Chemical synthesis of the W-glycans of gp63, the major surface glycoprotein from Leishmania mexicana amazonemis

Article abstract of DOI:10.1039/a809798d

The chemical synthesis of the conjugated heptasaccharide Man(α1-3)Man(α1-6)[GIc(α1-3)Man(α1-2)Man(α1-2) Man(α1-3)]Manβl-O[CH₂]₈CO₂Me 1 and four closely related biantennary oligomannose type structures derived from the glyc

Full text of DOI:10.1039/a809798d

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Chemical synthesis of the N-glycans of gp63, the major surface
glycoprotein from Leishmania mexicana amazonensis
Arno Düffels and Steven V. Ley*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
UK CB2 1EW
Received (in Cambridge) 16th December 1998, Accepted 18th January 1999
The chemical synthesis of the conjugated heptasaccharide
Man(á1-3)Man(á1-6)[Glc(á1-3)Man(á1-2)Man(á1-2)Man-
(á1-3)]Manâ1-O[CH2]8CO2Me 1 and four closely related
biantennary oligomannose type structures derived from the
glycoprotein 63 of Leishmania mexicana amazonensis is
described. Taking advantage of common structural motifs
found in these N-glycans, a strategy based on the principles
of reactivity tuning and orthogonal activation allowed
the rapid assembly of a whole class of complex oligo-
saccharides. Deprotection of all structures was achieved in
high yields by hydrogenolysis in one step.
Here we report the synthesis of the five oligomannose type
structures of L. mexicana amazonensis. All compounds contain
a β-linked methoxycarbonyl octyl chain,5 replacing the di-N-
acetylchitobiose (GlcNAc(β1-4)GlcNAc) moiety of the natural
occurring N-glycans which allows further derivatization of the
final glycoconjugates for use in biosynthesis studies of these
N-glycans.6
Results and discussion
Recently we reported the synthesis of a high mannose type
nonasaccharide7,8 as part of our research in the area of protect-
ing group mediated reactivity tuning in oligosaccharide syn-
thesis.9 In this context seleno- and thioglycosides have proven to
be versatile components for the stereoselective construction of
complex mannosides.
Based on this result the synthesis of the glycoconjugates 1–5
(Fig. 1) was addressed by stepwise addition of the respec-
tive mono- and disaccharide precursors 6–10 to the central
β-mannoside 118 (Fig. 2). This convergent approach allowed
the rapid assembly of the complete set of biantennary target
Introduction
Leishmania are digenetic parasites which are the causative
agents in a number of serious human diseases affecting popu-
lations throughout the tropical and subtropical world. The
parasite alternates between the promastigote, a free living fla-
gellate in the gut of the vector sandfly, and the amastigote, the
obligatory intracellular form, which resides in phagolysosomes
in mammalian macrophages. Essential for the establishment of
successful infection are four sequential events: recognition,
intracellular entry, survival and replication.1
Evidence exists that cell surface oligosaccharides of this
parasite play a role in the infection of human macrophages
by promastigotes.2 Investigations into the structure–activity
relationship of these carbohydrates, however, are restricted by
the poor availability of pure oligosaccharides and the micro-
heterogeneity of the naturally occurring glycoprotein.3
OBn
OBn
OBn
O
O
BnO
BnO
BnO
BnO
BnO
Ph
OBn
OBn
O
O
Ph
O
O
O
O
O
O
7
6
SePh
SePh
The N-linked oligosaccharides of gp63, the major surface
glycoprotein of L. mexicana amazonensis, were characterized in
1990.3 Interestingly, all the biantennary oligosaccharides iso-
lated from L. mexicana amazonensis lack the branched (3,6-
linked) α-mannosyl residue on the α-1,6 arm which is usually
observed in similar oligomannose structures.4
OBn
OBn
OAc
O
OBn
O
BnO
BnO
BnO
BnO
OBn
OBn
O
O
O
O
BnO
BnO
BnO
BnO
SEt
SEt
8
9
Man(α1-3)Man(α1-6)
1
Manβ1-O[CH2]8CO2Me
OBn
OTBDPS
OBn
Glc(α1-3)Man(α1-2)Man(α1-2)Man(α1-3)
OBn
O
O
BnO
BnO
BnO
BnO
O[CH2]8CO2Me
11
Man(α1-3)Man(α1-6)
F
10
2
3
Manβ1-O[CH2]8CO2Me
Manβ1-O[CH2]8CO2Me
Man(α1-2)Man(α1-2)Man(α1-3)
Fig. 2
OBn
OBn
Man(α1-3)Man(α1-6)
Man(α1-2)Man(α1-3)
OBn
O
Ph
O
O
i
BnO
BnO
O
6
+
+
HO
F
SePh
10
13
Man(α1-6)
OBn
4
5
Manβ1-O[CH2]8CO2Me
Manβ1-O[CH2]8CO2Me
OBn
O
Man(α1-2)Man(α1-3)
Ph
O
O
ii
BnO
BnO
O
7
HO
BnO
12
F
SePh
Man(α1-3)Man(α1-6)
Man(α1-3)
13
Scheme 1 Reagents and conditions: i, AgClO4, HfCp2Cl2, 4 Å molecu-
lar sieves, Et2O, 74%; ii, AgClO4, HfCp2Cl2, 4 Å molecular sieves, Et2O,
72%.
Fig. 1
J. Chem. Soc., Perkin Trans. 1, 1999, 375–378
375

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OMe
molecules 1–5 in a minimum number of steps from a common
set of building blocks.
OBn
OBn
OAc
O
i
O
O
O
BnO
BnO
BnO
BnO
The synthesis of the building blocks 6–11 began with the
preparation of the α-1,3 linked disaccharides 6 and 7. Start-
ing from the readily available benzylidene protected seleno-
mannoside 1310 (Scheme 1), orthogonal activation of 2,3,4,6-O-
benzyl-α--mannopyranosyl fluoride 10 and 2,3,4,6-O-benzyl-
SePh
14
15
ii
11
α--glucopyranosyl fluoride 12 using HfCp2Cl2–AgClO4 as
OBn
OR
O
the activation system in the presence of 13 gave the desired
disaccharides 6 (74%) and 7 (72%), respectively, as single
BnO
BnO
SEt
11
8
16 R = Ac
17 R = H
iii
OBn
OBn
O
15
BnO
BnO
OBn
OR
SePh
18
i
O
BnO
BnO
iv
OBn
O
O
BnO
BnO
OTBDPS
OBn
8
v
O
BnO
O
O[CH2]8CO2Me
9
19 R = Ac
20 R = H
Scheme 2 Reagents and conditions: i, HgBr2 (cat.), PhSeH, MeCN,
60 ЊC, 96%; ii, HgBr2 (cat.), EtSH, MeCN, 60 ЊC, 94%; iii, K2CO3,
MeOH, 85%; iv, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O
1:1, 75%; v, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1,
78%.
ii
Scheme 3 Reagents and conditions: i, NIS, TfOH (cat.), 4 Å molecular
sieves, DCM–Et2O 1:1, 81%; ii, K2CO3, MeOH, 88%.
7
18
20
OBn
O
BnO
BnO
i
ii
OBn
BnO
Ph
OBn
O
O
OBn
O
O
O
BnO
BnO
OBn
OBn
O
O
O
O
BnO
BnO
BnO
BnO
OBn
OBn
O
O
O
O
BnO
BnO
BnO
BnO
OR
OR
OBn
O
OBn
O
BnO
BnO
O
O
O[CH2]8CO2Me
O[CH2]8CO2Me
21 R = TBDPS
23 R = H
22 R = TBDPS
24 R = H
iv
iii
6
6
OBn
v
vi
O
BnO
BnO
OBn
OBn
OBn
O
OBn
BnO
Ph
OBn
OBn
O
O
OBn
O
O
O
BnO
BnO
O
BnO
BnO
BnO
O
BnO
OBn
OBn
O
OBn
OBn
O
O
Ph
Ph
O
O
O
O
O
O O
O
BnO
BnO
BnO
BnO
OBn
O
OBn
O
O
O
O
O
BnO
BnO
BnO
BnO
OBn
O
OBn
O
O
BnO
O
BnO
O[CH2]8CO2Me
O[CH2]8CO2Me
25
26
vii
viii
1
2
Scheme 4 Reagents and conditions: i, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1, 81%; ii, NIS, TfOH (cat.), 4 Å molecular sieves,
DCM–Et2O 1:1, 83%; iii, TBAF–AcOH (3%), THF, 88%; iv, TBAF–AcOH (3%), 92%; v, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1,
51%; vi, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1, 53%; vii, Pd(OH)2/C, H2 1 atm, DCM–MeOH–H2O 3:3:1, 84%; viii, Pd(OH)2/C,
H2 1 atm, DCM–MeOH–H2O 3:3:1, 98%.
376
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isomers. High α-selectivity for the glycosidation of anomeric
halides in the presence of AgClO4 has been reported.12
smoothly and was followed by the coupling of the resulting
glycosyl acceptors 23 and 24 with the seleno glycoside 6 to
complete the synthesis of the fully protected target molecules
25 and 26. The moderate yields for the last glycosidations (51
and 53%) are acceptable considering the complexity of the
product molecules. The saccharides 25 and 26 were deprotected
by hydrogenation in a homogeneous mixture of dichloro-
methane, methanol and water (3:3:1) using Pd(OH)2/C as a
catalyst. The solvent system was chosen to keep both the start-
ing material and the product in solution. The deprotections
were carried out on a 150 mg scale and the resulting saccharides
1 and 2 were isolated as colourless amorphous solids after size
exclusion chromatography on Sephadex G-15 (eluent: H2O–
nPrOH, 95:5) in 84 and 98% yield respectively.
As for the preparation of the hexa- and heptasaccharide 1
and 2, a common trisaccharide intermediate was chosen in the
synthesis of the high mannose type saccharides 3 and 4, taking
advantage of the common structural motifs in both target mole-
cules (Scheme 5). Thus stereoselective glycosidation of the cen-
tral β-mannoside 11 with the α-1,2 linked selenoglycoside 9
under NIS–TfOH conditions gave the trimannoside 27 in 68%
yield. Subsequent desilylation with TBAF in THF gave the key
glycosyl acceptor 28 which underwent glycosidation reactions
with fluoro mannoside 6 and the seleno dimannoside 10
(Scheme 6). The resulting high mannosides 29 and 30 were
isolated as single anomers in 64 and 67% yield respectively.
When the perbenzylated selenophenyl mannoside 18 was
reacted with the primary alcohol of trisaccharide 28 an ano-
meric mixture at the newly formed glycosidic linkage was
observed (82%; α:β = 3:1). Cleavage of the benzyl and benzyl-
idene protecting groups was achieved under the previously
described conditions and completed the synthesis of the glyco-
conjugates 3 and 4. The final products were isolated as colour-
less amorphous solids in 99 and 98% yield after size exclusion
chromatography on Sephadex G-15 (eluent: H2O–nPrOH,
95:5).
The α-1,2 linked disaccharides 8 and 9 were prepared using
the selectively acetylated seleno- and thiomannosides 15 and 17,
which in turn were derived from the known orthoester precur-
sor 1413 (Scheme 2). Following a protocol introduced by Sinäy
and co-workers14 orthoester 14 was reacted with benzene-
selenol or ethanethiol in the presence of catalytic amounts of
HgBr2 to yield the seleno- and thiomannosides 15 and 16 in
excellent 96 and 94% yields respectively. Deacetylation of 16
under standard conditions furnished the glycosyl acceptor 17
which was subsequently reacted with the selenoglycosyl donors
15 and 18 using van Boom’s NIS–TfOH activation conditions.15
Due to their inherent higher reactivity the selenoglycosyl
donors 15 and 18 were selectively activated in the presence
of the thioglycosyl donor 17 resulting in the stereoselective
formation of the disaccharides 8 and 9 in 75 and 78% yield
respectively.
With building blocks 6–11 in hand the target glycoconjugates
1–5 were prepared in four to six steps.
Synthesis of heptasaccharide 1 and hexasaccharide 2 was
accomplished via the common intermediary trisaccharide 20.
According to Scheme 3 the dimannose-thioglycoside 8 was
reacted with the selectively protected central β-mannoside 11
under NIS–TfOH activation conditions to give the fully pro-
tected product trisaccharide 19 in 81% yield on a gram scale.
Standard deacetylation of 19 followed by glycosidation of the
resulting acceptor saccharide 20 with the selenophenyl glyco-
side 18 and 7 under NIS–TfOH conditions yielded the desired
tetra- and pentasaccharides 21 and 22 in 81 and 83% yields
respectively and completed the construction of the α-1,3-arm
of the target compounds 1 and 2 (Scheme 4).
Desilylation of the fully protected glycoconjugates 21 and 22
with tetrabutylammonium fluoride (TBAF) in THF proceeded
11
9
Synthesis of the N-glycans of L. mexicana amazonensis was
completed with the four step preparation of tetramannoside 5
(Scheme 7). Thus NIS–TfOH mediated coupling of the per-
benzylated selenomannoside 18 with the free secondary alcohol
of the central β-mannoside 11 resulted in the α-stereoselective
formation of dimannoside 31 in 78% yield. Standard desilyl-
ation followed by glycosidation of the resulting primary alco-
hol with the α-1,3 linked dimannoside 6 furnished the fully
protected tetrasaccharide 33 as its α-anomer in 64% yield,
which was deprotected in one step by hydrogenation using the
conditions described above. The desired tetrasaccharide 5 was
isolated in 96% yield as a colourless amorphous solid after size
exclusion chromatography on Sephadex G-15 (eluent: H2O–
nPrOH, 95:5).
OBn
OBn
O
i
BnO
BnO
OBn
O
O
BnO
BnO
OR
OBn
O
BnO
O
O[CH2]8CO2Me
27 R = TBDPS
28 R = H
ii
Scheme 5 Reagents and conditions: i, NIS, TfOH (cat.), 4 Å molecular
sieves, DCM–Et2O 1:1, 68%; ii, TBAF–AcOH (3%), THF, 92%.
The structures of the final glycoconjugates 1–5 and their
6
10
28
ii
i
OBn
OBn
O
BnO
BnO
OBn
OBn
OBn
OBn
O
Ph
O
OBn
O
O
OBn
BnO
O
O
BnO
BnO
BnO
BnO
OBn
O
BnO
BnO
OBn
OBn
O
O
O
O
iv
O
BnO
BnO
BnO
BnO
O
OBn
OBn
O
O
O
BnO
O
O[CH2]8CO2Me
O[CH2]8CO2Me
30
4
29
iii
3
Scheme 6 Reagents and conditions: i, NIS, TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1, 64%; ii, AgClO4, HfCp2Cl2, 4 Å molecular sieves,
Et2O, 67%; iii, Pd(OH)2/C, H2 1 atm, DCM–MeOH–H2O 3:3:1, 99%; iv, Pd(OH)2/C, H2 1 atm, DCM–MeOH–H2O 3:3:1, 98%.
J. Chem. Soc., Perkin Trans. 1, 1999, 375–378
377

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Table 1 Selected 1H NMR and 13C NMR data (δH, δC in ppm) for the
deprotected oligosaccharide derivatives 1–5 (in D2O)
1
precursors were confirmed by H and 13C NMR spectroscopy
(DQF–COSY, TOCSY, HMQC and HMBC; see Table 1), and
by mass spectrometry (high resolution, ESMS).16
Residue
Mana
Manb
Manc
Mand
Gloe
Atom
1
2
3
4
5
To summarize, the first chemical synthesis of the N-glycans
of gp63 of L. mexicana amazonensis has been achieved using
a strategy based on the principles of reactivity tuning and
orthogonal activation.
H-1
C-1
H-1
C-1
H-1
C-1
H-1
C-1
H-1
C-1
H-1
C-1
H-1
C-1
4.58
99.7
5.28
100.6
5.23
101.0
4.97
102.7
5.19
102.2
4.83
100.2
5.08
4.47
100.2
5.38
100.6
5.28
101.0
4.98
102.7
4.49
100.3
5.48
100.8
5.00
102.7
4.47
100.2
5.37
100.8
4.99
102.8
4.49
100.2
5.07
102.5
Acknowledgements
We thank the Novartis Research Fellowship (to S. V. L.) and the
Zeneca Strategic Research Fund for financial support.
ManbЈ
MancЈ
4.83
100.2
5.08
102.3
4.89
100.2
5.10
102.3
4.83
100.0
4.83
100.1
5.08
102.3
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11
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i
OBn
OBn
O
BnO
BnO
OR
OBn
O
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O
BnO
O[CH2]8CO2Me
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31 R = TBDPS
32 R = H
ii
6
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iii
OBn
OBn
OBn
O
BnO
BnO
OBn
O
O
Ph
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O
O
OBn
O
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BnO
BnO
O
OBn
O
16
OH
O
BnO
O
O[CH2]8CO2Me
HO
HO
OH
OH
OH
O
HO
OH
O
e
HO
33
5
O
HO
HO
OH
OH
O
d
OH
c'
O
HO
O
O
HO
HO
iv
b'
c
OH
O
O
O
HO
HO
OH
O
b
O
HO
Scheme 7 Reagents and conditions: i, NIS, TfOH (cat.), 4 Å molecular
sieves, DCM–Et2O 1:1, 78%; ii, TBAF–AcOH (3%), THF, 95%; iii,
NIS–TfOH (cat.), 4 Å molecular sieves, DCM–Et2O 1:1, 64%; iv,
Pd(OH)2/C, H2 1 atm, DCM–MeOH–H2O 3:3:1, 96%.
O[CH2]8CO2Me
a
Communication 8/09798D
378
J. Chem. Soc., Perkin Trans. 1, 1999, 375–378