Chemo-enzymatic synthesis of the azasugars 1,4-dideoxyallonojirimycin and 1,4-dideoxymannojirimycin

Article abstract of DOI:10.1016/j.tetasy.2011.10.015

The enzymatic resolution of the N-phenylacetyl derivative of racemic homoserine lactone with penicillin G acylase immobilized on Eupergit C gave (R)-(+)-α-amino-γ-butyrolactone and (S)-(-)-α-N- phenylacetamido-γ-butyrolactone in high enantiomeric purity (ee >99%) and 46-47% yields for each enantiomer. The enantiomers were converted into azasugars 1,4-dideoxyallonojirimycin and 1,4-dideoxymannojirimycin using Wittig olefination, catalytic ring-closing metathesis (RCM), and stereoselective dihydroxylation with OsO₄ in 29% overall yield over 11 high yielding steps. Enzyme inhibition studies showed that 1,4-dideoxyallonojirimycin is a better β-glucosidase inhibitor (IC₅₀ 32.4 μM toward β-glucosidase from almonds) and a better β-galactosidase inhibitor (IC₅₀ 5.9 mM for β-galactosidase from Aspergillus oryzae) than 1,4-dideoxymannojirimycin (IC₅₀ 2.86 mM and 12.5 mM for β-glucosidase and β-galactosidase, respectively).

Full text of DOI:10.1016/j.tetasy.2011.10.015

Tetrahedron: Asymmetry 22 (2011) 1855–1860
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemo-enzymatic synthesis of the azasugars 1,4-dideoxyallonojirimycin
and 1,4-dideoxymannojirimycin
⇑
Mallam Venkataiah, Gowrisankar Reddipalli, Lakshmi Swarnalatha Jasti, Nitin W. Fadnavis
Biotransformation Laboratory, Indian Institute of Chemical Technology, Uppal Road, Habsiguda, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 September 2011
Accepted 28 October 2011
The enzymatic resolution of the N-phenylacetyl derivative of racemic homoserine lactone with penicillin
G acylase immobilized on Eupergit C gave (R)-(+)-
a
-amino-
c
-butyrolactone and (S)-(ꢀ)-
a-N-phenylace-
tamido- -butyrolactone in high enantiomeric purity (ee >99%) and 46–47% yields for each enantiomer.
c
The enantiomers were converted into azasugars 1,4-dideoxyallonojirimycin and 1,4-dideoxymannojiri-
mycin using Wittig olefination, catalytic ring-closing metathesis (RCM), and stereoselective dihydroxyla-
tion with OsO4 in 29% overall yield over 11 high yielding steps. Enzyme inhibition studies showed that
1,4-dideoxyallonojirimycin is a better b-glucosidase inhibitor (IC50 32.4 lM toward b-glucosidase from
almonds) and a better b-galactosidase inhibitor (IC50 5.9 mM for b-galactosidase from Aspergillus oryzae)
than 1,4-dideoxymannojirimycin (IC50 2.86 mM and 12.5 mM for b-glucosidase and b-galactosidase,
respectively).
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
O
NH2
O
Enantiomerically pure forms of homoserine lactone 1 (Fig. 1) are
important chiral intermediates. For example, D-homoserine lactone
is an useful building block for many pharmaceuticals such as
nocardicin antibiotics,1 antifungal peptides, syringotoxin B and
syringostatin A,2 and serine protease inhibitors.3 N-Methoxycar-
O
O
(S)-1
(R)-1
bonyl-
L
-homoserine is an useful precursor for herbicide
L-phosphi-
Figure 1.
nothricin.4 Similarly, N-acylated-
L
-homoserine lactones (AHLs) are
produced and used by Gram-negative bacteria such as
Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Vibrio
fischeri for intercellular communication (quorum sensing signals).5
The disruption of quorum sensing systems using AHLs represents
an attractive therapeutic approach for the treatment of bacterial
infections.6,7
pure
-homoserine. For example, Mochizuki et al.14 have carried out the
microbial resolution of DL-homoserine using the Arthrobacter
nicotinovorans strain wherein the -homoserine was degraded and
-homoserine was isolated. Similarly, Curran et al.15 have carried
out the resolution of Cbz-(DL)-homoserine to afford Cbz- -homo-
serine. Since both the - and -enantiomers of homoserine are valu-
able, it is preferable to perform a resolution in which both the
enantiomers are recovered. To the best of our knowledge, only
one report exists for homoserine resolution, which is based on
the hydrolysis of N-chloroacetyl amide by acylase I.16 Although
D-homoserine are mainly based on the selective destruction of
L
L
D
D
Considering their synthetic importance, several routes have
been developed for the synthesis of enantiopure homoserine
D
L
lactones. For example, D- and L-homoserine have been synthesized
from the corresponding methionine8,9 or aspartic acid.10,11
However, these methods are expensive. Since it is easy to prepare
racemic homoserine at low cost,12 resolution of racemic homoser-
the enantiomeric purity of the
D-homoserine was good, the enan-
ine lactone can be employed. The resolution of (RS)-a-amino-c-
tiomeric purity of the -enantiomer was only approximately 30%.
L
butyrolactone by preferential crystallization with a mixture of
ethanol and hydrochloric acid at 40 °C has been reported by
Shiraiwa et al.13 but the enantiomeric purities of the products were
not mentioned. Biocatalytic approaches to obtain enantiomerically
Herein we report an enzymatic resolution of racemic homoserine
lactone via enantioselective hydrolysis of its N-phenylacetyl deriv-
ative with commercially available immobilized penicillin G acylase
in high yields (92–94% of theoretical) and excellent ee (>99%) for
both the enantiomers. The resolved (S)-enantiomer was then used
for the synthesis of 2,3,6-trihydroxy-5-methyl piperidines
(1,4-dideoxy azasugars). It is well known that azasugars have high
⇑
Corresponding author. Fax: +91 40 27160512.
E-mail addresses: fadnavis@iict.res.in, fadnavisnw@yahoo.com (N.W. Fadnavis).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.10.015

1856
M. Venkataiah et al. / Tetrahedron: Asymmetry 22 (2011) 1855–1860
inhibitory activities against glycosidase enzymes17 and have po-
tential use in a wide range of therapeutic strategies, including
the treatment of viral infection (HIV), cancer, diabetes, tuberculo-
sis, and lysosomal storage diseases, and as inhibitors of the growth
of parasitic protozoa.18–22
OH
N
OH
HO
HO
OH
OH
N
H
H
2. Results and discussion
5
4
2.1. Resolution of racemic homoserine lactone with
immobilized penicillin G acylase
Figure 2.
be ineffective as an inhibitor of mammalian
biological activities of 4 and 5 toward glucosidase and galactosi-
dase were not reported.
a
-
L
-fucosidase,34 the
Penicillin acylases have been shown to resolve racemic
mixtures of chiral compounds such as amino acids,23–26 b-amino
esters,27 amines,28,29 amino alcohols,30 and secondary alcohols31,32
efficiently. The immobilized enzyme has further advantages of
easy separation and recycling. Since homoserine lactone is, in prin-
ciple, an amino acid, we expected that the enzyme would be able
to hydrolyze its N-phenylacetyl derivative stereospecifically and
provide the resolved enantiomerically pure product. Thus, racemic
homoserine lactone was prepared from inexpensive and easily
Only a few methods have been developed to access 1,4-dideoxy
azasugars.33,35 Azasugar 4 has been synthesized from 3-deoxy-
D-
ribo-hexose using glucose isomerase enzyme,33 and from isoxazo-
lines prepared via the Mukaiyama procedure.35a Azasugar 5 has
been synthesized from
D
-(ꢀ)-quinic acid using a Luche reduction
and SN2 reactions.35b These reported procedures are long and te-
dious. A retrosynthetic analysis showed that it should be much
simpler to construct the unsaturated piperidine chiral building
block tert-butyl 6-((tert-butyldimethylsilyloxy)methyl)-5,6-dihy-
dropyridine-1(2H)-carboxylate 6 from the resolved homoserine
lactone. The stereoselective dihydroxylation of 6 should lead to
the desired 1,4-dideoxy azasugars (Scheme 2).
available c
-butyrolactone by a reported procedure.12 The lactone
was converted to its N-phenylacetyl derivative 2 using phenyl
acetyl chloride in 4 M NaOH. Compound 2 (4.4 g, 20 mmol) was
suspended in water (10 mL) which slowly dissolved upon dropwise
addition of 10% aqueous ammonia, presumably by opening of the
lactone ring. The pH was adjusted to 7.5, after which immobilized
penicillin G acylase (5 g wet, 1000 units) was added and the reac-
tion mixture was shaken in a conical flask at 80 rpm (25 °C). The
reaction was followed by reverse phase HPLC analysis. The reaction
virtually stopped at 50% hydrolysis in 1 h. The enzyme was filtered,
after which the products were separated and treated with metha-
nolic HCl to obtain the cyclic lactones in 46–47% yield (Scheme 1).
The enantiomeric purities (ee) of the resolved cyclized products
2 and 1 (after derivatization to an N-phenylacetyl derivative) were
found to be >99%. The measurement of the specific rotation and
comparison with the literature values showed that the enzyme
had selectively hydrolyzed the (R)-amide derivative.
Thus, lactone (S)-1 was reduced with lithium aluminum hydride
in THF. Next the N-Boc protection of the diol and acetonide protec-
tion by treatment with 2,2-dimethoxy propane gave product 6
(70% yield over three steps). Oxidation of 6 under Swern conditions
followed by a Wittig olefination furnished compound 7. The aceto-
nide group was removed using CuCl2ꢁ2H2O in acetonitrile36 and
alcohol 8 was silylated (TBDMSCl/imidazole/CH2Cl2/rt) to give 9
(95%). Allylation of 9 with allyl bromide in DMF gave 10. Ring-clos-
ing metathesis (RCM) with Grubbs’ first generation catalyst in DCM
gave the desired piperidine intermediate 11 in a 97% yield. The
stereoselective dihydroxylation of the double bond of piperidine
11 with OsO4 gave 12 and 13 in an 80:20 ratio (determined on
the basis of isolated yields). The two diastereomers were easily
separated by column chromatography (the configuration was as-
signed on the basis of the configuration of the final product). The
formation of the major isomer 12 is most probably due to steric
hindrance caused by the bulky N-Boc group on the b-face, which
2.2. Synthesis of 1,4-dideoxy azasugars
The synthesis of polyhydroxylated piperidines and their syn-
thetic analogues has attracted a great deal of attention in recent
years.33 1,4-Dideoxymannojirimycin 5 and its isomer 1,4-dideoxy-
allonojirimycin 4 have been isolated34 from the Thai medicinal
plant Connarus ferrugineu (Fig. 2). Although 5 has been found to
causes a preferential approach of OsO4 from the less hindered
a-
face of the olefin.37 Deprotection of 12 and 13 with 6 M HCl in
O
O
-
Ph
Ph
O
O
OH
HN
+NH3
OH
HN
NH2
O
Immobilized
Ph
aq.NH3
pH 7.5
Pen G acylase
+
N
H
O
HO
HO
O
O
O
HO
HO
(R,S)-2
(S)-3
(R)-3
H+
CH3OH
Ph
HN
NH2·HCl
O
+
O
O
O
(R)-1 (46%, e.e.>99%)
(S)-2 (47%, e.e.>99%)
Scheme 1.

M. Venkataiah et al. / Tetrahedron: Asymmetry 22 (2011) 1855–1860
1857
Table 1
NH2
Boc
H
HO
HO
Inhibition of glucosidase and galactosidase activity by compounds 4 and 5
N
(O)
(O)
N
O
Compound
Enzyme
-Glucosidase (from Baker’s yeast, type-I)
b-Glucosidase inhibition (from almonds)
-Galactosidase from green coffee beans
b-Galactosidase from Aspergillus oryzae
-Glucosidase (from Baker’s yeast, type-I)
b-Glucosidase inhibition (from almonds)
-Galactosidase from green coffee beans
b-Galactosidase from Aspergillus oryzae
IC50
No inhibition
32.4
No inhibition
5.9 mM
O
4
a
OH
lM
OTBDMS
a
(S)-1
5
6
5
a
No inhibition
2.86 mM
No inhibition
12.5 mM
Scheme 2.
a
MeOH, followed by the treatment with ion-exchange resin DOWEX
50WX8 gave 5 and 4, respectively in an 89% yield. The spectro-
scopic and physical data were in good agreement with the litera-
ture values34 (Scheme 3).
4. Experimental
4.1. General
2.3. Determination of the glycosidase inhibition activity
All reagents were purchased from Sigma–Aldrich, USA. IR spec-
tra were recorded on a Perkin–Elmer RX-1 FT-IR system. 1H NMR
(200 MHz) spectra were recorded on a Varian Gemini-200 MHz
spectrometer. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra
were recorded on Bruker Avance-300 MHz spectrometer.
Optical rotations were measured with Horiba-SEPA-300 digital
polarimeter. Mass measurements were performed on a Q STAR
mass spectrometer (Applied Biosystems, USA). UV–visible spectro-
photometric measurements were performed on Perkin Elmer
Lambda 2 spectrophotometer equipped with temperature control
and PECSS software. All enzyme assays were performed at 25 °C.
All assay experiments were repeated three times and were repro-
ducible within 5%. HPLC analyses were carried out on Hewlett
Packard HP 1090 HPLC unit equipped with diode array detector
and HPCHEM software.
Compounds 4 and 5 were evaluated for their glucosidase and
galactosidase inhibition activity. The values of the inhibitor con-
centration at which the enzyme activity was inhibited by 50%
(IC50) are given in Table 1. Both 4 and 5 were found to be highly
specific toward the inhibition of b-pyroranosidase activity and do
not inhibit the
of b-glucosidase activity (IC50 32.4
ity is relatively low (5.9 mM). A similar trend was observed for
a-pyranosidases. Compound 4 is a good inhibitor
l
M) but its galactosidase activ-
the diastereomer
inhibitor.
5 but it was less efficient as an enzyme
3. Conclusion
In conclusion, we have demonstrated an efficient enzymatic
resolution of homoserine lactone with immobilized penicillin G
acylase and a simple, convenient and efficient approach to 1,4-
dideoxyallonojirimycin 4 and 1,4-dideoxymannojirimycin 5.
4.2. N-(2-Oxo-tetrahdrofuran-3-yl)-2-phenylacetamide 2
Racemic
a-amino-c-butyrolactone hydrobromide, prepared by
a known literature procedure12 (5 g, 27.4 mmol) was dissolved in
HO
a
b
d
c
e
OH
OTBDMS
(S)-1
BocN
BocN
BocHN
BocHN
O
O
9
8
6
7
OH
OH
HO
HO
g
f
OTBDMS
OTBDMS
+
OTBDMS
N
N
OTBDMS
N
Boc
Boc
N
Boc
Boc
10
11
12 (69%)
13 (17%)
h
h
OH
OH
HO
HO
OH
OH
N
H
N
H
4
5
Scheme 3. Reagents and conditions: (a) (i) LAH, THF, reflux; (ii) (Boc)2O; (iii) 2,2-DMP, DCM, rt, 70% over three steps; (b) (i) (COCl)2, DMSO, Et3N, ꢀ78 °C, 95%; (ii) CH3 +PPh3Iꢀ,
KOtBu, THF, 0 °C, 78%; (c) CuCl2ꢁ2H2O, CH3CN, rt, 90%; (d) TBDMSCl, imidazole, CH2Cl2, rt, 95%; (e) NaH, allyl bromide, DMF, 0 °C–rt, 92%; (f) 10 mol % of Grubbs’ first
generation catalyst, DCM, rt, 12 h, 93%; (g) OsO4, NMO monohydrate, acetone/water (3:1), rt, 12 h, 86%; (h), 6 M HCl, MeOH, rt, 12 h; DOWEX 50W X8, 89%.

1858
M. Venkataiah et al. / Tetrahedron: Asymmetry 22 (2011) 1855–1860
4 M NaOH (50 mL) and stirred in an ice bath. Phenylacetyl chloride
(4.7 g, 30.2 mmol) was added dropwise alternating with 4 M NaOH
(50 mL) with vigorous stirring. After complete addition, the reac-
tants were stirred overnight and extracted with dichloromethane.
The aqueous layer was then cooled in ice and acidified with 6 M
HCl. The precipitated phenylacetyl derivative was filtered, washed
with cold water, dried, and finally recrystallized from ethyl acetate
to obtain 2 (5.6 g, 93%). Mp 165–167 °C. 1H NMR (500 MHz, CDCl3):
d 1.91–2.11 (m, 1H), 2.61–2.79 (m, 1H), 3.58 (s, 2H), 4.11–4.25 (m,
1H), 4.30–4.56 (m, 2H), 6.02 (br s, 1H), 7.12–7.34 (m, 5H). ESIMS:
m/z 242 [M+Na].+
1253, 1172, 1072, 968, 770 cmꢀ1 1H NMR (CDCl3, 300 MHz): d
.
1.48–1.57 (m, 15H), 1.64–1.85 (m, 2H), 3.44–3.67 (m, 3H),
3.85–3.89 (m, 1H), 3.96–4.01 (m, 1H), 4.16–4.22 (m, 1H). 13C
NMR (CDCl3, 75 MHz): d 24.26, 27.66, 28.24, 37.54, 53.92, 58.59,
68.06, 80.82, 93.52, 153.70. ESIMS: m/z 268.6 [M+Na].+
4.5. (S)-tert-Butyl 4-allyl-2,2-dimethyloxazolidine-3-
carboxylate 7
At first, DMSO (1.3 mL, 18 mmol) was added to a stirred solution
of oxalyl chloride (0.8 mL, 9 mmol), in dry CH2Cl2 (25 mL) at ꢀ78 °C
and stirred at the same temperature for 30 min. A solution of 6
(2.0 g, 8.1 mmol) in dry CH2Cl2 (10 mL) was added at ꢀ78 °C to
the reaction mixture and stirred for 1 h at the same temperature.
Next, Et3N (6.8 mL, 49 mmol) was added at ꢀ78 °C and then stirred
for an additional 30 min at rt. The reaction mixture was diluted
with water (15 mL) and extracted with dichloromethane
(2 ꢃ 50 mL). The combined organic layers were washed with brine
(50 mL), dried over Na2SO4 and concentrated to obtain the aldehyde
as a pale yellow syrup. The aldehyde was used for the next reaction
without further purification.
4.3. Enzymatic hydrolysis
Compound 2 (5 g) was suspended in water (20 mL) and dissolved
by the dropwise addition of 5% aqueous ammonia solution. The pH
was adjusted to 7.5 after which immobilized penicillin G acylase
(5 g, wet) was added. The reaction mixture was shaken in a conical
flask at 80 rpm (25 °C) on an orbital shaker and the reaction was fol-
lowed by reverse phase HPLC analysis. The reaction virtually
stopped at 50% hydrolysis (1 h). The enzyme was filtered, after
which the aqueous filtrate was acidified to pH 2 with 1 M HCl and
extracted with cyclohexane to remove the phenylacetic acid. The
aqueous extract was freeze dried and the dry powder was extracted
with acetone to recover the unreacted (S)-N-phenylacetyl deriva-
tive. The residue after acetone extraction consisted of the hydro-
lyzed (R)-homoserine. The products were stirred separately with
methanolic HCl (1 M, 20 mL) overnight at room temperature.
Evaporation of methanol gave the cyclic lactone hydrochlorides 1
and 2.
At first, KOBut (2.60 g, 19.3 mmol) was added to a solution of
methyl triphenylphosphonium iodide (7.4 g, 23.2 mmol) in dry
THF (50 mL) at ꢀ15 °C and stirred for 3 h. A solution of crude alde-
hyde (1.88 g, 7.73 mmol) in THF (10 mL) was added dropwise and
stirred for 5 h at room temperature. Saturated aqueous NH4Cl solu-
tion (30 mL) was added and extracted with EtOAc (3 ꢃ 40 mL). The
organic layer was washed with water (50 mL), brine (50 mL), dried
over Na2SO4, and evaporated on a rotavapor. The residue was puri-
fied by column chromatography (silica gel 60–120 mesh, EtOAc/
hexane, 3:97) to furnish olefin 7 (1.49 g, 80%) as a pale yellow syr-
The phenylacetyl derivative (S)-2 was suspended in 6 M HCl
(10 mL) and refluxed for 6 h to hydrolyze the amide. The solution
was cooled in ice, extracted with ethyl acetate to remove phenyla-
cetic acid and the aqueous extract was evaporated to dryness on a
rotavapor to obtain (S)-1 as a hydrochloride in quantitative yield.
upy liquid. ½a 2D5
¼ þ32:0 (c 1, CHCl3). IR (neat): mmax 3448, 2977,
ꢂ
2931, 1697, 1454, 1386, 1253, 1175, 1092, 917, 850, 770 cmꢀ1
.
1H NMR (CDCl3, 400 MHz): d 1.43–1.59 (m, 15H), 2.16–2.32 (m,
1H), 2.38–2.62 (m, 1H), 3.69–3.99 (m, 3H), 5.02–5.11 (m, 2H),
5.64, 5.82 (m, 1H). 13C NMR (CDCl3, 75 MHz): d 23.67, 26.88,
28.24(ꢃ3), 37.40, 56.65, 66.22, 79.62, 93.46, 117.46, 134.42,
151.74. ESIMS: m/z 264 [M+Na]+. HRMS calcd for C13H23NO3Na:
[M+Na]+ 264.1575, found: 264.1582.
N-((S)-Tetrahydro-2-oxofuran-3-yl)-2-phenylacetamide (S)-2
Yield 47% (2.35 g). Mp 165–167 °C, ½a D25
¼ ꢀ15:0 (c 1, MeOH), ee
ꢂ
>99%.
(R)-3-Amino-dihydrofuran-2(3H)-one hydrochloride (R)-1
4.6. (S)-tert-Butyl 1-hydroxypent-4-en-2-ylcarbamate 8
Yield 46% (1.45 g). Mp 220–224 °C, ½a D25
ꢂ
¼ þ27:0 (c 1, H2O), lit16
½
a 2D5
ꢂ
¼ þ26:7 (c 5, H2O).
At first, CuCl2ꢁ2H2O (1.20 g, 7.5 mmol) was added to a solution of
acetonide 7 (1.40 g, 5.8 mmol,) in MeCN (10 mL) at 0 °C for 30 min.
The reaction was quenched by adding a saturated aq NaHCO3 solu-
tion, filtered through Celite and the Celite bed was washed with
acetonitrile (2 ꢃ 20 mL). The combined extracts were concentrated
on a rotavapor. The residue was extracted with ethyl acetate
(2 ꢃ 10 mL), the extract was dried over anhydrous Na2SO4 and con-
centrated on a rotavapor. The residue was chromatographed over
silica gel (60–120 mesh, EtOAc/hexane, 3:7) to give the correspond-
ing primary alcohol 8 (1.05 g, 90%) as a pale yellow oily syrup
4.4. (S)-tert-Butyl 4-(2-hydroxyethyl)-2,2-dimethyloxazolidine-
3-carboxylate 6
A solution of compound (S)-1 (1.57 g, 11.4 mmol) and triethyl-
amine (1.2 g, 12 mmol) in dry THF was added dropwise to a solu-
tion of lithium aluminum hydride (1.74 g, 45.6 mmol) in 20 mL
of dry THF at 0 °C for 20 min, after which the reaction mixture
was refluxed for 4 h. The reaction was quenched with a cold satu-
rated NH4Cl solution (10 mL), after which a 1 M NaOH solution
(5 mL) and Boc-anhydride (3.74 g, 17.1 mmol) were added to the
reaction mixture and stirred for 30 min. The reaction mixture
was filtered and the solvent was evaporated under reduced pres-
sure. The residue was extracted with EtOAc (2 ꢃ 20 mL) and dried
over anhydrous Na2SO4. After evaporation of the organic extract,
the residue was dissolved in dry CH2Cl2 (20 mL). 2,2-Dimethoxy-
propane (1.38 mL, 11.4 mmol) and cat. PTSA were added and the
reaction mixture was stirred at an ambient temperature for 3 h.
The organic layer was evaporated under reduced pressure to afford
the crude acetonide, which was purified by column chromatogra-
phy on silica gel (60–120 mesh, EtOAc/hexane, 2:8) to yield 6
(1.05 g, 90%). ½a D25
¼ þ13:5 (c 1, CHCl3). IR (neat): mmax 3415,
ꢂ
2976, 2930, 1689, 1509, 1456, 1366, 1249, 1171, 1055, 915,
774 cmꢀ1 1H NMR (CDCl3, 300 MHz): d 1.44 (s, 9H), 2.17–2.37 (m,
.
2H), 2.48 (br s, 1H), 3.50–3.70 (m, 3H), 4.64 (d, J = 6.2 Hz, 1H),
5.03–5.14 (m, 2H), 5.68–5.86 (m, 1H). 13C NMR (CDCl3, 75 MHz):
d 28.28(ꢃ3), 35.87, 52.02, 64.91, 79.57, 117.86, 134.16, 156.30.
ESIM: m/z 202 [M+1]+. HRMS calcd for C10H19NO3Na: [M+Na]+
224.1262, found: 224.1269.
4.7. (S)-tert-Butyl 1-(tert-butyldimethylsilyloxy)pent-4-en-2-
ylcarbamate 9
(1.96 g, 70%) as a colorless oil (1.96 g, 70%). ½a D25
ꢂ
¼ ꢀ10:0 (c 1,
Imidazole (1.02 g, 14.9 mmol) and TBDMSCl (0.83 g, 5.5 mmol)
were added to a stirred solution of alcohol 8 (1.0 g, 5 mmol) in
CHCl3). IR (neat): mmax 3446, 2931, 2856, 1667, 1451, 1395, 1369,

M. Venkataiah et al. / Tetrahedron: Asymmetry 22 (2011) 1855–1860
1859
CH2Cl2 (15 mL) and stirred for 2 h. The reaction mixture was trea-
ted with satd NH4Cl (10 mL) and extracted with CH2Cl2
(2 ꢃ 30 mL). The organic layer was washed with water
(2 ꢃ 20 mL), brine (2 ꢃ 20 mL), dried over Na2SO4, and evaporated.
The residue was purified by column chromatography (60–120 Sil-
ica gel, 4:96 EtOAc/hexane) to furnish silyl ether 9 (1.49 g, 95%) as
diastereomeric mixture. The two diastereomers 12 and 13
were obtained in an 80:20 ratio after separation by column
chromatography.
4.10.1. (2S,4R,5S)-tert-Butyl 2-((tert-butyl dimethylsilyloxy)
methyl)-4,dihydroxypiperidine-1-carboxylate 12
a colorless syrup. ½a D25
ꢂ
¼ ꢀ6:0 (c 1, CHCl3). IR (neat): mmax 3449,
Oil, 0.76 g, 69%. ½a D25
¼ þ14:0 (c 1, CHCl3). IR (neat): mmax 3419,
ꢂ
3357, 3078, 2955, 2931, 2859, 1703, 1643, 1498, 1472, 1391,
2955, 2930, 2859, 1690, 1469, 1420, 1365, 1253, 1174, 1135, 1079,
840, 774, 673, 609 cmꢀ1. 1H NMR (CDCl3, 400 MHz): d 0.05 (s, 6H),
0.89 (s, 9H), 1.46 (s, 9H), 1.77–1.89 (m, 2H), 2.27 (br s, 1H), 3.03 (br
d, J = 10.47 Hz, 1H), 3.56–3.70 (m, 2H), 3.78–3.92 (m, 2H), 4.03–
4.31 (m, 2H). 13C NMR (CDCl3, 75 MHz): d ꢀ5.52, 18.00, 25.84,
28.37, 45.15, 52.18, 60.08, 62.97, 66.49, 67.44, 80.04, 156.15.
ESIMS: m/z 362 [M+1]+. HRMS calcd for C17H35NO5NaSi: [M+Na]+
384.2182, found: 384.2188.
1366, 1253, 1173, 1113, 1005, 916, 838, 776, 669 cmꢀ1 1H NMR
.
(CDCl3, 300 MHz): d 0.05 (s, 6H), 0.90 (s, 9H), 1.43 (s, 9H), 2.21–
2.30 (m, 2H), 3.54–3.62 (m, 3H), 4.58 (d, J = 6.04 Hz, 1H), 5.02–
5.10 (m, 2H), 5.68–5.82 (m, 1H). 13C NMR (CDCl3, 75 MHz): d
ꢀ5.56, ꢀ3.64, 18.18, 25.98(ꢃ3), 28.32(ꢃ3), 35.92, 51.25, 64.03,
79.04, 117.36, 134.88, 155.44. ESIMS: m/z 316 [M+1]+. HRMS calcd
for C16H33NO3NaSi: [M+Na]+ 338.2127, found: 338.2129.
4.8. (S)-tert-Butyl allyl(1-(tert-butyldimethylsilyloxy)pent-4-en-
2-yl) carbamate 10
4.10.2. (2S,4S,5R)-tert-Butyl 2-((tert-butyldimethylsilyloxy)
methyl)-4,5-dihydroxypiperidine-1-carboxylate 13
Oil, 0.21 g, 19%. ½a D25
ꢂ
¼ ꢀ26:0 (c 1, CHCl3). 1H NMR (CDCl3,
At first, NaH (0.22 g, 8.9 mmol) was added to an ice-cold solu-
tion of olefin compound 9 (1.4 g, 4.4 mmol) in DMF (10 mL). After
30 min, allylbromide (0.45 mL, 5.3 mmol) was added and then stir-
red for 12 h at room temperature. The reaction was quenched with
saturated NH4Cl (25 mL) and extracted with diethyl ether
(3 ꢃ 50 mL). The combined organic layers were collected, dried
over anhydrous Na2SO4, and concentrated in vacuo to give a crude
oil. Purification of the residual product by chromatography
(hexane/ethyl acetate, 99:1) afforded diene 10 (1.45 g, 92%).
300 MHz): d 0.10 (s, 6H), 0.92 (s, 9H), 1.45 (s, 9H), 1.92–2.20 (m,
2H), 2.41 (d, J = 9.6 Hz, 1H), 2.86 (t, J = 12.06 Hz, 1H), 3.37–3.50
(m, 1H), 3.60 (dd, J = 2.1, 10.9 Hz, 1H), 3.77–3.88 (m, 1H), 3.90–
4.05 (m, 2H), 4.07–4.19 (m, 1H). 13C NMR (CDCl3, 75 MHz): d
ꢀ5.80, 18.25, 25.82, 28.34, 29.59, 32.86, 65.52, 66.61, 67.54,
67.56, 80.12, 154.71. ESIMS: m/z 362 [M+1]+.
4.11. (3S,4R,6S)-6-(Hydroxymethy)piperidine-3,4-diol (1,4-
Dideoxyallonojirimycin) 4
½
a 2D5
ꢂ
¼ ꢀ3:5 (c 1, CHCl3). IR (neat): mmax 3447, 3078, 2955, 2928,
2857, 1764, 1695, 1643, 1456, 1407, 1364, 1251, 1155, 1110,
993, 915, 840, 775 cmꢀ1 1H NMR (CDCl3, 300 MHz): d 0.03 (s,
At first, HCl (6 M, 10 mL) was added to a solution of 13 (200 mg,
0.55 mmol) in MeOH (10 mL) and the reaction mixture was stirred
at room temperature overnight. The solvent was evaporated under
reduced pressure. The residue was dissolved in water and treated
with Dowex 50 WX8 ion-exchange resin using a sequence of water
and 5% NH4OH as eluent. The ammoniacal eluent was freeze dried
to obtain 5a (72 mg, 89%) as a white solid 72 mg (89%). Mp 165–
.
6H), 0.88 (s, 9H), 0.39 (s, 9H), 2.25–2.37 (m, 2H), 3.54–3.97 (m,
5H), 4.96–5.19 (m, 4H), 5.63–5.88 (m, 2H). 13C NMR (CDCl3,
75 MHz): d ꢀ5.58(ꢃ2), 18.08, 25.77(ꢃ3), 28.32(ꢃ3), 33.93, 47.89,
57.53, 63.66, 79.18, 115.31, 116.66, 135.36, 136.33, 155.41. ESIMS:
m/z 356 [M+1]+. HRMS calcd for C19H37NO3NaSi: [M+Na]+
378.2440, found: 378.2449.
167 °C (lit34 166–170 °C).
½
a 2D5
ꢂ
¼ þ60:0 (c 1, H2O), lit34
½
a 2D5
ꢂ
¼ þ62:0 (c 0.92, H2O). IR (neat): mmax 3417, 3278, 2935,
4.9. (S)-tert-Butyl 6-((tert-butyldimethylsilyloxy)methyl)-5,6-
dihydropyridine-1(2H)-carboxylate 11
2862, 1634, 1424, 1358, 1283, 1217, 1121, 1073, 1041, 1000,
844, 641, 592, 539, 504 cmꢀ1 1H NMR (D2O, 400 MHz): d 1.43
.
(td, J = 1.76, 12.3 Hz, 1H), 1.80–1.82 (m, 1H), 2.77 (t, J = 11.4 Hz,
1H), 2.86 (dd, J = 5.2, 11.4 Hz, 1H), 2.90–2.96 (m, 1H), 3.42 (dd,
J = 7.0, 11.4 Hz, 1H), 3.53 (dd, J = 4.4, 11.4 Hz, 1H), 3.65–3.72 (m,
1H), 4.10 (m, 1H). 13C NMR (D2O, 75 MHz): d 36.0, 47.33, 53.19,
67.66, 70.11, 71.48. ESIMS: m/z 148 [M+1]+. HRMS calcd for
C6H14NO3: [M+H]+ 148.0973, found: 148.0976.
Diene 10 (1.3 g, 3.7 mmol) was dissolved in dry CH2Cl2 (50 mL).
Grubbs’s first generation catalyst (30 mg, 0.37 mmol) was added
and the resulting purple solution turned brown after 10 min. The
reaction mixture was stirred at room temperature for 12 h, and
then concentrated in vacuo. The residue was purified by column
chromatography (ethyl acetate/hexane; 3:97) and the title com-
pound 11 was obtained as a colorless oil (1.11 g, 93%). ½a D25
¼
ꢂ
4.11.1. (3R,4S,6S)-6-(Hydroxymethyl)piperidine-3,4-diol (1,4-
dideoxymanojirimycin) 5
ꢀ10:0 (c 1, CHCl3). IR (neat): mmax 3444, 2930, 2857, 1699, 1606,
1473, 1410, 1363, 1254, 1171, 1110, 839, 776, 665 cmꢀ1 1H NMR
.
Obtained by the deprotection of 12 (200 mg, 0.55 mmol) as de-
(CDCl3, 300 MHz): d 0.03 (s, 6H), 0.88 (s, 9H), 1.46 (s, 9H), 2.07–
2.38 (m, 2H), 3.56–3.58 (m, 3H), 4.02–4.50 (m, 2H), 5.54–5.78
(m, 2H). 13C NMR (CDCl3, 75 MHz): d ꢀ5.42, 18.20, 26.17(ꢃ3),
28.46(ꢃ3), 40.26, 58.00, 61.69, 79.55, 122.65, 123.36, 155.19.
ESIMS: m/z 328 [M+1]+. HRMS calcd for C17H33NO3NaSi: [M+Na]+
350.2127, found: 350.2132.
scribed above. Colorless syrup (73 mg, 89%). ½a D25
¼ ꢀ39:0 (c 1,
ꢂ
H2O), lit34
½
a 2D5
ꢂ
¼ ꢀ37:5 (c 0.14, H2O). 1H NMR (D2O, 400 MHz): d
1.51 (qt, J = 11.9, 12.6 Hz, 1H), 1.71–1.78 (m, 1H), 2.74–2.2.83 (m,
2H), 3.13 (dd, J = 1.5, 14.1 Hz, 1H), 3.58–3.67 (m, 2H), 3.88 (ddd,
J = 2.4, 4.6, 11.1 Hz, 1H), 3.93 (br s, 1H). 13C NMR (D2O, 75 MHz):
d 32.49, 51.41, 58.33, 66.99, 69.59, 71.80. ESIMS: m/z 148.1 [M+1]+.
4.10. Dihydroxylation using osmium tetraoxide
4.12. Reverse phase HPLC analysis
At first, OsO4 solution (16 mM in toluene, 3.89 mL, 0.06 mmol
OsO4) was added to a solution of compound 11 (1.0 g, 3 mmol)
in acetone (15 mL) and water (5 mL) at 0 °C. After 10 min,
NMO (2.06 g, 15.3 mmol) was added and the mixture was stirred
overnight at the same temperature. Next, Na2SO3 and Na2SO4
were added to the reaction mixture, filtered through a pad of
Celite and the filtrate evaporated to obtain an oily residue of a
The hydrolysis of phenylacetyl derivative 2 was followed by re-
verse phase HPLC on a C-8 column, 5 mm ꢃ 250 mm (Chrompack,
The Netherlands). Detection wavelength 220 nm, mobile phase
70% methanol in water containing 0.1% phosphoric acid. Flow rate
0.7 mL/min. Retention times: phenylacetyl amide 1 12.02 min,
phenyl acetic acid 16.03 min.

1860
M. Venkataiah et al. / Tetrahedron: Asymmetry 22 (2011) 1855–1860
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The enantiomeric purity of resolved 2 was determined by anal-
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flow rate 1 ml/min; detection wavelength 220 nm; retention times,
(S)-2 20.33 min, (R)-2 23.47 min.
4.14. Enzyme assay
14. Mochizuki, K.; Miyazaki, K. Enzyme Microb. Technol. 2007, 41, 318.
15. Curran, W. V. Prep. Biochem. 1981, 11, 269.
The
a
- and b-glucosidase activities were assayed using 2-nitro-
- and b- -glucopyranosides. Similarly the
- and b-galactosidase activities were assayed using p-nitrophenyl
phenyl derivatives of
a
D
16. Birnbaum, S. M.; Greenstein, J. P. Arch. Biochem. Biophys. 1953, 42, 212.
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19. Zechel, D. L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11.
20. Stutz, A. E. Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond; Wiley-
VCH: Weinheim, Germany, 1999.
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a
derivatives of a- and b-D-galactopyranosides. The enzyme activity
was measured at 25 °C using the specific substrate (1.6 mM) in a
phosphate buffer (0.25 M, pH 6.8). The assay solution (2.0 mL)
was placed in a cuvette, after which the enzyme solution (1 mg/
mL, 100 lL) was added and the change in absorbance was moni-
tored at 410 nm (molar extinction coefficient at 410 nm,
De
8800 Mꢀ1 cmꢀ1) for 3–5 min. The enzyme activity was expressed
as Vobsd for the native enzyme. For enzyme inhibition studies, a
concentrated stock solution of compound 4 or 5 was prepared
(0.1 M in phosphate buffer, pH 6.8) and was added to the substrate
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Tetrahedron: Asymmetry 2001, 12, 1645.
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4495.
31. Fuganti, C.; Grasselli, P.; Seneci, P. F.; Servi, S.; Casati, P. Tetrahedron Lett. 1986,
27, 2061.
solution at various concentrations (1 lM to 10 mM). Enzyme activ-
ity was then measured in the presence of the compound. The con-
centration of the compound at which the enzyme activity
decreased to 50% was designated as IC50
.
Acknowledgements
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We are thankful to CSIR-New Delhi, and UGC-New Delhi for
financial assistance.
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