Straightforward chemo-enzymatic synthesis of new aminocyclitols, analogues of valiolamine and their evaluation as glycosidase inhibitors

Article abstract of DOI:10.1016/j.tetasy.2006.09.010

An efficient fructose-1,6-bisphosphate aldolase mediated synthesis of new aminocyclitol analogues of valiolamine is described. The one-pot process where four stereocentres are created involves the formation of two carbon-carbon bonds. One is catalysed by

Full text of DOI:10.1016/j.tetasy.2006.09.010

Tetrahedron: Asymmetry 17 (2006) 2684–2688
Straightforward chemo-enzymatic synthesis
of new aminocyclitols, analogues of valiolamine and
their evaluation as glycosidase inhibitors
Lahssen El Blidi,a Mustapha Ahbala,b Jean Boltea and Marielle Lemairea,*
a
`
Laboratoire SEESIB, UMR 6504 du CNRS, Universite´ Blaise Pascal, 24 av. des Landais, 63177 Aubiere cedex, France
bLaboratoire de chimie organique et organome´tallique, Faculte´ des sciences, Universite´ Chouab Doukkali,
ˆ
km 1 Route Ben Machou BP 2400, El Jadida, Morocco
Received 30 August 2006; accepted 13 September 2006
Available online 17 October 2006
Abstract—An efficient fructose-1,6-bisphosphate aldolase mediated synthesis of new aminocyclitol analogues of valiolamine is described.
The one-pot process where four stereocentres are created involves the formation of two carbon–carbon bonds. One is catalysed by the
aldolase, coupling dihydroxyacetone phosphate to nitrobutyraldehydes. The other is the result of a highly stereoselective intramolecular
Henry reaction occurring on the intermediate nitroketones. Depending on the configuration of the hydroxyl which is a to the nitro group,
two series of configuration are accessible. The lipase resolution of the nitroalcohol ketal, precursor of the nitroaldehyde, is presented. The
inhibition properties of the aminocyclitols obtained after the reduction of the nitro group are evaluated towards five commercial
glycosidases.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
δ+
OH
δ+
H
Glycosidases are enzymes widely distributed in micro-
O
HO
HO
δ+
organisms, plants and animals. They selectively hydrolyse
glycosidic bonds and play important roles in crucial biolog-
ical pathways such as polysaccharide and glycoconjugate
anabolism and catabolism,1 cellular recognition2 and
eukaryotic glycoprotein processing.3 Glycosidases are also
involved in a variety of metabolic disorders and diseases
such as diabetes, viral or bacterial infections and cancer
formation. Consequently, their inhibitors have many
potential applications4 such as antidiabetic,5 antiviral
(VIH, influenza)6 and anticancer7 drugs.
O
R'
R
O
OH
OH
+
H
δ
OH
OH
OH
HO
HO
HO
HO
HO
NH2
NH2
HO
NH2
The design and synthesis of glycosidase inhibitors are
mainly focused on mimicking the transition state (TS) that
occurs in an enzymatic glycoside hydrolysis.8 A partial
positive charge develops at the anomeric carbon and at
the endocyclic oxygen (Fig. 1) or at the exocyclic oxygen.
Natural aminocyclitols (aminocarbasugars) such as valiol-
amine 1, validamine 2, valienamine 3 (Fig. 1) and their ana-
OH
OH
OH
1
2
3
Figure 1. Transition state model and natural aminocyclitols.
logues can be effective specific inhibitors of glycosidases
involved in intestinal degradation of carbohydrates. They
are supposed to be partially protonated in the active site
at physiological pH, mimicking the TS where the positive
charge is located on the exocyclic oxygen.
*
Corresponding author. Tel.: +33 4 73 40 75 84; fax: +33 4 73 40 77 17;
e-mail: marielle.lemaire@univ-bpclermont.fr
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.09.010

L. El Blidi et al. / Tetrahedron: Asymmetry 17 (2006) 2684–2688
2685
As a consequence, a large variety of synthetic approaches
have been used to develop this class of compounds, ranging
from chemical to enzymatic methods, and employing start-
ing materials ranging essentially from sugars to natural
carbocyclic compounds.9
tained using the procedure of Hoaglin et al.12 The nitro-
aldolisation (Henry reaction) between compound 6 and
nitromethane gave nitroalcohol 7 in an 80% yield. This
compound has been succinctly described by Yanovskaya
et al.,13 NMR and MS data have not been provided. We
fully characterised it to complete its analytical data.
Acid-mediated hydrolysis of the ketal furnished an inter-
mediate aldehyde (quantitative from TLC), which was used
directly in the aldolisation reaction after pH adjustment to
7.5. Rabbit muscle aldolase (RAMA) and DHAP14 were
added to the reaction mixture. After the DHAP was
consumed (checked by enzymatic assay),15 the pH was
adjusted to 3.9 and phytase10 added to hydrolyse the phos-
phate group. Two new nitrocyclitols 8 and 9 were isolated
in a 64% yield (from 7) in a 1:1 ratio. They were separated
by flash chromatography.
Recently we reported the first fructose-1,6-diphosphate
aldolase mediated synthesis of aminocyclohexitol
5
(Fig. 2).10 To access the target compound, our strategy
used the condensation of dihydroxyacetonephosphate
(DHAP) catalysed by the aldolase (RAMA) on 4-nitroal-
dehyde 4.
O
HO
HO
-
HO
OPO3
OH
6
NH2
DHAP
1
O
2
3
O2N
Their absolute configurations (Fig. 3) were determined
from NMR data (NOEs and coupling constants), based
on the aldolase stereoselectivity, which induces the forma-
tion of stereocentre 2 with an (S)-configuration. Interest-
ingly, the configuration of the alcohol functionality on 7
influenced nitrocyclisation. When the alcohol was (S),
nitrocyclitol 8, possessing the same (1S,6R)-configurations
as compound 5 (Fig. 2) was isolated. When the alcohol was
R, the configuration 1R,6S was obtained for compound 9.
We can reasonably assume that the reaction performed in
water and at rt was under thermodynamic control. The
two nitrocyclitols are the more stable isomers. In both
cases, the hydroxymethyl, the nitro and the hydroxyl at po-
sition 5 are equatorial. For compound 9, two hydroxyl
groups (on carbons 2 and 3) are in an axial position. The
reduction of the nitro group of 8 and 9 was then performed
over PtO2 under 50 psi of hydrogen. The two aminocycli-
tols 10 and 11 were isolated with the same 76% yield.
4
5
Aldolase RAMA
HO
Figure 2. Aldolase-mediated synthesis of aminocyclitol.
The enzyme controlled the configuration of two stereocen-
tres (2 and 3), forming the C2–C3 bond. The second
carbon–carbon bond formation (1 and 6) was the result of
a highly stereoselective intramolecular Henry reaction. In
such a process, an enantiomerically pure nitrocyclitol bear-
ing four asymmetric centres was formed. In this report,
following this new methodology, we document the synthesis
of two nitrocyclitols and their corresponding aminocyclitols
with an additional hydroxyl group on C5. The kinetic reso-
lution of the precursor via a lipase is presented. We further-
more report activities of the latter as glycosidase inhibitors.
2. Results and discussion
8
9
NOE
H
Aldehyde diethyl acetal 6 (Scheme 1) was prepared by
ozonolysis of the corresponding alkene11 previously ob-
OH
1
HO
5
H2
H6
OH
H
OH
H
HO
2
3
3
NO2
OH
5
OH
H
CH3NO2,
1
OH
O2N
OH
NaOH 10 M,
EtOH
OH
OEt
H6
HO
OEt
OEt
NOE
O2N
OEt
80%
1S,2S,3R,5S,6R
1R,2S,3R,5R,6S
O
7
6
1) Dowex 50x8 H+, H2O
2) DHAP, RAMA, pH 7.5
then phytase, pH 3.9
Figure 3. NOE’s, configurations and major conformations of compounds
8 and 9.
HO
HO
HO
OH
In order to improve yields and avoid the chromatographic
separation of nitrocyclitols 8 and 9, we decided to study the
lipase catalysed kinetic resolution of alcohol 7. We
expected to resolve 7 by direct enzymatic acylation of the
hydroxy group. All of the results are presented in the table
(Table 1).
OH
NO2
OH
HO
NO2
OH
+
HO
HO
1:1 64%
8
9
H2/PtO2,
MeOH/AcOH: 95/5
Three enzymes (entries 1–3) were tested and lipase B from
Candida antartica showed the best activity. Alcohol 7 was
not a substrate of lipase PS and PC (amano AK and PS,
respectively). Different solvents were screened (entries 4–
6) and diisopropylether (DIPE) gave the best results. Ester
12 underwent spontaneous elimination, giving a large
amount of alkene 13 (Scheme 2).
HO
HO
HO
OH
OH
HO
HO
NH2
OH
NH2
OH
HO
78% 10
80% 11
Scheme 1. Synthesis of aminocyclitols.

2686
L. El Blidi et al. / Tetrahedron: Asymmetry 17 (2006) 2684–2688
Table 1. Kinetic resolution of 7 at rt
Entry
Enzyme
Molarity
Acyl donor (equiv)
Solvent
t (d)
eesa (%)
ca (%)
E
1
2
3
4
5
6
7
8
9
CAL-Bb
PSLb
0.483
0.483
0.483
0.483
0.483
0.483
0.483
0.483
0.483
0.120
Vinylacetate (1.2)
Vinylacetate (1.2)
Vinylacetate (1.2)
Vinylacetate (1.2)
Vinylacetate
Vinylacetate (1.2)
Succinic anhydride (1.2)
Vinylbutyrate (1.2)
Vinylpalmitate (1.2)
Vinylbutyrate (2)
TBME
TBME
TBME
DIPE
Vinylacetate
CH2Cl2
DIPE
DIPE
DIPE
DIPE
6
—
—
10
10
—
—
7
55
—
—
86
25
—
—
97
9
60
—
—
55
30
—
—
52
24
50
3.6
—
—
15
—
—
—
75
—
78
PCLb
CAL-Bb
CAL-Bb
CAL-Bb
CAL-Bb
CAL-Bb
CAL-Bb
CAL-Bc
7
2
10
92
a Determined by HPLC using 1,3,5-trimethoxybenzene as an internal standard.
b Ratio in weight enzyme:alcohol (1:2.5).
c Ratio in weight enzyme:alcohol (1:4).
H
OH
OH
OEt
OEt
O2N
M
L
rac-7
1) dowex 50x8 H+, H2O
2) DHAP, RAMA, pH 7.5
then phytase, pH 3.9
novozym 435
vinylbutyrate
DIPE
HO
HO
OH
OEt
OEt
OH
NO2
O2N
OH
OEt
OEt
OCOR
OEt
OEt
OEt
OEt
+
O2N
O2N
O2N
R-7
OH
50%
HO
R or S-7
12
13
9
Scheme 3. Steric model for the preferentially converted enantiomer of
secondary alcohols by Candida antartica lipase and chemical correlation.
Scheme 2. Lipase resolution of alcohol 7.
pound 10 were 0.117 0.03 mM and 1.46 mM, and for
compound 11 2.27 and 2.62 mM, respectively for b-glu
and b-gal. Compound 10, mimicking the stereochemistry
of glucose more closely was the best inhibitor for b-gluco-
sidase. More generally, the selectivity for b-glycosidases
could be due to the equatorial position of the amine group.
On the contrary in valiolamine, a natural inhibitor of a-
glucosidases, this amino group is in the axial position.
This type of result was also observed by Sheldon et al. with
an analogous compound possessing a phenyl group at the
same place as our ketal functionality.16 Ester 12 was obser-
vable by HPLC, while the reaction medium was kept under
anhydrous conditions. Even after several attempts to purify
it with different workups, we always ended with alkene 13 as
the major product of the reaction. At this point, we turned
our attention to the influence of the acyl donor, trying to se-
lect a less good leaving group by increasing the alkyl chain
length (entries 7–9). No reaction was observed when succi-
nic anhydride was used as the acyl donor. This phenomenon
was also observed by Sheldon et al.16 with the phenyl ana-
logue cited above. The best result, with a largely improved
E, was obtained with vinylbutyrate (entry 8). On a prepara-
tive scale (entry 10), we recovered alcohol 7 in a 92% ee and
50% yield. Finally, we were never able to avoid the forma-
tion of alkene 13. From the steric model for the preferen-
tially converted enantiomer of secondary alcohols by
CAL-B, we expected to prepare alcohol 7 with an (R)-con-
figuration (Scheme 3). This was confirmed by chemical cor-
relation using the aldolase to convert 7 into a nitrocyclitol.
Cyclitol 9 was isolated in a 50% yield (Scheme 3), which
confirmed the R configuration for alcohol 7.
3. Conclusions
As a conclusion, we carried out another efficient synthesis
to prepare two new nitrocyclitols and aminocyclitols in a
highly stereoselective one-pot/two enzyme process. This
illustrates the versatility of our strategy to synthesise
aminocyclohexitols in just a few steps, reducing consider-
ably the laborious protection–deprotection steps always
found when sugars are used as starting material. Enzymatic
resolution of the alcohol precursor showed that the elimi-
nation reaction was difficult to avoid, and allowed us to
successfully prepare the enantiomerically pure ketal precur-
sor with R configuration. The amines were found to be
moderate and selective inhibitors towards b-glycosidases.
Finally, we comment on the inhibitory activities of the
amines prepared in this study against five commercial gly-
cosidases. Amines 10 and 11 were not active against the a-
glycosidase tested (see Experimental). On the contrary,
they moderately and selectively inhibited the b-glycosidases
(b-glucosidase and b-galactosidase). The Ki found for com-
4. Experimental
4.1. General procedures
All the reagents and solvents were of commercial quality
and were purchased from chemical companies. For chro-

L. El Blidi et al. / Tetrahedron: Asymmetry 17 (2006) 2684–2688
2687
matographic purification, technical grade solvents were dis-
tilled prior to use. Merck 60 F254 silica gel TLC plates and
Merck 60/230-400 and 60/40-63 mesh silica gel for column
chromatography were used. Visualisation of the developed
chromatogram was performed by oxidative staining by
either KMnO4–NaHCO3 solution or vanillin solution.
Optical rotations, reported in 10ꢀ1 deg cm2 gꢀ1, were
measured with a Jasco Dip-370 polarimeter. IR spectra
were recorded on an FT IR Perkin Elmer 881 spectropho-
tometer. 1H and 13C NMR spectra were recorded on a Bru-
ker Avance 400 spectrometer in CDCl3, D2O and CD3OD.
J values are given in Hz and d in ppm, referenced to the
internal solvent signals for 1H and 13C. Fructose-1,6-
diphosphate aldolase from rabbit muscle (RAMA; EC
4.1.2.13, suspension in ammonium sulfate) and phytase
from Aspergillus ficuum (EC 3.1.3.8, crude) were from Sig-
ma. C. Antartica lipase type B (CAL-B, Novozyme 435)
was acquired from Sigma. High performance liquid chro-
matography (HPLC) analyses were carried out in a Waters
590 chromatograph UV detector at 210 nm using a Daicel
Chiracel OD column (25 cm · 4.6 mm ID) (hexane/isopro-
panol 98/2, 0.7 mL/min). New cyclitols synthesised were
more or less hygroscopic. Final characterisation of most
of these compounds was therefore done by high resolution
was adjusted to 3.9 with 1 M HCl and phytase (92 U)
was added. The resulting solution was stirred at rt for
24 h, then concentrated under vacuum. The residue was
purified by chromatography on silica gel, eluting with
CH2Cl2/MeOH (9/1 then 8/2), to give the target com-
pounds 8 (96 mg, 32%) and 9 (95 mg, 32%) as brown solids.
Data for 8, Rf = 0.18 (CH2Cl2/MeOH: 85/15). F = 136 ꢁC.
23
½aꢁD = +27 (c 2.67, CH3OH). 1H NMR (400 MHz,
CD3OD): d 4.65 (d, 1H, J = 10.2 Hz); 4.5 (ddd, 1H, J =
10.2, 5.1, 12 Hz); 3.8 (ddd, 1H, J = 9.3, 12, 4.7 Hz); 3.78
(d, 1H, J = 11 Hz); 3.4 (d, 1H, J = 9.3 Hz); 3.25 (d, 1H,
J = 11 Hz); 2.27 (ddd, 1H, J = 4.7, 5.1, 12 Hz); 1.43
(ddd, 1H, J = 12, 12.2, 12 Hz). 13C NMR (100 MHz,
CD3OD): d 93.3; 76.6; 75; 69; 66.7; 62; 39.1. IR (KBr) m
(cmꢀ1) 3480; 1545; 1380; 1063. SM (IC): m/z: 246
(M+Na); 228. Anal. found, C, 37.01; H, 5.88; N, 5.95.
C7H13NO7 requires C, 37.67; H, 5.87; N, 6.28.
Data for 9, Rf = 0.36 (CH2Cl2/MeOH: 85/15). F = 153 ꢁC.
23
½aꢁD = ꢀ33.3 (c 4.25, CH3OH). 1H NMR (400 MHz,
CD3OD): d 4.67 (ddd, 1H, J = 11, 4, 11 Hz); 4.63 (d, 1H,
J = 11 Hz); 4.03 (ddd, 1H, J = 3, 3, 3 Hz); 3.96 (d, 1H,
J = 3 Hz); 3.77 (d, 1H, J = 11.5 Hz); 3.42 (d, 1H, J =
11.5 Hz); 2.15 (ddd, 1H, J = 3, 3, 13.5 Hz); 1.98 (ddd,
1H, J = 3, 11, 13.5 Hz). 13C NMR (100 MHz, CD3OD):
´
mass spectra (HRMS), recorded by the Centre Regional de
Mesures Physiques de Clermont-Fd, France.
d 93.5; 78.4; 72; 70.3; 65.4; 64.9; 36.4. IR (KBr) m (cmꢀ1
)
4.2. Experimental procedures
3480; 1544; 1378; 1063. SM (IC): m/z: 246 (M+Na); 228.
Anal. found, C, 37.66; H, 5.87; N, 6.28. C7H13NO7 requires
C, 37.67; H, 5.87; N, 6.28.
4.2.1. 4,4-Diethoxy-1-nitrobutan-2-ol 7. To a solution of
aldehyde 611,12 (2 g, 13.7 mmol, 1 equiv) in EtOH
(10 mL) was added nitromethane (750 lL, 13.7 mmol,
1 equiv) followed by NaOH 10 N (1.37 mL, 13.7 mmol,
1 equiv). The solution was stirred at 0 ꢁC for 45 min. The
mixture was neutralised with AcOH (785 lL, 13.7 mmol,
1 equiv) and diluted with 10 mL water, extracted with
3 · 100 mL ether. The combined organic phase was dried
over MgSO4, filtered and concentrated in vacuo. The resi-
due was purified by chromatography on silica gel, eluting
with cyclohexane/AcOEt (6/4), to afford the target com-
pound as a slightly yellow oil. Rf = 0.42 cyclohexane/
4.2.3.
(1S,2S,3R,5S,6R)-6-Amino-1-hydroxymethylcyclo-
hexane-1,2,3,5-tetraol 10. To a solution of nitrocyclitol
8 (80 mg, 0.32 mmol) in MeOH/AcOH (95/5) (40 mL)
was added PtO2 (20 mg). The mixture was submitted to
50 psi of H2 in a Parr apparatus. After stirring for 48 h
at rt, the catalyst was removed by ultrafiltration and
washed with MeOH. The filtrate was concentrated under
vacuum, and the crude product was purified by cation ex-
change chromatography (Dowexꢂ 50WX8, 200–400 mesh,
H+ form) eluted with 1 M NH4OH. Compound 10 was ob-
1
AcOEt (6/4). H NMR (400 MHz, CDCl3): d 4.74 (t, 1H,
tained as a white solid in 78% yield (48 mg). Rf = 0.22
23
J = 5 Hz); 4.57 (m, 1H); 4.45 (dd, 2H, J = 2.3, 7 Hz);
3.72 (m, 2H); 3.58 (m, 1H); 3.55 (m, 2H); 1.88 (m, 2H);
1.22 (m, 6H). 13C NMR (100 MHz, CDCl3): d 101.3;
(CH2Cl2/MeOH/NH4OH: 8/1/1). F = 101 ꢁC. ½aꢁD = ꢀ7.9
1
(c 1.1, H2O). H NMR (400 MHz, CD3OD): d 4.65 (d,
1H, J = 10.2); 4.5 (ddd, 1H, J = 10.2, 5.1, 11.7, 5.1 Hz);
3.8 (ddd, 1H, J = 9.3, 12, 4.7 Hz); 3.78 (d, 1H,
J = 11 Hz); 3.4 (d, 1H, J = 9.4); 3.25 (d, 1H, J = 11);
2.27 (ddd, 1H, J = 4.7, 5.1, 12 Hz); 1.43 (ddd, 1H,
J = 12, 12.2, 12 Hz). 13C NMR (100 MHz, CD3OD): d
80.4; 65.8; 62.8; 62.5; 37.2; 15.2. IR (thin film) m (cmꢀ1
)
3435; 1550; 1376; 1125. SM (IC): m/z 206; 190; 162; 144;
103.
4.2.2. (1S,2S,3R,5S,6R)-1-Hydroxymethyl-6-nitrocyclohex-
ane-1,2,3,5-tetraol
74.4; 70.1; 69.9; 65.7; 63.6; 57.7; 33.9. IR (KBr) m (cmꢀ1
3414; 1110. SM (IC): m/z: 193.
)
8 and (1R,2S,3R,5R,6S)-1-hydroxy-
methyl-6-nitrocyclohexane-1,2,3,5-tetraol 9. To a solu-
tion of 7 (400 mg, 2.65 mmol) in 5 mL water was added a
cation exchange resin (Dowex 50x8, H+ form, 1.5 g). The
suspension was stirred at 45 ꢁC for 2.5 h (quantitative by
TLC). The resin was filtered off, the pH was adjusted to
4.2.4. (1R,2S,3R,5R,6S)-6-Amino-1-hydroxymethylcyclo-
hexane-1,2,3,5-tetraol 11. Compound 11 was isolated as
a white solid following the same protocol as described
above, in 80% yield (49 mg). Rf = 0.32 (CH2Cl2/MeOH/
23
1
7.5 with 1 M NaOH. To this solution was added DHAP
(3.62 mL, 1.34 mmol, 1 equiv) followed by 30 mL water,
and the pH was adjusted to 7.5 with 1 M NaOH. The mix-
ture was bubbled with Ar and previously centrifuged aldol-
ase (60 U) was added. After stirring 24 h at rt, the mixture
was washed with 3 · 20 mL AcOEt. The water phase pH
NH4OH: 8/1/1). F = 76 ꢁC. ½aꢁ = +6.6 (c 1.2, H2O). H
NMR (400 MHz, CD3OD): dD4.00 (ddd, 1H, J = 3, 3,
3 Hz); 3.92 (ddd, 1H, J = 11, 4, 11 Hz); 3.75 (d, 1H,
J = 3 Hz); 3.73 (d, 1H, J = 11.5 Hz); 3.62 (d, 1H, J =
11.5 Hz); 3.16 (d, 1H, J = 10 Hz); 2.07 (ddd, 1H, J = 3,
3, 13.5 Hz); 1.83 (ddd, 1H, J = 3, 11, 13.5 Hz). 13C NMR

2688
L. El Blidi et al. / Tetrahedron: Asymmetry 17 (2006) 2684–2688
(100 MHz, CD3OD): d 74.8 70.1; 69.9; 65.5; 64.2;
57.3; 33.9. IR (KBr) m (cmꢀ1) 3414; 1110. SM (IC): m/z:
193.
according to the Hanes–Woolf method. Four substrate
concentrations (0.04–2.5 mM) and four inhibitor concen-
trations (0.005–0.8 mM) were chosen. The Ki was then cal-
culated from the Michaelis–Menten (KM and four K0M)
constants obtained in the presence or absence of inhibitor.
When the percentage of inhibition was between 33% and
10%, the Ki was determined with the following equation
Ki ¼ ½Iꢁ=ðK0M=KM ꢀ 1), using only one K0M value.
4.2.5. Lipase kinetic resolution of 7 by transesterification
4.2.5.1. General. In a typical procedure for analytical
study, the corresponding acyl donor was added to a sus-
pension of 7 and CAL-B in DIPE (or other solvent cited
in the table) and the mixture was shaken at RT following
the progress of the reaction by chiral HPLC (Chiracel
OD) and 1,3,5-trimethoxybenzene as an internal standard.
After removal of the enzyme by filtration and evaporation
of the solvent, the residue was purified by flash chromato-
graphy (cyclohexane/AcOEt (8/2)) to give alcohol (R)-7
and alkene 13.
Acknowledgements
The authors thank Agence Universitaire de la Francopho-
nie (AUF) for financial support (L. El Blidi grant).
4.2.5.2. For preparative scale. (R)-4,4-Diethoxy-1-
nitrobutan-2-ol 7. In the procedure described above, race-
mic alcohol 7 (0.5 g, 2.41 mmol, 1 equiv) and CAL-B
(2 g) in DIPE (20 mL) and vinylbutyrate (630 lL,
4.96 mmol, 2.06 equiv) as acyl donor were used. The alco-
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hol (R)-7 (460 mg) was obtained in a 49% yield, and 92% ee
23
after a flash chromatography purification. ½aꢁD = ꢀ10.4 (c
1.22, CHCl3). Spectral data were identical to the racemic
sample.
4,4-Diethoxy-1-nitrobut-1-ene 13. In the above procedure,
alkene 13 (190 mg) was obtained in a 41% yield.
1
Rf = 0.54 (cyclohexane/AcOEt: 7/3). H NMR (400 MHz,
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CD3OD): d 7.17 (m, 1H); 6.96 (d; 1H, J = 14 Hz); 4.53
(t, 2H, J = 5.3 Hz); 3.62 (qd, 2H, J = 7.1 Hz); 3.43 (qd,
2H, J = 7.1 Hz); 2.51 (dd, 2H, J = 5.3, 5.4 Hz); 1.16 (t,
6H, J = 7.2 Hz). 13C NMR (100 MHz, CD3OD): d 141.1;
137.4; 100.5; 62.0; 33.2; 15.2. IR (thin film) m (cmꢀ1
1652; 1528; 1349; 1122; 1061. SM (IC): m/z: 189.
)
4.2.6. Chemical correlation. Nitrocyclitol 9 was obtained
from (R)-7 following the protocol described above
(97 mg, 51%) as a brown solid.
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4.3. Inhibition studies
a-glucosidase from rice, a-glucosidase from baker’s yeast,
b-glucosidase from almond, a-galactosidase from green
coffee beans, b-galactosidase from Aspergillus oryzae, a-
mannosidase from jack beans and all substrates (4- or 2-
nitrophenyl a- or b-glycopyranosides) were purchased
from Sigma. Assays were run at 25 ꢁC in a phosphate buf-
fer (25 mM) at pH 6.8 using the corresponding 4-nitro-
phenyl-glycoside in a total volume of 1 mL. The potential
inhibitors were tested at a final concentration of 1 mM
and the amount of enzyme of each assay was adjusted so
that the system would give the initial rate. After two peri-
ods (5 min and 30 min) of incubation of the enzyme in
the presence of the tested molecule, the substrate was
added and the optical absorbance was followed at
400 nm. The initial rate was determined, compared to the
one obtained without the tested molecule, and the percent-
age of inhibition was calculated. When the percentage of
inhibition was higher than 33%, the Ki was determined
16. Sorgedrager, M. J.; Malpique, R.; van Rantwijk, F.; Sheldon,
R. A. Tetrahedron: Asymmetry 2004, 15, 1295–1299.