The acetates of p-nitrophenyl α-L-arabinofuranoside - Regioselective preparation by action of lipases

Article abstract of DOI:10.1016/j.bmc.2005.10.023

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl α-l-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitr

Full text of DOI:10.1016/j.bmc.2005.10.023

Bioorganic & Medicinal Chemistry 14 (2006) 1805–1810
The acetates of p-nitrophenyl
a-L-arabinofuranoside—Regioselective preparation
by action of lipases
*
´
´
´
Maria Mastihubova, Jana Szemesova and Peter Biely
Institute of Chemistry, Slovak Academy of Sciences, SK-845 38 Bratislava, Slovakia
Received 25 July 2005; revised 10 October 2005; accepted 18 October 2005
Available online 8 November 2005
Abstract—All possible di-O-acetates and mono-O-acetates of p-nitrophenyl a-L-arabinofuranoside were prepared by chemoenzy-
matic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acet-
ylated p-nitrophenyl a-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-
di-O-acetates were obtained by acetylation of p-nitrophenyl a-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in
organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl a-L-arabinofurano-
side catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl a-L-arabinofuranoside were obtained in
two steps. The enzymatic di-O-acetylation of p-nitrophenyl a-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis
of the primary acetyl group by CCL or LAY.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
alkyl arabinofuranosides7 or a-L-arabinofuranose-con-
taining disaccharides.8
Development of economically feasible and ecologically
friendly processes of bioconversion of renewable plant
biomass requires a detailed knowledge of catalytic prop-
erties of microbial glycosyl hydrolases and esterases in-
volved in biodegradation of plant cell walls. In
connection with this effort there is an increasing demand
for a variety of regioselectively acetylated p-nitrophenyl
glycosides1 that can serve as precursors for chromogenic
substrates important for investigation of catalytic prop-
erties of the enzymes and for elaboration of simple
methods for enzyme monitoring.2
The problem of selective modification of carbohydrates
as polyhydroxylated molecules is generally known. Dif-
ferent types of multi-step chemical protection/deprotec-
tion strategies had to be developed for structurally
different kinds of saccharides to accomplish reactions
at selected positions. Several approaches have also been
introduced in the case of a-L-arabinofuranosides.4,5,9,10
In order to shorten the lengthy protection/deprotection
sequences associated with the modification of monosac-
charides, we turned our attention to enzymes that can
selectively modify carbohydrates. Lipases are well-
known biocatalysts widely used for regioselective deac-
ylation or acylation of carbohydrates.11–17 Most of the
enzymatic studies have focused on hexopyranose deriv-
atives. Modifications of furanose derivatives by
lipases,11,18–22 esterases23–25 or proteases26–29 concern
mostly the primary position of furanoside ring. Excep-
a-L-Arabinofuranosyl residues are widely distributed in
plant cell-wall polysaccharides.3 These polymers4,5 and
enzymes hydrolysing their glycosidic linkages, such as
a-L-arabinofuranosidases,6 are gaining increasing atten-
tion. Recently, a few reports reported on transglycosyla-
tion reactions of a-L-arabinofuranosidases leading to
tions
are
some
D-furanosides11,18,21,23–25
or
ribonucleosides.20,26
Keywords: p-Nitrophenyl a-L-arabinofuranoside acetates; Lipases;
Regioselective acetylation and deacetylation.
Recently we synthesised 2-O- and 5-O-feruloylated
p-nitrophenyl a-L-arabinofuranoside30 as substrates for
feruloyl esterases.31 The starting di-O-acetates in this
*
Corresponding author. Tel.: +4212 59410 246; fax: +4212 59410
222; e-mail: chemjama@savba.sk
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.023

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M. Mastihubova et al. / Bioorg. Med. Chem. 14 (2006) 1805–1810
1806
synthesis were regioselectively prepared trough enzy-
matic steps. Here, we report summary of the optimised
enzymatic synthesis of three possible di-O-acetates and
three mono-O-acetates of p-nitrophenyl a-L-arabinofu-
ranoside. Regioselective acetylation or deacetylation
was accomplished by transesterification in organic med-
ia or hydrolysis catalysed by commercial lipases. We did
not find any paper, except our preliminary work,30 deal-
ing with enzymatic acylation or hydrolysis of L-arabino-
furanoside derivatives.
the reaction times in individual organic solvents (Table
1) with those of LPS-30-catalysed acetylation of p-nitro-
phenyl xylopyranoside under appropriate conditions1 is
in favour of arabinofuranoside 2. Solvent polarity
showed little effect on the final proportion of the diace-
tates. In most solvents, a mixture of 2,5- and 3,5-diace-
tate (4 and 5) was produced in a ratio about 1:3,
regardless of their hydrophobicity (Table 1). This fact
is in contradiction with our previous observation that
the regioselectivity of the di-O-acetylation of p-nitro-
phenyl xylopyranoside1 was affected by polarity of the
used organic solvents. Small amounts of the triacetate
1 and monoacetate 6 were also formed. Based on these
results, one may conclude that arabinofuranoside 2
has a weaker interaction with the enzyme active site than
pyranoside derivatives studied previously.1,32,33 We did
not find conditions to produce solely 5-O-acetate 6 upon
catalysis by LPS-30.
2. Results and discussion
2.1. Preparation of p-nitrophenyl 2,3-di-O-acetyl-a-L-
arabinofuranoside (3)
The starting compound p-nitrophenyl 2,3,4-tri-O-acetyl-
a-L-arabinofuranoside (1) was prepared by conventional
acetylation of p-nitrophenyl a-L-arabinofuranoside (2),
using acetanhydride in pyridine. p-Nitrophenyl 2,3-di-
O-acetyl-a-L-arabinofuranoside (3) (Scheme 1) was ob-
tained through hydrolysis of primary acetyl group of 1
by lipase from Candida cylindracea (CCL) or lipase from
Candida rugosa (LAY). In both cases, the starting triac-
etate 1 was completely consumed after 4 h and product 3
was isolated as a single crystalline compound in 90%
yield after purification. A similar selective removal of
the primary acyl in esterified monosaccharides was ob-
tained under the action of CCL or LAY.11,16 No acetyl
group migration1 in product 3 was observed in used
aqueous media, only a very slow spontaneous deacetyla-
tion took place during the reaction.
2.3. Preparation of p-nitrophenyl 5-O-acetyl-a-L-arabi-
nofuranoside (6)
We also focused on preparation of monoacetates of p-
nitrophenyl a-L-arabinofuranoside. The problem to ter-
minate the acetylation of 2 by LPS-30 at the stage of the
monoacetate 6 was overcome using the porcine pancreas
lipase (PPL) frequently applied for the acylation of pri-
mary hydroxyl group of monosaccharides.11,16 Using vi-
nyl acetate as an acetate donor, PPL in THF catalysed
selective synthesis of 5-monoacetate 6 from 2 in 95%
yield (Scheme 3) and satisfactory reaction time.
2.4. Preparation of p-nitrophenyl 2- and 3-O-acetyl-a-L-
arabinofuranoside (7 and 8)
2.2. Preparation of p-nitrophenyl 2,5- and 3,5-di-O-
acetyl-a-L-arabinofuranoside (4 and 5)
Among commercially available lipases and proteases, we
did not find any preparation able to catalyse acetylation
of unprotected monosaccharides selectively to position
2-OH or 3-OH. That is why two monoacetates 7 and 8
were obtained from 2,5- and 3,5-di-O-acetates by hydro-
lysis of the primary OAc moiety by CCL. The 2-O-ace-
tate 7 was produced from 2,5-diacetate 4 in 80% yield
(Scheme 4). Slightly acidic pH 6 was used to diminish
spontaneous hydrolysis of acetates. From Fig. 1A and
Table 2 (entry 4) it is evident that a small amount of
5-O-acetate 6 was formed beside the desired product 7.
The 5-O-acetate 6 was then rapidly converted to 2 by
CCL.
Of several enzymes, tested for preparation of the two
remaining di-O-acetates, the lipase from Pseudomonas
cepacia (LPS-30) was found to acetylate smoothly p-
nitrophenyl a-L-arabinofuranoside (2). A mixture of
di-O-acetates 4 and 5 was formed in high yields in differ-
ent organic solvents (Scheme 2 and Table 1). Solvents
possessing different polarity were examined. The reac-
tions were performed at 40 ꢁC and were stopped after al-
most all p-nitrophenyl 5-O-acetyl-a-L-arabinofuranoside
(6) was converted to diacetates. The course of acetyla-
tion of furanoside 2 was monitored by HPLC. Analysis
of the results presented in Table 1 suggests that activity
of LPS-30 depends on polarity of the solvents used. The
reaction proceeded more rapidly in less polar solvents.
The time of di-O-acetylation of 2 catalysed by LPS-30
in diisopropyl ether was comparable to the time of the
conventional chemical acetylation. The comparison of
The trend to hydrolyse the secondary OAc group beside
the primary one was stronger during treatment of 3,5-O-
diacetate 5 by CCL. The reaction led to formation of
several products—monoacetates 6, 8 and completely
deacetylated 2. The desired 3-O-acetate 8 was obtained
in 50–55% yield. These results are explained by parallel
deacetylation of 5-OAc and 3-OAc of 3,5-di-O-acetyl-a-
L-arabinofuranoside 5 (Scheme 5). The primary 5-OAc
group was hydrolysed away faster than the secondary
3-OAc group of 8, which seems to be a kinetically pre-
ferred product (Fig. 1B). Similar product composition
was observed at different pH (Table 2, entries 5 and
6). Decrease in the temperature did not reduce the for-
mation of by-products (Table 2, entry 7).
ONPh
ONPh
AcO
AcO
a
O
O
HO
AcO
OAc
OAc
3
1
Scheme 1. Reagents and conditions: (a) CCL or LAY, 10% DMF in
0.1 M phosphate buffer (pH 7), 37 ꢁC, 4 h.

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M. Mastihubova et al. / Bioorg. Med. Chem. 14 (2006) 1805–1810
1807
ONPh ONPh ONPh
HO
HO
AcO
O
O
O
a
+
6
+
1
+
HO
AcO
AcO
OH
OH
OAc
2
4
5
Scheme 2. Reagents: (a) LPS, vinyl acetate and organic solvent (Table 1).
Table 1. Results of acetylation of 2 by LPS-30 in organic solvents at 40 ꢁC
a
Entry
Organic solvent
LogPow
Reaction time (h)
Conversion (4 + 5) (%)
Ratio (4:5)
6 (%)
1 (%)
1
2
3
4
5
6
7
8
Cyclohexane
Toluene
3.40
2.69
76
75.5
23.5
77
64
91
97
96
93
96
97
93
1:1.1
1:3.6
1:2.7
1:2.3
1:3.1
1:3.6
1:3.0
1:3.2
0
0
0
2
1
0
1
1
36
9
Diisopropyl ether
Methyl isobutyl ketone
Ethyl acetate
Vinyl acetate
tert-Butanol
1.90
1.38
3
2
0.73
0.73
147
51
6
3
0.40
ꢀ0.33
146
146.5
2
Acetonitrile
6
a Octanol/water partition coefficient.
ONPh
ONPh
HO
The fact, that during hydrolysis of the primary acetyl
group of diacetates 4 or 5 the secondary OH groups
(3-OH more than 2-OH) were also partially deacetylat-
ed, motivated us to treat pure 2,3-diacetate 3 with
CCL under similar conditions like 4 and 5. Interestingly,
diacetate 3 exhibited high stability under these condi-
tions (Table 2, entry 3). No loss of 3 was recorded after
a 45.5 h treatment by CCL at 37 ꢁC.
HO
O
O
a
AcO
HO
OH
OH
2
6
Scheme 3. Reagents and conditions: (a) PPL, vinylacetate, THF, 15 h,
95%.
ONPh
ONPh
HO
HO
a
O
O
Next, to examine the effect of the acetyl migration or
spontaneous deacetylation on the results previously ob-
served, 3 was incubated several days in phosphate buffer
(pH 6) at 40 ꢁC according to our previous studies with
partially acetylated p-nitrophenyl xylopyranoside.1
AcO
HO
OAc
OAc
4
7
Scheme 4. Reagents and conditions: (a) CCL, 10% DMF in 0.1 M
phosphate buffer (pH 6), 37 ꢁC, 7 h, 80%.
100
100
80
60
40
20
0
A
B
80
60
40
20
0
0
2
4
6
8
0
2
4
6
8
Time (h)
Time (h)
Figure 1. Time course of conversion of di-O-acetates 4 (A), 5 (B) into compounds 6, 7, 8 and 2 during incubation with CCL in phosphate buffer
(pH 6) at 37 ꢁC. Symbols: (s), 4; (n), 5; (m), 6; (j), 7; (Ç), 8; (d), 2.
Table 2. HPLC results of enzymatic hydrolysis of acetates 1, 4 and 5 by LCC, resp. LAY at 37 ꢁC in 0.033 M solution of DMF/0.1 M phosphate
buffer, 1:9
Entry
SCa
pH
Lipase
Timeb (h)
1
3
4
5
6
7
8
2
1
2
3
4
5
6
7
1
1
3
4
5
5
5
6
7
6
6
6
6
7
CCL
CCL
CCL
CCL
CCL
LAY
CCL
5.8
3.7
4.7
4.2
94.4
95.0
0.9
0.8
0
0
45.5
7.3
100
4.7
1.9
8.3
6.8
8.0
80.7
12.7
17.3
18.8
16.5
4.6
4.9
6.2c
17.9
16.5
22.8
55.5
57.9
52.6
a Starting compound.
b Time of maximal concentration of the desired products in reaction mixtures.
c Reaction at 30 ꢁC.

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M. Mastihubova et al. / Bioorg. Med. Chem. 14 (2006) 1805–1810
1808
ONPh
ONPh
ONPh
HO
AcO
AcO
O
O
O
a
+
2
+
AcO
HO
AcO
OH
OH
OH
5
8
6
Scheme 5. Reagents and conditions: (a) CCL or LAY, 10% DMF in 0.1 M phosphate buffer (pH 6) (Table 2, entries 5–7).
from Amano (Japan). Lipase from C. cylindracea, syn.
100
80
60
40
20
0
C. rugosa (CCL) was a gift from Lyven (France). The
porcine pancreas lipase (PPL) was purchased from
Sigma. Solvents were distilled from the appropriate dry-
ing agents before use. The p-nitrophenyl a-L-arabinofu-
ranoside 2 was purchased from Sigma or prepared by
glycosylation of 1,2,3,5-tetra-O-acetyl-a,b-L-arabino-
furanose34 with p-nitrophenol using SnCl4 in CH2Cl2
followed by treatment with 0.09 mM MeONa/MeOH
in 60% yield. All reactions were monitored on TLC
plates (Silicagel 60, 0.25 mm, E. Merck, Darmstadt,
Germany). The compounds were detected by charring
the plates with 1% orcinol in 10% (v/v) ethanolic solu-
tion of H2SO4 at ca. 200 ꢁC. Column chromatography
was performed on Silica gel (0.035–0.070 mm, pore
0
100
200
Time (h)
300
Figure 2. Time course of conversion of acetate 3 during incubation in
phosphate buffer (pH 6) at 40 ꢁC. Symbols: (h), 3; (s), 4; (n), 5; (m),
6; (j), 7; (Ç), 8; (d), 2.
ꢀ
diameter ca. 6 nm, Acros Organics) in various solvent
systems. Melting points were determined with a Kofler
hot-stage and are uncorrected. Optical rotations were
measured with a Perkin-Elmer 241 polarimeter at
20 ꢁC. HPLC analysis was carried out on a chromato-
graph equipped with a silica gel column (Biospher Si
100 5 lm). The elution of acetates of p-nitrophenyl
a-L-arabinofuranoside was monitored with a calibrated
UV detector at 305 nm. The elution times in hexane/eth-
yl acetate, 3:2 and a rate of 1.0 mL/min were 2.0 min for
1, 2.5 min for 5, 3.1 min for 4, 3.6 min for 3, 4.7 min for
8, 9.1 min for 6, 12.33 min for 7 and 30.5 min for 2;
alternatively in hexane/ethyl acetate, 1:1 and a rate of
1.5 mL/min, they were 1.4 min for 1, 1.6 min for 5,
1.7 min for 4, 1.8 min for 3, 2.1 min for 8, 2.8 min for
6, 3.3 min for 7 and 12.4 min for 2. The quantitation
HPLC analysis showed (Fig. 2) that the acetyl migration
in 3 did not proceed or proceeded extremely slowly un-
der these conditions. Only spontaneous deacetylation
was monitored. The deacetylation from 2-OAc group
is more rapid due to a higher reactivity of O-2 resulting
from the inductive effect of the anomeric centre.
3. Conclusion
The regioselectivity of acetylation and deacetylation of
derivatives of p-nitrophenyl a-L-arabinofuranoside
using lipases was studied. All six possible partially acet-
ylated derivatives of p-nitrophenyl a-L-arabinofurano-
side have been prepared. The 2- and 3-O-acetates of
p-nitrophenyl a-L-arabinofuranoside were prepared in
two steps. The first step was the enzymatic di-O-acetyla-
tion and the second one was enzymatic hydrolysis of the
primary acetyl group. The prepared di-O-acetates have
been used for the syntheses of carbohydrate ferulates30
through enzymatic protection, feruloylation and chemi-
cal deacetylation. The monoacetates will be used to
study substrate specificity and substrate structure
requirements of a-L-arabinofuranosidases and carbohy-
drate esterases. These prepared partially acetylated ara-
binofuranosides can also serve as building blocks for the
synthesis of L-arabinooligosaccharides. Neither mono-
nor di-O-acetates of p-nitrophenyl a-L-arabinofurano-
side underwent significant spontaneous acetyl group
migration in aqueous media.
1
was done by integration of the peak areas. H NMR
spectra were recorded at 300 MHz with Bruker AM
300 (Me4Si as internal standard). Homonuclear correla-
tion two-dimensional technique COSY-45 was used for
complete assignment of protons. 13C NMR spectra were
recorded in CDCl3 at 75 MHz and shifts are referenced
to internal CDCl3. Microanalyses were performed with
a Fisons EA 1108 analyser.
4.2. p-Nitrophenyl 2,3-di-O-acetyl-a-L-arabinofuranoside
(3)
p-Nitrophenyl 2,3,5-tri-O-acetyl-a-L-arabinofuranoside
(1) (0.4 g, 1 mmol) was dissolved in DMF (3 mL) and
then 0.1 M phosphate buffer pH 7 (27 mL) was added
followed by addition of lipase CC (0.8 g). The reaction
mixture was shaken at 37 ꢁC and 200 rpm. After
220 min, the HPLC monitoring (hexane/ethyl acetate,
3:2, rate 1 mL/min) showed transformation of 1 to
95% of 3 and 0.8% of 7. The reaction was stopped by
extraction of saccharidic material from reaction mixture
to ethyl acetate. The organic phase was dried (Na2SO4)
and concentrated under reduced pressure. The product 3
was separated from the starting compound and traces of
4. Experimental
4.1. General
Lipase from C. rugosa (LAY-30) and lipase from
Burkholderia cepacia, syn. P. cepacia (LPS-30) were gifts

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M. Mastihubova et al. / Bioorg. Med. Chem. 14 (2006) 1805–1810
1809
0
7 by column chromatography (toluene/ethyl acetate,
2:1) to give 3 as a white solid (0.32 g, 90%). Mp =
OH), 4.29 (dd, 1H, J4,5 4.7 and J5;5 12.3 Hz, H-5),
4.41 (dd, 1H, J4;5 3.1 and J5;5 12.5 Hz, H-50), 4.45 (m,
1H, H-4), 4.48 (br d, 1H, H-2), 4.80 (dd, 1H, J2,3 2.7
and J3,4 6.3 Hz, H-3), 5.76 (s, 1H, H-1), 7.12 (d, 2H, J
9.3 Hz, H-20, H-60), 8.21 (d, 2H, J 9.3 Hz, H-30, H-50).
13C NMR (75 MHz CDCl3) d d 20.7 (2 · COCH3),
62.7 (C-5), 80.5, 80.8, 81.2 (C-2, C-3, C-4), 105.8 (C-
1), 2 · 116.4, 2 · 125.8, 142.6, 161.1 (6 · C-Ar), 170.5
(COCH3), 171.8 (COCH3). Anal. Calcd for
C15H17NO9: C, 50.71; H, 4.82, N, 3.94. Found: C,
0
0
77–79 ꢁC
(isopropyl
alcohol/diisopropyl
ether);
20
½aꢁ ꢀ141.0 (c 1.0, CHCl3); [lit. 35 77–79 ꢁC (diisopropyl
D
20
1
ether); ½aꢁ ꢀ142 (c 1.2, CHCl3)]. H NMR (300 MHz,
CDCl3) dDd 1.63 (br s, 1H, OH), 2.15 (s, 3H, COCH3),
0
2.16 (s, 3H, COCH3), 3.84 (dd, 1H, J4,5 4.0 and J5;5
0
0
12.3 Hz, H-5), 3.92 (dd, 1H, J4;5 3.4 and J5;5 12.3 Hz,
H-50), 4.25 (dt, 1H, H-4), 5.20 (dd, 1H, J3,4 5.2 and
J2,3 1.8 Hz, H-3), 5.43 (d, J1,2 0 and J2,3 1.8 Hz, H-2),
5.9 (d, 1H, H-1), 7.15 (d, 2H, J 9.2 Hz, H-20, H-60),
50.71; H, 5.00; N, 3.98. Next eluted was oily 4 (0.33 g
20
25%). ½aꢁ ꢀ156.0 (c 1.0, CHCl3). 1H NMR
8.21 (d, 2H,
J
9.2 Hz, H-30, H-50). 13C NMR
D
(75 MHz, CDCl3) d d 20.8 (2 · COCH3), 61.7 (C-5),
76.6 (C-3), 81.6 (C-2), 84.6 (C-4), 103.7 (C-1),
2 · 116.6, 2 · 125.8, 142.8, 160.8 (6 · C-Ar), 169.7
(COCH3), 170.5 (COCH3). Anal. Calcd for
C15H17NO9: C, 50.71; H, 4.82; N, 3.94. Found: C,
50.42; H, 5.17; N, 3.69.
(300 MHz, CDCl3) d d 2.11 (s, 3H, COCH3), 2.18 (s,
3H, COCH3), 3.32 (br s, 1H, OH), 4.12 (dd, 1H, J2,3
0
3.4 and J3;4 6.4 Hz, H-3), 4.27 (dd, 1H, J4,5 6.3 and
0
J5;5 12.9 Hz, H-5), 4.36 (m, 1H, H-4), 4.38 (dd, 1H,
J4;5 3.2 and J5;5 13.0 Hz, H-50), 5.18 (dd, J1,2 1.1 and
J2,3 3.4 Hz, H-2), 5.81 (d, J1,2 1.1 Hz, H-1), 7.14 (d,
2H, J 9.3 Hz, H-20, H-60), 8.21 (d, 2H, J 9.3 Hz, H-30,
0
0
4.3. HPLC test for the acetyl migration
H-50). 13C NMR (75 MHz, CDCl3)
d
d
20.7
(2 · COCH3), 62.8 (C-5), 76.9 (C-3), 82.1 (C-4), 86.1
(C-2), 103.7 (C-1), 2 · 116.5, 2 · 125.7, 142.7, 160.9
(6 · C-Ar), 170.8 (COCH3), 171.4 (COCH3). Anal.
Calcd for C15H17NO9: C, 50.71; H, 4.82, N, 3.94.
Found: C, 50.57; H, 5.07; N, 3.53.
Di-O-acetate 3 (0.002 mmol) was dissolved in DMSO
(80 lL) and mixed with 0.1 M sodium phosphate buffer
(1920 lL) of pH 6 preheated to 40 ꢁC. The reaction mix-
tures were incubated at 40 ꢁC for several days. The ali-
quots (50 lL) were withdrawn at time intervals and
directly injected to the HPLC system through a 20 lL
loop using a silica gel column eluted with hexane/ethyl
acetate, 3:2, rate 1 mL/min. The graphical illustration
of the monitored process is shown in Figure 2.
4.6. p-Nitrophenyl 5-O-acetyl-a-L-arabinofuranoside (6)
p-Nitrophenyl a-L-arabinofuranoside 2 (0.27 g, 1 mmol)
was dissolved in THF (25 mL) and vinyl acetate
(1.4 mL, 15 mmol) followed by PPL (1 g) was added.
The reaction mixture was shaken at 40 ꢁC and
200 rpm for 15 h. The reaction was stopped by filtering
off the lipase. The filtrate was concentrated and the res-
idue was purified by column chromatography (toluene/
ethyl acetate, 1:2) to give only monoacetate 6 (0.30 g,
4.4. Monitoring of di-O-acetylation of p-nitrophenyl a-L-
arabinofuranoside
p-Nitrophenyl a-L-arabinofuranoside (2) (27.1 mg,
0.1 mmol) was dissolved in a selected organic solvent
20
D
1
˚
(954 lL) and then molecular sieve (4 A, 250 mg), vinyl
95%) as a colourless oil. ½aꢁ ꢀ133 (c 1.0, CHCl3). H
acetate (46 lL, 5 equiv) and 270 mg LPS were added.
The reaction mixtures were shaken at 40 ꢁC at
200 rpm. Aliquots were withdrawn in different time
intervals and filtered (Durapore filter, 0.45 lm). The fil-
tered solutions were analysed by HPLC on a silica gel
column in hexane/ethyl acetate, 1:1, at an elution rate
of 1.5 mL/min. The ratios of compounds, calculated
from the integrated peak areas, are shown in Table 1.
NMR (300 MHz, CDCl3) d d 2.12 (s, 3H, COCH3),
2.81 (br s, 2H, OH), 4.10 (dd, 1H, J2,3 3.8 and J3,4
5.1 Hz, H-3), 4.29–4.34 (m, 3H, H-4, H-5, H-50), 4.49
0
0
(dd, 1H, J1;2 1.6 and J2;3 3.7 Hz, H-2), 5.71 (d, J1,2
1.5 Hz, H-1), 7.12 (d, 2H, J 9.2 Hz, H-20, H-60), 8.18
(d, 2H, J 9.2 Hz, H-30, H-50). 13C NMR (75 MHz,
CDCl3) d d 20.8 (COCH3), 63.4 (C-5), 77.8 (C-3), 81.4
(C-2), 83.6 (C-4), 106.0 (C-1), 2 · 116.5, 2 · 125.8,
142.7, 161.7 (C-Ar), 170.8 (COCH3). Anal. Calcd for
C13H15NO8: C, 49.84; H, 4.83, N, 4.47. Found: C,
50.06; H, 5.01; N, 4.05.
4.5. p-Nitrophenyl 2,5- and 3,5-di-O-acetyl-a-L-arabino-
furanoside (4 and 5)
p-Nitrophenyl a-L-arabinofuranoside (2) (1 g, 3.7 mmol)
was suspended in diisopropyl ether (300 mL). Molecular
4.7. p-Nitrophenyl 2-O-acetyl-a-L-arabinofuranoside (7)
˚
sieves (4 A, 5 g), vinyl acetate (3.4 mL, 37 mmol,
2,5-Diacetate 4 (0.18 g, 0.5 mmol) was dissolved in
DMF (1.5 mL), then 0.1 M phosphate buffer, pH 6
(13.5 mL), was added followed by addition of CCL
(0.1 g). The reaction mixture was shaken at 37 ꢁC and
200 rpm for 7 h. Then ethyl acetate was added to the
reaction mixture and products were extracted from the
aqueous mixture. The organic phase was dried (Na2SO4)
and concentrated under reduced pressure. The 2-acetate
7 was separated from 2 and traces of the starting com-
pound and 6 by column chromatography (toluene/ethyl
acetate, 1:1) to give 7 as a colourless oil (0.11 g, 76%).
10 equiv) and lipase PS (9 g) were added. The reaction
mixture was shaken at 37 ꢁC and 200 rpm for 30 h.
The reaction was stopped by filtration off the lipase,
which was then repeatedly used in four subsequent reac-
tion cycles. The filtrate was concentrated and the residue
was fractionated by column chromatography (toluene/
ethyl acetate, 5:1 ! 2:1) to give first 1 as an oil (0.06 g,
7%), then 5 as a white solid (0.48 g, 61%). Mp = 67–
20
68 ꢁC (diisopropyl ether/cyclohexane). ½aꢁ ꢀ200.0 (c
D
1
1.0, CHCl3). H NMR (300 MHz, CDCl3) d d 2.12 (s,
3H, COCH3), 2.18 (s, 3H, COCH3), 3.21 (br s, 1H,
20
D
1
½aꢁ ꢀ160 (c 1.0, CHCl3). H NMR (300 MHz, CDCl3)

´
M. Mastihubova et al. / Bioorg. Med. Chem. 14 (2006) 1805–1810
1810
0
4. Kaneko, S.; Kawabata, Y.; Ishii, T.; Gama, Y.; Kusakabe,
I. Carbohydr. Res. 1995, 268, 307.
5. Du, Y.; Pan, Q.; Kong, F. Carbohydr. Res. 2000, 329, 17.
6. Kaji, A. Adv. Carbohydr. Chem. Biochem. 1984, 42, 383.
d d 2.18 (s, 3H, COCH3), 3.80 (dd, 1H, J4,5 1.6 and J5;5
0
0
12.8 Hz, H-5), 3.96 (dd, 1H, J4;5 2.0 and J5;5 12.2 Hz,
H-50), 4.26 (m, 2H, H-3, H-4), 5.16 (br s, 1H, H-2),
5.84 (d, J1,2 1.2 Hz, H-1), 7.14 (d, 2H, J 9.3 Hz, H-20,
H-60), 8.22 (d, 2H, J 9.3 Hz, H-30, H-50). 13C NMR
(75 MHz, CDCl3) d d 20.8 (COCH3), 61.1 (C-5), 76.1
(C-3), 84.3 (C-4), 87.1 (C-2), 103.7 (C-1), 2 · 116.5,
2 · 125.8, 142.7, 161.0 (6 · C-Ar), 171.7 (COCH3).
Anal. Calcd for C13H15NO8: C, 49.84; H, 4.83; N,
4.47. Found: C, 50.11; H, 5.07; N, 4.09.
´
7. Remond, C.; Ferchichi, M.; Aubry, N.; Plantier-Royon,
R.; Portella, Ch.; OÕDonohue, M. J. Tetrahedron Lett.
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´
8. Remond, C.; Plantier-Royon, R.; Aubry, N.; Maes, E.;
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9. Czernecki, S.; Le Diguarher, T. Synthesis 1991, 683.
10. Kawabata, Y.; Kaneko, S.; Kusakabe, I.; Gama, Y.
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4.8. p-Nitrophenyl 3-O-acetyl-a-L-arabinofuranoside (8)
11. Hennen, W. J.; Sweers, H. M.; Wang, Y.-F.; Wong, C.-H.
J. Org. Chem. 1988, 53, 4939.
3,5-Diacetate 5 (0.36 g, 1 mmol) was dissolved in DMF
(3 mL) and then 0.1 M phosphate buffer, pH 6 (27 mL)
and lipase CC (0.8 g) were added. The reaction mixture
was shaken at 37 ꢁC and 200 rpm for 4.5 h when concen-
tration of 8 reached the maximum according to HPLC
(hexane/ethyl acetate, 1:1, rate 1.5 mL/min). The reac-
tion was stopped by extraction of the reaction material
to ethyl acetate. The organic phase was dried (Na2SO4)
and concentrated under reduced pressure. The product 8
was purified from starting compound 5, formed 2 and
traces of 6 by column chromatography (toluene/ethyl
12. Wang, Y. F.; Lalonde, J. J.; Momongan, M.; Bergbreiter,
D. E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200–
7205.
13. Drueckhammer, D. G.; Hennen, W. J.; Pederson, R. L.;
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Synthesis 1991, 499.
14. Reidel, A.; Waldmann, H. J. Prakt. Chem. 1993, 335, 109.
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Organic Synthesis—Regio- and Stereoselective Biotransfor-
mations; Wiley-VCH: Weinheim, 1999.
16. Kadereit, D.; Waldmann, H. Chem. Rev. 2001, 101, 3367.
17. La Ferla, B. Monatsh. Chem. 2002, 133, 351.
18. Kloosterman, M.; Mosmuller, E. W. J.; Schoemaker, H.
E.; Meijer, E. M. Tetrahedron Lett. 1987, 28, 2989.
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Tetrahedron Lett. 1995, 36, 6163.
acetate, 2:1) to give 8 as a white solid (0.16 g, 51%).
20
D
Mp = 107–108 ꢁC (diisopropyl ether); ½aꢁ ꢀ186 (c 1.0,
1
CHCl3). H NMR (300 MHz, CDCl3) d d 2.17 (s, 3H,
COCH3), 3.91 (dd, 1H, J4,5 2.0 and J5;5 11.9 Hz, H-5),
0
´
´
20. Garcıa, J.; Fernandez, S.; Ferrero, M.; Sanghvi, Y. S.;
Gotor, V. Tetrahedron: Asymmetry 2003, 14, 3533.
21. Fernandez-Lorente, G.; Palomo, J. M.; Cocca, J.; Mateo,
C.; Moro, P.; Terreni, M.; Fernandez-Lafuente, R.;
Guisan, J. M. Tetrahedron 2003, 59, 5705.
3.96 (dd, 1H, J4;5 2.3 and J5;5 11.9 Hz, H-50), 4.33 (m,
1H, H-4), 4.46 (br s, 1H, H-2), 5.01 (dd, 1H, J2,3 1.3
and J3,4 4.0 Hz, H-3), 5.79 (s, 1H, H-1), 7.12 (d, 2H, J
9.2 Hz, H-20, H-60), 8.21 (d, 2H, J 9.2 Hz, H-30, H-50).
13C NMR (75 MHz, CDCl3) d d 20.8 (COCH3), 61.5
(C-5), 79.6, 79.8 (C-2, C-3), 84.7 (C-4), 106.2 (C-1),
2 · 116.3, 2 · 125.8, 142.4, 161.2 (6 · C-Ar), 171.4
(COCH3). Anal. Calcd for C13H15NO8: C, 49.84; H,
4.83; N, 4.47. Found: C, 50.01; H, 4.96; N, 4.19.
0
0
22. Chien, T.-C.; Chern, J.-W. Carbohydr. Res. 2004, 339, 1215.
´
´
23. Kefurt, K.; Kefurtova, Z.; Jary, J. Carbohydr. Res. 1992,
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´
´
Stanek, J. J. Carbohydr. Chem. 1997, 16, 1011.
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25. Moravcova, J.; Hamernık, I.; Funkova, G.; Capkova, J.;
ˇ
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Kefurt, K. J. Carbohydr. Chem. 1998, 17, 1191.
´
´
´
26. Riva, S.; Chopineau, J.; Kieboom, A. P. G.; Klibanov, A.
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Acknowledgment
27. Wong, C.-H.; Chen, S.-T.; Hennen, W. J.; Bibbs, J. A.;
Wang, Y.-F.; Liu, J. L.-C.; Pantoliano, M. W.; Whitlow,
M.; Bryan, P. N. J. Am. Chem. Soc. 1990, 112, 945.
This work was supported by the Slovak Grant Agency
for Science VEGA No. 2/3079/23 and the Science and
Technology Assistance Agency under contract No.
APVT-51-032502.
´
28. Singh, H. K.; Cote, G. L.; Sikorski, R. S. Tetrahedron
Lett. 1993, 34, 5201.
´
29. Singh, H. K.; Cote, G. L.; Hadfield, T. M. Tetrahedron
Lett. 1994, 35, 1353.
´
´
30. Mastihubova, M.; Szemesova, J.; Biely, P. Tetrahedron
Lett. 2003, 44, 1671.
31. Biely, P.; Mastihubova, M.; van Zyl, W. H.; Prior, B. A.
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