Synthesis of 3-O-sulfonated heparan sulfate octasaccharides that inhibit the herpes simplex virus type 1 host-cell interaction

Article abstract of DOI:10.1038/nchem.1073

Cell surface carbohydrates play significant roles in a number of biologically important processes. Heparan sulfate, for instance, is a ubiquitously distributed polysulfated polysaccharide that is involved, among other things, in the initial step of herpes simplex virus type 1 (HSV-1) infection. The virus interacts with cell-surface heparan sulfate to facilitate host-cell attachment and entry. 3-O-Sulfonated heparan sulfate has been found to function as an HSV-1 entry receptor. Achieving a complete understanding of these interactions requires the chemical synthesis of such oligosaccharides, but this remains challenging. Here, we present a convenient approach for the synthesis of two irregular 3-O-sulfonated heparan sulfate octasaccharides, making use of a key disaccharide intermediate to acquire different building blocks for the oligosaccharide chain assembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugars blocked viral infection in a dosage-dependent manner with remarkable similarity to one another.

Full text of DOI:10.1038/nchem.1073

ARTICLES
PUBLISHED ONLINE: 19 JUNE 2011 | DOI: 10.1038/NCHEM.1073
Synthesis of 3-O-sulfonated heparan sulfate
octasaccharides that inhibit the herpes simplex
virus type 1 host–cell interaction
1,2
1,2
3
1
1
†
†
Yu-Peng Hu , Shu-Yi Lin , Cheng-Yen Huang , Medel Manuel L. Zulueta , Jing-Yuan Liu ,
Wen Chang3 and Shang-Cheng Hung1,4
*
Cell surface carbohydrates play significant roles in a number of biologically important processes. Heparan sulfate, for
instance, is a ubiquitously distributed polysulfated polysaccharide that is involved, among other things, in the initial step
of herpes simplex virus type 1 (HSV-1) infection. The virus interacts with cell-surface heparan sulfate to facilitate host-cell
attachment and entry. 3-O-Sulfonated heparan sulfate has been found to function as an HSV-1 entry receptor. Achieving a
complete understanding of these interactions requires the chemical synthesis of such oligosaccharides, but this remains
challenging. Here, we present a convenient approach for the synthesis of two irregular 3-O-sulfonated heparan sulfate
octasaccharides, making use of a key disaccharide intermediate to acquire different building blocks for the oligosaccharide
chain assembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugars blocked viral infection in a
dosage-dependent manner with remarkable similarity to one another.
eparan sulfate (HS) is a linear polysaccharide that is wide- (herpes virus entry mediator, nectin-1 and a unique 3-O-sulfonated
spread on the cell surface and in the extracellular matrix, as HS) are located12,13. The receptor interacts with gD (ref. 14), another
H
well as in the basement membrane1. Commonly found as a envelope glycoprotein, altering its conformation and initiating viral
component of proteoglycans, this sugar is polysulfated and consists penetration through the formation of a fusion-active complex that
of alternating 1 ꢀ 4-linked uronic acid (either b-D-glucuronic acid also includes gB and the gH/gL heterodimer15,16. In 2002, the litera-
(GlcUA) or a-L-iduronic acid (IdoUA)) and a-D-glucosamine ture reported the first gD-binding 3-O-sulfonated octasaccharide
(GlcN) residues2. The commercial anticoagulant heparin is a generated from a library of partially depolymerized HS by the
structural relative of HS that is typically sequestered in vivo by mas- HS-glucosaminyl-3-O-sulfotransferase isoform
3
(3-OST-3)17.
tocytes3. The biosynthesis of HS and heparin includes the formation Represented here by compound 1, the octasaccharide specially
of a bridging tetrasaccharide attached to serine side chains of a core carries the 3-O-sulfonate group as well as a free amino group at
protein, followed by the assembly of a long precursor chain charac- the reducing end residue (Fig. 1). The fourth residue from the redu-
terized by the GlcUA–N-acetyl-D-glucosamine (GlcNAc) repeating cing end was not fully established and could be either IdoUA2S or
pattern. Subsequent enzyme-mediated N-deacetylation/N-sulfona- GlcUA2S (2S: 2-O-sulfonate). Based on the identified structure, four
tion, uronic acid 5-C-epimerization, uronic acid 2-O-sulfonation likely variants could be extrapolated for the gD-binding sequence.
and glucosamine 6-O- and 3-O-sulfonations generate the mature Recently, compound 2, furnished by the 3-OST-3-mediated sulfo-
polysaccharide4. The incomplete nature of these modifications and nation of a heparin-degraded octasaccharide, was identified to
the varying specificities of participating enzyme isoforms deliver a inhibit both attachment and entry processes18. The specific 3-O-sul-
structure with extensive microheterogeneity5. Through the variable fonate group was located on the third residue from the reducing end.
make-up of its primary sequence, HS is able to mediate and/or The less defined non-reducing end residue in this structure offered
modulate the activity of a host of biomolecular agents directly affect- two possibilities for the natural HS chain sequence.
ing key physiological events, including blood coagulation, tissue
repair and remodelling, development, angiogenesis, and bacterial action necessitates structurally well-defined oligosaccharides acces-
and viral infections6.
sible mainly through chemical synthesis. Synthetic efforts directed
A complete understanding of the nature of HS–protein inter-
Herpes simplex virus type 1 (HSV-1), a prevalent human patho- towards the heparin–antithrombin interaction formed the basis of
gen causing chronic and recurrent mucocutaneous lesions, belongs the anticoagulant Fondaparinux, now in clinical use19. However,
to the neurotropic subgroup of the herpes virus family7,8. Cell- although the chemical synthesis of the regular sequences appearing
surface HS proteoglycans play dual roles not only in assisting cellu- in HS/heparin, particularly the IdoUA2S–GlcNS6S couple
lar attachment of HSV-1, but also in inducing viral entry into the (GlcNS6S: N- and 6-O-sulfonated GlcN), has been well documen-
host cell9,10. The virion envelope glycoproteins gB and gC bind ted20–28, the preparation of irregular structures remains a consider-
HS chains, which are richly expressed in filopodia-like projections able hurdle. In fact, before this article, no report had been made
on the cell surface11. HS-attached virions are then transported about the synthesis of irregular HS oligosaccharides longer than
onto the cell body, where three known viral entry receptor types six residues, and an attempt to construct
a gD-binding
1Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan, 2Department of Chemistry, National Tsing Hua University,
101, Section 2, Kuang-Fu Road, Hsinchu 300, Taiwan, 3Institute of Molecular Biology, Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan,
4Department of Applied Chemistry, National Chiao Tung University, 1001, Ta-Hsueh Road, Hsinchu 300, Taiwan; †These authors contributed equally to this
work. e-mail: schung@gate.sinica.edu.tw
*
NATURE CHEMISTRY | VOL 3 | JULY 2011 | www.nature.com/naturechemistry
557
© 2011 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
OH
O
OH
O
OH
O
–O2C
–
–O2C
–O2C
O
NHAc
–O3SO
OSO3
O
OH
OH
OH
O
O
O
HO
O
O
O
–
O
O
O
–
R1
NHSO3
–O3SO
HO
NHSO3
–O3SO
HO
–O3SO
R
NH2
–
CO2
–O2C
O
O
3: R=α- OMe, R1 =
1: R=α- or β-OH, R1 =
HO
HO
HO
OH
OH
OTBDPS
O
OAc
O-2-NAP
OAc
OTBDPS
O
O
OBn
O
O
STol
OLev
O
HO
BzO
LevO
PBBO
O
O
O
N
R1
BnO
BnO
HO
PBBO
R
OBz
OMe
3 BnO
CbzHN
BzO
6
9
5
7: R=N3, R1 =TCI
8: R=NAc2, R1 =α-STol
OTBDPS
O
OTBDPS
O
O
O
NH
+
TCI=
OAc
OCCCl3
STol
O
HO
BnO
O
O
2-NAPO
PBBO
2-NAPO
PBBO
N
N3
OBz
3 BnO
OBz
10
11
12
OTBDPS
O
OAc
OBn
OTBDPS
O
OTBDPS
O
O
OBn
BnO
O
O
O
2-NAPO
PBBO
O
OBz
N3
O
N
HO
BzO
HO
BnO
TCI
TCI
OMe
3 BnO
N3
BzO
BzO
13
14
15
16
–
–
–
–
OSO3
O
OSO3
O
OSO3
O
OSO3
O
–O2C
O
–O2C
O
–O2C
O
OH
OH
OH
R
O
O
O
O
–
O
–
O
HO
O
–
NHSO3
R1
NHSO3
–O3SO
–
NHSO3
–O3SO
HO
–O3SO
HO
NHSO3
–O3SO
–
–O2C
HO
CO2
OH
O
2: R=α- or β-OH, R1 =
4: R=α- OMe, R1 =
O
HO
–O3SO
–
OSO3
Figure 1 | Specific 3-O-sulfonated octasaccharides 1 and 2 and retro-synthetic design of the corresponding a-methylated glycosides 3 and 4. Synthetic
targets 3 and 4 were based on the structure of compounds 1 and 2, respectively. A common disaccharide intermediate 10, prepared from the D-glucosamine-
derived donor 11 and 1,6-anhydro-L-idopyranosyl acceptor 12, was used in the generation of several disaccharide building blocks for the total synthesis of
compounds 3 and 4. Ac, acetyl; Bn, benzyl; Bz, benzoyl; Cbz, benzyloxycarbonyl; Lev, levulinyl; 2-NAP, 2-naphthylmethyl; PBB, p-bromobenzyl; STol,
thiotoluenyl; TBDPS, tert-butyldiphenylsilyl; TCI, trichloroacetimidate.
octasaccharide based on compound 1 resulted in failure at the hexa- which the concept illustrated in Fig. 1 was eventually adopted
saccharide level29. In an effort to improve the current knowledge because of its robustness and simplicity.
about the interaction between HSV-1 and HS, we present herein a
We initially encountered some trouble in making the glycosidic
concise synthesis of the irregular a-methylated octasaccharides 3 bond between the third and fourth residues from the reducing
and 4, deducible from the recognized 3-O-sulfonated structures 1 end of octasaccharide 3. The trifluoroacetyl group used to block
and 2, respectively. The ability of these compounds to inhibit the amino group that would eventually become acetylated also pre-
HSV-1 infection of Vero cells has also been evaluated.
sented deprotection problems. After some exploration, a promising
route was carried out using disaccharide building blocks 6–8 for the
core residues and the monosaccharide derivatives 5 and 9 as non-
Results and discussion
Retrosynthesis. Notable concerns posed by the preparation of reducing and reducing end units, respectively. The chain would be
compounds 3 and 4 are the manner of chain backbone assembly, assembled by the initial couplings of compounds 8, 7 and 9 to
regio- and stereochemical control in glycosidic bond formation, form a pentasaccharide. Here, the levulinyl (Lev) groups would
the different functional make-up of the amino groups (that is, allow the generation of glycosyl acceptors for further condensation
NHSO32, NHAc and NH2), and the differentiation of alcohol with the trichloroacetimidate (TCI) and thiotoluenyl (STol) donors.
groups that would remain free, oxidized or O-sulfonated. For The octasaccharide would then be accessible by the introduction of a
octasaccharide 3, GlcUA was selected as the non-reducing end trisaccharide fashioned from compounds 5 and 6. The 1,2-trans
unit to ease the identification of a/b-stereochemistry, and stereoselectivity of these glycosidations would be ensured by the
IdoUA2S was arbitrarily chosen as the fourth residue from the adjacent Lev and benzoyl (Bz) groups via neighbouring-group par-
reducing end. Conversely, we decided to use IdoUA2S at the non- ticipation. The orthogonal azide (N3), N,N-diacetyl (NAc2) and
reducing end of octasaccharide 4 in view of the semi-repeating benzylcarbamate (NHCbz) were designated as masking groups,
pattern of compound 2. The difficulty inherent in the synthesis of leading to NHSO32, NHAc and NH2, respectively. Together with
the target compounds compelled us to try several approaches, of Bz, the primary acetyl (Ac) groups could be cleaved under basic
558
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© 2011 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
OTMS
O
OH
O
OAc
OBn
OTBDPS
O
OTBDPS
O
O
14
+
a
b
a
STol
STol
TMSO
TMSO
2-NAPO
HO
N3
N3
O
O
BzO
O
O
2-NAPO
PBBO
O
N
N3
3 BnO
OBz
15
17
18
BzO
26
OTBDPS
O
O
OTBDPS
O
OTBDPS
O
OAc
OBn
OTBDPS
O
c
O
O
O
STol
RO
N
2-NAPO
PBBO
N3
3 BnO
OBz
b
PBBO
O
OBz
O
BzO
O
O
O
PBBO
O
N
2-NAP
N3
3 BnO
10: R=2-NAP
11
h
i
f
BzO
6: R=H
2
OAc
OBn
21: R=Lev
27
OTBDPS
O
d
O
O
R1
OTBDPS
O
OAc
OBn
OTBDPS
O
OAc
OBn
RO
PBBO
N3
OTBDPS
O
O
BzO
c
20: R=2-NAP, R1 =OAc
22: R=Lev, R1 =OAc
14: R=2-NAP, R1 =TCI
7: R=Lev, R1 =TCI
O
O
BzO
O
O
O
PBBO
O
N3
2-NAP
N3
O
O
R
RO
N
OR1
g
3 BnO
R1O
BzO
BzO
2
g
19: R=2-NAP, R1 =H
15: R=H, R1 =Bz
e
28: R=OAc
29: R=TCI
d
g
j
32: R=α-STol
OAc
OAc
OTBDPS
O
OTBDPS
O
OTBDPS
O
h, i
OBn
OBn
STol
STol
f (for 29)
or
h (for 32)
O
O
O
O
2-NAPO
LevO
PBBO
R
R
HO
BnO
OMe
PBBO
N3
BzO
BzO
30: R=H
23: R=N3
24: R=NHAc
25: R=NHAc
8: R=NAc2
e
k
l
16: R=TBDPS
OAc
OBn
OTBDPS
O
OAc
OBn
OTBDPS
O
OTBDPS
O
Figure 2 | Preparation of disaccharide building blocks 6, 7, 8, 14 and 15.
.
a, 2-C10H9CHO, TMSOTf, CH2Cl2, 2 h; BH3 THF, TMSOTf, 77% (one pot);
b, TBDPSCl, DMAP, Et3N, CH2Cl2; NaH, p-bromobenzyl bromide, DMF,
71% (one pot); c, 12, NIS, TfOH, CH2Cl2, 278 8C ꢀ 220 8C, 4 h, 79%;
d, (1) Pd2(dba)3, N-methylaniline, (o-biphenyl)P(t-Bu)2, sodium tert-
butoxide, toluene, 80 8C, 5 h; (2) SnCl4, CH2Cl2, 10 min, 63% (two steps);
e, (1) Benzoyl chloride, Et3N, DMAP, CH2Cl2, 89%; (2) DDQ, CH2Cl2/H2O
(18/1, v/v), 87%; f, Cu(OTf)2, Ac2O, 20: 95%, 22: 94%; g, (1) Saturated
NH3(g) in MeOH/THF solution; (2) CCl3CN, K2CO3, CH2Cl2, 14: 88% (two
steps), 7: 90% (two steps); h, DDQ, CH2Cl2/H2O (18/1, v/v), 4 h, 6: 93%;
i, Lev2O, Et3N, DMAP, CH2Cl2, 21: 95%, 25: 81% (two steps); j, TMSSTol,
ZnI2, CH2Cl2, 3 h, 92%; k, Thioacetic acid, Pyr, CHCl3, 87%; l, Isopropenyl
acetate, p-toluenesulfonic acid, 65 8C, 78%. Ac2O, acetic anhydride;
Cu(OTf)2, copper(II) trifluoromethanesulfonate; DDQ, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone; DMAP, 4-(N,N-dimethylamino)pyridine;
DMF, N,N-dimethylformamide; Lev2O, levulinic anhydride; NIS,
N-iodosuccinimide; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium;
Pyr, pyridine; TBDPSCl, tert-butylchlorodiphenylsilane; TfOH,
O
O
O
O
BnO
O
O
PBBO
O
N3
OMe
R
N3
N3
BzO
BzO
BzO
2
31: R=2-NAP
33: R=H
i
Figure 3 | Preparation of heptasaccharide acceptor 33. a, TMSOTf, CH2Cl2,
278 8C ꢀ 240 8C ꢀ 0 8C, 2 h, 83%; b, (1) DDQ, CH2Cl2/H2O (18/1, v/v),
2 h, 82%; (2) 14, TMSOTf, CH2Cl2, 278 8C ꢀ 240 8C ꢀ 0 8C, 2 h, 76%;
c, Cu(OTf)2, Ac2O, 83%; d, (1) Saturated NH3(g) in MeOH/THF (1/10, v/v),
76%; (2) CCl3CN, K2CO3, CH2Cl2, 92%; e, TBDPSCl, Et3N, DMAP, CH2Cl2,
3.5 h, 92%; f, TMSOTf, CH2Cl2, 278 8C ꢀ 0 8C, 2 h, 76%; g, TMSSTol,
ZnI2, CH2Cl2, 2 h, 92%; h, NIS, TfOH, CH2Cl2, 278 8C ꢀ 0 8C, 2 h, 76%;
i, DDQ, CH2Cl2/H2O (18/1, v/v), 2 h, 74%.
1,6-anhydro ring in 15, which could allow facile incorporation of
a leaving group for further coupling. In addition to the hydroxyl
positions protected by the Bz groups, the orthogonal TBDPS moi-
eties also mask the primary alcohols that would ultimately hold sul-
fonate groups. PBB, Bn, N3 and Ac groups have roles similar to
those stated for compound 3.
trifluoromethanesulfonic acid; THF, tetrahydrofuran; TMS, trimethylsilyl;
TMSOTf, trimethylsilyl trifluoromethanesulfonate; TMSSTol,
trimethyl(4-methylphenylthio)silane.
conditions and the primary hydroxyls would be regioselectively
All the disaccharide building blocks 6, 7, 8, 14 and 15 would be
oxidized to the corresponding carboxylate. The 2-naphthylmethyl accessible through simple transformations from the common inter-
(2-NAP) group temporarily protects the sole primary alcohol that mediate 10, which could be prepared by coupling monosaccharide
would be sulfonated simultaneously with the debenzoylated second- units 11 and 12. Conceptually, the bulky TBDPS group and the
ary alcohols. All the ether groups (benzyl (Bn), p-bromobenzyl non-participating 2-C-N3 group would enhance the stereoselective
(PBB) and tert-butyldiphenylsilyl (TBDPS)) act as permanent a-glycosidation of the D-glucosamine-derived donor 11. Aside from
protection and would be removed in the late stage of the synthesis this, the N3 group could be readily manipulated into various amine
to reveal the free hydroxyls.
derivatives. Both the PBB and 2-NAP groups could be selectively
The envisioned strategy for octasaccharide 4 assembly involved cleaved and transformed into other functional moieties as necessary.
building blocks 13–16. A hexasaccharide, formed by two successive
elongations of the disaccharide acceptor 15 with the disaccharide Disaccharide building block syntheses. The preparative routes
donor 14, would be capped by the monosaccharide derivatives 13 for the disaccharide derivatives are depicted in Fig. 2. Through our
and 16 at the non-reducing and reducing ends, respectively. recently conceived regioselective one-pot protection strategy30–32
,
Chain elongation would be made possible by the 2-NAP group, the 3,4,6-tri-O-trimethylsilylated thioglycoside 17 was transformed
enabling transformation to the glycosyl acceptor and the into the 3,6-diol 18 by a trimethylsilyl trifluoromethanesulfonate
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© 2011 Macmillan Publishers Limited. All rights reserved.
559

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
OAc
OBn
OTBDPS
O
OAc
OBn
OTBDPS
O
OAc
OBn
O
O
BzO
O
O
STol
O
O
PBBO
O
N3
R
N3
b
BnO
O
BzO
BzO
RO
BnO
2
O
OBz
TCI
BzO
32: R=2-NAP
36: R=H
d
13
12: R=H
34: R=Bn
a
c
e
(13+33)
(13+36+16 in one-pot)
OR1
OBn
OR1
OBn
OR1
OBn
OTBDPS
O
OTBDPS
O
OTBDPS
O
g
O
O
RO
O
O
BnO
O
O
PBBO
O
OMe
N3
O
BnO
RO
N3
N3
RO
RO
2
35: R=Bz, R1 =Ac
37: R=R1 =H
f
OTBDPS
O
OTBDPS
OTBDPS
O
O
h
BnO
BnO
O
O
BnO
BnO
O
HO
O
O
PBBO
BnO
O
N3
O
O
N3
OMe
N3
O
O
O
O
O
O
2
38
OR
O
OR
O
OR
O
–O2C
O
–O2C
O
–O2C
BnO
j
OBn
OBn
OBn
4
O
O
RO
O
BnO
O
O
PBBO
OMe
N3
O
N3
N3
RO
RO
RO
2
39: R=H
40: R=SO3
i
–
Figure 4 | Synthesis of target octasaccharide 4. a, Ag2O, BnBr, CH2Cl2, 83%; b, (1) Cu(OTf)2, Ac2O, 90%; (2) saturated NH3(g) in MeOH/THF (1/10, v/v),
83%; (3) CCl3CN, K2CO3, CH2Cl2, 97%; c, 13, TMSOTf, CH2Cl2, 278 8C ꢀ 240 8C ꢀ 0 8C, 2 h, 78%; d, DDQ, CH2Cl2/H2O (18/1, v/v), 2 h, 71%;
e, 13, TfOH, CH2Cl2, 278 8C ꢀ 240 8C ꢀ 220 8C, 2 h; 16, NIS, TfOH, 278 8C ꢀ 0 8C, 3 h, 31% (one pot); f, NaOMe, MeOH/CH2Cl2 (1/1, v/v), 78%;
.
g, TEMPO, BAIB, CH2Cl2/H2O (1/1, v/v), 68%; h, (1) tetra-n-butylammonium fluoride, AcOH, THF; (2) LiOH, THF, 1 h, 67% (two steps); i, SO3 Et3N, DMF,
.
60 8C, 62%; j, (1) Pd(OH)2/C, H2, phosphate buffer pH ¼ 7, MeOH; (2) SO3 Pyr, pH ¼ 9.5, H2O, 3 h, 72% (two steps). AcOH, acetic acid; BAIB,
bis(acetoxy)iodo-benzene; BnBr, benzyl bromide; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxyl free radical.
(TMSOTf)-promoted two-step process. Regioselective 6-O-silylation the fully protected disaccharide 22. Treatment of 22 with saturated
of 18 catalysed by 4-(N,N-dimethylamino)pyridine (DMAP), NH3(g) followed by subsequent imidation provided the correspond-
followed by 3-O-p-bromobenzylation under Williamson’s conditions, ing TCI derivative 7.
furnished the fully protected thioglycoside 11 (71%) in a one-pot
Compound 8, with NAc2 protection and a STol leaving group,
manner. Coupling of the glycosyl donor 11 with alcohol 12 (ref. 33) was generated via intermediate 20. We opted to form the thioglyco-
via an N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid side, because NAc2 would not withstand the anomeric deacetylation
(TfOH) promoter combination led to the desired a-linked required before imidate formation. Thus, conversion of the 1-C
disaccharide 10 (79%, J ¼ 3.8 Hz for 1′-H) as a single isomer. acetate of 20 into the thioether was carried out by treatment with
Removal of the PBB group at the 3′-O position of 10 was carried out trimethyl(4-methylphenylthio)silane (TMSSTol) and ZnI2, generat-
through palladium-catalysed amination with N-methylaniline, ing the corresponding thioglycoside 23 in excellent 92% yield. This
followed by treatment with tin tetrachloride34. Cleavage of the 2-O- ZnI2-promoted thioether substitution at the anomeric centre toler-
Bz group also occurred in this transformation, giving the 2,3′-diol 19 ated different functional groups in the substrate, unlike the conven-
.
in 63% yield. Dibenzoylation of 19 followed by 2,3-dichloro-5,6- tional BF3 Et2O/p-thiocresol tandem. Subsequently, azido group
dicyano-1,4-benzoquinone (DDQ)-mediated cleavage of the 4′-O-2- transformation in 23 using thioacetic acid yielded the N-acetylated
NAP group provided the desired alcohol 15.
derivative 24 (87%). Successive 4′-O-denaphthylmethylation and
1,6-Anhydro ring opening in compound 10 with acetic anhy- levulinylation led to compound 25 (81% in two steps), which, on
dride (Ac2O) using copper(II) trifluoromethanesulfonate reaction with isopropenyl acetate in catalytic p-toluenesulfonic
(Cu(OTf)2) as catalyst35 afforded the 1,6-diacetate 20 in excellent acid, provided the desired building block 8 in 78% yield.
yield. The 2-NAP and TBDPS moieties were stable under this con-
dition, but the p-methoxybenzyl group did not survive when used in Synthesis of compound 4. The preparation of the target
place of 2-NAP. Regioselective 1-O-deacetylation of 20 with satu- octasaccharide 4 started from the TMSOTf-promoted coupling of
rated NH3(g) gave the 1-alcohol, which was reacted with CCl3CN the disaccharide donor 14 with the 4′-alcohol 15 (Fig. 3). The
and K2CO3 to obtain the glycosyl donor 14.
fully protected tetrasaccharide 26, isolated in 83% yield as a single
The 2-NAP group in compound 10 was removed by DDQ, isomer, was treated with DDQ delivering the corresponding
leaving the 4′-alcohol 6 in 93% yield. Installation of Lev in the 4′′′-alcohol. Further elongation with 14 afforded the desired
free hydroxyl of 6 using levulinic anhydride in the presence of hexasaccharide 27 (76%). Acetolysis of the 1,6-anhydro ring in 27
DMAP delivered ester 21, which underwent acetolysis to furnish furnished the ester 28 (83%), which underwent regioselective 1-O-
560
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
OAc
OBn
OR2
O
OTBDPS
O
OTBDPS
O
O
OAc
O
OAc
O
STol
c
e
O
R1O
R1O
O
STol
O
BnO
O
O
N3
O
PBBO
OBz
N3
OR
PBBO
BnO
BnO
BnO
BnO
BzO
OR
41: R=R1 =R2 =TMS
42: R=R2 =H, R1 =Bn
5: R=Lev, R1 =Bn, R2 =Ac
BnO
a
b
43: R=Lev
44: R=Bn
45
j
d
OAc
OBn
OAc
OBn
OAc
OBn
OTBDPS
O
OTBDPS
O
O-2-NAP
O
O-2-NAP
OTBDPS
O
7
O
h
f
O
O
BzO
O
+
O
O
O
BzO
O
NAc2
O
O
PBBO
RO
PBBO
N3
N3
OMe
RO
PBBO
OMe
9
CbzHN
CbzHN
BzO
BzO
BzO
46: R=Lev
47: R=H
48: R=Lev
49: R=H
g
i
OAc
OBn
OAc
OBn
OTBDPS
O
OAc
OBn
OTBDPS
O
O-2-NAP
O
OTBDPS
O
OAc
k
O
O
O
BzO
O
NAc2
O
O
O
O
O
PBBO
O
PBBO
N3
O
PBBO
N3
OMe
CbzHN
BnO
BnO
BzO
BzO
BzO
BnO
50
OTBDPS
O
O-2-NAP
OTBDPS
O
OTBDPS
O
O
l
BnO
BnO
BnO
O
O
O
HO2C
O
O
O
O
O
HO
PBBO
OMe
PBBO
O
O
O
N3
AcHN
CbzHN
N3
BnO
BnO
PBBO
BnO
O
O
O
O
O
O
51
OTBDPS
OTBDPS
OTBDPS
OR
O
MeO2C
MeO2C
OBn
O
MeO2C
O
OBn
O
OBn
n
O
O
O
RO
O
NHAc
MeO2C
O
O
O
O
O
PBBO
O
PBBO
N3
O
PBBO
N3
OMe
CbzHN
BnO
BnO
RO
RO
RO
BnO
52: R=H
53: R=SO3
m
–
OH
–
OH
OSO3
O
OH
–O2C
–O2C
O
–O2C
OBn
O
OBn
O
OBn
p
–O2C
BnO
O
O
–O3SO
3
O
O
O
NHAc
–O3SO
O
O
PBBO
O
O
O
PBBO
R
O
PBBO
OMe
R
–O3SO
CbzHN
–O3SO
BnO
BnO
54: R=N3
55: R=NH2
o
.
Figure 5 | Synthesis of target octasaccharide 3. a, Benzaldehyde, TMSOTf, CH2Cl2, 278 8C, 2 h; benzaldehyde, Et3SiH, TMSOTf, 278 8C, 4 h; BH3 THF,
TMSOTf, 5 h, 86% (one pot); b, (1) Ac2O, Et3N, CH2Cl2, 83%; (2) Lev2O, Et3N, DMAP, CH2Cl2, 98%; c, 6, TfOH, NIS, CH2Cl2, 278 8C ꢀ 220 8C, 3 h,
92%; d, (1) NH2NH2, acetic acid, Pyr, 5 h, 93%; (2) NaH, BnBr, CH2Cl2/DMF (30/1, v/v), 0.5 h, 82%; e, (1) Cu(OTf)2, Ac2O, 88%; (2) TMSSTol, ZnI2,
CH2Cl2, 3 h, 95%; f, TMSOTf, CH2Cl2, 240 8C, 3 h, 70%; g, NH2NH2, AcOH, Pyr, 5 h, 85%; h, 8, NIS, TfOH, CH2Cl2, 278 8C ꢀ 220 8C, 4 h, 91%;
i, NH2NH2, AcOH, Pyr, CH2Cl2, 1.5 h, 81%; j, NIS, TfOH, CH2Cl2, 278 8C ꢀ 220 8C, 4 h, 89%; k, (1) NaOMe, MeOH/CH2Cl2 (1/1.3, v/v), 85%;
(2) TEMPO, BAIB, CH2Cl2/H2O (1/1, v/v), 80%; l, (1) DDQ, CH2Cl2/H2O (18/1, v/v), 3.5 h, 71%; (2) LiOH, THF, 1 h; (3) CH2N2, CH2Cl2, Et2O, 81%
.
.
(two steps); m, SO3 Et3N, DMF, 60 8C, 76%; n, (1) HFPyr, Pyr, 83%; (2) LiOH, H2O2, H2O, THF, MeOH, 37 8C, 74%; o, 1,3-propanedithiol, Et3N, Pyr/H2O
.
(4/1, v/v), 50 8C, 81%; p, (1) SO3 Pyr, NaOH(aq), Et3N, MeOH; (2) Pd(OH)2/C, H2, phosphate buffer pH ¼ 7, 47% (two steps).
deacetylation to provide the corresponding 1-alcohol in 76% yield. thioether was carried out by treatment with TMSSTol and ZnI2, fur-
Other acetyl groups were presumably cleaved in this reaction, nishing the thioglycoside 32 in 92% yield. The expected heptasac-
causing a yield drop in comparison with the preparation of charide 31 was acquired in 76% yield through the condensation of
donors 14 and 7. Reaction of this hemiacetal with CCl3CN and 32 and 16 in the presence of NIS and TfOH. This two-step pro-
K2CO3 led to the expected TCI donor 29. We next turned our cedure is shorter than the TCI route, with an improved 70%
attention to the synthesis of the reducing end sugar unit 16. overall yield from compound 28. Cleavage of 2-NAP in 31 by
Methyl 2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranoside (30) DDQ generated the heptasaccharide acceptor 33.
was prepared in 49% overall yield through a six-step protocol
Preparation of the target molecule 4 is summarized in Fig. 4. The
starting from N-acetyl-D-glucosamine (see Supplementary non-reducing end L-ido sugar 13 was prepared through 4-O-benzy-
Information). Regioselective installation of TBDPS in 30 led to lation (Ag2O, benzyl bromide) of alcohol 12, forming ether 34 fol-
4-alcohol 16 (92%), which was glycosidated by donor 29 under lowed by the usual acetolysis–deacetylation–imidation protocol. On
TMSOTf activation to give heptasaccharide 31 in 76% yield.
TMSOTf activation, coupling of 13 and acceptor 33 gave the fully
An alternative coupling procedure was also studied. protected octasaccharide 35 in 78% yield. Alternatively, a one-pot
Transformation of the 1-C-acetate of the hexasaccharide 28 to the [1 þ 6 þ 1] glycosylation was investigated. First, the 2-NAP group
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561
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
120
100
80
60
40
20
0
of thioglycoside 32 was cleaved to provide alcohol 36. TfOH-pro-
moted coupling of 13 with 36 followed by addition of alcohol 16
in the presence of NIS and TfOH afforded the octasaccharide 35
in a moderate yield of 31%.
Compound 3
Compound 4
HS
With compound 35 in hand, a series of functional group trans-
formations was carried out. Removal of all ester groups using
sodium methoxide gave alcohol 37 (78%), which underwent regiose-
lective 2,2,6,6-tetramethyl-1-piperidinyloxyl free radical (TEMPO)-
oxidation of the primary hydroxyl groups to yield lactone 38 (68%).
The structure of 38 was confirmed by several two-dimensional
nuclear magnetic resonance (NMR) experiments, indicating the sig-
nificant downfield shifts of the 2-H signals on all four L-ido rings (see
Supplementary Information). Desilylation by tetra-n-butylammo-
nium fluoride in the presence of acetic acid followed by lactone
ring opening with LiOH supplied the corresponding carboxylate
39. Per-O-sulfonation of all hydroxyls with sulfur trioxide triethyl-
Blank
1
5
10
20
40
100
100
Sugar concentration (µgml–1
)
Figure 6 | Inhibition of HSV-1 infection of Vero cells by compounds 3 and
4. Vero cells were infected with HSV-1 in the absence or presence of either
octasaccharide 3 or 4 for 60 min on ice. A commercially acquired HS was
also tested for comparison. The cells were then washed with phosphate-
buffered saline and overlaid with 1% agarose for plaque determination
assays as described in the Methods. The number of plaques obtained in the
absence of sugars was assigned as 100% and referred to as blank. The
results displayed are means of duplicate batches, each batch carried out in
two trials. Error bars are standard deviations of the means of the
individual batches.
.
amine complex (SO3 Et3N) led to the corresponding sulfate deriva-
tive 40 in 62% yield. Hydrogenolysis was carried out to cleave all
Bn and PBB groups simultaneously with the conversion of the
azido into the amino groups. The amino-alcohol was subjected to
.
N-sulfonation with sulfur trioxide pyridine complex (SO3 Pyr)
through pH control at ꢁ9.5 to afford the crude product 3.
Purification through Sephadex G25 and ion-exchange with Dowex
50WX8-Naþ resin furnished the target molecule 4 in 72% yield
from 40. Analysis of one- and two dimensional NMR spectra (see
Supplementary Information) confirmed the structure of compound
4. High-resolution electrospray ionization–mass spectrum (ESI-MS) resulted in cleavage of all O-acyl (Bz and Ac) groups and the con-
gave a m/z value of 871.5614 corresponding to [M þ 9Na–12H]32 version of NAc2 into NHAc, fashioning a polyhydroxylated com-
and the formula C49H71N4O80S13Na9 (calculated value, 871.5608).
pound (85%) that, on TEMPO oxidation, gave the lactone and
carboxylate derivative 51 in 80% yield. DDQ-promoted removal
of 2-NAP provided the corresponding 6-alcohol, which underwent
successive lactone ring opening with LiOH, and methylation of the
generated carboxylate groups using diazomethane furnished com-
pound 52. The material obtained after LiOH treatment was diffi-
cult to purify, so methyl ester formation was deemed necessary to
afford a homogeneous compound for O-sulfonation, a critical step
in the transformation process. Treatment of pentaol 52 with
Synthesis of compound 3. Figure 5 depicts the synthetic method
used for the acquisition of compound 3. The per-O-
trimethylsilylated thioglycoside 41 was converted to the 2,6-diol
42 in an excellent 86% yield via the TMSOTf-promoted one-pot
process30–32, which included 4,6-O-benzylidene formation, 3-O-
benzylation and 6-O-ring opening. Regioselective 6-O-acetylation
followed by 2-O-levulinylation generated the non-reducing end
monosaccharide synthon 5, which was coupled with the glycosyl
.
SO3 Et3N smoothly generated the desired sulfate derivative 53 in
76% yield after Sephadex LH-20 column purification. The
TBDPS and methyl ester functionalities were then consecutively
removed, supplying product 54. To allow N-sulfonation, the
azido groups were selectively reduced to the free amine by 1,3-pro-
panedithiol and the desired derivative 55 was obtained in 81%
acceptor
6 under NIS/TfOH promotion to furnish the
trisaccharide 43 (92%) as a single isomer. The 2′′-O-Lev group,
used only in driving the 1,2-trans stereoselective glycosidation,
needs to be replaced in consideration of the final structure. Thus,
hydrazine treatment of 43 led to the 2′′-alcohol (93%), which was
.
yield. Immediate sulfonation of the amino groups using SO3 Pyr
benzylated under the NaH/benzyl bromide tandem in
a
in the presence of Et3N and NaOH followed by global hydrogeno-
lysis finally delivered target molecule 3 in 47% yield from
diamine 55, after gel filtration and Naþ cation exchange. As with
compound 4, the structure of 3 was analysed and supported by
one- and two-dimensional NMR experiments together with high-
resolution ESI-MS measurements (see Supplementary Information).
CH2Cl2/DMF (30/1, v/v) mixed solvent to afford the ether 44
(82%). In this etherification, the solvent combination, 30 min
reaction time and acetic acid quenching assisted in preserving the
installed ester moieties. The 1,6-anhydro ring of 44 was then
opened by acetolysis, and subsequent reaction with TMSSTol and
ZnI2 brought about the formation of the thioether 45.
The 4-alcohol 9 was prepared from the known methyl 2-N-benzyl-
oxycarbonyl-2-deoxy-a-D-glucopyranoside36 in three steps, with an Inhibition of viral infection. The capability of the synthesized
overall yield of 70% (see Supplementary Information). compounds in disrupting HSV-1 infection was next investigated.
Condensation of this acceptor with the TCI donor 7 delivered trisac- HSV-1 was mixed with various concentrations of octasaccharides
charide 46, which underwent hydrazine-mediated Lev cleavage to 3 and 4, and a commercially acquired HS was also tested for
afford acceptor 47. Further coupling with thioglycoside 8 under comparison. The resulting mixtures were used to infect Vero cells
NIS/TfOH activation led to pentasaccharide 48 (91%). Our attempts at 37 8C for 60 min, and plaque numbers were counted 3 days
at generating this glycosidic linkage gave very low yields when we used after infection. Both octasaccharides reduced plaque formation in
different lengths of TCI and STol donors having trifluoroacetyl as the a dosage-dependent manner and gave strikingly similar inhibition
protecting group for the amine destined for acetylation. Complete profiles (Fig. 6). Approximately 80% of the infection was inhibited
replacement of all hydrogen-bonding protons in the amine by acetyl at 40 mg ml21 and at 100 mg ml21; this closely resembles the
groups favoured the coupling reaction. Hydrazinolysis of 48 supplied results for compound 2 (40 mM) inhibition of HSV-1 infection of
alcohol 49, which was condensed with glycosyl donor 45 to obtain the HeLa cells18. The half-maximal inhibitory concentrations (IC50)
desired octasaccharide 50 in 89% yield.
for compounds 3 and 4 were determined to be 5.4 mg ml21 and
Compound 50 was next subjected to functional group trans- 3.9 mg ml21, respectively. In contrast, no significant inhibition was
formations en route to the final compound. Saponification of 50 observed with commercial HS at 100 mg ml21. These results
562
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1073
ARTICLES
15. Campadelli-Fiume, G. et al. The multipartite system that mediates entry of
herpes simplex virus into the cell. Rev. Med. Virol. 17, 313–326 (2007).
16. Reske, A., Pollara, G., Krummenacher, C., Chain, B. M. & Katz, D. R.
Understanding HSV-1 entry glycoproteins. Rev. Med. Virol. 17, 205–215 (2007).
17. Liu, J. et al. Characterization of a heparan sulfate octasaccharide that binds
to herpes simplex virus type 1 glycoprotein D. J. Biol. Chem. 277,
33456–33467 (2002).
18. Copeland, R. et al. Using a 3-O-sulfated heparin octasaccharide to inhibit the
entry of herpes simplex virus type 1. Biochemistry 47, 5774–5783 (2008).
19. Petitou, M. & van Boeckel, C. A. A. A synthetic antithrombin III binding
pentasaccharide is now a drug! What comes next? Angew. Chem. Int. Ed. 42,
3118–3133 (2004).
clearly demonstrate that the synthesized 3-O-sulfonated HS
octasaccharides competed with cell surface HS and blocked HSV-1
infections of Vero cells. It has been shown previously that
enzymatically generated compounds 1 and 2 have nearly identical
binding affinity with HSV-1 gD (refs 17, 18). These findings, and
our own data, suggest that differences in HS fine structures and
the location of the requisite 3-O-sulfonate group do not have a
sizeable effect on the extent of inhibition of HSV-1 infection, or
on gD binding for that matter. These assumptions therefore offer
greater flexibility in designing 3-O-sulfonate-containing HS
oligosaccharides that specifically prevent gD-mediated HSV-1
entry into the host cell.
20. de Paz, J. L. et al. The activation of fibroblast growth factors by heparin:
synthesis, structure, and biological activity of heparin-like oligosaccharides.
ChemBioChem 2, 673–685 (2001).
21. Laremore, T. N., Zhang, F., Dordick, J. S., Liu, J. & Linhardt, R. J. Recent progress
and applications in glycosaminoglycan and heparin research. Curr. Opin. Chem.
Biol. 13, 633–640 (2009).
22. Hung, S-C. et al. 1,6-anhydro-b-L-hexopyranoses as potent synthons in the
synthesis of the disaccharide units of bleomycin A2 and heparin. J. Am. Chem.
Soc. 123, 3153–3154 (2001).
23. Lee, J-C. et al. From D-glucose to biologically potent L-hexose derivatives:
synthesis of a-L-iduronidase fluorogenic detector and the disaccharide moieties
of bleomycin A2 and heparan sulfate. Chem. Eur. J. 10, 399–415 (2004).
Conclusions
We have successfully developed a convenient route for the synthesis
of the specific 3-O-sulfonated octasaccharides 3 and 4 correspond-
ing to the previously suggested gD-binding structures. The
preparation made use of key disaccharide intermediate 10, which
was effectively converted into various building blocks. Overall,
this is the first report of irregular HS octasaccharides acquired
through chemical synthesis. The in vitro inhibition of HSV-1
infection exhibited by these oligosaccharides is a step towards a
better understanding of viral attachment and entry. Interaction
between these sugars and HSV-1 glycoprotein gD, and particularly
the complex structure at the molecular level, will be further
studied to provide valuable information for the discovery of new
anti-HSV-1 drugs.
´
24. Codee, J. D. C. et al. A modular strategy toward the synthesis of heparin-like
oligosaccharides using monomeric building blocks in a sequential glycosylation
strategy. J. Am. Chem. Soc. 127, 3767–3773 (2005).
25. Noti, C., de Paz, J. L., Polito, L. & Seeberger, P. H. Preparation and use of
microarrays containing synthetic heparin oligosaccharides for the rapid analysis
of heparin–protein interactions. Chem. Eur. J. 12, 8664–8686 (2006).
26. Baleux, F. et al. A synthetic CD4–heparan sulfate glycoconjugate inhibits CCR5
and CXCR4 HIV-1 attachment and entry. Nat. Chem. Biol. 5, 743–748 (2009).
27. Arungundram, S. et al. Modular synthesis of heparan sulfate oligosaccharides for
structure–activity relationship studies. J. Am. Chem. Soc. 131, 17394–17405 (2009).
28. Lu, L-D. et al. Synthesis of 48 disaccharide building blocks for the assembly of a
heparin and heparan sulfate oligosaccharide library. Org. Lett. 8, 5995–5998 (2006).
29. Lohman, G. J. S. & Seeberger, P. H. A stereochemical surprise at the late stage of
the synthesis of fully N-differentiated heparin oligosaccharides containing
amino, acetamido, and N-sulfonate groups. J. Org. Chem. 69, 4081–4093 (2004).
30. Wang, C-C. et al. Regioselective one-pot protection of carbohydrates. Nature
446, 896–899 (2007).
Methods
Inhibition of HSV-1 infection by octasaccharides 3 and 4. Vero cells were seeded
at a density of 2 × 105 cells per well in six-well plates. Next day, the synthesized
sugars (compounds 3 or 4 at 1, 5, 10, 20, 40 and 100 mg ml21) or a commercially
acquired HS (Sigma, from bovine kidney, 100 mg ml21) were mixed with 200
plaque-forming units per well of HSV-1 (KOS) tk12 (ref. 37) recombinant virus on
ice for 30 min. The mixtures were then added to Vero cells on ice for 1 h. The
infected cells were subsequently washed with phosphate-buffered saline and overlaid
with 1% agarose at 37 8C for 3 days. The cells were then fixed with 10%
formaldehyde at room temperature for 15 min and stained with crystal violet to
visualize plaques for counting. The numbers of plaques obtained in the absence of
compounds 3 or 4 were assigned as 100%.
31. Wang, C-C., Kulkarni, S. S., Lee, J-C., Luo, S-Y. & Hung, S-C. Regioselective
one-pot protection of glucose. Nat. Protoc. 3, 97–113 (2008).
32. Chang, K-L., Zulueta, M. M. L., Lu, X-A., Zhong, Y-Q. & Hung, S-C.
Regioselective one-pot protection of D-glucosamine. J. Org. Chem. 75,
7424–7427 (2010).
33. Lee, J-C., Lu, X-A., Kulkarni, S. S., Wen, Y-S. & Hung, S-C. Synthesis of
heparin oligosaccharides. J. Am. Chem. Soc. 126, 476–477 (2004).
34. Plante, O. J., Buchwald, S. L. & Seeberger, P. H. Halobenzyl ethers as protecting
groups for organic synthesis. J. Am. Chem. Soc. 122, 7148–7149 (2000).
35. Kulkarni, S. S. & Hung, S-C. Metal tifluoromethanesulfonates as versatile
catalysts in carbohydrate synthesis. Lett. Org. Chem. 2, 670–677 (2005).
36. van den Berg, R. J. B. H. N., Noort, D., van der Marel, G. A., van Boom, J. H.,
Benschop, H. P. Synthesis of pseudo-disaccharide analogues of Lipid A: haptens
for the generation of antibodies with glycosidase activity towards Lipid A.
J. Carbohydr. Chem. 21, 167–168 (2002).
Received 25 October 2010; accepted 17 May 2011;
published online 19 June 2011
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Acknowledgements
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Author contributions
S-C.H. conceived the idea of HS synthesis, supervised students to carry out the experiments,
drew and summarized the figures, and finalized the preparation of the manuscript. Y-P.H.
and S-Y.L. synthesized the irregular HS octasaccharides 3 and 4, respectively. C-Y.H.
carried out the inhibition experiments of HSV-1 with Vero cells. M.M.L.Z. participated
in the discussion and wrote the manuscript. J-Y.L. initiated the work on the preparation
of the oligosaccharide skeleton. W.C. supervised C-Y.H. on the inhibition study of
HSV-1 infection.
Additional information
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints/. Correspondence and requests for materials should be addressed to S.C.H.
14. Akhtar, J. & Shukla, D. Viral entry mechanisms: cellular and viral mediators of
herpes simplex virus entry. FEBS J. 276, 7228–7236 (2009).
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