Systematic synthesis of bisubstrate-type inhibitors of TV- acetylglucosaminyltransferases

Article abstract of DOI:10.1002/chem.200501348

Bisubstrate-type inhibitors for N-acetylglucosaminyltransferase (GnT)-V and -IX were designed and synthesized. These compounds carry both an acceptor trisaccaride and an UDP-GlcNAc component tethered by a linker of variable length. The acceptor trisaccharide unit was constructed using a combination of a polymer support and a resin capture-release strategy. Namely, starting with a β-mannoside bound to low molecular weight monomethyl PEG (MPEG), successive glycosylations with donors having chloroacetyl group produced the trisaccharide, which was subjected to the capture-release purification using cysteine loaded resin. UDP-GlcNAc units carrying phosphate moieties were separately synthesized from the bromoacetamide-containing glucosamine derivative. Ligation between the acceptor thiol and each alkyl bromide on the donor unit readily proceeded, and produced the coupling product. The introduction of the UMP component gave target compounds. All of the synthesized compounds had significant activities to GnT-V and -IX. Their potencies were dependent upon the linkers length. GnT-IX was more sensitive to these inhibitors and optimum linker length was clearly different between these GnTs. The most potent inhibitor of GnT-V had K_i = 18.3 μM, while that of GnT-IX had K_i = 4.7 μM.

Full text of DOI:10.1002/chem.200501348

FULL PAPER
Chem. Eur. J. 2006, 12, 3449 – 3462
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3449

DOI: 10.1002/chem.200501348
Systematic Synthesis of Bisubstrate-Type Inhibitors of
N-Acetylglucosaminyltransferases
Shinya Hanashima,[a, b] Kei-ichiro Inamori,[c] Shino Manabe,[a, d] Naoyuki Taniguchi,[c] and
Yukishige Ito*[a]
Abstract: Bisubstrate-type inhibitors
for N-acetylglucosaminyltransferase
chloroacetyl group produced the trisac-
charide, which was subjected to the
capture–release purification using cys-
teine loaded resin. UDP–GlcNAc units
carrying phosphate moieties were sepa-
rately synthesized from the bromoace-
tamide-containing glucosamine deriva-
tive. Ligation between the acceptor
thiol and each alkyl bromide on the
donor unit readily proceeded, and pro-
duced the coupling product. The intro-
duction of the UMP component gave
target compounds. All of the synthe-
sized compounds had significant activi-
ties to GnT-V and -IX. Their potencies
were dependent upon the linkers
length. GnT-IX was more sensitive to
these inhibitors and optimum linker
length was clearly different between
these GnTs. The most potent inhibitor
of GnT-V had Ki =18.3 mm, while that
of GnT-IX had Ki =4.7 mm.
(GnT)-V and -IX were designed and
synthesized. These compounds carry
both an acceptor trisaccaride and an
UDP–GlcNAc component tethered by
a linker of variable length. The accep-
tor trisaccharide unit was constructed
using a combination of a polymer sup-
port and a resin capture–release strat-
egy. Namely, starting with a b-manno-
side bound to low molecular weight
monomethyl PEG (MPEG), successive
glycosylations with donors having
Keywords:
glycoproteins
·
glycosylation · glycosyltransferase ·
inhibitors · solid-phase synthesis
Introduction
plex-type asparagine (Asn)-linked (N-linked) glycoproteins.
They share the ability to transfer an N-acetylglucoamine
(GlcNAc) residue to the appropriate hydroxyl group of gly-
cans or serine/threonine residues using uridine-5’-diphos-
phate-a-d-N-acetylglucosamine (UDP-GlcNAc) as a donor
substrate. Among a variety of glycan structures made by
GnTs, the characteristic branch produced by GnT-V, in
which a GlcNAc residue is linked via a b1,6-linkage to the
core a1,6-mannose (Man) arm on complex type N-glycan
(Figure 1a), is highly intriguing, because of its relationship
with various biomedically important phenomena (see
below).[2] The more recently discovered GnT-IX is a closely
related homologue of GnT-V, being exclusively expressed in
brain.[3] It has a broader substrate specificity than GnT-V
and transfers a GlcNAc residue to both a1,3- and a1,6-
linked mannose structures, as well as to O-linked
GlcNAcb1,2-Man.[4]
Elevated levels of GnT-V and the GlcNAcb1,6-Man-
branched glycans produced by this enzyme correlate with
the malignant transformation and metastaticpotential of
tumor cells.[5,6a–h] Therefore, the suppression of GnT-V
might have clinical potential in the treatment of cancer.[7] In
addition, GnT-V affects T-cell activation and angiogene-
sis.[6f,g] The ubiquitous expression of GnT-V is in sharp con-
Glycosyltransferases are a group of enzymes responsible for
the biosynthesis of glycoconjugate oligosaccharides.[1] Mam-
malian N-acetylglucosaminyltransferases (GnTs) are of par-
ticular importance, because they are key enzymes in the
production of highly diverse and variously branched com-
[a] Dr. S. Hanashima, Dr. S. Manabe, Dr. Y. Ito
RIKEN (The Institute of Physical and Chemical Research)
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
Fax : (+81)48-462-4680
E-mail: yukito@riken.jp
[b] Dr. S. Hanashima
Present address:
Laboratorium für Organische Chemie, ETH Hçnggerberg
8093 Zürich (Switzerland)
[c] Dr. K.-i. Inamori, Prof. Dr. N. Taniguchi
Department of Biochemistry
Osaka University Medical School
2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)
[d] Dr. S. Manabe
PRESTO, Japan Science and Technology Agency (JST)
Kawaguchi, Saitama 332-1102 (Japan)
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Glucosyltransferase Inhibitors
FULL PAPER
nent, we planned to incorporate
GlcN-a-phosphate
using
a
thiol-based ligation strategy, be-
cause of its technical simplicity
and chemoselectivity, which was
to be followed by coupling with
uridine-5’-monophosphate
(UMP).
With our approach, the ac-
ceptor trisaccharide unit and
GlcNAc unit are constructed
separately, and coupled togeth-
er by chemoselective ligation
(Scheme 1). The acceptor tri-
saccharide unit was synthesized
using a soluble polymer sup-
port.[13] Preliminary results on
the synthesis of 1a and evalua-
tion of its inhibitory activity
against GnT-V and -IX were re-
ported previously.[14] We now
wish to fully disclose the further
systematicpreparation and in-
hibitory activities of a series of
derivatives (1a–e) toward GnT-
V and -IX.
Co-crystallization and multi-
dimensional NMR spectroscopy
are direct and powerful meth-
ods of elucidating the substrate-
binding structure of enzymes.[15]
By contrast, the results ob-
tained with tunable synthetic
probes in combination with a
traditional inhibition assay can
Figure 1. a) Substrate specificities of GnT-V and GnT-IX; b) bisubstrate-type inhibitors introduced various
lengths of linkers, consisting of acceptor trisaccharide and UDP-GlcNAc moieties.
trast to the restricted distribution of GnT-IX in brain, sug-
gesting that GnT-IX plays an important role in neural devel-
opment and functions.[8]
not be directly visualized. However, they should provide re-
liable information on active site structures.
Several GnT-V inhibitors have been reported, the design
of which has relied upon the modification of the acceptor
substrate oligosaccharide.[9] Because the incorporation of a
donor component into an acceptor mimetic is expected to
augment the binding and result in a potent and specific in-
hibitor of glycosyltransferases, we turned our attention to bi-
substrate-type derivatives, 1a–e (Figure 1b).[10] these com-
pounds were designed to include both donor (UDP-
GlcNAc) and acceptor units, taking into consideration the
proposed mechanism of inverting GnTs.[11] This design ena-
bled us to alter the lengths of linkers, in order to define the
optimal distance between these components. These com-
pounds are expected to help provide an insight into the mo-
lecular basis of the glycosyl transfer mechanism and specific-
ity of GnT-V and -IX.
Results and Discussion
The acceptor trisaccharide was synthesized based on a solu-
tion-phase polymer-support strategy, which was combined
with resin capture–release purification, as depicted in
Scheme 2.[14] As described previously, monomethyl polyethy-
leneglycol (MPEG: average MW ꢀ750 Da) was used as a
support, which functioned as a polar tag; the product could
be readily isolated from the reaction mixture by passage
through a short silica gel column.[13,16] Namely, non MPEG-
containing fractions (e.g. excess donor) were first eluted
with ethyl acetate (hexane/EtOAc). With subsequent elution
with a more polar solvent such as EtOAc/MeOH, MPEG-
bound materials can be retrieved.
As an acceptor component, GlcNAcb1,2-Mana1,6-Manb
was chosen a priori, because this trisaccharide was reported
to be an efficient acceptor substrate of GnT-V.[12] In order to
achieve conjugation with a donor (UDP-GlcNAc) compo-
To start with, the 6-O-trityl (Tr)-protected monosacchar-
ide 2a[13d] was synthesized using Kovµcꢁs anomer locked b-
mannosylation strategy.[17] After the removal of Tr with tri-
fluoroacetic acid (TFA) and Et3SiH, the liberated alcohol
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Y. Ito et al.
Scheme 1. Route for the synthesis of bisubstrate-type derivatives 1a–e.
Scheme 2. Polymer-resin hybrid-based synthesis of trisaccharide 8.
2b was glycosylated with phenylthio mannoside 3 by the
action of N-iodosuccinimide (NIS) and trifluoromethanesul-
fonic acid (TfOH, 0.7 equiv) to afford disaccharide 4.[18,19]
The crude mixture was capped with Ac2O/pyridine so that
any unreacted 2b would not be carried over to the next gly-
cosylation. Cleavage of the chloroacetyl group[20] and further
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Glucosyltransferase Inhibitors
FULL PAPER
glycosylation with 5 provided trisaccharide 6.[21] At this
stage, the product was subjected to capture–release purifica-
tion. Namely, 6 was captured with cysteine-conjugated Wang
resin 7 through a selective reaction between chloroacetyl
and thiol groups. Liberation of an amino group by Fmocde-
protection with 4-aminomethylpiperidine[22] initiated cycliza-
tion and trisaccharide 8 was obtained in 49% overall yield
from 2.
Further deprotection and introduction of a thiol group
were conducted as depicted in Scheme 3. Acidic cleavage of
the benzylidene acetal, dephthaloylation with ethylenedi-
amine, and acetylation afforded 9. Subsequent deacetylation
and esterification gave 10 in 85% yield. Tosylation of the
primary hydroxy group was performed using 4-dimethylami-
nopyridine (DMAP) as a base to afford 11; removal of the
benzyl groups gave 12. The tosyl group was substituted with
thioacetate by using AcSK in DMF at 708C.[23,24] Deacetyla-
tion under mildly basicocnditions using Et 3N in MeOH/
H2O caused a spontaneous oxidation to provide disulfide
13a.
Glucosamine-1-phosphate derivatives 19, 22, 23, 28, and
29 carrying various linkers were prepared as depicted in
Scheme 4. To begin with, the common starting material 14
was transformed into 15 in a standard manner. Namely, the
anomeric position was selectively deacetylated and repro-
tected with a tert-butyldimethylsilyl (TBS) group to provide
15. After hydrogenolyticremoval of the benzyloxycarbonyl
(Cbz) group, a reactive bromoacetoamide group was intro-
duced to furnish 16. For the synthesis of the glucosamine
phosphate unit 19, 16 was desilylated and reacted with the
amidite 17[25] to introduce a phosphite group, which was oxi-
dized to the phosphate 18.[26] Deprotection of the allyl ester
using [PdCAHTRE(UGN PPh3)4] furnished 19 in 85% yield.
Scheme 3. Synthesis of disulfide 13a. a) 60% AcOH/H2O, 60 8C, 77%; b) i) 1m KOH, EtOH/THF, reflux, ii) ethylenediamine, 1-BuOH, 1008C, iii) Ac2O,
pyridine, 70%; c) i) 0.05m NaOMe/MeOH, ii) TMSCHN2, PhH, MeOH, 85%; d) p-TsCl, DMAP, CH2Cl2, 60%; e) H2, 20% Pd(OH)2/C, AcOH, MeOH,
88%; f) i) AcSK, DMF 708C, ii) Et3N, MeOH, H2O, 75%.
Scheme 4. Synthesis of GlcN phosphates. a) H2NNH2·AcOH, THF, 94%; b) TBSCl, imidazole, DMF, 95%; c) i) H2, 10% Pd/C, EtOAc, ii) (BrAc)2O,
pyridine, CH2Cl2, 98%; d) AcSH, iPr2NEt, CH3CN, 48C, 93%; e) i) HOCAHTRE(UGN CH2)nSH, iPr2NEt, CH3CN, ii) NBS, Ph3P, CH2Cl2, 24: 96%, 25: 95%; f) 47%
HFaq, CH3CN, from 16: 93%; from 24: 70%; from 25: 90%; g) HF-pyridine, THF, from 20: 78%; h) i) 17, 1H-tetrazole, CH2Cl2, ꢁ108C, ii) TBHP,
Me2S, ꢁ40 ! 08C, 18: 83%; 21: 77%; 26: 68%; 27: 78%; i) [Pd
ACHTRE(UNG PPh3)4], Et3SiH, AcOH, toluene, 19: 85%; 22: 51%; 28: 86%, 29: 56%; j) Et3N,
MeOH, H2O.
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Y. Ito et al.
In order to convert bromoacetamide 16 to the mercapto-
acetamide 23, compound 16 was treated with thioacetic acid
and Hünigꢁs base to give 20. It was then desilylated, phos-
phitylated, and oxidized to afford 21. Deprotection of the
allyl ester gave 22, which was deacetylated to afford 23. On
the other hand, for the preparation of compounds 28 and
29, 2-mercaptoethanol and 3-mercaptopropanol were intro-
duced into 16. Subsequent treatment with N-bromosuccin-
imide (NBS) and Ph3P gave 24 and 25. These compounds
were transformed to 28 and 29 in moderate overall yield, via
26 and 27, respectively.
Three components, namely acceptor trisaccharide (13a),
linker-conjugated GlcNAc-phosphate (19, 28, 29) and UMP
were sequentially coupled as shown in Scheme 5. For in-
stance, disulfide 13a was first reduced with triscarboxyethyl-
phosphine (TCEP) in MeOH/H2O to liberate thiol, which
was subjected to chemoselective ligation with 19 having a
bromoacetamide linker. It proceeded smoothly to give the
coupled product that was deacetylated to afford 30a in 58%
yield. Finally, the UMP unit was introduced into 30a by
using UMP–morpholidate[27] in pyridine to afford 1a in 78%
yield.
al of excess phosphine and phosphine oxide by ether extrac-
tion, ligation between thiol and bromide 28 or 29 was con-
ducted in the presence of Cs2CO3 in dry DMF.[28] This was
followed by the removal of acetyl groups with Et3N to
afford 30d and 30e in 37 and 63% yield, respectively. Cou-
pling with the UMP unit readily produced 1d and 1e in
moderate yield.
Synthesis of the disulfide-linked congener 1b was ach-
ieved as shown in Scheme 6. The thiol derived from 13a
(nBu3P) was converted to pyridyl disulfide under acidic con-
ditions[29] to afford 31. A reaction with mercaptoacetamide
23 in MeOH/1m NH4OAc[30] smoothly produced the mixed
disulfide 30b in 47% yield from 13, which was coupled with
UMP to give 1b.
The synthesis of 1c called for the introduction of a C1
linker, which was less straightforward, because of the insta-
bility of the halomethylthio moiety. This task was achieved
by the route depicted in Scheme 7. Namely, compound 20
was selectively deacetylated to give 32 and chloromethylat-
ed by using a large excess of bromochloromethane[31] in the
presence of iPr2NEt to give 33.
Coupling between the thiol derived from 13a and
GlcNAc derivative 33 was conducted in the presence of a
stoichiometric amount of Cs2CO3. The coupled product was
acetylated to give 34, which was
For the synthesis of 30d and 30e, nBu3P was used for re-
ducing the disulfide bond instead of TCEP. After the remov-
desilylated by using a HF/pyri-
dine complex to afford the hem-
iacetal 35 in 31% yield from
the disulfide 13a. A phosphite
group was introduced into 35 at
higher temperature (458C), and
subsequent oxidation afforded
the phosphate 36. Then, the
allyl ester was cleaved using
[PdACHTRE(UNG PPh3)4]. Although NaOMe-
mediated deacetylation was ac-
companied by a partial loss of
the anomericphosphate, the de-
sired compound 30c could be
isolated in 45% yield from 36.
Subsequent coupling with UMP
gave 1c in 63% yield.
Scheme 5. Synthesis of 1a, 1d, and 1e. a) i) TCEP-HCl, MeOH/H2O, ii) 19, iPr2NEt, iii) Et3N, 30a; 58%; b) i)
nBu3P, THF, H2O, ii) 28 or 29, Cs2CO3, DMF, iii) Et3N, MeOH/H2O, 30d; 37%, 30e; 63%; c) UMP-morpholi-
date, 1H-tetrazole, pyridine, 1a; 78%, 1d; 58%, 1e; 61%.
Scheme 6. Synthesis of 1b. a) nBu3P, THF, H2O, b) pyridyl disulfide, 0.5m HCl, MeOH/H2O, c) 23, MeOH/1m NH4OAc, 47%, d) UMP-morpholidate,
1H-tetrazole, pyridine, 56%.
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Glucosyltransferase Inhibitors
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Table 1. Inhibitory activity for GnT-V and IX.
Inhibitor
GnT-V
GnT-IX
Ki [mm]
1a
1b
1c
1d
1e
71.9
119.3
47.1
26.9
18.3
10.1
4.7
17.6
21.5
15.1
Conclusion
Among various types of glycoconjugates, complex-type gly-
coproteins are particularly important. GnTs are key en-
zymes to produce highly branched N-glycans having diverse
structures. Therefore, potent inhibitors of GnTs are expect-
ed to be valuable tools in glycobiology. Because the three-
dimensional structures of GnTs are yet to be explored, bi-
substrate-type inhibitor would help clarifying the active site
structure and mechanism of action of this class of enzyme.
Here we designed and synthesized bisubstrate-type inhibi-
tors for GnTs having both an acceptor trisaccaride and a
donor UDP–GlcNAc tethered by a linker of various lengths.
For the construction of the trisaccharide component, we ap-
plied the oligosaccharide synthesis strategy using MPEG as
a support. Coupling reactions with various GlcNAc phos-
phates were conducted under chemoselective ligation condi-
tions. Finally, incorporation of the UDP component provid-
ed designed bisubstrate-type derivatives. It was clearly ob-
served that their potencies toward GnT-V and -IX were sen-
sitive to the linker length. Interestingly, the modes of corre-
lation between linker length and activities were markedly
different between these enzymes. These results suggest that,
although they are homologous to each other, the distances
of donor- and acceptor-binding sites are quite different.
Scheme 7. Synthesis of 1c. a) H2NNH2·AcOH, THF; b) bromochlorome-
thane, iPr2NEt, CH3CN; c) nBu3P, THF/H2O; d) 33, Cs2CO3, DMF; e)
Ac2O, pyridine; f) HF/pyridine, DMF, 31% form 13a; g) i) 17, 1H-tetra-
zole, ClACHTREUNG(CH2)2Cl, 458C, ii) TBHP, Me2S, 85%; h) [PdCAHTRE(UGN PPh3)4], Et3SiH,
AcOH, toluene, 408C, i) 0.05m NaOMe, MeOH, 45%; j) UMP-morpholi-
date, 1H-tetrazole, pyridine, 63%.
Analysis of enzyme inhibition: The inhibitory activities of
compounds 1a–e against GnT-V and IX are summarized in
Table 1. Enzyme assays were conducted using pyridylami-
nated substrates (GnGn-bi-PA and GnMSer-PAES) as de-
scribed previously.[32,33] All had significant inhibitory effects
on the two GnT-V and -IX.[34] In both cases, the extent of in-
hibition was clearly dependent upon the length of the linker.
For GnT-V, compounds having a longer linker showed stron-
ger activity, with a single exception (1a vs 1b), 1e being the
most active (Ki =18.3 mm). Since the Km value of GnT-V
toward acceptor and donor substrates was reported to be
0.150 mm and 4–6 mm, respectively, an enhancement in bind-
ing was evident, except for 1b. On the other hand, GnT-IX
was uniformly more sensitive to inhibition by 1a–e. The
strongest activity (Ki =4.7 mm) against GnT-IX was exhibited
by 1b, which was least active toward GnT-V. Compounds
1c–e having longer linkers all had similar levels of activity.
From the results, it is suggested that these enzymes differ
significantly in the relative orientations of donor- and ac-
ceptor-binding sites, in spite of their high homology.
Experimental Section
General procedure: 1H and 13C NMR spectra were measured by JEOL
EX-400 or ECP-500 spectrometer. 1H and 13C chemical shifts in CDCl3
were revealed in ppm relative to the CHCl3 or TMS signal adjusted to
7.24 or 0 ppm, and the CDCl3 signal adjusted to 77.0 ppm, respectively.
Chemical shifts in CD3OD were revealed relative to solvent peaks adjust-
ed to 3.30 (1H) and 49.5 ppm (13C), respectively. Chemical shifts in D2O
1
are relative to DOH (4.65 ppm, H) and CH3OH (49.0 ppm, 13C), respec-
tively. Optical rotations were measured by JASCO DIP-301. MALDI-
TOF MS spectra were measured by Shimadzu AXIMA-CFR using
DHBA and CHCA as matrix. ESI-TOF MS spectra were measured by
JEOL JMS-T100 LC spectrometer. Molecular Sieves 4 Š were purchased
from Nakalai Tesque Inc(Kyoto) and dried at 170 8C under vacuum im-
mediately prior to use.
PEG-supported products were obtained in a following manner: The mix-
ture was loaded on a pad of silica gel and was first washed with hexanes/
AcOEt 1:4 to remove non-PEG supported materials. Subsequently, PEG-
supported fractions were eluted by EtOAc/MeOH 1:3. The fractions
were evaporated and thoroughly dried under high vacuum before used
for the next reaction.
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Y. Ito et al.
Synthetic procedures
washed with brine (2”), dried over Na2SO4, filtered, and concentrated.
Purification by short silica gel chromatography (hexane/EtOAc 1:4, then
EtOAc/MeOH 3:1) yielded 6 (281.0 mg, 0.125 mmol, 93%). 1H NMR
(400 MHz, CDCl3): d=7.88–6.96 (m, 34H), 5.42 (s, 1H), 5.28–5.23 (2H,
8-(MPEG)oxycarbonyloctyl
2,3,4-tri-O-benzyl-b-d-mannopyranoside
(2b): Et3SiH (3.5 mL, 22 mmol) and TFA (3.5 mL, 45 mmol) were added
dropwise at 48C to a solution of trityl ether 2a (1.65 g, 1.10 mmol) in
CH2Cl2 (50 mL). After stirring for 30 min at 48C under Ar atmosphere,
the mixture was neutralized with sat. NaHCO3 solution. The aqueous
phase was extracted with CHCl3 (2”), and the combined organic layers
were washed with brine (2”), dried over Na2SO4, filtered, and concen-
trated. Purification by short silica gel chromatography (hexanes/EtOAc
1:4, then EtOAc/MeOH 3:1) afforded 6-O-unprotected mannoside deriv-
H-1GN, H-4GN), 5.02 (d, 1H, 2J
2J(H,H)=12.9 Hz; PhCH), 4.78 (d, 1H, 2J
(d, 1H, 2J(H,H)=13.0 Hz; PhCH), 4.67 (d, 1H, 2J
PhCH), 4.60–4.54 (m, 3H, H-1aMan, PhCH), 4.46–4.33 (m, 4H, PhCH),
4.32 (s, 1H, H-1bMan), 4.29 (d, 1H, 2J
(H,H)=11.0 Hz; PhCH), 4.08 (m,
1H, CHalkyl), 3.92–3.34 (overlapped with PEG-CH2O signal), 3.19 (m,
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
2
1
1H, H-5bMan), 3.02 (t, 1H, J
2H, JACHTREUNG
A
ACHTRE(UNG H,H)=10.2 Hz; H-6baMan), 2.30 (t,
ative 2b (1.38 g, 1.03 mmol, 94%). H NMR (400 MHz, CDCl3): d=7.46–
3
7.26 (m, 15H), 4.96 (d, 1H, 2J
ACHTREUNG
2J
A
ACHTREUNG
8-(MPEG)oxycarbonyloctyl 3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-a-
d-mannopyranosyl-(1!6)-2,3,4-tri-O-benzyl-b-d-mannopyranoside (8)
E
ACHTREUNG
ACHTREUNG
Capture: Fmoc-Cys on Wang resin 7 (0.58 mmolgꢁ1, 710 mg, 0.412 mmol)
and iPr2NEt (150 mL, 2.87 mmol) were added to a solution of trisacchar-
ide 6 (281 mg, 0.125 mmol) in CH3CN/CH2Cl2 (1:1, 4 mL). The mixture
was shaken for 24 h at room temperature, and resin was rinsed with
CHCl3/MeOH, and dried under reduced pressure.
ACHTREUNG
8-(MPEG)oxycarbonyloctyl 3-O-benzyl-4,6-O-benzylidene-2-O-chloro-
acetyl-a-d-mannopyranosyl-(1!6)-2,3,4-tri-O-benzyl-b-d-mannopyrano-
side (4): MS 4 Š (3.7 g) was added to
a solution of 2b (1.226 g,
0.916 mmol) and donor 3 (1.294 g, 2.194 mmol) in CH2Cl2 (40 mL), and
the reaction mixture was stirred at room temperature for 10 min under
Ar atmosphere. Then, the mixture was cooled to ꢁ208C, to which were
added NIS (612 mg, 2.72 mmol) and TfOH (180 mL, 2.03 mmol). After
stirring for 10 h at ꢁ208C, the mixture was filtered through Celite, and
the filtrate was washed with 5% Na2S2O3 solution. The aqueous phase
was extracted with CHCl3 (2”), and the combined organic layers were
washed with brine (2”), dried over Na2SO4, filtered, and concentrated.
Purification by short silica gel chromatography (hexanes/EtOAc 1:4, then
Release: The trisaccharide-loaded resin was swelled in THF (5 mL), and
4-aminomethylpiperidine (727 mg, 6.37 mmol) was added. The mixture
was shaken for 24 h, and resin was rinsed with CHCl3/MeOH. The organ-
icphase was washed with 10% ictricaicd solution, and the aqueous
phase was extracted with CHCl3 (2”). The combined organic layers were
washed with brine (2”), dried over Na2SO4, filtered, and concentrated.
Purification by short silica gel chromatography (hexane/EtOAc 1:4, then
EtOAc/MeOH 3:1) yielded
1H NMR (400 MHz, CDCl3): d=7.88–6.95 (m, 34H), 5.40 (s, 1H), 5.23
(d, 1H, 3J(H,H)=8.1 Hz; H-1GN), 5.02 (d, 1H, 2J
(H,H)=12.9 Hz;
PhCH), 4.90 (d, 1H, 2J
(H,H)=12.7 Hz; PhCH), 4.81–4.75 (2H, PhCH),
4.72 (d, 1H, 2J(H,H)=12.9 Hz; PhCH), 4.64 (d, 1H, 2J
(H,H)=12.9 Hz;
(H,H)=
(H,H)=11.7 Hz; PhCH), 4.48 (s, 1H,
8 (171.5 mg, 0.080 mmol, 49% from 2).
EtOAc/MeOH 3:1) yielded
(400 MHz, CDCl3): d=7.47–7.20 (m, 25H), 5.59 (s, 1H), 5.52 (m, 1H;
H-2aMan), 4.99 (d, 1H, 2J(H,H)=12.7 Hz; PhCH), 4.94 (d, 1H, 2J
(H,H)=
11.2 Hz; PhCH), 4.87 (s, 1H, H-1aMan), 4.85 (d, 1H, 2J
(H,H)=12.4 Hz;
PhCH), 4.65 (d, 1H, 2J(H,H)=12.2 Hz; PhCH), 4.59 (d, 1H, 2J
(H,H)=
12.2 Hz; PhCH), 4.35 (s, 1H, H-1bMan), 4.23 (dd, 1H, 2J
(H,H)=10.0,
3J
(H,H)=4.4 Hz; H-6aaMan), 4.15 (s, 2H, ClCH2COO), 4.00–3.40 (over-
4
(1.592 g, 0.91 mmol, 98%). 1H NMR
A
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
A
ACHTREUNG
PhCH), 4.58 (d, 1H, 2J(H,H)=12.4 Hz; PhCH), 4.54 (d, 1H, 2J
ACTHERUGN ACHTREUNG
AHCTREUNG
12.4 Hz; PhCH), 4.50 (d, 1H, 2J
H-1aMan), 4.44 (d, 1H, J
A
A
ACHTREUNG
2
(H,H)=11.7 Hz; PhCH), 4.37–4.28 (5H, H-1bMan
,
AHCTREUNG
H-2GN, H-3GN, PhCH), 4.09 (m, 1H, CHalkyl), 3.92–3.34 (overlapped with
PEG-CH2O signal), 3.18 (m, 1H, H-5bMan), 3.00 (t, 1H, 2J
(H,H)=
3J(H,H)=10.2 Hz; H-6baMan), 2.29 (t, 2H, 3J
(H,H)=7.3 Hz), 1.62 (br,
4H), 1.30 (br, 8H).
ACHTREUNG
ACHTREUNG
lapped with PEG-CH2O signal), 3.35 (2H, H-5bMan, OCH2-alkyl).
A
ACHTREUNG
8-(MPEG)oxycarbonyloctyl 3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-2-
phthalimido-b-d-glucopyranosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-2-
O-chloroacetyl-a-d-mannopyranosyl-(1!6)-2,3,4-tri-O-benzyl-b-d-man-
nopyranoside (6): Freshly prepared hydrazinedithiocarbonate (HDTC)[20]
(0.42m, 6.5 mL) was added to a solution of 4 (1.592 g, 0.907 mmol) in
CH3CN (50 mL), and stirred for 1 h at room temperature. The mixture
was concentrated and dissolved in CHCl3. The organicphase was washed
with 10% citric acid solution, and the aqueous phase was extracted with
CHCl3 (2”), and the combined organic layers were washed with brine
(2”), dried over Na2SO4, filtered, and concentrated. Purification by short
silica gel chromatography (hexane/EtOAc 1:4, then EtOAc/MeOH 3:1)
yielded 2-O-unprotected disaccharide (1.474 g, 0.878 mmol, 97%).
1H NMR (400 MHz, CDCl3): d=7.47–7.22 (m, 25H), 5.59 (s, 1H), 5.00
8-Carboxyoctyl 2-acetamido-3,6-di-O-benzyl-4-O-chloroacetyl-2-deoxy-b-
d-glucopyranosyl-(1!2)-4,6-di-O-acetyl-3-O-benzyl-a-d-mannopyrano-
syl-(1!6)-2,3,4-tri-O-benzyl-b-d-mannopyranoside (9):
A solution of
MPEG-ester 8 (47.9 mg, 0.0221 mmol) in 80% aqueous AcOH (5 mL)
was stirred for 2 h at 608C. After cooling to room temperature, the mix-
ture was concentrated and purified by silica gel chromatography (EtOAc/
MeOH 100:0 ! 3:1) to afford triol (43.7 mg, 0.0215 mmol, 97%).
A solution of crude triol (139.4 mg, 0.0671 mmol) in THF/EtOH (1:1,
10 mL) was added 1n KOH (1 mL). The mixture was stirred for 6 h at
808C, and cooled to room temperature. After neutralized with Amberlyst
15E, the resin was removed by filtration, and the filtrate was concentrat-
ed, and dried under reduced pressure.
(d, 1H, 3J
PhCH), 4.92 (d, 1H, J
PhCH), 4.73 (d, 1H, J=12.0 Hz PhCH), 4.62 (d, 1H, 2J
PhCH), 4.56 (d, 1H, 2J(H,H)=12.0 Hz; PhCH), 4.52 (d, 1H, 2J
11.0 Hz; PhCH), 4.47 (d, 1H, 2J
(H,H)=11.7 Hz; PhCH), 4.35 (s, 1H,
H-1bMan), 4.23 (dd, 1H, 2J(H,H)=9.3, 3J
(H,H)=3.9 Hz; H-6aaMan), 4.13
(dd, 1H, 3J(H,H)=1.2, 3.2 Hz; H-2aMan), 4.08 (t, 1H, 3J
(H,H)=9.3 Hz;
ACHTREUNG ACHTRENUG
(H,H)=1.5 Hz; H-1aMan), 4.97 (d, 1H, 2J
2
ACHTREUNG
The residue was dissolved in nBuOH (6 mL), to which was added ethyle-
nediamine (1.5 mL). The mixture was stirred for 10 h at 1008C, cooled to
room temperature, and evaporated in vacuo. The residue was suspended
in pyridine (6 mL) and Ac2O (3 mL) was added under ice-water cooling.
After stirring for 20 h at room temperature, the mixture was concentrat-
ed, co-evaporated with toluene, and purified by preparative TLC (tol-
uene/EtOAc/AcOH 5:5:0.1) to give 9 (64.5 mg, 0.0466 mmol, 70%).
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
A
ACHTREUNG
H-4aMan), 3.92–3.50 (overlapped with PEG-CH2O signal), 3.37 (m, 1H,
OCH2-b), 3.35 (m, 1H, H-5bMan), 2.28 (t, 2H, J=7.3 Hz), 1.58 (br, 4H),
1.28 (br, 8H).
8-Methoxycarbonyloctyl 2-acetamido-3,6-di-O-benzyl-4-O-chloroacetyl-2-
deoxy-b-d-glucopyranosyl-(1!2)-3-O-benzyl-a-d-mannopyranosyl-(1!
6)-2,3,4-tri-O-benzyl-b-d-mannopyranoside (10): A solution of carboxylic
acid 9 (64.5 mg, 0.0466 mmol) in 0.05m NaOMe in MeOH (5.0 mL) was
stirred at room temperature under Ar atmosphere for 20 h. The mixture
was neutralized with Amberlyst 15E, filtered, and the filtrate was concen-
trated, and dried under reduced pressure. The residue was dissolved in
MeOH/benzene (1:1, 4 mL) and a hexane solution of TMSCHN2 (2.0m)
was added dropwise until the yellow color persisted for more than 5 min.
After quenching with AcOH, the solution was concentrated and purified
4 Š MS (500 mg) was added to a solution of the disaccharide (226.3 mg,
0.135 mmol) and donor 5 (162.5 mg, 0.273 mmol) in CH2Cl2 (6 mL), and
the reaction mixture was stirred at room temperature for 10 min under
Ar atmosphere. Then, the mixture was cooled to ꢁ208C, to which were
added NIS (73.9 mg, 0.329 mmol) and TfOH (5 mL, 0.06 mmol). After
stirring for 19 h at ꢁ108C, the mixture was filtered through Celite, and
the filtrate was washed with 5% Na2S2O3 solution. The aqueous phase
was extracted with CHCl3 (2”), and the combined organic layers were
3456
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3449 – 3462

Glucosyltransferase Inhibitors
FULL PAPER
with preparative TLC (CHCl3/MeOH 25:1) to afford 10 (50.2 mg,
3.2 Hz), 4.00–3.30 (m, 44H), 2.83 (dd, 2H, 3J
(H,H)=9.3, 2J
ACHRTUNEG ACHTREUNG(H,H)=
0.03945 mmol, 85%). [a]D23
=
ꢁ40 (c=1.13 in chloroform); 1H NMR
13.9 Hz), 2.37 (t, 4H, 3J
ACHTREUNG
(400 MHz, CDCl3): d=7.45 (d, 2H, 3J
A
1.31 (m, 16H); 13C NMR (100 MHz, D2O, 40 8C): d=174.3, 171.7, 97.5,
96.9, 94.4, 73.8, 73.3, 72.3, 70.9, 70.7, 68.3, 68.1, 67.4, 67.34, 67.27, 67.1,
64.6, 64.1, 58.2, 52.8, 46.4, 38.9, 31.3, 26.3, 26.0–25.7, 22.7, 21.9, 20.1; HR
ESI-MS: m/z: calcd for C60H104N2O34S2Na: 1483.5810, found 1483.5834
[M+Na]+.
28H), 5.85 (br, 1H), 4.99 (d, 1H, 2J
(m, 3H), 4.83 (d, 1H, 3J(H,H)=8.3 Hz), 4.73 (d, 2H, 2J
PhCH), 4.66 (d, 1H, 2J
(H,H)=11.7 Hz; PhCH), 4.52–4.39 (m, 6H), 4.34
(s, 1H), 4.22 (m, 1H), 4.01 (m, 1H), 3.96–3.85 (4H), 3.71–3.56 (11H),
3.54–3.44 (m, 4H), 3.37 (m, 1H), 3.31 (m, 1H), 2.29 (t, 2H, 3J
(H,H)=
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
AHCTREUNG
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-(benzyloxycarbonyl)amino-2-
deoxy-b-d-glucopyranoside (15): Hydrazine acetate (916 mg, 9.95 mmol)
was added at 48C to a solution of 14 (4.53 g, 9.41 mmol) in THF
(120 mL). After stirring for 24 h at room temperature, ice-water was
added and the mixture was extracted with CHCl3 (3”). The combined or-
ganiclayers were washed with brine (2”), dried over Na 2SO4, filtered,
and concentrated. Separation by silica gel chromatography (toluene/
EtOAc5:1 ! 1:1) gave hemiacetal (3.86 g, 8.79 mmol, 93%).
7.6 Hz), 1.86 (s, 3H), 1.61 (m, 4H), 1.31 (m, 8H); 13C NMR (100 MHz,
CDCl3): d=174.0, 171.1, 138.2–137.4, 128.3–127.3, 101.8, 99.4, 98.3, 82.5,
80.0, 76.1, 75.1, 74.9, 74.9, 74.2, 73.7, 73.6, 73.43, 73.38, 73.0, 72.9, 72.5,
71.4, 71.2, 70.3, 69.8, 66.5, 65.5, 62.0, 56.2, 51.5, 34.1, 29.7, 29.34, 29.32,
29.2, 26.2, 25.0, 23.6; HR ESI-MS: m/z: calcd for C72H89NO18Na:
1278.5978; found 1278.5973 [M+Na]+.
8-Methoxycarbonyloctyl 2-acetamido-3,6-di-O-benzyl-4-O-chloroacetyl-2-
deoxy-b-d-glucopyranosyl-(1!2)-3-O-benzyl-6-O-tosyl-a-d-mannopyra-
A solution of the hemiacetal (3.85 g, 8.76 mmol) in DMF were added
TBSCl (2.00 g, 13.3 mmol) and imidazole (1.79 g, 26.3 mmol) at 48C.
After stirring for 11 h at room temperature, the mixture was quenched
with 10% aqueous citric acid. Aqueous phase was extracted with CHCl3
(3”). The combined organic layers were washed with brine (2”), dried
over Na2SO4, filtered, and concentrated. Purification by silica gel chro-
matography (toluene/EtOAc15:1 ! 4:1) gave 15 (4.61 g, 8.33 mmol,
95%). [a]2D2 = +9.9 (c=1.15 in chloroform); 1H NMR (400 MHz, C6D6,
nosyl-(1!6)-2,3,4-tri-O-benzyl-b-d-mannopyranoside
(11):
DMAP
(3.5 mg, 0.029 mmol) and TsCl (4.2 mg, 0.022 mmol) were added at 48C
to a solution of triol 10 (26.0 mg, 0.021 mmol) in CH2Cl2. After stirring
for 10 h at room temperature under Ar atmosphere, the second portions
of DMAP (3.0 mg, 0.025 mmol) and TsCl (3.2 mg, 0.017 mmol) were
added. After the additional stirring for 13 h, the mixture was diluted with
CHCl3 and washed with 0.5m HCl. The aqueous phase was extracted
with CHCl3 (2”), and the combined organic layers were washed with
brine (2”), dried over Na2SO4, filtered, and concentrated. Purification
with preparative TLC (CHCl3/MeOH 20:1) produced 11 (17.6 mg,
708C): d=7.25–7.00 (m, 5H), 5.28 (t, 1H, 3J
(t, 1H, 3J(H,H)=9.6 Hz; H-4), 5.04 (s, 2H), 4.57 (d, 1H, 3J
7.6 Hz; NH), 4.33 (br, 1H, H-1), 4.17 (dd, 1H, 3J(H,H)=5.6, 2J
12.0 Hz; H-6a), 4.11 (dd, 1H, 3J(H,H)=2.8, 2J
(H,H)=12.0 Hz; H-6b),
ACHTREUNG
R
ACHTREUNG
A
ACHTREUNG
0.0125 mmol, 60%). [a]D23
=
ꢁ41 (c=0.69 in chloroform); 1H NMR
A
ACHTREUNG
(400 MHz, CDCl3): d=7.71 (d, 2H, 3J
(H,H)=8.5 Hz), 7.45–7.14 (m,
3.58 (m, 1H, H-2), 3.40 (m, 1H, H-5), 1.74–1.68 (9H), 0.96 (s, 9H), 0.16
(s, 3H), 0.11 (s, 3H); 13C NMR (100 MHz, C6D6, 608C): d=170.1, 169.7,
169.0, 155.8, 137.3, 128.6–127.8, 96.7, 72.8, 72.4, 69.9, 67.0, 62.6, 58.9, 26.0,
20.31, 20.26, 18.3, ꢁ3.9, ꢁ4.8; elemental analysis calcd (%) for
C26H39NO10Si: C 56.40, H 7.10, N 2.53; found: C 56.39, H 7.11, N 2.46.
3
3
32H), 6.08 (d, 1H, J
A
A
(d, 1H, 2J
ACHTREUNG ACHTRENUG
(H,H)=12.7 Hz; PhCH), 4.89 (d, 1H, 2J
PhCH), 4.87 (d, 1H, 2J
T
ACHTREUNG
AHCTREUNG
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-bromoacetamido-2-deoxy-b-d-
glucopyranoside (16): 10% Pd/C (96 mg) was added to a solution of 15
(536.0 mg, 0.968 mmol) in EtOAc, and the mixture was stirred under H2
atmosphere for 2.5 h. After additional stirring for 15 min under N2 atmos-
phere, the catalyst was filtered off through Celite, and the filtrate was
concentrated, and exposed to high-vacuum for 2 h. The resulting solid
was dissolved in CH2Cl2 (10 mL), and treated with bromoacetic anhy-
dride (336 mg, 1.29 mmol) and pyridine (100 mL, 1.24 mmol) at 48C.
After stirring for 30 min at 48C, the mixture was neutralized with 10%
aqueous citric acid. The aqueous phase was extracted with CH2Cl2 (2”).
The combined organic layers were washed with brine (2”), dried over
Na2SO4, filtered, and concentrated. Purification by silica gel chromatog-
ACHTREUNG
ACHTREUNG
13C NMR (100 MHz, CDCl3): d=174.0, 171.9, 144.5, 138.6–137.4, 133.1,
129.7–127.3, 102.0, 98.4, 97.4, 82.5, 79.2, 77.2, 75.7, 74.8, 74.5, 74.2, 73.7,
73.6, 73.5, 73.2, 71.9, 71.3, 71.2, 70.3, 70.1, 69.7, 68.8, 67.2, 65.0, 58.6, 51.5,
34.2, 29.8, 29.39, 29.36, 29.2, 26.2, 25.1, 23.8, 21.7; HR ESI-MS: m/z:
calcd for C79H95NO20SNa: 1432.6066, found 1432.6059 [M+Na]+.
8-Methoxycarbonyloctyl 2-acetamido-2-deoxy-b-d-glucopyranosyl-(1!2)-
6-O-tosyl-a-d-mannopyranosyl-(1!6)-b-d-mannopyranoside (12): A sol-
ution of tosylate 11 (286.7 mg, 0.191 mmol) in MeOH (20 mL) containing
AcOH (0.5 mL) was hydrogenated over Pd(OH)2/C (20%, 253 mg)
under vigorous stirring for 12 h. After additional stirring under an N2 at-
mosphere for 30 min, the catalyst was filtered off through Celite, and the
filtrate was concentrated. Purification by silica gel chromatography
(CHCl3/MeOH 10:1 to CHCl3/MeOH/H2O 6:2:0.2) gave 12 (147.0 mg,
raphy (toluene/EtOAc15:1
98%). [a]2D4 +2.5 (c=0.97 in chloroform); 1H NMR (400 MHz,
CDCl3): d=6.33 (d, 1H, 3J(H,H)=8.8 Hz; NH), 5.19 (dd, 1H, 3J
(H,H)=
9.6, 10.8 Hz; H-3), 4.92 (t, 1H, 3J
(H,H)=9.6 Hz; H-4), 4.79 (d, 1H,
3J(H,H)=8.0 Hz; H-1), 4.08 (dd, 1H, 3J(H,H)=6.0, 2J
(H,H)=12.4 Hz;
H-6a), 4.01 (dd, 1H, 3J(H,H)=2.8, 2J
(H,H)=12.4 Hz; H-6b), 3.73 (m,
!
4:1) gave 16 (513.2 mg, 0.950 mmol,
=
A
ACHTREUNG
AHCTREUNG
0.169 mmol, 88%). [a]D22
=
ꢁ12 (c=1.12 in methanol); 1H NMR
A
N
ACHTREUNG
(400 MHz, CD3OD): d=7.79 (d, 2H, 3J
ACHTRE(UNG H,H)=8.3 Hz), 7.43 (d, 2H,
A
ACHTREUNG
3J
H-1GN), 4.46 (s, 1H, H-1bMan), 4.27 (dd, 1H, 3J
(dd, 1H, 3J(H,H)=7.3, 10.4 Hz), 4.01 (dd, 1H, 3J
3.89–3.27 (m, 20H), 2.45 (s, 3H), 2.30 (t, 2H, 3J
(H,H)=7.6 Hz), 1.92 (s,
T
ACHTRE(UNG H,H)=8.3 Hz;
1H, H-2), 3.69 (s, 2H), 3.61 (m, 1H, H-5), 1.96–1.91 (9H), 0.86 (s, 9H),
0.11 (m, 6H); 13C NMR (100 MHz, CDCl3): d=170.3, 170.2, 169.0, 165.1,
95.4, 77.2, 71.7, 70.2, 68.7, 62.3, 57.1, 28.6, 25.5, 20.67, 20.65, 20.60, 17.8,
ꢁ4.2, ꢁ5.3; elemental analysis calcd (%) for C20H34BrNO9Si: C 44.44, H
6.34, N 2.59; found: C 44.37, H 6.27, N 2.53.
AHCTREUNG
N
ACHTREUNG
AHCTREUNG
3H), 1.58 (m, 4H), 1.31 (8H); 13C NMR (100 MHz, CD3OD): d=175.9,
174.1, 146.3, 134.2, 131.0, 129.0, 101.8, 100.8, 98.5, 77.8, 77.6, 76.9, 75.3,
75.2, 72.4, 72.0, 71.6, 70.7, 68.4, 67.8, 62.6, 57.1, 52.0, 49.3, 34.8, 30.7, 30.4,
30.3, 30.1, 27.0, 26.0, 23.4, 21.6; HR ESI-MS: m/z: calcd for
C37H59NO20SNa: 892.3249, found 892.3289 [M+Na]+.
3,4,6-Tri-O-acetyl-2-bromoacetamido-2-deoxy-a-d-glucopyranosyl phos-
phate diallyl ester (18): A solution of 16 (394.5 mg, 0.7299 mmol) in
CH3CN (5.0 mL) was added 47% aq. HF (750 mL), and stirred for 11 h at
room temperature. The mixture was neutralized with aq. NaHCO3, and
the aqueous phase was extracted with CHCl3 (3”). The combined organ-
iclayers were washed with brine (2”), dried over Na 2SO4, filtered, and
concentrated. Purification by silica gel chromatography (toluene/EtOAc
5:1 ! 1:1) gave the hemiacetal (290.3 mg, 0.6811 mmol, 93%). 1H NMR
Compound 13a: A solution of tosylate 12 (22.3 mg, 0.0256 mmol) in
DMF (1.5 mL) was added AcSK (9.2 mg, 0.081 mmol), and stirred for 9 h
at 658C under N2 atmosphere. The mixture was cooled to room tempera-
ture, to which were added MeOH/H2O 7:3 (3 mL) and Et3N (0.3 mL).
After stirring for 15 h at room temperature, the mixture was concentrat-
ed. Purification by size-exclusion chromatography (Sephadex LH-20,
CHCl3/MeOH 1:4) gave 13a (13.4 mg, 9.2 mmol, 72%). [a]2D2 = +31 (c=
0.71 in methanol); 1H NMR (400 MHz, D2O, 40 8C): d=4.93 (2H), 4.59–
(400 MHz, CDCl3): d=6.65 (1H, 3J
A
A
3J
3J
ACHTREUNG
AHCTREUNG
2H), 3.19 (d, 1H, 3J
A
4.57 (m, 4H, overlapped with HOD), 4.11 (dd, 2H, 3J
A
(100 MHz, CDCl3): d=170.7, 170.3, 168.9, 165.5, 91.2, 77.2, 70.3, 67.9,
Chem. Eur. J. 2006, 12, 3449 – 3462
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3457

Y. Ito et al.
67.7, 61.8, 52.7, 28.3, 20.74, 20.70, 20.59; elemental analysis calcd (%) for
C14H20BrNO9: C 39.45, H 4.73 N 3.29; found: C 39.68, H 4.69, N3.24.
under Ar atmosphere, the mixture was cooled to ꢁ408C, and Me2S
(600 mL) and TBHP (300 mL; 5–6m in decane) were added. The mixture
was stirred for 2.5 h at ꢁ40 to 08C and quenched with 5% Na2S2O3 solu-
tion. The aqueous phase was extracted with CHCl3 (3”). The combined
organiclayers were washed with brine (2”), dried over Na 2SO4, filtered,
and concentrated. Purification by silica gel chromatography (CHCl3/
EtOAc6:1 ! 2:1) gave 21 (173.6 mg, 0.307 mmol, 77%). [a]2D4 = +91
(c=0.96 in chloroform); 1H NMR (400 MHz, CDCl3): d=6.53 (d, 1H,
Amidite 17 (175 mL, 0.662 mmol) and tetrazole (96.3 mg, 1.37 mmol)
were added at ꢁ208C to
a solution of the hemiacetal (183.9 mg,
0.431 mmol) in CH2Cl2 (2.5 mL). After stirring at ꢁ108C for 50 min
under Ar atmosphere, the mixture was cooled to ꢁ408C and added tert-
butyl hydroperoxide (TBHP, 5.0–6.0m in decane, 300 mL). After stirring
for 6 h at ꢁ40 to 08C, the mixture was quenched with 5% aq. Na2S2O3
and extracted with CHCl3 (3”). Combined organiclayers were washed
with brine (2”), dried over Na2SO4, filtered, concentrated, and purified
with silica gel chromatography (toluene/EtOAc 2:1 ! 1:2) to give 18
3J
3J
A
ACHTRE(UGN H,H)=3.4,
AHCTREUNG
4.25 (dd, 1H, J
1H, J
ACHTREUNG
(181.2 mg, 0.358 mmol, 83%). [a]D22
=
+65 (c=0.66 in chloroform);
A
ACHTREUNG
1H NMR (400 MHz, CDCl3): d=6.98 (br, 1H, NH), 5.97 (m, 2H), 5.74
AHCTREUNG
3
3
(dd, J
3J
(H,H)=9.6 Hz; H-4), 4.61 (m, 4H), 4.38 (m, 1H, H-2), 4.29–4.22 (m,
2H, H-5, H-6a), 4.11 (d, 1H, 2J
(H,H)=10.8 Hz; H-6b), 3.85 (d, 1H,
2J(H,H)=13.2 Hz), 3.78 (d, 1H, 3J
(H,H)=13.2 Hz), 2.11–2.00 (3s, 9H);
ACHTREUNG(H,H)=2.8, JACHTREUNG(P,H)=5.6 Hz; H-1), 5.44–5.29 (m, 5H), 5.20 (t, 1H,
A
ACHTREUNG
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
32.6, 30.2, 20.73, 20.70, 20.6; HR ESI-MS: m/z: calcd for
C22H32NO13PSNa: 604.1211, found 604.1230 [M+Na]+.
A
ACHTREUNG
13C NMR (100 MHz, CDCl3): d=170.6, 170.1, 168.9, 166.0, 131.7 (d,
3J
2J
A
ACHTREUNG
3,4,6-Tri-O-acetyl-2-(acetylthio)acetamido-2-deoxy-a-d-glucopyranosyl
phosphate (22): Et3SiH (480 mL, 3.00 mmol), AcOH (175 mL, 3.05 mmol),
and [PdCAHTER(UNG PPh3)4] (33 mg, 0.029 mmol) were added to a solution of 21
G
A
ACHTREUNG
3
5.8 Hz), 67.1, 61.2, 52.5 (d, JCAHTREUNG
(170.8 mg, 0.3020 mmol) in toluene (3 mL). The mixture was stirred for
14 h at 408C under Ar atmosphere, and concentrated. Purification by
silica gel chromatography (CHCl3/MeOH/AcOH 30:1:0 ! 8:4:0.1) was
followed by size-exclusion chromatography (Sephadex LH-20, CHCl3/
MeOH 1:4) to give 22 (77.4 mg, 0.154 mmol, 51%). 1H NMR (400 MHz,
3,4,6-Tri-O-acetyl-2-bromoacetamido-2-deoxy-a-d-glucopyranosyl phos-
phate (19): Et3SiH (350 mL, 2.19 mmol), AcOH (130 mL, 2.27 mmol), and
[PdACHTREUNG(PPh3)4] (14 mg, 0.012 mmol) were added to a solution of 18
(133.0 mg, 0.2268 mmol) in toluene (4 mL), and the reaction mixture was
stirred for 2.5 h at 408C under Ar atmosphere. Then, the mixture was
cooled to room temperature, concentrated, and purified by silica gel
CD3OD): d=5.46 (dd, 1H, 3J
(dd, 1H, J
(H,H)=9.5, 10.7 Hz; H-3), 5.00 (t, 1H, 3J
4), 4.21–4.17 (2H, H-2, H-6a), 4.12 (m, 1H, H-5), 4.02 (dd, 1H, J
2.2, 12.2 Hz; H-6b), 3.06 (d, 1H, (H,H)=15.3 Hz), 3.43 (d, 1H,
(H,H)=15.4 Hz), 2.27 (s, 3H), 1.95 (s, 3H), 1.92 (s, 3H), 1.90 (s, 3H);
13C NMR (100 MHz, CD3OD): d=195.9, 172.2, 171.1, 170.9, 95.8 (d,
2J(P,C)=5.8 Hz), 71.4, 70.3, 69.5, 53.4 (d, 3J
(P,C)=8.3 Hz), 33.5, 30.1,
(H,H)=3.4, 3J
ACHRTEUNG ACHTREUNG
A
ACHTREUNG
chromatography (CHCl3/MeOH/AcOH 30:1:0
(97.9 mg, 0.193 mmol, 85%). 1H NMR (400 MHz, CD3OD): d=5.84 (br,
1H, H-1), 5.24 (t, 1H, 3J(H,H)=9.2 Hz; H-3), 5.02 (t, 1H, 3J
(H,H)=
10.0 Hz; H-4), 4.25–4.13 (3H, H-2, H-5, H-6a), 4.04 (d, 1H, 2J
(H,H)=
11.6 Hz; H-6b), 3.83 (d, 1H, J(H,H)=10.8 Hz), 3.67 (d, 1H, J(H,H)=
10.8 Hz), 1.97 (s, 3H), 1.91 (s, 3H), 1.89 (s, 3H); 13C NMR (100 MHz,
CD3OD): d=172.3, 171.8, 171.1, 169.9, 95.5 (d, 2J
(P,C)=5.8 Hz), 71.7,
70.2, 69.6, 62.8, 53.5 (d, 3J
(P,C)=9.1 Hz), 28.4, 20.7, 20.63, 20.61; HR
!
6:2:0.1) to give 19
ACHTREUNG
JACHTREUNG
A
ACHTREUNG
JACHTREUNG
AHCTREUNG
A
ACHTREUNG
A
ACHTREUNG
20.74, 20.65, 20.59; HR ESI-MS: m/z: calcd for C16H23NO13PS: 500.0628,
found 500.0663 [MꢁH]ꢁ.
AHCTREUNG
AHCTREUNG
ESI-MS: m/z: calcd for C14H20BrNO12P: 503.9907, found 503.9926
[MꢁH]ꢁ.
2-Deoxy-2-mercaptoacetamido-a-d-glucopyranosyl phosphate triethylam-
monium salt (23): Et3N (0.75 mL) was added to a solution of 22 (32.8 mg,
0.0654 mmol) in degassed MeOH/H2O (5 mL, 7:3), and stirred for 1 d
under Ar atmosphere. The mixture was concentrated, and dried under re-
duced pressure. Purification was not conducted because of the instability
of the material.
tert-Butyldimethylsilyl
deoxy-b-d-glucopyranoside (20):
3,4,6-tri-O-acetyl-2-(acetylthio)acetamido-2-
solution of bromoacetamide 16
A
(1.10 g, 2.04 mmol) in CH3CN (10 mL) were added AcSH (180 mL,
2.52 mmol) and iPr2NEt (550 mL, 3.16 mmol) at 48C. The mixture was
stirred for 1 h at 48C, and quenched with aq. NaHCO3. The aqueous
phase was extracted with CHCl3 (3”). The combined organic layers were
washed with brine (2”), dried over Na2SO4, filtered, and concentrated.
Purification by silica gel chromatography (toluene/EtOAc 2:1) to give 20
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-(2-bromoethylthio)acetamido-
2-deoxy-b-d-glucopyranoside
(24):
2-Mercaptopropanol
(190 mL,
2.709 mmol) and iPr2NEt (0.9 mL, 5.17 mmol) were added to a solution
of bromoacetamide 16 (1.134 g, 2.099 mmol) in CH3CN (15 mL). After
stirring for 4 h, the mixture was concentrated and dissolved in CHCl3.
The solution was washed with 10% citric acid solution and brine (2”),
dried over Na2SO4, filtered, concentrated, and dried under vacuum.
Then, the crude alcohol was dissolved in CH2Cl2 (20 mL), to which were
added NBS (453 mg, 2.54 mmol) and Ph3P (667 mg, 2.54 mmol) at 48C.
The mixture was stirred for 1 h at 48C under Ar atmosphere and diluted
with CHCl3. The organicphase was washed with 5% Na 2S2O3 solution
and brine (2”), dried over Na2SO4, filtered, and concentrated. Purifica-
tion with silica gel chromatography (toluene/EtOAc 6:1 ! 2:1) gave bro-
1
(1.01 g, 1.88 mmol, 93%). [a]D22 = +33 (c=0.91 in chloroform); H NMR
3
(400 MHz, CDCl3): d=6.31 (d, 1H, J
A
3J
ACHTREUNG ACHTRENUG
(H,H)=9.3, 10.7 Hz; H-4), 5.01 (t, 1H, 3J
1H, 3J
A
ACHTREUNG
ACHTREUNG
A
ACHTREUNG
2.41 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 0.88 (s, 9H), 0.10 (s,
6H); 13C NMR (100 MHz, CDCl3): d=195.9, 170.6, 170.4, 169.2, 167.9,
95.9, 72.0, 71.8, 68.9, 62.4, 56.4, 32.8, 30.1, 25.6, 20.71, 20.68, 20.61, 17.9,
ꢁ4.2, ꢁ5.2; elemental analysis calcd (%) for C22H37BNO10SSi: C 49.33,
H 6.96, N 2.91; found: C 49.3, H 6.96, N 2.91.
mide 24 (1.206 g, 2.01 mmol, 96%). [a]D24
form); 1H NMR (400 MHz, CDCl3): d=6.71 (d, 1H, 3J
NH), 5.30 (dd, 1H, 3J(H,H)=9.2, 10.8 Hz; H-3), 5.04 (t, 1H, 3J
9.2 Hz; H-4), 4.93 (d, 1H, 3J
(H,H)=8.0 Hz; H-1), 4.23–4.11 (m, 2H,
H-6a, H-6b), 3.84 (m, 1H, H-2), 3.73 (m, 1H, H-5), 3.49 (t, 2H,
3J
(H,H)=7.6 Hz), 3.19 (s, 2H), 2.94 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H),
=
+5.9 (c=0.95 in chloro-
(H,H)=9.2 Hz;
(H,H)=
AHCTREUNG
H
ACHTREUNG
3,4,6-Tri-O-acetyl-2-(acetylthio)acetamido-2-deoxy-a-d-glucopyranosyl
phosphate diallyl ester (21): HF/pyridine (100 mL) was added at 48C to a
solution of 20 (275.3 mg, 0.514 mmol) in THF (2 mL). After stirring for
5.5 h at room temperature, the mixture was neutralized with sat NaHCO3
solution. The aqueous phase was extracted with CHCl3 (3”). The com-
AHCTREUNG
AHCTREUNG
2.03 (s, 3H), 0.87 (s, 9H), 0.09 (s, 6H); 13C NMR (100 MHz, CDCl3): d=
170.7, 170.5, 169.3, 168.0, 95.8, 72.1, 71.9, 69.0, 62.5, 57.0, 35.9, 34.9, 29.7,
25.7, 25.6, 20.9, 20.8, 20.7, ꢁ4.1, ꢁ5.0; HR ESI-MS: m/z: calcd for
C22H38BrNO9SSiNa: 622.1118, found 622.1143 [M+Na]+; elemental anal-
ysis calcd (%) for C22H38BrNO9SSi: C 44.00, H 6.38, N 2.33; found: C
44.11, H 6.31, N 2.32.
bined organiclayers were washed with brine (2”), dried over Na SO4, fil-
2
tered, and concentrated. Purification by silica gel chromatography
(CHCl3/MeOH 100:1
0.400 mmol, 78%).
!
30:1) gave the hemiacetal (168.6 mg,
To a solution of the hemiacetal (168.6 mg, 0.400 mmol) in CH2Cl2 (2 mL)
were added 1H-tetrazole (123.1 mg, 1.76 mmol) and diallylphosphorami-
dite 17 (190 mL, 0.719 mmol) at ꢁ208C. After stirring for 1 h at ꢁ108C
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-(3-bromopropylthio)acetami-
do-2-deoxy-b-d-glucopyranoside (25): 3-Mercaptopropanol (210 mL,
3458
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3449 – 3462

Glucosyltransferase Inhibitors
FULL PAPER
2.43 mmol) and iPr2NEt (1.25 mL, 7.18 mmol) were added to a solution
of the bromoacetamide 16 (1.096 g, 2.028 mmol) in CH3CN (10 mL).
After stirring for 7.5 h, the mixture was concentrated, and dissolved in
CHCl3. The mixture was washed with 10% citric acid solution and brine
(2”), dried over Na2SO4, filtered, concentrated, and dried under vacuum.
Then, the crude alcohol was dissolved in CH2Cl2 (15 mL), to which were
added NBS (444 mg, 2.49 mmol) and Ph3P (645 mg, 2.46 mmol) at 48C.
The mixture was stirred for 1 h at 48C under Ar atmosphere, and diluted
with CHCl3. The organicphase was washed with 5% aqueous Na 2S2O3
solution and brine (2”), dried over Na2SO4, filtered, and concentrated.
Purification with silica gel chromatography (toluene/EtOAc 6:1 ! 2:1)
gave bromide 25 (1.178 g, 1.917 mmol, 95%). [a]2D4 = +5.0 (c=0.75 in
TBHP (750 mL, 5–6m in decane) were added. The mixture was stirred at
ꢁ40 to 08C for 1.5 h. After the reaction, mixture was quenched with 5%
Na2S2O3 solution and extracted with CHCl3 (3”). The combined organic
layers were washed with brine (2”), dried over Na2SO4, filtered, and
concentrated. Purification with silica gel chromatography (toluene/
EtOAc4:1 ! 2:3) gave 27 (442.2 mg, 0.669 mmol, 78%). [a]2D2 =+44 (c=
0.76 in chloroform); 1H NMR (400 MHz, CDCl3): d=6.93 (d, 1H,
3J
2J
A
ACHTRE(UGN H,H)=3.4,
A
ACHTREUNG
4.64–5.58 (m, 4H), 4.40 (m, 1H), 4.25 (dd, 1H, J
4.20 (m, 1H), 4.10 (dd, 1H, J
6.3 Hz), 3.20 (d, 1H, J
2.66 (t, 2H,
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
chloroform); 1H NMR (400 MHz, CDCl3): d=6.67 (d, 1H, 3J
8.8 Hz), 5.19 (dd, 1H, 3J
(H,H)=9.3, 10.7 Hz; H-3), 4.91 (t, 1H,
3J(H,H)=10.0 Hz; H-4), 4.82 (d, 1H, 3J
(H,H)=7.8 Hz; H-1), 4.07 (dd,
1H, 3J(H,H)=5.9, 2J(H,H)=12.2 Hz; H-6a), 4.02 (dd, 1H, 3J
(H,H)=2.7,
2J
(H,H)=12.2 Hz; H-6b), 3.71 (m, 1H, H-2), 3.62 (m, 1H, H-5), 3.38
(ddd, 2H, 3J(H,H)=1.2, 6.3, 2J
(H,H)=12.7 Hz; CHsBr), 3.04 (s, 2H),
2.55 (t, 2H, 3J(H,H)=7.1 Hz), 1.99 (t, 2H, 3J
(H,H)=6.8 Hz), 1.95 (s,
ACHTRE(UNG H,H)=
JACHTREUNG
AHCTREUNG
JACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
N
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
MS: m/z: calcd for C23H35BrNO12PSNa: 682.0699, found 682.0651
[M+Na]+.
A
ACHTREUNG
3H), 1.91 (s, 3H), 1.90 (s, 3H), 0.75 (s, 9H), ꢁ0.02 (s, 6H); 13C NMR
(100 MHz, CDCl3): d=170.5, 170.2, 169.3, 168.2, 95.8, 72.1, 71.9, 69.1,
62.5, 57.0, 35.9, 35.9, 31.7, 31.5, 31.0, 25.6, 20.82, 20.76, 20.69, 18.0, ꢁ4.1,
ꢁ5.0; HR ESI-MS: m/z: calcd for C23H40BrNO9SSi: 636.1274, found
636.1264 [M+H]+.
3,4,6-Tri-O-acetyl-2-(2-bromoethylthio)acetamido-2-deoxy-a-d-glucopy-
ranosyl phosphate (28): Et3SiH (240 mL, 1.50 mmol), AcOH (85 mL,
1.5 mmol), and [PdACHTRE(UNG PPh3)4] (16.9 mg, 0.015 mmol) were added to a solu-
tion of the phosphate 26 (95.1 mg, 0.147 mmol) in toluene (2 mL). After
stirring for 24 h at 408C under Ar atmosphere, the mixture was concen-
trated, and purified with silica gel chromatography (CHCl3/MeOH/
AcOH 30:1:0 ! 4:1:0.1). Final purification with size-exclusion chroma-
tography (Sephadex LH-20, CHCl3/MeOH 1:4) yielded 28 (71.5 mg,
0.1263 mmol, 86%). 1H NMR (400 MHz, CDCl3): d=5.69 (br, 1H), 5.32
3,4,6-Tri-O-acetyl-2-(2-bromoethylthio)acetamido-2-deoxy-a-d-glucopy-
ranosyl phosphate diallyl ester (26): 47% Aqueous HF (1.0 mL) was
added at 48C to a solution of 25 (496.2 mg, 0.826 mmol) in CH3CN
(9 mL). The mixture was stirred for 10 h at room temperature, and neu-
tralized with sat. NaHCO3 solution, and the aqueous phase was extracted
with CHCl3 (3”). Combined organiclayers were washed with brine
(2”), dried over Na2SO4, filtered, and concentrated. Purification by silica
(t, 1H, JACTHERNGU(H,H)=10.0 Hz), 5.19 (t, 1H, JACHTRE(UGN H,H)=9.5 Hz), 4.50–4.07 (m,
4H), 3.57 (m, 2H), 3.29 (m, 2H), 2.74 (m, 2H); 13C NMR (100 MHz,
CDCl3): d=175.9, 171.4, 170.8, 169.3, 94.1 (br), 71.7, 70.1, 71.7, 70.1, 69.2,
67.9, 61.0, 58.6, 52.0 (br), 35.5, 34.6, 31.9, 30.2, 20.9–20.7; HR ESI-MS:
m/z: calcd for C16H24BrNO12PS: 563.9940, found 563.9967 [MꢁH]ꢁ.
gel chromatography (toluene/EtOAc 2:1
(281.8 mg, 0.579 mmol, 70%).
!
1:1) gave hemiacetal
To a solution of the hemiacetal (279.2 mg, 0.574 mmol) in CH2Cl2 (5 mL)
were added amidite 17 (310 mL, 1.17 mmol) and 1H-tetrazole (162.1 mg,
2.31 mmol) at ꢁ208C. After stirring for 1 h at ꢁ108C under Ar atmos-
phere, the mixture was cooled to ꢁ408C, and Me2S (1 mL) and TBHP
(500 mL, 5–6m in decane) were added. The mixture was stirred at ꢁ40 to
08C for 2.5 h. The mixture was quenched with 5% Na2S2O3 solution and
extracted with CHCl3 (3”). The combined organic layers were washed
with brine (2”), dried over Na2SO4, filtered, and concentrated. Purifica-
tion with silica gel chromatography (toluene/EtOAc 4:1 ! 2:3) gave 26
3,4,6-Tri-O-acetyl-2-(3-bromoethylthio)acetamido-2-deoxy-a-d-glucopy-
ranosyl phosphate (29): Et3SiH (490 mL, 3.07 mmol), AcOH (180 mL,
3.14 mmol), and [PdACHTRE(UNG PPh3)4] (36.0 mg, 0.0312 mmol) were added to a sol-
ution of the allyl ester 27 (200.8 mg, 0.3040 mmol) in degassed toluene
(4 mL). After stirring for 14 h at 408C under Ar atmosphere, the mixture
was concentrated and purified with silica gel chromatography (CHCl3/
MeOH/AcOH 30:1:0 ! 6:3:0.1), following size-exclusion chromatogra-
phy (Sephadex LH-20, CHCl3/MeOH 1:4) gave 29 (99.4 mg,
0.1713 mmol, 56%). 1H NMR (400 MHz, CD3OD): d=5.55 (dd, 1H,
(251.5 mg, 0.389 mmol, 68%). [a]D23
1H NMR (400 MHz, CDCl3): d=6.89 (d, 1H, J
(m, 2H), 5.73 (dd, 3J(H,H)=3.2, 3J
(P,H)=5.9 Hz; H-1), 5.44–5.27 (m,
5H), 5.20 (t, 1H, 3J
(H,H)=9.8 Hz), 4.64–4.57 (m, 4H), 4.39 (m, 1H, H-
2), 4.26 (dd, 1H, 3J(H,H)=3.8, 2J
(H,H)=12.2, H-6a), 4.22 (m, 1H, H-5),
4.10 (dd, 1H, 3J(H,H)=2.0, 2J
(H,H)=12.4 Hz; H-6b), 3.48 (t, 2H,
3J
(H,H)=7.6 Hz), 3.21 (s, 2H), 2.95 (t, 2H, J(H,H)=7.6 Hz), 2.09 (s,
3H), 2.05 (s, 6H); 13C NMR (100 MHz, CDCl3): d=171.0, 170.4, 169.0,
168.7, 132.0, 131.9 (d, 3J(P,C)=6.6 Hz), 131.8, (d, 3J
(P,C)=6.6 Hz), 119.1,
119.0, 95.6 (d, 2J(P,C)=5.8 Hz), 69.9, 69.7, 68.9 (d, 2J
(P,C)=5.0 Hz), 68.7
(d, 2J(P,C)=5.0 Hz), 67.3, 61.4, 52.3 (d, 3J
(P,C)=8.3 Hz), 35.6, 34.8, 29.6,
= +55 (c=0.95 in chloroform);
3J
11.0 Hz; H3), 5.07 (t, 1H, J
H-5, H-6a), 4.09 (dd, (H,H)=2.0, 12.2 Hz), 3.50 (t, 2H,
6.3 Hz), 3.19 (d, 1H, J(H,H)=14.5 Hz), 3.13 (d, 1H, J(H,H)=14.5 Hz),
2.68 (m, 2H), 2.10–1.94 (m, 11H); 13C NMR (100 MHz, CD3OD): d=
172.7, 172.3, 171.9, 171.1, 95.9 (2J
(P,C)=5.8 Hz), 71.5, 70.3, 69.5, 62.7,
A
(P,H)=6.1 Hz; H-1), 5.27 (dd, 1H, 3J
(H,H)=10.2 Hz), 4.28–4.4.15 (m, 3H, H-2,
(H,H)=
ACHTRE(UGN H,H)=9.5,
3
AHCTRE(UNG H,H)=8.5 Hz; NH), 5.97
AHCTREUNG
A
ACHTREUNG
J
G
JACHTREUNG
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
49.5 (3J
ACHTRE(UNG P,C)=9.1 Hz), 35.6, 33.0, 32.7, 31.7, 20.8, 20.7, 20.6; HR ESI-MS:
m/z: calcd for C17H26BrNO12PS: 578.0097, found 578.0087 [MꢁH]ꢁ.
A
ACHTREUNG
A
ACHTREUNG
Compound 30a: TCEP-HCl (10.8 mg, 0.0377 mmol) was added to a solu-
tion of the disulfide 13a (10.8 mg, 0.00740 mmol) in MeOH/H2O (7:3;
1 mL) and the mixture was stirred for 3 h at room temperature under Ar
atmosphere. Then, bromoacetamide 19 (12.8 mg, 0.0254 mmol) and
iPr2NEt (20 mL, 0.12 mmol) were added. After stirring for 3 h at room
temperature under Ar atmosphere, Et3N (150 mL) was added and stirring
continued for additional 10 h, and Et3N (150 mL) was added to complete
the reaction. The mixture was further stirred for 9 h, and concentrated.
Purification by SepPak C-18 cartridge (MeOH/H2O 0:100 ! 50:50), and
size-exclusion chromatography (Sephadex G-15, H2O) gave 30a (8.8 mg,
6.5 mmol, 58%). 1H NMR (400 MHz, D2O, 40 8C): d=5.42 (dd, 1H,
A
ACHTREUNG
20.8, 20.7, 20.6; elemental analysis calcd (%) for C22H33BrNO12PS: C
40.88, H 5.15, N 2.17; found: C 41.02, H 5.11, N 2.19.
3,4,6-Tri-O-acetyl-2-(3-bromopropylthio)acetamido-2-deoxy-a-d-gluco-
pyranosyl phosphate diallyl ester (27): 47% Aqueous HF was added at
48C to a solution of 25 (809.3 mg, 1.317 mmol) in CH3CN (8.5 mL) and
the reaction mixture was stirred for 13 h at room temperature. Then, the
mixture was neutralized with a saturated NaHCO3 solution, and the
aqueous phase was extracted with CHCl3 (3”). The combined organic
layers were washed with brine (2”), dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography (toluene/EtOAc
3:1 ! 1:1) gave hemiacetal (597.3 mg, 1.19 mmol, 90%).
To a solution of the hemiacetal (431.6 mg, 0.863 mmol) in CH2Cl2 (5 mL)
were added amidite 17 (460 mL, 1.74 mmol) and 1H-tetrazole (302.2 mg,
4.31 mmol) at ꢁ208C. After stirring for 1 h at ꢁ108C under Ar atmos-
phere, the mixture was cooled to ꢁ408C, to which Me2S (1.5 mL) and
3J
(2H, H-1GN
3J
(H,H)=1.5, 3.4 Hz; H-2aMan), 4.03–3.35 (25H), 3.68 (s, 3H, CH3COO),
3.11 (dd, 1H, 3J(H,H)=2.4, 2J
(H,H)=13.9 Hz; H-6aaMan), 2.74 (dd, 1H,
3J(H,H)=8.3, 2J(H,H)=13.9 Hz; H-6baMan), 2.38 (t, 2H, 3J
(H,H)=
7.3 Hz; CH2COO), 2.05 (s, 3H, AcN), 1.59 (br, 4H), 1.30 (br, 8H); HR
A
ACHTREUNG
,
,
AHCTREUNG
A
ACHTREUNG
A
N
ACHTREUNG
Chem. Eur. J. 2006, 12, 3449 – 3462
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3459

Y. Ito et al.
ESI-MS: m/z: calcd for C36H66N2O26SP: 1029.3362, found 1029.3314
[MꢁH]ꢁ.
CH3COO), 3.06 (dd, 1H, 2J
SCH2), 2.63 (dd, 1H, 3J(H,H)=8.8, 2J
2H, 3J
(H,H)=7.6 Hz; CH2COO), 2.05 (s, 3H, AcN), 1.92 (m, 2H), 1.59
ACHTREUNG
G
ACHTREUNG
AHCTREUNG
Compound 1a: Phosphate 30a (2.7 mg, 2.4 mmol) and UMP-morpholi-
date (5.3 mg, 7.7 mmol) were co-evaporated with dry pyridine 3” and
dried under vacuum for 1 h. The residue was redissolved in dry pyridine
(0.5 mL), to which was added 1H-tetrazole (1.2 mg, 0.017 mmol), and stir-
red for 3 d under Ar atmosphere. Resultant mixture was quenched with
H2O and concentrated. The residue was applied to SepPak C-18 cartridge
(MeOH/H2O 0:100 ! 60:40). Subsequent purification with size-exclusion
chromatography (Sephadex G-15, H2O) gave 1a (2.5 mg, 1.9 mmol,
(br, 4H), 1.30 (br, 8H); HR ESI-MS: m/z: calcd for C41H72N2O26PS2:
1103.3552, found 1103.3558 [MꢁH]ꢁ.
Compound 1e: Compound 30e (4.9 mg, 4.4 mmol) was treated with
UMP-morpholidate (9.6 mg, 0.014 mmol) and 1H-tetrazole (1.6 mg,
0.023 mmol), in a manner as described for 1a. Successive chromatograph-
icpurifaiction afforded
1e (3.8 mg, 0.0027 mmol, 61%). 1H NMR
3
(400 MHz, D2O, 40 8C): d=7.93 (d, 1H, J
N
5.95 (m, 2H, H-5Uracil, H-1Rib), 5.52 (dd, 1H, 3J
A
ACHTREUNG
78%). 1H NMR (400 MHz, D2O, 40 8C): d=7.93 (d, 1H, 3J
8.1 Hz; H-6Uracil), 5.98–5.95 (2H, H-5Uracil H-1Rib), 5.52 (dd, 1H,
3J(H,H)=3.2, 3J
(P,H)=6.3 Hz; H-1GN1P), 4.91 (s, 1H, H-1aMan), 4.65–4.57
(2H, H-1GN, H-1bMan, overlapped with HOD signal), 4.36–4.20 (5H, H-
Rib, H-3Rib, H-4Rib, H-5aRib, H-5bRib), 4.13 (br, 1H, H-2aMan), 4.02–3.13
ACHTRE(UNG H,H)=
,
,
,
A
ACHTREUNG
AHCTREUNG
3
2
A
ACHTREUNG(H,H)=
2
2
AHCTREUNG
(25H), 3.64 (s, 3H, CH3COO), 3.11 (d, 1H, JACHTREUNG
2.73 (dd, 1H, JACHTREUNG(H,H)=8.3, JACHTRENUG
3
2
A
ACHTREUNG
2.05 (s, 3H, AcN), 1.90 (m, 2H), 1.59 (br, 4H), 1.30 (br, 8H); HR ESI-
MS: m/z: calcd for C50H82N4O34P2S2Na: 1431.3625, found 1431.3672
[Mꢁ2H+Na]ꢁ.
7.6 Hz; CH2COO), 2.05 (s, 3H, AcN), 1.59 (br, 4H), 1.30 (br, 8H); HR
ESI-MS: m/z: calcd for C47H76N4O34P2SNa: 1357.3435, found 1357.3417
[Mꢁ2H+Na]ꢁ.
Compound 31: nBu3P (10 mL, 0.040 mmol) was added to a solution of di-
sulfide 13a (9.7 mg, 6.6 mmol) in degassed THF/H2O (1 mL, 9:1). After
stirring for 5 h at room temperature, the mixture was concentrated and
dissolved in H2O. The aqueous phase was washed with Et2O (3”), con-
centrated, and dried under vacuum. Resultant thiol was dissolved in de-
gassed MeOH/H2O (6 mL, 1:1), to which were added 0.5m HCl
(0.15 mL) and pyridyl disulfide (15.1 mg, 0.069 mmol) at ꢁ208C under
Ar atmosphere under vigorous stirring. The mixture was stirred for 2 h at
ꢁ208C to room temperature and evaporated in vacuo. The residue was
re-dissolved in H2O, and the solution was washed with Et2O (3”) to
remove excess pyridyl disulfide, concentrated, and dried under vacuum.
No further purification was attempted because of the instability of this
compound.
Compound 30d: nBu3P (10 mL, 0.040 mmol) was added to a solution of
disulfide 13a (10.0 mg, 6.8 mmol) in degassed THF/H2O (1 mL, 9:1).
After stirring for 5 h at room temperature, the mixture was concentrated
and dissolved in H2O. The aqueous phase was washed with Et2O (3”),
concentrated, and dried under vacuum. Resultant thiol was dissolved in
degassed DMF (0.7 mL), to which were added GlcNAc derivative 28
(12.2 mg, 0.0215 mmol) and Cs2CO3 (10.8 mg, 0.033 mmol). The mixture
was stirred at 408C under Ar atmosphere for 10 h. The mixture was di-
rectly applied to size-exclusion chromatography (Sephadex LH-20,
CHCl3/MeOH 1:4). Appropriate fractions were collected, evaporated and
1
lyophilized to give 30d (5.5 mg, 0.0051 mmol, 37%). H NMR (400 MHz,
D2O, 40 8C): d=5.44 (dd, 1H, 3J(H,H)=3.2, 3J
ACTHERNGU ACHTREU(GN P,H)=6.8 Hz; H-1GN1P),
4.89 (s, 1H, H-1aMan), 4.65–4.56 (m, 2H, H-1GN, H-1bMan, overlapped with
HOD signal), 4.12 (br, 1H, H-2aMan), 4.02–3.35 (25H), 3.69 (s, 3H,
Compound 30b: A solution of crude thiol 23 in degassed MeOH/1m aq.
NH4OAc(1.5 mL, 1:1) was added crude 31 dissolved in 0.5 mL MeOH/
1m aq. NH4OAcsolution at ꢁ208C under Ar atmosphere. After stirring
for 6 h at ꢁ208C to room temperature, the mixture was concentrated. Pu-
CH3COO), 3.08 (d, 1H, 2J
(dd, 1H, 3J(H,H)=9.0, 2J
3J
(H,H)=7.6 Hz; CH2COO), 2.06 (s, 3H, AcN), 1.59 (br, 4H), 1.30 (br,
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
8H); HR ESI-MS: m/z: calcd for C40H70N2O26PS2: 1089.3396, found
1089.3423 [MꢁH]ꢁ.
Compound 1d: Compound 30d (5.1 mg, 0.0047 mmol) was treated with
UMP-morpholidate (9.8 mg, 0.014 mmol) in the presence of 1H-tetrazole
(1.6 mg, 0.023 mmol), as described for 1a. Successive purification with
SepPak C-18 cartridge, Sephadex G-15, H2O, preparative TLC, and
SepPak C-18 cartridge afforded 1d (3.8 mg, 0.00272 mmol, 58%).
rification by SepPak C-18 cartridge column (MeOH/H2O 0:100
!
50:50), and gel filtration chromatography (Sephadex G-15, H2O) gave
30b (6.6 mg, 6.2 mmol, 47%). 1H NMR (400 MHz, D2O, 40 8C): d=5.40
(dd, 1H, 3J(H,H)=2.9, 3J
ACTHERNGU CAHRTEU(NG P,H)=7.1 Hz; H-1GN1P), 4.90 (s, 1H, H-1aMan),
4.65–4.56 (m, 2H, H-1GN, H-1bMan, overlapped with HOD signal), 4.13 (br,
1H, H-2aMan), 4.03–3.35 (m, 25H), 3.68 (s, 3H, CH3COO), 3.37 (dd, 1H,
3J
2J
(H,H)=2.2, 3J(H,H)=13.9 Hz; H-6aaMan), 2.87 (dd, 1H, 3J
ACHTREUNG ACHTREUNG ACHTREUNG
ACHRTEUGN ACHTREUNG
(H,H)=13.9 Hz; H-6baMan), 2.38 (t, 2H, 3J
1H NMR (400 MHz, D2O, 40 8C): d=7.93 (d, 1H, 3J
Uracil), 5.98–5.95 (m, 2H, H-5Uracil, H-1Rib), 5.52 (dd, 1H, 3J
3J
(P,H)=6.8 Hz; H-1GN1P), 4.90 (s, 1H, H-1aMan), 4.65–4.56 (m, 2H, H-
GN, H-1bMan, overlapped with HOD signal), 4.38–4.17 (m, 5H, H-2Rib, H-
3Rib, H-4Rib, H-5aRib, H-5bRib), 4.12 (br, 1H, H-2aMan), 4.02–3.33 (m, 25H),
(H,H)=8.1 Hz; H-
(H,H)=3.4,
2.05 (s, 3H, AcN), 1.59 (br, 4H), 1.31 (br, 8H); HR ESI-MS: m/z: calcd
6
ACHTREUNG
for C38H66N2O26PS2: 1061.3083, found 1061.3049 [MꢁH]ꢁ.
ACHTREUNG
1
Compound 1b: Disulfide linked 30b (4.7 mg, 0.0044 mmol) and UMP-
morpholidate (9.3 mg, 0.014 mmol) were co-evaporated with dry pyridine
(3”) and dried under vacuum for 1 h. Then, the mixture was dissolved in
dry pyridine (0.5 mL), to which was added 1H-tetrazole (1.7 mg,
0.024 mmol). The mixture was stirred for 3 d under Ar atmosphere,
quenched with H2O and concentrated. The residue was purified as descri-
bed for 1a to give 1b (3.4 mg, 0.0025 mmol, 56%). 1H NMR (400 MHz,
3.68 (s, 3H, CH3COO), 3.08 (d, 1H, 2J
ACHTREUNG
(br, 4H, SCH2CH2S), 2.66 (dd, 1H, 3J
A
ACHTREUNG
3
6baMan), 2.38 (t, 2H, J
A
(br, 4H), 1.30 (br, 8H); HR ESI-MS: m/z: calcd for C49H80N4O34P2S2Na
1417.3468, found 1417.3516 [Mꢁ2H+Na]ꢁ.
D2O, 40 8C): d=7.93 (d, 1H, 3J
H-5Uracil, H-1Rib), 5.53 (dd, 1H, 3J
4.90 (d, 1H, 3J
(H,H)=1.5 Hz; H-1aMan), 4.65–4.57 (m, 2H, H-1GN
H-1bMan, overlapped with HOD signal), 4.37–4.18 (m, 5H, H-2Rib, H-3Rib
H-4Rib, H-5aRib, H-5bRib), 4.13 (dd, 1H, 3J
(H,H)=1.7, 3.4 Hz; H-2aMan),
4.02–3.42 (m, 25H), 3.64 (s, 3H, CH3COO), 3.37 (dd, 1H, 3J
(H,H)=2.4,
2J(H,H)=14.1 Hz; H-6aaMan), 2.86 (dd, 1H, 3J(H,H)=8.5, 2J
(H,H)=
13.9 Hz; H-6baMan), 2.38 (t, 2H, J(H,H)=7.6 Hz; CH2COO), 2.05 (s, 3H,
ACHTREUNG
Compound 30e: nBu3P (10 mL, 0.040 mmol) was added to a solution of
disulfide 13a (10.1 mg, 0.0069 mmol) in degassed THF/H2O (0.7 mL,
9:1). After stirring for 5 h at room temperature, the mixture was concen-
trated and dissolved in H2O. The aqueous phase was washed with Et2O
(3”) and concentrated, and dried under reduced pressure. Resultant
thiol was dissolved in degassed DMF (0.7 mL), to which were added
GlcNAc derivative 29 (12.4 mg, 0.0214 mmol), and Cs2CO3 (10.2 mg,
0.031 mmol). The mixture was stirred at 408C under Ar atmosphere for
12 h, and applied to size-exclusion chromatography (Sephadex LH-20,
CHCl3/MeOH 1:4). The appropriate fraction was collected and lyophi-
lized to give 30e (9.7 mg, 0.088 mmol, 63%). 1H NMR (400 MHz, D2O,
A
ACHTREUNG
A
,
,
AHCTREUNG
AHCTREUNG
A
G
ACHTREUNG
3
AHCTREUNG
AcN), 1.59 (br, 4H), 1.30 (br, 8H); HR ESI-MS: m/z: calcd for
C47H76N4O34P2S2Na: 1389.3155, found 1389.3111 [Mꢁ2H+Na]ꢁ.
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-deoxy-2-mercaptoacetamido-
b-d-glucopyranoside (32): Hydrazine acetate (248 mg, 1.61 mmol) was
added to a solution of the thioacetate 20 (744 mg, 1.39 mmol) in THF
(18 mL). After stirring for 3.5 h at room temperature, the mixture was di-
3
3
408C): d=5.44 (dd, 1H, JACTHREGNU(H,H)=2.9, JHCARTE(UGN P,H)=7.1 Hz; H-1GN1P), 4.89 (s,
1H, H-1aMan), 4.64–4.59 (m, 2H, H-1GN, H-1bMan, overlapped with HOD
signal), 4.11 (br, 1H, H-2aMan), 4.01–3.30 (m, 25H), 3.68 (s, 3H,
3460
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3449 – 3462

Glucosyltransferase Inhibitors
FULL PAPER
luted with 10% citric acid solution. The solution was extracted with
CHCl3 (2”), and the combined organic layers were washed with brine
(2”), dried over Na2SO4, concentrated, and dried under vacuum to pro-
duce thiol 32 (683 mg, 1.39 mmol, quant.) This compound was not puri-
fied to avoid dimerization; HR ESI-MS: m/z: calcd for
C20H35N2O9SSiNa: 516.1700, found 516.1684 [M+Na]+.
AcN), 1.59 (br, 4H), 1.30 (br, 8H); HR ESI-MS: m/z: calcd for
C39H68N2O26PS2: 1075.3239, found 1075.3211 [MꢁH]ꢁ.
Compound 1c: Compound 30c (2.7 mg, 2.5 mmol) and UMP-morpholi-
date (5.2 mg, 7.6 mmol) were co-evaporated with dry pyridine (3”) and
dried under reduced pressure for 1 h. Then, the mixture was dissolved in
dry pyridine (0.5 mL) and added 1H-tetrazole (1.2 mg, 0.019 mmol), and
stirred for 3 d under Ar atmosphere. The mixture was quenched with
H2O and concentrated. The residue was purified as described for 1a to
give 1c (2.2 mg, 0.0016 mmol, 63%). 1H NMR (400 MHz, D2O, 40 8C):
tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-(chloromethylthio)acetamido-
2-deoxy-b-d-glucopyranoside (33): iPr2NEt (45 mL, 0.26 mmol) was added
at 48C to a solution of the thiol 32 (42.5 mg, 0.0861 mmol) in CH3CN/
BrCH2Cl (1:1, 1 mL). After stirring for 5 h at 48C under Ar atmosphere,
the mixture was concentrated, and dried under vacuum for 1 h to afford
33, which was used for the next reaction without purification.
d=7.93 (d, 1H, 3J
H-1Rib), 5.52 (dd, 1H, 3J
1H, 3J
(H,H)=1.4 Hz; H-1aMan), 4.65–4.53 (m, 2H, H-1GN, H-1bMan, over-
lapped with HOD signal), 4.37–4.15 (m, 5H, H-2Rib, H-3Rib H-4Rib
H-5aRib, H-5bRib), 4.12 (dd, 1H, 3J
(H,H)=1.5, 3.4 Hz; H-2aMan), 4.01–3.40
(m, 27H), 3.68 (s, 3H, CH3COO), 3.08 (dd, 1H, J
13.7 Hz; H-6aaMan), 2.72 (dd, 1H, 3J(H,H)=8.8, 2J
H-6baMan), 2.38 (t, 2H, 3J
(H,H)=7.3 Hz; CH2COO), 2.05 (s, 3H, AcN),
A
,
A
ACHTREUNG
AHCTREUNG
,
,
Compound 34: nBu3P (35 mL, 0.14 mmol) was added to a solution of di-
sulfide 13 (38.6 mg, 0.0264 mmol) in degassed THF/H2O (9:1, 1 mL).
After stirring for 5 h at room temperature under Ar atmosphere, the mix-
ture was concentrated, and dissolved in H2O. The aqueous phase was
washed with Et2O (3”), concentrated, and dried under reduced pressure
to afford crude thiol. It was then dissolved in DMF (1 mL), to which
were added crude compound 33 (0.08610 mmol) and Cs2CO3 (26.5 mg,
0.0813 mmol). The reaction mixture was stirred for 24 h at 408C under
Ar atmosphere, Ac2O (1 mL) and pyridine (2 mL) were added, and the
mixture was stirred further for 20 h. The mixture was concentrated, and
purified with silica gel chromatography (chloroform/EtOAc 3:1 ! 1:3) to
give 34.
AHCTREUNG
3
2
A
ACHTREUNG(H,H)=
A
ACHTREUNG
AHCTREUNG
1.59 (br, 4H), 1.30 (br, 8H); HR ESI-MS: m/z: calcd for
C48H78N4O34P2S2Na: 1403.3312, found 1403.3271 [Mꢁ2H+Na]ꢁ.
Inhibitor assays: GnT-V activity was assayed using the pyridylaminated
acceptor substrate and purified recombinant soluble GnT-V.[32] GnT-IX
activity was assayed using the pyridylaminoethylsuccinamyl acceptor sub-
strate and partial purified recombinant soluble GnT-IX.[4] For kinetic
analyses, these enzyme sources were incubated at 378C for 2 h with
20 mm acceptor substrate (GnGn-bi-PA[33] or GnM-S-PAES[4]) and various
concentrations of UDP-GlcNAc and the inhibitor in 100 mm MES
(pH 6.25) or MOPS (pH 7.5) buffer containing 200 mm GlcNAc, 0.5%
Triton X-100, and 10 mm EDTA. The reaction was terminated by boiling
for 3 min and then centrifuged at 15000 rpm for 5 min. The resulting su-
pernatant was analyzed by HPLC.
Compound 35: A solution of crude 34 in DMF (2 mL) was added HF/
pyridine complex (100 mL), and stirred for 14 h at room temperature. The
mixture was neutralized with sat. NaHCO3 solution under cooling. The
aqueous phase was extracted with CHCl3 (3”), and the combined organ-
iclayers were washed with brine (2”), dried over Na 2SO4, filtered and
concentrated. Purification by preparative TLC (toluene/acetone 3:2)
gave 35 (23.8 mg, 0.016 mmol, 31% from 13).
Compound 36: A solution of hemiacetal 35 (23.1 mg, 0.0158 mmol) in di-
chloroethane (1 mL) were added amidite 17 (10 mL, 0.038 mmol) and
1H-tetrazole (4.0 mg, 0.057 mmol). After stirring for 7 h at 458C under
Ar atmosphere, the mixture was cooled to ꢁ208C, Me2S (200 mL) and
TBHP (100 mL, 5–6m in decane) were added, and stirred for 13 h at ꢁ20
to 08C, and then quenched with 5% Na2S2O3 solution. The aqueous
phase was extracted with CHCl3 (3”), and the combined organic layers
were washed with brine (2”), dried over Na2SO4, filtered and concentrat-
ed. Purification by silica gel chromatography (toluene/acetone 4:1 ! 3:2
with 1% Et3N) gave 36 (21.8 mg, 0.0135 mmol, 85%). 1H NMR
(400 MHz, CDCl3): d=6.90 (d, 1H, J=8.5 Hz; NH), 6.65 (d, 1H,
Acknowledgements
This work was supported by the Special Postdoctoral Researchers Pro-
gram at RIKEN and by a Grant-in-Aid for Scientific Research for Young
Scientists B (16710165, to S.H.) and Grant-in-Aid for Creative Scientific
Research (17GS0420, to Y.I.) from the Japan Society for the Promotion
of Science (JSPS). We thank Dr. Teiji Chihara and associates for the ele-
mental analyses and Ms. Tamiko Chijimatsu for the NMR measurements.
We also thank Ms. Akemi Takahashi for technical assistance.
3J
3J
A
ACHTRE(UGN H,H)=3.4,
[1] Handbook ofGlycosyltransferases and Related Genes (Eds.: N. Tani-
guchi, K. Honke, M. Fukuda), Springer, Tokyo, 2002.
[2] a) I. Brockhausen, J. P. Carver, H. Schachter, Biochem. Cell Biol.
1988, 66, 1134–1151; b) R. D. Cummings, I. S. Trowbridge, S. Korn-
feld, J. Biol. Chem. 1982, 257, 13421–13427.
[3] a) K.-i. Inamori, T. Endo, Y. Ide, S. Fujii, J. Gu, K. Honke, N. Tani-
guchi, J. Biol. Chem. 2003, 278, 43102–43109; b) M. Kaneko, G. Al-
varez-Manilla, M. Kamar, I. Lee, J. K. Lee, K. Troupe, W. Zang, M.
Osawa, M. Pierce, FEBS Lett. 2003, 554, 515–519.
[4] K.-i. Inamori, T. Endo, J. Gu, I. Matsuo, Y. Ito, S. Fujii, H. Iwasaki,
H. Narimatsu, E. Miyoshi, K. Honke, N. Taniguchi, J. Biol. Chem.
2004, 279, 2337–2340.
A
ACHTREUNG
15H), 4.63 (m, 4H), 4.38–3.21 (m, 18H), 2.92 (dd, 1H, 3J
A
2J
A
C
ACHTREUNG
14.4 Hz; H-6baMan), 2.30 (t, 2H, JAHCTREUNG
1.59 (m, 4H), 1.29 (m, 8H); HR ESI-MS: m/z: calcd for
C67H99N2O37PS2Na: 1641.5003, found 1641.4910 [M+H]+.
Compound 30c: A solution of allyl ester 36 (10.8 mg, 6.7 mmol) in de-
gassed toluene (0.5 mL) were added Et3SiH (22 mL, 0.14 mmol), AcOH
(8 mL, 0.14 mmol), and [PdACHTRE(UGN PPh3)4] (1.8 mg, 0.0016 mmol). The mixture
was stirred for 14 h at 408C under Ar atmosphere, and cooled to room
temperature. After evaporation, the mixture was applied to size exclusion
chromatography (Sephadex LH-20, MeOH). Appropriate fractions were
concentrated, and dried under reduced pressure. Then, the deallylated
product was dissolved in 0.05m NaOMe in MeOH (3 mL), and stirred for
10 h at room temperature under Ar atmosphere. The mixture was sepa-
rated by size exclusion chromatography (Sephadex G-15, H2O). Further
purification by SepPak C-18 cartridge (MeOH/H2O 0:100 ! 60:40) pro-
vided 30c (3.2 mg, 3.0 mmol, 45%). 1H NMR (400 MHz, D2O, 40 8C): d=
[5] J. W. Dennis, M. Granovsky, C. E. Warren, Biochim. Biophys. Acta
1999, 1473, 21–34.
[6] a) J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman, R. S.
Kerbel, Science 1987, 236, 582–585; b) S. Ihara, E. Miyoshi, J. Ko,
K. Murata, S. Nakahara, K. Honke, R. B. Dickson, C.-Y. Lin, N. Ta-
niguchi, J. Biol. Chem. 2002, 277, 16960–16967; c) M. Demetriou,
I. R. Nabi, M. Coppolino, S. Dedhar, J. W. Dennis, J. Cell Biol. 1995,
130, 383–392; d) H.-B. Guo, I. Lee, M. Kamar, S. K. Akiyama, M.
Pierce, Cancer Res. 2002, 62, 6837–6845; e) H.-B. Guo, I. Lee, B. T.
Bryan, M. Pierce, J. Biol. Chem. 2005, 280, 8332–8342; f) M. Deme-
triou, M. Granovsky, S. Quaggin, J. W. Dennis, Nature 2001, 409,
733–739; g) T. Saito, E. Miyoshi, K. Sasai, N. Nakano, H. Eguchi, K.
Honke, N. Taniguchi, J. Biol. Chem. 2002, 277, 17002–17008;
5.38 (dd, 1H, 3J
3J
(H,H)=1.5 Hz; H-1aMan), 4.65–4.56 (2H, H-1GN, H-1bMan, overlapped
with HOD signal), 4.12 (dd, 1H, 3J
(H,H)=1.5, 3.4 Hz; H-2aMan), 4.02–
3.42 (m, 25H), 3.68 (s, 3H, CH3COO), 3.08 (dd, 1H, 3J
(H,H)=2.4,
2J(H,H)=13.9 Hz; H-6aaMan), 2.74 (dd, 1H, 3J(H,H)=8.8, 2J
(H,H)=
(H,H)=7.3 Hz; CH2COO), 2.06 (s, 3H,
ACHTREUNG HCARTEU(GN P,H)=7.3 Hz; H-1GN1P), 4.89 (d, 1H,
(H,H)=3.2, 3J
ACHTREUNG
AHCTREUNG
AHCTREUNG
A
G
ACHTREUNG
3
14.1 Hz; H-6baMan), 2.38 (t, 2H, J
ACHTREUNG
Chem. Eur. J. 2006, 12, 3449 – 3462
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3461

Y. Ito et al.
h) E. A. Partridge, C. L. Roy, G. M. Di Guglielmo, J. Pawling, P.
Cheung, M. Granovsky, I. R. Nabi, J. L. Wrana, J. W. Dennis, Sci-
ence 2004, 306, 120–124.
[18] a) P. Konradsson, D. R. Mootoo, R. E. McDevitt, B. Fraser-Reid, J.
Chem. Soc. Chem. Commun. 1990, 270–272; b) G. H. Veeneman,
S. H. van Leeuwen, J. H. van Boom, Tetrahedron Lett. 1990, 31,
1331–1334.
[19] In this particular case, use of a catalytic amount of TfOH afforded
the corresponding orthoester exclusively.
[7] M. Granovsky, J. Fata, J. Pawling, W. J. Muller, R. Khokha, J. W.
Dennis, Nat. Med. 2000, 6, 306–312.
[8] a) T. Endo, Biochim. Biophys. Acta 1999, 1473, 237–246; b) T.
Willer, M. C. Valero, W. Tanner, J. Cruces, S. Strahl, Curr. Opin.
Struct. Biol. 2003, 13, 621–631.
[9] a) P.-P. Lu, O. Hindsgaul, H. Li, M. M. Palcic, Carbohydr. Res. 1997,
303, 283–291; b) I. Brockhausen, F. Reck, W. Kuhns, S. Khan, K. L.
Matta, E. Meinjohanns, H. Paulsen, R. N. Shah, M. A. Baker, H.
Schachter, Glycoconjugate J. 1995, 12, 371–379.
[10] Previous reports on bisubstrate-type glycosyltransferase inhibitors;
a) M. M. Palcic, L. D. Heerze, O. P. Srivastava, O. Hindsgaul, J. Biol.
Chem. 1989, 264, 17174–17181; b) H. Hashimoto, T. Endo, Y. Kaji-
hara, J. Org. Chem. 1997, 62, 1914–1915; c) B. Waldscheck, M.
Streiff, W. Notz, W. Kinzy, R. R. Schmidt, Angew. Chem. 2001, 113,
4120–4124; Angew. Chem. Int. Ed. 2001, 40, 4007–4011; d) H.
Hinou, X.-L. Sun, Y. Ito, J. Org. Chem. 2003, 68, 5602–5613;
e) D. V. Filippov, H. van den Elst, C. M. Tromp, G. A. van der
Marel, C. A. A. van Boeckel, H. S. Overkleeft, J. H. van Boom, Syn-
lett 2004, 773–778.
[20] C. A. A. van Boeckel, T. Beetz, Tetrahedron Lett. 1983, 24, 3775–
3778.
1
[21] Ratio GlcNAc a/b 1:20, determined by H NMR spectrum.
[22] T. Høeg-Jensen, M. H. Jakobsen, A. Holm, Tetrahedron Lett. 1991,
32, 6387–6390.
[23] C. L. R. Zaliz, O. Varela, J. Carbohydr. Chem. 2001, 20, 689–701.
[24] The 3,6-anhydro-type side product 13b was produced in this step
(~15%); 13b was removed in the next step with size-exclusion chro-
matography (Sephadex LH-20, CHCl3/MeOH 1:4).
[25] Preparation of bisallyloxydiisopropylaminophosphine (17): W. Bann-
warth, E. Kung, Tetrahedron Lett. 1989, 30, 4219–4222.
[26] M. M. Sim, H. Kondo, C.-H. Wong, J. Am. Chem. Soc. 1993, 115,
2260–2267.
[27] V. Wittmann, C.-H. Wong, J. Org. Chem. 1997, 62, 2144–2147.
[28] M. Bengtsson, J. Broddefalk, J. Dahmꢂn, K. Henriksson, J. Kihlberg,
H. Lçnn, B. R. Srinivasa, K. Stenvall, Glycoconjugate J. 1998, 15,
223–231.
[29] Under basic or neutral conditions, the dimerized product was
mainly obtained.
[30] W. M. Macindoe, A. H. van Oijen, G.-J. Boons, Chem. Commun.
1998, 847–848.
[31] a) S. Nakamura, Y. Ito, L. Wang, T. Toru, J. Org. Chem. 2004, 69,
1581–1589; b) X. Zhu, K. Pachamuthu, R. R. Schmidt, Org. Lett.
2004, 6, 1083–1085.
[32] K. Sasai, Y. Ikeda, T. Fujii, T. Tsuda, N. Taniguchi, Glycobiology
2002, 12, 119–127.
[11] I. Tvaroska, I. Andre, J. P. Carver, J. Am. Chem. Soc. 2000, 122,
8762–8776.
[12] S. H. Tahir, O. Hindsgaul, Can. J. Chem. 1986, 64, 1771–1780.
[13] a) H. Ando, S. Manabe, Y. Nakahara, Y. Ito, J. Am. Chem. Soc.
2001, 123, 3848–3849; b) H. Ando, S. Manabe, Y. Nakahara, Y. Ito,
Angew. Chem. 2001, 113, 4861–4864; Angew. Chem. Int. Ed. 2001,
40, 4725–4728; c) Y. Ito, S. Manabe, Chem. Eur. J. 2002, 8, 3076–
3084; d) S. Hanashima, S. Manabe, Y. Ito, Synlett 2003, 979–982.
[14] S. Hanashima, S. Manabe, K.-i. Inamori, N. Taniguchi, Y. Ito,
Angew. Chem. 2004, 116, 5792–5795; Angew. Chem. Int. Ed. 2004,
43, 5674–5677.
[15] a) Y. Hu, L. Chen, S. Ha, B. Gross. B, Falcone, D. Walker, M.
Mokhtarzadeh, S. Walker, Proc. Natl. Acad. Sci. USA 2003, 100,
845–849; b) C. A. Lepre, J. M. Moore, J. W. Peng, Chem. Rev. 2004,
104, 3641–3675.
[33] N. Taniguchi, A. Nishikawa, S. Fujii, J. Gu, Methods Enzymol. 1989,
179, 397–408.
[34] Km values of UDP-GlcNAc were 5.0 mm (to GnT-V) and 0.68 mm
(to GnT-IX) (K. Inamori et al. unpublished results).
[16] L. Jiang, R. C. Hartley, T.-H. Chan, Chem. Commun. 1996, 2193–
2194.
Received: October 31, 2005
[17] G. Hodosi, P. Kovµc, Carbohydr. Res. 1998, 308, 63–75.
Published online: March 14, 2006
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