Synthesis of mucin glycans from the protozoon parasite Trypanosoma cruzi

Article abstract of DOI:10.1055/s-2008-1078249

A short, blockwise, 2+2-glycosylation approach to the synthesis of a tetrasaccharide component of Trypanosoma cruzi mucin is reported. Despite the use of a 1,2-linked disaccharide donor, high yield (79%) and good stereocontrol (>10:1, β:α) were achieved in the key glycosylation step. Preliminary studies indicate that this branched tetrasaccharide can serve as a substrate for enzymatic sialylation by the parasite cell surface trans-sialidase. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2008-1078249

LETTER
2175
Synthesis of Mucin Glycans from the Protozoon Parasite Trypanosoma cruzi
P
arasite
MucinG
e
lycans nate M. van Well,a Beatrice Y. M. Collet,a Robert A. Field*a,b
a
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Fax +44(1603)450018; E-mail: rob.field@bbsrc.ac.uk
b
Received 2 June 2008
Dedicated to Professor Sir Jack Baldwin FRS, in honour of his many and varied contributions to organic chemistry
better understand how TcTS binds and performs chemis-
Abstract: A short, blockwise, 2+2-glycosylation approach to the
synthesis of a tetrasaccharide component of Trypanosoma cruzi
mucin is reported. Despite the use of a 1,2-linked disaccharide do-
nor, high yield (79%) and good stereocontrol (>10:1, b:a) were
try on its natural substrates, the parasite-cell-surface mu-
cin glycoproteins.3 Given the heterogeneity of T. cruzi
mucins, and the issues associated with culturing quantities
achieved in the key glycosylation step. Preliminary studies indicate of human pathogen, we set out to synthesise T. cruzi mu-
that this branched tetrasaccharide can serve as a substrate for enzy-
matic sialylation by the parasite cell surface trans-sialidase.
cin glycopeptides. The de Lederkremer lab, in particular,
have made substantial efforts in the chemical synthesis
Key words: oligosaccharide, synthesis, parasite, mucin, trans-sial-
idase, drug target
and assessment of the mucin glycans from T. cruzi G
strain.12 We have recently reported on the action of TcTS
on synthetic glycopeptide fragments of mucins from
T. cruzi Y strain.13 Herein we report approaches to the
The South American protozoon parasite Trypanosoma synthesis of a more complex T. cruzi Y strain mucin tet-
cruzi is the causative agent of Chagas’ disease, one of the rasaccharide, in a form suitable for elaboration into gly-
most serious parasitic diseases in tropical regions. Ap- coamino acid building blocks for glycopeptide production
proximately 18 million people are infected with T. cruzi and for direct assessment as a substrate for TcTS.
and a further 100 million are at risk of infection; 2–3 mil-
In developing a strategy for the synthesis of the tetrasac-
lion chronic cases are current, with around 45000 deaths
charide component of the Y strain mucin glycan, a key
each year.1 The repertoire of current drugs for treating
consideration was the compatibility of the synthesis with
Chagas’ disease is poor: new leads are needed. As with
subsequent use of the glycan product for glycopeptide
many pathogenic microorganisms, the nature and extent
synthesis (Figure 1). The ability to control glycosylation
of cell-surface glycosylation impacts directly on their in-
stereochemistry is the key. The challenge with the struc-
ture shown is that the acetamido group at C-2 of the core
fectivity.2 In the case of T. cruzi, the cell surface is deco-
rated with a complex glycocalyx composed of very
a-GlcNAc has the potential to participate in glycosyla-
heavily glycosylated mucins (ca. 60% carbohydrate by
tion, directing the reaction to give the undesired b-linked
weight).3 The mucin glycans are the major substrate for a
product. We have recently reported the direct synthesis of
parasite trans-sialidase (TcTS)4 which, like the mucins
a-linked GlcNAc-threonine from a 2-acetamido donor,13
themselves, is attached to the parasite surface via a GPI
although modest yields mean that the method is only prac-
anchor.5 trans-Sialidase catalyses the transfer of sialic
tical for a cheap GlcNAc donor but not for complex syn-
acid from host glycoproteins onto the parasite surface as
thetic oligosaccharides (for a recent review of hexosamine
part of its effort to evade the host immune response. Try-
glycoside syntheses, see ref.15). This led us to consider a
reducing terminus based on 2-azido-2-deoxy-glucose, in
the form of a thioglycoside, since we have already shown
panosoma cruzi is unable to synthesise sialic acid de novo
so the trans-sialidase represents a potential drug target.
Numerous studies have aimed to understand TcTS struc-
ture and action6 and to exploit it in enzymatic synthesis.7
OH
HO
In our work,8 this enzyme has proved invaluable for the
OH
O
OH
HO
HO
preparation of isotopically enriched glycans for NMR
structural studies, in particular.7f,9 However, despite its
broad similarity to microbial neuraminidases, for which
several potent inhibitors are known (and which form the
basis of current drugs, such as tamiflu),10 this potential
parasite drug target remains rather enigmatic. Despite nu-
merous efforts, only very limited progress has been made
with inhibitor development.11 There is therefore a need to
Thr
Thr
O
Gal-β-1,2-Gal-β-1,6
O
OH
OH
O
Gal-β-1,4-GlcNAc-α Thr
O
HO
O
Thr
Thr
HO
O
AcHN
O
HO
OH
OH
Me
O
Me
O
HO
Me
O
H
H
N
N
N
H
N
H
N
H
O
O
HO
Me
HO
Me
SYNLETT 2008, No. 14, pp 2175–2177
2
2
.0
8
.2
0
0
8
Advanced online publication: 05.08.2008
DOI: 10.1055/s-2008-1078249; Art ID: D19408ST
© Georg Thieme Verlag Stuttgart · New York
Figure
1
Fragment of Trypanosoma cruzi
Y
strain mucin
structure14

2176
R. M. van Well et al.
LETTER
OAc
OAc
AcO
OAc
OAc
O
AcO
AcO
OH
OH
AcO
AcO
HO
OBn
OBn
OH
O
HO
HO
O
O
BnO
CCl3
O
O
O
AcO
O
2 + 1 + 1
2 + 2
AcO
O
O
OH
OH
HO
HN
O
CCl3
HN
BzO
O
OH
O
HO
HO
O
BzO
SPh
HO
O
O
O
BzO
BzO
O
BzO
BzO
O
SPh
AcHN
SPh
BzO
O
BzO
O
OH
Tetrasaccharide (1)
AcHN
AcHN
OBz
OBz
(2 x 1) + 2
OBn
BnO
OBn
OBn
BnO
OAc
AcO
AcO
OBn
O
O
O
HO
AcO
Br
AcO
O
CCl3
O
OH
BzO
(x 2)
O
O
HO
HN
BzO
BzO
O
SPh
BzO
SPh
BzO
O
N3
AcHN
OBz
Scheme 1 Outline of potential disconnections of the target tetrasaccharide 1
that 2-azido-2-deoxy-lactose donors can be convenient To summarise, we have developed a convenient synthesis
for the installation of a-linked LacNAc disaccharide of T. cruzi Y strain mucin tetrasaccharide in the form of a
units.13,16
thioglycoside. Preliminary experiments (Scheme 3) show
that this thioglycoside is capable of acting as an acceptor
substrate for Trypanosoma cruzi trans-sialidase. In con-
Target tetrasaccharide 1 presents a number of potential
disconnections (Scheme 1). In initial efforts, we explored
both a 2+1+1 and a (2×1)+2 approach. The former route
was unsuccessful due to our inability to selectively de-O-
acetylate the 2-position of the 1,6-linked galactose in the
presence of benzoate protecting groups17 elsewhere in a
trisaccharide intermediate. Attempts to perform double
glycosylation of a 1,6-linked diol acceptor in a (2×1)+2
approach yielded only 1,2-glycosylated product, the 4-
OH of the azido-glucose unit proving unreactive. Con-
cerns about the difficulty of effecting stereocontrolled
glycosylation with a 1,2-linked glycosyl donor18 initially
put us off a blockwise 2+2 approach, but in light of the
failure of other approaches this strategy was adopted.
trast to expectations from the biological literature,14
a
doubly sialylated glycan product is evident in such
biotransformations; full details will be reported in due
course.
OAc
OAc
OAc
OAc
AcO
OAc
AcO
OAc
AcO
AcO
AcO
AcO
i, ii
O
O
O
O
O
O
O
AcO
OAc
OAc
2 (ref. 20)
3
NH
CCl3
OH
OAc
O
AcO
AcO
O
A preference for a thioglycoside at the reducing terminus
of the target tetrasaccharide 1 dictated the use of glyco-
sylation chemistry that would not result in thioglycoside
activation. With this in mind, the acid-activated trichlor-
acetimidate donor system was employed.19 Hence, known
digalactoside 220 was subjected to selective anomeric
deprotection and conversion to the corresponding tri-
chloracetimidate 3. TMS triflate-catalysed glycosylation
with imidate 3 in MeCN, as a participating solvent,21 was
used to convert disaccharide acceptor 4, which was pre-
pared by conventional means (full details will be reported
elsewhere), into tetrasaccharide thioglycoside 5. The tet-
rasaccharide was obtained in 79% yield as an inseparable
mixture of diastereoisomers, with the required b-linked
compound dominant (>10:1, b:a), as judged by 1H NMR
spectroscopy.22,23 Subsequent reductive acetylation13,24 of
azide 5 and final de-O-acetylation gave the target tetrasac-
charide thioglycoside 1 (Scheme 2).
SPh
BzO
O
iii
N3
4
OAc
OH
OH
OAc
OAc
HO
AcO
OH
O
HO
HO
OAc
AcO
AcO
O
O
O
>10:1, β:α
O
O
OH
OH
OAc
OAc
O
O
iv, v
O
O
HO
HO
O
AcO
AcO
O
SPh
SPh
HO
BzO
O
O
AcHN
N3
1
OH
OAc
5
Scheme 2 Reagents and conditions: (i) H2NNH2·OAc (1 equiv),
DMF, 5 h, quant.; (ii) Cl3CCN (4 equiv), DBU (0.3 equiv), CH2Cl2,
0 °C, 1 h, 88%; (iii) TMSOTf (0.1 equiv), MeCN, 0 °C, 1 h, 79%;
(iv) Zn powder (13 equiv), CuSO4, THF–AcOH–Ac2O (3:1:2), 24 h,
45%; (v) Na, MeOH, 18 h, quant.
Synlett 2008, No. 14, 2175–2177 © Thieme Stuttgart · New York

LETTER
Parasite Mucin Glycans
2177
OH
HO
OH
OH
OH
OH
HO
CO2H
OH
O
OH
HO
HO
O
OH
O
HO
HO
O
O
O
AcHN
(p-nitrophenyl)sialic acid
trans-sialidase
O
O
HO
HO
OH
OH
O
OH
OH
O
AcHN
O
O
O
O
O
HO
HO
O
SPh
HO
O
SPh
HO
O
OH
OH
HO
CO2H
AcHN
HO
AcHN
OH
OH
Scheme 3 Action of recombinant T. cruzi trans-sialidase on synthetic mucin tetrasaccharide 1.7d
(12) For instance, see: (a) Agusti, R.; Giorgi, M. E.; Mendoza, V.
M.; Gallo-Rodriguez, C.; de Lederkremer, R. M. Bioorg.
Med. Chem. 2007, 15, 2611. (b) Mendoza, V. M.; Agusti,
R.; Gallo-Rodriguez, C.; de Lederkremer, R. M. Carbohydr.
Res. 2006, 341, 1488. (c) Agusti, R.; Mendoza, V. M.;
Gallo-Rodriguez, C.; de Lederkremer, R. M. Tetrahedron:
Asymmetry 2005, 16, 541; and references cited therein.
(13) Campo, V. L.; Carvalho, I.; Allman, S.; Davis, B. G.; Field,
R. A. Org. Biomol. Chem. 2007, 5, 2645.
Acknowledgment
This study was supported by an EU Marie Curie Intra-European
Fellowship (RMvW) and a studentship from the University of East
Anglia (BYMC). We thank the EPSRC Mass Spectrometry Service
Centre, Swansea, for invaluable support.
References and Notes
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M. Nature Rev. Microbiol. 2006, 4, 229.
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V. Cell 1991, 65, 1117. (b) Cross, G. A. M.; Takle, G. B.
Annu. Rev. Microbiol. 1993, 47, 385; and citations thereof.
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A.; Guther, L. S.; Homans, S. W.; McConville, M. J.;
Mehlert, A.; Milne, K. G.; Ralton, J. E.; Roy, Y. A.;
Schneider, P.; Zitzmann, N. Parasitology 1994, 108, S45.
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F.; Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Watts, A.;
Withers, S. G. Biochemistry 2008, 47, 3507. (b) Blume, A.;
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Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Withers, S. G.
J. Am. Chem. Soc. 2003, 125, 7532.
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W.; Schenkman, S.; Crout, D. H. G. Chem. Commun. 2000,
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(16) The studies reported herein have been conducted in parallel
with efforts that replace the reducing terminal N-acetyl-
glucosamine unit with mannose, which offers more
straightforward entry to an a-linked amino acid–glycan
linkage.
(17) Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L. V.;
Kochetkov, N. K. Carbohydr. Res. 1983, 124, C8.
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Carbohydr. Res. 1993, 246, 23.
(22) In the parallel series with a mannose at the reducing
terminus, the corresponding coupling gave complete
b-stereocontrol albeit in lower yield (54%).
(23) Selected Analytical Data
Imidate 3: [a]D20 +41.0 (c 1, CHCl3). 1H NMR (400MHz,
CDCl3): d = 8.65 (s, 1 H, NH), 6.51 (d, 1 H, H1, J1,2 = 3.6
Hz), 4.66 (d,1 H, H1¢, J1¢,2¢ = 7.8 Hz). 13C NMR (100 MHz,
CDCl3): d = 160.7 (C=NH), 101.3 (C1¢), 94.9 (C1). HRMS:
m/z calcd for C28O18NCl3H36 [M + NH4]: 797.1336; found:
797.1339.
Tetrasaccharide 5: [a]D20 –4.0 (c 1 in CHCl3). 1H NMR (600
MHz, CDCl3): d = 4.78 (d, 1 H, H1c, J1,2 = 8.0 Hz), 4.70 (d,
1 H, H1a, J1,2 = 10.0 Hz), 4.60 (d, 1 H, H1b, J1,2 = 7.7 Hz),
4.55 (d, 1 H, H1d, J1,2 = 7.9 Hz). 13C NMR (100 MHz,
CDCl3): d = 101.7 (C1b), 100.9 (C1c), 100.3 (C1d), 85.5
(C1a). MS (ES): m/z = 1372.5 [M + Na]+.
Tetrasaccharide 1: [a]D20 +15.0 (c 1 in H2O). 1H NMR, (400
MHz, D2O): d = 4.87 (d, 1 H, H1a, J1,2 = 10.4 Hz), 4.37 (d,
1 H, H1b, J1,2 = 6.8 Hz), 4.34 (d, 1 H, H1c, J1,2 = 8.0 Hz),
4.31 (d, 1 H, H1d, J1,2 = 7.9 Hz). 13C NMR (75 MHz, D2O):
d = 175.1 (C=ONHAc), 104.2 (C1c), 103.8 (C1d), 102.2
(C1b), 85.9 (C1a). HRMS: m/z calcd for C32H49N3O20S [M +
Na]+: 822.2461; found: 822.2487.
(24) Winans, K. A.; King, D. S.; Rao, V. R.; Bertozzi, C. R.
Biochemistry 1999, 38, 11700.
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