Synthesis, Evaluation, and Mechanism of N,N,N-Trimethyl-D-glucosamine-(1→4)-chitooligosaccharides as Selective Inhibitors of Glycosyl Hydrolase Family 20 β-N-Acetyl-D-hexosaminidases

Article abstract of DOI:10.1002/cbic.201000561

GH20 β-N-acetyl-D-hexosaminidases are enzymes involved in many vital processes. Inhibitors that specifically target GH20 enzymes in pests are of agricultural and economic importance. Structural comparison has revealed that the bacterial chitindegrading β-

Full text of DOI:10.1002/cbic.201000561

DOI: 10.1002/cbic.201000561
Synthesis, Evaluation, and Mechanism of N,N,N-Trimethyl-
d-glucosamine-(1!4)-chitooligosaccharides as Selective
Inhibitors of Glycosyl Hydrolase Family 20 b-N-Acetyl-d-
hexosaminidases
You Yang,[a] Tian Liu,[b] Yongliang Yang,[b] Qingyue Wu,[b] Qing Yang,*[b, c] and Biao Yu*[a]
GH20 b-N-acetyl-d-hexosaminidases are enzymes involved in
many vital processes. Inhibitors that specifically target GH20
enzymes in pests are of agricultural and economic importance.
Structural comparison has revealed that the bacterial chitin-
degrading b-N-acetyl-d-hexosaminidases each have an extra
+1 subsite in the active site; this structural difference could be
exploited for the development of selective inhibitors. N,N,N-
trimethyl-d-glucosamine (TMG)-chitotriomycin, which contains
three GlcNAc residues, is a natural selective inhibitor against
bacterial and insect b-N-acetyl-d-hexosaminidases. However,
our structural alignment analysis indicated that the two
GlcNAc residues at the reducing end might be unnecessary. To
prove this hypothesis, we designed and synthesized a series of
TMG-chitotriomycin analogues containing one to four GlcNAc
units. Inhibitory kinetics and molecular docking showed that
TMG-(GlcNAc)2, is as active as TMG-chitotriomycin [TMG-
(GlcNAc)3]. The selective inhibition mechanism of TMG-chito-
triomycin was also explained.
Introduction
b-N-Acetyl-d-hexosaminidases (EC 3.2.1.52) catalyze the remov-
al of b-linked N-acetyl-d-glucosamine (GlcNAc) or N-acetyl-d-
galactosamine (GalNAc) from the nonreducing ends of a varie-
ty of oligosaccharides and glycoconjugates. These enzymes
belong to glycosyl hydrolase families 3, 20, and 84 according
to the CAZy database (http://www.cazy.org/). Glycosyl hydro-
lase family 20 (GH20) b-N-acetyl-d-hexosaminidases are widely
distributed in mammals, microorganisms, plants, and insects.
However, they differ from on another in physiological func-
tions. Mammalian b-N-acetyl-d-hexosaminidases are located in
lysosome and are responsible for the catabolism of the glyco-
syl components of proteins and lipids.[1] Human HexA is re-
sponsible for the removal of terminal nonreducing GalNAc
from GM2 gangliosides. Dysfunction of HexA results in massive
accumulation of GM2 gangliosides, which can result in Tay–
Sachs and Sandhoff diseases.[1] Bacterial b-N-acetyl-d-hexosami-
nidases degrade chitin, the b-(1!4)-linked GlcNAc polymer,
which is used as a source of nutrient by the bacteria.[2] Fungal
and insect b-N-acetyl-d-hexosaminidases play a role in the
recycling of chitin during growth and development.[3,4] Plant b-
N-acetyl-d-hexosaminidases exhibit the highest activity in ger-
minating seeds, suggesting the importance of these enzymes
in the storage of glycoproteins.[5]
been designed on the basis of this mechanism. Currently
known highly efficient inhibitors, with Ki values in the nm
range, include 1,2-dideoxy-2’-methyl-a-d-glucopyranoso-[2,1-
d]-D2’-thiazoline (NGT),[13] N-acetylglucosaminono-1,5-lactone
O-(phenylcarbamoyl)oxime (PUGNAc),[14] and nagstatin.[15] How-
ever, these inhibitors are not specific, because their targets
cover most of the known GH20 b-N-acetyl-d-hexosaminidases.
It is always a challenge to design selective inhibitors against
different GH20 b-N-acetyl-d-hexosaminidases, which can have
different functions. We compared the crystal structures of
three known b-N-acetyl-d-hexosaminidases—SmCHB (chito-
biase from Serratia marcescens, PDB ID: 1QBB),[6] SpHex (b-N-
acetyl-d-hexosaminidase from Streptomyces plicatus, PDB ID:
1HP5),[7] and HsHexB (b-N-acetyl-d-hexosaminidaseB from
[a] Y. Yang,+ Prof. B. Yu
State Key Laboratory of Bio-organic and Natural Products Chemistry
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
354 Fenglin Road, 200032 Shanghai (P. R. China)
Fax: (+86)21-64166128
E-mail: byu@mail.sioc.ac.cn
[b] T. Liu,+ Y. Yang, Q. Wu, Prof. Q. Yang
Department of Bioscience and Biotechnology
Dalian University of Technology
2 Linggong Road, 116024 Dalian (P. R. China)
Fax: (+86)411-84707245
E-mail: qingyang@dlut.edu.cn
Although GH20 b-N-acetyl-d-hexosaminidases vary in phys-
iological functions, they share the same substrate-assisted re-
taining catalytic mechanism.[6–12] In this mechanism, the 2-acet-
amido group of the nonreducing end sugar of the substrate
acts as a nucleophile to attack C-1, leading to the formation of
a bicyclic oxazolinium intermediate. The intermediate is then
attacked by a water molecule activated by the catalytic gluta-
mate. Inhibitors that mimic the oxazolinium intermediate have
[c] Prof. Q. Yang
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy
East China University of Science and Technology
130 Meilong Road, 200032 Shanghai (P. R. China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000561.
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457

Q. Yang, B. Yu et al.
Scheme 1. Synthesis of the TMG-a-GlcNAc derivatives 10 and 11. a) PPh3AuOTf, 96%; b) H2NCH2CH2NH2, EtOH, reflux; c) Ac2O, DMAP, pyridine, RT, 93%; d) pro-
pane-1,3-dithiol, Et3N, pyridine, H2O, RT, 92%; e) MeI, iPr2NEt, THF, RT, 80%; f) K2CO3, MeOH, CH2Cl2, RT, 94%; g) H2, Pd(OH)2/C, MeOH, CH2Cl2, aq. HCl, RT, 89%;
h) Ag(DPAH)2, CH3CN, H2O, RT, 63%.
Homo sapiens, PDB ID: 1NP0)[8,9]—and found that the bacterial
Results
enzymes each have an extra +1 subsite that consists of two
Synthesis
conserved residues, valine and tryptophan, at the entrance of
the active pocket. We postulated that this structural difference
might thus provide important clues for the designing of spe-
cies-specific drugs.
The TMG-GlcNAc, TMG-chitobiose, and TMG-tetraose deriva-
tives 10/11, 17, 22/23, and 30/31 were readily synthesized by
modifying previously reported synthetic approaches for TMG-
chitotriomycin (1) and its a-TMG congener 2,[17] as shown in
Schemes 1–4, below.
To validate the hypothesis, TMG-chitotriomycin[16] was
chosen as the starting inhibitor, from which N,N,N-trimethyl-d-
glucosamine (TMG) derivatives with one to four b-(1!4)-linked
GlcNAc residues were designed and synthesized. TMG-chito-
triomycin is a recently disclosed and efficient inhibitor for bac-
terial, fungal, and insect b-N-acetyl-d-hexosaminidases, but has
The a-(1!4)-disaccharide 5 (Scheme 1) was readily prepared
by coupling the glycosyl ortho-hexynylbenzoate 3 with the
glucosamine-4-OH derivative 4 in the presence of PPh3AuOTf
as catalyst in Et2O at ꢀ308C to room temperature.[17] Removal
of the N-Phth group on 5 with ethylenediamine, followed by
acetylation of the resulting amine, provided the acetamide 6 in
93% yield.[18] In the presence of propane-1,3-dithiol and Et3N in
aqueous pyridine,[19] the azido group on 6 was readily convert-
ed into an amine group in 92% yield. Use of other conditions,
such as PPh3/silica gel/THF/H2O[20] and Zn/HOAc,[21] led to mi-
gration of the O-acetyl groups to the nascent NH2 group.
Treatment of the resulting amine derivative 7 with excess CH3I
in the presence of (iPr)2NEt readily yielded the ammonium
compound 8, in 80% yield after purification by column chro-
matography (Sephadex LH-20). Subsequently, compound 8 was
subjected to basic conditions (K2CO3, MeOH/CH2Cl2) to give the
ammonium compound 9, followed by hydrogenolysis over
Pd(OH)2/C catalyst under acidic conditions to furnish the de-
sired TMG-a-GlcNAc MP (p-methoxyphenyl) glycoside 10 (84%
for two steps). Attempts to remove the anomeric MP group
with CAN under various conditions were found to be futile.
However, it was readily removed by the mild oxidizing agent
no effect on the enzymes from plants and mammals,[16]
a
promising feature that might allow for the development of
novel and eco-friendly fungicides as well as insecticides. It is
also a pseudotetrasaccharide containing three b-(1!4)-linked
GlcNAc units linked to TMG at the nonreducing end. Although
the structure of TMG-chitotriomycin is known, its inhibition
mechanism remains to be clarified. It has been speculated that
the charged nitrogen atom in TMG is vital for its inhibitory
activity,[16] but Kanzaki et al. have shown that TMG itself is inac-
tive against b-N-acetyl-d-hexosaminidase from the insect Spo-
doptera litura.[16] This surprising result led them to presume
that the (GlcNAc)3 moiety is important for inhibition of b-N-
acetyl-d-hexosaminidase. The question arising from this is why
and how the sugar component affects the interaction between
TMG-chitotriomycin and its target enzymes. On the other
hand, TMG was originally postulated by Kanzaki et al.[16] to be
a-(1!4)-linked to (GlcNAc)3, but was later found by our group
to be b-(1!4)-linked.[17] It is thus important to confirm if the
a-conformation indeed has no activity toward b-N-acetyl-d-
hexosaminidase. In this study, two series of both a- and b-(1!
4)-linked TMG-chitotriomycin analogues with different number
of GlcNAc units were synthesized and their inhibition of GH20
b-N-acetyl-d-hexosaminidases from different organisms was
evaluated. Furthermore, an inhibition mechanism that partially
supported our hypothesis was postulated in terms of the
enzymes’ structures.
[22]
Ag(DPAH)2 to convert the MP glycoside 10 into the TMG-a-
GlcNAc derivative 11, in a satisfactory yield of 63%.
BF3·Et2O-promoted glycosylation of the glucosamine-4-OH
derivative 13 (Scheme 2) with the 2-azido-glucopyranosyl-a-
imidate 12 afforded the b-(1!4)-disaccharide 14 in 72%
yield.[17,23] A procedure similar to that used for the previous
conversion of disaccharide 5!8 was then employed to con-
vert 14 into the disaccharide ammonium compound 16 (77%
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TMG-(GlcNAc)1–4 as Selective Inhibitors
Scheme 2. Synthesis of the TMG-b-GlcNAc derivative 17. a) BF3OEt2, 72%; b) H2NCH2CH2NH2, EtOH, reflux; c) Ac2O, DMAP, pyridine, RT, 88% for 2 steps; d) pro-
pane-1,3-dithiol, Et3N, pyridine, H2O, RT; e) MeI, iPr2NEt, THF, RT, 87% for 2 steps; f) H2, Pd(OH)2/C, MeOH, CH2Cl2, aq. HCl, RT, 81%.
for four steps). Hydrogenolysis of 16 over Pd(OH)2/C under
acidic conditions provided the desired TMG-b-GlcNAc MP gly-
coside 17 in 81% yield.
d=5.23~5.42 ppm with small J1,2 values of 0~4.5 Hz, implying
that a twist-boat conformation was adopted by the TMG resi-
due rather than the normal chair conformation assumed by
the glucosamine residue.[17]
The TMG-b-chitobiose derivatives 22 and 23 were synthe-
sized as shown in Scheme 3. Coupling of the disaccharide
ortho-hexynylbenzoate 18[17] with the glucosamine-4-OH deriv-
ative 4 in the presence of PPh3AuOTf in CH2Cl2 at ꢀ308C af-
forded the b-linked trisaccharide 19 in an excellent 91% yield.
Steps similar to those used for 5!10 were then employed for
the conversion of 19 into the TMG-b-chitobiose MP glycoside
22 (six steps, 37%). Final removal of the anomeric MP group
with Ag(DPAH)2 gave the TMG-b-chitobiose 23 in 70% yield.
The TMG-tetraose derivatives 30 and 31 were assembled by
use of a convergent 3+2 glycosidic coupling as a key step
(Scheme 4). Treatment of the MP glycoside 19 with CAN, fol-
lowed by condensation with ortho-hexynylbenzoic acid,[24] led
to the trisaccharide ortho-hexynylbenzoate 24 (69% yield for
two steps). Coupling of 24 with the disaccharide acceptor
25[17] with PPh3AuOTf catalysis in CH2Cl2 provided the b-linked
pentasaccharide 27 in a modest yield of 51%, with the trisac-
charide glycal 26 obtained as the major by-product (32%).[24]
Steps similar to those used for 5!10 were then employed for
the conversion of 27 into the TMG-b-tetraose MP glycoside 30
(six steps, 49%). Final removal of the anomeric MP group with
Ag(DPAH)2 furnished the TMG-b-tetraose 31 in 71% yield. The
solubilities of the pentasaccharides 30 and 31 in methanol
were poor, so MeOH/H2O (1:1) was used as eluent for column
chromatography (Sephadex LH-20).
Activity evaluation of TMG
TMG was found to be inactive against b-N-acetyl-d-hexosami-
nidase from the insect Spodoptera litura,[16] but whether TMG is
inactive against b-N-acetyl-d-hexosaminidases from other or-
ganisms was unclear. We evaluated its inhibitory activity
against b-N-acetyl-d-hexosaminidases from bacteria (SpHex),
insect (OfHex1), plant (CeHex), and mammal (BtHex). TMG
showed no inhibition against any of the tested enzymes
(Table 1). This result confirmed that it was the sugar compo-
nent of TMG-chitotriomycin that was essential for its inhibitory
activity against b-N-acetyl-d-hexosaminidases.
Inhibitory activity comparison of a-linked TMG-chitotrio-
mycin analogues
All of the synthesized a-linked and b-linked TMG-chitotriomy-
cin analogues with one to four GlcNAc components were
tested for inhibition against representatives of bacterial, insect,
plant, and mammal GH20 b-N-acetyl-d-hexosaminidases
(Table 1). The a-linked TMG-chitotriomycin analogues all
showed no inhibition against all the tested enzymes. On the
other hand, all of the b-linked TMG-chitotriomycin analogues
inhibited the bacterial and insect enzymes in a competitive
mode with Ki values in the micro- to submicromolar ranges,
respectively, whereas no inhibition of the plant and mammal
It should be noted that the chemical shifts of the anomeric
protons of the b-TMG residues in these final oligosaccharide
compounds (1,[17] 17, 22, 23, 30, 31, and 33[17]) appeared at
Scheme 3. Synthesis of the TMG-b-GlcNAc-GlcNAc derivatives 22 and 23. a) PPh3AuOTf (0.2 equiv), 4 ꢁ MS, CH2Cl2, ꢀ308C–RT, 91%; b) H2NCH2CH2NH2, EtOH,
reflux; c) Ac2O, DMAP, pyridine, RT, 72% for 2 steps; d) propane-1,3-dithiol, Et3N, pyridine, H2O, RT; e) MeI, iPr2NEt, THF, RT, 84% for 2 steps; f) K2CO3, MeOH,
CH2Cl2, RT; g) H2, Pd(OH)2/C, MeOH, CH2Cl2, aq. HCl, RT, 62% for 2 steps; h) Ag(DPAH)2, CH3CN, H2O, RT, 70%.
ChemBioChem 2011, 12, 457 – 467
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459

Q. Yang, B. Yu et al.
Scheme 4. Synthesis of the TMG-b-(GlcNAc)3-GlcNAc derivatives 30 and 31. a) CAN, toluene, CH3CN, H2O, RT, 77%; b) o-hexnylbenzoic acid, DCC, DMAP,
CH2Cl2, RT, 90%; c) PPh3AuOTf (0.2 equiv), 4 ꢁ MS, CH2Cl2, ꢀ308C–RT; d) H2NCH2CH2NH2, EtOH, reflux; e) Ac2O, DMAP, pyridine, RT, 75% for 2 steps; f) propane-
1,3-dithiol, Et3N, pyridine, H2O, RT; g) MeI, iPr2NEt, THF, RT, 89% for 2 steps; h) K2CO3, MeOH, CH2Cl2, RT; i) H2, Pd(OH)2/C, MeOH, CH2Cl2, aq. HCl, RT, 74% for 2
steps; j) Ag(DPAH)2, CH3CN, H2O, RT, 71%.
Inhibitory activity of b-linked
Table 1. Inhibitory activities of b-linked TMG-chitooligosaccharides against b-N-acetyl-d-hexosaminidases from
different organisms.
TMG-chitotriomycin analogues
To evaluate how the GlcNAc
Origins of b-N-acetyl-d-hexosaminidases
components affected the selec-
tive inhibition potency of TMG-
chitotriomycin, we synthesized
TMG-b-(GlcNAc)2 (23), TMG-b-
(GlcNAc)3 (1), and TMG-b-
(GlcNAc)4 (31) and evaluated
them against b-N-acetyl-d-hexo-
saminidases from bacterium
(SpHex), insect (OfHex1), and
plant (CeHex), as well as
mammal (BtHex) (Table 1). All of
TMG-chito-
oligosaccharides
Chitin-processing organism
Non-chitin-containing organism
bacterium[a]
Ki [mm]
insect[b]
plant[c]
Ki [mm]
mammal[d]
Ki [mm]
Ki [mm]/IC50
TMG (32)[25]
TMG-b-(GlcNAc)2 (23)
TMG-b-(GlcNAc)3 (1)
TMG-b-(GlcNAc)4 (31)
n.d.[e]
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.1
1.0
1.1
0.077
0.065
0.058
n.d.
n.d.
n.d.
[a] Streptomyces plicatus. [b] Ostrinia furnacalis. [c] Canavalia ensiformis. [d] Bos taurus. [e] n.d.=inhibition not
detected at 0.1 mm.
enzymes was observed, even at 0.1 mm concentration. The re-
sults therefore demonstrated that only the b-linked TMG-chito-
triomycin analogues could act as efficient inhibitors against
GH20 b-N-acetyl-d-hexosaminidases.
these analogues selectively inhibited SpHex and OfHex1 but
not CeHex and BtHex, meaning that the number of GlcNAc
units had no effect on the selective potency. However, TMG-b-
(GlcNAc)3 (1) and TMG-b-(GlcNAc)4 (31) inhibited SpHex and
OfHex1 with the same magnitude of Ki as TMG-b-(GlcNAc)2
(23), thus implying that a minimum of two GlcNAc units were
needed for full activity, but that additional GlcNAc units (up to
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ChemBioChem 2011, 12, 457 – 467

TMG-(GlcNAc)1–4 as Selective Inhibitors
two) can also be accommodated
with no effect on the activities
of these inhibitors (Table 1). Fur-
thermore, the Ki values varied
Table 2. Inhibitory activity of p-methoxyphenyl-derivatized b-linked TMG-chitooligosaccharides against b-N-
acetyl-d-hexosaminidases from different organisms.
Origins of b-N-acetyl-d-hexosaminidases
TMG-chito-
oligosaccharides
Chitin-processing organism
Non-chitin-containing organism
over the different organisms,
suggesting the existence of se-
lective inhibition of b-N-acetyl-
d-hexosaminidases by TMG-chi-
totriomycin and its analogues.
TMG-b-(GlcNAc)3 (1) inhibited
bacterium[a]
insect[b]
plant[c]
Ki [mm]
mammal[d]
Ki [mm]
Ki [mm]
Ki [mm]
TMG (32)[25]
TMG-b-GlcNAc-OMP (17)
TMG-b-GlcNAc-GlcNAc-OMP (22)
TMG-b-(GlcNAc)2-GlcNAc-OMP (33)
TMG-b-(GlcNAc)3-GlcNAc-OMP (30)
n.d.[e]
5.7
1.3
1.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.86
0.073
0.07
0.066
n.d.
n.d.
n.d.
n.d.
1.1
OfHex1 with
a Ki value of
[a] Streptomyces plicatus. [b] Ostrinia furnacalis. [c] Canavalia ensiformis. [d] Bos taurus. [e] n.d.=inhibition not
detected at 0.1 mm.
0.065 mm, which was more than
ten times lower than the Ki
values with b-N-acetyl-d-hexosa-
minidases
from
bacterium
(Streptomyces plicatus) and fungus (Aspergillus oryzae).[16]
GlcNAc-OMP (33) inhibited SpHex and OfHex1 with the same
magnitude as TMG-b-(GlcNAc)3 (1) and TMG-b-(GlcNAc)4 (31),
as revealed by the range of Ki values (Tables 1 and 2), suggest-
ing that the third and the fourth GlcNAc residues can be re-
placed by other substituents with little effect on the inhibitory
activities of these compounds.
TMG-chitiotriomycin analogues with p-methoxyphenyl
substituents
We also investigated the effects of the sugar component at
the reducing end of TMG-chitiotriomycin. p-Methoxyphenyl-
substituted (MP-substituted) analogues, the intermediates in
the synthesis of TMG-chitiotriomycin, were chosen. TMG-b-
GlcNAc-OMP (17) inhibited SpHex and OfHex1 with Ki values
five and 11 times higher, respectively, than those achieved by
TMG-b-(GlcNAc)2 (23; Tables 1 and 2), demonstrating that the
MP group could not serve as the second GlcNAc. On the other
hand, TMG-b-GlcNAc-GlcNAc-OMP (22) and TMG-b-(GlcNAc)2-
Binding modes of TMG-chitotriomycin in the SpHex and
HsHexB by molecular docking
The modes by which TMG-chitotriomycin binds to SpHex and
to human HsHexB were compared by molecular docking
(Figure 1). The best docking results were obtained by setting
the side chains of several amino acid residues of the enzymes
Figure 1. Binding modes of TMG-chitotriomycin in A) SpHex, and B) HsHexB. Upper figures show the active pocket surface and the bottom figures show the
residues in the active pocket. Hydrogen bonds and salt bridges are shown as dashed and solid lines, respectively.
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461

Q. Yang, B. Yu et al.
(including Glu314, Trp361, and Trp408 of SpHex, and Glu355
and Trp424 of HsHexB) as flexible. From the free energies of
binding, the binding between TMG-chitotriomycin and SpHex
was more favorable (DG=ꢀ7.48 kcalmolꢀ1) than that between
TMG-chitotriomycin and HsHexB (DG=ꢀ0.13 kcalmolꢀ1).
In both SpHex and HsHexB, the TMG component of TMG-
chitotriomycin is bound to the ꢀ1 subsite, which is enclosed
by three conserved Trp residues (Trp344, Trp361, and Trp442
in SpHex, and Trp405, Trp424, and Trp489 in HsHexB) in a
skewed chair conformation, positioning TMG in direct interac-
tion with the conserved residues (Figure 1). TMG-chitotriomy-
cin seemed to mimic the substrate because the acid/base cata-
lyst (Glu314 in SpHex, Glu355 in HsHexB) is close to the
oxygen atom forming the glycosidic bond between TMG and
the first GlcNAc (Figure 1). It is noteworthy that the positively
charged nitrogen atom on the N(CH3)3 group is close to
Asp313 in the case of SpHex and to Asp354 in the case of
HsHexB (Figure 1), suggesting that a salt bridge might form
when this catalytic Asp is deprotonated during catalysis. The
main difference in the binding between TMG-chitotriomycin
and SpHex-37 versus TMG-chitotriomycin and HsHexB-2 at the
ꢀ1 subsite is that the stacking of the pyranose ring (in TMG)
against Trp442 of SpHex was tighter than that against Trp489
in HsHexB (Figure 1).
By comparing the structures of two bacterial chitinolytic b-
N-acetyl-d-hexosaminidases—SmCHB[6] and SpHex[7]—with
that of lysosomal HsHexB,[8,9] we found that the bacterial chiti-
nolytic enzymes each have an extra +1 subsite that contains
two highly conserved amino acid residues in the active site.
We speculated that this structural discrepancy between the
bacterial and human enzymes could be a determining factor
for selective inhibition.
TMG-chitotriomycin has recently been found to be an inhibi-
tor that shows selective inhibition toward b-N-acetyl-d-hexosa-
minidases from chitin-containing organisms.[16] It is surprising
that this naturally occurring compound contains four sugar
units, because the extra two sugar units at the reducing end
were not essential for selective inhibition. Although the struc-
ture of TMG-chitotriomycin has been established, its inhibition
mechanism remains to be clarified. In addition, the complex
structure of TMG-chitotriomycin makes it difficult to synthesize
on large scales.
In this work, two series of a- and b-(1!4)-linked TMG-
(GlcNAc)1–4 derivatives were synthesized and their inhibitory
potencies against several GH20 b-N-acetyl-d-hexosaminidases
from different organisms were evaluated. The enzymatic assay
showed that only b-(1!4)-linked TMG-(GlcNAc)1–4 derivatives
were effective. Glycosidases can be classified in term of their
preference either for a- or for b-glycoside bonds in the sub-
strates, so it is easy to understand why b-(1!4)- rather than a-
(1!4)-linked TMG-chitotriomycin has been selected in nature
as an inhibitor for the GH20 b-N-acetyl-d-hexosaminidase b-
glycosidases. In addition to the previously reported NMR
data,[17] here we have confirmed the structure of this inhibitor
through an enzymatic approach.
In the +1 subsite, which is present in SpHex but absent in
HsHexB, the GlcNAc adjacent to TMG is stacked against Trp408
and the oxygen atom on the 2-acetamido group is hydrogen-
bonded to the nitrogen atom of the indolyl group of Trp408
(Figure 1A). The binding of this GlcNAc seemed to push the
TMG component into the ꢀ1 subsite so as to form tighter
stacking against Trp442. This is consistent with our observation
that a conserved Trp408 at the +1 subsite of a TMG-chitotrio-
mycin-sensitive enzyme is essential for binding with TMG-chi-
totriomycin.
The activity assay of the b-(1!4)-linked TMG-(GlcNAc)1–4 de-
rivatives showed that either one or two GlcNAc units are re-
quired for inhibition. Crystal structure comparison of bacterial
(SmCHB and SpHex)[6,7] and human (HsHexB)[8,9] b-N-acetyl-d-
hexosaminidases indicated that the bacterial enzymes have
larger and longer active pockets than the human enzyme. The
exceptional +1 subsite with conserved residues (Val493 and
Trp685 of SmCHB and Val276 and Trp408 of SpHex) in the bac-
terial enzyme is ideal for binding long and linear substrates. It
is interesting to note that the reducing end GlcNAc (NAGB) of
chitobiose is tightly bound in the +1 subsite through a stack-
ing interaction with the indolyl group of Trp685 in the SmCHB-
chitobiose complex.[6] NAGB is twisted around 908 relative to
the nonreducing end GlcNAc (NAGA). In SpHex, a glycerol is
located at the +1 subsite and superimposed onto half of the
NAGB in the SpHec-NAG-thiazoline complex,[7] so the binding
of +1 GlcNAc might be required for stabilizing the binding
conformation of the ꢀ1 sugar. We thus presume that the
GlcNAc component next to TMG binds at the +1 subsite and
stabilizes the binding conformation of TMG in TMG-chitotrimy-
cin-sensitive enzymes. Furthermore, the docking of TMG-chito-
triomycin to SpHex suggested that TMG-chitotriomycin could
be tightly bound through interaction with the amino acid resi-
dues in both the ꢀ1 and the +1 subsites of SpHex. Because
HsHexB does not contain a +1 subsite, docking of TMG-chito-
triomycin at the ꢀ1 subsite in HsHexB probably occurred
As for SpHex-37 and HsHexB-2, no intermolecular interaction
was found between the second or third GlcNAc units of TMG-
chitotriomycin and the enzymes (Figure 1). This is in good
agreement with our experimental data. It is interesting to note
that in the case of SpHex, the Ki value for TMG-b-(GlcNAc)2 (23)
was five times lower than that for TMG-b-GlcNAc-OMP (17).
The reason is not known. We deduced that there might be a
+2 subsite in SpHex for binding the second GlcNAc of TMG-b-
(GlcNAc)2 through hydrogen bonding. Because the MP group
does not contain hydroxy substituents, MP was not able to
bind and be stabilized by the +2 subsite. However, the precise
mechanism requires further work.
Discussion
GH20 b-N-acetyl-d-hexosaminidases are enzymes with different
physiological roles. To achieve selective inhibition for each of
these enzymes would provide insight into the specialized func-
tions of these enzymes in vivo. Selective inhibition would also
be an advantage for disease control and plant protection.
Little progress has been made as far as selective inhibitors are
concerned.
462
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 457 – 467

TMG-(GlcNAc)1–4 as Selective Inhibitors
0.78 mmol) in pyridine/H2O (40 mL/10 mL). The solution was stirred
at room temperature for 1 h and then concentrated in vacuo to
give a residue, which was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH/Et3N 80:1:1) to furnish 7 (532 mg, 92%) as a
white solid.1H NMR (300 MHz, CDCl3): d=7.31 (m, 5H), 6.97 (d, J=
9.3 Hz, 2H), 6.78 (d, J=9.0 Hz, 2H), 5.94 (d, J=9.3 Hz, 1H), 5.20 (d,
J=3.3 Hz, 1H), 5.15 (t, J=8.7 Hz, 1H), 4.93 (m, 3H), 4.58 (dd, J=
11.7, 19.8 Hz, 2H), 4.25 (m, 1H), 4.20 (m, 1H), 4.11 (m, 2H), 3.83 (m,
2H), 3.74 (m, 5H), 2.89 (dd, J=3.3, 9.9 Hz, 1H), 2.08 (s, 3H), 2.06 (s,
3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.96 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=171.6, 170.8, 170.5, 170.2, 169.6, 155.4, 151.2, 137.8,
128.4, 127.7, 127.5, 118.4, 114.5, 100.5, 100.3, 75.0, 74.3, 73.5, 69.1,
68.7, 68.4, 61.8, 55.6, 54.3, 54.0, 23.2, 21.4, 20.8, 20.7, 20.6 ppm;
HRMS (MALDI): m/z: calcd for C36H46N2O15Na: 769.2798 [M+Na]+;
found: 769.2790.
through weak interaction. The higher DG value obtained for
the binding of TMG-chitotriomycin to HsHexB confirmed that
this binding was unstable. To this end, we speculate that the
GlcNAc component of TMG is positioned at the +1 subsite
through stacking against the conserved Trp residues and is
hydrogen-bonded to the conserved Val residues of TMG-chito-
trimycin-sensitive enzymes. In this way, TMG is stabilized and
can interact with the catalytic residues at the ꢀ1 subsite.
TMG-chitotriomycin is a stronger inhibitor against insect b-
N-acetyl-d-hexosaminidases (OfHex1 and b-N-acetyl-d-hexosa-
minidase from Spodoptera litura[16]) than against bacterial b-N-
acetyl-d-hexosaminidase (SpHex) and fungal b-N-acetyl-d-hex-
osaminidase (b-N-acetyl-d-hexosaminidase from A. oryzae[16]).
We deduced that the insect enzyme might contain a narrower
substrate binding cleft than the bacterial and fungal enzymes.
This study has shed more light on the mechanisms of the
selectivity of TMG-chitotriomycin with respect to the inhibition
it exerts on GH20 b-N-acetyl-d-hexosaminidases. Furthermore,
the inhibition of these enzymes by TMG-b-(GlcNAc)2, which has
one GlcNAc unit fewer than TMG-chitotriomycin, also validated
our hypothesis that selective inhibitors can be designed ac-
cording to the structural difference at the active pockets of
GH20 b-N-acetyl-d-hexosaminidases.
(iPr)2Net (0.4 mL) and CH3I (2 mL) were added to a solution of the
amine 7 (103 mg, 0.14 mmol) in dry THF (3 mL). After having been
stirred at room temperature for 40 h, the mixture was filtered, con-
centrated in vacuo, and passed through a chromatography column
(Sephadex LH-20, CH2Cl2/MeOH 1:1) to afford 8 (87 mg, 80%) as a
slightly yellow solid. [a]2D7 =+40.4 (c=0.30, CHCl3); 1H NMR
(300 MHz, CDCl3): d=7.29 (m, 5H), 7.01 (d, J=8.7 Hz, 2H), 6.76 (d,
J=9.3 Hz, 2H), 5.96 (t, J=8.4 Hz, 1H), 5.69 (m, 2H), 5.58 (d, J=
7.5 Hz, 1H), 4.99 (t, J=9.0 Hz, 1H), 4.67 (d-like, J=11.7 Hz, 1H),
4.53 (d-like, J=11.4 Hz, 1H), 4.40 (m, 2H), 4.24 (m, 2H), 4.10 (m,
1H), 3.88 (m, 2H), 3.80 (m, 2H), 3.72 (s, 3H), 3.53 (s, 9H), 2.16 (s,
3H), 2.12 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.90 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=171.9, 170.6, 170.4, 169.3, 169.2,
155.4, 150.9, 137.6, 128.4, 127.9, 127.4, 118.9, 114.5, 99.4, 92.3, 75.7,
73.7, 73.1, 70.5, 69.9, 69.1, 68.9, 68.3, 68.0, 60.6, 55.6, 55.3, 54.8,
23.2, 21.9, 21.2, 20.7, 20.4 ppm; HRMS (MALDI): m/z: calcd for
C39H53N2O15: 789.3452 [M]+; found: 789.3441.
Experimental Section
Synthesis
p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-b-d-gluco-
pyranosyl-(1!4)-3-O-acetyl-6-O-benzyl-2-acetamino-2-deoxy-b-
d-glucopyranoside (6): Compound 5 (1.00 g, 1.16 mmol) was dis-
solved in EtOH (13 mL) at room temperature, followed by addition
of ethylenediamine (4 mL). After having been heated at reflux at
1008C for 14 h, the mixture was concentrated in vacuo to give a
residue, which was used in the next step without further purifica-
tion.
p-Methoxyphenyl 2-trimethylammonium-2-deoxy-b-d-glucopyra-
nosyl-(1!4)-2-acetamino-2-deoxy-b-d-glucopyranoside
(10):
K2CO3 (23 mg, 0.17 mmol) was added to a solution of 8 (102 mg,
0.13 mmol) in MeOH/CH2Cl2 (4 mL/0.8 mL). The mixture was stirred
at room temperature for 4 h and then neutralized with Dowex-50
(H+). Filtration, concentration in vacuo, and elution through a
chromatography column (Sephadex LH-20, CH2Cl2/MeOH 1:1) pro-
vided 9 (75 mg, 94%) as a white solid. [a]2D5 =+30.1 (c=1.00,
Ac2O (8 mL) was added to a solution of the residue and DMAP
(80 mg, 0.66 mmol) in pyridine (14 mL). After having been stirred
at room temperature overnight, the solution was diluted with
CH2Cl2 and washed with aqueous HCl (1n), saturated aqueous
NaHCO3, and brine. The organic layer was dried over Na2SO4, fil-
tered, and concentrated in vacuo to give a residue, which was pu-
rified by silica gel column chromatography (petroleum ether/EtOAc
1:1!1:1.5) to give 6 (841 mg, 93% for two steps) as a white solid.
[a]2D7 =+54.7 (c=0.50, CHCl3); 1H NMR (300 MHz, CDCl3): d=7.32
(m, 5H), 6.94 (d, J=9.0 Hz, 2H), 6.78 (d, J=8.7 Hz, 2H), 5.93 (d, J=
9.3 Hz, 1H), 5.38 (t, J=10.2 Hz, 1H), 5.30 (d, J=3.9 Hz, 1H), 5.17 (t,
J=9.0 Hz, 1H), 5.01 (t, J=9.9 Hz, 1H), 4.92 (d, J=7.5 Hz, 1H), 4.56
(dd, J=12.0, 14.7 Hz, 2H), 4.33 (dd, J=9.6, 16.8 Hz, 1H), 4.18 (dd,
J=3.6, 12.6 Hz, 1H), 4.05 (m, 1H), 3.97 (m, 1H), 3.78 (m, 7H), 3.42
(dd, J=3.9, 10.8 Hz, 1H), 2.08 (s, 6H), 2.03 (s, 3H), 2.02 (s, 3H),
1.97 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=170.8, 170.4,
170.1, 170.0, 169.5, 155.4, 151.1, 137.7, 128.4, 127.8, 127.6, 118.3,
114.5, 100.1, 98.0, 74.8, 73.7, 73.6, 70.3, 69.0, 68.5, 68.1, 61.3, 61.0,
55.6, 53.2, 23.2, 20.8, 20.6, 20.5 ppm; HRMS (MALDI): m/z: calcd for
C36H44N4O15Na: 795.2706 [M+Na]+; found: 795.2695.
1
MeOH); H NMR (300 MHz, CD3OD): d=7.32 (m, 5H), 6.99 (d, J=
8.7 Hz, 2H), 6.79 (d, J=9.3 Hz, 2H), 6.34 (d, J=2.1 Hz, 1H), 5.07 (d,
J=7.5 Hz, 1H), 4.56 (s, 2H), 4.11 (m, 2H), 4.03 (m, 2H), 3.84 (m,
2H), 3.73 (s, 3H), 3.63 (m, 5H), 3.40 (s, 9H), 2.04 ppm (s, 3H);
13C NMR (100 MHz, CD3OD): d=172.8, 155.5, 151.4, 138.1, 127.9,
127.5, 117.3, 118.1, 114.1, 100.0, 94.3, 75.3, 74.0, 73.0, 72.9, 72.5,
71.4, 70.5, 68.9, 68.4, 60.2, 56.7, 54.7, 53.6, 21.7 ppm; HRMS
(MALDI): m/z: calcd for C31H45N2O11: 621.3019 [M]+; found:
621.3018.
Compound 9 (45 mg, 0.072 mmol) was dissolved in MeOH (5 mL)
at room temperature, followed by addition of aqueous HCl (37%,
two drops) and Pd(OH)2/C (200 mg, 20%). After having been
hydrogenated at room temperature for 22 h, the suspension was
filtered and neutralized with Dowex-1X8 (OHꢀ). Filtration, concen-
tration in vacuo, and elution through a chromatography column
(Sephadex LH-20, MeOH) gave 10 (34 mg, 89%) as a colorless
solid. [a]2D7 =+61.8 (c=1.00, MeOH); 1H NMR (300 MHz, CD3OD):
d=6.97 (d, J=9.0 Hz, 2H), 6.82 (d, J=9.0 Hz, 2H), 6.30 (d, J=
2.1 Hz, 1H), 5.03 (d, J=7.5 Hz, 1H), 4.11 (m, 2H), 3.92 (m, 5H), 3.73
(m, 5H), 3.62 (m, 2H), 3.47 (m, 1H), 3.41 (s, 9H), 2.02 ppm (s, 3H);
13C NMR (100 MHz, CD3OD): d=172.8, 155.4, 151.6, 117.8, 114.1,
100.0, 94.5, 75.2, 74.8, 73.0, 72.6, 71.1, 70.9, 68.9, 60.6, 60.4, 56.7,
p-Methoxyphenyl
3,4,6-tri-O-acetyl-2-trimethylammonium-2-
deoxy-b-d-glucopyranosyl-(1!4)-3-O-acetyl-6-O-benzyl-2-acet-
amino-2-deoxy-b-d-glucopyranoside (8): Et3N (2 mL) and pro-
pane-1,3-dithiol (4 mL) were added to a solution of 6 (600 mg,
ChemBioChem 2011, 12, 457 – 467
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
463

Q. Yang, B. Yu et al.
54.7, 53.6, 21.7 ppm; HRMS (MALDI): m/z: calcd for C24H39N2O11:
531.2552 [M]+; found: 531.2548.
glucopyranosyl-(1!4)-3-O-acetyl-6-O-benzyl-2-N-phthalimido-2-
deoxy-b-d-glucopyranoside (19): Newly prepared PPh3AuOTf in
CH2Cl2 (0.05m, 2.4 mL) was added dropwise under argon at ꢀ308C
to a stirred mixture of the donor 18[17] (775 mg, 0.68 mmol), the ac-
ceptor 4 (312 mg, 0.57 mmol), and freshly activated MS (4 ꢁ, 1 g)
in dry CH2Cl2 (20 mL). After 0.5 h, the mixture was allowed to warm
up naturally to room temperature and the stirring was continued
overnight. The mixture was then filtered and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (petroleum ether/EtOAc 3:1!2:1) to afford 19 (762 mg, 91%)
as a white solid: [a]2D5 =+7.3 (c=0.5, CHCl3); 1H NMR (300 MHz,
CDCl3): d=7.71 (m, 8H), 7.27 (m, 25H), 6.92 (m, 2H), 6.75 (m, 5H),
6.63 (d, J=9.0 Hz, 2H), 5.73 (t, J=9.9 Hz, 1H), 5.71 (d, J=9.0 Hz,
1H), 5.25 (d, J=8.7 Hz, 1H), 4.80 (m, 4H), 4.62 (d, J=11.4 Hz, 1H),
4.53 (d, J=10.8 Hz, 1H), 4.39 (m, 8H), 4.19 (m, 2H), 4.08 (m, 2H),
3.91 (dd, J=2.1, 10.5 Hz, 1H), 3.67 (m, 6H), 3.56 (m, 2H), 3.45 (m,
2H), 3.36 (m, 2H), 3.23 (t, J=9.3 Hz, 1H), 3.12 (d-like, J=9.6 Hz,
1H), 1.87 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=170.2, 155.5,
150.7, 138.7, 138.2, 138.1, 138.0, 137.9, 137.8, 134.2, 133.6, 131.6,
128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4,
126.9, 123.5, 123.2, 118.9, 114.3, 100.9, 97.5, 97.4, 83.2, 77.7, 77.5,
77.3, 75.4, 74.8, 74.7, 74.6, 74.5, 74.2, 73.3, 73.1, 72.6, 71.4, 68.3,
67.8, 67.6, 66.9, 56.1, 55.5, 55.0, 20.6 ppm; HRMS (MALDI): m/z:
calcd for C85H81N5O19Na: 1498.5435 [M+Na]+; found: 1498.5418.
2-Trimethylammonium-2-deoxy-b-d-glucopyranosyl-(1!4)-2-
acetamino-2-deoxy-d-glucopyranose (11): Ag(DPAH)2 (30 mg,
0.065 mmol) was added to a solution of 10 (16 mg, 0.03 mmol) in
CH3CN/H2O (0.7 mL/0.7 mL). After having been stirred at room tem-
perature for 4 min, the solution was filtered and neutralized with
Dowex-1X8 (OHꢀ). The mixture was filtered and concentrated in
vacuo. The residue was eluted through a chromatography column
(Sephadex LH-20, MeOH) and then a Dowex-1X8 (Clꢀ) column
(H2O) to afford 11 (8 mg, 63%) as a white solid. [a]2D6 =+79.4 (c=
0.30, MeOH); 1H NMR (300 MHz, CD3OD): d=6.28 (brs, 1H), 5.08
(d, J=3.3 Hz, 0.5H), 4.68 (d, J=8.1 Hz, 0.4H), 4.15 (m, 1H), 3.96 (m,
3H), 3.83 (m, 3H), 3.70 (m, 2H), 3.54 (m, 1H), 3.40 (s, 9H), 2.00 ppm
(s, 3H); 13C NMR (100 MHz, CD3OD): d=172.5, 94.6, 90.9, 72.9, 72.6,
72.5, 71.7, 70.9, 69.8, 68.9, 60.6, 60.5, 54.8, 54.4, 53.4, 21.2 ppm;
HRMS (MALDI): m/z: calcd for C17H33N2O10: 425.2139 [M]+; found:
425.2130.
p-Methoxyphenyl
3,4,6-tri-O-benzyl-2-trimethylammonium-2-
deoxy-b-d-glucopyranosyl-(1!4)-3,6-di-O-benzyl-2-acetamino-2-
deoxy-b-d-glucopyranoside (16): Et3N (0.2 mL) and propane-1,3-
dithiol (0.4 mL) were added to
a
solution of 15[17] (97 mg,
0.10 mmol) in pyridine/H2O (4 mL/1 mL). The solution was stirred
at room temperature for 1 h and was then concentrated in vacuo
to give a residue, which was purified by silica gel column chroma-
tography (petroleum ether/EtOAc/Et3N 1:2:0.03) to give a white
solid (88 mg, 94%). (iPr)2Net (0.3 mL) and CH3I (2 mL) was added to
a solution of the white solid (85 mg, 0.09 mmol) in dry THF (3 mL).
After having been stirred at room temperature for 40 h, the mix-
ture was filtered, concentrated in vacuo, and eluted through a
chromatography column (Sephadex LH-20, CH2Cl2/MeOH 1:1) to
provide 16 (83 mg, 93%) as a white solid. [a]2D5 =ꢀ20.3 (c 0.70,
p-Methoxyphenyl 2-trimethylammonium-2-deoxy-b-d-glucopyra-
nosyl-(1!4)-2-acetamino-2-deoxy-b-d-glucopyranosyl)-(1!4)-2-
acetamino-2-deoxy-b-d-glucopyranoside (22): Compound 19
(200 mg, 0.14 mmol) was dissolved in EtOH (6 mL) at room temper-
ature, followed by addition of ethylenediamine (1.2 mL). After
having been heated at reflux at 1008C for 14 h, the mixture was
concentrated in vacuo to give a residue, which was used in the
next step without further purification.
1
Ac2O (2 mL) was added to a solution of the residue and DMAP
(36 mg, 0.30 mmol) in pyridine (6 mL). After having been stirred at
room temperature overnight, the solution was diluted with CH2Cl2
and washed with aqueous HCl (1n), saturated aqueous NaHCO3,
and brine. The organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo to give a residue, which was purified by
silica gel column chromatography (CH2Cl2/MeOH 80:1!60:1) to
give 20 (126 mg, 72% for two steps) as a slightly yellow solid:
[a]2D6 =ꢀ39.4 (c=0.3, CHCl3); 1H NMR (400 MHz, CDCl3): d=7.25
(m, 30H), 6.92 (d, J=8.8 Hz, 2H), 6.77 (d, J=9.2 Hz, 2H), 6.09 (d-
like, J=9.2 Hz, 1H), 5.07 (t, J=8.0 Hz, 1H), 4.88 (m, 2H), 4.79 (m,
3H), 4.56 (m, 5H), 4.45 (m, 2H), 4.35 (m, 4H), 4.07 (t, J=8.8 Hz,
1H), 3.96 (t, J=7.2 Hz, 1H), 3.89 (dd, J=2.8, 10.8 Hz, 1H), 3.76 (m,
4H), 3.62 (m, 7H), 3.49 (m, 1H), 3.38 (m, 2H), 3.24 (t, J=9.2 Hz,
1H), 3.14 (dd, J=1.2, 9.6 Hz, 1H), 1.97 (s, 3H), 1.96 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=171.2, 170.4, 170.3, 155.2, 151.3,
139.1, 138.0, 137.9, 137.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 118.2, 114.5, 100.9, 100.2,
100.0, 83.0, 79.1, 77.6, 77.3, 76.5, 75.4, 74.9, 74.8, 74.7, 74.4, 73.8,
73.7, 73.3, 73.2, 73.1, 72.5, 68.4, 68.2, 66.9, 55.9, 55.6, 52.9, 23.3,
23.2, 20.8 ppm; HRMS (MALDI): m/z: calcd for C73H81N5O17Na:
1322.5537 [M+Na]+; found: 1322.5520.
CHCl3); H NMR (300 MHz, CDCl3): d=7.29 (m, 21H), 7.14 (m, 4H),
6.97 (d, J=8.7 Hz, 2H), 6.77 (d, J=8.7 Hz, 2H), 5.33 (m, 2H), 4.76 (s,
2H), 4.64 (m, 2H), 4.53 (m, 3H), 4.45 (m, 5H), 4.07 (m, 5H), 3.74 (m,
6H), 3.59 (m, 1H), 3.49 (m, 1H), 3.33 (m, 1H), 2.95 (s, 9H), 1.99 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3): d=170.8, 155.3, 151.3, 138.4,
137.8, 137.7, 136.7, 136.3, 128.7, 128.5, 128.4, 128.2, 128.0, 127.9,
127.8, 127.7, 118.8, 114.5, 99.8, 93.5, 79.5, 78.3, 76.5, 74.9, 74.0, 73.6,
73.4, 73.3, 72.9, 72.6, 72.3, 69.8, 69.6, 55.7, 53.8, 23.6 ppm; HRMS
(MALDI): m/z: calcd for C59H69N2O11: 981.4906 [M]+; found:
987.4896.
p-Methoxyphenyl 2-trimethylammonium-2-deoxy-b-d-glucopyra-
nosyl-(1!4)-2-acetamino-2-deoxy-b-d-glucopyranoside
(17):
Compound 16 (68 mg, 0.07 mmol) was dissolved in MeOH (5 mL)
at room temperature, followed by addition of aqueous HCl (37%,
two drops) and Pd(OH)2/C (200 mg, 20%). After having been hy-
drogenated at room temperature for 4 h, the suspension was fil-
tered and neutralized with Dowex-1X8 (OHꢀ). Filtration, concentra-
tion in vacuo, and elution through a chromatography column (Se-
phadex LH-20, MeOH) gave 17 (30 mg, 81%) as a colorless solid.
1
[a]2D7 =ꢀ9.6 (c=0.90, MeOH); H NMR (300 MHz, CD3OD): d=6.94
(d, J=9.0 Hz, 2H), 6.82 (d, J=9.0 Hz, 2H), 5.39 (d, J=4.5 Hz, 1H),
4.94 (d, J=7.5 Hz, 1H), 3.93 (m, 5H), 3.83 (m, 2H), 3.73 (m, 6H),
3.58 (m, 2H), 3.34 (s, 9H), 2.01 ppm (s, 3H); 13C NMR (100 MHz,
CD3OD): d=172.5, 155.4, 151.6, 117.8, 114.1, 100.4, 95.7, 78.6, 77.6,
77.2, 75.0, 71.0, 69.9, 69.5„ 61.5, 60.6, 56.5, 54.7, 53.4, 21.7 ppm;
HRMS (MALDI): m/z: calcd for C24H39N2O11: 531.2561 [M]+; found:
531.2548.
Et3N (0.3 mL) and propane-1,3-dithiol (0.6 mL) were added to a so-
lution of the trisaccharide azide 20 (120 mg, 0.09 mmol) in pyri-
dine/H2O (4 mL/1 mL). The solution was stirred at room tempera-
ture for 3 h and then concentrated in vacuo to give a residue,
which was purified by silica gel column chromatography (CH2Cl2/
MeOH/Et3N 100:2:1) to give the corresponding amine as a white
solid. (iPr)2Net (0.6 mL) and CH3I (4 mL) was added to a solution of
the above white solid in dry THF (6 mL). After having been stirred
p-Methoxyphenyl 3,4,6-tri-O-benzyl-2-azido-2-deoxy-b-d-gluco-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-N-phthalimido-2-deoxy-b-d-
464
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 457 – 467

TMG-(GlcNAc)1–4 as Selective Inhibitors
at room temperature for 40 h, the mixture was filtered, concentrat-
ed in vacuo, and eluted through a column (Sephadex LH-20,
CH2Cl2/MeOH 1:1) to provide 21 (102 mg, 84% for two steps) as a
slightly yellow solid: [a]2D6 =ꢀ25.1 (c=0.3, CHCl3); 1H NMR
(400 MHz, CDCl3): d=7.26 (m, 30H), 6.95 (d, J=8.8 Hz, 2H), 6.74
(d, J=8.8 Hz, 2H), 6.54 (d-like, J=9.2 Hz, 1H), 5.26 (d, J=5.2 Hz,
1H), 5.20 (t, J=8.0 Hz, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.78 (dd, J=4.8,
13.2 Hz, 1H), 4.70 (brs, 2H), 4.59 (m, 4H), 4.45 (m, 6H), 4.27 (dd,
J=8.8, 16.8 Hz, 1H), 4.20 (t, J=8.4 Hz, 1H), 4.09 (m, 1H), 4.01 (m,
4H), 3.82 (m, 3H), 3.70 (m, 6H), 3.52 (m, 2H), 3.43 (m, 1H), 3.33
(brs, 1H), 2.92 (s, 9H), 1.95 (s, 3H), 1.94 (s, 3H), 1.88 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=169.9, 169.7, 169.6, 154.2, 150.2,
137.4, 136.8, 136.5, 135.7, 135.4, 127.6, 127.5, 127.4, 127.3, 127.0,
126.9, 126.8, 126.7, 126.6, 126.5, 126.4, 117.5, 113.5, 98.8, 92.4, 78.4,
78.1, 76.4, 75.4, 73.9, 73.6, 73.2, 72.6, 72.3, 72.2, 72.1, 71.8, 71.6,
71.3, 68.7, 68.0, 54.9, 54.6, 52.6, 52.2, 22.4, 22.2, 19.9 ppm; HRMS
(MALDI): m/z: calcd for C76H90N3O17: 1316.6244 [M]+; found:
1316.6265.
to a solution of the MP glycoside 19 (400 mg, 0.27 mmol) in tolu-
ene/CH3CN/H2O (2:3:2 mL). After having been stirred at room tem-
perature for 30 min, the solution was poured into ice water and ex-
tracted with CH2Cl2. The organic layer was washed with saturated
aqueous NaHCO3 and brine and dried over Na2SO4. Filtration, con-
centration in vacuo, and purification by silica gel column chroma-
tography (petroleum ether/EtOAc 3:2) gave the corresponding
lactol as a yellow solid (285 mg, 77%). o-Hexynylbenzoic acid
(51 mg, 0.25 mmol), DCC (64 mg, 0.32 mmol), and DMAP (39 mg,
0.32 mmol) were added to a solution of the above yellow solid in
dry CH2Cl2 (7 mL). The mixture was stirred at room temperature
overnight and filtered. The filtrate was concentrated in vacuo and
purified by silica gel column chromatography (petroleum ether/
EtOAc 3:1) to give 24 (292 mg, 90%) as a white solid: [a]2D7 =+1.7
(c=2.0, CHCl3); 1H NMR (300 MHz, CDCl3): d=7.70 (m, 8H), 7.30
(m, 29H), 6.92 (m, 2H), 6.75 (m, 3H), 6.56 (d, J=9.0 Hz, 1H), 5.83
(dd, J=9.3, 10.8 Hz, 1H), 5.26 (d, J=8.4 Hz, 1H), 4.82 (m, 4H), 4.50
(m, 10H), 4.19 (m, 3H), 4.08 (m, 1H), 3.90 (m, 1H), 3.68 (m, 4H),
3.55 (m, 2H), 3.39 (m, 3H), 3.24 (t, J=9.6 Hz, 1H), 3.12 (d-like, J=
9.6 Hz, 1H), 2.38 (t, J=7.5 Hz, 2H), 1.85 (s, 3H), 1.54 (m, 2H), 1.39
(m, 2H), 0.89 ppm (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=170.1, 167.5, 163.1, 138.7, 138.2, 138.1, 138.0, 137.9, 137.8,
134.5, 134.1, 133.6, 132.2, 131.7, 131.4, 130.7, 129.6, 128.5, 128.4,
128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.0,
126.9, 125.7, 125.5, 123.6, 100.9, 97.3, 97.1, 90.2, 83.2, 78.9, 77.7,
77.6, 77.3, 77.2, 75.4, 75.0, 74.8, 74.7, 74.5, 73.4, 73.1, 73.0, 72.7,
71.0, 68.3, 67.9, 67.5, 67.0, 56.1, 54.0, 30.6, 22.0, 20.5, 19.4,
13.6 ppm; HRMS (MALDI): m/z: calcd for C91H87N5O19Na: 1576.5887
[M+Na]+; found: 1576.5888.
K2CO3 (50 mg, 0.36 mmol) was added to a solution of 21 (95 mg,
0.072 mmol) in MeOH/CH2Cl2 (5 mL/2.5 mL). The mixture was
stirred at room temperature for 2 h and was then neutralized with
Dowex-50 (H+). The mixture was filtered and concentrated in
vacuo to give a white solid for the next step, which was used with-
out further purification.
The white solid was dissolved in MeOH/CH2Cl2 (3 mL/2 mL) at
room temperature, followed by addition of aqueous HCl (37%, two
drops) and Pd(OH)2/C (300 mg, 20%). After having been hydrogen-
ated at room temperature for 14 h, the suspension was filtered
and neutralized with Dowex-1X8 (OHꢀ). Filtration, concentration in
vacuo, and elution through a column (Sephadex LH-20, CH2Cl2/
MeOH 1:1) provided 22 (32 mg, 62% for two steps) as a white
solid: [a]2D4 =ꢀ13.3 (c=0.25, MeOH); 1H NMR (400 MHz, CD3OD):
d=6.85 (d, J=9.2 Hz, 2H), 6.72 (d, J=9.2 Hz, 2H), 5.28 (d, J=
4.4 Hz, 1H), 4.80 (d, J=8.4 Hz, 1H), 4.48 (d, J=8.0 Hz, 1H), 3.85 (m,
2H), 3.76 (m, 6H), 3.63 (m, 7H), 3.55 (m, 3H), 3.46 (m, 2H), 3.37 (m,
1H), 3.24 (s, 9H), 1.95 (s, 3H), 1.90 ppm (s, 3H); 13C NMR (100 MHz,
CD3OD): d=172.5, 172.3, 155.3, 151.6, 117.7, 114.1, 100.6, 100.5,
95.6, 79.6, 78.6, 77.6, 77.1, 75.1, 72.7, 70.9, 69.9, 69.3, 61.4, 60.5,
60.0, 56.3, 55.2, 54.7, 53.4, 21.7, 21.6 ppm; HRMS (MALDI): m/z:
calcd for C32H52N3O16: 734.3365 [M]+; found: 734.3342.
p-Methoxyphenyl 3,4,6-tri-O-benzyl-2-azido-2-deoxy-b-d-gluco-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-N-phthalimido-2-deoxy-b-d-
glucopyranosyl-(1!4)-3-O-acetyl-6-O-benzyl-2-N-phthalimido-2-
deoxy-b-d-glucopyranosyl-(1!4)-3-O-acetyl-6-O-benzyl-2-N-
phthalimido-2-deoxy-b-d-glucopyranosyl-(1!4)-3-O-acetyl-6-O-
benzyl-2-N-phthalimido-2-deoxy-b-d-glucopyranoside
(27):
Newly prepared PPh3AuOTf in CH2Cl2 (0.05m, 0.17 mL) was added
dropwise under argon at ꢀ308C to a stirred mixture of the donor
24 (130 mg, 0.084 mmol), the acceptor 25[17] (81 mg, 0.084 mmol),
and freshly activated MS (4 ꢁ, 250 mg) in dry CH2Cl2 (5 mL). As the
temperature was allowed to warm up naturally to ꢀ158C, further
newly prepared PPh3AuOTf in CH2Cl2 (0.05m, 0.17 mL) was added
dropwise to the mixture. The mixture was then allowed to warm
up naturally to room temperature and the stirring was continued
for 2 h. The mixture was then filtered and concentrated in vacuo.
The residue was purified by silica gel column chromatography (tol-
uene/EtOAc 6:1) to afford 27 (98 mg, 51%) and the glycal 26
(37 mg, 32%) as white solids.
2-Trimethylammonium-2-deoxy-b-d-glucopyranosyl-(1!4)-2-
acetamino-2-deoxy-b-d-glucopyranosyl-(1!4)-2-acetamino-2-
deoxy-d-glucopyranose (23): Ag(DPAH)2 (25 mg, 0.054 mmol) was
added to a solution of the MP glycoside 22 (10 mg, 0.014 mmol) in
CH3CN/H2O (0.5 mL/0.5 mL). After having been stirred at room tem-
perature for 4 min, the solution was filtered and neutralized with
Dowex-1X8 (OHꢀ). The mixture was filtered and concentrated in
vacuo. The residue was eluted through columns [Sephadex LH-20,
MeOH and then Dowex-1X8 (Clꢀ), H2O] to afford 23 (6 mg, 70%) as
a white solid: [a]2D1 =ꢀ7.5 (c=0.35, MeOH); 1H NMR (400 MHz,
CD3OD): d=5.36 (d, J=4.0 Hz, 1H), 5.08 (d, J=2.8 Hz, 0.3H), 4.52
(d, J=7.6 Hz, 1H), 4.49 (d, J=8.4 Hz, 0.7H), 3.92 (m, 2H), 3.80 (m,
10H), 3.61 (m, 6H), 3.32 (s, 9H), 2.03 (s, 3H), 2.00 ppm (s, 3H);
13C NMR (100 MHz, CD3OD): d=173.2, 172.4, 101.6, 95.7, 78.6, 77.6,
77.2, 75.0, 71.0, 69.9, 69.5, 69.2, 61.4, 60.4, 60.3, 56.6, 56.4, 54.0,
53.4, 53.3, 21.6, 21.1 ppm; HRMS (ESI): m/z: calcd for C25H46N3O15:
628.2938 [M]+; found: 628.2923.
Trisaccharide glycal 26: 1H NMR (400 MHz, CDCl3): d=7.81 (m,
8H), 7.25 (m, 25H), 6.93 (m, 2H), 6.75 (m, 3H), 6.55 (s, 1H), 5.83 (d,
J=5.2 Hz, 1H), 5.36 (d, J=8.0 Hz, 1H), 4.82 (m, 3H), 4.73 (m, 2H),
4.54 (d, J=10.8 Hz, 1H), 4.41 (m, 5H), 4.30 (m, 1H), 4.16 (m, 6H),
3.93 (dd, J=2.4, 11.2 Hz, 1H), 3.68 (m, 4H), 3.52 (m, 3H), 3.35 (dd,
J=8.4, 13.6 Hz, 1H), 3.25 (t, J=9.2 Hz, 1H), 3.15 (m, 1H), 1.86 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3): d=169.3, 166.7, 147.9, 137.6,
137.1, 137.0, 136.9, 136.7, 133.1, 132.6, 130.7, 127.4, 127.3, 127.2,
126.9, 126.8, 126.7, 126.6, 126.5, 126.4, 125.9, 122.5, 122.1, 105.2,
99.9, 96.8, 82.1, 76.7, 76.4, 76.1, 76.0, 74.4, 74.0, 73.8, 73.7, 73.6,
72.4, 72.3, 71.9, 68.5, 67.2, 66.7, 66.0, 65.9, 54.8, 19.7 ppm; HRMS
(ESI): m/z: calcd for C78H73N5O17Na: 1374.4958 [M+Na]+; found:
1374.4894.
3,4,6-Tri-O-benzyl-2-azido-2-deoxy-b-d-glucopyranosyl-(1!4)-
3,6-di-O-benzyl-2-acetamino-2-deoxy-b-d-glucopyranosyl-(1!4)-
3,6-O-benzyl-2-N-phthalimido-2-deoxy-b-d-glucopyranosyl
ortho-hexynylbenzoate (24): CAN (740 mg, 1.35 mmol) was added
Pentasaccharide 27: [a]2D4 =ꢀ12.7 (c=0.3, CHCl3); 1H NMR
(400 MHz, CDCl3): d=7.78 (m, 16H), 7.24 (m, 29H), 7.05 (t, J=
ChemBioChem 2011, 12, 457 – 467
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
465

Q. Yang, B. Yu et al.
7.6 Hz, 2H), 6.96 (t, J=7.6 Hz, 2H), 6.88 (m, 3H), 6.74 (m, 5H), 6.61
(m, 3H), 5.67 (d, J=8.4 Hz, 1H), 5.65 (dd, J=9.2, 10.8 Hz, 1H), 5.45
(dd, J=10.0, 19.6 Hz, 2H), 5.23 (d, J=8.0 Hz, 1H), 5.16 (d, J=
8.4 Hz, 1H), 5.12 (d, J=8.8 Hz, 1H), 4.76 (m, 4H), 4.54 (dd, J=11.2,
15.6 Hz, 2H), 4.39 (m, 10H), 4.30 (m, 2H), 4.05 (m, 8H), 3.83 (m,
1H), 3.66 (m, 6H), 3.52 (m, 4H), 3.30 (m, 6H), 3.10 (t-like, J=8.4 Hz,
2H), 3.00 (d-like, J=10.0 Hz, 1H), 2.71 (d-like, J=9.6 Hz, 1H), 1.78
(s, 3H), 1.77 (s, 3H), 1.68 ppm (s, 3H); 13C NMR (100 MHz, CDCl3):
d=169.3, 169.2, 167.1, 166.9, 166.7, 166.3, 154.5, 149.7, 137.7,
137.2, 137.1, 137.0, 136.9, 136.8, 133.1, 132.7, 130.7, 130.5, 127.5,
127.4, 127.3, 127.2, 127.0, 126.9, 126.8, 126.7, 126.6, 126.5, 126.4,
126.3, 126.2, 126.1, 126.0, 125.8, 122.6, 122.5, 122.4, 117.9, 113.4,
99.8, 96.4, 95.8, 95.6, 95.0, 82.2, 76.7, 76.5, 76.2, 74.4, 73.8, 73.7,
73.5, 73.3, 73.0, 72.8, 72.5, 72.4, 72.0, 71.8, 71.6, 71.3, 71.2, 70.2,
69.9, 69.8, 67.4, 66.9, 66.7, 66.6, 66.5, 66.0, 55.1, 54.5, 54.4, 54.3,
53.9, 19.5, 19.4 ppm; HRMS (MALDI): m/z: calcd for
C131H123N7O33Na: 2344.8098 [M+Na]+; found: 2344.8054.
20, CH2Cl2/MeOH 1:1) to provide 29 (70 mg, 89%) as a white solid:
1
[a]2D6 =ꢀ39.2 (c=0.3, CHCl3); H NMR (400 MHz, CDCl3/CD3OD 1:1):
d=7.22 (m, 40H), 6.86 (d, J=9.2 Hz, 2H), 6.68 (d, J=8.8 Hz, 2H),
5.21 (d, J=4.8 Hz, 1H), 5.04 (t, J=9.6 Hz, 1H), 4.96 (m, 2H), 4.88 (d,
J=8.4 Hz, 1H), 4.59 (m, 8H), 4.44 (m, 10H), 4.30 (dd, J=3.2,
12.0 Hz, 2H), 4.07 (m, 1H), 3.98 (m, 4H), 3.85 (m, 4H), 3.65 (m,
13H), 3.48 (m, 2H), 3.39 (m, 2H), 3.29 (m, 1H), 3.18 (m, 2H), 2.75 (s,
9H), 1.91 (s, 3H), 1.86 (s, 3H), 1.85 (s, 6H), 1.83 (s, 3H), 1.75 (s, 3H),
1.74 ppm (s, 3H); 13C NMR (100 MHz, CDCl3/CD3OD 1:1): d=171.3,
171.1, 170.9, 170.8, 170.5, 170.4, 170.3, 154.7, 150.6, 137.7, 137.4,
137.3, 137.2, 136.9, 136.7, 136.0, 135.9, 127.9, 127.8, 127.7, 127.6,
127.5, 127.3, 127.2, 127.1, 127.0, 126.9, 126.8, 126.7, 126.6, 126.5,
117.6, 113.7, 99.5, 99.4, 98.7, 98.4, 93.1, 78.8, 78.7, 77.1, 75.9, 74.5,
74.1, 73.9, 73.7, 73.5, 73.4, 73.3, 73.0, 72.6, 72.5, 72.4, 72.3, 72.2,
72.0, 71.9, 71.8, 69.2, 68.9, 67.5, 67.3, 67.2, 56.1, 54.6, 54.2, 53.9,
53.1, 52.3, 22.0, 21.7, 21.6, 21.5, 19.8, 19.7, 19.6 ppm; HRMS
(MALDI): m/z: calcd for C110H132N5O29: 1986.9026 [M]+; found:
1986.9003.
p-Methoxyphenyl 2-trimethylammonium-2-deoxy-b-d-glucopyra-
nosyl-(1!4)-2-acetamino-2-deoxy-b-d-glucopyranosyl-(1!4)-2-
acetamino-2-deoxy-b-d-glucopyranosyl-(1!4)-2-acetamino-2-
deoxy-b-d-glucopyranosyl-(1!4)-2-acetamino-2-deoxy-b-d-glu-
copyranoside (30): Compound 27 (150 mg, 0.065 mmol) was dis-
solved in EtOH (4.5 mL) at room temperature, followed by addition
of ethylenediamine (0.9 mL). After having been heated at reflux at
1008C for 14 h, the mixture was concentrated in vacuo to give a
residue, which was used in the next step without further purifica-
tion.
K2CO3 (60 mg, 0.43 mmol) was added to a solution of 29 (100 mg,
0.050 mmol) in MeOH/CH2Cl2 (6:3 mL). The mixture was stirred at
room temperature for 6 h and was then neutralized with Dowex-
50 (H+). Filtration, concentration in vacuo, and elution through a
column (Sephadex LH-20, CH2Cl2/MeOH 1:1) gave a white solid
(83 mg, 88%) for the next step. The white solid (70 mg,
0.038 mmol) was dissolved in MeOH/CH2Cl2 (3:2 mL) at room tem-
perature, followed by addition of aqueous HCl (two drops, 37%)
and Pd(OH)2/C (300 mg, 20%). After having been hydrogenated at
room temperature for 14 h, the suspension was filtered and neu-
tralized with Dowex-1X8 (OHꢀ). Filtration, concentration in vacuo,
and elution through a column (Sephadex LH-20, MeOH/H2O 1:1)
provided 30 (36 mg, 84%) as a white solid: [a]2D2 =ꢀ27.6 (c=0.1,
MeOH/H2O 2:1); 1H NMR (400 MHz, CD3OD/D2O 1:1): d=7.02 (d,
J=7.6 Hz, 2H), 6.94 (d, J=8.0 Hz, 2H), 5.42 (brs, 1H), 5.00 (d, J=
7.2 Hz, 1H), 4.61 (brs, 3H), 3.80 (m, 33H), 3.37 (s, 9H), 2.09 (s, 9H),
2.06 ppm (s, 3H); 13C NMR (100 MHz, CD3OD/D2O 1:1): d=173.9,
154.9, 151.3, 118.1, 114.7, 101.3, 100.4, 95.6, 79.2, 79.1, 78.3, 77.0,
76.6, 74.8, 74.6, 72.3, 72.2, 70.8, 69.6, 69.1, 61.3, 60.4, 59.9, 56.1,
55.5, 55.1, 53.8, 44.5, 22.0, 21.9 ppm; HRMS (ESI): m/z: calcd for
C48H78N5O26: 1140.4944 [M]+; found: 1140.4930.
Ac2O (1.5 mL) was added to a solution of the residue and DMAP
(27 mg, 0.23 mmol) in pyridine (4.5 mL). After having been stirred
at room temperature overnight, the solution was diluted with
CH2Cl2 and washed with aqueous HCl (1n), saturated aqueous
NaHCO3, and brine. The organic layer was dried over Na2SO4, fil-
tered, and concentrated in vacuo to give a residue, which was pu-
rified by silica gel column chromatography (CH2Cl2/MeOH 50:1!
30:1) to give 28 (95 mg, 75% for two steps) as a white solid:
[a]2D7 =ꢀ45.5 (c=0.3, CHCl3); 1H NMR (400 MHz, CDCl3): d=7.30
(m, 40H), 6.92 (d, J=9.2 Hz, 2H), 6.78 (d, J=9.2 Hz, 2H), 6.24 (d,
J=9.6 Hz, 1H), 5.44 (d, J=8.8 Hz, 1H), 5.06 (t, J=7.6 Hz, 1H), 4.94
(d, J=12.4 Hz, 1H), 4.88 (d, J=6.8 Hz, 1H), 4.79 (m, 6H), 4.68 (m,
2H), 4.52 (m, 5H), 4.36 (m, 8H), 4.23 (d, J=8.4 Hz, 1H), 4.17 (d, J=
8.0 Hz, 1H), 4.02 (t, J=8.4 Hz, 1H), 3.89 (m, 5H), 3.76 (m, 4H), 3.60
(m, 12H), 3.33 (dd, J=8.8, 17.6 Hz, 2H), 3.21 (m, 3H), 3.10 (m, 1H),
2.05 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.90 (s, 3H), 1.75 (s, 3H), 1.67
(s, 3H), 1.66 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=170.3,
170.2, 169.9, 169.5, 169.3, 168.9, 168.8, 154.1, 150.3, 138.3, 137.0,
136.9, 136.8, 136.6, 136.3, 128.1, 128.0, 127.9, 127.8, 127.6, 127.5,
127.4, 127.3, 127.2, 127.0, 126.9, 126.8, 126.7, 126.6, 126.5, 126.3,
117.1, 113.5, 99.9, 99.8, 99.7, 99.2, 98.4, 82.0, 78.4, 76.6, 76.3, 75.4,
74.4, 73.9, 73.8, 73.6, 73.1, 72.9, 72.6, 72.5, 72.3, 72.2, 72.1, 72.0,
71.9, 71.8, 71.4, 67.5, 67.2, 66.5, 66.3, 65.9, 55.2, 54.6, 52.8, 51.5,
22.4, 22.2, 22.1, 22.0, 19.8, 19.6 ppm; HRMS (MALDI): m/z: calcd for
C107H123N7O29Na: 1992.8244 [M+Na]+; found: 1922.8258.
2-Trimethylammonium-2-deoxy-b-d-glucopyranosyl-(1!4)-2-
acetamino-2-deoxy-b-d-glucopyranosyl-(1!4)-2-acetamino-2-
deoxy-b-d-glucopyranosyl-(1!4)-2-acetamino-2-deoxy-b-d-glu-
copyranosyl-(1!4)-2-acetamino-2-deoxy-d-glucopyranose (31):
Ag(DPAH)2 (25 mg, 0.054 mmol) was added to a solution of the MP
glycoside 30 (11 mg, 0.0096 mmol) in CH3CN/H2O (0.7:0.7 mL).
After having been stirred at room temperature for 3 min, the solu-
tion was filtered and neutralized with Dowex-1X8 (OHꢀ). The mix-
ture was filtered and concentrated in vacuo. The residue was
eluted through columns [Sephadex LH-20 MeOH/H2O 1:1 and then
Dowex-1X8 (Clꢀ), H2O] to afford 31 (7 mg, 71%) as a white solid:
[a]2D3 =ꢀ12.4 (c=0.2, MeOH/H2O 1:1); 1H NMR (400 MHz, CD3OD/
D2O 1:1): d=5.42 (d, J=4.0 Hz, 1H), 5.18 (d, J=1.6 Hz, 0.4H), 4.68
(d, J=8.0 Hz, 0.4H), 4.60 (m, 3H), 4.05 (t, J=8.0 Hz, 1H), 3.91 (m,
15H), 3.67 (m, 12H), 3.55 (m, 2H), 3.37 (s, 9H), 2.10 (s, 3H), 2.09 (s,
6H), 2.07 ppm (s, 3H); 13C NMR (125 MHz, CD3OD/D2O 1:1): d=
175.3, 175.2, 102.7, 97.0, 80.6, 79.7, 78.4, 78.0, 76.0, 73.6, 72.2, 71.4,
70.9, 70.6, 70.4, 62.8, 61.7, 61.3, 57.5, 56.5, 55.1, 46.1, 23.4 ppm;
HRMS (MALDI): m/z: calcd for C41H72N5O25: 1034.4547 [M]+; found:
1034.4511.
Et3N (0.2 mL) and propane-1,3-dithiol (0.4 mL) were added to a so-
lution of the pentasaccharide azide 28 (78 mg, 0.040 mmol) in pyri-
dine/H2O (3 mL/0.8 mL). The solution was stirred at room tempera-
ture for 3 h and then concentrated in vacuo to give a residue,
which was purified by silica gel column chromatography (CH2Cl2/
MeOH/Et3N 90:3:1) to give the corresponding amine as a white
solid. (iPr)2Net (0.45 mL) and CH3I (3 mL) were added to a solution
of the above white solid in dry THF (4.5 mL). After having been
stirred at room temperature for 40 h, the mixture was filtered, con-
centrated in vacuo, and eluted through a column (Sephadex LH-
Expression and purification of recombinant OfHex1: OfHex1 was
overexpressed in Pichia pastoris strain GS115 as a C-terminal His6-
tagged fusion protein and purified by ammonium sulfate precipita-
466
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ChemBioChem 2011, 12, 457 – 467

TMG-(GlcNAc)1–4 as Selective Inhibitors
[1] D. J. Mahuran, Biochim. Biophys. Acta Mol. Basis Dis. 1999, 1455, 105–
138.
[2] D. E. Hunt, D. Gevers, N. M. Vahora, M. F. Polz, Appl. Environ. Microbiol.
2008, 74, 44–51.
[3] O. Plihal, J. Sklenar, K. Hofbauerova, P. Novak, P. Man, P. Pompach, D.
Kavan, H. Ryslava, L. Weignerova, A. Charvatova-Pisvejcova, V. Kren, K.
Bezouska, Biochemistry 2007, 46, 2719–2734.
[4] H. Merzendorfer, L. Zimoch, J. Exp. Biol. 2003, 206, 4393–4412.
[5] R. Strasser, J. Singh, J. S. Bondili, J. Schoberer, B. Svoboda, E. Liebminger,
J. Glossl, F. Altmann, H. Steinkellner, L. Mach, Plant Physiol. 2007, 145,
5–16.
[6] I. Tews, A. Perrakis, A. Oppenheim, Z. Dauter, K. S. Wilson, C. E. Vorgias,
Nat. Struct. Biol. 1996, 3, 638–648.
[7] B. L. Mark, D. J. Vocadlo, S. Knapp, B. L. Triggs-Raine, S. G. Withers,
M. N. G. James, J. Biol. Chem. 2001, 276, 10330–10337.
[8] T. Maier, N. Strater, C. G. Schuette, R. Klingenstein, K. Sandhoff, W.
Saenger, J. Mol. Biol. 2003, 328, 669–681.
[9] B. L. Mark, D. J. Mahuran, M. M. Cherney, D. L. Zhao, S. Knapp, M. N. G.
James, J. Mol. Biol. 2003, 327, 1093–1109.
[10] N. Ramasubbu, L. M. Thomas, C. Ragunath, J. B. Kaplan, J. Mol. Biol.
2005, 349, 475–486.
tion, metal chelating chromatography, and anion exchange chro-
matography as described previously.[26]
Enzymatic assay: The activities of b-N-acetyl-d-hexosaminidases
were measured with p-nitrophenyl N-acetyl-d-glucosaminide (pNP-
b-GlcNAc, Sigma–Aldrich, USA) as substrate at 258C. b-N-Acetyl-d-
hexosaminidase (BtHex) from Bos taurus and b-N-acetyl-d-hexosa-
minidase (CeHex) from Canavalia ensiformis were purchased from
Sigma–Aldrich. b-N-Acetyl-d-hexosaminidase (SpHex) from Strepto-
myces plicatus was purchased from New England Biolabs (USA).
BtHex, CeHex, and SpHex were assayed in sodium citrate buffer
(40 mm) at their optimal pH values (BtHex 4.5; CeHex 5.0;
SpHex 4.0). OfHex1 was assayed in sodium phosphate buffer
(40 mm, pH 7.0).[26,27] The reactions were terminated by addition of
equal amounts of Na2CO3 (0.5m) and absorbance at 405 nm was
monitored with a microplate reader (TECAN, Switzerland). For
determinations of Ki values, different amount of inhibitors were
preincubated with the enzymes for 10 min before the addition of
substrate. The Ki values were calculated with Dixon plots. The
values represent the means of three independent experiments.
[11] M. J. Lemieux, B. L. Mark, M. M. Cherney, S. G. Withers, D. J. Mahuran,
M. N. G. James, J. Mol. Biol. 2006, 359, 913–929.
Molecular docking: The crystal structures of SpHex from bacteria
(PDB ID: 1HP5) and HsHexB from human (PDB ID: 1NOU) were
used as 3D models for docking studies. The newest AutoDock 4.2
program was applied to dock the TMG-chitotriomycin into the
binding sites of the two enzymes.[28] The Gasteiger charges were
used for TMG-chitotriomycin and the active torsions were assigned
with the aid of the newest ADT program. The residues at the en-
trance of each binding pocket were designated as flexible (SpHex:
Glu314, Trp361, and Trp408, and HsHexB: Glu355 and Trp424) and
allowed for conformational search in the docking process. The geo-
metric centers of the binding sites were chosen as the grid center,
the grid size was set to 80ꢂ80ꢂ80, and the used grid space was
the default value of 0.375 ꢁ. In the docking process, a conforma-
tional search was performed for the TMG-chitotriomycin by the
Solis and Wets local search method, and the Lamarkian genetic
algorithm (LGA) was applied for conformational searching of the
binding complexes of TMG-chitotriomycin with SpHex and HsHexB.
To achieve optimal docking results, the population size of the
Lamarkian genetic algorithm was set as 150 and the maximum
number of evaluations was set as 25000000. The interaction ener-
gies that resulted from probing the TMG-chitotriomycin with
SpHex and HsHexB were assessed by the newest adopted Auto-
Dock scoring function. Cluster analysis was performed on a set of
50 candidates for the docked complex structures, and the best one
was selected according to the interaction energy and the comple-
mentarity in the binding pocket inspected visually.
[12] T. Sumida, R. Ishii, T. Yanagisawa, S. Yokoyama, M. Ito, J. Mol. Biol. 2009,
392, 87–99.
[13] S. Knapp, D. Vocadlo, Z. N. Gao, B. Kirk, J. P. Lou, S. G. Withers, J. Am.
Chem. Soc. 1996, 118, 6804–6805.
[14] M. Horsch, L. Hoesch, A. Vasella, D. M. Rast, Eur. J. Biochem. 1991, 197,
815–818.
[15] T. Aoyagi, H. Suda, K. Uotani, F. Kojima, T. Aoyama, K. Horiguchi, M.
Hamada, T. Takeuchi, J. Antibiot. 1992, 45, 1404–1408.
[16] H. Usuki, T. Nitoda, M. Ichikawa, N. Yamaji, T. Iwashita, H. Komura, H.
Kanzaki, J. Am. Chem. Soc. 2008, 130, 4146–4152.
[17] Y. Yang, Y. Li, B. Yu, J. Am. Chem. Soc. 2009, 131, 12076–12077.
[18] a) O. Kanie, S. C. Crawley, M. M. Palcic, O. Hindsgaul, Carbohydr. Res.
1993, 243, 139; b) J. Hansson, P. J. Garegg, S. Oscarson, J. Org. Chem.
2001, 66, 6234.
[19] a) T. Stauch, U. Greilich, R. R. Schmidt, Liebigs Ann. 1995, 2101; b) M. C.
Galan, A. P. Venot, J. Glushka, A. Imberty, G. J. Boons, J. Am. Chem. Soc.
2002, 124, 5964.
[20] a) H. Staudinger, J. Meyer, Helv. Chim. Acta 1919, 2, 635–646; b) E.
Saxon, C. R. Bertozzi, Science 2000, 287, 2007–2010.
[21] a) Y. H. Zhang, J. Gaekwad, M. A. Wolfert, G. J. Boons, J. Am. Chem. Soc.
2007, 129, 5200–5216; b) Y. Y. Yang, S. Ficht, A. Brik, C. H. Wong, J. Am.
Chem. Soc. 2007, 129, 7690–7701.
[22] a) K. Kloc, J. Mlochowski, L. Syper, Chem. Lett. 1980, 725; b) T. Noshita, T.
Sugiyama, Y. Kitazumi, T. Oritani, Tetrahedron Lett. 1994, 35, 8259.
[23] a) W. Kinzy, R. R. Schmidt, Liebigs Ann. Chem. 1985, 1537–1545; b) L. X.
Wang, C. Li, Q. W. Wang, Y. Z. Hui, Tetrahedron Lett. 1993, 34, 7763–
7766; c) L. Cirillo, E. Bendini, A. Molinaro, M. Parrilli, Tetrahedron Lett.
2010, 51, 1117–1120.
[24] a) Y. Li, Y. Yang, B. Yu, Tetrahedron Lett. 2008, 49, 3604–3608; b) Y. Li, X.
Yang, Y. Liu, C. Zhu, Y. Yang, B. Yu, Chem. Eur. J. 2010, 16, 1871–1882;
c) Y. Yang, Y. Li, B. Yu, Tetrahedron Lett. 2010, 51, 1504–1507.
[25] a) W. M. Reckendorf, S. Sander, Tetrahedron Lett. 1988, 29, 2047–2050;
b) L. Falkowski, M. Beszczynski, A. Jarzebski, B. Stefanska, Pol. J. Chem.
1983, 57, 1353–1355.
[26] T. Liu, F. Y. Liu, Q. Yang, J. Yang, Protein Expression Purif. 2009, 68, 99–
103.
[27] Q. Yang, T. Liu, F. Y. Liu, M. B. Qu, X. H. Qian, FEBS J. 2008, 275, 5690–
5702.
[28] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K.
Belew, A. J. Olson, J. Am. Chem. Soc. 1998, 120, 1639–1662.
Acknowledgements
Financial support was provided by the National Special Fund for
State Key Laboratory of Bioreactor Engineering (ECUST) (Shang-
hai, China), the National Key Project for Basic Research
(2010CB126100 and 2010CB833202), and the National Natural
Science Foundation of China (20932009, 20921091). The authors
thank Dr. Alan K. Chang (Dalian University of Technology) for his
help in the final revision of the manuscript.
Keywords: enzymes
·
hexosaminidase
·
hydrolases
·
Received: September 18, 2010
inhibition · TMG-chitotriomycin
Published online on December 23, 2010
ChemBioChem 2011, 12, 457 – 467
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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