Synthesis, conformational study, glycosidase inhibitory activity and molecular docking studies of dihydroxylated 4- and 5-amino-iminosugars

Article abstract of DOI:10.1016/j.carres.2015.03.004

An efficient methodology for the synthesis of new amino iminosugars 6a, 7a and 8, starting from d-glucose, is reported. The conformational study using ¹H NMR data showed that the amino iminosugar 6a exists in the ²C₅ while

Full text of DOI:10.1016/j.carres.2015.03.004

Carbohydrate Research 408 (2015) 25e32
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis, conformational study, glycosidase inhibitory activity and
molecular docking studies of dihydroxylated 4- and 5-amino-
iminosugars
Vijay M. Kasture a, Navnath B. Kalamkar a, Roopa J. Nair b, Rakesh S. Joshi c,
,
*
Sushma G. Sabharwal b, Dilip D. Dhavale a
a Department of Chemistry, Garware Research Centre, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India
b Division of Biochemistry, Department of Chemistry, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India
c Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient methodology for the synthesis of new amino iminosugars 6a, 7a and 8, starting from D-
glucose, is reported. The conformational study using 1H NMR data showed that the amino iminosugar 6a
exists in the 2C5 while; the 7a and 8 exist in the 5C2 conformation. The inhibition activities with different
glycosidases showed that 6a and 7a are poor glycosidase inhibitors. However, amino iminosugar 8
Received 29 December 2014
Received in revised form
3 March 2015
Accepted 4 March 2015
Available online 12 March 2015
showed selective inhibition against the
b-galactosidase (IC50¼43
m
M, Ki¼153
mM). These results are
substantiated by the molecular docking studies.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Iminosugar
Amino piperidine
Glycosidase inhibitor
Chiron approach
Reductive amino-cyclization
Molecular docking
1. Introduction
deoxynojirimycin (trade name Zavesca) are being used for the
treatment of type-II diabetes and Gaucher diseases, respectively.14
In the search for promising glycosidase inhibitors, modification of
natural and unnatural iminosugars with the variation of stereo-
chemistry and replacement of hydroxyl substituent with hydrox-
yalkyl,15e22 aminoalkyl,23,24 amine,25e31 halogen32e34 alkyl
groups35e39 and study of their structureeactivity relationship is the
established protocol in synthetic carbohydrate chemistry. Amongst
these, mono- and bi-cyclic iminosugars in which hydroxyl group is
substituted with amino group-amino iminosugars are known to be
selective glycosidase inhibitors.40,41 For example, Pandey et al. re-
ported the synthesis of amino iminosugar 1 (Fig. 1) that showed
Polyhydroxylated cyclic compounds with the nitrogen atom in
the ring, commonly called iminosugars, are the most notable car-
bohydrate mimetics, which comprises the low molecular weight
heterocyclic molecules such as azitidines, pyrrolidines, piperidines,
azepanes and bi-cyclic (nitrogen atom at the ring fusion) pyrroli-
zidines, indolizidines and quinolizidines etc.1e7 This class of mole-
cules are valuable for the basic understanding of glycobiology due
to their action as inhibitors of glycosidases in the biological
processes such as digestion, endoplasm reticulum associated
degradations and lysosomal catabolism of glycoconjugates.8e11 As a
result, iminosugars are investigated as promising candidates for
the treatment of carbohydrate disorder mediated diseases such
as diabetes, obesity, cancer and viral infections.12,13 For
example, piperidine iminosugars namely N-hydroxyethyl-1-
deoxynojirimycin (trade name Miglitol) and N-butyl-1-
good
synthesis of amino dihydroxypiperidine 2-an amino analog of iso-
a
-glucosidase inhibitory activity.42 Bols et al. reported the
fagomine, which showed selective inhibition activity against the b-
glycosidase.43 Fleet. et al. reported the C-2 amino analog of 1-deoxy
nojirimycin 3a (R¼H) that showed a potent anticancer activity with
50% inhibition at 6
inhibition of
mM with Ki 0.9 mM and also showed a selective
b
-N-acetylaminoglucosaminidase.44 Tropak et al. and
Wong. et al. reported a series of potent amino-piperidine com-
* Corresponding author.
E-mail address: ddd@chem.unipune.ac.in (D.D. Dhavale).
pounds 3aec for Tay-Sachs and Sandhoff diseases.45,46 In addition,
http://dx.doi.org/10.1016/j.carres.2015.03.004
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

26
V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
synthesis and glycosidase inhibitory activity of iminosugars
H
N
H2
N
(piperidine triols) 6b and 7b.53 The structureeactivity relationship
(SAR) activity study presented here that the substitution of C4eOH
in 6b and 7b with the amino group having same configuration di-
minishes the glycosidase inhibitory activity. While, substitution of
the C3eOH in 7b with the amino group having inverted orientation
Cl
O
Cl
NH2
NH3
HO
OH
OH
OH
led to the potent selective inhibition against the
Our results are depicted herein.
b-Galactosidase.
1
2
N
R1
N
OH
NH2
OH
R2
2. Results and discussion
HO
O
NH
As shown in Scheme 1,
deoxy-5-O-p-toluenesulphonyl-
D
-glucose was converted to a 3-azido-3-
NHAc
HO
N
a-D
-xylofuranose 9 using reported
OH
method.54 Treatment of 9 with NaN3 in DMF at 90 ꢀC afforded a
diazido-compound 10 in 93% yield.55,56 Hydrolysis of 1,2-acetonide
group in 10 using TFA/H2O provided hemiacetal that was directly
subjected for reductive amino-cyclization using 10% Pd/C, H2, (80
psi) in methanol to afford amino-iminosugar 6a in 78% (in two
steps).
3a, R1 = R2=H
3b, R1 = alkyl, R2=H
3c, R1=H, R2=OH
Cl
H
N
N
N
NH2
N
5 Cisapride
4 BMS-690514
OMe
In order to synthesize amino iminosugar 7a,
converted to the 1,2:5,6-di-O-isopropylidene-3-deoxy-3-
benzyloxycarbonylamino- -allofuranose 11 using the reported
D-glucose was
H
H
N
H
N
a-D
N
method (Scheme 1).57 Selective deprotection of 5,6-acetonide
functionality with 1% H2SO4 afforded diol 12 in 88% yield.58 Diol
12 on treatment with sodium metaperiodate in acetoneewater
followed by reduction with sodium borohydride in methanole-
water afforded a primary alcohol 13 in 87% yield (two steps).59 The
primary hydroxyl group of 13 was converted to a tosyl derivative 14
(TsCl, Py)59 and then to a C-5 azido compound 15 (NaN3, DMF) in
87% yield. Hydrolysis of the 1,2-acetonide group in 15 using TFA/
H2O followed by reductive amino-cyclization (10% Pd/C, H2, 80 psi)
afforded an amino iminosugar 7a in 68% yield (two steps).
1
2
3
6
5
4
OH
HO
HO
OH
HO
NH2
R
R
OH
7a, R=NH2
7b, R=OH
6a, R=NH2
6b, R=OH
8
Fig. 1. Natural and synthesized iminosugars.
NH2
1. TFA/H2O
2. H2, Pd/C, MeOH,
12 h
N3
TsO
NaN3, DMF,
90 oC, 8 h,
Ref. 54
O
HO
OH
O
D-Glucose
O
O
93%
two steps 78 %
N
H
N3
N3
O
O
Ref. 57
9
10
1. NaIO4,
Acetone;Water,
rt, 3h
6a
R
i. TFA/H2O
ii. H2, Pd/C,
MeOH, 24 h
O
O
HO
HO
1% H2SO4,
NH2
O
MeOH:Water,
O
O
O
HO
OH
O
O
2.NaBH4,
two steps
68 %
rt, 12h, 88%
HN
Cbz
HN
Cbz
HN
Cbz
MeOH:Water,
O
N
H
7a
O
O
0 oC-rt , 3 h,
11
12
two steps 87%
13 R = OH
14 R = OTs
15 R = N3
Ts-Cl, Py, DCM,
rt, 8 h, 93%
NaN3, DMF,90oC,
12h, 94%
Scheme 1. Syntheses of amino iminosugars 6a, 7a.
the amino iminosugar framework is sometimes also part of drug
candidates, such as, BMS-690514 4 (developed for the treatment of
non-small cell lung cancer) and Cisapride 5 (a potent gastric pro-
kinetic agent with reduced dopamine D2 receptor antagonist ac-
tivity).47,48 As a part of our continuous efforts in the area of
iminosugars,49e52 we now report a modest approach, in gram scale,
for the synthesis of new amino iminosugars 6a, 7a and 8 from D-
glucose and study of their glycosidase inhibitory activity along with
correlation using molecular docking. Earlier we have reported the
For the synthesis of amino iminosugar 8, D-glucose was trans-
formed to 6-O-tosyl derivative 16 as per reported method
(Scheme 2).60 Intermolecular SN2 displacement of a 6-O-tosyl
group in 16 with NaN3 in DMF at 90 ꢀC afforded an azido-alcohol 17
in 92% yield.61,62 The secondary C-5 hydroxy functionality in 17 was
protected as benzyl ether using benzyl bromide and sodium hy-
dride to get compound 18 in 93% yield. Hydrolysis of 1,2-acetonide
group in 18 with TFA/H2O provided hemiacetal that was directly
subjected for the oxidative cleavage using NaIO4/H2O to afford

V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
27
Scheme 2. Synthesis of amino iminosugar 8.
unstable aldehyde, which on reductive amino cyclization (10% Pd/C,
H2, 80 psi) in methanol afforded amino-iminosugar 8 in 52% yield
(in three steps).
While, in case of the symmetrical amino iminosugar 7a, the H2a
and H6a (both protons are equivalent) showed a doublet of doublet
at 2.98
d with J values 14.5 (J2ae2e, J6ae6e) and 1.4 (J2ae3e, J6ae5e) Hz,
indicating the equatorial orientation of H3 and H5 (equivalent
protons), respectively. In accordance with this, the H3 appeared as a
narrow multiplet (WH¼9.6 Hz) and the H4 appeared as a triplet at
2.1. Conformational analysis
3.35 d with small coupling constant value of 3.5 Hz indicating their
It is known that configuration and conformation of the sub-
stituents in iminosugars are the decisive factors in their specific
enzymatic activities.63,64 Therefore, the conformations of amino
iminosugars 6a, 7a and 8 were studied based on the 1H NMR data
wherein; the assignment of chemical shifts to different protons and
coupling constant values were determined using the spin-
decoupling experiments and are given in Table 1. In case of the
amino-iminosugars 6a, 7a and 8 two conformations 2C5 and 5C2
were considered. In conformation 5C2, the J values between axially
orientated H2a, H3, H4, H5 and H6a is expected to be large
(~8e11 Hz) as compared to the conformation 2C5 in which the H3,
H4, H5 and H6 are equatorially oriented and expected to show
small J values (~1e4 Hz). In case of a symmetrical amino-
iminosugar 6a, the equivalent protons H2a and H6a showed
relative equatorial orientation. Based on this data the 2C5 confor-
mation was assigned for compound 7a. In case of amino iminosugar
8, all protons were found to be non-equivalent. The H6a proton
appeared as a doublet of doublet at 2.23
and 10.0 Hz (J6ae5a), indicating the axial orientation of H5. In
analogy with this, H4 showed a doublet of doublet at 3.38 with J
d with J values 12.9 (J6ae6e)
d
values 9.5 and 2.8 Hz, indicating the axial orientation of H5 and
equatorial orientation of H3. This requires the 2C5 conformation for
the compound 8. This fact is supported by the appearance of H2a as
doublet of doublet at 2.68
d with J values 14.3 (J2ae2e) and 1.4
(J2ae3e) indicates the equatorial orientation of the H3 proton, con-
firming the 2C5 conformation for the compound 7a.
Thus, it is interesting to note that an amino iminosugar 6a exists
in 5C2 conformation while, 7a and 8 exist in 2C5. In all the preferred
conformations the amino group attains an equatorial position. This
fact can be rationalized as follows. In compound 6a, all substituents
are equatorial thus stabilizing the 5C2 conformation. In compound
7a the C-4 amino substituent is residing axially in 5C2 conformation
that destabilizes the conformation by 1.2 kcal/mol65 however, in
case of 2C5 conformation the equatorial amino functionality stabi-
lizes the conformation, which is augmented by the intramolecular
hydrogen bonding between the 1,3-diaxial hydroxyl groups as
shown in Fig. 2. An analogous observation was noticed in amino
iminosugar 8 wherein an equatorial orientation of amino group
stabilizes the 2C5 over 5C2 conformation.
doublet of doublet at 2.36 d with J values 11.9 (J2ae2e, J6ae6e) and 10.9
(J2ae3a, J6ae5a) Hz indicating the axial orientation of H3 and H5
(equivalent protons), respectively. This suggests the 5C2 confor-
mation for a compound 6a. In agreement with this, the H3 (axial
proton) showed a doublet of doublet of doublet at 3.35
values 10.9, 9.5 and 4.5 Hz and the H4 (axial proton) showed a
triplet at 2.55
with J value 10.9 Hz thus confirming the 5C2
d with J
d
conformation for 6a (Fig. 2).
Table 1
1H NMR chemical shifts (
d
) and coupling constants (J) in compounds 6a, 7a and 8
Compound 7a Compound 8
H2a, 2.98 (dd) H2a, 2.68 (dd)
Compound 6a
2.2. Biological activities
H2a, 2.36
d
(dd)
d
d
H2a-H2e¼11.9
H2a-H2e¼14.5
H2a-H3e¼1.4
H2a-H2e¼14.3
H2a-H3e¼1.4
2.2.1. Glycosidase inhibition
H2a-H3a¼10.9
H6a, 2.36
d
(dd)
H6a, 2.98
d
(dd)
H2e, 2.83e3.08
H5a, 2.83e3.08
H6e, 2.83e3.08
H3e, 3.87e3.93
d
d
d
d
(m)
(m)
(m)
(m)
Synthesized amino-iminosugars 6a, 7a, 8 were screened for the
H6a-H6e¼11.9
H6a-H6e ¼ 14.5
H6a-H5e ¼ 1.4
H2e, 3.17 (dd)
H2e-H2a¼14.5
H2e-H3e¼3.5
H6e, 3.17 (dd)
H6e-H6a ¼ 14.5
H6e-H5e ¼ 3.5
glycosidase inhibitory profile against
dase, -mannosidase and -galactosidase (isolated from almond
seeds), -mannosidase and N-acetyl- -glucosaminidase (isolated
from Jack bean seeds), -glucosidase(isolated from Rice seeds),
galactosidase (isolated from Bovine liver), -fucosidase and N-
acetyl- -glucosaminidase (isolated from Bovine kidney), -amy-
lase(isolated from Geobacillus JN704808) and -glucosidase (Yeast
b-galactosidase, b-glucosi-
H6a-H5a¼10.9
a
a
H2e, 3.03
d (dd)
a
b-D
H2e-H2a¼11.9
H4a, 3.38 d (dd)
a
b-
H2e-H3a¼4.5
H4a-H3a¼2.8
H6e, 3.03
d (dd)
H4a-H5a¼9.5
a
H6e-H6a ¼ 11.9
H6a, 2.23 d (dd)
b
-D
a
H6e-H5a ¼ 4.5
H6a-H6e¼12.9
a
H3a, 3.35
d
(ddd)
H3e, 4.02e4.08
H5e, 4.02e4.08
d
(m)
(m)
H6a-H5a¼10.0
-procured from Sigma Chemical Co). The IC50 values and Ki values
were determined only for the amino-iminosugars showing high
inhibitory activity. Amongst three amino iminosugars 6a, 7a and 8,
the 6a, 7a showed no inhibition against any of the enzyme under
study. However, the amino iminosugar 8 found to be selective in-
H3a-H2a¼10.9
H3a-H2e¼4.5
H3a-H4a¼9.5
d
H4a, 3.35
d (t)
H4a-H3a¼3.5
H5a, 3.35
d (ddd)
H4a-H5a¼3.5
H5a-H6a ¼ 10.9
H5a-H6e ¼ 4.5
H5a-H4a ¼ 9.5
hibitor of b-galactosidase.
H4a, 2.55
d (dd)
These results were compared with the parent iminosugars
namely piperidines triols 6a, 7a that were synthesized by us. The
SAR study indicated that the substitution of C4eOH group in 6b as
H4a-H3a¼9.5
H4a-H5a¼9.5

28
V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
Fig. 2. 2C5 and 5C2 conformations of amio-iminosugars 6a, 7a and 8.
well as in 7b, with the amino group having same orientation
(retention of configuration), giving amino iminosugars 6a and 7a,
diminished the glycosidase inhibitory activity. The substitution of
C3eOH in 7b, with amino group with inverted orientation giving
amino iminosugar 8, showed potent selective inhibition against the
intermolecular hydrogen bonds and presence of electrostatic pi-
interaction augments the stronger binding of the compound 8 to
the active site of beta-galactosidase.
Binding energy is calculated after multiple interactions using
Lamarckian Genetic Algorithm. It is well accepted method for
binding energy calculation. Although, the difference among the
binding energy is low, the density of intermolecular hydrogen
bonds is higher in compound 8. According to the thermodynamics
of binding, hydrogen bond mainly contributes to the strong bind-
ing. This is supported by docking studies.
In case of compound 8, axial eOH makes polar contact with
GLU267 and thus projects remaining equatorial functional groups
to establish the maximum contact. In compound 8, axial eOH and
other equatorial groups lead to synergistic effect on binding.
Furthermore, in case 6a all of the groups are equatorial, giving to
least number of contacts. Similarly, 7a makes polar contact via axial
eOH group, thus resulted ineNH2 equatorial unreactive in terms of
polar contact.
b
-Galactosidase (Bovine liver) (IC50¼43
m
M, Ki¼153
mM).
The glycosidase assays were repeated three times. All the
compounds were tested for their inhibitory activity initially at
10 mM concentration (the highest concentration used). At this
concentration no significant inhibition was observed due to 6a and
7a, although compound 8 showed inhibitory activity. Hence, for 8
inhibitions was studied at different concentration between 20
mM
and 10 mM. Even at 20 M compound 8 showed good inhibitory
m
activity with IC 50 value of 43 mM and Ki value 153 mM.
2.2.2. Molecular docking
Interactions of synthesized amino iminosugars 6a, 7a and 8 with
the binding pocket of bovine liver -galactosidase were analyzed
b
and depicted using molecular docking studies. Galactosidase
inhibitory activity of compound 8 showed its efficiency as selective
3. Conclusion
b
-galactosidase inhibitors. Homology model for the Bovine liver b-
An efficient process from
grams quantity is reported for the synthesis of amino iminosugars
6a, 7a and 8 in overall yields of 23%, 16% and 14% from -glucose,
respectively. The conformational study indicates that the amino-
iminosugar 6a exists in 2C5 conformation while; 7a and 8 prefer
5C2 conformation. It has been noticed that the amino group in
amino iminosugar dictates the conformation and the stable
conformation is the one in which the amino group is equatorially
orientated. Out of all three amino iminosugars, compound 8
D-glucose that can be scaled up to
galactosidase was predicted and quality of model was assessed
using ProSA and Ramchandran plot analysis. Docking studies of the
amino-iminosugars 6a, 7a and 8 showed that, 5-amino iminosugar
D
8 showed strong binding affinity (ꢁ5.8 kcal/mol) with
b-galacto-
sidase. Binding score for the compound 6a and 7a is ꢁ5.0
and ꢁ5.1 kcal/mol, respectively, indicating that these derivatives
have lower binding affinity as compared to the compound 8.
Binding free energies are corroborated with inhibition kinetic
studies, where the compound
competitive inhibitor of bovine liver
of 6a, 7a and 8 are conserved, but there are significant variations in
term of molecular interactions with active site of -galactosidase
8
was found to be selective
showed selective b-galactosidase inhibitory activity, while; com-
pounds 6a and 7a were found to be poor inhibitors of glycosidases.
b-galactosidase. Binding poses
b
(Fig. 3). Analysis of polar contacts and other interactions such as
electrostatic and pi-interaction displayed that amino iminosugar 8
form multiple hydrogen bonds with GLU128, ASN186, GLU187,
4. Experimental section
4.1. General experimental methods
GLU267 and TYR269 residues of
electrostatic interactions with GLU128, GLU187 and GLU267. Strong
binding of the compound 8 with active site of -galactosidase is
also contributed by its position in the pi-interaction in space of
TRP272 and TYR484. Whereas the compound 6a form hydrogen
bond with ASN186, GLU187 and GLU267, and the compound 7a
contributes to hydrogen bonds with GLU187 only resulting into
weak binding. Although all three compounds are forming hydrogen
b-galactosidase. There are also
Melting points were recorded with melting point apparatus and
are uncorrected. IR spectra were recorded with an FTIR as a thin
b
film or using KBr pellets and are expressed in cmꢁ1 1H (500/
.
300 MHz) and 13C (125/75 MHz) and NMR spectra were recorded
using CDCl3, D2O as solvent. Chemical shifts were reported in
d
units (ppm) with reference to TMS as an internal standard and J
values are given in Hz. Elemental analyses were carried out with a
C, H-analyzer. Thin layer chromatography was performed on pre-
bonds with binding pocket of b-galactosidase, the higher number of

V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
29
Fig. 3. Molecular docking study of bovine liver
b-galactosidase with derivatives of amino iminosugar (6a, 7a and 8) (A), (C) and (E) binding poses of compound 6a, 7a and 8, while
(B), (D) and (F). Intermolecular hydrogen bonding and other interactions between
b-galactosidase and amino iminosugar derivatives complex.
coated plates. Column chromatography was carried out with silica
gel (100e200 mesh). The reactions were carried out in oven-dried
glassware under dry N2. Methanol and THF were purified and dried
before use. Distilled n-hexane and ethyl acetate were used for
column chromatography. After quenching of the reaction with
water, the work-up involves washing of combined organic layers
with water, brine, drying over anhydrous sodium sulphate and
evaporation of the solvent at reduced pressure. We have used AR
grade methanol and tetrahydrofuran. For drying THF: AR grade THF
was stirred for 12 h on LAH. Then it was refluxed over sodium wire
and benzophenone (indicator) and distilled whenever needed in
presence of nitrogen atmosphere. Methanol was refluxed over
CaCO3 for 6 h, distilled out. Then refluxed with magnesium in the
presence of catalytic amount of iodine and distilled out. The imi-
nosugar was dissolved in double distilled water and filtered
4.1.2. (3R, 4r, 5S)-4-Amino-3,5-dihydroxy-piperidine (6a)
An ice-cold solution of 10 (0.20 g, 0.83 mmol) in TFA-H2O (2 mL,
3:2) was stirred for 15 min at 0 ꢀC and at 25 ꢀC for 3 h. Trifluoro-
acetic acid (TFA) was co-evaporated with toluene at rotary evapo-
rator using high vacuum to furnish hemiacetal as a thick liquid
(crude wt¼0.150 g). A solution of hemiacetal (0.150 g, 0.75 mmol)
and 10% Pd/C (0.030 g) in methanol (12 mL) was hydrogenolyzed at
80 psi for 12 h at 25 ꢀC. The catalyst was filtered through Celite.
Evaporation of solvent and column purification (MeOH/30%
NH4OH¼20/1) afforded 6a (0.086 g, 78%) as a pale yellow solid. Mp
146 ꢀC (decomposition); ½aꢂD26 0.0 (c 1.0, H2O); Rf 0.2 (MeOH/30%
NH4OH): 10/1); IR (KBr): 3347e2989 (broad), 1110, cmꢁ1 1H NMR
;
(300 MHz, D2O): 3.35 (ddd, J¼10.9, 9.5, 4.5 Hz, 2H), 3.03 (dd, J¼10.9,
4.3 Hz, 2H), 2.55 (t, J¼9.5 Hz, 1H), 2.35 (dd, J¼11.9, 10.9 Hz, 2H); 13
C
NMR (75 MHz, D2O): 71.0, 60.4, 49.4; Anal. Calcd. For C5H12N2O2: C,
45.44; H, 9.15; Found: C, 45.40; H, 9.11.
through the syringe filters (NY0.45 mm, make-ALLPURE).
4.1.1. 3,5-Diazido-3,5-dideoxy-1,2-O-isopropylidene-
xylofuranose (10)
a
-
D
-
4.1.3. 3-Benzyloxycarbonylamino-3-deoxy-1,2-O-isopropylidene-
-allofuranose (12)
A solution of compound 11 (5.00 g, 12.72 mmol) in MeOH
a-
D
To a stirred solution of compound 9 (2.00 g, 5.42 mmol) in DMF
(20 mL), NaN3 (0.53 g, 8.13 mmol) was added. Reaction mixture was
stirred at 90 ꢀC for 8 h. Reaction mixture was cooled, diluted with
ethyl acetate (20 mL) and reaction mixture was extracted with
water. Evaporation of solvent and column purification (n-hexane/
ethyl acetate¼8.0/2.0) afforded 10 (1.20 g, 93%) as pale yellow oil.
½aꢂD26 ꢁ55 (c 1.0, CHCl3); [lit56 ½aꢂDꢁ60 (c 1.0, CHCl3); Rf 0.4 (ethyl
acetate/n-hexane: 4/6); IR (neat): 2989, 2105, 1083 cmꢁ1; 1H NMR
(300 MHz, CDCl3): 5.91 (d, J¼3.8 Hz, 1H), 4.69 (d, J¼3.8 Hz, 1H), 4.34
(dt, J¼6.6, 3.4 Hz,1H), 4.01 (d, J¼3.4 Hz,1H), 3.62 (dd, J¼12.4, 6.6 Hz,
1H), 3.48(dd, J¼12.4, 6.6 Hz, 1H), 1.52 (s, 3H), 1.35 (s, 3H); 13C NMR
(75 MHz, CDCl3):112.4,104.6, 83.3, 77.5, 65.9, 49.5, 26.5, 26.11; Anal.
Calcd. For C8H12N6O3: C, 40.00; H, 5.04; Found: C, 40.03; H, 5.08.
(50 mL) and 1% H2SO4 in water (2 mL) was stirred at room tem-
perature for 12h. Reaction was quenched with saturated potassium
carbonate. Methanol was evaporated and reaction mixture was
extracted with chloroform (3ꢃ20 mL). Evaporation of solvent and
column purification (n-hexane/ethyl acetate¼7.0/3.0) afforded 12
as thick liquid (3.92 g, 88%). ½aꢂ2D4 þ29.3(c¼2.3, CH2Cl2); Rf 0.5 (ethyl
acetate); IR (neat): 3440e3190,1724,1521,1379, 875 cmꢁ1; 1H NMR
(500 MHz, CDCl3þdrop of D2O) 7.31e7.51 (m, 5H), 5.84 (d, J¼3.8 Hz,
1H), 5.15 (ABq, J¼12.1 Hz, 2H), 4.65 (dd, J¼5.1, 4.0 Hz,1H), 4.07e3.96
(m, 1H), 3.80 (dd, J¼9.5, 4.3 Hz, 3H), 3.66 (dd, J¼11.2, 5.9 Hz, 1H),
1.55 (s, 3H), 1.35 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.4, 26.5, 55.2,
63.3, 67.6, 72.3, 79.3, 79.9, 103.8, 112.7, 128.3, 128.4 (str), 128.6 (str),

30
V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
135.8, 156.9; Anal. Calcd. For C17H23NO7: C, 57.78; H, 6.56; Found: C,
57.81; H, 5.59.
Trifluoroacetic acid was co-evaporated with toluene at rotary
evaporator using high vacuum to furnish hemiacetal as a thick
liquid (crude wt¼0.24 g). A solution of hemiacetal (0. 24 g,
0.78 mmol) and 10% Pd/C (0.040 g) in methanol (12 mL) was
hydrogenolyzed at 80 psi for 24 h at 25 ꢀC. The catalyst was filtered
through Celite. Evaporation of solvent and column purification
(MeOH/CHCl3/30%NH4OH¼20/5/1) afforded 7a (0.077 g, 68%) as a
off white solid. Mp 245 ꢀC (decomposition); ½aꢂ2D6 0.0 (c 1.1, H2O); Rf
0.2 (MeOH/30% NH4OH): 10/1); IR (KBr): 3447e2992 (broad), 1198,
4.1.4. 3-Benzyloxycarbonylamino-3-deoxy-1,2-O-isopropylidene-
-ribofuranose (13)
To an ice-cooled solution of 12 (3.00 g, 8.4 mmol) in acetonee-
a-
D
water (30 mL, 5:1) was added sodium metaperiodate (2.73 g,
12.74 mmol) and the solutionwas stirred for 3h at 25 ꢀC. Solvent was
evaporated on rotary evaporator and the residue was extracted with
chloroform (10 mLꢃ3). Solvent was evaporated to get crude alde-
hyde (2.52 g). To an ice cooled solution of crude aldehyde (2.52 g,
7.8 mmol) in MeOH-water (25 mL, 4:1), sodium borohydride (0.44 g,
11.8 mmol) was added in portions. Reaction was stirred at room
temperature for 3h. Reaction was quenched with saturated ammo-
nium chloride. Methanol was evaporated and reaction mixture was
extracted with chloroform (3ꢃ15 mL). Evaporation of solvent and
column purification (ethyl acetate/n-hexane: 4/6) afforded 13 as
liquid (2.38 g, 87%). ½aꢂD26 þ116 (c 1.0, MeOH); [lit59 ½aꢂ2D6 þ109.4 (c 1.0,
MeOH); Rf 0.4 (ethyl acetate/n-hexane; 5:5); IR (neat): 3456, 3279,
1712,1529,1186 cmꢁ1; 1H NMR (300 MHz, CDCl3) 7.28e7.48 (m, 5H),
5.85 (d, J¼3.8 Hz, 1H), 5.38 (bd, J¼8.6 Hz, 1H), 5.14 (s, 2H), 4.62 (t,
J¼4.7 Hz, 1H), 4.09 (dt, J¼9.1, 5.3 Hz, 1H), 3.60e3.98 (m, 3H), 2.96 (s,
exchangeable with D2O, 1H), 1.46 (s, 3H), 1.28 (s, 3H); 13C NMR
(75 MHz, CDCl3): 26.3, 26.4, 52.9, 60.5, 67.5, 78.9, 80.2, 104.1, 112.6,
128.3,128.4 (str),128.6 (str),135.9,156.6 Anal. Calcd. For C16H21NO6:
C, 59.43; H, 6.55; Found: C, 59.47; H, 6.58.
1005 cmꢁ1 1H NMR (300 MHz, D2O): 4.02e4.08 (m, 2H), 3.35 (t,
;
J¼3.5, 1H), 3.17 (dd, J¼14.5, 3.5 Hz, 2H), 2.98 (dd, J¼14.5, 1.4, 2H);
13C NMR (75 MHz, D2O): 65.3, 50.6, 48.1; Anal. Calcd. For
C5H12N2O2: C, 45.44; H, 9.15; Found: C, 45.49; H, 9.20.
4.1.8. 3,6-Diazido-3,6-dideoxy-1,2-O-isopropylidene-a-D-
glucofuranose (17)
To a stirred solution of compound 16 (5.0 g, 12.53 mmol) in DMF
(50 mL), NaN3 (1.22 g, 18.79 mmol) was added. Reaction mixture
was stirred at 90 ꢀC for 12 h. Reaction mixture was cooled, diluted
with ethyl acetate (30 mL) and combined organic layer was
extracted with water (3ꢃ10 mL). Evaporation of solvent and col-
umn purification (n-hexane/ethyl acetate¼7/3) afforded compound
17 as a thick liquid (3.11 g, 92%). ½aꢂ2D4 ꢁ35.6 (c 0.7, CH2Cl2): Rf 0.4
(ethyl acetate/n-hexane: 3/7); IR (neat): 3400, 2106, 1163 cmꢁ1; 1H
NMR (300 MHz, CDCl3þdrop of D2O) 5.86 (d, J¼3.4 Hz, 1H), 4.64 (d,
J¼3.4 Hz, 1H), 4.09e4.22 (m, 2H), 3.98 (m, 1H), 3.65 (dd, J¼12.6,
2.4 Hz, 1H), 3.5 (dd, J¼12.6, 6.2 Hz, 1H), 1.52 (s, 3H), 1.32 (s, 3H); 13
C
4.1.5. 3-Benzyloxycarbonylamino-3-deoxy-5-O-p-
NMR (75 MHz, CDCl3): 26.2, 26.8, 54.8, 66.6, 68.6, 79.0, 83.2, 104.9,
112.4 Anal. Calcd. For C9H14N6O4: C, 40.00; H, 5.22; Found: C, 40.03;
H, 5.24.
toluenesulphonyl-1,2-O-isopropylideneꢁ
a-D-ribofuranose (14)
To a stirred solution of compound 13 (2.0 g, 6.19 mmol) in dry
dichloromethane (20 mL) and pyridine (2 mL), p-toluenesulphonyl
chloride (1.53 g, 8.05 mmol) was added at 0 ꢀC. Reaction mixture
was stirred for 8 h. Evaporation of solvent and column purification
(n-hexane/ethyl acetate¼8/2) afforded tosyl derivative 14 as a thick
colorless liquid (2.75 g, 93%); ½aꢂ2D8 þ55.3 (c 0.57, CH2Cl2); Rf 0.5
(ethyl acetate/n-hexane:4/6); IR (neat): 3389, 1739, 1353,
1186 cmꢁ1; 1H NMR (300 MHz, CDCl3) 7.81 (d, J¼8.2 Hz, 2H), 7.32 (d,
J¼8.2 Hz, 2H), 7.39 (s, 5H), 5.73 (d, J¼3.3 Hz, 1H), 5.22 (bd, J¼8.6 Hz,
1H), 5.10 (s, 2H), 4.55 (t, J¼3.8 Hz, 1H), 4.38 (bd, J¼11.0 Hz, 1H),
3.82e4.15 (m, 3H), 2.22 (s, 3H), 1.26 (s, 3H), 1.15 (s, 3H); 13C NMR
(125 MHz, CDCl3): 21.6, 26.4, 26.5, 53.8, 67.4, 68.8, 77.7, 78.5, 104.2,
112.8, 128.1, 128.4, 128.5, 128.7, 129.8, 132.8, 136.0, 144.8, 155.7 Anal.
Calcd. For C23H27NO8S: C, 57.85; H, 5.70; Found: C, 57.83; H, 5.68.
4.1.9. 5-O-Benzyl-3,6-diazido-3,6-dideoxy-1,2-O-isopropylidene-
-glucofuranose (18)
To an ice cooled suspension of NaH (0.33 g, 13.88 mmol) in dry
a-
D
THF (20 mL) compound 17 (2.50 g, 9.25 mmol) was added in THF
(20 mL) under nitrogen atmosphere over 10 min. Benzyl bromide
(1.6 ml, 13.88 mmol) was added dropwise to the reaction mixture.
The reaction was stirred at room temperature for 12h. The reaction
mixture was quenched by saturated aqueous ammonium chloride,
THF was evaporated and reaction mixture was extracted with ethyl
acetate (3ꢃ15 mL). Evaporation of solvent and column chroma-
tography (ethyl acetate/n-hexane: 20/80) afforded benzyl ether 18
as a white solid (3.10 g, 93%). m. p.73e74 ꢀC; ½aꢂD28 ꢁ48.9 (c 1.6,
CH2Cl2); Rf 0.5 (ethyl acetate/n-hexane: 30/70); IR (KBr): 2985,
4.1.6. 5-Azido-5-deoxy-3-benzyloxycarbonylamino-3-deoxy-1,2-O-
2100, 1452, 1377, 873 cmꢁ1 1H NMR (300 MHz, CDCl3): 7.23e7.45
;
isopropylidene
a
-
D
-ribofuranose (15)
(m, 5H), 5.84 (d, J¼3.6,1H), 4.77 (d, J¼10.9 Hz,1H), 4.64 (d, J¼3.6 Hz,
1H), 4.58 (d, J¼10.9 Hz, 1H), 4.29 (dd, J¼9.1, 2.9 Hz, 1H), 4.09 (d,
J¼2.9 Hz, 1H), 3.77e3.82 (m, 1H), 3.73 (dd, J¼13.2, 2.6 Hz, 1H), 3.41
(dd, J¼13.2, 4.1 Hz, 1H), 1.45 (s, 3H), 1.26 (s, 3H); 13C NMR (75 MHz,
CDCl3): 26.8, 26.1, 50.7, 65.5, 72.0, 75.2, 77.4, 82.7, 104.1, 112.0, 127.6
(str), 128.0 (str), 136.8; Anal. Calcd. For C16H20N6O4: C, 53.33; H,
5.59; Found: C, 53.38; H, 5.64.
To a stirred solution of compound 14 (2.50 g, 5.23 mmol) in DMF
(25 mL), NaN3 (0.51 g, 7.84 mmol) was added. Reaction mixture was
stirred at 90 ꢀC for 12 h. Reaction mixture was cooled, diluted with
ethyl acetate (20 mL) and combined organic layer was extracted
with water (3ꢃ10 mL). Evaporation of solvent and column purifi-
cation (n-hexane/ethyl acetate¼8/2) afforded compound 15 as a
thick liquid (1.72 g, 94%). ½aꢂ2D8 þ74.5¼(c 0.55, CH2Cl2); Rf 0.4 (eth-
ylacetate/n-hexane: 30/70); IR (neat): 3300e3190 (broad), 2987,
2102, 1728, 1518,1379, 873 cmꢁ1; 1H NMR (300 MHz, CDCl3): 7.18 (s,
5H), 5.78 (d, J¼3.3 Hz, 1H), 5.42 (bd, J¼8.6 Hz, 1H), 5.10 (s, 2H), 4.52
(d, J¼3.5 Hz, 1H), 4.18e3.88 (m, 1H), 3.88e3.74 (m, 1H), 3.64e3.52
(m, 1H), 3.38e3.24 (m, 1H), 1.25 (s, 3H), 1.45 (s, 3H); 13C NMR
(75 MHz, CDCl3): 26.3, 26.5, 51.15, 54.2, 67.3, 78.8, 78.9, 104.2, 112.6,
128.4 (str), 128.6 (str), 136.0, 155.9; Anal. Calcd. For C16H20N4O5: C,
55.17; H, 5.79; Found: C, 55.14; H, 5.77.
4.1.10. (3R, 4S, 5R)-5-Amino-3,4-dihydroxy-piperidine (8)
An ice-cold solution of 18 (0.30 g, 0.86 mmol) in TFA-H2O
(3 mL, 3:2) was stirred for 15 min at 0 ꢀC and at 25 ꢀC for 3 h.
Trifluoroacetic acid was co-evaporated with toluene at rotary
evaporator using high vacuum to furnish hemiacetal as a thick
liquid (crude wt¼0.24 g). To an ice-cooled solution of hemiacetal
(0.24 g, 0.75 mmol) in acetoneewater (5 mL, 5:1) was added so-
dium metaperiodate (0.24 g, 1.12 mmol) and the solution was
stirred for 1 h at 25 ꢀC. Solvent was evaporated on rotary evapo-
rator and the residue was extracted with chloroform (10 mLꢃ3).
Usual workup afforded a thick liquid (0.20 g, 95%). Asolution of
aldehyde (0.20 g, 0.68 mmol) and 10% Pd/C (0.040 g) in methanol
4.1.7. (3R, 4s, 5S)-4-Amino-3,5-dihydroxy-piperidine (7a)
An ice-cold solution of 15 (0.30 g, 0.86 mmol) in TFA-H2O (3 mL,
3:2) was stirred for 15 min at 0 ꢀC and at 25 ꢀC for 3 h.

V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32
31
(12 mL) was hydrogenolyzed at 80 psi for 24 h at 25 ꢀC. The
Acknowledgements
catalyst was filtered through Celite. Evaporation of solvent and
column purification (MeOH/30%NH4OH¼20/1) afforded 8 (0.057 g,
52%) as a pale yellow syrup. ½aꢂ2D8ꢁ9.2 (c 1.3, H2O); Rf 0.2 (MeOH/
CHCl3/30% NH4OH) 10/2/1); IR (neat): 3347, 2989, 1110,
1005 cmꢁ1; 1H NMR (300 MHz, D2O): 3.87e3.97 (m, 1H), 3.38 (dd,
J¼9.5, 2.8 Hz, 1H), 2.83e3.08 (m, 3H), 2.68 (dd, J¼14.3, 1.4 Hz, 1H),
2.23 (dd, J¼12.9, 10.0 Hz, 1H); 13C NMR (75 MHz, D2O): 74.9, 68.2,
49.2, 48.9, 48.9; Anal. Calcd. For C5H12N2O2: C, 45.44; H, 9.15;
Found: C, 45.49; H, 9.21.
We are grateful to Prof. M. S. Wadia for helpful discussions. V. M.
K. and N. B. K. are thankful to the CSIR, New Delhi for the senior
research fellowship. We are thankful to the Department of Science
and Technology, New Delhi (Project File No. SR/S1/OC-20/2010) for
the financial support.
Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra of
compounds 10, 12, 13, 14, 15, 17, 18, 6a, 7a, 8) associated with this
article can be found at http://dx.doi.org/10.1016/j.carres.2015.03.
004.
4.2. Glycosidase inhibition assay
The substrates p-nitrophenyl-
phenyl- -glucopyranoside, p-nitrophenyl-
side, p-nitrophenyl- -galactopyranoside, p-nitrophenyl-
mannopyranoside, p-nitrophenyl- -fucopyranoside and p-nitro-
phenyl N-acetyl- -glucosaminide were procured from sigma
a
-D-glucopyranoside, p-nitro-
b
-D
a-D-galactopyrano-
b-
D
a-D-
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