Chemoenzymatic synthesis of the sialyl-α-(2→3′)-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end

Article abstract of DOI:10.1016/S0008-6215(03)00167-8

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic acid-(2→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy- β-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acce

Full text of DOI:10.1016/S0008-6215(03)00167-8

Carbohydrate Research 338 (2003) 1265ꢀ1270
/
www.elsevier.com/locate/carres
Chemoenzymatic synthesis of the sialyl-a-(20
/
3?)-lactosamine
trisaccharide with a 3-aminopropyl group as a spacer at the
reducing end
Indrani Choudhury (Mukherjee), Norihiko Minoura, Hirotaka Uzawa*
Laboratory of Advanced Bioelectronics, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba,
Ibaraki 305-8565, Japan
Received 25 January 2003; accepted 2 April 2003
Abstract
The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-
topyranosyl-(104)-2-acetamido-2-deoxy-b- -glucopyranoside has been synthesized chemoenzymatically for the first time. First,
the acceptor, 3-aminopropyl b- -galactopyranosyl-(10 -glucopyranoside was synthesized in a conven-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonic acid-(20
/
3)-b-
D-galac-
/
D
D
/
4)-2-acetamido-2-deoxy-b-
D
tional chemical manner, and then it was coupled with CMP-sialic acid using a-(20/3)-(N)-sialyltransferase to afford the desired
trisaccharide by an enzymatically stereocontrolled manner.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Lactosamine; Sialic acid; Trisaccharide; Chemoenzymatic
1. Introduction
moenzymatic11 methods have been used for stereospe-
cific synthesis of a-linked sialo-oligosaccharides.
However, the chemoenzymatic method has been found
more advantageous compared to its chemical counter-
part in that it often minimizes the number of synthetic
steps and provides high yields of products. There are a
Sialo glycoproteins, glycolipids and sialo-oligosacchar-
ides play crucial roles in biological functions.1ꢀ3 They
are found as excellent natural ligands for the selectin
family,1 pathogenic bacteria and viruses,1 and other
carbohydrate receptor proteins like lectins.4ꢀ6 Recently,
we have reported a rapid detection for Shiga toxins
secreted from Escherichia coli O-157 by applying the
artificial carbohydrate ligand.7 Bacterial toxins and
number of reports on the synthesis of Neu5Ac-a-(20
/
3?)-lactosamine glycoside carrying different groups at
the reducing end using chemical9 and chemo-
enzymatic12ꢀ14 synthetic strategies. However, to the
best of our knowledge, we report herein for the first
other speciesꢀspecific proteins that recognize the spe-
/
time, the chemoenzymatic synthesis of sialyl-a-(203?)-
/
cific sialo-oligosaccharides on their binding sites might
have potential application for the detection of patho-
gens and also for new drug discovery associated with
inflammation and cancer.8 These important phenomena
have provided the impetus for the scientist to synthesize
sialo-oligosaccharides for more precise studies. Most of
the naturally occurring sialo-oligosaccharides contain a-
linked sialic acids. Both chemical9,10 as well as che-
lactosamine carrying a 3-aminopropyl group as the
spacer at the reducing end so that the final compound
can be utilized for further biological studies. It should be
noted here that Nifantiev and co-workers9,15 have used
the 3-aminopropyl group as a spacer by a different
chemical manipulation for their chemical synthesis of
Neu5Ac-a-(20
/
6?)-lactosamine 3-aminopropyl glyco-
6?)-lactosamine 3-aminopropyl
side,15 Neu5Gc-a-(20
/
glycoside15 and Neu5Gc-a-(20
3?)-lactosamine 3-ami-
/
nopropyl glycoside.9 However, their chemical ap-
proaches yielded a byproduct and poor yields during
the coupling of sialic acid in the final step.9 To improve
* Corresponding author. Tel.: ꢁ
298-614680.
E-mail address: h.uzawa@aist.go.jp (H. Uzawa).
/
81-298-614431; fax: ꢁ81-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00167-8

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I. Choudhury (Mukherjee) et al. / Carbohydrate Research 338 (2003) 1265ꢀ1270
/
1
the yield of sialylation and to avoid the byproduct, we
first chemically synthesized the disaccharide acceptor,
lactosamine 3-aminopropyl glycoside (13), and then
stereospecifically coupled it with CMP-sialic acid using
3 was determined by its H NMR spectrum, with H-1
appearing at d 4.64 as a wide doublet (J 8.4 Hz) and by
its 13C NMR spectrum that showed C-1 at d 101.0.
Compound 3 was O-deacetylated with sodium methox-
ide18 in methanol, and the resulting 3-azidopropyl 2-
a-(20
/
3)-(N)-sialyltransferase (E.C. 2.4.99.5) to afford
the desired trisaccharide 14 in high yield. This chemoen-
zymatic synthesis provided a better yield of the desired
trisaccharide 14 and also minimized the number of
synthetic steps.
acetamido-2-deoxy-b-
D-glucopyranoside (4) was treated
with benzaldehyde dimethylacetal in 1:1 acetonitrileꢀ
/
N,N-dimethylformamide in the presence of a catalytic
amount of p-toluenesulfonic acid monohydrate to
provide 3-azidopropyl 2-acetamido-4,6-O-benzylidene-
2-deoxy-b-
D-glucopyranoside (5) in 87% yield. To avoid
N-benzylation, compound 5 was benzylated by using
phase-transfer conditions in the presence of tetrabuty-
lammonium bromide as a phase-transfer catalyst to
obtain 3-azidopropyl 2-acetamido-3-O-benzyl-4,6-O-
2. Results and discussion
The known 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-
D-
glucopyranosyl trichloroacetimidate16 (1) was allowed
(Scheme 1) to react with 3-bromo-1-propanol in the
presence of trimethylsilyl trifluoromethanesulfonate17 to
obtain 3-bromopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-
benzylidene-2-deoxy-b-D-glucopyranoside (6) in 84%
yield. Removal of the benzylidene group of compound
6 with 80% aqueous acetic acid gave the diol derivative
7. Selective benzylation19 of compound 7 was performed
via its stannylene derivative to afford 3-azidopropyl 2-
deoxy-b-
D-glucopyranoside (2) in 76% yield. Treatment
of compound 2 with NaN3 in N,N-dimethylformamide
acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
side (8) in 52% yield. The presence of a free OH group at
C-4 of 8 was confirmed by acetylation of 8 with acetic
at 60 8C afforded 3-azidopropyl 2-acetamido-3,4,6-tri-
O-acetyl-2-deoxy-b-
D-glucopyranoside (3) in 94% yield.
The b linkage at the anomeric position of the compound
˚
Scheme 1. Reagents and conditions: (i) Br(CH2)3OH, Me3SiOTf, CH2Cl2, 4 A MS, 20 min, ꢂ
/
10 8C; (ii) NaN3, DMF, 3 h, 60 8C;
(iii) NaOMe, MeOH; (iv) PhCH(OMe)2, 1:1 CH3CNꢀ
80% AcOH, 3 h, 80 8C; (vii) Bu2SnO, toluene, BnBr, Bu4NBr; (viii) Ac2O, pyridine; (ix) AgOTf, CH2Cl2, 4 A MS, ꢂ
(x) H2, 10% PdꢀC, 2:1 MeOHꢀH2O; (xi) CMP-b- -sialic acid, disodium salt, rat liver a-(203)-(N)-sialyltransferase, calf intestine
alkaline phosphatase, HEPES buffer.
/
DMF, p-TsOH, 48 h; (v) BnBr, Bu4NBr, 10% NaOH, CH2Cl2, 48 h, rt; (vi)
˚
/
300
/
ꢂ10 8C;
/
/
/
D
/

I. Choudhury (Mukherjee) et al. / Carbohydrate Research 338 (2003) 1265ꢀ
/1270
1267
anhydride and pyridine. In the 1H NMR spectrum of the
resulting acetate 9, the signal of H-4 had shifted down-
field to d 5.00 compared with its position at d 3.66 in the
spectrum of 8. Commercially available 2,3,4,6-tetra-O-
various sialo-oligosaccharides binding lectins like E, P,
and L-selectins.1,4 Application to these studies are in
progress and will be reported in due course.
acetyl-a-
D-galactopyranosyl bromide (10) was used as
donor and was reacted20 with the acceptor 8 using
AgOTf as activator in dichloromethane to afford the
3. Experimental
disaccharide 3-azidopropyl 2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(104)-2-acetamido-3,6-di-O-benzyl-
2-deoxy-b-
D-
3.1. General methods
/
D
-glucopyranoside (11) in 76% yield. The b
All reactions were monitored by thin-layer chromato-
graphy (TLC) on Silica Gel 60 F254 (E. Merck Japan
Ltd.) and 1:2:1 n-butanolꢀ
linkages of compound 11 were confirmed from its 1H (d
4.70, J1,2 5.6 Hz, H-1 and d 4.50, J1?,2? 8.8 Hz, H-1?) and
13C NMR [100.2 (C-1), 99.8, (C-1?)] spectra. Deacetyla-
tion of compound 11 with sodium methoxide, followed
/
n-propanolꢀ
/
0.1 M aq HCl
(BPH), 1:1:1 MeCNꢀMeOHꢀ
/
/water (MMW) were used
as elution solvents for the deblocked trisaccharide 14.
Column chromatography was performed on Silica Gel
60 N (Cica Reagent). Toyopearl HW-40S gel was
purchased from Tosoh Corporation (Japan) and was
used for gel-permeation chromatography. 2,3,4,6-Tetra-
by catalytic hydrogenolysis with Pdꢀ
afforded 3-aminopropyl b- -galactopyranosyl-(10
acetamido-2-deoxy-b- -glucopyranoside (13) in 91%
/
C in MeOHꢀ/H2O,
D
/4)-2-
D
overall yield in two steps. Compound 13 was character-
ized from its 1H NMR and FAB mass spectra
(FABMS). Compound 13 was enzymatically12 sialylated
using commercially available cytidine-5?-monophospho-
O-acetyl-a-
from Aldrich Chemical Co. CMP-b-
odium salt, and a-(203)-(N)-sialyltransferase were
D
-galactopyranosyl bromide was purchased
D-sialic acid, dis-
/
b-
donor in N-2-hydroxyethylpiperazine-N?-2-ethanesulfo-
nic acid (HEPES) buffer in the presence of a-(203)-
D-sialic acid (CMP-b-
D-sialic acid), disodium salt as
purchased from Calbiochem (USA). Alkaline phospha-
tase was purchased from Wako Pure Chemical Indus-
tries Ltd. (Japan). Optical rotations were measured with
a JASCO DIP-1000 digital polarimeter at 25 8C. NMR
spectra were recorded on a Varian INOVA 400 MHz
/
(N)-sialyltransferase, isolated from rat liver, and alka-
line phosphatase (E.C. 3.1.3.1), isolated from calf
intestine, to obtain exclusively 3-aminopropyl 5-acetam-
spectrometer. For the H and 13C NMR spectra, Me4Si
1
ido-3,5-dideoxy-
osylonic acid-(20
acetamido-2-deoxy-b-
D
-glycero-a-
3)-b- -galactopyranosyl-(10
-glucopyranoside (14) in 78%
D
-galacto -2-nonulopyran-
was used as the internal standard in CDCl3 and CD3OD
solutions. tert-Butyl alcohol (d 1.24 (1H)) was used as
the internal standard in D2O solution for the NMR
spectrum of compound 13. The 13C NMR spectrum of
compound 14 in D2O solution was measured by fixing
HOD peak at d 4.70 in the 1H NMR spectrum followed
by measuring 13C NMR spectra, and thus 13C chemical
shifts in D2O solution was reported in ppm without any
reference standard. FABMS were recorded using a
JEOL DX 303 mass spectrometer, and high-resolution
EI mass spectra (EIMS) were recorded using a Hitachi
M 80 mass spectrometer.
/
D
/
4)-2-
D
1
yield. The structure of 14 was determined by H and
13C NMR spectroscopy. The characteristic peaks at d
2.62 (H-3ƒe), 1.65 (H-3ƒa) in the 1H NMR spectrum and
peaks at d 174.0 (C-1ƒ), 72.3 (C-6ƒ) in the 13C NMR
spectrum confirmed the a coupling of the Neu5Ac
residue in trisaccharide 14. The peak appearing at d
78.5 (C-3?) in the 13C NMR spectrum of compound 14
was clearly shifted towards downfield compared to other
ring carbons [C-2? (71.9), C-4? (69.5) and C-6? (61.2)] in
the Gal residue, which are well consistent with the data
reported for Neu5Gc-a-(203?)-lactosamine 3-amino-
/
3.2. 3-Azidopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-D-glucopyranoside (3)
propyl glycoside.9 In the H NMR of 14, the doublet
of doublets peak appearing at d 3.97 (H-3?) in the Gal
moiety was also shifted downfield, supporting the a-
1
A solution of compound 1 (4.00 g, 8.13 mmol) and 3-
bromo-1-propanol (7.4 mL, 81 mmol) in dry CH2Cl2 (20
˚
mL) containing freshly activated 4 A MS (400 mg) was
(20
Neu5Ac and Gal moieties are connected via the sialyl
a-(203?)-Gal linkage.
/
3?) regioselectivity. These results show that the
/
stirred for 1 h at 25 8C under N2. The reaction mixture
In conclusion, we have chemoenzymatically synthe-
sized the sialylated lactosamine 3-aminopropyl glycoside
14 in high yield, which is readily accessible for sub-
sequent polymerization and immobilization for further
biological studies with a surface plasmon resonance
(SPR) technique and a quartz crystal microbalance
(QCM) method.7,21 The trisaccharide synthesized here
is a promising candidate for ligands of, for instance,
was then cooled at ꢂ
mmol) was added. The reaction mixture was stirred at
10 8C for 20 min and then filtered over a Celite bed.
The organic layer was washed with satd aq NaHCO3
and then water. The organic layer was dried (Na2SO4),
filtered and concentrated. Column chromatography of
/
10 8C, and Me3SiOTf (0.20 mL, 1.1
ꢂ
/
the product on silica gel with 5:1 EtOAcꢀ
/hexane
afforded compound 2 as a white solid (2.90 g, 76%). 3-

1268
I. Choudhury (Mukherjee) et al. / Carbohydrate Research 338 (2003) 1265ꢀ1270
/
Bromopropyl glycoside 2 (2.90 g, 6.19 mmol) was then
dissolved in DMF (5 mL), and NaN3 (2.01 g, 30.9
mmol) was added to it. The reaction mixture was stirred
at 60 8C for 3 h, and it was then cooled at 25 8C. The
insoluble material was filtered off. The organic layer was
diluted with EtOAc, washed with brine solution, dried
(Na2SO4), filtered and concentrated. Column chroma-
3.4. 3-Azidopropyl 2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-b- -glucopyranoside (6)
D
A mixture of compound 5 (781 mg, 1.99 mmol), 10% aq
NaOH (5 mL), and BnBr (0.35 mL, 3.0 mmol) in
CH2Cl2 (8 mL) was stirred at 25 8C for 30 min, and then
Bu4NBr (126 mg, 0.391 mmol) was added. The reaction
mixture was stirred at 25 8C for 48 h. The reaction
mixture was then diluted with CH2Cl2. The organic
layer was washed with water, dried (Na2SO4), filtered
and concentrated. Column chromatography of the
tography of the product on silica gel with 2:1 EtOAcꢀ
/
hexane gave compound 3 as white foam (2.50 g, 94%):
1
[a]D
ꢂ8.38 (c 0.6, CHCl3); NMR (CDCl3): H, d 5.81
/
(d, 1 H, JNꢀH,2 8.8 Hz, NH), 5.25 (dd, 1 H, J2,3 10.8, J3,4
9.6 Hz, H-3), 5.07 (t, 1 H, J4,5 9.6 Hz, H-4), 4.64 (d, 1 H,
J1,2 8.4 Hz, H-1), 4.26 (dd, 1 H, J6a,6b 12.4, J5,6a 4.8 Hz,
product on silica gel with 8:1 EtOAcꢀ
white foam (810 mg, 84%): [a]D 60.58 (c 0.6, CHCl3);
7.31 (m, 10 H, aromatic
/hexane gave 6 as
ꢂ
/
1
NMR (CDCl3): H, d 7.49ꢀ
/
H-6a), 4.15 (dd, 1 H, J5,6b 2.4 Hz, H-6b), 3.99ꢀ
2 H, H-5, OCH2CH2CH2N3), 3.74ꢀ3.69 (m, 1 H,
OCH2CH2CH2N3), 3.64ꢀ3.57 (m, 1 H, H-2), 3.39ꢀ
3.36 (m, 2 H, OCH2CH2CH2N3), 2.09, 2.04, 2.03 (3 s,
1.83 (m,
/
3.89 (m,
protons), 5.56 (s, 1 H, PhCH), 5.54 (d, 1 H, JNꢀH,2 8.0
Hz, NH), 4.88 (dd, 2 H, J 11.6, J1,2 8.0 Hz, PhCH2, H-
1), 4.65 (d, 1 H, PhCH2), 4.35 (dd, 1 H, J6a,6b 10.4, J5,6a
/
/
/
5.2 Hz, H-6a), 4.14 (t, 1 H, J2,3
3.94ꢀ3.89 (m, 1 H, OCH2CH2CH2N3), 3.78 (t, 1 H, J5,6b
10.3 Hz, H-6b), 3.68 (t, 1 H, J4,5 9.2 Hz, H-4), 3.61ꢀ3.55
(m, 1 H, H-5), 3.53ꢀ3.47 (m, 1 H, OCH2CH2CH2N3),
3.44ꢀ3.34 (m, 3 H, H-2, OCH2CH2CH2N3), 1.89 (s, 3 H,
NHCOCH3), 1.87ꢀ1.76 (m, 2 H, OCH2CH2CH2N3);
13C, 170.4 (CO), 138.3ꢀ
126.0 (Ph), 101.2, 100.8 (PhCH,
ꢃ/J3,4
ꢃ/9.2 Hz, H-3),
9 H, CH3CO), 1.96 (s, 3 H, NHCOCH3), 1.89ꢀ
/
/
2 H, OCH2CH2CH2N3); 13C, d 171.0, 170.7, 170.3,
169.4 (4 CO), 101.0 (C-1), 72.4, 71.8, 68.6, 66.2, 62.1,
54.5, 48.0 (OCH2CH2CH2N3), 28.9 (OCH2CH2-
CH2N3), 23.3 (NHCOCH3), 20.8, 20.7, 20.6 (3
CH3CO). EIMS: Anal. Calcd for [C17H26N4O9
/
/
/
/
/
(430.1700)ꢁ
H]ꢁ, 431.1779. Found: m/z 431.1766.
/
C-1), 82.7, 76.5, 74.4, 68.8, 66.4, 66.0, 57.3, 48.1
(OCH2CH2CH2N3), 29.0 (OCH2CH2CH2N3), 23.5
(NHCOCH3). EIMS: Anal. Calcd for [C25H30N4O6
(482.2165)ꢁ
H]ꢁ, 483.2244. Found: m/z 483.2260.
/
3.3. 3-Azidopropyl 2-acetamido-4,6-O-benzylidene-2-
deoxy-b- -glucopyranoside (5)
D
3.5. 3-Azidopropyl 2-acetamido-3,6-di-O-benzyl-2-
deoxy-b- -glucopyranoside (8)
D
Treatment of compound 3 (2.40 g, 5.58 mmol) with 0.1
M NaOMe for 2 h, followed by neutralization with
Dowex 50W (Hꢁ) resin, afforded compound 4 (1.60 g,
94%) as a white solid. To a solution of compound 4
A suspension of compound 6 (357 mg, 0.740 mmol) in
80% aq AcOH (2 mL) was heated at 80 8C for 3 h. The
reaction mixture was then concentrated and flashed
three times with toluene. The residue was dried over-
night under vacuum to afford compound 7 (280 mg,
96%) as white foam. To a solution of 7 (280 mg, 0.710
mmol) in toluene, Bu2SnO (199 mg, 0.799 mmol) was
added, and the mixture was refluxed with azeotropic
removal of water for 18 h. The reaction mixture was
concentrated to dryness. Fresh toluene (3 mL), BnBr
(0.40 mL, 3.6 mmol) and Bu4NBr (114 mg, 0.354 mmol)
were added, and the mixture was stirred overnight at
60 8C. The solvent was evaporated under reduced
pressure, MeOH was added, and the mixture was
cooled. Solids that separated were removed by filtration,
and the filtrate was concentrated. Column chromato-
(1.60 g, 5.26 mmol) in 1:1 CH3CNꢀDMF (20 mL),
/
benzaldehyde dimethylacetal (0.90 mL, 6.2 mmol) and a
catalytic amount of p-TsOH were added. The reaction
mixture was stirred at 25 8C for 48 h and then
neutralized with Et3N. The solution was concentrated
under reduced pressure. Column chromatography on
silica gel with EtOAc gave compound 5 as a white foam
(1.80 g, 87%): [a]D
ꢂ
/
358 (c 0.8, CHCl3); NMR (1:1
CDCl3): H, d 7.65ꢀ7.34 (m, 5 H, aromatic
protons), 5.58 (s, 1 H, PhCH), 4.53 (d, 1 H, J1,2 8.4 Hz,
H-1), 4.36ꢀ4.32 (m, 1 H, H-6a), 3.96ꢀ3.91 (m, 1 H,
OCH2CH2CH2N3), 3.83ꢀ3.73 (m, 3 H, H-4, H-6b,
OCH2CH2CH2N3), 3.63ꢀ3.59 (m, 1 H, H-5), 3.55 (t, 1
H, J2,3 J3,4 9.6 Hz, H-3), 3.48ꢀ3.43 (m, 1 H, H-2),
3.42ꢀ3.36 (m, 2 H, OCH2CH2CH2N3), 2.01 (s, 3 H,
NHCOCH3), 1.84ꢀ1.82 (m, 2 H, OCH2CH2CH2N3);
13C, 172.1 (CO), 136.8ꢀ
125.7 (Ph), 101.3, 101.2 (C-1,
1
CD3ODꢀ
/
/
/
/
/
/
ꢃ
/
ꢃ
/
/
graphy of the product on silica gel with 5:1 EtOAcꢀ
hexane gave pure 8 as white foam (180 mg, 52%): [a]D
12.28 (c 0.8, CHCl3); NMR (CDCl3): 1H, d 7.32ꢀ
7.26
/
/
/
ꢂ
/
/
/
(m, 10 H, aromatic protons), 5.68 (d, 1 H, JNꢀH,2 8.0 Hz,
NH), 4.79 (d, 1 H, J 12.0 Hz, PhCH2), 4.72 (d, 1 H, J1,2
8.4 Hz, H-1), 4.69 (d, 1 H, PhCH2), 4.57 (dd, 2 H,
PhCH), 81.0, 70.6, 68.0, 65.7, 56.2, 48.0
(OCH2CH2CH2N3), 28.3 (OCH2CH2CH2N3), 21.7
(NHCOCH3). EIMS: Anal. Calcd for [C18H24N4O6
PhCH2), 3.91ꢀ
/
3.84 (m, 2 H, H-3, OCH2CH2CH2N3),
J4,5 9.6
(392.1696)ꢁ
/
H]ꢁ, 393.1775. Found: m/z 393.1746.
3.74ꢀ3.70 (m, 2 H, H-6), 3.66 (t, 1 H, J3,4
/
ꢃ
/
ꢃ
/

I. Choudhury (Mukherjee) et al. / Carbohydrate Research 338 (2003) 1265ꢀ
/1270
1269
Hz, H-4), 3.56ꢀ
3.48ꢀ3.42 (m,
OCH2CH2CH2N3), 1.89 (s, 3 H, NHCOCH3), 1.87ꢀ
1.76 (m, 2 H, OCH2CH2CH2N3); 13C, 170.4 (CO),
138.4ꢀ127.7 (Ph), 100.2 (C-1), 80.5, 74.0, 73.8, 73.6,
/
3.49 (m, 2 H, OCH2CH2CH2N3, H-5),
3.7. 3-Aminopropyl b- -galactopyranosyl-(10
D
/
4)-2-
/
1
H, H-2), 3.36ꢀ3.30 (m, H,
/
2
acetamido-2-deoxy-b-D-glucopyranoside (13)
/
Compound 11 (46 mg, 0.06 mmol) was deacetylated
with 0.1 M NaOMe according to Zemple´n18 to give the
deacetylated product 12, which was then dissolved in 2:1
/
73.0, 70.5, 66.0, 56.3, 48.1 (OCH2CH2CH2N3), 28.9
(OCH2CH2CH2N3), 23.5 (NHCOCH3). FABMS: Anal.
MeOHꢀ
/
H2O (3 mL) and was stirred with 10% PdꢀC (30
/
Calcd for [C25H32N4O6 (484)ꢁ
/
H]ꢁ, 485. Found: m/z
485. Compound 8 (10 mg) was acetylated in the usual
mg) under H2 for 48 h at 25 8C. The reaction mixture
was then filtered through Celite, and the pad was
washed thoroughly with water. The combined filtrate
and washings was concentrated to afford compound 13
way with Ac2O and Py to afford the acetate 9 (9 mg):
1
NMR (CDCl3): H, d 7.32ꢀ/7.26 (m, 10 H, aromatic
protons), 5.57 (d, 1 H, JNꢀH,2 7.6 Hz, NH), 5.00 (t, 1 H,
J3,4 10.4, J4,5 9.6 Hz, H-4), 4.96 (d, 1 H, J1,2 8.4 Hz, H-
1), 4.62 (d, 1 H, J 11.2 Hz, PhCH2), 4.56 (d, 1 H,
PhCH2), 4.52 (s, 2 H, PhCH2), 4.26 (t, 1 H, J2,3 9.2 Hz,
as an amorphous powder (24 mg, 91%): [a]D
ꢂ
/
6.78 (c
0.25, water); NMR (D2O): H, d 4.38 (d, 1 H, J1,2 7.6
Hz, H-1), 4.33 (d, 1 H, J1?,2? 7.6 Hz, H-1?), 4.01ꢀ3.84 (m,
2 H, H-4?, OCH2CH2CH2NH2), 3.81ꢀ3.77 (m, 1 H, H-
6a), 3.75ꢀ3.67 (m, 1 H, H-6b), 3.65ꢀ3.51 (m, 8 H, H-3,
H-4, H-5, H-2?, H-3?, H-5?, H-6?), 3.48ꢀ3.43 (m, 1 H,
OCH2CH2CH2NH2), 3.42ꢀ3.38 (m, 1 H, H-2), 3.00ꢀ
2.93 (m, 2 H, OCH2CH2CH2NH2), 1.91 (s, 3 H,
1
/
/
H-3), 3.95ꢀ
(m, 4 H, H-5, H-6, OCH2CH2CH2N3), 3.38ꢀ
H, OCH2CH2CH2N3), 3.30ꢀ3.23 (m, 1 H, H-2), 1.90,
1.88 (2 s, 6 H, COCH3, NHCOCH3), 1.87ꢀ1.76 (m, 2 H,
/
3.92 (m, 1 H, OCH2CH2CH2N3), 3.67ꢀ
/
3.55
/
/
/3.34 (m, 2
/
/
/
/
/
OCH2CH2CH2N3).
NHCOCH3), 1.87ꢀ
FABMS: Anal. Calcd for [C17H32N2O11 (440)ꢁ
/
1.76 (m, 2 H, OCH2CH2CH2NH2);
H]ꢁ,
/
441. Found: m/z 441.
3.6. 3-Azidopropyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl-(10
/
4)-2-acetamido-3,6-di-O-benzyl-2-
3.8. 3-Aminopropyl 5-acetamido-3,5-dideoxy-
D
-glycero-
deoxy-b-D-glucopyranoside (11)
a-
galactopyranosyl-(10
glucopyranoside (14)
D
-galacto-2-nonulopyranosylonic acid-(20
/
3)-b-D-
4)-2-acetamido-2-deoxy-b-D-
/
A solution of the acceptor 8 (50 mg, 0.10 mmol) and
donor 10 (85 mg, 0.21 mmol) in CH2Cl2 (2 mL)
˚
containing 4 A MS (218 mg) was stirred under N2 for
The disaccharide acceptor 13 (3 mg, 0.007 mmol), CMP-
b- -sialic acid, disodium salt (4.6 mg, 0.007 mmol),
alkaline phosphatase (17.6 U), rat liver a-(203)-(N)-
sialyltransferase (18.6 mU) was incubated with HEPES
buffer (1 mL, 50 mM, pH 7.4) at 35 8C for 5 days. On
the third day of the incubation another portion of CMP-
2 h and cooled to ꢂ
was then added. Stirring was continued in the dark for 1
h at ꢂ30 8C and then for 2 h at ꢂ10 8C. The reaction
/
30 8C. AgOTf (81 mg, 0.32 mmol)
D
/
/
/
mixture was diluted with CH2Cl2, filtered and washed
with satd aq NaHCO3 and water. The organic layer was
dried (Na2SO4), filtered and concentrated. Column
chromatography of the product on silica gel with 3:2
b-
D-sialic acid, disodium salt (2.3 mg, 0.003 mmol) was
added. On the fifth day TLC analysis (using 1:1 BPHꢀ
/
EtOAcꢀ
/
hexane afforded recovered acceptor 8 (8 mg,
16%) and disaccharide 11 (54 mg, 76%) as a white foam:
MMW as solvents) showed that all the donor was
consumed. The reaction mixture was heated at 100 8C to
deactivate the enzyme, and the crude product was
subjected to a gel-permeation column (Toyopearl HW-
1
[a]D
ꢁ
/
168 (c 0.6, CHCl3); NMR (CDCl3): H, d 7.32ꢀ
/
7.26 (m, 10 H, aromatic protons), 5.83 (d, 1 H, JNꢀH,2
8.0 Hz, NH), 5.32 (br d, 1 H, J 2.8 Hz, H-4?), 5.12 (dd, 1
H, J2?,3? 10.4, J1?,2? 8.0 Hz, H-2?), 4.91 (dd, 1 H, J3?,4? 3.6
Hz, H-3?), 4.76 (d, 1 H, J 11.6 Hz, PhCH2), 4.70 (d, 1 H,
J1,2 5.6 Hz, H-1), 4.65 (dd, 2 H, PhCH2), 4.50 (d, 1 H,
40S, 1.5ꢄ50 cm), and the column was eluted with 0.1 M
/
aq AcOH. The fractions containing the desired product
were collected, combined and concentrated. The pro-
duct was passed again through similar two Toyopearl
HW-40S columns to obtain the desired trisaccharide 14
J
1?,2? 8.8 Hz, H-1?), 4.47 (d, 1 H, PhCH2), 4.15ꢀ
11 H, H-2, H-3, H-4, H-5, H-6, H-5?, H-6?,
OCH2CH2CH2N3), 3.37ꢀ3.28 (m, 2 H, OCH2CH2-
CH2N3), 2.11, 2.03, 2.01, 1.98, 1.93 (5 s, 15 H,
COCH3, NHCOCH3), 1.87ꢀ1.76 (m, 2 H, OCH2CH2-
CH2N3); 13C, 170.3, 170.2, 170.1, 170.0, 169.8 (CO),
138.4ꢀ127.7 (Ph), 100.2, 99.8 (C-1, C-1?), 75.2, 74.4,
/3.47 (m,
as an amorphous powder (4 mg, 78%): [a]D
0.25, water); NMR (D2O): 1H, d 4.41 (d, 1 H, J1?,2? 8 Hz,
ꢂ3.38 (c
/
/
H-1?), 4.37 (d, 1 H, J1,2 7.6 Hz, H-1), 3.97 (dd, 1 H, J2?,3?
/
10, J3?,4? 3.2 Hz, H-3?), 3.91ꢀ
OCH2CH2CH2NH2), 3.81 (d, 1 H, H-4?), 3.78ꢀ
(m, 5 H, H-5, H-5?, H-5ƒ, H-6b, H-8ƒ), 3.62ꢀ3.54 (m, 5
H, H-2, H-3, H-4, H-4ƒ, H-6ƒ), 3.52ꢀ3.40 (m, 7 H, H-2?,
H-6?, H-7ƒ, H-9ƒ, OCH2CH2CH2NH2), 3.00ꢀ2.87 (m, 2
H, OCH2CH2CH2NH2), 2.62 (dd, 1 H, J 12.8 Hz, J3ƒe,4ƒ
4.8 Hz, H-3ƒe); 1.89, 1.87 (2 s, 6 H, NHCOCH3), 1.81ꢀ
1.79 (m, 2 H, OCH2CH2CH2NH2), 1.65 (t, 1 H, H-3aƒ);
/3.85 (m, 2 H, H-6a,
/3.68
/
/
73.6, 73.2, 70.7, 70.6, 69.3, 68.7, 66.8, 66.0, 60.8, 53.1,
48.2 (OCH2CH2CH2N3), 29.0 (OCH2CH2CH2N3), 23.4
(NHCOCH3), 20.8, 20.6, 20.5 (CH3CO). FABMS:
Anal. Calcd for [C39H50N4O15 (814)ꢁ
m/z 815.
/
/
/
H]ꢁ, 815. Found:
/

1270
I. Choudhury (Mukherjee) et al. / Carbohydrate Research 338 (2003) 1265ꢀ1270
/
13C, 175.2, 174.9 (2 CO), 174.0 (C-1ƒ), 102.8 (C-1?),
101.4 (C-1), 99.9 (C-2ƒ), 78.5 (C-3?), 75.6 (C-5?), 75.4 (C-
4), 74.9 (C-5), 73.1 (C-3), 72.3 (C-6ƒ), 71.9 (C-8ƒ, C-2?),
69.5 (C-4?), 68.3 (C-7ƒ), 68.1 (C-4ƒ), 67.6 (OCH2CH2-
CH2NH2), 63.0 (C-9ƒ), 61.2 (C-6?, C-6), 55.2 (C-2), 51.9
(C-5ƒ), 40.0 (C-3ƒ), 37.8 (OCH2CH2CH2NH2), 26.8
(OCH2CH2CH2NH2), 22.2 (NHCOCH3). FABMS:
7. Uzawa, H.; Kamiya, S.; Minoura, N.; Dohi, H.; Nishida,
Y.; Taguchi, K.; Yokoyama, S.; Mori, H.; Shimizu, T.;
Kobayashi, K. Biomacromolecules 2002, 3, 411ꢀ
8. Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.;
Wong, C.-H. Chem. Rev. 1998, 98, 833ꢀ862.
/
414.
/
9. Sherman, A. A.; Yudina, O. N.; Shashkov, A. S.;
Menshov, V. M.; Nifantiev, N. E. Carbohydr. Res. 2002,
337, 451ꢀ/457.
Anal. Calcd for [C28H49N3O19 (731)ꢁ
H]ꢁ, 732. Found:
m/z 732.
/
10. Marra, A.; Sinay, P. Carbohydr. Res. 1990, 195, 303ꢀ
/
308.
11. Gaudino, J. J.; Paulson, J. C. J. Am. Chem. Soc. 1994,
116, 1149ꢀ1150.
12. Limberg, G.; Slim, G. C.; Compston, C. A.; Stangier, P.;
Palcic, M. M.; Furneaux, R. H. Liebigs Ann. 1996, 1773ꢀ
¨
/
Acknowledgements
/
1784.
13. Matsuda, M.; Nishimura, S.-I. J. Med. Chem. 2001, 44,
This work was partly supported by subsidies from the
Ministry of Economy, Trade and Industry of Japan. We
thank Dr. Xiaoxiong Zeng of AIST for enzymatic
synthesis.
715ꢀ724.
/
14. Izumi, M.; Shen, G.-J.; Wacowich-Sgarbi, S.; Nakatani,
T.; Plettenburg, O.; Wong, C.-H. J. Am. Chem. Soc. 2001,
123, 10909ꢀ10918.
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15. Sherman, A. A.; Yudina, O. N.; Shashkov, A. S.;
Menshov, V. M.; Nifantiev, N. E. Carbohydr. Res. 2001,
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