Chemical and chemoenzymatic synthesis of glycosyl-amino acids and glycopeptides related to Trypanosoma cruzi mucins

Article abstract of DOI:10.1039/b707772f

This study describes the synthesis of the α- and β-linked N-acetyllactosamine (Galp-β-1,4-GlcNAc; LacNAc) glycosides of threonine (LacNAc-Thr). LacNAc-α-Thr was prepared by direct chemical coupling of a 2-azido-2-deoxy-lactose disaccharide donor to a suitable partially protected threonine unit. In contrast, stepwise chemical generation of β-linked N-acetylglucosamine followed by enzymatic galactosylation to give LacNAc-β-Thr proved effective, whereas use of a 2-azido-2-deoxy-lactose donor in acetonitrile failed to give the desired β-linked disaccharyl glycoside. This study illustrates that it is possible to overcome the inherent stereoselection for 1,2-trans chemical glycosylation with a GlcNAc donor, and that the well-established preference of bovine β-1,4-galactosyltransferase for β-linked acceptor substrates can also be overcome. Using this knowledge, short glycopeptide fragments based on T. cruzi mucin sequences, Thr-Thr-[LacNAcThr]-Thr-Thr-Gly, were synthesised. All LacNAc-based compounds outlined were shown to serve as acceptor substrates for sialylation by T. cruzi trans-sialidase. The Royal Society of Chemistry.

Full text of DOI:10.1039/b707772f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Chemical and chemoenzymatic synthesis of glycosyl-amino acids and
glycopeptides related to Trypanosoma cruzi mucins
Vanessa Leiria Campo,a,b Ivone Carvalho,*b Sarah Allman,c Benjamin G. Davisc and Robert A. Field*a,d
Received 23rd May 2007, Accepted 29th June 2007
First published as an Advance Article on the web 16th July 2007
DOI: 10.1039/b707772f
This study describes the synthesis of the a- and b-linked N-acetyllactosamine (Galp-b-1,4-GlcNAc;
LacNAc) glycosides of threonine (LacNAc-Thr). LacNAc-a-Thr was prepared by direct chemical
coupling of a 2-azido-2-deoxy-lactose disaccharide donor to a suitable partially protected
threonine unit. In contrast, stepwise chemical generation of b-linked N-acetylglucosamine followed by
enzymatic galactosylation to give LacNAc-b-Thr proved effective, whereas use of a
2-azido-2-deoxy-lactose donor in acetonitrile failed to give the desired b-linked disaccharyl glycoside.
This study illustrates that it is possible to overcome the inherent stereoselection for 1,2-trans chemical
glycosylation with a GlcNAc donor, and that the well-established preference of bovine
b-1,4-galactosyltransferase for b-linked acceptor substrates can also be overcome. Using this
knowledge, short glycopeptide fragments based on T. cruzi mucin sequences,
Thr-Thr-[LacNAcThr]-Thr-Thr-Gly, were synthesised. All LacNAc-based compounds outlined were
shown to serve as acceptor substrates for sialylation by T. cruzi trans-sialidase.
but contain few hydrophobic amino acids.9,10 The tightly regulated
Introduction
expression of a large repertoire of mucin structures helps the
Parasitic infections remain a major cause of mortality in the de-
parasite to interact with the infected host on one hand, whilst
veloping world. However, commercial and socioeconomic factors
on the other it also serves to mask the presence of the parasite
mean that very few new drugs reach the clinic.1 Identification
of new leads and drug-able targets would enable the design of
inhibitors of parasite-specific metabolic processes and help in the
search for affordable new medicines.2 Chagasꢀ disease, caused by
from the host immune response.
The O-glycans of T. cruzi mucins resemble those of mam-
malian mucins, although the oligosaccharides are linked to the
threonine or serine of the parasite protein via an a-linked N-
the protozoan parasite Trypanosoma cruzi, is endemic in South
acetyl-glucosamine (GlcNAc) rather than an a-linked N-acetyl-
galactosamine (GalNAc), as in vertebrate mucins.9–11 The a-
GlcNAc is substituted by galactose (Gal) on O-4 and O-6, with
further galactosylation leading to complex branched structures.
In the more infective Y and CL strains of T. cruzi, Gal residues are
only present in the b-pyranose form (b-Galp),12–14 whereas in the
less infective G strain the mucins also contain b-galactofuranose
(b-Galf ).15 The mucin glycans are also significant in being the
major substrate for trans-sialidase which catalyzes transfer of
sialic acid on the parasite cell surface.16 Decoration of cell surface
architecture with this negatively charged sugar is common practice
for microbial pathogens.17 In this case, T. cruzi is unable to
synthesize sialic acid de novo and so uses a cell surface trans-
sialidase enzyme (TcTS)18 to scavenge this monosaccharide from
host glycoconjugates in order to generate a-2,3-linked sialylated-b-
galactopyranose units on the parasite mucins. The similarity19 of
the parasite trans-sialidase to the numerous microbial sialidases
(neuraminidases) that have been investigated suggests potential
for this enzyme as a therapeutic target. In addition, any of the
other enzymes involved in mucin glycan biosynthesis might also
be suitable targets for chemotherapeutic intervention: a-GlcNAc
transferase activity has been characterised in T. cruzi microsomal
membranes20 and b-Galp transferase activities have been detected
in Golgi preparations.21,22
and Central America, Mexico and the southern United States. It
has been classified after malaria and schistosomiasis as one of
the most serious parasite diseases in tropical regions: there are
currently 18 million people infected and a further 100 million,
mainly in Latin America, are at risk of infection. Typically 2–3
million individuals show clinical symptoms of the chronic phase
of the disease at any one time, and 45000 die every year.3 There is
a clear need for new therapeutics.4–6
Recent sequencing shows that over 50% of the genome of
Trypanosoma cruzi (strain CL Brener) is composed of repeated
sequences.7 These sequences included genes for large families
of surface molecules, including trans-sialidases, mucins, gp63
proteases and novel mucin-associated surface proteins. As with
many other pathogenic microorganisms, the nature and extent
of cell surface glycosylation can have a profound impact on cell
surface protein structure and biological function.8 T. cruzi mucins
are highly glycosylated (about 60% carbohydrate by weight),
polyanionic glycoproteins that are rich in Thr, Ser and Pro residues,
aSchool of Chemical Sciences and Pharmacy, University of East Anglia,
Norwich, UK NR4 7TJ
bFaculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de
Sa˜o Paulo, Ribeira˜o Preto 14040 930, Brazil. E-mail: carronal@usp.br
cDepartment of Chemistry, University of Oxford, Chemistry Research
Laboratory, Mansfield Road, Oxford, UK OX1 3TA
Given the heterogeneity of T. cruzi mucins, there are no suitable
natural sources of homogeneous mucin glycopeptides with which
to investigate mucin biosynthesis. The purpose of the study
dDepartment of Biological Chemistry, John Innes Centre, Colney Lane,
Norwich, UK NR4 7UH. E-mail: rob.field@bbsrc.ac.uk
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reported herein was to synthesise simple glycosylated amino acid
and glycopeptide acceptor substrate analogues (Fig. 1) with which
to study T. cruzi mucin glycosyltransferase and trans-sialidase
activities.20–22 In the latter case, we set out to complement the
work of de Lederkremer and co-workers, who have synthesised
and assessed various glycans from T. cruzi G strain mucins, with
the preparation of fragments of the mucin glycopeptides found in
the pathogenic T. cruzi Y strain.23–25
amino acids and galactosylated versions thereof.26 Here we
report chemical and chemoenzymatic syntheses of GlcNAc-
a-Thr (1), GlcNAc-b-Thr (3) and the corresponding a- and
b-linked N-acetyllactosamine (Galp-b-1,4-GlcNAc; LacNAc)
derivatives, (2) and (4) respectively, along with mucin-related N-
acetyllactosamine-based glycopeptides, (5) and (6) (Fig. 2). Each
of these structures was assessed for their ability to be glycosylated
by recombinant T. cruzi trans-sialidase.
Results and discussion
In the context of the preparation of LacNAc-a-Thr (2) and
diastereomeric LacNAc-b-Thr (4), linear glycosylation disconnec-
tions require the sequential glycosylation of suitably protected
threonine with glucosamine and then galactose units, or a conver-
gent approach would utilize direct introduction of an intact lac-
tosamine disaccharide. Both strategies allow scope for enzymatic
glycosylation steps, particularly the use of readily available, and
well studied, bovine b-1,4-galactosyltransferase (b-1,4-GalT)27 to
effect the sequential galactosylation. Hence, this study exploited
chemical methods for the synthesis of monosaccharyl-amino
acids (1) and (3), and compared chemical and chemoenzymatic
approaches to prepare disaccharyl-amino acids (2) and (4). Results
from these studies informed the choice of methods for preparation
of glycopeptides (5) and (6).
Fig. 1 (a) Structure of a T. cruzi CL/Y strain mucin glycopeptide
fragment, and (b) the corresponding mucin tandem repeat peptide
sequence.
Chemical synthesis of glyco-amino acids GlcNAc-a-Thr (1) and
GlcNAc-b-Thr (3)
Although it is counter intuitive to use a glycosyl donor possessing
a C-2 participating group (in this case an acetamide) when
attempting to synthesise a 1,2-cis-linked glycoside, 3,4,6-tri-O-
acetyl-2-acetamido-2-deoxy-a-D-glucopyranosyl chloride (7) is
We previously reported the a-selective iodine-promoted gly-
cosylation of serine and threonine (Thr) with 2-azido-2-deoxy-
glycosyl iodides, but sought a shorter route to a-linked GlcNAc-
Fig. 2 (a) Structure of target a-linked glyco-amino acid fragments and the corresponding b-linked controls; (b) target mucin-related glycopeptides,
prospective trans-sialidase substrates.
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a shelf-stable crystalline solid that is readily accessible on a
gram scale from cheap glucosamine.28,29 We previously reported
the glycosylation of suitably protected serine and threonine
derivatives with glycosyl chloride (7) in the presence of mercuric
bromide, isolating the expected b-linked glyco-amino acids in
respectable yield (∼60–65%).29 Whilst these were practical yields
for preparative purposes, it raised the question of mass balance:
what happened to the remaining 35–40% of the material? On
closer inspection, we were able to identify quantities of the
unexpected a-linked glycoside from these reactions. On tweaking
the reaction conditions (reaction at room temperature for 10 h,
followed by reflux for 9 h, in contrast to just refluxing the
reaction mixture for 9 h), glycosylation of N-Fmoc-Thr benzyl
ester (8) with glycosyl chloride (7) in 1,2-dichloroethane in the
presence of HgBr2 gave the expected b-glycoside (10)29 in 52%
yield, accompanied by the corresponding a-linked isomer (9)
in a low but reproducible 20% yield.† This method provided a
straightforward and direct approach to the a-linked GlcNAc-
Thr system that, given that there is no need to manipulate C-
2 functionality, is competitive with other approaches to such
a-linked 2-amino-2-deoxy-glucosides.30,31 Removal of the benzyl
ester and Fmoc groups from protected glyco-amino acids (9)
and (10) was performed by standard hydrogenation (10% Pd-
C/H2); subsequent removal of the acetate groups was realised
in the presence of 1 M NaOMe in MeOH. In this way, 20–30 mg
quantities of the completely deprotected glyco-amino acids (1) and
(3) were obtained (Scheme 1).
Preparation of disaccharide glycosides LacNAc-a-Thr (2) and
LacNAc-b-Thr (4)
The chemical synthesis of the protected a-linked disaccharyl
amino acid (12) was achieved by convergent glycosylation of N-
Fmoc-Thr benzyl ester (8) with known a-linked glycosyl chloride
(11)32,33 under the same conditions described for synthesis of (9)
and (10) (Scheme 2).29 The only isolable glycosyl amino acid
product obtained was the a-isomer, aLacN3-FmocThrOBn (12), in
† The a-glycoside appears to be formed directly, not through equilibration
of the b-glycoside. Addition of pure b-glycoside to the reaction mixture
did not result in additional a-glycoside formation.
Scheme 1 Reagents and conditions: (a) HgBr2, ClCH2CH2Cl, rt 10 h then reflux 9 h; (b) i. H2-Pd/C, MeOH–AcOH (10 : 1), 10 h, ii. NaOMe, MeOH.
Scheme 2 Reagents and conditions: (a) HgBr2, ClCH2CH2Cl, rt 10 h then reflux 9 h; (b) Zn powder, AcOH, Ac2O, THF; (c) i. H2-Pd/C, MeOH–AcOH
(10 : 1), 10 h, ii. NaOMe, MeOH.
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60% yield.‡ The configuration of the newly-formed sugar-amino
acid glycosidic centre was confirmed by 1H NMR spectroscopy (d
4.50 ppm, d, J1,2 3.7 Hz, H-1). Subsequently, the sugar azido group
in (12) was reductively acetylated using zinc powder in THF–acetic
anhydride–acetic acid,34 affording a-LacNAc-FmocThrOBn (13)
in 65% yield. Complete deprotection, giving a-linked disaccharyl
amino acid glycoside (2) in 90% yield, was realized by standard
hydrogenation (to remove the O-Bn and N-Fmoc groups) followed
by Zemplen deacetylation.
In an attempt to obtain the b-linked isomer of protected
LacNAc-a-Thr (13) whilst limiting the number of building blocks
in use, the glycosylation of (8) and (11) was performed in the
presence of the participating solvent acetonitrile.35–37,§ However,
repeated attempts at this reaction gave, at best, only poor yields
on the unwanted a-glycoside (12) (up to 10% yield), none of the
desired b-glycoside and much hemiacetal. Hence we resorted to a
chemo-enzymatic approach.
performed on a 5 mg acceptor scale using commercial UDP-
galactose as the donor substrate in the presence of bovine b-
1,4-GalT (0.5 U) and alkaline phosphatase, the latter to remove
potentially inhibitory UDP (Scheme 3). In relation to the b-linked
acceptor (3), complete conversion to the desired galactosylated
product, LacNAc-b-Thr (4), was observed after 24 h reaction.
In contrast, for a-linked acceptor (1), conversion to LacNAc-a-
Thr (2) is much slower and product was not observable at this
time point. This result shows the substantially lower reactivity
of the b-1,4-GalT enzyme towards the a-linked acceptor. Hence,
enzymatic galactosylation of (1) was repeated with high b-1,4-
GalT concentration (2 U) for longer (96 h), at which point TLC
indicated that conversion to galactoside (2) had progressed to
>60%. The disaccharyl amino acids (2) and (4) were subsequently
isolated by gel filtration chromatography in 57% and 85%
yields, respectively, and their structures were confirmed by NMR
1
spectroscopy and mass spectrometry. H NMR spectra showed
Whilst its natural preference is for glycosylation of b-linked
acceptor substrates,27 the relatively loose acceptor substrate
tolerance of bovine b-1,4-galactosyltransferase (b-1,4-GalT) has
previously been exploited in the 4-O-galactosylation of a-
linked acceptors, such as maltose (Glc-a-1,4-Glc)39 and uridine-
diphospho-N-acetylglucosamine (GlcNAc-a-UDP).40 We were
therefore prompted to consider the enzymatic galactosylation of
the glycosylated amino acids (1) and (2). These reactions was
doublets at d 4.77 ppm (J1,2 3.8 Hz) or 4.39 ppm (J1,2 8.1 Hz),
corresponding to GlcNAc H-1 of (2) and (4), respectively. ESI-
MS analysis showed characteristic adducts for the a-glycoside (2)
(m/z [M + Na]+ 507.17) and the corresponding b isomer (4) (m/z
[M + H]+ 485.19).
Solid-phase and chemo-enzymatic glycopeptide synthesis
Moving towards more complex mimetics of the mucin glycopep-
tides present on the T. cruzi parasite cell surface we next em-
ployed solid-phase glycopeptide synthesis, coupled with enzymatic
glycosylation. Commercial Wang resin pre-loaded with Fmoc-
Gly (0.65 mmol/g resin) was employed as the solid support
for the synthesis of glycopeptides (5) and (6). Syntheses were
performed manually in a fritted glass vessel fitted with a three-
way tap so that argon purging could be used to effect mixing.41
‡ Paulsen and Holck described the use of the corresponding b-linked glyco-
syl chloride donor, which gave excellent yields (>80%) of a-glycosyl amino
acid as the sole glycoside product. Clearly the anomeric configuration
of the glycosyl chloride plays little role in determining the stereochemical
outcome of the glycosylation reaction with these particular sugar donors.33
§ Recent studies exploiting acetonitrile to influence glycosylation stereo-
chemistry show that use in limited quantity can significantly outperform
its use as a solvent.38
Scheme 3 Reagents and conditions: (a) bovine b-1,4-GalT (2 U), UDP-galactose, alkaline phosphatase, 30 ◦C, 96 h; (b) bovine b-1,4-GalT (0.5 U),
UDP-galactose, alkaline phosphatase, 30 ◦C, 24 h.
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After removal of N-Fmoc groups with 20% piperidine in DMF,
the amino acid building blocks were coupled in DMF in the
presence of the coupling reagents PyBOP–HOBt and N,N-
diisopropylethylamine (DIPEA). The progress of amide coupling
was monitored spectrophotometrically, based on quantitation of
dibenzofulvene released (with piperidine) from a dried sample of
resin-bound (glyco)peptide. Optimal coupling times ranged from
3 h to 24 h for the incorporation of Fmoc-Thr, depending on
the preceding peptide sequence. For incorporation of the more
sterically and electronically demanding glycosylated amino acids
(14) (Scheme 4) and (15) (Scheme 5) the coupling times typically
ranged from 24 h to 72 h.
careful hydrogenation of benzyl ester (13) without competing
removal of the Fmoc group. Introduction of two further threonine
residues completed preparation of the protected, resin-bound gly-
copeptide (Scheme 4). The peptide coupling efficiencies obtained,
as judged by measuring released dibenzofulvene from the product,
varied with the position in the peptide chain: Thr1 (90%), Thr2
(90%), LacNAc-a-Thr3 (54%), Thr4 (55%) and Thr5 (80%).
Cleavage from the resin with aqueous TFA, concentration in
vacuo and trituration with cold ether gave crude glycopeptide. Fi-
nally, removal of the sugar acetate protecting groups with catalytic
NaOMe in MeOH and purification by reverse-phase HPLC gave
glycopeptide (5) in 22% overall yield. The structure of compound
(5) was confirmed by NMR spectroscopy [characteristic 1H signals
for sugar H-1 at d 4.71 ppm (J1,2 3.7 Hz), 1 × NHAc and
5 × CH3Thr units] and ESI-MS analysis (observed [M + H]+
946.4).
Synthesis of the glycopeptide
H2N-(Thr)2-(LacNAc-a-Thr)-(Thr)2-Gly-OH (5)
The synthesis of a-LacNAc-based glycopeptide (5) was initiated
by cleavage of the Fmoc group from Fmoc-Gly-Wang resin,
followed by two rounds of coupling–deprotection with Fmoc-Thr.
Subsequent coupling employed glyco-amino acid building block
aLacNAc-FmocThrOH (14), which was successfully prepared by
Chemoenzymatic synthesis of
H2N-(Thr)2-(LacNAc-b-Thr)-(Thr)2-Gly-OH (6)
Following on from our observations on chemical and chemoen-
zymatic approaches to LacNAc-b-Thr (4), a chemoenzymatic
Scheme 4 Reagents and conditions: (a) H2–Pd/C, MeOH–AcOH (10 : 1), 1 h; (b) 20% piperidine in DMF; (c) Fmoc-Thr, PyBOP, HOBt, DIPEA;
(d) Glyco-amino acid 14, PyBOP, HOBt, DIPEA; (e) TFA; (f) NaOMe, MeOH.
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Scheme 5 Reagents and conditions: (a) H2–Pd/C, MeOH–AcOH (10 : 1), 1 h; (b) 20% piperidine in DMF; (c) Fmoc-Thr, PyBOP, HOBt, DIPEA; (d)
Glyco-amino acid 15, PyBOP, HOBt, DIPEA; (e) TFA; (f) NaOMe, MeOH; (g) bovine b-1,4-GalT, UDP-galactose, alkaline phosphatase, 30 ◦C, 24 h.
approach to glycopeptide (6) was explored. That is, GlcNAc-b-
Thr-containing glycopeptide (16) was prepared first in a similar
manner to glycopeptide (2). The peptide coupling efficiencies ob-
tained, as judged by measuring released dibenzofulvene from the
product, varied with the position in the peptide chain: Thr1 (90%),
Thr2 (90%), GlcNAc-b-Thr3 (61%), Thr4 (58%) and Thr5 (85%).
Cleavage from resin with aqueous TFA, concentration in vacuo and
trituration with cold ether gave crude glycopeptide. Removal of the
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sugar acetate protecting groups with catalytic NaOMe in MeOH
and purification by reverse-phase HPLC gave the glycopeptide
(16) in 14% overall yield. The structure of compound (16) was
Conclusion
In summary, we have developed chemical and chemoenzymatic
routes to glyco-amino acids and glycopeptides related to T.
cruzi mucin structures. The early phase of the study highlights
the difficulty in predicting the outcome of both chemical and
enzymatic glycosylation reactions, particularly in relation to
stereochemistry. The latter part of the study shows that T. cruzi
trans-sialidase has relaxed acceptor substrate specificity, acting
efficiently on both its natural LacNAc-a-Thr-based substrates but
also on the diastereomeric b-linked compounds.
1
confirmed by NMR spectroscopy [characteristic H signals for
sugar H-1 at d 4.41 ppm (J1,2 8.1 Hz), 1 × NHAc and 5 × CH3Thr
units] and ESI-MS analysis (observed [M + H]+ 847.1).
Subsequent solution-phase enzymatic b-galactosylation of
GlcNAc-glycopeptide (16) with bovine b-1,4-GalT and UDP-
galactose gave the crude LacNAc-glycopeptide which, following
purification by Toyopearl HW-40S gel filtration chromatogra-
phy with 0.1% aqueous TFA, gave glycopeptide (6) in 80%
yield from the corresponding b-GlcNAc-based glycopeptide (16).
The structure of compound (6) was confirmed by NMR spec-
Experimental
1
troscopy [characteristic H signals for sugar H-1ꢀ at d 4.43 ppm
(J1,2 7.5 Hz)] and ESI-MS analysis (observed [M + Na]+
General
968.4).
All chemicals were purchased as reagent grade and used without
further purification. Solvents were dried according to standard
methods.47 Reagents for glycopeptide synthesis, including FmocG-
lyWang resin (loading 0.65 mmol/g), FmocThrOH and PyBOP,
were purchased from Novabiochem. HOBt was purchased from
Acros. Bovine b-1,4-galactosyltransferase (EC 2.4.1.90), UDP-
galactose and calf intestinal alkaline phosphatase (EC 3.1.3.1)
were purchased from Sigma Chemical Co. Fetuin (calf serum) was
purchased from Sigma Chemical Co or Quest Biomedical.
Reactions were monitored by thin layer chromatography (TLC)
on 0.25 nm precoated silica gel plates (Whatman, AL SIL G/UV,
aluminium backing) with the indicated eluents. Compounds were
visualized under UV light (k = 254 nm) and/or dipping in ethanol–
sulfuric acid (95 : 5, v/v) or orcinol (2%) in aqueous sulfuric acid
(10%), followed by heating the plate for a few minutes. Column
chromatography was performed on silica gel 60 (Fluorochem, 35–
70 mesh) or on a Biotage Horizon High-Performance FLASH
Chromatography system using 12 mm or 25mm flash cartridges
with the eluents indicated.
Nuclear magnetic resonance spectra were recorded on a Varian
Gemini spectrometer. 1H NMR spectra recorded at 400 MHz were
referenced to dH 7.27 for CDCl3, dH 3.35 for CD3OD, dH 4.63 ppm
for D2O, and 13C NMR spectra recorded at 100 MHz were
referenced to dC 77.0 for CDCl3 and dC 49.15 for CD3OD. Chemical
shifts of NMR signals recorded in D2O are reported with respect
to the methyl resonance of internal acetone at dH 2.22 ppm and
dC 30.89 ppm. Assignments were made with the aid of HSQC
and COSY experiments. Optical rotations were measured at
ambient temperature on a Perkin-Elmer model 141 polarimeter
using a sodium lamp. Accurate mass electrospray ionization mass
spectra (ESI-MS) were obtained from the EPSRC National Mass
Spectrometry Service Centre, Swansea using positive ionization
mode on a Finningan MAT 900 XLT mass spectrometer or from
the John Innes Centre metabolite analysis service on a Thermo
Finningan DecaXPplus mass spectrometer.
Enzymatic sialylation of glycopeptides with Trypanosoma cruzi
trans-sialidase (TcTS)
Each of the LacNAc-containing glyco-amino acids (2 and 4) and
glycopeptides (5 and 6) prepared in this study were subjected to the
action of recombinant Trypanosoma cruzi trans-sialidase (TcTS)
in the presence of a sialic acid donor substrate. In contrast to
the use of sialidases, which require long incubation times and/or
excess of donor substrate,42 trans-glycosylation with TcTS takes
place with equimolar amounts of substrates and much shorter
reaction times.18,43 It is therefore a very useful biocatalyst for
this difficult glycosylation. Although a variety of TcTS sialyl
donor substrates have been reported in the literature,43–45 including
sialic acid para-nitrophenyl and methylumbelliferyl glycosides and
a-2,3-sialyl-lactose, amongst several others, the TcTS transfer
activity depends strongly on reaction conditions, including pH,
temperature and time. Each of the a-sialosides has its own
problems, with cost, rate of reaction and complications with by-
product separation limiting utility. Considering the disadvantages
of the cited donors, and following the work of Kim et al.,46
the glycoprotein fetuin from calf serum was chosen as a sialic
acid donor for the experiments reported in this study. This
glycoprotein contains ∼8% a-2,3-linked sialic acid by weight
and the glycoprotein by-product of the sialyl transfer reaction
is easily removed by precipitation with ethanol or with the aid
of a 10 kDa cut-off spin filter.46 It therefore provides a highly
competitive source of sialic acid in terms of availability and ease
of use.
All of the LacNAc glycosides synthesized in this study (2, 4,
5 and 6) proved to be effective acceptor substrates for TcTS,
giving complete conversion to the corresponding a-2,3-sialylated
structures in the presence of a three fold excess of fetuin. Careful
monitoring proved essential as in all cases prolonged incubation
resulted in hydrolysis of the sialylated products, regenerating the
LacNAc acceptors. From milligram scale reactions, the sialylated
products (17–20) were readily isolated by removal of protein
using a spin filter, followed by gel filtration (Toyopearl HW-
40S column) in 5 mM NH4HCO3 buffer. Using this approach,
the sialylated products were obtained in >80% isolated yield.
NMR spectroscopy and mass spectrometry confirmed the product
structures (Table 1).
N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-deoxy-3,4,6-tri-
O-acetyl-a-D-glucopyranosyl)-L-threonine benzyl ester (9) and
N-(fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-deoxy-3,4,6-tri-
O-acetyl-b-D-glucopyranosyl)-L-threonine benzyl ester (10)29
A
mixture of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
glucopyranosyl chloride (7)29 (400 mg, 1.09 mmol) and
N-(fluoren-9-ylmethoxycarbonyl)-L-threonine benzyl ester (8)
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Table 1 Data for TcTS-catalyzed sialylation of LacNAc-based acceptors
LacNAc
acceptor
Glyco-amino
acid linkage
Isolated
yield
Sialylated product
Characteristic 1H NMR data (D2O)
ESI-HRMS data
(2)
(4)
(5)
a
b
a
Neu5Ac-a-2,3-LacNAc-
a-Thr (17)
92%
90%
85%
d 4.77 (1H, d, J1,2 = 3.7 Hz, H-1 GlcNAc), 4.30
(1H, d, J1,2 = 7.8 Hz, H-1 Gal), 4.28 (1H, m,
bCHThr), 2.60 (1H, dd, J3eq,3ax = 12.0 Hz, J3eq,4
Calcd. for
C29H48N3O20Na+ [M +
Na]+: 798.2756, found:
798.2756.
=
4.4 Hz, H-3eq Neu5Ac), 1.92, 1.90 (6H, 2 × s, 2 ×
NHAc), 1.60 (1H, t, J3eq,3ax = J3ax,4 12.0 Hz, H-3ax
Neu5Ac), 1.24 (3H, d, J = 6.5 Hz, CH3 Thr)
d 4.42 (1H, d, J1,2 = 8.1 Hz, H-1 GlcNAc), 4.36
(1H, d, J1,2 = 7.8 Hz, H-1 Gal), 4.20 (1H, m,
Neu5Ac-a-2,3-LacNAc-
b-Thr (18)
Calcd. for
C29H48N3O20Na+ [M +
Na]+: 798.2756, found:
798.2756.
bCHThr), 2.60 (1H, dd, J3eq,3ax = 12.1 Hz, J3eq,4
=
4.4 Hz, H-3eq Neu5Ac), 1.92, 1.90 (6H, 2s,
NHAc), 1.60 (1H, t, J3eq,3ax = J3ax,4 12.0 Hz, H-3ax
Neu5Ac), 1.24 (3H, d, J = 6.5 Hz, CH3 Thr).
d 4.68 (1H, d, J1,2 = 3.7 Hz, H-1 GlcNAc), 4.36
(1H, d, J1,2 = 8.1 Hz, H-1 Gal), 4.40–3.80 (10H,
H2N-(Thr)2-[Neu5Ac-a-
2,3-LacNAc-a-Thr]-
(Thr)2-Gly-OH (19)
Calcd. for C47H80N8O30
[M + H]+: 1237.38,
found: 1237.38.
m, aCHThr, bCHThr), 2.62 (1H, dd, J3eq,3ax
=
12.0 Hz, J3eq,4 = 4.4 Hz, H-3eq Neu5Ac), 1.92, 1.90
(6H, 2 × s, 2 × NHAc), 1.65 (1H, t, J3eq,3ax
=
J3ax,4 = 12.0 Hz, H-3ax Neu5Ac), 1.20–1.00 (15H,
m, 5 × CH3 Thr)
(6)
b
H2N-(Thr)2-[Neu5Ac-a-
2,3-LacNAc-b-Thr]-
(Thr)2-Gly-OH (20)
82%
d 4.42 (1H, d, J1,2 = 8.1 Hz, H-1 GlcNAc), 4.35
(1H, d, J1,2 = 7.8 Hz, H-1 Gal), 4.40–3.90 (10H,
Calcd. for C47H80N8O30
[M + H]+: 1237.39,
found: 1237.39.
m, aCHThr, bCHThr), 2.62 (1H, dd, J3eq,3ax
=
12.1 Hz, J3eq,4 = 4.4 Hz, H-3eq Neu5Ac), 1.93, 1.90
(6H, 2 × s, 2 × NHAc), 1.65 (1H, t, J3eq,3ax
=
J3ax,4 = 12.0 Hz, H-3ax Neu5Ac), 1.20–1.00 (15H,
m, 5 × CH3 Thr)
(235 mg, 0.54 mmol) (prepared from commercially available
amino acid Fmoc-threonine by treatment with caesium carbonate
and benzyl bromide in DMF)48 in 1,2-dichloroethane (4 cm3)
was stirred for 10 h at room temperature with mercuric bromide
(430 mg, 1.17 mmol) and then refluxed for 9 h,29 at which point
TLC (hexane–EtOAc, 3 : 7 v/v) showed the disappearance of the
donor (7). The resulting amber mixture was concentrated in vacuo
and the residue was purified by silica gel column chromatography
(eluent: hexane–EtOAc, 3 : 7 v/v). The a-glycoside (9) eluted
first, followed by the b-glycoside (10): both were obtained as
amorphous solids.
125.3, 120.2 (CH Ph), 99.5 (C-1), 77.6 (bCHThr), 71.4 (C-3), 68.5,
68.4 (C-5, C-4), 68.0 (OCH2Ph), 67.7 (CH2 Fmoc), 62.2 (C-6),
58.7 (aCHThr), 51.8 (C-2), 47.3 (CH Fmoc), 23.3, 21.0, 20.9, 20.8
(COCH3), 18.5 (CH3 Thr). ESI-HRMS: calcd. for C40H45N2O13.
[M + H]+: 761.2916, found 761.2918.
b-Glycoside (10). (200 mg, 52%); Rf 0.26 [EtOAc–Hex (7 : 3)];
[a]D −15.6 (c 1.0, CHCl3); dH (CDCl3) 7.76 (2H, d, J = 7.6 Hz,
CH Fmoc Ph), 7.64 (2H, d, J = 7.8 Hz, CH Fmoc Ph), 7.41–7.26
(9H, m, Fmoc Ph, OCH2Ph), 5.83 (1H, d, J = 9.1 Hz, NHThr),
5.54 (1H, d, J = 8.4 Hz, NHAc), 5.25 (1H, t, J2,3 = 9.9 Hz, J3,4
=
9.9 Hz, H-3), 5.21, 5.14 (2H, AB, JAB = 12.2 Hz, OCH2Ph), 5.00
(1H, t, J3,4, J4,5 = 9.9 Hz, H-4), 4.65 (1H, d, J1,2 = 8.1 Hz, H-1),
4.47–4.40 (3H, m, CH2 Fmoc, bCHThr), 4.35 (1H, dd, J = 7.3 Hz,
J = 10.7 Hz, aCHThr), 4.24 (1H, t, J = 7.3 Hz, CHFmoc), 4.19
a-Glycoside (9). (77 mg, 20%); Rf 0.31 [EtOAc–Hex (7 : 3)];
25
[a]D +30.4 (c 1.0 CHCl3); dH (CDCl3) 7.77 (2 H, d, J = 7.5 Hz,
CH Fmoc Ph), 7.64 (2 H, d, J = 6.0 Hz, CH Fmoc Ph), 7.49–
7.26 (9H, m, Fmoc Ph, OCH2Ph), 5.94 (1 H, d, J = 9.3 Hz,
NHAc), 5.73 (1 H, d, J = 9.5 Hz, NHThr), 5.20–5.14 (1 H, m,
H-3), 5.19, 5.09 (2 H, AB, JAB = 12.2 Hz, OCH2Ph), 5.08 (1 H,
t, J3,4 = 9.4 Hz, J4,5 = 9.4 Hz, H-4), 4.70 (1 H, d, J1,2 = 3.6 Hz,
H-1), 4.55–4.44 (3 H, m, CH2 Fmoc, aCHThr), 4.31–4.07 (5 H,
m, bCHThr, CHFmoc, H-2, H-6, H-6ꢀ), 4.01–3.97 (1 H, m, H-5),
2.06, 2.04, 2.03, 1.96 (12H, 4s, COCH3), 1.28 (3 H, d, J = 6.2,
CH3 Thr); dC (CDCl3) 171.5, 170.9, 170.8, 170.6 (COCH3), 169.5
(COCH2Ph), 156.7 (CO Fmoc), 144.0, 143.8, 141.5 (Cquat Fmoc
Ph), 134.5 (Cquat. OCH2Ph), 129.2, 129.1, 128.8, 128.0, 127.3,
ꢀ
ꢀ
,
(1 H, dd, J5,6 = 4.4 Hz, J6,6 = 12.3 Hz, H-6), 4.02 (1H, dd, J6,6
J5,6 = 2.2 Hz, H-6ꢀ), 3.67 (1H, dd, J1,2, J2,3, H-2), 3.51–3.47 (1H,
m, H-5), 2.03, 2.02, 1.99, 1.93 (12H, s, COCH3), 1.20 (3H, d,
J = 6.3 Hz, CH3 Thr); dC (CDCl3) 170.9, 170.6, 170.4, 170.0
(COCH3), 169.4 (COCH2Ph), 156.8 (CO Fmoc), 144.0, 141.3
(Cquat. Fmoc Ph), 135.5 (Cquat. OCH2Ph), 128.6, 128.5, 128.3,
127.7, 127.1, 125.5, 119.9 (CH Ph), 98.5 (C-1), 74.5 (bCHThr),
71.9, 71.7 (C-5, C-3), 68.5 (C-4), 67.3 (OCH2Ph, CH2 Fmoc),
61.9 (C-6), 58.7 (aCHThr), 55.3 (C-2), 47.3 (CH Fmoc), 23.3,
20.7, 20.5, 20.6 (COCH3), 17.0 (CH3 Thr). ESI-HRMS: calcd.
ꢀ
2652 | Org. Biomol. Chem., 2007, 5, 2645–2657
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+
For C40H44N2O13NH4 [M + NH4 ]: 778.3187, found: 778.3187. The
(CDCl3) 7.77 (2 H, d, J = 7.5 Hz, CH Fmoc Ph), 7.64 (2 H, d, J =
6.0 Hz, CH Fmoc Ph), 7.49–7.26 (9H, m, Fmoc Ph, OCH2Ph),
physical and analytical data for compound (10) were in accordance
with the literature.29
5.79 (1 H, d, J 9.4 Hz, NH), 5.39 (1 H, dd, J3 ,4 = 3.4 Hz, J4 ,5
=
ꢀ
ꢀ
ꢀ ꢀ
0.9 Hz, H-4ꢀ), 5.37 (1 H, dd, J1 ,2 = 7.8 Hz, J2 ,3 = 10.4 Hz, H-2ꢀ),
ꢀ
ꢀ
ꢀ ꢀ
5.30 (2 H, AB, JAB = 12.2 Hz, OCH2Ph), 5.13 (1 H, dd, J2,3
=
2-Acetamido-2-deoxy-a-D-glucopyranosyl-L-threonine (1)
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
10.5 Hz, J3,4 = 9.8 Hz, H-3), 4.96 (1 H, dd, J2 ,3 , J3 ,4 , H-3 ), 4.80
ꢀ
ꢀ
ꢀ
A solution of N-(fluoren-9-ylmethoxycarbonyl)-(2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)-L-threonine ben-
zyl ester (9) (76 mg, 0.09 mmol) in MeOH (3 cm3) was treated
with glacial AcOH (0.3 cm3) and 10% Pd/C (40 mg) for removal
of the O-Bn and N-Fmoc groups. The reaction mixture was stirred
and kept under H2 (∼1.5 atm) for 8 h. The reaction mixture was
then filtered through Celite, concentrated in vacuo and purified by
column chromatography (DCM–MeOH 9 : 1 v/v). The product
obtained was then dissolved in MeOH (1.5 cm3) and made basic
with 1 M NaOMe in MeOH. The reaction mixture was stirred for
2 h, and then neutralized with Dowex 50WX8–200 resin. Filtration
and concentration of the reaction mixture gave the product (1)
(1 H, d, J1,2 = 3.7 Hz, H-1), 4.47 (1 H, d, J1 ,2 , H-1 ), 4.40 (3 H,
m, aCHThr, bCHThr, H-6b), 4.31 (1 H, t, J = 7.3 Hz, CHFmoc),
4.26 (1 H, dd, J6 a,6 b = 11.0 Hz, J5 ,6 b = 5.9 Hz, H-6ꢀb), 4.21 (1
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
H, dd, J6a,6b = 12.0 Hz, J5,6a = 5.3 Hz, H-6a), 4.13 (1 H, J6 a,6 b
=
11.0 Hz, J5 ,6 a = 7.4 Hz, H-6ꢀa), 3.99 (1 H, ddd, J4, 5 = 10.0 Hz,
ꢀ
ꢀ
J
5,6a, J5,6b = 1.9 Hz, H-5), 3.97 (2 H, m, CH2 Fmoc), 3.85 (1 H,
dd, J5 ,6 a, J5 ,6 b, H-5ꢀ), 3.66 (1 H, dd, J3,4, J4, 5 = 9.5 Hz, H-4), 3.09
(1 H, dd, J1,2, J2,3, H-2), 2.16–1.96 (18 H, 6 s, 6 CH3CO), 1.33 (3
H, d, J = 6.5 Hz, CH3 Thr); dC (CDCl3) 170.5–169.3 (COCH3,
COCH2Ph), 157.1 (CO Fmoc), 144.0, 141.2 (Cquat. Fmoc Ph),
135.1 (Cquat. OCH2Ph), 128.8, 127.9, 127.3, 125.5, 120.1 (CH Ph),
101.3 (C-1ꢀ), 99.2 (C-1), 76.6 (C-4), 71.2 (C-3ꢀ), 70.9 (C-5ꢀ), 70.1 (C-
3), 69.3 (C-2ꢀ), 69.1 (C-5), 68.0 (C-4ꢀ), 66.7 (CH2 Fmoc, OCH2Ph),
62.1 (C-6), 61.6 (C-6ꢀ), 60.8 (aCHThr, bCHThr), 59.1 (C-2), 47.3
(CH Fmoc), 21.2–20.8 (COCH3), 18.9 (CH3 Thr). ESI-HRMS:
ꢀ
ꢀ
ꢀ ꢀ
25
(28 mg, 86%) as colourless amorphous solid. [a]D +61.0 (c 0.2,
CH3OH); dH (D2O), 4.80 (1H, J1,2 = 3.7 Hz, H-1), 4.40 (1H, m,
bCH Thr), 3.82 (1H, m, aCH Thr), 3.72–3.65 (2H, m, H-2, H-6a),
3.62–3.48 (3H, m, H-3, H-5, H-6b), 3.33 (1 H, t, J3,4 = 10.1 Hz,
J4,5 = 10.1 Hz, H-4), 1.85 (3H, s, NHAc), 1.21 (3H, d, J 6.5 Hz,
CH3 Thr). dC (CD3OD, 100 MHz) 99.3 (C-1), 73.2 (bCHThr),
73.0 (C-5), 70.8 (C-4), 69.0 (C-3), 61.0 (C-6), 56.8 (aCHThr), 53.0
(C-2), 21.3 (NCOCH3), 17.3 (CH3 Thr). ESI-HRMS: calcd. for
C12H23N2O8Na+ [M + Na]+: 345.1269, found: 345.1270.
+
+
calcd. for C50H56N4O20NH4 [M + NH4 ]: 1050.3824, found:
1050.3831.
3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-acetamido-2-deoxy-a-D-glucopyranosyl-N-
(fluoren-9-ylmethoxycarbonyl)-L-threonine benzyl ester (13)
2-Acetamido-2-deoxy-b-D-glucopyranosyl-L-threonine (3)
Azidosugar (12) (150 mg, 0.145 mmol) was added to a mixture
of THF–acetic anhydride–acetic acid (7.0 cm3) and zinc dust
(247 mg, 3.8 mmol) was added.33 Saturated aqueous CuSO4
solution (0.4 cm3) was added to the reaction mixture and it was
stirred for 30 min at room temperature. The mixture was filtered
through Celite and co evaporated with toluene. Purification by
column chromatography (hexane–EtOAc 3 : 7 v/v) afforded the
The above procedure for the deprotection of compound (9)
was applied to compound (10). Starting from compound (10)
(60.0 mg, 0.078 mmol), the product (3) was obtained as a colourless
25
amorphous solid (20.0 mg, 80%). [a]D +81.1 (c 1.0, CH3OH); dH
(D2O) 4.40 (1H, d, J1,2 = 8.3 Hz, H-1), 4.18 (1H, m, bCH Thr),
3.82–3.78 (1H, m, H-6a), 3.65–3.62 (1H, m, H-2), 3.60 (1H, t,
J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.52–3.50 (1H, m, 3.92, aCH
Thr), 3.44 (1H, t, J3,4, J4,5 = 9.8 Hz, H-4), 3.37–3.30 (2H, m, H-5,
H-6b), 1.95 (3H, s, NHAc), 1.22 (3H, d, J = 6.5 Hz, CH3 Thr);
dC (CD3OD) 101.9 (C-1), 77.6 (bCHThr, aCHThr), 76.7 (C-5),
74.7 (C-3), 70.9 (C-4), 61.5 (C-6), 56.5 (C-2), 22.8 (NCOCH3),
18.9 (CH3 Thr). ESI-HRMS: calcd. for C12H24N2O8 [M + H]+:
323.1449, found: 323.1449.
25
product (13) as an amorphous solid (99 mg, 65%). [a]D + 28.8
(c 1.0, CHCl3); dH (CDCl3) 7.77 (2 H, d, J = 7.5 Hz, CH Fmoc
Ph), 7.64 (2 H, d, J = 6.0 Hz, CH Fmoc Ph), 7.49–7.26 (9H, m,
Fmoc Ph, OCH2Ph), 5.85–5.62 (2 H, 2 d, J = 9.4 Hz, 2NH), 5.28
(1 H, dd, J3 ,4 = 3.4 Hz, J4 ,5 = 0.8 Hz, H-4ꢀ), 5.13 (1 H, dd, J2,3
=
=
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
ꢀ ꢀ
10.5 Hz, J3,4 = 8.6 Hz, H-3), 5.10 (1 H, d, J1 ,2 7.8 Hz, J2 ,3
10.4 Hz, H-2ꢀ), 5.02 (2 H, AB, JAB = 12.2 Hz, OCH2Ph), 4.90 (1
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
H, dd, J2 ,3 , J3 ,4 , H-3 ), 4.57 (1 H, d, J1,2 = 3.7 Hz, H-1), 4.48 (1 H,
d, J1 ,2 7.8 Hz, H-1ꢀ), 4.37 (3 H, m, aCHThr, CH2 Fmoc, H-6b),
ꢀ
ꢀ
3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-azido-2-deoxy-a-D-glucopyranosyl-N-
(fluoren-9-ylmethoxycarbonyl)-L-threonine benzyl ester (12)
4.22 (1 H, t, J = 7.3 Hz, CHFmoc), 4.14–4.09 (3H, m, bCHThr,
H-2, H-6aꢀ), 4.06 (1 H, dd, J6 a,6 b = 11.0 Hz, J5 ,6 b = 5.9 Hz, H-
6bꢀ), 4.01 (1 H, dd, J6a,6b = 11.0 Hz, J5,6a = 5.3 Hz, H-6a), 3.86 (1
H, ddd, J4,5 = 10.0 Hz, J5,6a, J5,6b = 1.9 Hz, H-5), 3.80 (1 H, dd,
ꢀ
ꢀ
ꢀ ꢀ
A
mixture of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
J5 ,6 a = 7.4 Hz, J5 ,6 b, H-5ꢀ), 3.69 (1 H, dd, J3,4, J4,5, H-4), 2.15–
1.93 (21 H, 7 s, 7 CH3CO), 1.21 (3 H, d, J = 6.5 Hz, CH3 Thr);
dC (CDCl3) 170.5–169.3 (COCH3, COCH2Ph), 157.1 (CO Fmoc),
144.0, 141.2 (Cquat. Fmoc Ph), 135.1 (Cquat. OCH2Ph), 128.8,
127.9, 127.3, 125.5, 120.1 (CH Ph), 101.3 (C-1ꢀ), 99.3 (C-1), 76.6
(C-4), 71.2 (C-3ꢀ), 70.9 (C-3), 70.1 (C-5ꢀ), 69.3 (C-2ꢀ), 69.1 (C-5),
68.0 (CH2 Fmoc, OCH2Ph), 66.7 (C-4ꢀ), 62.1 (C-6), 61.6 (C-6ꢀ),
60.8 (aCHThr, bCHThr), 59.1 (C-2), 47.3 (CH Fmoc), 21.2–20.8
(COCH3), 18.9 (CH3 Thr). ESI-HRMS: calcd. for C52H60N2O21H+
[M + H]+: 1049.3761, found: 1049.3768.
ꢀ
ꢀ
ꢀ ꢀ
galactopyranosyl)-2-azido-2-deoxy-a-D-glucopyranosyl chloride
(11)32,33 (200 mg, 0.31 mmol) and N-(fluoren-9-ylmethoxy-
carbonyl)-L-threonine benzyl ester (8) (135.4 mg, 0.31 mmol) in
1,2-dichloroethane (2.5 cm3) was refluxed with mercuric bromide29
(112 mg, 0.31 mmol) for 8 h, when TLC (hexane–EtOAc, 3 : 7 v/v)
showed the disappearance of the donor (11). The resulting amber
mixture was concentrated in vacuo and the residue was purified
by silica gel column chromatography (eluent: hexane–EtOAc, 1 :
1 v/v). The desired azido-sugar glycoside (12) was obtained as an
25
amorphous solid (195 mg, 60%). [a]D +32.2 (c 1.0, CHCl3); dH
This journal is
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4-O-b-D-Galactopyranosyl-(2-acetamido-2-deoxy-a-D-
glucopyranosyl)-L-threonine (2)
(1H, d, J1 ,2 = 7.8 Hz, H-1ꢀ), 4.23 (1H, m, bCHThr), 3.78 (1H,
d, JaCH, bCH = 12.1 Hz, aCH Thr) 3.77–3.74 (2H, m, H-4ꢀ, H-4),
3.70–3.62 (2H, m, H-6a, H-6b), 3.60–3.50 (5H, m, H-6aꢀ, H-6bꢀ,
ꢀ
ꢀ
A solution of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-acetamido-2-deoxy-a-D-glucopyranosyl-N-
(fluoren-9-ylmethoxycarbonyl)-L-threonine benzyl ester (13)
(48 mg, 0.045 mmol) in MeOH (3.6 mL) was treated with glacial
AcOH (0.36 cm3) and 10% Pd/C (51mg) for removal of the O-Bn
and N-Fmoc groups. The reaction mixture was stirred under H2
(∼1.5 atm) for approx. 10 h, filtered in Celite, concentrated in vacuo
and the crude product was purified by column chromatography
(DCM–MeOH 9 : 1 v/v). The product obtained (43 mg,
0.058 mmol) was dissolved in MeOH (1.5 mL) and treated with
NaOMe (1 M in MeOH) until the solution was basic. The reaction
mixture was stirred for 8 h and then neutralized with Dowex
50WX8–200 ion exchange resin. Filtration and concentration of
the mixture gave the deprotected a-linked disaccharyl threonine
H-5ꢀ, H-3, H-2), 3.47 (1H, dd, J2 ,3 = 9.9 Hz, J3 ,4 = 3.3 Hz, H-3ꢀ),
3.38–3.34 (2H, m, H-5, H-2ꢀ), 1.87 (3H, s, NHAc), 1.24 (3H, d,
J = 6.5 Hz, CH3 Thr). dC (D2O) 103.7 (C-1ꢀ), 100.5 (C-1), 79.0
(C-4), 76.1 (C-5ꢀ), 75.4 (C-5), 74.5 (bCHThr), 73.3 (C-3ꢀ), 72.9 (C-
3), 71.7 (C-2ꢀ), 69.3 (C-4ꢀ), 61.7 (C-6ꢀ), 60.6 (C-6), 59.0 (aCHThr),
55.6 (C-2), 22.8 (NCOCH3), 17.9 (CH3Thr). ESI-HRMS: calcd.
for C18H33N2O13 [M + H]+: 485.1977, found: 485.1977.
ꢀ
ꢀ
ꢀ ꢀ
General method for glycopeptide synthesis41
The glycopeptides (5) and (6) were assembled manually using a frit-
ted glass reaction vessel with nitrogen purging for effective mixing.
Pre-loaded FmocGly-Wang resin (typically 100 mg) was swollen in
CH2Cl2 for 1 h and then washed with DMF (3×). Deprotection of
the N-a-Fmoc group was carried out using 20% piperidine–DMF
followed by filtration, washing with DMF (3×) and subsequently
with DIPEA. Coupling reactions were performed with 2.0 mol
equiv of Fmoc-amino acid and coupling agents (PyBOP and
HOBt) dissolved in DMF. Coupling times were variable, ranging
from 3 h to 24 h for the threonine amino acids and between 24 h
and 72 h for the glycosylated amino acid (13) and the disaccharide
glycoside (15). The reaction mixtures were filtered after each
coupling and the resin washed three times with DMF, CH2Cl2 and
MeOH. After drying in vacuo, small aliquots of resin (1 mg) were
treated with 20% piperidine in DMF for removal of N-Fmoc group
and consequent generation of dibenzofulvene product which
absorb UV strongly (290 nm), offering potential for monitoring of
coupling reactions by spectrophotometer. After removal of the N-
Fmoc group of the last amino acid, the glycopeptides were cleaved
from resin in the presence of 95% TFA in water and after cleavage
was complete, the solutions were filtered to remove the cleaved
resin and concentrated to yellowish oil. The crude glycopeptides
were triturated with cold Et2O, dried in vacuo and O-deprotected
with 1 M sodium methoxide solution in MeOH. The purification of
glycopeptides (5) and (16) was carried out on Perkin-Elmer HPLC
equipment by reverse-phase chromatography using a Phenomenex
Luna C18 column (250 × 10 mm) with a linear gradient of 0–30%
CH3CN in 0.1% aq TFA at 1.0 cm3 min−1. Under these conditions
the retention times for glycopeptides (5) and (16) were 13.5 min
and 13.0 min, respectively.
25
(2) as an amorphous solid (20 mg, 90%); [a]D +10.0 (c 0.1,
MeOH); dH (D2O), 4.77 (1H, d, J1,2 = 3.7 Hz, H-1), 4.30 (1H,
d, J1 ,2 = 7.8 Hz, H-1ꢀ), 4.29 (1H, m, bCHThr), 3.80–3.65 (7H,
m, H-2, H-3, H-6a, H-6b, H-3ꢀ, H-4ꢀ, aCHThr), 3.62–3.52 (4H,
ꢀ
ꢀ
H-4, H-5, H-6aꢀ, H-6bꢀ,), 3.50 (1H, m, H-5ꢀ), 3.36 (1H, dd, J1 ,2
ꢀ
ꢀ
,
J2 ,3 = 10.1 Hz, H-2ꢀ), 1.87 (3H, s, NHAc), 1.24 (3H, d, J =
6.5 Hz, CH3 Thr). dC (D2O) 103.7 (C-1ꢀ), 99.5 (C-1), 79.5 (C-4),
76.1 (C-5ꢀ), 75.6 (bCHThr), 73.3 (C-5), 71.92, 71.91 (C-3ꢀ, C-3),
71.7 (C-2ꢀ), 70.1 (aCHThr), 69.3 (C-4ꢀ), 61.8 (C-6ꢀ), 61.7 (C-6),
53.9 (C-2), 22.9 (NCOCH3), 18.9 (CH3 Thr). ESI-HRMS: calcd.
for C18H32N2O13.Na+ [M + Na]+: 507.1797, found: 507.1798.
ꢀ
ꢀ
Enzymatic synthesis of 4-O-b-D-galactopyranosyl-(2-acetamido-2-
deoxy-a-D-glucopyranosyl)-L-threonine (2)
To a solution of 2-acetamido-2-deoxy-a-D-glucopyranosyl-L-
threonine (1) (5 mg, 0.015 mmol), UDP-galactose (5.0 mg,
0.008 mmol), and alkaline phosphatase (1.2 mg) in HEPES buffer
(0.4 cm3, 50 mM, pH 7.0, containing 100mM MnCl2) was added
the enzyme bovine b-1,4-galactosyltransferase (2.0 U), and the
mixture was incubated for 96 h at 30 ◦C. After completion of
the reaction the enzyme was precipitated by the addition of cold
ethanol, the mixture was centrifuged and the supernatant was
concentrated to dryness. The resulting residue was dissolved in
water and fractionated by gel filtration chromatography (15 ×
850 mm Toyopearl HW-40S column, eluted with water containing
0.1% TFA, at a flow rate of 0.5 cm3 min−1). Lyophilization of
relevant fractions, typically eluting in the 175–185 cm3 range, gave
deprotected a-linked disaccharyl threonine (2) as an amorphous
solid (3.9 mg, 57%). Analytical data for this compound were
identical to those reported for the same compound produced solely
by chemical means.
3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-acetamido-2-deoxy-a-D-glucopyranosyl-N-
(fluoren-9-ylmethoxycarbonyl)-L-threonine (14)
A solution of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-acetamido-2-deoxy-a-D-glucopyranosyl-N-
(fluoren-9-ylmethoxycarbonyl)-L-threonine benzyl ester (13)
(110 mg, 0.1 mmol) in MeOH (7.0 mL) was treated with glacial
AcOH (0.7 cm3) and Pd/C 10% (55 mg) for removal of the O-
Bn group. The reaction mixture was stirred and kept under H2
(∼1.5 atm) for 1 h, with careful monitoring by TLC (longer
reaction times resulted removal of the N-Fmoc group). The
reaction mixture was then filtered through Celite, concentrated
in vacuo and purified by column chromatography (DCM–MeOH
9 : 1 v/v). The product (14) was obtained as an amorphous solid
Enzymatic synthesis of 4-O-b-D-galactopyranosyl-(2-acetamido-
2-deoxy-b-D-glucopyranosyl)-L-threonine (4)
The general procedure described for the preparation of compound
(2) was applied to the galactosylation of b-linked glycosyl amino
acid 3. However, less b-1,4-galactosyltransferase (0.5 U) was
employed and the reaction was incubated for a shorter time (24 h).
Starting from compound (3) (5 mg, 0.015 mmol) the product (4)
25
was obtained as an amorphous solid (6.35 mg, 85%). [a]D −5.0
(c 0.1, MeOH). dH (D2O), 4.39 (1H, d, J1,2 = 7.5 Hz, H-1), 4.27
2654 | Org. Biomol. Chem., 2007, 5, 2645–2657
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(59.5 mg, 60%). [a]D25 +47.7 (c 0.8, CHCl3); dH (CDCl3) 7.77 (2 H,
d, J = 7.5 Hz, CH Fmoc Ph), 7.64 (2 H, d, J = 6.0 Hz, CH Fmoc
Ph), 7.45–7.28 (9H, m, Fmoc Ph), 6.58 (1H, d, J = 9.4 Hz, NH),
6.12 (1H, d, J = 9.4 Hz, NH), 5.36 (1 H, m, H-4ꢀ), 5.16 (1 H, dd,
H2N-(Thr)2-(GlcNAc-b-Thr)-(Thr)2-Gly-OH (16)
Using the standard peptide synthesis procedure outlined, followed
by preparative reverse-phase chromatography, gave compound
(16) as an amorphous solid (4.5 mg, 14%). [a]D +6.0 (c 0.1,
MeOH); dH (D2O) 4.42 (1H, d, J1,2 = 8.1 Hz, H-1), 4.40–4.25
(5H, m, aCHThr), 4.20–4.00 (5H, m, bCHThr), 3.85–3.55 (5H, m,
CH2Gly, H-6a, H-6b, H-5), 3.50 (1H, t, J = 9.8 Hz, H-3), 3.36 (1H,
t, J = 9.8 Hz, H-4), 3.25 (1H, m, H-2), 1.87 (3H, s, NHAc), 1.20–
1.00 (15H, m, 5 × CH3Thr). ESI-HSMS: calcd. for C34H70N7O17
[M + H]+: 847.4705, found: 847.4705.
25
ꢀ
ꢀ
J2, 3 = 10.5 Hz, J3, 4 = 9.4 Hz, H-3), 5.10 (1 H, d, J1 ,2 7.8 Hz, J2, 3
=
10.4 Hz, H-2ꢀ), 4.94 (1 H, dd, J2 ,3 = 10.4 Hz, J3 ,4 = 3.4 Hz, H-3ꢀ),
ꢀ
ꢀ
ꢀ ꢀ
4.57 (1 H, d, J1,2 = 3.7 Hz, H-1), 4.48 (1 H, d, J1 ,2 7.8 Hz, H-1ꢀ),
4.44–4.32 (3 H, m, aCHThr, CH2 Fmoc, H-6b), 4.25 (1 H, t, J =
7.3 Hz, CHFmoc), 4.16–4.08 (3H, m, bCHThr, H-2, H-6aꢀ), 4.06–
4.01 (2H, m, H-6bꢀ, H-6a), 3.96–3.82 (2 H, m, H-5, H-5ꢀ), 3.72 (1
H, t, J3, 4 = 9.8 Hz, H-4), 2.18–1.90 (21 H, 7 s, 7 CH3CO), 1.26 (3
H, d, J = 6.5 Hz, CH3 Thr); dC (CDCl3) 171.0–169.5 (COCH3),
157.1 (CO Fmoc), 144.0, 141.2 (Cquat. Fmoc Ph), 128.0–120.1
(CH Ph), 101.4 (C-1ꢀ), 99.0 (C-1), 76.1 (C-4), 71.2 (C-3ꢀ), 70.9
(C-3), 70.1 (C-5ꢀ), 69.3 (C-2ꢀ), 69.1 (C-5), 67.5 (CH2 Fmoc), 66.8
(C-4ꢀ), 62.1 (C-6), 61.6 (C-6ꢀ), 60.8 (aCHThr, bCHThr), 58.5 (C-
2), 47.4 (CH Fmoc), 21.4–20.8 (COCH3), 18.8 (CH3 Thr). ESI-
HSMS: calcd. for C45H55N2O21H+ [M + H]+: 959.3228, found:
959.3229.
ꢀ
ꢀ
H2N-(Thr)2-(LacNAc-b-Thr)-(Thr)2-Gly-OH (6)
To a solution of the glycopeptide H2N-(Thr)2-(bGlcNAc-Thr)-
(Thr)2-Gly-OH (16) (4.0 mg, 0.005 mmol) UDP-galactose (2.9 mg,
0.005 mmol), and alkaline phosphatase (0.4 mg) in HEPES buffer
(0.4 mL, 50 mM, pH 7.0, containing 100 mM MnCl2) was added
the enzyme bovine b-1,4-galactosyltransferase (1.0 U), and the
mixture was incubated for 24 h at 30 ◦C. After completion of the
reaction the enzyme was denatured with ethanol and centrifuged
before the supernatant was lyophilised. The dry residue was
dissolved in water and purified by gel filtration (Toyopearl HW-
40 S column, 15 × 850 mm, eluting with 0.1% aqueous TFA
at 0.5 mL min−1). Lyophilization of relevant fractions afforded
H2N-(Thr)2-(LacNAc-a-Thr)-(Thr)2-Gly-OH (5)
Using the standard peptide synthesis procedure outlined, followed
by preparative reverse-phase chromatography, glycopeptide (5)
25
25
was obtained as an amorphous solid (7.0 mg, 22%). [a]D +3.0
glycopeptide (6) as an amorphous solid (3.5 mg, 80%). [a]D −2.2
(c 0.1, MeOH). dH (D2O) 4.45–4.40 (2H, m, H-1, H-1ꢀ), 4.38–4.21
(5H, m, bCHThr), 4.15–4.00 (5H, m, aCHThr), 3.85–3.45 (13H,
m, CH2Gly, H-6a, H-6b, H-6ꢀa, H 6ꢀb, H-5, H-5ꢀ, H-4, H-4ꢀ, H-
3, H-3ꢀ, H-2), 3.37 (1H, m, H-2), 1.88 (3H, s, NHAc), 1.20–1.00
(15H, m, 5 × CH3Thr). ESI-HSMS: calcd. for C36H63N7O22 [M +
H]+: 946.4099, found: 946.4103.
(c 0.1, MeOH). dH (D2O) 4.71 (1H, d, J1,2 = 3.7 Hz, H-1), 4.28
(1H, d, J1,2 = 8.1 Hz, H-1ꢀ), 4.50–4.30 (5H, m, aCHThr), 4.10–3.95
(5H, m, bCHThr), 3.90–3.45 (12H, m, CH2Gly, H-6a, H-6b, H-
6ꢀa, H-6ꢀb, H-2, H-3, H-4, H-4ꢀ, H-5), 3.50 (1H, m, H-3ꢀ), 3.35 (1H,
m, H-2ꢀ), 1.85 (3H, s, NHAc), 1.20–1.00 (15H, m, 5 × CH3Thr).
ESI-HSMS: calcd. for C36H63N7O22 [M + H]+: 946.4099, found:
946.4103.
Cell culture and expression of T. cruzi trans-sialidase
N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-deoxy-3,4,6-tri-
O-acetyl-b-D-glucopyranosyl)-L-threonine (15)
SOB media (Hanahan’s broth)
Tryptone (20.0 g), yeast extract (5.0 g), sodium chloride (0.5 g)
were dissolved in de-ionised water (1000 cm3). The media was
autoclaved and stored until required. Immediately prior to use,
potassium chloride (10 cm3, 250 mM stock solution, pH 7.0, sterile
filtered) and magnesium chloride (5 cm3, 2 mM stock solution,
sterile filtered) were added.
A solution of N-(fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-
deoxy-3,4,6-tri-O-acetyl-b-D-glucopyranosyl)-L-threonine benzyl
ester (10) (119 mg, 0.157 mmol) in MeOH (5.0 mL) was treated
with glacial AcOH (0.2 cm3) and Pd/C 10% (27 mg) for removal of
the O-Bn group. The reaction mixture was stirred and kept under
H2 (∼1.5 atm) for 1 h. After this time, it was filtered in Celite,
concentrated in vacuo and purified by column chromatography
(DCM–MeOH 9 : 1 v/v). The product (15) was obtained as an
amorphous solid (101 mg, 96%). [a]D25 +14.4 (c 1.0, MeOH) (lit.,49
[a]D + 14.7); dH (CDCl3) 7.73 (2H, d, J = 7.3 Hz, CH Fmoc
Ph), 7.61 (2H, d, J = 6.6 Hz, CH Fmoc Ph), 7.38–7.24 (4H,
m, CH Fmoc Ph), 6.12 (1H, d, J = 9.2 Hz, NH), 6.03 (1H, d,
J = 9.2 Hz, NH), 5.26 (1H, t, J2,3 = 9.6 Hz, H-3), 5.09 (1H, t,
J3,4 = 9.6 Hz, H-4), 4.64 (1H, d, J1,2 = 8.3 Hz, H-1), 4.50–3.60
(9H, m, CH2 Fmoc, 2 CH Thr, H-6, CH Fmoc, H-6ꢀ, H-2, H-5),
2.04, 1.98, 1.97, 1.93 (12H, s, COCH3), 1.18 (3H, d, J = 6.3 Hz,
CH3 Thr); dC (CDCl3) 171.3, 170.9, 170.8, 169.3 (COCH3), 156.8
(CO Fmoc), 143.7, 141.1 (Cquat.), 127.6, 127.0, 125.1, 119.9 (CH
Ph), 99.4 (C-1), 71.7, 71.5, 70.9, 68.2, 67.3 (C-3, C-4, C-5, 2 CH
Thr, CH2 Fmoc), 62.0 (C-6), 52.3 (C-2), 47.0 (CH Fmoc), 22.9,
20.8, 20.7, 20.5 (COCH3), 17.3 (CH3 Thr). ESI-HRMS: calcd. for
C33H39N2O13 [M + NH4]+: 671.2452, found 671.2453.
Cell culture
Clones of TcTS (pTrcTs611/2)50 in E. coli (XL1-Blue) were
selected and raised on ampicillin (100 lg cm−3) LB agar slopes.
Single colonies were selected for the generation of glycerol stocks
(20% glycerol; stored at −80 ◦C) using culture conditions given
below. Overnight cultures were prepared directly from glycerol
stocks in sterile SOB media (25 cm3 in 50 ml culture tubes)
with ampicillin (100 ll cm−3, sterile filtered). The cultures were
incubated overnight at 37 ◦C with shaking (200 rpm). SOB media
(1000 cm3 containing 100 ll cm−3 ampicillin, sterile filtered prior
to addition, 2 L baffled plastic culture flasks) was warmed to
37 ◦C and inoculated to an optical density of 0.05–0.1 OD600 units.
Cultures were left to incubate at 37 ◦C with shaking (250 rpm)
until OD600 = 0.6–0.8 (4–6 h). Cells were induced with 1 mM
IPTG (sterile filtered) and left for 12 h at 23 ◦C. The cells were
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then harvested by centrifugation at 4 ◦C and the cell pellet was
Wickstead, J. Wortman, O. White, C. M. Fraser, K. D. Stuart and B.
Andersson, Science, 2005, 309, 409–415.
8 R. G. Spiro, Glycobiology, 2002, 12, 43R–56R.
stored at −20 ◦C until required.
9 A. Acosta-Serrano, I. C. Almeida, L. H. Freitas, N. Yoshida and S.
Schenkman, Mol. Biochem. Parasitol., 2001, 114, 143–150.
10 C. A. Buscaglia, V. A. Campo, A. C. C. Frasch and J. M. Di Noia, Nat.
Rev. Microbiol., 2006, 4, 229–236.
11 O. A. Agrellos, C. Jones, A. R. Todeschini, J. O. Previato and L. M.
Previato, Mol. Biochem. Parasitol., 2003, 126, 93–96.
12 M. L. Salto, C. Gallo-Rodriguez, C. Lima and R. M. de Lederkremer,
Anal. Biochem., 2000, 279, 79–84.
13 G. D. Pollevick, J. M. Di Noia, M. L. Salto, C. Lima, M. S. Leguizamon,
R. M. de Lederkremer and A. C. C. Frasch, J. Biol. Chem., 2000, 275,
27671–27680.
14 A. R. Todeschini, E. X. da Silveira, C. Jones, R. Wait, J. O. Previato
and L. Mendonc¸a-Previato, Glycobiology, 2001, 11, 47–55.
15 J. O. Previato, C. Jones, L. P. B. Goncalves, R. Wait, L. R. Travassos
and L. Mendonc¸a-Previato, Biochem. J., 1994, 301, 151–159.
16 G. A. M. Cross and G. B. Takle, Annu. Rev. Microbiol., 1993, 47, 385–
411 and citations thereof.
Preparation of a cell-free extract
The cell pellet was thawed on ice and re-suspended in 30 ml of
phosphate buffer saline (PBS). After 30 minutes incubation on ice,
the viscosity of the solution was decreased by addition of DNase
(20 lg cm−3). The cells were then sonicated on ice for 5 × 1 minutes
(cycle 2, 50% power) with 1 minute intervals between bursts. The
suspension was then centrifuged (10000 g for 30 min) to remove
cell debris. The supernatant was then filtered through a 0.2 lm
filter. The cleared protein lysate obtained was then employed in
biotransformations.
General procedure for enzymatic reactions with trans-sialidase
17 A. K. Chava, S. Bandyopadhyay, M. Chatterjee and C. Mandal,
Glycoconjugate J., 2003, 20, 199–206.
18 S. Schenkman, M.-S. Jiang, G. W. Hart and V. Nussenzweig, Cell, 1991,
65, 1117–25.
19 M. F. Amaya, A. G. Watts, I. Damager, A. Wehenkel, T. Nguyen, A.
Buschiazzo, G. Paris, A. C. Frasch, S. G. Withers and P. M. Alzari,
Structure, 2004, 12, 775–784 and citations therein.
20 J. O. Previato, M. Sola-Penna, O. A. Agrellos, C. Jones, T. Oeltmann,
L. R. Travassos and L. Mendonca-Previato, J. Biol. Chem., 1998, 273,
14982–14988.
21 J. A. Morgado-Diaz, C. V. Nakamura, O. A. Agrellos, W. B. Dias, J. O.
Previato, L. Mendonca-Previato and W. De Souza, Parasitology, 2001,
123, 33–43.
22 M. J. McConville, K. A. Mullin, S. C. Ilgoutz and R. D. Teasdale,
Microbiol. Mol. Biol. Rev., 2002, 66, 122–154.
Reactions were typically conducted with 1–2 mg of LacNAc-
based acceptor substrate. To a solution of acceptor (1 eq.) and
the calf serum fetuin (3 mol eq with respect to sialic acid), in
phosphate buffer (0.2 M, pH 7.0) was added sufficient TcTS to
effect complete conversion within 2 h at 30 ◦C. After completion
of the reaction, as judged by TLC (DCM–MeOH–H2O 10 : 10 :
3 v/v/v), protein was removed with a VIVASPIN 500 spin filter
(MW cut-off 10000), and the filtrate was directly applied to a gel
filtration column (Toyopearl HW-40 S column, 15 × 850 mm)
using 5 mM NH4HCO3 buffer as eluent at 0.5 cm3 min−1. Data for
sialylated compounds (17–20) can be found in Table 1.
23 R. Agusti, V. M. Mendoza, C. Gallo-Rodriguez and R. M. de
Lederkremer, Tetrahedron: Asymmetry, 2005, 16, 541–551.
24 V. M. Mendonza, R. Agusti, C. Gallo-Rodriguez and R. M. de
Lederkremer, Carbohydr. Res., 2006, 341, 1488–1497.
25 R. Agusti, M. E. Giorgi, V. M. Mendoza, C. Gallo-Rodriguez and
R. M. de Lederkremer, Bioorg. Med. Chem., 2007, 15, 2611–2616.
26 R. M. van Well, K. P. R. Kartha and R. A. Field, J. Carbohydr. Chem.,
2005, 24, 463–474 and citations therein.
27 A. Pisvejcova, C. Rossi, L. Husakova, V. Kren, S. Riva and D. Monti,
J. Mol. Catal. B: Enzym., 2006, 39, 98–104, and references cited therein.
28 D. Horton, Methods Carbohydr. Chem., 1972, 6, 282–285.
29 I. Carvalho, S. L. Scheuerl, K. P. R. Kartha and R. A. Field, Carbohydr.
Res., 2003, 338, 1039–1043.
Acknowledgements
These studies were supported by FAPESP, the University of East
Anglia, BBSRC and Basic Technology Grant GR/S79268/02
from Research Councils UK (http://www.glycoarrays.org.uk/).
We thank Prof. A. C. C. Frasch, Universidad Nacional General
San Martın, Argentina, for kindly providing the TcTS clone50 used
in these studies. The EPSRC Mass Spectrometry Service Centre,
Swansea and Dr Lionel Hill of the John Innes Centre are thanked
for invaluable assistance.
30 R. J. Kerns and P. Wei, ACS Symp. Ser., 2006, 932, 205–236.
31 F. G. Bongat and A. V. Demchenko, Carbohydr. Res., 2007, 342, 374–
406.
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