Microwave-assisted construction of triazole-linked amino acid-glucoside conjugates as novel PTP1B inhibitors

Article abstract of DOI:10.1039/c0nj00835d

There has been increasing interest in the development of protein tyrosine phosphatase 1B (PTP1B) inhibitors for the treatment of type 2 diabetes, obesity and breast cancer. We report here the identification of a series of mono- and bis-phenylalaninyl and

Full text of DOI:10.1039/c0nj00835d

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PAPER
Microwave-assisted construction of triazole-linked amino acid–glucoside
conjugates as novel PTP1B inhibitorsw
Xiao-Peng He,abc Cui Li,c Xiao-Ping Jin,b Zhuo Song,b Hai-Lin Zhang,b
Cheng-Jiang Zhu,ab Qiang Shen,d Wei Zhang,d Li Sheng,d Xiao-Xin Shi,c
Yun Tang,c Jia Li,*d Guo-Rong Chen*b and Juan Xie*a
Received (in Montpellier, France) 27th October 2010, Accepted 4th December 2010
DOI: 10.1039/c0nj00835d
There has been increasing interest in the development of protein tyrosine phosphatase 1B
(PTP1B) inhibitors for the treatment of type 2 diabetes, obesity and breast cancer. We report here
the identification of a series of mono- and bis-phenylalaninyl and tyrosinyl glucoside derivatives
as novel PTP1B inhibitors. The designed compounds bearing one or two phenylalanine or
tyrosine derivatives on the 6-, 2,3-, 2,6-, 3,4- and 4,6-positions of the glucosyl scaffolds were
efficiently constructed via the microwave-assisted Cu(I)-catalyzed azide–alkyne cycloaddition in
moderate-to-excellent yields. Successive biological assays identified these compounds as novel
PTP1B inhibitors, with the 4,6-disubstituted tyrosinyl glucoside being the most potent. A kinetic
study established that both mono- and bis-triazole-linked glycosyl acids act as typical competitive
inhibitors whereas the bis-triazolyl ester that also exhibited inhibitory activity on PTP1B
displayed a mixed-type inhibition pattern. Furthermore, docking simulation plausibly proposed
the diverse binding modes of these compounds with the enzymatic target.
to the increase of insulin sensitivity and resistance to diet-induced
Introduction
obesity while treatment with PTP1B antisense oligonucleotides
The prevalence of diabetes, obesity and cancer is dramatically
resulted in the improvement of hyperglycaemia in diabetes
rodent models.3–5 In addition, more recent reports indicated
increasing, while the morbidity and mortality related with
secondary complications of such diseases have similarly risen.
that PTP1B might act as an oncogene in the context of breast
cancer.6 These data suggest that inhibition of PTP1B may
There is consequently an urgent need for effective approaches
toward the disease therapeutics. Belonging to the superfamily
represent a viable strategy for the treatment of type 2 diabetes,
of protein tyrosine phosphatases (PTPs) that are pivotal
obesity as well as cancer.
regulators of tyrosine phosphorylation-dependent cellular
As a consequence, extensive efforts have been devoted to the
events,1,2 protein tyrosine phosphatase 1B (PTP1B) has been
development of PTP1B inhibitors by both academia and the
pharmaceutical industry.7,8 The numerous bioactive compounds
well-characterized as a promising and versatile therapeutic
target. It is reported that PTP1B-knockout in mice could lead
disclosed over the past few years have mainly fallen into the
competitive nonhydrolyzable phosphotyrosine (pTyr) surrogates9
a PPSM, Institut d’Alembert, ENS de Cachan, CNRS UMR 8531,
61 Avenue du Pt Wilson, F-94235 Cachan, France.
E-mail: joanne.xie@ens-cachan.fr; Fax: +33 1-47402454;
Tel: +331-47405586
including the difluoromethylene phosphonate (DFMP),10
isoxazole acid,11 2-oxalylamino benzoic acid (OBA),12
hydroxylpropionic acid13 and thiophene diacid derivatives.14
Meanwhile, the identification of the noncompetitive inhibitors
that trigger the open conformation of WPD loop,15 such as the
recently revealed aryl diketoacid derivatives,16 has provided an
alternative way for selectively achieving PTP1B inhibition.
However, there are only limited PTP1B inhibitors entering
clinical trials due principally to the low oral bioavailability
of most pTyr mimetics/analogs. In addition, the highly
homologous active site of PTP1B (for instance, T-cell
protein tyrosine phosphatase (TCPTP) owns a 74% sequence
identity with PTP1B) and the relatively shallow nature
of the surrounding protein surface largely impeded the
productive yielding of its potent and selective inhibitors. In a
b Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, East China University of Science and Technology,
130 Meilong Road, Shanghai 200237, P. R. China.
E-mail: mrs_guorongchen@ecust.edu.cn; Fax: +86 21-64252758;
Tel: +86 21-64253016
c School of Pharmacy, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, P. R. China
d National Center for Drug Screening, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, P. R. China.
E-mail: jli@mail.shcnc.ac.cn; Fax: +86 21-50800721;
Tel: +86 21-50801552
w Electronic supplementary information (ESI) available: General
synthetic procedure and characterization of reported compounds.
See DOI: 10.1039/c0nj00835d
c
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binding sites adjacent to the active pocket (such as the YRD
motif),7,8 we decided to use the benzylated sugar alkynes as the
glycoscaffold.
Indeed, a multitude of sugar-based functionalized triazoles
have been reported lately.23,24 In the present study, we decided
to employ the microwave-assisted click chemistry25 toward the
construction of the designed glycosyl amino acid derivatives
comprising of one or two phenylalanine or tyrosine derivatives
on different positions of a benzylated glucosyl scaffold.
Successive biological assessments including the inhibitory
assay and the kinetic study were performed which characterized
the newly constructed glycosyl acids as micromole-ranged
competitive or non-specific PTP1B inhibitors. Furthermore,
docking simulation was conducted to plausibly illustrate the
binding manners of the identified enzymatic inhibitors.
Fig. 1 Reported and novel phosphotyrosine (pTyr) surrogates.
word, fresh and promising PTP1B inhibitor entities are of
great demand.
Results and discussion
Carbohydrate-based drug development has recently attracted
considerable interest and is gradually proving indispensable
for boosting the modern drug discovery due to their structural
diversity, high biocompatibility and natural abundance.17 For
example, the two drugs (namely zanamivir and oseltamivir)
approved to date to combat both influenza A and B were
derived from the sialic acid scaffold via a computational
study.18 Consequently, the construction of new glycomimetics
that are biologically active has become extremely desirable.
Two independent research groups (i.e. Dondoni’s19a and
Rutjes’s19b) first unveiled a novel series of glycoconjugates in
which the bioactive glycosides and amino acids were rigidly
coupled via the click chemistry.20 Numerous studies related to
this work have successively been reported.21 Enlightened by
this conjugation pattern, we sought to design glycosyl triazolyl
acids as PTP1B inhibitors.
Synthesis of bidentate glycosides
The azido derivatives a22a and b22b (Fig. 1) as well as the
mono-O-propynyl glucoside 126 (Scheme 1) were prepared
according to the previous literature reports and the benzylated
di-O-propargyl glucosides were readily synthesized via facile
protection–deprotection and O-propargylation strategies.
The click reaction was then realized between the sugar
alkynes and the azides. As shown in Scheme 1, glucosyl amino
acid derivatives bearing one triazolyl moiety have been firstly
prepared. Methyl 6-O-propargyl glucopyranoside 1 and azide
a or b were dissolved in a biphasic solvent of CH2Cl2/H2O
(1 : 1, v/v) followed by the addition of catalytic amount of Na
ascorbate (0.4 equiv.) and CuSO4 (0.2 equiv.). The mixture
was then transferred into a Yalian (YL8023B1) microwave
oven system at 55 1C for 14 min, yielding the desired triazoles 2
and 3 in excellent yields of 97.0% and 96.7%, respectively.
Saponification of 2 and 3 with LiOH gave the corresponding
acids 4 and 5.
As shown in Fig. 1, L-phenylalanine and L-tyrosine derivatives
(a and b, respectively)22 where the amine group was substituted
by an azide functionality were chosen as the azido donor for
the Cu(I)-catalyzed azide–alkyne cycloaddition. We envisioned
that the triazolyl acid bearing a benzyl or p-OH benzyl group
would perform as a new pTyr mimetic similar to the reported
heterocyclic carboxylates by Abbott’s11 and Novo Nordisk’s12
laboratories (Fig. 1). In addition, Zhang et al. have proposed a
dual binding strategy10 to form ‘bidentate’ PTP1B inhibitors
that simultaneously access the active site (A-site) and an
adjacent pocket such as the second phosphotyrosine site
(B-site) and/or the YRD motif (C-site), which has proven
efficacious to yield fairly potent and selective ligands. We were
then encouraged to create bidentate PTP1B ligands that
contain two triazolyl acid residues. Unlike the previously
utilized linear alkyl linkages for connecting two bioactive
molecules, a monosaccharide (methyl a-D-glucopyranoside,
Fig. 1) core was employed in the present study. Such linkage
owns naturally endowed primary (C6) or secondary hydroxyl
groups (C2, C3 and C4) with distinct reactivities and spatial
orientations, enabling flexible modifications to fabricate
structurally diverse products. Moreover, since it’s reported
that aryl groups covalently attached to the catalytic site-
directed pharmacophore are crucial for PTP inhibition possibly
by generating hydrophobic interactions with periphery
We then tended to prepare benzylated di-O-propynyl gluco-
sides. The 2,6-di-O-propynyl glucosides were synthesized by
introducing propargyl bromide onto the 2,6-positions of 3,4-
di-O-benzyl glucoside 627 in the presence of NaH. In a similar
way, we have realized the synthesis of 4,6-di-O-propynyl
glucoside 9 from methyl 2,3-di-O-benzyl-a-D-glucopyranoside
716 (Scheme 2). The preparation of 2,3-di-O-propynyl
glucoside was achieved by O-propargylation of the known
benzylidene derivative 1028 which was then converted into the
benzyl derivative 11 by removal of the benzylidene group with
TFA followed by O-benzylation (Scheme 2).
Scheme 1 Reagents and conditions: (i) CuSO4, Na ascorbate, CH2Cl2,
H2O, MW; (ii) LiOH, MeOH, H2O.
c
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Scheme 2 Reagents and conditions: (i) propargyl bromide, NaH, DMF; (ii) TFA, MeOH; (iii) BnBr, NaH, DMF; (iv) CuSO4, Na ascorbate,
CH2Cl2, H2O, MW; (v) LiOH, MeOH, H2O.
With the di-O-propargyl materials ready, we then studied
the click reaction for preparing bis-triazoles. However, our
initial attempt under the same condition used for the
formation of 2 and 3 (0.4 equiv. Na ascorbate/0.2 equiv.
CuSO4 under microwave irradiation during 14 min) failed to
give the dual triazoles. This could possibly attribute to the
spatial hindrance of the previously formed mono-triazolyl
intermediates which impede the further formation of the
difunctional final adducts.29 We then attempted to increase
the catalyst loading with a prolonged reaction time.
To our delight, as shown in Scheme 2, in the presence of
2 equiv. Na ascorbate and 1 equiv. CuSO4, the desired 2,6-,
4,6- and 2,3-disubstituted phenylalanine and tyrosine derivatives
were successfully obtained within 30 min under microwave
irradiation (55 1C) in good-to-excellent yields (12—88.1%;
13—89.8%; 17—95.8%; 18—97.5%; 21—89.6%; 22—77.7%).
The corresponding acids 14, 15, 19, 20, 23 and 24 were then
obtained after saponification. Finally, we prepared benzylated
3,4-di-O-propynyl glucoside 27 from the selectively silylated
2530 via O-propargylation, desilylation and O-benzylation.
Nevertheless, the click reactions between compound 27 and
azides a and b were incomplete under the optimized condition
(2 equiv. Na ascorbate, 1 equiv. CuSO4 in CH2Cl2/H2O within
30 min) due probably to the spatial hindrance caused by the
closely situated functional groups on C3 and C4 positions of
the glucoside. We then found that the desired 3,4-disubstituted
glucosides could be obtained in moderate yields (28—61.4%;
Scheme
3 Reagents and conditions: (i) propargyl bromide,
29—67.3%) under microwave irradiation (55 1C) for 2 h with
an excessive catalyst loading (6 equiv. Na ascorbate and
3 equiv. CuSO4) (Scheme 3), whereas further increase of the
NaH, DMF; (ii) AcCl, MeOH; (iii) BnBr, NaH, DMF;
(iv) CuSO4, Na ascorbate, CH2Cl2, H2O, MW; (v) LiOH, MeOH,
H2O.
c
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reaction time rendered the formation of unknown byproducts.
Saponification led to the free acids 30 and 31. Meanwhile, the
fully deprotected 33 for a SAR (structure–activity relationship)
study was also synthesized by treating silylated alkyne 26 with
azide b, followed by acidic desilylation and saponification
reactions, with a total yield of 72%.
a reverse inhibitory pattern was observed by comparing
the 2,3-disubstituted compound 24 (tyrosine derivative,
IC50 = 23.6 mM) and compound 23 (phenylalanine derivative,
IC50 = 13.1 mM). In addition, these triazolyl acids were not
inhibitors of LAR, SHP1 and SHP2 (IC50 > 112 mM) and
showed moderate selectivity over TCPTP, with the 2,3- and
4,6-phenylalanine derivatives being most selective (4.6-fold).
The inhibitory potencies of the glucosyl esters were also
determined (Table 1). Notably, non-negatively charged and
hydrophobic compounds have been recognized previously as
promiscuous PTP1B inhibitors9 whereas the identification of
aryl diketoacid derivative–PTP1B co-crystal well exemplified
that this specific compound series may also inhibit PTP1B with
unconventional binding manners, such as targeting the WPD
loop open.16 Obviously, all the di-esters exhibited micro-
mole-ranged PTP1B inhibitory activity, with the mono-esters
being less potent (2—IC50 > 112 mM; 3—IC50 = 51.0 mM).
The 2,6- and 4,6-disubstituted esters showed similar IC50
Inhibitory assay
The freshly prepared triazole-linked glycosyl acid derivatives
were then assayed toward PTP1B and a panel of homologous
PTPs including TCPTP, SHP1, SHP2 and LAR according to
our previously established methods.31 As listed in Table 1, in
general, the majority of the benzylated triazolyl glucosides
exhibited moderate-to-reasonable inhibitory activities on
PTP1B whereas the fully deprotected acid 33 as well as its
corresponding ester 32 was observed to be inactive on both
PTP1B and other PTPs, indicating that the lack of aryl groups
on sugar moiety led to inhibitory deficiency.
values (12—IC50
= 15.6 mM; 13—IC50 = 13.6 mM;
The mono-acids 4 and 5 possessed moderate PTP1B
inhibitory activity with IC50 values of 40.7 and 29.5 mM,
respectively, with the tyrosine-containing triazolyl glucoside
5 being more active. For the di-acids that have been expected
to perform as bidentate PTP1B inhibitors, the 2,6- (14, 15) and
3,4-disubstituted (30, 31) glucosides were first determined
unsatisfactory (IC50 > 50 mM).
In contrary, the 2,3- and 4,6-substitutions on the glucoside
scaffold were found preferential for achieving bidentate
PTP1B inhibitors as the synthesized 2,3- (19, 20) and 4,6-
(23, 24) disubstituted glucosides exhibited up to 4-fold
enhanced inhibitory activity compared to the monoacids 4
and 5 (20—IC50 = 10.8 mM). Interestingly, the 4,6-tyrosine
derivative 20, which is the most potent di-acid compound,
owns a slightly lowered IC50 value (10.8 mM) compared to its
4,6-phenylalanine counterpart (19—IC50 = 16.9 mM) whereas
17—IC50 = 18.6 mM; 18—IC50 = 16.9 mM) with no
significant difference between the tyrosine and phenylalanine
derivatives. In contrast, the inhibitory potencies of 2,3- and
3,4-disubstituted esters were slightly better (21—IC50 = 14.1 mM;
22—IC50 = 9.9 mM; 28—IC50 = 12.9 mM; 29—IC50 = 9.6 mM)
with the tyrosine derivatives 22 and 29 being more active.
Furthermore, esters 22 and 29 displayed better selectivity over
other PTPs (22: 7.4-fold over TCPTP; inactive on SHP-1,
SHP-2 and LAR; 29: 6.2-fold over TCPTP; inactive on SHP-1,
SHP-2 and LAR).
Docking simulation
We then tended to propose the plausible binding manners of
this triazolyl glycosyl acid series with PTP1B. Compounds 5,
20 and 29 were first selected for the kinetic study. The effect of
Table 1 Inhibitory activity of synthesized glycosyl acids on PTPs
IC50a/mM
PTP1B
TCPTP
LAR
SHP-1
SHP-2
Compound
OBn-acids
4
5
40.7 Æ 2.5
>112
98.2 Æ 9.2
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
>112
29.5 Æ 4.5 (Ki = 16.9 mM)
14
15
19
20
23
24
30
31
2
>112
79.6 Æ 3.3
>112
>112
16.9 Æ 3.1
78.0 Æ 10.0
31.7 Æ 9.6
60.2 Æ 2.5
74.2 Æ 5.5
>112
10.8 Æ 0.8 (Ki = 14.4 mM)
13.1 Æ 1.2
23.6 Æ 0.8
>112
51.0 Æ 5.3
>112
>112
OBn-esters
>112
51.0 Æ 27.6
15.6 Æ 3.4
3
109.4 Æ 13.8
62.3 Æ 0.3
46.6 Æ 3.9
49.5 Æ 5.7
50.6 Æ 0.5
64.1 Æ 5.9
73.1 Æ 5.1
47.1 Æ 8.7
59.6 Æ 1.6
>112
12
13
17
18
21
22
28
29
32
33
13.6 Æ 0.8
18.6 Æ 0.5
16.9 Æ 1.0
14.1 Æ 0.5
9.9 Æ 1.3
12.9 Æ 1.8
9.6 Æ 1.8 (Ki = 8.7 mM)
OH-ester
OH-acid
>112
>112
>112
a
Values are means of three experiments.
c
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Two additional hydrogen bonds were made between Arg24
and the oxygen atom on C3 position of the glucosyl scaffold
for co-immobilizing this molecule. Similarly, the proposed
binding mode of 20 (Fig. 3B) revealed hydrogen-bonding-
dominated interactions between 20 and the enzyme. In addition
to such interaction of the C6-tyrosine residue with the active
site of PTP1B, the other triazole-linked C4-tyrosine residue
concomitantly targeted the second phosphotyrosine site,
generating hydrogen bonds with Gly259, Arg24 and Asp29.
This may plausibly suggest that the designed 4,6-disubstituted
glycosyl acid derivatives are bidentate PTP1B inhibitors.
Furthermore, the hydroxyl group of the phenol precursor
appended on C4 position made one hydrogen bond with
Asp48 in the YRD motif.
More interestingly, the plausible binding mode of the
mixed-type inhibitor 29 is quite different from that of the
competitive inhibitors 5 and 20. The hydroxyl group of
the phenol moiety and the oxygen atom of carboxylic ester
on the C4-tyrosine residue generated several hydrogen bonds
with Arg221 and Gln262 of the active site and Arg47 of the
YRD motif, respectively (Fig. 3C). Hydrogen-bonding
interactions were also made by the C3-tyrosine residue with
Asp48 and Arg24. In addition, the triazole ring that links the
C3-tyrosine residue simultaneously contributed to the binding
affinity by generating one hydrogen bond with Arg24. Non-
polar interactions were made between the distal benzene group
on C2 position and Gln21 while the existence of the C6-distal
benzene moiety possibly induced the open conformation of the
WPD loop. Similar to the previously reported aryl diketoacid
derivatives,6 the key distal benzene group on C6 position of
the glucosyl scaffold is nearly parallel to the benzyl residue of
Phe182 which spatially impedes the WPD loop closure.
Indeed, as shown in Fig. 3D, the WPD loop of PTP1B
usually adopts a closed conformation when binding to its
substrate32 with a shallow and narrow active site cleft. The
design of its competitive inhibitors is consequently somewhat
challenging due to this unique structural feature. In contrary,
the binding surface would be greatly broadened with an
inhibitor that may prevent the WPD loop closure (Fig. 3E),
thus providing substantially flexible space for the design of
unconventional PTP1B inhibitors. Thus, this unique approach
may represent a promising strategy toward the development of
potent PTP1B ligands as suggested by Zhang et al.16
Fig. 2 Inhibition of PTP1B catalyzed pNPP hydrolysis by compounds 5
(A), 20 (B) and 29 (C). Experiments were performed at 30 1C and
pH 6.5.
the inhibitors on PTP1B-catalyzed reaction was studied at
5 different concentrations. The Eadie–Hofstee plots are given
for each compound in Fig. 2 (A for 5, B for 20 and C for 29).31
Clearly, plots A and B indicated that the acids 5 and 20 are
typical competitive PTP1B inhibitors as the Vmax values
remained approximately constant and the Km values gradually
increased with the increasing concentration of inhibitors.
Meanwhile, plot C (for ester 29) is consistent with a mixed-
type inhibition pattern as the Vmax values decreased regularly
and the Km values increased with the gradual decrease of
compound concentration. The Ki values of each compound
were then calculated to be 16.9 (5), 14.4 (20) and 8.7 (29) mM,
respectively (Table 1).31
Docking simulation was sequentially conducted to propose
plausible binding modes of the three selected inhibitors (5, 20
and 29) with a crystal structure in complex with a reference
inhibitor (PDB code: 3EB1). Water was removed from the
original structure and the rest of the protein prepared using
the Protein preparation wizard (Schrodinger, LLC, New
York, NY, 2005). Then the compounds were docked to the
active site of the protein using the Induced Fit Docking
workflow (Schrodinger, LLC, New York, NY, 2005). The
center atom was set to be a virtual center of referenced key
residues: Phe182, Cys215 and Gly259.
Conclusions
To summarize, with the assist of microwave irradiation, we
have successfully established a facile method for the efficient
and rapid synthesis of difunctional glycosyl triazoyl acids via
the click reaction. The synthesized glycosyl mono- and
di-phenylalanine and tyrosine derivatives were identified as
novel PTP1B inhibitors with at least several fold selectivity
over other homologous PTPs. We have also demonstrated the
glucosyl scaffold to be suitable as a chiral linkage toward the
construction of structurally versatile PTP1B inhibitors. A
docking study then suggested that 4,6-substituted glucosyl
As illustrated in Fig. 3A, a densely functionalized hydrogen-
bonding network was generated between the carboxylic group
of monomer 5 and amino acid residues (Ser216, Ala217,
Lys116, Asp181 and Lys 120) of the PTP1B catalytic site while
the appended phenol group provided additional hydrophobic
interaction with Tyr46.
acid (20) possibly performed as
a bidentate inhibitor
which simultaneously targets the active site and the second
phosphotyrosine site of PTP1B. Furthermore, the glucosyl
c
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Fig. 3 The docking study. Carbon atoms are in gray for PTP1B and pink for ligands, nitrogen atoms are in blue and oxygen atoms are in red.
(A) PTP1B in complex with compound 5; (B) PTP1B in complex with compound 20; (C) PTP1B in complex with compound 29; (D) WPD close
conformation of PTP1B; (E) WPD open conformation of PTP1B.
3,4-tyrosine ester (29) which is a mixed-type inhibitor might
presumably inhibit the enzyme in an unconventional manner,
targeting the WPD loop open. Our work should thus provide
new insights into the development of new triazolyl glycosyl
amino acid-based PTP1B inhibitors via the microwave-assisted
click reaction.
Na ascorbate (0.4–6 equiv.) and CuSO4Á5H2O (0.2–3 equiv.)
were added. Then the mixture was transferred to the micro-
wave oven (a Yalian (YL8023B1) system) for a ramp time of
2 min and a hold time of 12 to 118 min at 55 1C. After
completion of the reaction, the resulting mixture was diluted
with CH2Cl2, washed with water, dried over MgSO4, filtered
and evaporated to give a crude residue which was purified by
column chromatography.
Experimental section
3-Phenyl-2(S)-[4-(methyl 2,3,4-tri-O-benzyl-a-D-glucopyranosid-
6-yloxy)methyl-1H-1,2,3-triazole-1-yl]propanoate methyl ester
(2). From compound 1 (78.4 mg, 0.16 mmol), a (22.4 mg,
0.11 mmol), Na ascorbate (8.5 mg, 0.06 mmol) and
CuSO4Á5H2O (6.1 mg, 0.03 mmol), 14 min, column chromato-
graphy (petroleum ether/EtOAc = 1 : 1) afforded 2 as a
yellow syrup (74.4 mg, 97.0%). Rf = 0.58 (petroleum ether/
General
Solvents were purified by standard procedures. 1H and 13C
NMR spectra were recorded on Bruker AC-300, AM-500
or JEOL DX-400 spectrometers in CDCl3, CD3OD, or
DMSO-d6 solutions. Microwave-assisted reactions were
performed in a Yalian (YL8023B1) system. Optical rotations
were measured using a Perkin-Elmer 241 polarimeter at room
temperature and a 10 cm 1 mL cell. Analytical thin-layer
chromatography was performed on E. Merck aluminium perco-
lated plates of Silica Gel 60F-254 with detection by UV and by
spraying with 6N H2SO4 and heating at 300 1C. High resolution
mass spectra (HRMS) were recorded on a Waters LCT Premier
XE spectrometer using standard conditions (ESI, 70 eV).
1
EtOAc = 1 : 1). [a]D = +11.5 (c = 1.0/CH2Cl2). H NMR
(400 MHz, CDCl3) d 7.56 (s, 1H, triazole-H), 7.34–7.19 (m,
18H, PhH), 7.00–6.99 (m, 2H, PhH), 5.50 (t, 1H, J = 7.6 Hz,
CHCO2), 4.98 (d, 1H, J = 10.8 Hz, PhCH), 4.84–4.78 (m,
3H), 4.68–4.57 (m, 4H), 4.51 (d, 1H, J = 10.8 Hz, PhCH), 3.98
(t, 1H, J = 9.2 Hz), 3.77–3.65 (m, 3H), 3.70 (s, 3H, CO2CH3),
3.61–3.52 (m, 2H), 3.46 (dd, 1H, J = 6.0, 14.0 Hz, PhCH),
3.36–3.32 (m, 1H), 3.36 (s, 3H, OCH3). 13C NMR (100 MHz,
CDCl3) d 168.6, 144.9, 138.8, 138.3, 138.2, 134.8, 128.9, 128.8,
128.5, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6,
122.7, 98.2, 82.1, 79.8, 77.6, 75.8, 75.1, 73.4, 70.0, 68.9, 64.9,
General procedure for the Huisgen [3+2] cycloaddition. To a
biphasic solution of alkynyl sugar (1.0 equiv.) and azido ester
a or b (1.0 or 2.2 equiv.) in CH2Cl2 (5 mL) and H2O (5 mL),
c
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64.0, 55.3, 53.1, 38.8. HR-ESI-MS m/z: calcd for C41H45N3O8 +
5.57 (dd, 1H, J = 5.2, 9.2 Hz, CHCO2), 5.46 (dd, 1H, J = 6.0 Hz,
8.4 Hz, CHCO2), 5.06 (d, 1H, J = 11.6 Hz, PhCH), 4.87 (d,
1H, J = 11.6 Hz, PhCH), 4.74 (d, 1H, J = 12.0 Hz, PhCH),
4.69 (d, 1H, J = 12.0 Hz, PhCH), 4.64–4.62 (m, 2H), 4.55 (d,
1H, J = 3.2 Hz, H-1), 4.48 (d, 1H, J = 12.0 Hz, PhCH), 4.36
(d, 1H, J = 11.2 Hz, PhCH), 3.91 (t, 1H, J = 9.2 Hz), 3.76 (s,
3H, CO2CH3), 3.74–3.65 (m, 3H), 3.70 (s, 3H, CO2CH3), 3.50
(t, 1H, J = 9.2 Hz), 3.46 (dd, 1H, J = 3.2, 9.6 Hz, PhCH),
3.38 (dd, 1H, J = 4.2, 14.4 Hz, PhCH), 3.32 (s, 3H, OCH3),
3.29–3.25 (m, 2H), 3.20 (dd, 1H, J = 8.8, 14.0 Hz, PhCH).
13C NMR (100 MHz, CDCl3) d 168.8, 168.6, 156.4, 156.0,
144.9, 144.8, 144.8, 138.4, 138.1, 123.0, 129.8, 128.4, 128.3,
128.2, 127.9, 127.7, 127.6, 125.4, 125.0, 123.1, 122.9, 115.9,
115.8, 98.1, 81.8, 79.8, 77.2, 74.7, 73.2, 69.8, 68.9, 65.8, 64.6,
64.2, 63.9, 55.2, 53.1, 53.0, 37.9. HR-ESI-MS m/z: calcd for
C47H52N6O12 + Na 915.3541, found 915.3540.
Na 730.3104, found 730.3128.
3-p-Hydroxyphenyl-2(S)-[4-(methyl 2,3,4-tri-O-benzyl-a-D-
glucopyranosid-6-yloxy)-methyl-1H-1,2,3-triazole-1-yl]propanoate
methyl ester (3). From compound 1 (73.8 mg, 0.15 mmol), b
(21.3 mg, 0.10 mmol), Na ascorbate (9.0 mg, 0.06 mmol) and
CuSO4Á5H2O (6.3 mg, 0.03 mmol), 14 min, column chromato-
graphy (petroleum ether/EtOAc = 3 : 2) afforded 3 as a
yellow-brown syrup (67.4 mg, 96.7%). Rf = 0.63 (petroleum
ether/EtOAc = 1 : 2). [a]D = +22.1 (c = 0.3/CH2Cl2).
1H NMR (400 MHz, CDCl3) d 7.40–7.24 (m, 16H, PhH,
triazole-H), 6.76 (d, 2H, J = 7.6 Hz, (p-OH)Ph-H), 6.64 (d,
2H, J = 7.6 Hz, (p-OH)Ph-H), 5.57 (t, 1H, J = 6.0 Hz,
CHCO2), 4.99 (d, 1H, J = 10.8 Hz, PhCH), 4.85 (d, 1H,
J = 9.6 Hz, PhCH), 4.84–4.80 (m, 2H), 4.68–4.65 (m, 2H),
4.57–4.49 (m, 3H), 4.01 (t, 1H, J = 8.8 Hz), 3.76 (s, 3H,
CO2CH3), 3.74–3.67 (m, 4H), 3.64–3.54 (m, 2H), 3.41–3.35 (m,
1H), 3.35 (s, 3H, OCH3), 3.29 (dd, 1H, J = 5.6, 14.4 Hz,
PhCH). 13C NMR (100 MHz, CDCl3) d 168.7, 156.1, 144.8,
138.5, 138.2, 138.1, 130.1, 128.5, 128.4, 128.3, 128.0, 127.9,
127.9, 125.5, 123.0, 115.9, 98.1, 82.2, 79.8, 77.5, 76.1, 75.0,
73.4, 70.0, 69.0, 64.9, 64.3, 55.3, 53.1, 38.1. HR-ESI-MS m/z:
calcd for C41H45N3O9 + Na 746.3048, found 746.3049.
Methyl 3,4-di-O-benzyl-2,6-di-O-{1-[(1(S)-methoxycarbonyl-
2-phenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-glucopyrano-
side (17). From compound 9 (42 mg, 0.09 mmol) and a
(46.3 mg, 0.23 mmol), Na ascorbate (35.8 mg, 0.18 mmol) and
CuSO4Á5H2O (22.6 mg, 0.09 mmol), 30 min, column chromato-
graphy (petroleum ether/EtOAc, 2 : 1 to 1 : 1) afforded 17 as a
yellow syrup (76.9 mg, 95.8%). Rf
= 0.74 (EtOAc).
[a]D = +46.9 (c
=
0.7/CH2Cl2). 1H NMR (CDCl3,
Methyl 3,4-di-O-benzyl-2,6-di-O-{1-[(1(S)-methoxycarbonyl-
2-phenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-glucopyrano-
side (12). From compound 8 (38.9 mg, 0.09 mmol), a
(45.0 mg, 0.20 mmol), Na ascorbate (32.6 mg, 0.18 mmol)
and CuSO4Á5H2O (24.0 mg, 0.09 mmol), 30 min, column
chromatography (petroleum ether/EtOAc, 2 : 3) afforded 12
as a white solid (65.5 mg, 88.1%). Rf = 0.50 (petroleum
ether/EtOAc, 2 : 3). [a]D = +2.0 (c = 0.1/CH2Cl2). 1H
NMR (400 MHz, CDCl3) d 7.57 (s, 1H, triazole-H), 7.51 (s,
1H, triazole-H), 7.38–7.19 (m, 16H, PhH), 7.01–7.00 (m, 4H,
PhH), 5.51 (t, 1H, J = 7.6 Hz, CHCO2), 5.45 (t, 1H,
J = 7.6 Hz, CHCO2), 5.08 (d, 1H, J = 12.0 Hz, PhCH),
4.94 (d, 1H, J = 12.0 Hz, PhCH), 4.82 (d, 1H, J = 11.2 Hz,
PhCH), 4.77 (d, 1H, J = 12.0 Hz, PhCH), 4.67 (t, 2H,
J = 12.0 Hz), 4.62–4.58 (m, 2H), 4.50 (d, 1H, J = 10.8 Hz),
3.93 (t, 1H, J = 9.2 Hz), 3.76–3.65 (m, 3H), 3.71 (s, 3H,
CO2CH3), 3.69 (s, 3H, CO2CH3), 3.57–3.39 (m, 5H), 3.37 (s,
3H, OCH3), 3.32 (dd, 1H, J = 8.0, 13.6 Hz, PhCH). 13C NMR
(100 MHz, CDCl3) d 168.6, 168.5, 145.6, 144.9, 138.3, 134.8,
134.7, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 122.6, 122.4, 98.1, 82.0, 79.6,
74.9, 73.3, 69.9, 68.8, 66.7, 64.9, 64.0, 63.9, 55.2, 53.5, 53.0,
53.0, 38.8, 38.6. HR-ESI-MS m/z: calcd for C47H52N6O10 + H
861.3823, found 861.3815.
400 MHz) d 7.72 (s, 1H, triazole-H), 7.55 (s, 1H, triazole-H),
7.39–7.17 (m, 16H, PhH), 7.06–7.03 (m, 4H, PhH), 5.56–5.49
(m, 2H, 2ÂCHCO2), 4.97 (d, 1H, J = 10.8 Hz, PhCH), 4.89
(d, 1H, J = 11.6 Hz, PhCH), 4.83–4.77 (m, 2H), 4.69–4.60
(m, 4H), 4.56 (d, 1H, J = 12.8 Hz, PhCH), 3.94 (t, 1H,
J = 9.2 Hz), 3.72–3.61 (m, 3H), 3.71 (s, 3H, CO2CH3), 3.70 (s,
3H, CO2CH3), 3.56–3.41 (m, 6H), 3.37 (s, 3H, OCH3).
13C NMR (100 MHz, CDCl3) d 168.7, 144.9, 144.8, 138.8,
138.1, 134.9, 134.8, 129.0, 128.9, 128.8, 128.5, 128.4, 128.2,
128.0, 127.9, 127.6, 127.5, 122.9, 122.9, 98.2, 81.9, 79.7, 77.6,
77.3, 75.6, 73.4, 69.8, 68.8, 66.0, 64.8, 64.0, 55.2, 53.0, 53.0,
36.6, 36.5. HR-ESI-MS m/z: calcd for C47H52N6O10 + H
861.3823, found 861.3826.
Methyl 2,3-di-O-benzyl-4,6-di-O-{1-[(1(S)-methoxycarbonyl-
2-p-hydroxyphenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-
glucopyranoside (18). From compound 9 (45.5 mg, 0.10 mmol),
b (54.1 mg, 0.24 mmol), Na ascorbate (38.8 mg, 0.20 mmol)
and CuSO4Á5H2O (24.4 mg, 0.10 mmol), column chromato-
graphy (petroleum ether/EtOAc, 2 : 1 to 1 : 1) afforded 18
as a yellow syrup (87.9 mg, 97.5%). Rf = 0.70 (EtOAc).
[a]D
=
À33.2 (c
=
0.7/CH2Cl2). 1H NMR (CDCl3,
400 MHz) d 7.53 (s, 1H, triazole-H), 7.47 (s, 1H, triazole-H),
7.36–7.23 (m, 10H, PhH), 6.74 (d, 2H, J = 8.0 Hz, (p-OH)Ph-H),
6.73 (d, 2H, J = 8.0 Hz, (p-OH)Ph-H), 6.65 (d, 2H,
J = 8.4 Hz, (p-OH)Ph-H), 6.62 (d, 2H, J = 8.4 Hz,
(p-OH)Ph-H), 5.55 (t, 1H, J = 7.2 Hz, CHCO2), 5.48 (dd,
1H, J = 6.0, 8.8 Hz, CHCO2), 4.95 (d, 1H, J = 10.8 Hz,
PhCH), 4.89 (d, 1H, J = 12.0 Hz, PhCH), 4.79 (d, 1H, J =
10.8 Hz, PhCH), 4.76 (d, 1H, J = 12.0 Hz, PhCH), 4.64 (d,
1H, J = 12.0 Hz, PhCH), 4.59–4.56 (m, 3H), 4.46 (d, 1H, J =
12.4 Hz, PhCH), 3.92 (t, 1H, J = 9.2 Hz), 3.75 (s, 3H,
CO2CH3), 3.74 (s, 3H, CO2CH3), 3.66–3.64 (m, 1H),
3.53–3.50 (m, 3H), 3.48 (t, 1H, J = 6.4 Hz), 3.40–3.35
Methyl 3,4-di-O-benzyl-2,6-di-O-{1-[(1(S)-methoxycarbonyl-
2-p-hydroxyphenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-
glucopyranoside (13). From compound 8 (39.2 mg, 0.09 mmol),
b (47.3 mg, 0.20 mmol), Na ascorbate (37.4 mg, 0.18 mmol)
and CuSO4Á5H2O (22.7 mg, 0.09 mmol), column chromato-
graphy (petroleum ether/EtOAc, 1 : 4) afforded 13 as a white
solid (69.8 mg, 89.8%). Rf = 0.62 (petroleum ether/EtOAc,
1
1 : 6). [a]D = À3.3 (c = 0.1/CH2Cl2). H NMR (400 MHz,
CDCl3) d 7.57 (s, 1H, triazole-H), 7.42 (s, 1H, triazole-H),
7.35–7.21 (m, 10H, PhH), 6.73–6.59 (m, 8H, (p-OH)Ph-H),
c
628 New J. Chem., 2011, 35, 622–631
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(m, 3H), 3.33 (s, 3H, OCH3), 3.31–3.27 (m, 1H). 13C NMR
(100 MHz, CDCl3) d 168.6, 168.5, 156.2, 156.1, 144.8, 144.5,
138.4, 138.1, 130.0, 129.9, 128.5, 128.2, 128.2, 127.9, 127.8,
125.3, 125.3, 123.6, 123.3, 115.9, 115.8, 98.0, 81.8, 79.8, 77.6,
77.3, 75.8, 73.4, 70.0, 69.0, 65.7, 64.6, 64.1, 55.3, 53.5, 53.2,
38.0, 37.9. HR-ESI-MS m/z: calcd for C47H52N6O10 + H
893.3721, found 893.3721.
(53.6 mg, 0.26 mmol), Na ascorbate (118.9 mg, 0.6 mmol)
and CuSO4Á5H2O (75 mg, 0.3 mmol), 120 min, column
chromatography (petroleum ether/EtOAc, 3 : 2) afforded 28
as a brown syrup (55.3 mg, 61.4%). Rf = 0.45 (petroleum
ether/EtOAc, 1 : 1). [a]D = +68.0 (c = 0.2/CH2Cl2).
1H NMR (400 MHz, CDCl3) d 7.56 (s, 1H, triazole-H),
7.40–7.13 (m, 17H, triazole-H, PhH), 7.04–7.00 (m, 4H,
PhH), 5.51–5.45 (m, 2H, CHCO2), 4.94 (d, 1H, J = 11.6 Hz,
PhCH), 4.90 (d, 1H, J = 11.2 Hz, PhCH), 4.84–4.79 (m, 3H),
4.75 (d, 1H, J = 3.6 Hz, H-1), 4.68 (d, 1H, J = 11.6 Hz,
PhCH), 4.64 (d, 1H, J = 12.0 Hz, PhCH), 4.54 (d, 1H, J =
12.0 Hz, PhCH), 3.93 (t, 1H, J = 9.2 Hz), 3.78–3.62 (m, 4H),
3.74 (s, 3H, CO2CH3), 3.71 (s, 3H, CO2CH3), 3.54 (dd, 1H,
J = 3.2, 9.6 Hz, PhCH), 3.46 (dd, 1H, J = 6.8, 14.4 Hz,
PhCH), 3.41–3.35 (m, 2H), 3.39 (s, 3H, OCH3), 3.21 (dd, 1H,
J = 8.8, 14.4 Hz, PhCH). 13C NMR (100 MHz, CDCl3) d
168.5, 145.0, 138.8, 138.1, 134.8, 134.7, 128.9, 128.8, 128.8,
128.5, 128.4, 129.0, 127.8, 127.6, 127.5, 122.7, 122.5, 97.6, 81.7,
79.4, 77.9, 75.6, 73.5, 70.1, 68.6, 66.1, 64.4, 64.0, 63.9,
55.1, 53.0, 52.9, 38.7, 38.5. HR-ESI-MS m/z: calcd for
C47H52N6O10 + Na 883.3643, found 883.3637.
Methyl 4,6-di-O-benzyl-2,3-di-O-{1-[(1(S)-methoxycarbonyl-
2-phenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-glucopyrano-
side (21). From compound 11 (41.5 mg, 0.10 mmol), a
(45.7 mg, 0.22 mmol), Na ascorbate (35.4 mg, 0.18 mmol) and
CuSO4Á5H2O (22.3 mg, 0.09 mmol), 30 min, column chromato-
graphy (petroleum ether/EtOAc, 2 : 1 to 1 : 1) afforded 21 as a
yellow syrup (68.8 mg, 89.6%). Rf = 0.77 (EtOAc). [a]D
=
+19.5 (c = 1.0/CH2Cl2). 1H NMR (CDCl3, 400 MHz) d 8.00 (s,
1H, triazole-H), 7.62 (s, 1H, triazole-H), 7.34–7.15 (m, 16H,
PhH), 7.07–7.00 (m, 4H, PhH), 5.57 (dd, 1H, J = 6.0, 9.2 Hz,
CHCO2), 5.51 (dd, 1H, J = 6.8, 8.4 Hz, CHCO2), 5.04 (d, 1H,
J = 11.6 Hz, PhCH), 4.88 (d, 1H, J = 12.0 Hz, PhCH),
4.83–4.76 (m, 4H), 4.63 (d, 1H, J = 12.0 Hz, PhCH), 4.51 (d,
2H, J = 12.4 Hz, PhCH), 3.92 (t, 1H, J = 9.2 Hz), 3.76–3.66 (m,
3H), 3.73 (s, 3H, CO2CH3), 3.69 (s, 3H, CO2CH3), 3.61 (t, 1H,
J = 9.2 Hz), 3.55–3.50 (m, 2H), 3.48–3.42 (m, 2H), 3.40 (s, 3H,
OCH3), 3.38–3.35 (m, 1H). 13C NMR (100 MHz, CDCl3) d
168.7, 145.3, 144.9, 138.3, 138.0, 134.9, 134.8, 128.9, 128.9, 128.7,
128.4, 128.3, 127.9, 127.9, 127.7, 127.5, 126.2, 123.4, 122.8, 97.5,
81.8, 79.3, 77.5, 74.9, 73.5, 70.1, 68.5, 66.6, 64.3, 64.2, 63.9, 55.2,
Methyl 2,6-di-O-benzyl-3,4-di-O-{1-[(1(S)-methoxycarbonyl-
2-p-hydroxyphenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-
glucopyranoside (29). From compound 27 (54.7 mg, 0.12 mmol),
b (67.2 mg, 0.30 mmol), Na ascorbate (142.6 mg, 0.72 mmol) and
CuSO4Á5H2O (90 mg, 0.36 mmol), 120 min, column chromato-
graphy (petroleum ether/EtOAc, 1 : 3) afforded 29 as a white
solid (72.9 mg, 67.3%). Rf = 0.45 (petroleum ether/EtOAc,
53.1, 53.0, 38.6, 38.5. HR-ESI-MS m/z: calcd for C47H52N6O10
+
1
H 861.3823, found 861.3828.
1 : 4). [a]D = À22.7 (c = 0.1/CH2Cl2). H NMR (400 MHz,
CDCl3) d 7.42–7.27 (m, 11H, triazole-H, PhH), 7.12 (s, 1H,
triazole-H), 6.72 (d, 4H, J = 8.4 Hz, (p-OH)Ph-H), 6.68 (d, 2H,
J = 8.4 Hz, (p-OH)Ph-H), 6.62 (d, 2H, J = 8.4 Hz, (p-OH)Ph-H),
5.36 (dd, 1H, J = 4.8, 10.4 Hz, CHCO2), 5.27 (dd, 1H, J = 4.8,
10.0 Hz, CHCO2), 4.80 (d, 1H, J = 11.6 Hz, PhCH), 4.79–4.70
(m, 5H), 4.64 (d, 1H, J = 12.0 Hz, PhCH), 4.55 (d, 1H,
J = 11.6 Hz, PhCH), 4.05–4.04 (m, 1H), 3.83 (t, 1H, J =
9.2 Hz), 3.79 (s, 3H, CO2CH3), 3.77 (s, 3H, CO2CH3), 3.71–3.65
(m, 2H), 3.55 (dd, 1H, J = 5.2 Hz, 10.8 Hz, PhCH), 3.43 (dd,
1H, J = 4.8, 14.4 Hz, PhCH), 3.41–3.33 (m, 2H), 3.29 (s, 3H,
OCH3), 3.24–3.18 (m, 3H). 13C NMR (100 MHz, CDCl3) d
168.5, 168.5, 156.2, 156.1, 144.6, 144.3, 138.7, 137.6, 129.9, 129.9,
128.4, 128.2, 127.9, 127.8, 127.6, 125.5, 125.4, 123.6, 123.4, 116.0,
115.9, 97.7, 81.7, 78.7, 77.4, 75.4, 73.5, 69.8, 68.7, 65.0, 64.4, 64.3,
63.8, 55.1, 53.1, 53.0, 37.8, 37.6. HR-ESI-MS m/z: calcd for
C47H52N6O12 + H 893.9721, found 893.9719.
Methyl 4,6-di-O-benzyl-2,3-di-O-{1-[(1(S)-methoxycarbonyl-
2-p-hydroxyphenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-
glucopyranoside (22). From compound 11 (73 mg, 0.16 mmol)
and b (89.6 mg, 0.41 mmol), Na ascorbate (64.2 mg, 0.32 mmol),
CuSO4Á5H2O (40.4 mg, 0.16 mmol), column chromatography
(petroleum ether/EtOAc, 2 : 1 to 1 : 1) afforded 22 as a yellow
syrup (112.3 mg, 77.7%). Rf = 0.66 (EtOAc). [a]D = +5.2
(c = 0.6/CH2Cl2). 1H NMR (CDCl3, 400 MHz) d 7.60 (s, 1H,
triazole-H), 7.49 (s, 1H, triazole-H), 7.33–7.25 (m, 8H, PhH),
7.16–7.15 (m, 2H, PhH), 6.77–6.73 (m, 4H, (p-OH)Ph-H),
6.64–6.61 (m, 4H, (p-OH)Ph-H), 5.51–5.45 (m, 2H,
2ÂCHCO2), 4.91 (d, 1H, J = 12.0 Hz, PhCH), 4.87 (d, 1H,
J = 12.0 Hz, PhCH), 4.76 (d, 1H, J = 10.8 Hz, PhCH), 4.71
(d, 1H, J = 13.6 Hz, PhCH), 4.66 (d, 1H, J = 12.8 Hz,
PhCH), 4.62 (d, 1H, J = 12.0 Hz, PhCH), 4.50 (d, 2H, J =
12.8 Hz, PhCH), 4.46 (d, 1H, J = 10.8 Hz, PhCH), 3.86 (t, 1H,
J = 9.2 Hz), 3.77 (s, 3H, CO2CH3), 3.73 (s, 3H, CO2CH3),
3.69–3.65 (m, 3H), 3.54 (t, 1H, J = 8.8 Hz), 3.46–3.39 (m, 2H),
3.35–3.27 (m, 3H), 3.33 (s, 3H, OCH3). 13C NMR (100 MHz,
CDCl3) d 168.6, 168.6, 156.0, 145.2, 144.6, 144.4, 138.1, 137.4,
130.0, 129.9, 128.5, 128.4, 128.4, 128.2, 128.0, 127.9, 127.8,
126.2, 125.6, 124.0, 123.2, 116.1, 115.9, 97.7, 82.2, 78.9, 77.6,
77.3, 74.9, 73.6, 69.8, 68.4, 66.6, 64.5, 64.2, 62.3, 55.1, 53.2,
53.1, 37.9, 37.7. HR-ESI-MS m/z: calcd for C47H52N6O10 À H
891.3565, found 891.3558.
Inhibitory assay
The recombinant human PTP1B catalytic domain was expressed
and purified according to procedures described previously.31a
Enzymatic activity of PTP1B was determined at 30 1C by
monitoring the hydrolysis of pNPP. Dephosphorylation of
pNPP generates the product pNP, which can be monitored at
405 nm. In a typical 100 mL assay, a mixture containing
50 mM MOPS, pH 6.5, 2 mM pNPP and recombinant
enzymes, PTP1B activities were continuously monitored on a
SpectraMax 340 microplate reader at 405 nm for 2 min at 30 1C
and the initial rate of the hydrolysis was determined using the
early linear region of the enzymatic reaction kinetic curve. For
Methyl 2,6-di-O-benzyl-3,4-di-O-{1-[(1(S)-methoxycarbonyl-
2-phenylethyl)]-4-methyl-1H-1,2,3-triazole-4-yl}-a-D-glucopyrano-
side (28). From compound 27 (47.1 mg, 0.10 mmol), a
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 622–631 629

View Article Online
calculating IC50, inhibition assays were performed with 30 nM
recombinant enzyme, 2 mM pNPP in 50 mM MOPS at pH 6.5,
and the inhibitors diluted around the estimated IC50 values.
IC50 was calculated from the nonlinear curve fitting of percent
inhibition (inhibition (%)) vs. inhibitor concentration [I] by using
the following equation: inhibition (%) = 100/{1 + (IC50/[I])k},
where k is the Hill coefficient.
7 S. Zhang and Z.-Y. Zhang, Drug Discovery Today, 2007, 12,
373–381.
8 V. V. Vintonyak, A. P. Antonchick, D. Rauh and H. Waldmann,
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To study the inhibition selectivity on other PTP family
members, human TCPTP, SHP-1, SHP-2 and LARD1 were
prepared, and assays were performed according to procedures
described previously.31b
Kinetic study
To determine the inhibitor modality, assay was carried out in a
100 mL assay mixture containing 50 mM MOPS at pH 6.5,
30 nM PTP1B, pNPP in two-fold dilution up to 80 mM, and
different concentrations of the inhibitor. In the presence of the
competitive inhibitor, the Michaelis–Menten equation is
described as 1/v = [Km/(Vmax[S])](1 + [I]/Ki) + 1/Vmax, where
v is the initial rate, Vmax is the maximum rate, and [S] is the
substrate concentration. The Ki value was obtained by a linear
re-plot of apparent Km/Vmax (slope) from a primary reciprocal
plot vs. inhibitor concentration [I] according to the equation
Km/Vmax = 1 + [I]/Ki.
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Acknowledgements
X.-P. He gratefully acknowledges the French Embassy in
Beijing, China, for a doctorate fellowship. C.-J. Zhu thanks
the French Ministry of Foreign Affairs for an Eiffel Doctorate
fellowship. This project is supported by CNRS and ENS Cachan.
Grants were also from the National Natural Science Foundation
of China (Grant No. 20876045 and 30801405), National Basic
Research Program of China (No. 2007CB914201), National
Science & Technology Major Project of China ‘‘Key New
Drug Creation and Manufacturing Program’’ (No.
2009ZX09302-001), Shanghai Science and Technology
Community (No. 10410702700 and 09DZ2291200), and the
Chinese Academy of Sciences (No. KSCX2-YW-R-168).
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