Synthesis of a fluorescence-labeled K30 antigen repeating unit using click chemistry

Article abstract of DOI:10.1016/j.carres.2007.01.015

An N-dansyl-labeled K30 antigen repeating unit, {4-[5-(N,N′-dimethylamino)naphthalene-1-sulfonamine]-1H-1,2,3-triazol-1-yl}hexyl β-d-glucopyranosyluronate-(1→3)-α-d-galactopyranosyl-α-d-mannopyranosyl-(1→3)-β-d-galactopyranoside, was synthesized using cli

Full text of DOI:10.1016/j.carres.2007.01.015

Carbohydrate Research 342 (2007) 975–981
Note
Synthesis of a fluorescence-labeled K30 antigen repeating
unit using click chemistry
Lijian Cheng,a,b Qi Chen,a Jun Liua and Yuguo Dua,*
aThe State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, Beijing 100085, China
bSchool of Chemical and Environmental Engineering, China University of Mining and Technology-Beijing, Beijing 100083, China
Received 31 December 2006; received in revised form 23 January 2007; accepted 25 January 2007
Available online 1 February 2007
Abstract—An N-dansyl-labeled K30 antigen repeating unit, {4-[5-(N,N0-dimethylamino)naphthalene-1-sulfonamine]-1H-1,2,3-tri-
azol-1-yl}hexyl b-D-glucopyranosyluronate-(1!3)-a-D-galactopyranosyl-a-D-mannopyranosyl-(1!3)-b-D-galactopyranoside, was
synthesized using click chemistry, the copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of an azide and an alkyne. The target
compound could further facilitate the studies of interactions among K30 oligosaccharides and proteins.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Glycosylation; Click chemistry; K30 Antigen; Fluorescence; Thioglycosides
Bacterial cells are capable of secreting a range of large
molecules, including both complex polysaccharides and
proteins. Some of these macromolecules are required
for cell viability, whereas others are involved in interac-
tions between a bacterial pathogen and its host. Among
these cell-surface polysaccharides, K30 antigen1 is an
interesting one. The K30 capsular antigen is a member
of the group I, or heat-stable, capsules.2 It may be
responsible for resistance against phagocytosis.3 Unlike
most of the K-antigen, formation of the K30 capsule in
Escherichia coli does not require attachment to a lipo-
polysaccharide lipid A-core.4 A mutation in wza
(K30), an integral membrane lipoprotein, severely
restricts the formation of the K30 capsular structure
on the cell surface, but does not interfere with biosyn-
thesis or polymerization of the K30 repeating unit.5
Generally, carbohydrates do have not effective absorp-
tion and fluorescence for sensitive detection. Further-
more, the available amounts in most analyses are
minute, and the direct detection of weak interaction
between carbohydrate chains and their conjugates in real
samples is very difficult.6 Fluorescence-labeling of enzyme
substrates is a valuable tool, as it enables the sensitive
detection of the small quantities of material involved in
an enzymatic assay.7 This technique has been used in this
capacity for single sugar glycosyltransferase systems.8
In a collaborative project for an investigation into the
biological functions of the K30 antigen, we were re-
quired to prepare a fluorescence-labeled K30 repeating
unit, compound 1, as shown in Figure 1.
Initially, we thought the a-linkage between sugar units
II and III (Scheme 1) could be prepared based on our
previous results.9 Accordingly, disaccharide donor A
and acceptor B were prepared. We expected that the
b-bond in donor A, although it had an acetyl group
for neighboring-group participation at C-2, would pre-
dominantly give a 1,2-cis outcome in glycosylation with
acceptor B.10 Unfortunately, the desired a product was
obtained in less than 30% yield, and we had to turn
our attention on a new strategy. We describe herein a
step-by-step, but more efficient synthesis of a fluores-
cence-labeled K30 repeating unit.
As outlined in Scheme 2, building blocks isopropyl
2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-b-D-galacto-
pyranoside (3), 6-azidohexyl 2-O-acetyl-4,6-O-benzyl-
idene-b-D-galactopyranoside (8), isopropyl 2,4,6-tri-O-
acetyl-3-O-benzyl-1-thio-a-D-mannopyranoside (9), and
*
Corresponding author. Tel.: +86 10 62849126; fax: +86 10
62923563; e-mail: duyuguo@rcees.ac.cn
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.01.015

976
L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
OH
HO
O
HOOC
HO
HO
O
O
HO
HO
HO
OH
O
OH
O
HO
HO
N
O
O
NMe2
N
N
O
HO
SO2
NH
Figure 1. Structure of a fluorescence-labeled K30 repeating unit, compound 1.
Ph
O
AcO
II
AcO
AcO
HO
O
MeOOC
BzO
BzO
O
OAc
III
IV
OAc
O
O
+
O
SCHMe2
O
I
O
O
BzO
AcO
N3
AcO
A
B
Scheme 1. Attempted preparation of K30 tetrasaccharide.
methyl 2,3,4-tri-O-benzoyl-a-D-glucuronosyl trichloro-
acetimidate (16)11 were selected for the synthesis of tar-
get compound 1. Thus, isopropyl 4,6-O-benzylidene-1-
thio-b-D-galactopyranoside (2)12 was benzylated with
benzyl bromide and sodium hydride in DMF at 0 ꢁC
obtaining synthon 3 in a yield of 95%. Selection of benzyl
protecting group on C-2 is to favor the a-glycoside
bond formation in the ongoing coupling reaction.
Meanwhile, 2 was acetylated with acetic anhydride in
pyridine to give isopropyl 2,3-di-O-acetyl-4,6-O-benzyl-
idene-1-thio-b-D-galactopyranoside (4), which was re-
acted with 6-azido-1-hexanol13 in the presence of NIS
and TMSOTf in CH2Cl2 at ꢀ20 ꢁC for 4 h affording 6-
azidohexyl 2,3-di-O-acetyl-4,6-O-benzylidene-b-D-galac-
topyranoside (5) in good yield (85%). Deacetylation of 5
(!6) with NaOMe in MeOH, followed by regioselective
protection of C-3 with 9-fluorenylmethyloxycarbonyl
chloride (FmocCl) in pyridine,14 and acetylation of the
C2–OH with Ac2O in a single step gave 6-azidohexyl
2-O-acetyl-3-O-(9-fluorenylmethyloxycarbonyl)-4,6-O-
benzylidene-b-D-galactopyranoside (7). Removal of
Fmoc from 7 with triethylamine in dichloromethane at
rt afforded synthon 8 in an overall yield of 83% for four
steps. Isopropyl 2,4,6-tri-O-acetyl-3-O-benzyl-1-thio-
a-D-mannopyranoside (9) was prepared according to
the literature procedures.15 Coupling of 9 and 8 in
dry CH2Cl2 in the presence of TMSOTf and NIS at
ꢀ20 ꢁC for 30 min afforded 6-azidohexyl 2,4,6-tri-O-
acetyl-3-O-benzyl-a-D-mannopyranosyl-(1!3)-2-O-
to generate disaccharide acceptor, 6-azidohexyl 2,4,6-tri-
O-acetyl-a-D-mannopyranosyl-(1!3)-2,4,6-tri-O-acetyl-
b-D-galactopyranoside (12, 80%). To favor the a glyco-
syl bond as required in the target molecule, donor 3
was prepared and condensed with 12, using the same
method as described in the preparation of 10, to form
6-azidohexyl
2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-D-manno-
pyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galactopyran-
oside (13) in 78% isolated yield. Debenzylidenation and
acetylation of 13 as described in the preparation of 11,
followed by debenzylation, afforded trisaccharide accep-
tor 6-azidohexyl 4,6-di-O-acetyl-a-D-galactopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-a-D-mannopyranosyl-(1!3)-2,
4,6-tri-O-acetyl-b-D-galactopyranoside (15, 70% yield
over three steps). Regioselective glycosylation of 15
and 16 in dry dichloromethane at ꢀ20 ꢁC, in the pres-
ence of a catalytic amount of TMSOTf, gave tetra-
saccharide 6-azidohexyl (methyl 2,3,4-tri-O-benzoyl-b-
D-glucopyranosyluronate)-(1!3)-2,4,6-tri-O-acetyl-a-D-
galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-D-manno-
pyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galacopyrano-
side (17) in 89% yield. The desired (1!3)-linkage of 17
was further confirmed by 2D NMR spectral analysis
of its acetylated compound 18, showing a downfield-
shifted doublet of doublets at d 4.86 ppm (J1,2 3.7 Hz,
J2,3 10.4 Hz) corresponding to the H-2III unit. On the
basis of the copper(I)-catalyzed 1,3-dipolar cycloaddi-
tion reaction of the azide and alkyne,17 18 was con-
densed with 5-(dimethylamino)-N-(2-propynyl)-1-naph-
thalene-sulfonamide (19)18 to obtain {4-[5-(N,N0-di-
methylamino)naphthalene-1-sulfonamine]-1H-1,2,3-tri-
azol-1-yl}hexyl (methyl 2,3,4-tri-O-benzoyl-b-D-gluco-
pyranosyluronate)-(1!3)-2,4,6-tri-O-acetyl-a-D-galacto-
pyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-D-mannopyrano-
syl-(1!3)-2,4,6-tri-O-acetyl-b-D-galactopyranoside (20)
in 90% yield. Global deprotection of 20 in 0.5 N NaOH
acetyl-4,6-O-benzylidene-b-D-galactopyranoside
(10).
Hydrolysis of 4,6-O-benzylidene of 10 with 80%
aqueous acetic acid, followed by acetylation with
Ac2O in pyridine, gave 6-azidohexyl 2,4,6-tri-O-acetyl-
3-O-benzyl-a-D-mannopyranosyl-(1!3)-2,4,6-tri-O-acet-
yl-b-D-galactopyranoside (11) in 72% yield for three
steps. NaBrO3/Na2S2O4 catalyzed debenzylation16 un-
der two-phase reaction conditions carried out smoothly

L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
977
Ph
Ph
O
D-Mannose
g
O
O
c
O
O
O
R1O
RO
SCHMe2
b
O
N3
R2O
OAc
O
AcO
AcO
BnO
OR
5 R1 = R2 = Ac
2 R = H
3 R = Bn
4 R = Ac
a
d
6 R1 = R2 = H
SCHMe2
9
e
f
7 R1 = Fmoc, R2 = Ac
8 R1 = H, R2 = Ac
Ph
OAc
AcO
AcO
RO
OAc
AcO
AcO
BnO
k
i
h
O
O
OAc
O
AcO
O
9 + 8
O
O
O(CH2)6N3
O
O(CH2)6N3
O
AcO
11 R = Bn
12 R = H
AcO
10
j
OR1
R1O
R2O
O
l
OAc
O
AcO
AcO
OAc
AcO
O
R2O
MeOOC
BzO
BzO
O
O
O
O(CH2)6N3
AcO
BzO
OC(NH)CCl3
NMe2
13 R1, R1 = PhCH, R2 = Bn
i
j
14 R1 = Ac, R2 = Bn
16
15 R1 = Ac, R2 = H
OAc
AcO
MeOOC
BzO
BzO
O
O
OAc
O
AcO
AcO
O
SO2NHCH2C CH
OAc
O
19
AcO
RO
BzO
O
O(CH2)6N3
m
O
AcO
17 R = H
b
18 R = Ac
R3OOC
R2O
R1O
O
OR1
O
O
R1O
R1O
OR1
O
R2O
R2O
IV
R1O
OR1
O
OR1
O
III
II
N
O
O
N
N
NMe2
R1O
I
NH SO2
20 R1 = Ac, R2 = Bz, R3 = Me
n
1 R1 = R2 = R3 = H
Scheme 2. Regents and conditions: (a) BnBr, NaH, DMF, 95%; (b) Ac2O, Py; (c) 6-azido-hexanol, NIS, TMSOTf, CH2Cl2, ꢀ20 ꢁC, 85%; (d)
NaOMe, MeOH; (e) (i) FmocCl, Py, overnight; (ii) Ac2O, Py; (f) Et3N, CH2Cl2, 83% for four steps; (g) (i) Ac2O, Py; (ii) HSCHMe2, BF3ÆEt2O,
CH2Cl2; (iii) NaOMe, MeOH; (iv) Bu2SnO, MeOH, reflux, 4 h; (v) Bu4NI, BnBr, toluene; (vi) Ac2O, Py, 46% for six steps; (h) NIS, TMSOTf,
CH2Cl2, ꢀ20 ꢁC; (i) (i) 80% HOAc, 60 ꢁC, 6 h; (ii) Ac2O, Py, 72% for three steps; (j) NaBrO3, Na2S2O4, EtOAc/H2O, rt, 80% for 12; 70% for 15 for
three steps; (k) 3, NIS, TMSOTf, CH2Cl2, ꢀ20 ꢁC, 78%; (l) TMSOTf, CH2Cl2, ꢀ20 ꢁC, 89%; (m) CuSO4Æ5H2O, sodium ascorbate, 1:1 THF–H2O,
50–60 ꢁC, 90%; (n) 0.5 N NaOH, 1:1 CH2Cl2–MeOH, 0 ꢁC–rt, 2.5 h, 95%.
aqueous solution at 0 ꢁC for 2.5 h furnished fluores-
cence-labeled K30 unit 1.
actopyranoside, using isopropyl thioglycosides as do-
nors in NIS/TMSOTf-catalyzed glycosylations and
Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of
azide and alkyne. The compound thus prepared could
be further used for the studies of interactions among
K30 oligosaccharides and proteins, and the results will
be reported in due course.
In conclusion, we have synthesized a N-dansyl fluores-
cence-labeled K30 antigen repeating unit, {4-[5-(N,N0-
dimethylamino)naphthalene-1-sulfonamine]-1H-1,2,3-
triazol-1-yl}hexyl b-D-glucopyranosyluronate-(1!3)-a-
D-galactopyranosyl-a-D-mannopyranosyl-(1!3)-b-D-gal-

978
L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
1. Experimental
which was purified by silica gel column chromatography
(2:1 petroleum ether–EtOAc) to give compound 5 as a
25
1
1.1. General methods
syrup (5.64 g, 85%): ½aꢁD +20 (c 1, CHCl3); H NMR
(400 MHz, CDCl3): d 1.35–1.39 (m, 4H), 1.57–1.61 (m,
4H), 2.06, 2.07 (2s, 2 · 3H, 2CH3CO), 3.26 (t, 2H, J
6.9 Hz, CH2N3), 3.45–3.51 (m, 2H, one proton of
OCH2 and H-5), 3.89–3.95 (m, 1H, one proton of
OCH2), 4.06 (dd, 1H, J 1.6, 12.4 Hz, H-6a), 4.33 (dd,
1H, J 1.6, 12.4 Hz, H-6b), 4.37 (br d, 1H, J 3.6 Hz, H-
4), 4.49 (d, 1H, J 8.0 Hz, H-1), 4.96 (dd, 1H, J 3.6,
10.4 Hz, H-3), 5.38 (dd, 1H, J 8.0, 10.4 Hz, H-2), 5.51
(s, 1H, PhCH), 7.36–7.53 (m, 5H, Ph). Anal. Calcd for
C23H31N3O8: C, 57.85; H, 6.54. Found: C, 58.09; H, 6.39.
Optical rotations were determined at 25 ꢁC with a Per-
kin–Elmer Model 241-Mc automatic polarimeter, and
[a]D-values are in units of 10ꢀ1 deg cm2 gꢀ1. H NMR,
1
1
1
13C NMR and H–1H, H–13C COSY spectra were re-
corded with a Bruker ARX 400 spectrometer for solu-
tions in CDCl3 or D2O. Chemical shifts are given in
parts per million downfield from internal Me4Si. Mass
spectra were measured using a MALDI-TOF-MS with
a-cyano-4-hydroxycinnamic acid (CCA) as the matrix.
Thin-layer chromatography (TLC) was performed on sil-
ica gel HF254 with detection by charring with 30% (v/v)
H2SO4 in MeOH or in some cases by UV detection. Col-
umn chromatography was conducted by the elution of a
column of silica gel (100–200 mesh) with EtOAc–petro-
leum ether (60–90 ꢁC) as the eluent. Solutions were con-
centrated at <60 ꢁC under reduced pressure.
1.4. 6-Azidohexyl 2-O-acetyl-4,6-O-benzylidene-b-D-
galactopyranoside (8)
A solution of 5 (5.10 g, 10.7 mmol) in MeOH (100 mL)
was treated with NaOMe (2.0 mL, 1 M in MeOH) at rt
for 6 h. The mixture was neutralized with IR-120 (H+)
resin, and filtered, and the filtrate was evaporated. The
syrup was dissolved in pyridine (20 mL), and FmocCl
(3.04 g, 11.77 mmol) was added to the solution in an
ice-water bath. The mixture was stirred at rt for 15 h,
then Ac2O (5 mL) and a catalytic amount of DMAP
(50 mg) were added. The mixture was stirred at rt for
3 h, then co-evaporated with toluene to dryness under re-
duced pressure. The residue was subsequently dissolved
in CH2Cl2 (80 mL), to which was added Et3N (2 mL)
at rt and stirred under these conditions for 10 h. The sol-
vents were evaporated in vacuo to give a residue, which
was purified by silica gel column chromatography (3:2
petroleum ether–EtOAc) to give compound 8 as a syrup
1.2. Isopropyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-
b-D-galactopyranoside (3)
To a vigorously stirred solution of 2 (1.273 g, 3.90 mmol)
in DMF (15 mL) at 0 ꢁC was added NaH (50% content,
0.75 g, 15.6 mmol). BnBr (1.00 mL, 8.58 mmol) was
added 20 min later, and allowed to stir at rt for 2 h. It
was then poured into ice-cold water (50 mL) and ex-
tracted with EtOAc (3 · 50 mL). The combined organic
phases were dried over anhyd Na2SO4 and concentrated.
Purification of the residue by silica gel column chroma-
tography (3:1 petroleum ether–EtOAc) gave 3 (1.87 g,
25
25
95%) as an amorphous solid: ½aꢁD ꢀ70 (c 1, CHCl3); 1H
(3.86 g, 83%): ½aꢁD +24 (c 1, CHCl3); 1H NMR
NMR (400 MHz, CDCl3): d 1.33, 1.37 (2s, 6H, 2CH3),
3.26–3.33 (m, 2H, H-5, SCH), 3.58 (dd, 1H, J 3.5,
9.2 Hz, H-3), 3.86 (t, 1H, J 9.2 Hz, H-2), 3.94 (dd, 1H,
J 1.7, 12.2 Hz, H-6a), 4.14 (d, 1H, J 3.5 Hz, H-4), 4.28
(br d, 1H, J 1.7, 12.2 Hz, H-6b), 4.49 (d, 1H, J 9.2 Hz,
H-1), 4.74 (s, 2H, PhCH2), 4.80, 4.89 (2d, 2H, PhCH2),
5.46 (s, 1H, PhCH), 7.27–7.56 (m, 15H, Ph). Anal. Calcd
for C30H34O5S: C, 71.12; H, 6.76. Found: 71.44; H, 6.57.
(400 MHz, CDCl3): d 1.37–1.40 (m, 4H), 1.57–1.61 (m,
4H), 2.04 (s, 3H, CH3CO), 3.26 (t, 2H, J 6.9 Hz,
CH2N3), 3.47–3.51 (m, 2H, one proton of OCH2 and
H-5), 3.73 (dd, 1H, J 3.8, 9.9 Hz, H-3), 3.88–3.94 (m,
1H, one proton of OCH2), 4.07 (dd, 1H, J 1.7, 12.4 Hz,
H-6a), 4.21 (d, 1H, J 3.8 Hz, H-4), 4.31 (dd, 1H, J 1.7,
12.4 Hz, H-6b), 4.41 (d, 1H, J 8.0 Hz, H-1), 5.08 (dd,
1H, J 8.0, 9.9 Hz, H-2), 5.55 (s, 1H, PhCH), 7.36–7.52
(m, 5H, Ph). Anal. Calcd for C21H29N3O7: C, 57.92; H,
6.71. Found: C, 58.27; H, 6.60.
1.3. 6-Azidohexyl 2,3-di-O-acetyl-4,6-O-benzylidene-b-D-
galactopyranoside (5)
1.5. Isopropyl 2,4,6-tri-O-acetyl-3-O-benzyl-1-thio-a-D-
mannopyranoside (9)
To a solution of compound 4 (5.70 g, 13.8 mmol) and
6-azido-1-hexanol (1.80 g, 12.58 mmol) in dry CH2Cl2
˚
(60 mL) was added 4 A molecular sieves (2 g). The mix-
The mixtures of isopropyl 1-thio-a-D-mannopyranoside
(7.60 g, 31.9 mmol) and dibutyltin oxide (8 g, 32 mmol)
were dissolved into anhyd MeOH (300 mL).15 The reac-
tion mixture was refluxed for 4 h, concentrated to dry-
ness under reduced pressure. The syrup was suspended
into anhyd toluene (250 mL), and Bu4NI (11.8 g,
31.9 mmol) and BnBr (5.73 mL, 47.9 mmol) were added.
The mixture was stirred at 70 ꢁC for 16 h, then concen-
ture was stirred at ꢀ20 ꢁC for 20 min under an N2 atmo-
sphere, then NIS (3.70 g, 16.44 mmol) and TMSOTf
(125 lL, 0.69 mmol) were added. The mixture was stirred
under these conditions for 30 min, quenched by Et3N, di-
luted with CH2Cl2 (100 mL), and washed with aq
Na2S2O3. The organic phase was dried over Na2SO4.
The solvents were evaporated in vacuo to give a residue,

L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
979
trated to dryness. The residue was treated with Ac2O
(15 mL) in pyridine (40 mL) at rt for 6 h, and co-evapo-
rated with toluene under diminished pressure. Purifica-
tion of the residue by silica-gel column chromato-
graphy (3:1 petroleum ether–EtOAc) gave compound 9
as a syrup (8.12 g, 56%): ½aꢁD ꢀ37 (c 1, CHCl3); H
NMR (400 MHz, CDCl3): d 1.30, 1.32 (d, 6H, 2CH3),
2.03, 2.07, 2.16 (3s, 3 · 3H, 3CH3CO), 3.02–3.10 (m,
1H, SCH), 3.75 (dd, 1H, J 3.3, 9.6 Hz, H-3), 4.05–4.09
(m, 1H, H-6a), 4.23–4.30 (m, 2H, H-5, H-6b), 4.40,
4.60 (2d, 2H, J 12.1 Hz, PhCH2), 5.23 (t, 1H, J 9.7 Hz,
H-4), 5.36 (d, 1H, J 1.4 Hz, H-1), 5.42 (dd, 1H, J 1.4,
3.3 Hz, H-2), 7.24–7.35 (m, 5H, Ph). Anal. Calcd for
C22H30O8S: C, 58.13; H, 6.65. Found: C, 58.29; H, 6.51.
wise over 10 min at rt. After completion of the reaction
(TLC), the mixture was diluted with EtOAc, and the or-
ganic phase was washed with aq Na2S2O3. The crude
product was then purified by silica-gel chromatography
(1:1 petroleum ether–EtOAc) to give syrupy compound
25
25
1
12 (0.91 g, 80%): ½aꢁD +24 (c 1, CHCl3); 1H NMR
(400 MHz, CDCl3): d 1.36–1.38 (m, 4H), 1.58–1.60 (m,
4H), 2.05, 2.09, 2.11, 2.12, 2.16, 2.18 (6s, 6 · 3H,
6CH3CO), 3.27 (t, 2H, J 6.8 Hz, CH2N3), 3.43–3.49
(m, 1H, one proton of OCH2), 3.79–3.90 (m, 4H, H-5I,
H-5II, one proton of OCH2, H-6aI), 3.92 (dd, 1H, J
3.5, 10.0 Hz, H-3II), 4.12 (dd, 1H, J 7.1, 11.2 Hz, H-
6aII), 4.17–4.26 (m, 3H, H-3I, H-6bI, H-6bII), 4.37 (d,
1H, J 7.9 Hz, H-1I), 4.96 (dd, 1H, J 2.9, 3.5 Hz, H-
2II), 5.06 (t, 1H, J 10.0 Hz, H-4II), 5.08 (d, 1H, J
1.0 Hz, H-4I), 5.14 (dd, 1H, J 7.9, 10.2 Hz, H-2I), 5.39
(d, 1H, J 2.9 Hz, H-1II). Anal. Calcd for C30H45N3O17:
C, 50.07; H, 6.30. Found: C, 49.75; H, 6.23.
1.6. 6-Azidohexyl 2,4,6-tri-O-acetyl-3-O-benzyl-a-D-
mannopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galacto-
pyranoside (11)
To a solution of compounds 9 (2.25 g, 4.95 mmol) and 8
1.8. 6-Azidohexyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-
D-galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-D-manno-
pyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galactopyran-
oside (13)
(1.96 g, 4.50 mmol) in dry CH2Cl2 (30 mL) was added
˚
4 A molecular sieves (1 g). The mixture was stirred at
ꢀ20 ꢁC for 10 min under an N2 atmosphere, then NIS
(1.34 g, 5.95 mmol) and TMSOTf (80 lL, 0.5 mmol) were
added. The mixture was stirred under these conditions for
30 min, quenched by Et3N, diluted with CH2Cl2 (40 mL),
and washed with aq Na2S2O3. The organic phase was
dried over Na2SO4 and concentrated. The residue was
purified by silica-gel column chromatography (2:1 petro-
leum ether–EtOAc) to give syrupy compound 10, which
was treated with aq 80% HOAc (60 mL) at 60 ꢁC
for 6 h, then co-evaporated with toluene. The residue
was reacted with Ac2O (5 mL) in pyridine (15 mL) at rt
overnight, and co-evaporated with toluene under dimin-
ished pressure. Purification of the residue by silica-gel col-
umn chromatography (2:1 petroleum ether–EtOAc) gave
To a solution of compounds 3 (260 mg, 0.51 mmol) and
12 (310 mg, 0.43 mmol) in dry CH2Cl2 (10 mL) was
added NIS (270 mg, 0.60 mmol) and TMSOTf (15 lL,
0.08 mmol) at ꢀ20 ꢁC under an N2 atmosphere. The mix-
ture was stirred under these conditions for 45 min,
quenched by Et3N, diluted with CH2Cl2 (40 mL), and
washed with aq Na2S2O3. The organic phase was dried
over Na2SO4, the solvents were evaporated in vacuo,
and the residue was purified by silica-gel column chroma-
tography (1:1 petroleum ether–EtOAc) to give compound
25
1
13 (386 mg, 78%) as a syrup: ½aꢁD +52 (c 1, CHCl3); H
NMR (400 MHz, CDCl3): d 1.16–1.52 (m, 8H), 1.68,
2.04, 2.05, 2.08, 2.09, 2.16 (6s, 6 · 3H, 6CH3CO), 3.26
(t, 2H, J 6.8 Hz, CH2N3), 3.44–3.48 (m, 1H, one proton
of OCH2), 3.61 (br s, 1H, H-5III), 3.79–3.94 (m, 6H,
one proton of OCH2, H-5I, H-5II, H-6aII, and 2H-6III),
4.01 (dd, 1H, J 3.5, 10.2 Hz, H-3II), 4.09–4.20 (m, 6H,
H-2III, H-3III, H-3I, 2H-6I, H-6bII), 4.37 (d, 1H, J
8.0 Hz, H-1I), 4.63, 4.75 (2d, 2H, J 11.6 Hz, PhCH2),
4.69, 4.78 (2d, 2H, J 11.9 Hz, PhCH2), 4.99 (d, 1H, J
1.5 Hz, H-1II), 5.08–5.11 (m, 2H, H-1III, H-2II), 5.14
(dd, 1H, J 8.0, 10.4 Hz, H-2I), 5.26 (t, 1H, J 10.0 Hz,
H-4II), 5.37 (d, 1H, J 2.9 Hz, H-4I), 5.43 (s, 1H, PhCH),
7.25–7.47 (m, 15H, Ph). Anal. Calcd for C57H71N3O22:
C, 59.52; H, 6.22. Found: C, 59.39; H, 6.06.
25
1
11 as a syrup (2.62 g, 72%): ½aꢁD +21 (c 1, CHCl3); H
NMR (400 MHz, CDCl3): d 1.36–1.62 (m, 8H), 1.91,
1.98, 2.06, 2.10, 2.14, 2.19 (6s, 6 · 3H, 6CH3CO), 3.27
(t, 2H, J 6.8 Hz, CH2N3), 3.45–3.48 (m, 1H, one of
OCH2), 3.65 (dd, 1H, J 3.3, 9.8 Hz, H-3I), 3.79–3.90 (m,
4H, one of OCH2, H-5I, H-3II, H-6aII), 4.13–4.22 (m,
4H, H-5II, H-6bII, H-6aI, H-6bI), 4.33 (d, H, J 11.9 Hz,
PhCH2), 5.08 (d, 1H, J 1.5 Hz, H-1II), 5.13–5.19 (m, 3H,
H-2I, H-2II, H-4II), 5.38 (d, 1H, J 2.9 Hz, H-4I), 7.24–
7.34 (m, 5H, Ph). Anal. Calcd for C37H51N3O17: C,
54.88; H, 6.35. Found: C, 54.58; H, 6.40.
1.7. 6-Azidohexyl 2,4,6-tri-O-acetyl-a-D-mannopyran-
osyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galactopyranoside (12)
1.9. 6-Azidohexyl 4,6-di-O-acetyl-a-D-galactopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-a-D-mannopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-galactopyranoside (15)
Compound 11 (1.28 g, 1.55 mmol) was dissolved in
EtOAc (25 mL) and then a solution of NaBrO3
(2.30 g, 15.5 mmol) in water (60 mL) was added. To
the well-stirred two-phase system aq Na2S2O4 (2.70 g,
15.5 mmol, dissolved in 60 mL water) was added drop-
Compound 13 (385 mg, 0.33 mmol) in 80% HOAc–
H2O (10 mL) was heated at 60 ꢁC for 6 h, and then

980
L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
co-evaporated with the help of toluene. The residue was
acetylated with Ac2O (1 mL) in pyridine (4 mL), then
purified by silica-gel column chromatography (2:1 petro-
leum ether–EtOAc). The residue was dissolved in EtOAc
(10 mL), and then a solution of NaBrO3 (0.99 g,
6.6 mmol) in water (30 mL) was added. To the well-
stirred two-phase system aq Na2S2O4 (1.15 g, 6.6 mmol,
dissolved in 25 mL water) was added dropwise over
10 min at rt. The reaction was monitored by TLC until
all starting material was consumed. The mixture was
diluted with EtOAc, and the organic phase was washed
with aq Na2S2O3. The crude product was then purified
by silica-gel column chromatography (1:2 petroleum
ether–EtOAc) to give compound 15 as a syrup (228
3.75–3.90 (m, 4H), 3.95 (dd, 1H, J 2.1, 9.8 Hz, H-3I),
3.98 (dd, 1H, J 4.0, 11.2 Hz, H-6), 4.05 (dd, 1H, J 2.2,
9.6 Hz, H-3III), 4.10–4.28 (m, 5H), 4.30 (d, 1H, J
9.9 Hz, H-5IV), 4.30–4.33 (m, 2H, J 7.9 Hz, H-1I, H-6),
4.86 (dd, 1H, J 3.7, 10.4 Hz, H-2III), 4.96 (d, 1H, J
7.5 Hz, H-1IV), 5.02–5.05 (m, 3H, H-2I, H-1II, H-4I),
5.14 (t, 1H, J 9.3 Hz, H-4II), 5.20 (d, 1H, J 3.7 Hz, H-
1III), 5.38 (br d, 1H, J 3.1 Hz, H-2II), 5.42 (dd, 1H, J
7.5, 9.3 Hz, H-2IV), 5.49 (d, 1H, J 3.0 Hz, H-4III), 5.71
(t, 1H, J 9.3 Hz, H-4IV), 5.83 (t, 1H, J 9.3 Hz, H-3IV),
7.27–7.92 (m, 15H, Ph); 13C NMR (100 MHz, CDCl3):
d 20.1, 20.4, 20.5, 20.55, 20.58, 20.66, 20.68, 20.7, 20.8,
25.3, 26.3, 28.7, 29.1, 51.2, 52.8, 60.3, 61.1, 62.1, 62.4,
64.8, 67.6, 68.4, 69.0, 69.2, 69.6, 69.7, 69.8, 69.9, 70.1,
70.4, 70.5, 71.7, 72.2, 72.8, 73.0, 94.6, 96.0, 100.8,
101.3, 128.2, 128.3, 128.6, 128.8, 129.0, 129.5, 129.7,
133.3, 164.8, 165.6, 166.7, 168.5, 169.6, 169.7, 170.0,
170.2, 170.29, 170.31, 170.4, 170.6. Anal. Calcd for
C70H83N3O34: C, 55.66; H, 5.54. Found: C, 55.29; H,
5.35. MALDITOF-MS: calcd for C70H83N3O34: 1509.5
[M]+; found: 1532.4 [M+Na]+.
25
1
mg, 70%): ½aꢁD +27 (c 1, CHCl3); H NMR (400 MHz,
CDCl3): d 1.37–1.62 (m, 8H), 2.05 (s, 3H, CH3CO),
2.09 (s, 9H, 3CH3CO), 2.11 (s, 6H, 2CH3CO), 2.15 (s,
3H, CH3CO), 2.17 (s, 3H, CH3CO), 3.27 (t, 2H, J
6.8 Hz, CH2N3), 3.45–3.47 (m, 1H, one proton of
OCH2), 3.69 (dd, 1H, J 3.6, 11.9 Hz, H-6), 3.77 (dd,
1H, J 3.3, 11.9 Hz, H-6), 3.81 (br t, 1H, J 7.0 Hz,
H-5II), 3.85–3.91 (m, 3H, one proton of OCH2, H-5I,
H-5III), 3.95 (dd, 1H, J 2.8, 10.2 Hz, H-3I), 4.05–4.10
1.11. {4-[5-(N,N0-Dimethylamino)naphthalene-1-sulfon-
amine]-1H-1,2,3-triazol-1-yl}hexyl (methyl 2,3,4-tri-O-
benzoyl-b-D-glucopyranosyluronate)-(1!3)-2,4,6-tri-O-
acetyl-a-D-galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-
D-mannopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galacto-
pyranoside (20)
(m, 3H, 3H-6), 4.19–4.23 (m, 4H, H-2III, H-3II, H-3III
,
H-6), 4.37 (d, 1H, J 7.9 Hz, H-1I), 4.99 (d, 1H, J
3.7 Hz, H-1III), 5.04–5.05 (m, 2H, H-2II, H-1II), 5.12
(dd, 1H, J 7.9, 10.2 Hz, H-2I), 5.27 (t, 1H, J 10.0 Hz,
H-4II), 5.34 (d, 1H, J 2.2 Hz, H-4III), 5.39 (d, 1H, J 2.8
Hz, H-4I); 13C NMR (100 MHz, CDCl3): d 20.4, 20.5,
20.6, 20.7, 20.8, 25.3, 26.3, 28.7, 29.2, 51.2, 61.3, 61.5,
62.2, 65.1, 67.5, 67.6, 68.9, 69.1, 69.7, 69.75, 69.82,
70.5, 73.4, 75.0, 77.2, 94.7, 101.1, 101.2, 168.8, 169.5,
170.1, 170.3, 170.4, 170.5, 170.6, 170.8. Anal. Calcd for
C40H59N3O24: C, 49.74; H, 6.16. Found: C, 49.52; H, 6.30.
To a mixture of 18 (50 mg, 0.033 mmol) and 19 (15 mg,
0.04 mmol) in 1:1 H2O–THF (6 mL) were added freshly
prepared 1 M aq sodium ascorbate (2 mg, 0.01 mmol)
and CuSO4Æ5H2O (1 mg, 0.0033 mmol). The hetero-
geneous mixture was stirred vigorously in a dark
room at 50 ꢁC until complete consumption of the reac-
tants was indicated by TLC analysis. After removal of
THF under reduced pressure, water (6 mL) was added,
and the product was extracted with EtOAc
(3 · 15 mL). The combined organic layers were dried
over anhyd Na2SO4 and evaporated in vacuo. The crude
product was subjected to column chromatography (1:1
petroleum ether–EtOAc) to give 20 as a foamy solid
1.10. 6-Azidohexyl (methyl 2,3,4-tri-O-benzoyl-b-D-
glucopyranosyluronate)-(1!3)-2,4,6-tri-O-acetyl-a-D-
galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-a-D-manno-
pyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-galactopyran-
oside (18)
To a solution of 16 (45 mg, 0.067 mmol) and 15 (54 mg.
0.056 mmol) in dry CH2Cl2 (3 mL) was added TMSOTf
(1.8 lL, 0.01 mmol) at ꢀ20 ꢁC under an N2 atmosphere.
The mixture was stirred under these conditions for
30 min, then quenched with Et3N. The solvent was evap-
orated, and the residue was dissolved into pyridine
(4 mL). To this mixture was added Ac2O (1 mL) and
then co-evaporated with toluene after 6 h. Purification
of the residue by silica-gel column chromatography
(2:3 petroleum ether–EtOAc) obtained compound 18
25
(54 mg, 90%): ½aꢁD +16 (c 1, CHCl3); 1H NMR
(400 MHz, CDCl3): d 1.50–1.55 (m, 3H), 1.75–1.83 (m,
5H), 1.64, 1.96, 2.04, 2.05, 2.09, 2.11, 2.12, 2.13, 2.17
(9s, 9 · 3H, 9CH3CO), 2.91 (s, 6H, 2CH3), 3.38–3.43
(m, 1H, one proton of OCH2), 3.74 (s, 3H, CH3),
3.80–3.88 (m, 2H), 3.90 (dd, 1H, J 2.3, 9.8 Hz, H-3I),
3.97 (dd, 1H, J 2.4, 9.6 Hz, H-3III), 4.00 (dd, 1H, J
4.0, 10.9 Hz, H-6), 4.05 (dd, 1H, J 3.6, 11.3 Hz, H-6),
4.10–4.30 (m, 8H), 4.31–4.38 (m, 3H, H-1I, H-5IV, H-
6), 4.86 (dd, 1H, J 3.6, 10.5 Hz, H-2III), 4.97 (d, 1H, J
7.5 Hz, H-1IV), 5.00–5.05 (m, 3H, H-2I, H-1II, H-4I),
5.15 (t, 1H, J 9.2 Hz, H-4II), 5.20 (d, 1H, J 3.7 Hz, H-
1III), 5.38 (br d, 1H, J 2.9 Hz, H-2II), 5.41 (dd, 1H, J
7.5, 9.5 Hz, H-2IV), 5.48 (d, 1H, J 3.2 Hz, H-4III),
25
1
as a syrup (75 mg, 89%): ½aꢁD +18 (c 1, CHCl3); H
NMR (400 MHz, CDCl3): d 1.35–1.60 (m, 8H), 1.65,
1.97, 2.03, 2.04, 2.09, 2.11, 2.12, 2.13, 2.17 (9s, 9 · 3H,
9CH3CO), 3.26 (t, 2H, J 6.8 Hz, CH2N3), 3.41–3.45
(m, 1H, one proton of OCH2), 3.74 (s, 3H, CH3),

L. Cheng et al. / Carbohydrate Research 342 (2007) 975–981
981
5.50–5.55 (m, 1H, NHSO2), 5.70 (t, 1H, J 9.5 Hz, H-
References
4IV), 5.86 (t, 1H, J 9.5 Hz, H-3IV), 7.26–8.27 (m, 22H,
Ph and C@CH); Selected 13C NMR (100 MHz, CDCl3):
d 50.0, 52.8, 60.3, 61.1, 62.2, 62.4, 64.9, 67.6, 68.4, 68.9,
69.2, 69.6, 69.8, 69.9, 70.0, 70.4, 71.7, 72.1, 72.8, 73.0,
94.6, 96.0, 100.8, 101.3, 115.3, 118.7, 164.5, 164.9,
165.6, 166.7, 168.6, 169.7, 169.8, 170.0, 170.3, 170.6.
Anal. Calcd for C85H99N5O36S: C, 56.76; H, 5.55.
Found: C, 57.02; H, 5.41. MALDITOF-MS: calcd for
C85H99N5O36S: 1797.6 [M]+; found: 1820.5 [M+Na]+.
1. Chakraborty, A. K.; Friebolin, H.; Stirm, S. J. Bacteriol.
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2. Keenleyside, W. J.; Jayaratne, P.; MacLachlan, P. R.;
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1.12. {4-[5-(N,N0-Dimethylamino)naphthalene-1-sulfon-
amine]-1H-1,2,3-triazol-1-yl}hexyl b-D-glucopyranosyl-
uronate-(1!3)-a-D-galactopyranosyl-a-D-mannopyran-
osyl-(1!3)-b-D-galactopyranoside (1)
To a mixture of compound 20 (50 mg, 0.027 mmol) in 1:1
MeOH–CH2Cl2 (40 mL) was added aq 0.5 N NaOH
at rt. After stirring for 2.5 h, the mixture was neutralized
with IR-120 (H+) resin, and filtered, and the filtrate was
concentrated to dryness under diminished pressure to
25
give compound 1 (28 mg, 95%) as a syrup: ½aꢁD +21 (c
1
0.5, H2O); H NMR (400 MHz, D2O): d 0.90–0.92 (m,
2H), 1.10–1.17 (m, 2H), 1.30–1.45 (m, 4H), 3.37 (s, 6H,
2CH3), 3.40–4.20 (m, 31H), 4.93 (s, 1H, H-1III), 5.20 (s,
1H, H-1II), 7.31 (s, 1H, C@CH), 7.67–7.74 (m, 2H),
7.93 (d, 1H, J 7.2 Hz, Ph), 8.14 (d, 1H, J 7.2 Hz, Ph),
8.23 (d, 1H, J 8.4 Hz, Ph), 8.44 (d, 1H, J 8.4 Hz, Ph);
13C NMR (100 MHz, D2O): d 13.2, 19.3, 20.5, 24.3,
25.0, 28.3, 29.0, 36.9, 46.8, 49.7, 60.8, 60.9, 61.3, 61.7,
62.5, 64.3, 65.9, 67.7, 69.1, 69.2, 69.9, 70.2, 71.2, 71.3,
72.8, 72.9, 74.9, 75.1, 76.3, 78.4, 79.3, 95.9, 100.4,
102.6, 103.7, 119.3, 123.9, 125.2, 125.5, 126.2, 126.9,
128.0, 128.4, 131.0, 135.6, 138.5, 142.6, 172.8. MALDI-
TOF-MS: calcd for C45H67N5O24S: 1093.3897 [M]+;
found: 1116.3843 [M+Na]+.
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Acknowledgment
This work was supported in partial by NNSF of China
(Project 20572128).
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.01.015.