Synthesis, computational study and glycosidase inhibitory activity of polyhydroxylated conidine alkaloids - A bicyclic iminosugar

Article abstract of DOI:10.1039/c004690f

New bicyclic conidine iminosugars 1d and 1e were synthesized from d-glucose. Thus, d-glucose was converted to sugar β-amino acids 3a and 3b in good yields. Individual treatment of 3a/3b with the Mukaiyama reagent afforded sugar β-lactams 4a/4b that on reduction with LiAlH ₄/AlCl₃ gave azetidines 5a/5b with a sugar appendage. Reductive aminocyclization of sugar azetidines 5a/5b afforded the corresponding conidine iminosugars 1d/1e. Based on the ¹H NMR and DFT calculation studies the conformation of 1d was assigned as half chair A2 and that of 1e as a boat B2. The glycosidase inhibitory activities of 1d and 1e such as α-mannosidase, α-glucosidase and α-galactosidase were studied. The α-amylase activity was compared with acarbose. Compound 1d was found to be a moderate inhibitor of glycosidases while 1e was noticed to be a good inhibitor of α-mannosidase and a moderate inhibitor of other glycosidases. These results were substantiated by molecular docking studies using WHAT IF software and the AUTODOCK 3.0 program. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c004690f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis, computational study and glycosidase inhibitory activity of
polyhydroxylated conidine alkaloids—a bicyclic iminosugar†
Shrihari P. Sanap,a Sougata Ghosh,b Amit M. Jabgunde,a Rahul V. Pinjari,c Shridhar P. Gejji,c Shailza Singh,b
Balu A. Chopadeb and Dilip D. Dhavale*a
Received 26th March 2010, Accepted 13th May 2010
First published as an Advance Article on the web 2nd June 2010
DOI: 10.1039/c004690f
New bicyclic conidine iminosugars 1d and 1e were synthesized from D-glucose. Thus, D-glucose was
converted to sugar b-amino acids 3a and 3b in good yields. Individual treatment of 3a/3b with the
Mukaiyama reagent afforded sugar b-lactams 4a/4b that on reduction with LiAlH4/AlCl3 gave
azetidines 5a/5b with a sugar appendage. Reductive aminocyclization of sugar azetidines 5a/5b
afforded the corresponding conidine iminosugars 1d/1e. Based on the 1H NMR and DFT calculation
studies the conformation of 1d was assigned as half chair A2 and that of 1e as a boat B2. The
glycosidase inhibitory activities of 1d and 1e such as a-mannosidase, a-glucosidase and
a-galactosidase were studied. The a-amylase activity was compared with acarbose. Compound 1d was
found to be a moderate inhibitor of glycosidases while 1e was noticed to be a good inhibitor of
a-mannosidase and a moderate inhibitor of other glycosidases. These results were substantiated by
molecular docking studies using WHAT IF software and the AUTODOCK 3.0 program.
inhibitors includes competitive, non-/un-competitive and mixed
Introduction
inhibition of enzymes. In general, enzyme catalyzed glycosidic
Glycosidic bond cleavage plays a pivotal role in a wide range
bond cleavage of pyranosides is mediated by two catalytic car-
of metabolic processes that include digestion, glycoconjugate
boxylic acid residues and typically proceeds through a positively
charged oxo-carbenium ion transition state.10 Many natural and
synthetic glycosidase inhibitors mimic the charge buildup and/or
the conformational distortion of the transition state and thus
act as glycosidase inhibitors. In this direction, the search for
an a-glucosidase inhibitor led to the discovery of acarbose
(trade names: Glucobay/Precose/Prandase)—a pseudotetrasac-
charide as a potent sucrase inhibitor, voglibose (trade name:
Voglib)—a polyhydroxylated aminocyclitol and N-hydroxyethyl-
1-deoxynojirimycin (Trade name: Glyset/Miglitol)—a synthetic
iminosugar used in the treatment of type II diabetes mellitus.11
Iminosugars are polyhydroxylated mono- or bi-cyclic com-
pounds with the nitrogen atom in the ring for monocyclic and at
the ring fusion for bicyclic compounds with promising glycosidase
inhibitory activity.12 A number of bi-cyclic iminosugars such as
six–five ring fused indolizidine (castanospermine, swainsonine,
lentiginosine), six–six ring fused quinolizidine, five–five ring fused
pyrrolizidine iminosugars (australine, casuarine, hyacinthacine)
and seven–five ring fused perhydroazaazulenes have been either
isolated or synthesized and evaluated for their glycosidase in-
hibitory activity. However, six–four ring fused iminosugars are
rare and only one example of a b-lactam-iminosugar hybrid 1a
(Fig. 1), which is a competitive potent galactosidase inhibitor,
has been reported by Pandey et al.13 In general, the six–four ring
fused compounds with nitrogen atom at the ring fusion of type
1b are called conidine14 alkaloids and its 25 oligomer is known
to be heparin antagonist.15 Although, a few examples of alkyl
substituted conidines are known;16 polyhydroxylated conidines of
general type 1c that resembles bi-cyclic iminosugars, to the best of
our knowledge, are unknown. As a part of our continuing interest
in mono- and bi-cyclic iminosugars,17 we are reporting herewith
the synthesis, computational studies and glycosidase inhibitory
lysosomal catabolism and glycoprotein biosynthesis/degradation
within the endoplasmic reticulum (ER).1 However, increased
levels of glycosidases like a-mannosidase, a-galactosidase and a-
glucosidase leads to severe pathological complications. For exam-
ple, a-mannosidase is involved in the N-glycosylation pathway2
by trimming Glc3Man9GlcNAc2 moieties in human ER, how-
ever, abnormalities in this pathway lead to disease progression,
metastasis and poor clinical outcome in the case of breast,
colon and skin cancers.3 a-Galactosidase regulates catabolism
of glycosphingolipids in the lysosome and any disorder in this
process causes lysosomal storage disease (LSD)4 and Fabry’s
disease.5 a-Glucosidase is involved in non-insulin dependent
diabetes mellitus (NIDDM) or type II diabetes6 and inhibition
of a-glucosidase/a-amylase significantly decreases post-prandial
hyperglycemia (PPHG) in the treatment of type II diabetic
patients. Thus, discovery of a suitable inhibitor of the glycosidase
process is a challenge in the search for a potent therapeutic
agent for the treatment of diseases such as cancer,7 diabetes8 and
viral infections including AIDS.9 The mechanism of glycosidase
aGarware Research Centre, Department of Chemistry, University of Pune,
Pune-411007, India. E-mail: ddd@chem.unipune.ernet.in; Fax: +91-20-
25691728
bInstitute of Bioinformatics and Biotechnology, University of Pune, Pune-
411007, India
cDivision of Physical Chemistry, Department of Chemistry, University of
Pune, Pune-411007, India
† Electronic supplementary information (ESI) available: General experi-
mental methods, copies of the 1H and 13C NMR spectra of compounds 3a,
3b, 4a, 4b, 5a, 5b, 1d and 1e, data for DFT calculations of compound 1d
and 1e, calculated 1H NMR and coupling constants for compound 1d and
1e, molecular docking of compound 1e with human mannosidase (PDB:
1FO3). See DOI: 10.1039/c004690f
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Fig. 1 Conidine iminosugars.
activity of new analogues of constrained polyhydroxylated coni-
dine iminosugars 1d and 1e. It was anticipated that in 1d and 1e,
the ring nitrogen atom at physiological pH will mimic the positive
charge of the oxo-carbenium ion intermediate and the con-
formationally constrained azetidine ring A will induce the poly-
hydroxylated piperidine ring B to adopt a nearly half-chair
conformation—a transition state required to mimic the enzymes in
the glycosidase inhibition process. Such a restricted conformation
with a polyhydroxylated framework, recognized by an enzyme,
may increase the potency by lowering the entropic barrier to
complex formation making these molecules promising glycosidase
inhibitors.
Result and discussion
Our synthetic approach starts from the L-ido and D-gluco config-
ured b-aminoesters 2a and 2b (Scheme 1), which were prepared in
grams quantity from D-glucose as reported earlier by us.18 In the
next step, the L-ido configured b-aminoester 2a was hydrolyzed
with lithium hydroxide in methanol–water at 0 ◦C to room
temperature to give b-amino acid 3a19 in 98% yield as a white
solid. Treatment of 3a with 2-chloro-1-methylpyridinium iodide
(Mukaiyama reagent)20 and triethylamine in dichloromethane at
Scheme
1
Reagents and conditions: (a) Ref. 18; (b) LiOH·H2O,
MeOH–H2O, 0 ◦C to rt, 2 h; (c) 2-chloro-1-methylpyridinium iodide, Et3N,
dry CH2Cl2, 25 ◦C, 2 h; (d) LiAlH4/AlCl3, anhyd. THF, -10 ◦C to 0 ◦C,
10 min; (e) (i) TFA/H2O (3 : 2), 0 ◦C to rt, 4 h; (ii) H2, 10% Pd/C, MeOH,
80 psi, rt, 5 days.
◦
25 C afforded sugar b-lactam 4a as a white solid in 89% yield.
Reduction of b-lactam 4a was attempted with various reagents
such as DIBAL-H, LiAlH4, 9-BBN and BH3–THF. However, a
better result in high yield was achieved with chlorohydroalane21
reagent, prepared in situ from LiAlH4 : AlCl3 (3 : 1). Thus, reaction
of 4a with chlorohydroalane in anhydrous THF at -10 ◦C
to 0 ◦C, yielded four membered azetidine compound 5a with
sugar appendage in 83% yield. In the final step, opening of the
as a white solid in 85% yield. In the final step, hydrolysis of the
1,2-acetonide functionality in 5b and reductive aminocyclization
using H2, 10% Pd/C afforded conidine iminosugar 1e in 71% yield.
Conformational study
◦
1,2-acetonide functionality in 5a with TFA : water (3 : 2) at 0 C
In bicyclic iminosugars, the binding ability and their function
as glycosidase inhibitors depend on their conformation. For
example, an indolizidine iminosugar (six–five ring fused system)
to room temperature afforded the hemiacetal (as evident from
the 1H NMR of the crude product) which on hydrogenation
with H2, 10% Pd/C at room temperature undergoes reductive
aminocyclization22 to give polyhydroxylated conidine iminosugar
8
namely 1-deoxy-castanospermine exists in the C5 conformation
I (Fig. 2) and exhibits potent enzyme inhibition toward the a-
and b-glucosidase and a-mannosidase; whereas 1-deoxy-8a-epi-
castanospermine exists in the 5C8 conformation II and shows se-
lective inhibition of a- and b-galactosidase and b-glucosidase.23,24
In the case of conidine iminosugars 1d and 1e (six–four ring fused
1
1d in 78% yield. The H and 13C NMR spectra were found to
be consistent with the structure. While targeting the synthesis
of polyhydroxylated 6a-epi-conidine 1e, the same sequence of
reaction was repeated with D-gluco configured b-aminoester 2b.
Thus, reaction of 2b with LiOH in methanol–water at 0 ◦C to
room temperature afforded b-amino acid 3b in 96% yield that on
treatment with Mukaiyama reagent at 25 ◦C gave b-lactam 4b
as a white solid in 91% yield. Reduction of b-lactam 4b using
1
systems), the H NMR spectra were found to be dramatically
different and knowing that the four membered fused azetidine
ring system will change the conformation of the six membered
piperidine ring, it is expected that both target molecules could
exist in different conformations.
◦
◦
chlorohydroalane at -10 C to 0 C afforded sugar azetidine 5b
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Fig. 2 Conformations of compounds 1d and 1e.
Table 1 1H NMR Spectra of 1d and 1e
conformation A and ruled out conformation A1. In the precursor
5a, the relative stereochemistry of H-2/H-3 and H-3/H-4 is
trans and assuming that the same stereochemistry is retained in
compound 1d, the H-3/H-4 and H-4/H-5 should have axial–axial
orientation. In accordance with this, H-4 appeared as a triplet
(J4a,3a = J4a,5a = 6.9 Hz). The relatively small coupling constant for
axially oriented H-3/H-4 (J3a,4a = 6.9 Hz) required little distortion
from the usual chair conformation of the piperidine ring. Thus, the
dihedral angle of ~160◦ between the H-3 and H-4 protons (rather
than the 180◦ in axial–axial) accounts for coupling constant of
6.9 Hz. In agreement with this, the axially oriented H-5 showed
a doublet of doublets with J4a,5a = 6.9 Hz and J5a,6e = 6.0 Hz
wherein, the relatively large coupling constant for axial–equatorial
protons indicates a dihedral angle between H-5a and H-6e ~10◦.
This ruled out conformation A1. To account for these features, the
half chair conformation A2 was assigned to 1d wherein, the fused
four membered strained azetidine ring forced the piperidine ring
to adopt a distorted half chair conformation.
1d
1e
H-2a
H-2e
3.34 (dd)
3.25 (dd)
J2a,2e = 12.6 Hz
J2a,3a = 11.0 Hz
3.63 (dd)
J2a,2e = 12.6 Hz
J2e,3a = 5.0 Hz
4.88–5.05 (m)
3.90 (t)
J2a,2e = 13.5 Hz
J2a,3a = 6.0 Hz
3.43 (dd)
J2a,2e = 13.5 Hz
J2e,3a = 3.3 Hz
4.06–4.14 (m)
3.56 (dd)
J4,5 = 8.2 Hz
J4,3 = 4.8 Hz
4.14–4.27 (m)
H-3
H-4
J4,3 = J4,5 = 6.9 Hz
H-5
3.95 (dd)
J5,4 = 6.9 Hz
J5,6 = 6.0 Hz
3.67–3.76 (m)
2.44–2.58 (m)
2.78–2.96 (m)
3.82 (dt)
H-6
4.27–4.36 (m)
2.34–2.50 (m)
2.74–2.90 (m)
4.14–4.27 (m)
H-7a
H-7e
H-8a
J8a,8e = J8a,7a = 10.0 Hz
J8a,7e = 4.7 Hz
4.45 (dt)
In case of 1e, three conformers B, B1 and B2 (Fig. 2) were
H-8e
4.14–4.27 (m)
1
J8a,8e = J8a,7e = 10.0 Hz
considered. In the H NMR of 1e, one of the H-2 appeared as a
J8e,7a = 8.8 Hz
doublet of doublets with geminal coupling constants of 13.5 Hz
(J2a,2e) and a vicinal coupling constant of 6.0 Hz (J2a,3) while
the other H-2 appeared as a doublet of doublets with coupling
constants of 13.5 Hz and 3.3 Hz. The small vicinal coupling
In order to assign the conformations, we studied the 1H NMR
spectra of 1d and 1e and the chemical shifts as well as the coupling
constants obtained by the decoupling experiments and are given
in Table 1. For compound 1d, we assumed three conformations
A, A1 and A2 (Fig. 2). In the 1H NMR spectrum of 1d, the H-2a
proton appeared as a doublet of doublets with geminal and vicinal
coupling constants of 12.6 Hz and 11.0 Hz, respectively. The
large vicinal coupling constant J2a,3a = 11.0 Hz requires the axial–
axial relative orientation of H-2 and H-3 and therefore H-3 was
assigned the axial orientation. This established the possibility of
5
constant of J2e,3e = 3.3 Hz ruled out the C2 conformation B as
it requires a large coupling constant between the axially oriented
H-2/H-3 protons. To decide between conformations B1 and B2,
we considered the H-4 proton. In conformation B1, the H-4 is
equatorial and is expected to show a doublet of doublets with small
coupling constants. However, H-4 showed a doublet of doublets
with J4,5 = 8.2 Hz and J4,3 = 4.8 Hz. The appearance of H-4
with a large coupling constant of J4,5 = 8.2 Hz ruled out the 2C5
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conformation B1. To account for this feature, we assumed the most
probable boat conformation B2 wherein the quasi-axially oriented
H-4 has a large coupling constant of 8.2 Hz with the quasi-axial H-
5 and a small coupling constant of 4.8 Hz with the quasi-equatorial
H-3. In accordance with this, the H-5 showed a doublet of doublets
with large coupling constants of 8.2 Hz with H-4 and 9.3 Hz with
H-6 suggesting that the ring fusion proton (H-6) is quasi-axial.
Thus, the boat conformation B2 was assigned to compound 1e
wherein the two fused rings are the farthest away from each other
and –OH groups are positioned either quasi-equatorial or quasi-
axial. Thus, based on the 1H NMR studies, conidine iminosugars
1d and 1e were found to exist in the half chair (A2) and boat or
twist boat (B2) conformations respectively. This observation was
substantiated by employing density functional theory (DFT).
Thus, the different conformers of 1d and 1e were generated by
varying the C4–C5–C6–N1 and C2–N1–C6–C5 dihedral angles
from -80◦ to +80◦ within the framework of HF/3-21G(d) theory.
The corresponding energy profiles are displayed in Fig. 19S and
20S, of the supporting information. As revealed from these profiles,
ring conformers with chair (C), half chair (HC), twisted chair (TC)
and twisted boat (TB) were encompassed. Minima (24 for 1d and
20 for 1e) on the potential energy surface using the dihedral angle
as reaction coordinate, were optimized at the B3LYP/6-31G(d,p)
level of theory. Stationary point geometries thus obtained were
confirmed to be local minima from the vibrational frequencies,
all of which turn out to be real. Relative stabilization energies
(DERel ) were calculated by subtracting the energy of lowest energy
conformer from those of rest of the conformers (Table 2S in
Supporting Information). Some of these geometries from the scan
of both the C4–C5–C6–N1 and C2–N1–C6–C5 dihedral angles
exhibiting different hydrogen bonding patterns finally converged
to identical conformers. Out of several local minima possessing
similar six membered ring conformations as well as the orien-
tation of OH group, one conformer was chosen for subsequent
calculations. The gas phase structures were reoptimized employing
the SCRF-PCM model using water as solvent. The DERel and
Boltzmann contribution (BC) values in the gas phase as well
as in water are given in Table 3S in the supporting information
specifying the ring conformation. As may be noted 1d is present
in chair (C) and half chair (HC) forms and 1e exhibits twist boat
(TB) and twist chair (TC) ring conformations. Furthermore 1d
1
conformers with the BC values > 5% in water possess the C4
2
ring conformation, whereas the TB5 conformation is noticed in
the case of 1e. The conformers 1d_A112, 1d_A119, 1d_A211 and
1d_D111 reveal similar structures except for the orientation of the
hydroxyl group. Similar inferences may be drawn for 1e_A206,
1e_B206, and 1e_D207 conformers. Lowest energy conformers
1d_A119 and 1e_A206 are shown in Fig. 3 where both views top
Fig. 3 Optimized geometries of 1d and 1e in water. Views from (i) the top, (ii) along the C2–C3 bond are shown (iii) ring conformations (1C4, 3HC and
2TB5 respectively) are displayed.
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Table 2 IC50 values of 1d and 1e in mM
Compound a-Galactosidase a-Mannosidase a-Glucosidase a-Amylase
and side along the C2–C3 bond are displayed along with the ring
conformations. Thus the C4 conformation of 1d is transparent;
1
1e on the other hand exhibits the 2TB5 ring conformation.
It should be remarked here that the configuration around the
C2–C3 bond is different in 1d and 1e diastereomers. The dihedral
angle H–C3–C4–H turns out to be 67◦ here compared to 98◦ in
1e with the near eclipsed conformer. Furthermore, the conformer
1d_C232 is predicted to possess the half chair ring conformation
(3HC) as inferred from the experiments. The calculated coupling
constants for the minimum energy 1d_A112 conformer and that
inferred from experiments 1d_C232 and 1e_A206 are given in
Table 5S in the supporting information. In case of conformer
1d
1e
NI
60 0.03
322.6 0.12
33.6 0.09*
465.8 0.22 110.6 0.03
270.3 0.06 126.5 0.02
NI = No inhibition under assay conditions. The data is indicated as the
mean SEM; (n = 3). (*) denotes a more significant value (P < 0.05).
relaxation in the presence of solvent. The NMR chemical shifts
were calculated from the gauge invariant atomic orbital method
by subtracting the nuclear magnetic shielding tensors of protons
in conformers 1d and 1e from that of the protons in TMS.29 The
DFT investigations revealed that the calculated 1H NMR chemical
shifts in solvent using the SCRF-PCM model agree better with
the experimental ones than those from the gas phase.30–32 In the
1d_C232, coupling constants for H-2a and H-2e (J2a,3 and J2e,3
)
are 9.11 and 4.20 Hz which matches well with 11.0 and 5.0 Hz,
respectively, as observed in the experimental NMR spectra of 1d.
While for 1d_A112, the calculated values of the coupling constants
were found to be 1.37 and 3.27 Hz and thus deviate largely
from experimental values. In addition, for conformer 1d_C232
the calculated J3,4 = 7.52 Hz and J4,5 = 7.33 Hz values for the
H-4 were matching well with the experimentally observed value of
6.9 Hz. The large coupling constant values suggested that the H-3
and H-5 protons are trans◦to H-4 (dihedral angle H–C3–C4–H and
H–C4–C5–H being ~180 ). Thus, the 1H NMR data of 1d_C232
in the 3HC ring conformation matches well with the experimental
values corresponding to the half chair conformation of 1d.
In case of compound 1e, the experimental coupling constants for
H-2 were observed to be J2a,3 = 6.0 Hz and J2e,3 = 3.3 Hz. A similar
trend has been observed for the calculated coupling constant of
1e_A206 conformer for which the coupling constants for these
protons are predicted to be 3.45 Hz and 1.41 Hz, respectively.
The lower coupling constant for the J2e,3 is in agreement with
the experiments. Similar observation may also be made for the
coupling constant of H-4. Thus, coupling constant data suggests
that the compound 1e (1e_A206) is present in the twisted boat
(2TB5) conformation.
1
case of glucose, it has been pointed out that the accuracy of H
chemical shifts is affected significantly by the solute geometry
and the SCRF models suffice to describe the effect of solvent on
the shielding constants.30 Boltzmann contributions (BC) of these
conformers were estimated from the relative stabilization energy
values in the gas phase and water. The calculated BC values provide
the valuable information regarding relative population of different
conformers.
Biological activity
The glycosidase inhibitory activity was studied with different
glycosidases.33 As shown in Table 2, glycosidase inhibition studies
of the target compounds revealed that compound 1d showed no
inhibition with a-galactosidase (E.C. 3.2.1.22) and moderate inhi-
bition with both a-mannosidase (E.C. 3.2.1.24) and a-glucosidase
(E.C. 3.2.1.20) while 1e showed selective inhibition against
a-mannosidase and moderate inhibition against a-galactosidase
and a-glucosidase in the millimolar range. Compounds 1d and 1e
were also found to have moderate inhibition against a-amylase
(E.C. 3.2.1.1) (Table 2). As a reference, porcine pancreatic a-
amylase34 with 0.21 U min-1 was taken as 100% enzymatic activity.
Computational methods
The conformations of 1d and 1e possessing different intramolec-
ular hydrogen bonded networks were optimized at the Hartree–
Fock (HF/3-21G) level of theory using the Gaussian03 package.25
The conformers possessing unique hydrogen bonding patterns
were labeled with notations A, B, C, and D (cf. Fig. 17S in
supporting information). Other ring conformations viz. chair,
twist chair, half chair, twist boat, and boat forms, were generated
by scanning the C5–C6–N1–C2 and C4–C5–C6–N1 dihedral
angle from -80.0◦ to 80.0◦. Minima on the scanned potential
energy surface of both the dihedral angles were identified and
further optimized at B3LYP/6-31G(d,p) level of theory by relaxing
all the geometrical constraints.26,27 Relative stabilization energies
(DERel ) were calculated by subtracting the energy of lowest
energy conformer from those of rest of the conformers. Amongst
the conformers having different DERel only one conformer that
possesses the same DERel values and the same geometry is
considered for the calculations hereafter. Self consistent reaction
field theory was utilized to investigate the influence of solvent on
the conformational energies of 1d and 1e.
Molecular docking
Biological activity was supported by a docking study in order to
understand the interactions between compound 1e and the amino
acid residues of mannosidase. Mannosidases are lectin binding
proteins and N-glycans of a-mannosidase have unique topologies,
important functions in protein folding, oligomerization or enzyme
activity.35 The complete sequence of jack bean mannosidase is
yet to be reported. Therefore, it is difficult to compare the active
sites of the mannosidase from jack bean and ER. Hence, for this
study, we have chosen human mannosidase (PDB: 1FO3) as our
target structure to perform the molecular docking studies. The
template structure of human mannosidase is used to dock the
ligand 1e into the binding pocket of mannosidase. This might
provide an insight into the interaction of 1e with mannosidase
from Jack Bean. The active site was predicted by using WHAT IF
software. The AUTODOCK 3.036 program was used to perform
an automatic docking exploration for different conformations of
the ligand in the model. The interactions between compound 1e
and human mannosidase (PDB: 1FO3) are given in the Fig. 23S in
The gas phase structures were optimized in water employing the
polarizable continuum model (PCM)28 to account for the geometry
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Table 3 The total energy (Etotal), Van der Waals energy (Evdw) and
electrostatic energy (Eele) between interacting residues of compound 1e
and human mannosidase (PDB: 1FO3)
on pre-coated plates (0.25 mm, silica gel 60 F254). Column
chromatography was carried out with silica gel (100–200 mesh).
The reactions were carried out in oven-dried glassware under dry
N2. Methanol, THF, dichloromethane were purified and dried
before use. After decomposition of the reaction mixture, the work-
up involves extraction with solvent, washing of combined organic
layer with water, brine, drying over anhydrous sodium sulfate and
evaporation of solvent under reduced pressure below 50 ◦C.
Residue
E
total/kcal mol-1
E
vdw/kcal mol-1
E
elec/kcal mol-1
Ser158
Thr446
Lys229
Ser222
Glu225
-50.101
25.131
254.120
92.476
-1.45
-12.27
-15.71
-6.65
-48.65
24.06
257.61
82.41
256.171
-20.30
262.41
1,2-O-Isopropylidene-3-O-benzyl-5,6-dideoxy-5-(N-benzylamino)-
b-L-ido-heptofuranuronic acid (3a)
the supporting information. Interactions were observed between
1e and Ser158, Thr446, Lys229, Ser222, Glu225. It shows that 1e
may have significant interaction with mannosidase and may act
as an inhibitor. The total binding energies are given in Table 3.
The total energy of the complex is 133.50 kcal mol-1 with the total
electrostatic energy being -27.07 kcal mol-1 and total Van der
Waals interaction energy -106.44 kcal mol-1.
To an ice cooled solution of b-L-ido-b-amino ester 2a (0.200 g,
0.44 mmol) in methanol–water (5 cm3, 4/1) was added lithium
hydroxide monohydrate (0.112 g, 2.66 mmol) at 0 ◦C. The mixture
was brought to 25 ◦C, stirred for 2.5 h and neutralized to
pH 7 by addition of 0.5 M H3PO4. Methanol was evaporated
and residue was extracted with chloroform (3 ¥ 7 cm3), the
combined chloroform layer was washed with water, dried with
anhydrous Na2SO4 and the solvent removed under reduced
pressure to give a pale yellow solid which on purification by col-
umn chromatography (chloroform–methanol = 9.5/0.5) afforded
b-L-ido-heptofuranuronic acid 3a (0.184 g, 98%) as a white solid
(Found: C, 67.09; H, 6.94; N, 3.23. Calc for C24H29NO6: C,
67.43; H, 6.84; N, 3.28%); Rf 0.30 (chloroform–methanol = 9/1);
[a]3D2 -32.17 (c 1.08, in CHCl3); mp 165–166 ◦C; nmax (neat)/cm-1
3445 (COOH), 1611 (NH), 2928, 1084; dH (300 MHz; CDCl3;
Me4Si) 1.32 (3H, s, CH3), 1.48 (3H, s, CH3), 2.20 (1H, dd, J 17.1
and 5.1 Hz, H-6a), 2.58 (1H, dd, J 17.1 and 4.95 Hz, H-6b), 3.30–
3.56 (1H, m, H-5), 3.88 (1H, d, J 12.7 Hz, NCH2Ph), 3.98 (1H,
d, J 3.0 Hz, H-3), 4.02 (1H, d, J 12.7 Hz, NCH2Ph), 4.25 (1H,
dd, J 9.5 and 3.0 Hz, H-4), 4.48 (1H, d, J 11.7 Hz, OCH2Ph),
4.67 (1H, d, J 3.6 Hz, H-2), 4.69 (1H, d, J 11.7 Hz, OCH2Ph),
5.94 (1H, d, J 3.6 Hz, H-1), 7.23–7.45 (10H, m, Ar-H) 7.40–8.20
(2H, br, Exchangeable with D2O, NH and COOH); dC (75 MHz;
CDCl3; Me4Si) 26.1 (CH3), 26.7 (CH3), 31.3 (C-6), 50.2 (C-5),
53.3 (NCH2Ph), 72.0 (OCH2Ph), 80.3, 81.1, 81.7 (C-2/C-3/C-4),
101.8 (C-1), 112.2 (OCO), 128.2, 128.4, 128.7, 128.9, 135.9, 136.5
(Ar-C), 172.6 (COOH).
Conclusion
We have synthesized a new class of iminosugars viz. conidine
iminosugars 1d and 1e with an overall yield of 41% and
37%, respectively, from D-glucose. Reduction of sugar derived
b-lactam to azetidine was achieved which is less common in sugar
moiety due to acid labile 1,2-acetonide protection. Hydrogenolysis
was achieved without destruction of the constrained azetidine
ring which is otherwise already reported in the literature. The
experimental and DFT calculated 1H coupling constant data
suggests that compound 1d should be assigned the half chair
conformation whereas compound 1e has the twist boat conforma-
tion. Compound 1e was found to have inhibitory activity against
a-mannosidase at the mM range whereas compound 1d was found
to be a moderate inhibitor of a-mannosidase and a-glucosidase
at the same concentration. Molecular docking studies could suc-
cessfully correlate interaction energies calculated from the docked
poses of the ligands with experimental binding affinities which
offer a possible structural rationale for the inhibition mechanism
of the conidine iminosugars, 1d and 1e. It is noteworthy that
1d and 1e inspite of being diastereomers of the same conidine
iminosugar showed differential inhibition against a-mannosidase
that supports that stereochemistry of the ring fusion proton in
conidine iminosugars is significant in glycosidase inhibition.
1,2-O-Isopropylidene-3-O-benzyl-5,6-dideoxy-5-(N-benzylamino)-
a-D-gluco-heptofuranuronic acid (3b)
Following the same procedure, a-D-gluco-b-amino ester 2b
(0.250 g, 0.55 mmol) was hydrolyzed with LiOH·H2O (0.140 g,
3.34 mmol) in methanol–water (5 cm3, 4/1) for 3 h to give the
a-D-gluco-heptofuranuronic acid 3b (0.225 g, 96%) after silica gel
column purification (chloroform–methanol = 9.5/0.5) as a white
solid (Found: C, 66.94; H, 6.78; N, 3.11. Calc for C24H29NO6: C,
67.43; H, 6.84; N, 3.28%); Rf 0.30 (chloroform–methanol = 9/1);
[a]3D2 -5.75 (c 1.02, in CHCl3); mp 162–163 ◦C; nmax(neat)/cm-1
3438 (COOH), 1625 (NH), 2938, 1062; dH (300 MHz; CDCl3;
Me4Si) 1.34 (3H, s, CH3), 1.49 (3H, s, CH3), 2.17 (1H, dd, J 15.3
and 11.0 Hz, H-6a), 2.48 (1H, dd, J 15.3 and 4.0 Hz, H-6b), 3.55
(1H, dt, J 11.0 and 4.0 Hz, H-5), 3.82 (1H, d, J 12.3 Hz, NCH2Ph),
4.09 (1H, d, J 3.3 Hz, H-3), 4.16 (1H, d, J 12.3 Hz, NCH2Ph), 4.18
(1H, dd, J 4.7 and 3.3 Hz, H-4), 4.46 (1H, d, J 11.4 Hz, OCH2Ph),
4.64 (1H, d, J 3.9 Hz, H-2), 4.68 (1H, d, J 11.4 Hz, OCH2Ph),
6.05 (1H, d, J 3.9 Hz, H-1), 6.20–6.90 (2H, br, Exchangeable with
Experimental section
General experimental methods
Melting points were recorded with a Thomas Hoover melting
point apparatus and are uncorrected. IR spectra were recorded
with a Shimadzu FTIR-8400 as a thin film or in nujol mull or
using KBr pellets and are expressed in cm-1. H (300 MHz) and
13C (75 MHz) NMR spectra were recorded on a Varian Mercury
300 using CDCl3/D2O as solvent. Chemical shifts were reported in
d unit (ppm) with reference to TMS as an internal standard and J
values are given in Hz. Assignments of the signals were confirmed
by decoupling experiments. Elemental analyses were carried
out with an Elemental Analyser Flash 1112. Optical rotations
were measured using a JASCO P-1020 polarimeter with sodium
light (589.3 nm). Thin layer chromatography was performed
1
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D2O, NH and COOH), 7.15–7.38 (10H, m, Ar-H); dC (75 MHz;
CDCl3; Me4Si) 26.1 (CH3), 26.7 (CH3), 33.2 (C-6), 51.4 (C-5),
53.6 (NCH2Ph), 71.9 (OCH2Ph), 79.3, 81.2, 82.1 (C-2/C-3/C-4),
104.5 (C-1), 112.1 (OCO), 128.2, 128.3, 128.5, 128.8, 128.9, 135.9,
135.9 (Ar-C), 173.2 (COOH).
1,2-O-Isopropylidene-3-O-benzyl-5,6,7-trideoxy-5,7-(N-
benzylimino)-b-L-ido-sept-1,4-furanose (5a)
To a cooled suspension (ice–salt mixture) of LiAlH4 (0.014 g,
0.36 mmol) in anhydrous THF (2 cm3) was added a solution of
AlCl3 (0,016 g, 0.12 mmol) in anhydrous THF (2 cm3) and stirred
at -10 ◦C for 30 min. Then a solution of b-L-ido-b-lactam 4a
(0.100 g, 0.24 mmol) in anhydrous THF (3 cm3) was added drop
wise at -10 ◦C for 5 min and further stirred for 10 min at 0 ◦C. The
reaction was quenched with saturated aqueous Na2SO4 at 0 ◦C
slowly. The reaction mixture was filtered through celite, solvent
removed under reduced pressure to give a colorless oil which
was chromatographed (n-hexane–ethyl acetate = 8.8/1.2) to give
b-L-ido-azetidine 5a (0.082 g, 83%) as a white solid (Found: C,
72.97; H, 7.46; N, 3.60. Calc for C24H29NO4: C, 72.89; H, 7.39;
N, 3.54%); Rf 0.50 (n-hexane–ethyl acetate = 7/3); [a]3D2 -131.68
(c 2.14, in CHCl3); mp 94–96 ◦C; nmax (neat)/cm-1 1060, 1375; dH
(300 MHz; CDCl3; Me4Si) 1.33 (3H, s, CH3), 1.53 (3H, s, CH3),
1.82–1.94 (2H, m, H-6a and H-6b), 2.76–2.88 (1H, m, H-7a),
3.13–3.24 (1H, m, H-7b), 3.37 (1H, d, J 12.6 Hz, NCH2Ph), 3.60
(1H, m, H-5), 3.89 (1H, d, J 3.3 Hz, H-3), 4.09 (1H, d, J 12.6 Hz,
NCH2Ph), 4.38 (1H, dd, J 4.9 and 3.3 Hz, H-4), 4.42 (1H, d, J
12.0 Hz, OCH2Ph), 4.59 (1H, d, J 3.7 Hz, H-2), 4.66 (1H, d, J
12.0 Hz, OCH2Ph), 5.99 (1H, d, J 3.7 Hz, H-1), 7.18–7.40 (10H, m,
Ar-H); dC (75 MHz; CDCl3; Me4Si) 20.0 (C-6), 26.4 (CH3), 26.8
(CH3), 51.2 (C-7), 62.4 (C-5), 64.3 (NCH2Ph), 71.8 (OCH2Ph),
81.6, 81.8, 84.8 (C-2/C-3/C-4), 105.4 (C-1), 111.5 (OCO), 126.8,
127.7, 127.8, 128.1, 128.3, 128.5, 128.9, 137.1, 137.6 (Ar-C).
1,2-O-Isopropylidene-3-O-benzyl-5,6-dideoxy-5,7-(N-
benzylimino)-b-L-ido-sept-1,4-furan-7-ulose (4a)
To a suspension of 2-chloro-1-methylpyridinium iodide (0.329 g,
1.29 mmol) and Et3N (0.359 cm3, 2.58 mmol) in dry
dichloromethane (6 cm3) at 25 ◦C was added a solution of
b-L-ido-heptofuranuronic acid 3a (0.500 g, 1.17 mmol) in dry
dichloromethane (2 cm3) drop wise. The solution was stirred at
25 ◦C for 2 h and water (2 cm3) was added. The reaction mixture
was extracted with ethyl acetate (3 ¥ 10 cm3), the organic layer dried
on anhydrous Na2SO4 and the solvent removed under reduced
pressure to give a thick liquid which on column purification
(n-hexane–ethyl acetate = 8.5/1.5) afforded b-L-ido-b-lactam 4a
(0.430 g, 89%) as white crystalline solid (Found: C, 70.58; H, 6.55;
N, 3.38. Calc for C24H27NO5: C, 70.40; H, 6.65; N, 3.42%); Rf 0.45
(n-hexane–ethyl acetate = 7/3); [a]3D2 -39.86 (c 2.61, in CHCl3);
mp 129–131 ◦C; nmax (neat)/cm-1 1748 (C O), 1065, 1380; dH
=
(300 MHz; CDCl3; Me4Si) 1.27 (3H, s, CH3), 1.42 (3H, s, CH3),
2.37 (1H, dd, J 14.6 and 2.5 Hz, H-6a), 2.83 (1H, dd, J 14.6 and
5.2 Hz, H-6b), 3.79 (1H, ddd, J 8.5, 5.2 and 2.5 Hz, H-5), 3.87 (1H,
d, J 3.6 Hz, H-3), 4.11 (1H, dd, J 8.5 and 3.6 Hz, H-4), 4.22 (1H,
d, J 14.7 Hz, NCH2Ph). 4.35 (1H, d, J 12.3 Hz, OCH2Ph), 4.58
(1H, d, J 3.6 Hz, H-2), 4.64 (1H, d, J 14.7 Hz, NCH2Ph), 4.67 (1H,
d, J 12.3 Hz, OCH2Ph), 5.98 (1H, d, J 3.6 Hz, H-1), 7.18–7.40
(10H, m, Ar-H); dC (75 MHz; CDCl3; Me4Si) 26.2 (CH3), 26.7
(CH3), 39.0 (C-6), 45.6 (NCH2Ph), 49.7 (C-5), 71.7 (OCH2Ph),
81.6, 81.8,83.7 (C-2/C-3/C-4), 105.7 (C-1), 111.8 (OCO), 127.3,
128.1, 128.3, 128.5, 128.5, 128.6, 136.5, 136.6 (Ar-C), 165.9 (CO).
1,2-O-Isopropylidene-3-O-benzyl-5,6,7-trideoxy-5,7-
(N-benzylimino)-a-D-gluco-sept-1,4-furanose (5b)
The reduction of a-D-gluco-b-lactam 4b (0.500 g, 1.22 mmol) with
LiAlH4 (0.070 g, 1.83 mmol) and AlCl3 (0.082 g, 0.61 mmol)
in anhydrous THF (10 cm3) at -10 ◦C to 0 ◦C in 15 min gave
the colorless oil which on column purification (n-hexane–ethyl
acetate = 9/1) afforded a-D-gluco-azetidine 5b (0.410 g, 85%)
as a white solid (Found: C, 72.74; H, 7.28; N, 3.43. Calc for
C24H29NO4: C, 72.89; H, 7.39; N, 3.54%); Rf 0.50 (n-hexane–
ethyl acetate = 7/3); [a]D32 -24.43 (c 2.12, in CHCl3); mp 101–
103 ◦C; nmax (neat)/cm-1 1070, 1355; dH (300 MHz; CDCl3; Me4Si)
1.31 (3H, s, CH3), 1.44 (3H, s, CH3), 2.08–2.16 (1H, m, H-6a),
2.24–2.41 (1H, m, H-6a), 2.84-2.88 (1H, m, H-7a), 3.35 (1H, td, J
9.0, 2.4 Hz, H-7b), 3.50 (1H, d, J 12.9 Hz, NCH2Ph), 3.56–3.70
(2H, m, NCH2Ph, H-5), 3.81 (1H, d, J 3.0 Hz, H-3), 4.06 (1H,
dd, J 6.0 and 3.0 Hz, H-4), 4.38 (1H, d, J 12.0 Hz, OCH2Ph), 4.60
(1H, d, J 3.9 Hz, H-2), 4.62 (1H, d, J 12.0 Hz, OCH2Ph), 5.91 (1H,
d, J 3.9 Hz, H-1), 7.18-7.40 (10H, m, Ar-H), dC (75 MHz; CDCl3;
Me4Si) 21.1 (C-6), 26.4 (CH3), 26.7 (CH3), 52.4 (C-7), 62.7 (C-5),
63.4 (NCH2Ph), 71.2 (OCH2Ph), 81.8, 82.0, 82.6 (C-2/C-3/C-4),
104.8 (C-1), 111.5 (OCO), 126.9, 127.4, 127.8, 128.0, 128.2, 128.4,
128.7, 137.4, 138.4 (Ar-C).
1,2-O-Isopropylidene-3-O-benzyl-5,6-dideoxy-5,7-
(N-benzylimino)-a-D-gluco-sept-1,4-furan-7-ulose (4b)
As above, the reaction of a-D-gluco-heptofuranuronic acid 3b
(0.700 g, 1.64 mmol) with 2-chloro-1-methylpyridinium iodide
(0.461 g, 1.80 mmol) and Et3N (0.503 cm3, 3.61 mmol) in dry
dichloromethane (10 cm3) at 25 ◦C for 3 h gave a thick liquid
which on silica gel column purification (n-hexane–ethyl acetate =
8.7/1.3) afforded a-D-gluco-b-lactam 4b (0.610 g, 91%) as a white
solid (Found: C, 70.68; H, 6.43; N, 3.35. Calc for C24H27NO5: C,
70.40; H, 6.65; N, 3.42%); Rf 0.45 (n-hexane–ethyl acetate = 7/3);
[a]3D2 -38.38 (c 2.58, in CHCl3); mp 126–127 ◦C; nmax (neat)/cm-1
=
1744 (C O), 1080, 1385; dH (300 MHz; CDCl3; Me4Si) 1.23 (3H, s,
CH3), 1.40 (3H, s, CH3), 3.03 (1H, dd, J 14.7 and 4.9 Hz, H-6a),
3.15 (1H, dd, J 14.7 and 2.4 Hz, H-6b), 3.66 (1H, d, J 3.6 Hz,
H-3), 3.83 (1H, dt, J 4.9 and 2.4 Hz, H-5), 4.18 (1H, dd, J 4.9 and
3.6 Hz, H-4), 4.28 (1H, d, J 15.6 Hz, NCH2Ph), 4.30 (1H, d, J
12.0 Hz, OCH2Ph), 4.45 (1H, d, J 15.6 Hz, NCH2Ph), 4.57 (1H,
d, J 3.9 Hz, H-2), 4.59 (1H, d, J 12.0 Hz, OCH2Ph), 5.89 (1H, d,
J 3.9 Hz, H-1), 7.18–7.40 (10H, m, Ar-H); dC (75 MHz; CDCl3;
Me4Si) 26.2 (CH3), 26.7 (CH3), 40.2 (C-6), 44.8 (NCH2Ph), 50.0
(C-5), 71.4 (OCH2Ph), 78.6, 81.8, 82.3 (C-2/C-3/C-4), 104.7
(C-1), 111.7 (OCO), 127.2, 127.5, 127.6, 127.9, 128.4, 128.6, 136.1,
137.0 (Ar-C), 168.0 (CO).
(3S,4R,5R,6S)-3,4,5-Trihydroxy-conidine (1d)
A cooled (0 ◦C) solution of 5a (0.41 g, 1.04 mmol) in TFA–H2O
(5 cm3, 3 : 2) was stirred for 1 h, brought to 25 ◦C, and stirred
for additional 2 h. TFA was evaporated at high vacuum to yield
the crude hemiacetal (0.32 g). A solution of the above product
in dry methanol (8 cm3) was hydrogenated in the presence of
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10% Pd/C (0.07 g) at 80 psi for 5 days. The catalyst was filtered
and washed with methanol, and the filtrate was concentrated
to afford a thick liquid. Purification by column chromatography
on silica gel chloroform–methanol (5.5/4.5) afforded 1d (0.13 g,
78%) as a moisture sensitive colorless solid (Found: C, 52.76; H,
8.16; N, 8.87. Calc for C7H13NO3: C, 52.82; H, 8.23; N, 8.80%);
Rf 0.30 (methanol); [a]D32 -36.43 (c 1.12, MeOH); nmax (neat)/cm-1
3600–3200 (broad, OH); dH (300 MHz; D2O; Me4Si) 2.44–
2.58 (1H, m, H-7a), 2.78–2.96 (1H, m, H-7b), 3.34 (1H,
General procedure for a-amylase inhibition assay
Amylase activity was assayed using a modified Bernfeld method
(1955) using starch as substrate. 50 mg cm-3 (O. D. adjusted to
0.4 at 280 nm) of porcine pancreatic a-amylase was incubated with
5 mg cm-3 samples at 37 ◦C for 10 min. One percent starch was used
as substrate. The samples without a-amylase were used as controls
and the test reading was subtracted from the absorbance of these
controls. The reducing sugar was estimated using DNSA assay at
540 nm and the enzyme units were expressed as micro-molar per
minute. One unit of enzyme was defined as the amount of enzyme
required to liberate 1 mM of maltose under assay conditions. The
final inhibition shown by different samples were compared with
the standard inhibitor, acarbose.
dd,
J 12.6 and 11.0 Hz, H-2a), 3.63 (1H, dd, J 12.6
and 5.0 Hz, H-2b), 3.67–3.76 (1H, m, H-6), 3.82 (1H, dt,
J 10.0 and 4.7 Hz, H-8a), 3.90 (1H, t, J 6.9 Hz, H-4), 3.95 (1H,
dd, J 6.9 Hz, H-5), 4.45 (1H, dt, J 10.0 and 8.8 Hz, H-8b), 4.88–
5.05 (1H, m, merged with HDO signal, H-3); dC (75 MHz; D2O;
Me4Si) 22.2 (C-7), 49.3 (C-8), 53.7 (C-6), 62.9 (C-2), 68.3 (C-3),
71.5 (C-5), 73.7 (C-4).
Statistical analysis
The statistical analysis was performed using one way analysis of
variance (ANOVA) and two tailed t-test (P < 0.05). Results are
expressed as means SEM.
(3S,4R,5R,6R)-3,4,5-Trihydroxy-conidine (1e)
Following a similar procedure, compound 5b (0.35 g, 0.89 mmol)
was treated with TFA–H2O (5 cm3, 3 : 2), and the resultant hemiac-
etal was hydrogenated over 10% Pd/C (0.05 g) for 5 days to furnish,
after isolation and purification by column chromatography on
silica gel (chloroform–methanol = 7.5/2.5), compound 1e (0.1 g,
71%) as a moisture sensitive colorless solid (Found: C, 52.89; H,
8.33; N, 8.77. Calc for C7H13NO3: C, 52.82; H, 8.23; N, 8.80%); Rf
0.45 (chloroform–methanol = 5/5); [a]3D2 +44.21 (c 1.12, MeOH);
nmax (neat)/cm-1 3600–3200 (broad, OH); dH (300 MHz; D2O;
Me4Si) 2.34–2.50 (1H, m, H-7a), 2.74–2.90 (1H, m, H-7b), 3.25
(1H, dd, J 13.5 and 6.0 Hz, H-2a), 3.43 (1H, dd, J 13.5 and 3.3 Hz,
H-2b), 3.56 (1H, dd, J 8.2 and 4.8 Hz, H-4), 4.06–4.14 (1H, m,
H-3), 4.14–4.27 (3H, m, H-5, H-8a, H-8b), 4.27–4.36 (1H, m, H-
6); dC (75 MHz; D2O; Me4Si) 18.8 (C-7), 50.2 (C-8), 55.0 (C-6),
61.3 (C-2), 67.2 (C-3), 68.3 (C-5), 73.4 (C-4).
Molecular docking
Interactions of compound 1e against amino acid residues of
human mannosidase (PDB: 1FO3) were found out by employing
molecular docking studies. The active site was predicted by
using WHAT IF software. The AUTODOCK 3.0 program was
used to perform an automatic docking exploration for different
conformations of the ligand in the model. AUTODOCK 3.0
uses three search methods [a genetic algorithm, a local search
method, and an adaptive global–local search method based on
Lamarckian genetics (LGA)] in conjunction with an empirical
force field that allows the prediction of binding free energies for
docked ligand. The optimized autodocking run parameters are as
follows: the maximum number of energy evaluations was increased
to 2 500 000 per run; the maximum number of generation in the
genetic algorithm was increased to 100 000; and the number of
GA run was 100. The binding pocket consists mainly of glutamate
residues that make the site very active. Distances between polar
General procedure for glycosidase inhibition assay
Glycosidase inhibition assay of conidine iminosugars 1d and 1e
was carried out by mixing 0.1 U cm-3 each of a-galactosidase,
a-mannosidase and a-glucosidase with the samples and incubated
for 1 h at 37 ◦C. Enzyme action for a-galactosidase was initiated by
addition of 10 mM p-nitrophenyl-a-D-galactopyranoside (pNPG)
as a substrate in 200 mM sodium acetate buffer. The reaction was
˚
atoms less than 3.6 A may be regarded as hydrogen bonds and
˚
interactions less than 4.5 A may be taken as Van der Waals
interactions.
◦
incubated at 37 C for 10 min and stopped by adding 2 cm3 of
Acknowledgements
200 mM borate buffer of pH 9.8. a-Mannosidase activity was ini-
tiated by addition of 10 mM p-nitrophenyl-a-D-mannopyranoside
as a substrate in 100 mM citrate buffer of pH 4.5. The reaction
We are grateful to Prof. M. S. Wadia for helpful discussions.
SPS and RVP are thankful to CSIR, New Delhi, for the Junior
Research Fellowship and Senior Research Fellowship, respectively.
SG is thankful to University of Pune for Research Stipend. AMJ
is thankful to UGC for Senior Research Fellowship. We are also
thankful to the University of Pune, (BCUD Research Project,
2009-2011) Pune for the financial support.
◦
was incubated at 37 C for 10 min and stopped by adding 2 cm3
of 200 mM borate buffer of pH 9.8. Initiation of a-glucosidase
activity was done by addition of 10 mM p-nitrophenyl-a-D-
glucopyranoside in 100 mM phosphate buffer of pH 6.8 and
stopped by adding 2 cm3 of 0.1 M Na2CO3 after an incubation
of 10 min at 37 ◦C. a-Glycosidase activity was determined by
measuring absorbance of the p-nitrophenol released from pNPG
at 420 nm using Shimadzu Spectrophotometer UV-1601. One unit
of glycosidase activity is defined as the amount of enzyme that
hydrolyzed 1 mM of p-nitrophenyl pyranoside per minute under
assay condition.
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