3.87
4.01
3.95
3.84
3.71–3.81
3.88
3.84
4.00–4.01
4.15
3.61–3.68
3.75
2.071
2.063
2.036
2.066
2.055
/
4.12
4.07
3.88
3.84
4.27
3.87–3.88
3.43–3.45
3.68–3.69
3.60–3.62
3.77
4.12–4.13
3.92
3.79–3.80
3.82–3.84
3.82–3.85
3.69
4.09
3.98
ManNAc e
GlcNAc
4.06
4.15
3.88–3.89
3.93
3.89
3.92
4.06
4.07
4.03
4.04
4.16
3.82
3.98
4.20
4.22
4.15
2.047–2.048
2.056
b-Galf-(1“4)- f
b-Galf-(1“4)- f
ManNAc
3.92
3.80
3.78
3.79
3.95
3.94
3.84
2.033
2.063
2.032
2.057
b-Galp-(1“4)- f, b-Galp-(1“6)- or b-Glcp-(1“6)- GlcNAc
4.33
3.95
328
f
b-Galp-(1“4)- , b-Galp-(1“6)- or b-Glcp-(1“6)- ManNAc
4.12
4.12
n.d.
3.83
b-Galf-(1“4)-; b-Galp-(1“6)- f
b-Galf-(1“4)-; b-Galp-(1“6)- f
GlcNAc
ManNAc
4.17
3.93
)
Compound not available
321
a Figures in bold are for those resonances which are likely to be resolved.
3
b Include data obtained for PO3“6)-a-Glc-(1“6)-b-Glc-(1“3)-b-Galf-(1“3)-b-Glc-(1“3)-GlcNAc-ol isolated from the pneumococcal Type 20 CPS [8].
c Includes data obtained for a-NeuNAc-(2“3)-b-Galp-(1“4)-GlcNAc-ol and a-NeuNAc-(2“3)-b-Galp-(1“4)-GlcNAc-ol.
d Includes data for b-Galp-(1“6)-b-Galp-(1“6)-GalNAc-ol.
e Includes data for b-Galp-(1“6)-b-Galp-(1“6)-ManNAc-ol.
f Data from unpublished work on biological samples, or Refs. [1] or [2].

C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
329
ing 1 or 0.25% aq NH3 were carried out with
b-Galp-(1“3)-GlcNAc.
with 20% aq HOAc, and the monosaccharide
alditols desalted by HPLC on PGC using a
gradient of MeCN between 2 and 15% over
20 min, essentially as described below.
NMR spectroscopy.—NMR spectra were
obtained on a Varian Unity 500 NMR spec-
trometer equipped with a 5 mm triple reso-
nance PFG probe, running VNMR version
5.3 or 6.1B. Samples were deuterium ex-
changed twice before redissolution in D2O
(ca. 250 mL) and introduction into the NMR
tube. Low volume susceptibility-matched
tubes (Shigemi, Tokyo) were used, and spec-
tra were obtained at an indicated probe tem-
perature of 30 °C. Standard pulse sequences
as supplied by Varian were used except for
the introduction of an echo sequence into the
TOCSY sequence, and the use of the Wider
and Wu¨thrich implementation of the HSQC
sequence [20]. Chemical shifts are referenced
against internal TSP-d4 acid at 0 ppm (1H) or
−1.80 ppm (13C) [21].
Preparation of the glucosylated and galacto-
sylated monosaccharide alditols.—The deriva-
tives were prepared by incubating either
GalNAc, GlcNAc or ManNAc (20 mg)
with either Glcb1-OPNP or Galb1-OPNP (10
mg) in 100 mL of 40% 50 mM Na acetate
buffer pH 4.5/60% (EtO)3PO in the presence
of either b-glucosidase (from almonds) or b-
galactosidase (from E. coli or A. oryzae) at
30 °C. After 2–5 h the reaction was stopped
by freezing and aliquots injected onto a
PGC column (‘HyperCarb’, 4.6 mm×10
cm, Life Science International, Basingstoke,).
The column was eluted with a gradient of
MeCN (2–100%) in water, containing 0.05%
TFA over a period of 40 min, and elution
monitored at 206 nm. Disaccharide fractions
were collected and dried, resuspended in
water and reduced with NaBH4 (ca. 5 mg).
Excess NaBH4 was destroyed by the addition
of 20% HOAc until evolution of H2 ceased,
and the reaction mixture evaporated to dry-
ness. Monosaccharide alditols were isolated
by chromatography on a PGC column
(4.6 mm×20 cm), eluted with a gradient of
2–25% MeCN containing 0.05% TFA, over
a period of 30 min. Fractions still impure
were further purified by chromatography on
a 3 mm Phenomosphere NH2 aminopropyl
column (4.6×150 mm, Phenomonex, Mac-
clesfield, UK), using a gradient of 15–40%
3 mM KH2PO4 in MeCN [19], with elution
monitored at 206 nm. UV-absorbing frac-
tions were collected, dried in vacuo and
desalted by chromatography on Hyper-
Carb.
Gas chromatography, GC–MS and methy-
lation analysis.—b-Galp-(1“X)-HexNAc-ol
in Me2SO was methylated by the method of
Ciucanu and Kerek [22] for 40 min with soni-
cation, the reaction diluted with aq Na2S2O3
and permethylated material extracted with
CHCl3. The product was methanolysed (0.5
M HCl in MeOH, 18 h, 80 °C), solvent evap-
orated and the material acetylated with
HOAc/C5H5N for 24 h at 18 °C. The result-
ing mixture of permethylated Gal methyl gly-
cosides and partially methylated, partially
acetylated N-acetylated N-methylhexosamini-
tol was analysed by GC on DB-1 column (25
m×0.2 mm i.d.) using a linear temperature
gradient from 120 to 180 °C (2 °C min−1
,
then hold). Peaks were identified by GC–MS
as previously described [1].
Epimerisation of GlcNAc-containing disac-
Acknowledgements
charides.—b-Galp-(1“3)-GlcNAc
or
b-
Galp-(1“4)-GlcNAc (5 mg, Dextra, Reading
UK) were treated with NaOH (50 mM, 50
mL) for 18 h at r.t. [10], reduced with
NaBH4, desalted on BioRad AG50x8 100–
200 mesh H+ form, and purified as a mixture
by HPLC on PGC. Products were analysed
by NMR and HPLC on PGC using a 2–15%
MeCN gradient. Additional experiments us-
We thank Merck and Co. Inc. (West Point,
PA) for the sample of the pneumococ-
cal Type 20 CPS. This work was supported
by grants from PRONEX, FINEP, CNPq
and PADCT. We are grateful for support for
our collaborative work from the Sir Halley
Stewart Trust. L.M.-P. is a Howard Hughes
International scholar.

330
C. Jones et al. / Carbohydrate Research 328 (2000) 321–330
[11] T. Sugai, A. Kuboki, S. Hiramasu, H. Okazaki, H.
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