Lipase-catalysed preparation of acetates of 4-nitrophenyl β-D-xylopyranoside and their use in kinetic studies of acetyl migration

Article abstract of DOI:10.1016/j.carres.2004.02.016

Di-O-acetates and mono-O-acetates of 4-nitrophenyl β-D-xylopyranoside were prepared by use of lipase PS-30. Polarity of organic solvents and reaction time affected the regioselectivity of the di-O-acetylation as well as the yields of monoacetates. The kin

Full text of DOI:10.1016/j.carres.2004.02.016

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1353–1360
Lipase-catalysed preparation of acetates of 4-nitrophenyl b-
D
-
xylopyranoside and their use in kinetic studies of acetyl migration
ꢀ
ꢀ*
Maria Mastihubova and Peter Biely
Institute of Chemistry, Slovak Academy of Sciences, SK-845 38 Bratislava, Slovakia
Received 30 November 2003; received in revised form 16 February 2004; accepted 16 February 2004
Available online 15 April 2004
Abstract—Di-O-acetates and mono-O-acetates of 4-nitrophenyl b-
D
-xylopyranoside were prepared by use of lipase PS-30. Polarity
of organic solvents and reaction time affected the regioselectivity of the di-O-acetylation as well as the yields of monoacetates. The
kinetics of acetyl groups migration in these derivatives was studied in aqueous media using HPLC. Migration of the acetyl group
strongly depended on pH. The highest rate of acetyl migration was observed from O-2 to O-3in both 2,4-di-O-acetate and 2-O-
acetate. On the contrary, acetyl exchange between O-3 and O-4 in both directions was slower than between O-2 and O-3. The 2,3-di-
O-acetate and 4-O-acetate showed to be the most stable towards acetyl migration. The 3,4-di-O-acetate and 4-O-acetate were
dominant in the corresponding equilibration mixtures.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: 4-Nitrophenyl b-D-xylopyranoside acetates; Lipase PS-30; Regioselectivity; Acetyl migration kinetics; Acetylesterase assay
1. Introduction
Studies of acetylesterases operating on plant polysac-
charides are complicated by the phenomenon of acetyl
group migration (AcM) observed in partially acetylated
carbohydrates in aqueous media. AcM has also been
often observed in partially acetylated products in the
process of preparative enzymatic hydrolysis of per-O-
acetylated carbohydrates by lipases. Obviously, such
phenomenon negatively affects the regioselectivity of the
reaction.8 This AcM can be avoided by application of
controlled enzymatic hydrolysis at low pH (4–5),9 by use
of enzymatic alcoholysis in organic solvents10 or by using
bulkier acyl moieties. However, these conditions usually
lead to a decrease in the rate of product formation.
The kinetics of irreversible consecutive acetyl migra-
tion from secondary to primary hydroxyl group of 2,3,4-
During evolution, nature has created many examples of
carbohydrate esters with special distribution of acyl
groups between hydroxyls at C-2 and C-3of saccharide
units. Partially O-acetylated plant polysaccharides (e.g.,
4-O-methylglucuronoxylan,1 glucomannan,2 pectic
polysaccharides3) or 20(30)-O-aminoacyl derivatives of
D
-ribose in t-RNA terminal adenosine, important in
peptide synthesis,4 can serve as suitable examples. The
interest in systematic study of microbial esterases5
involved in biodegradation of plant cell walls requires
detailed knowledge of their catalytic properties. In this
connection, there is a growing demand for a variety of
modified6 or regioselectively acetylated 4-nitrophenyl
glycosides that could serve as precursors for chromo-
genic substrates important for investigation of catalytic
properties of these enzymes7 and for elaboration of
simple enzyme assays.
tri-O-acetylated methyl a- and b-
D
-glucopyranoside
-hexofura-
(4 fi 6 AcM)11 and 1,2-O-isopropylidene-a-
D
nose acetates (5 fi 6 AcM)12 in neutral condition is well
documented. Although examples of reversible trans acyl
migration were discussed in literature (e.g., 2 fi 3AcM
on Bn-4-O-Me-b-
xylan1b or on xylo-oligosaccharides,1e acyl migration
around the ring on cyclitols13 or -glucose derivatives14)
D
-Xylp,1a on partially acetylated
* Corresponding author. Tel.: +421-2-59410246; fax: +421-2-594102-
22; e-mail: chemjama@savba.sk
D
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.02.016

1354
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
the positional, kinetic and thermodynamic aspects of
AcM on a pyranoid ring have not been investigated in
detail. It is known that AcM is significantly influenced
by temperature and pH and that vicinal trans-migration
in some cases occurs with nearly the same ease as vicinal
cis-migration.13–15
i
O
O
AcO
AcO
HO
AcO
ONPh
ONPh
OAc
OAc
1
Scheme 1. Reagents and conditions: (i) LPS-30, n-butanol, toluene,
40 °C, 68%.
The aim of this work is to present results on pre-
parative transesterification in organic media catalysed
by lipase PS-30 (from Burkholderia cepacia, Amano,
further as LPS-30), leading to three possible di-O-ace-
tates (compounds 1–3) and three mono-O-acetates
(Scheme 1). The transesterification condition (toluene
and n-butanol) was used to eliminate migration of the
acetyl group along the xylopyranoside ring. The reac-
tion was slow but highly regioselective and 1 was isol-
ated in 68% yield.
(compounds 4–6) of 4-nitrophenyl b-
D-xylopyranoside
(NPh-b- -Xylp). The availability of acetates 1–6, bear-
D
Some general trends regarding the regioselectivity of
acetylation of D-glycopyranosides at O-2 and O-3by
ing the 4-nitrophenyl chromophore, led us to investigate
the kinetics of AcM between equatorial oxygen atoms
along the pyranoid ring in aqueous media. The results
LPS-30 have already been reported. The regioselectivity
can be dependent, for example, on the anomeric con-
figuration of the aglycon17 or on the relationship be-
tween hydrophobicity and polarity of the solvent.18;19
The enzyme can also catalyse acetylation of NPh-
show that partially acetylated b-D-xylopyranoside
compounds, depending on pH, are prone to vicinal
trans-AcM in aqueous media. This fact may have an
impact on acetylesterase enzymatic studies.
The choice to synthesise compounds 1–6 was sup-
ported by the following considerations:
(i) Microbial acetylxylan esterases deacetylate par-
b-
octyl-b-
D
-Xylp. Based on earlier studies of the acetylation of
-xylopyranoside18 and phenyl 6-O-trityl-b-
D-
D
glucopyranoside,19 we assumed that the hydrophobic
character of the 4-nitrophenyl aglycon and the polarity
of the solvent could affect the final proportion of diac-
etates 2 and 3. Therefore, the effect of organic solvents
with different polarity on the regioselectivity of the
tially acetylated 4-O-methyl-
fragments generated upon the action of endo-b-(1 fi 4)-
xylanases at positions O-2 and O-3of the b- -xylopyr-
D-glucuronoxylan, or its
D
anosyl residues.5 Compounds 1–6 could serve as model
substrates to investigate the specificity of acetylxylan
esterases and the role of acetyl group migration in their
mechanism of action; (ii) The 4-nitrophenyl aglycon in
compounds 1–6 could mimic another xylopyranoside
unit similarly as in 4-nitrophenyl aglycon-containing
substrates of glycosidases and thus partly aid to proper
orientation of the substrate in the enzyme binding site.
Our recent study of the mode of action of Streptomyces
lividans acetylxylan esterase indicated that acetylated
acetylation of NPh-b-
D-Xylp was evaluated with the aim
to increase the proportion of the 2,4-diacetate 2. The
course of acetylation of NPh-b-D-Xylp was monitored
by HPLC.
In all solvents used, ranging from more to less
hydrophobic character, a mixture of 2,4- and 3,4-diac-
etates (2 and 3) (Scheme 2) was produced in high yields
but in different ratios. Table 1 shows a clear increase in
the formation of the 2,4-diacetate in more hydrophobic
solvents. A small amount of water in a hydrophobic
solvent such as toluene (solvents 2 and 9) had a positive
effect on 3,4-diacetate formation. We did not observe
any appreciable production of per-O-acetylated NPh-b-
methyl b-D-xylopyranosides could form productive
complexes in two different orientations;7 (iii) The com-
pounds and their products of deacetylation could be
easily monitored and quantified by HPLC using a
spectrophotometric detector.
D
-Xylp. This observation is in a good agreement with
the results reported on Me-b- -Xylp conversion to the
3,4-diacetate in acetonitrile, and with the results with
octyl-b- -xylopyranoside affording the 2,4-diacetate as
D
2. Results and discussion
D
the major product of acetylation by LPS-30 in hexane.18
Toluene was chosen as a solvent to obtain 2 and 3 in
preparative scale and 62% of 3 and 25% of 2 were isol-
ated by column chromatography after 72 h of shaking at
40 °C.
2.1. Lipase-catalysed preparation of NPh-b- -Xylp
acetates 1–6
D
According to published data for Me-b-
catalyses the deacetylation of per-O-acetylated NPh-
D
-Xylp,16 LPS-30
No AcM was observed during fractionation of acet-
b-
D
-Xylp exclusively at O-4 to give the 2,3-diacetate 1
ylated products by column chromatography on silica
ii
O
O
O
AcO
+
HO
HO
AcO
HO
ONPh
AcO
ONPh
ONPh
OH
OH
OAc
2
3
Scheme 2. Reagents and conditions: (ii) LPS-30, vinyl acetate, organic solvent (Table 1), 40 °C.

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
1355
Table 1. Solvent effects on regioselectivity of the enzymatic acetylation of NPh-b- -Xylp by LPS-30 at 40 °C
D
a
No
Organic solvent
log Pow
Reaction time (h)
Conversion (%)
Ratio (2:3)
1
2
Cyclohexane
Toluene
3.4
2.69
193
75
85
94
1:1.6
1:2.1
3Diisopropyl ether
1.9
50
100
192
1:2.8
88
4
5
6
7
8
9
Methyl isobutyl ketone
1.38
0.73195
0.7375
0.4
1:2.9
Ethyl acetate
Vinyl acetate
t-Butanol
90
95
.1
1:3
1:3
1:3
87
.3
19389 .8
194
7385
Acetonitrile
Tolueneb
À0.33
1:4.3
1:4.8
aOctanol/water partition coefficient.
b
ꢁ
Without drying, without MS 4 A (0.05% water).
gel. Acetyl migration due to contact with silica gel was
also excluded by HPLC while monitoring the solutions
of diacetates 1–3 shaken with silica gel in ethyl acetate
(concentration 210 mg/mL) for several days.
(Table 2). Solvent effect was less pronounced in the case
of the 3-acetate 5.
2.2. Kinetic study of acetyl migration
Monoacetates 4, 5 and 6 (Scheme 3) were obtained on
-Xylp by LPS-30 in several sol-
acetylation of NPh-b-
D
Solutions (1 mM) of diacetates 1–3 (pH 5, 6 and 7) and
monoacetates 4–6 (pH 6) in phosphate buffer (0.1 M,
containing 4% Me2SO) were prepared and the time
course of AcM in 1–6 was monitored by HPLC over a
period of several hours at 40 °C until a thermodynamic
equilibrium was reached. The proposed reaction
sequence of AcM products is shown in Scheme 4. The
3,4-diacetate 3 was generated from the 2,3-diacetate 1
vents. The reactions were monitored by HPLC. The
composition of the reaction mixtures corresponding to
maximum yields of monoacetates is shown in Table 2.
The relative percentage of monoacetates showed the
tendency of the 2-acetate 4 to be formed preferentially in
more hydrophobic solvents whereas the 4-acetate 6 was
formed in a greater proportion in more polar solvents
O
O
O
AcO
HO
HO
HO
AcO
ONPh
HO
ONPh
ONPh
OH
OAc
OH
6
4
5
Scheme 3.
Table 2. Monoacetylation of NPh-b- -Xylp by LPS-30 at 40 °C
D
Organic solvent
Reaction time (h)
Yield of monoacetates (%)
Relative proportion in the mixture (%)
4
5
6
Cyclohexane
Toluene
15357
5
46
54
0
51
38
49
34
533
34
32
34
27
51
56
0
10
Diisopropyl ether
Methyl isobutyl ketone
Vinyl acetate
Ethyl acetate
t-Butanol
5.5
50
67
54
54
43
42
8
9
25.5
75
51
50
30
29
15
18
36
44
45.5
75
Acetonitrile
O
O
O
k2,3
k3,2
k1,2
HO
AcO
AcO
HO
AcO
AcO
ONPh
ONPh
ONPh
ONPh
ONPh
k2,1
OAc
OAc
OH
1
2
5
3
6
O
O
O
k4,5
k5,4
k5,6
k6,5
HO
HO
HO
AcO
AcO
HO
ONPh
OAc
OH
OH
4
Scheme 4.

1356
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
through the 2,4-diacetate 2 by first-order, reversible and
consecutive reactions, and the same is true for the
reverse reaction. On the other hand the 2,4-diacetate 2
produced 1 and 3 by two parallel, reversible and first-
order AcM reactions. A similar reaction scheme
occurred with monoacetates 4, 5 and 6. These processes
could be described by eight rate constants indicated in
Scheme 4.
The corresponding first-order rate constants were
computed from exponential dependences between time
and molar concentration of the starting compounds and
migration products using equations applicable for the
given kinetic systems.20 Based on the average values of
calculated four rate constants important in each case,
the quantitative prediction of three migration products
could be illustrated at any reaction time. The amounts
of products of spontaneous hydrolysis (competitive
reaction to AcM) close to thermodynamic equilibriums
were small (about 10%) and therefore neglected. Figures
1 and 2 give examples of time course of AcM on diac-
etates 1–3 and monoacetates 4–6 at pH 6, respectively.
The experimental points fit well the curves generated
from average rate constants.
According to the rate constants calculated from
measurements at pH 5, 6 and 7 at 40 °C (summarised in
Table 3), AcM strongly depends on pH. The highest rate
of AcM (k2;3, k4;5) at pH 6 was observed from O-2 to O-3
in diacetate 2 and in monoacetate 4. Generally, the
acetyl exchange between O-2 and O-3in both directions
is easier than between O-3and O-4 (Table 3).
Tsuda and Yoshimoto14 explained a similar phe-
nomenon occurring in partially acylated methyl a-D-
glucopyranoside by a smaller dihedral angle between
O-2 and O-3as compared to the angle between O-3and
4
O-4 of the glucopyranose ring in C1 conformation and
also by the smaller value of the torsion energy of a five-
membered orthoacid intermediate between the neigh-
bouring OH-groups. Based on crystallographic data for
NPh-b-D
-Xylp in 4C1 conformation,21 it appears that the
torsion angles between O-2 and O-3or O-3and O-4 are
1.0
0.8
0.6
0.4
0.2
0.0
1.0
1.0
0.8
0.6
0.4
0.2
0.0
2
3
1
0.8
0.6
0.4
0.2
0.0
0
1000
2000
0
1000
2000
0
2000
4000
Time (min)
Time (min)
Time (min)
Figure 1. Interconversion of diacetates 1, 2 and 3 at pH 6 as a function of time. The experimental points are fitted by curves generated using the rate
constants k1;2, k2;1, k2;3 and k3;2 (Table 3). Symbols: ꢀ, 1; s, 2; M, 3.
1.0
0.8
0.6
0.4
0.2
0 0
1.0
0.8
0.6
0.4
0.2
0.0
1
0.8
0.6
0.4
0.2
0
6
4
5
0
2000
Time (min)
4000
0
2000
Time (min)
4000
0
2000
Time (min)
4000
Figure 2. Interconversion of monoacetates 4, 5 and 6 at pH 6 as a function of time. The experimental points are fitted by curves generated using the
rate constants k4;5, k5;4, k5;6 and k6;5 (Table 3). Symbols: d, 4; j, 5; N, 6.
Table 3. Average values of calculated rate constants (105 minÀ1) shown in reaction in Scheme 4
pH
k1;2
k2;1
5.5
k2;3
45.6
529.2
k3;2
28.3
336.1
k4;5
k5;4
k5;6
k6;5
5
6
7
6.8
122.4
1172.4
75.8
670.2
266.5
105.9
45.4
37.8
4657.9
2777.4

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
1357
similar (72.0° or 73.4°, respectively). Consequently, one
may assume that the higher rate of AcM from O-2 to
O-3is due to a steric repulsive effect of the aglycon. On
the other hand, a backward process could take place due
to a higher reactivity of O-2 resulting from the inductive
effect of the anomeric centre.
Derivatives 1 and 6 proved to be the most stable
towards AcM. The corresponding rate constant k2;1 and
k6;5 were depressed by one order of magnitude. As can
be seen in Figures 1 and 2, the acetyl group at O-4 of
selected position in diacetates is approximately 2–3
times faster than in monoacetates. This can be a con-
sequence of steric and electronic factors associated with
the presence of the second acetyl group on the b-xylo-
pyranoside ring.
We are planning to use the prepared monoacetates
and diacetates of NPh-b-D-Xylp as new acetylxylan
esterase substrates to establish the positional specificity
of this extremely variable group of carbohydrate ester-
ases and thus contribute to their classification. The
information on the rate of AcM in individual com-
pounds dissolved in buffers suitable for the enzyme as-
says is of considerable importance for the development
of the assays. It is to be expected from our results that
length in substrate solution preparation and incubation
with enzymes may result in interconversion of the sub-
strates due to AcM. Therefore, the ratios of all three
NPh-b-D-Xylp appears thermodynamically more stable
because the 3,4-diacetate 3 and the 4-acetate 6 prevail in
the equilibrium mixtures. On the other hand, the 2,3-
diacetate 1 and 2-acetate 4 were found at equilibration
in the lowest concentrations (Table 4). The final ratio of
acetylated NPh-b-D-Xylp-s was not significantly influ-
enced by pH.
The values of rate constants determined at pH 6
(Table 3) also show that the AcM from between two
mono- and diacetates of NPh-b-D-Xylp formed from
individual compounds during a relatively short time at
three different pH values at 40 °C have been calculated
and are presented in Tables 5 and 6.
Table 4. Ratio of acetylated NPh-b-
equilibrium at 40 °C
D
-Xylp-s at thermodynamic
Data in Tables 5 and 6 show clearly that, with regard
to regioselectivity, the acetylxylan esterase assays should
be as short as possible so the transformation of sub-
strates due to AcM could be neglected. Stock substrate
solutions will have to be prepared in solvents in which
AcM does not proceed. Such solvent could be, for
example, Me2SO.
pH
Ratio of diacetates
1:2:3
Ratio of monoacetates
4:5:6
5
6
7
24:29:47
20:31:49
18:31:51
15:39:46
Table 5. Ratio of positional isomers of diacetates of NPh-b-
D
-Xylp in 1 mM solutions of compounds 1–3 incubated in 0.1 M sodium phosphate
buffer (pH 5.0–7.0) at 40 °C
Time of incubation
(min)
pH
5
AcM products from 1 AcM products from 2 AcM products from 3
2 (%)
3 (%)
1 (%)
3 (%)
1 (%)
2 (%)
10
30
0.07
0.20
0.40
0.79
0.0002
0.001
0.006
0.02
0.06
0.16
0.32
0.64
0.45
1.35
2.67
5.22
0.00009
0.0007
0.003
0.28
0.84
1.66
3.24
60
120
0.01
10
30
1.18
3.30
0.030.735.05
0.26
0.95
0.01 .21
3
6
7
2.05
3.71
6.22
13.83
24.24
37.92
0.10
0.37
1.23
8.78
15.39
24.08
60
120
5.99
10.04
3.14
10
30
8.72
16.81
22.02
26.88
2.034.991.86
10.99
23.81
3
9.61
0.69
19.00
51.72
54.89
53.32
3.75
8.12
30.84
32.73
31.79
60
120
12.59
15.37
39.07
13.32
Table 6. Ratio of positional isomers of monoacetates of NPh-b-
buffer (pH 6.0) at 40 °C
D-Xylp in 1 mM solutions of compounds 4–6 incubated in 0.1 M sodium phosphate
Time of incubation (min)
AcM products from 4
6 (%)
AcM products from 5
6 (%)
AcM products from 6
5 (%)
5 (%)
4 (%)
4 (%)
10
30
2.61
7.51
0.006
0.05
0.20
1.04
2.99
5.62
0.45
1.33
2.58
0.002
0.02
0.07
0.38
1.10
2.15
60
120
14.14
25.09
0.739.97
4.90
0.24
4.08

1358
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
3. Conclusion
30 (5 g). The reaction mixture was shaken for 7 days at
40 °C and 200 rpm. The reaction was stopped by filtrating
off the lipase. The solvent was eliminated under dimin-
ished pressure and the product was separated from the
starting compound by column chromatography (1:1 tol-
All three possible diacetates 1–3 and monoacetates 4–6
were prepared from NPh-b- -Xylp by the catalytic
action of LPS-30. Both mono- and diacetates of NPh-b-
-Xylp will be used as substrates for classification of
acetylxylan esterases on the basis of positional specific-
ity. The diacetates will also serve as precursors for the
synthesis of chromogenic substrates for hemicellulolytic
hydrolases and esterases such as a-glucuronidase and
feruloyl esterase. The reversible trans-migrations of the
D
D
uene–EtOAc) to give 1 as a white solid (0.60 g, 68%):
20
D
mp 135–136 °C (EtOAc–diisopropyl ether); ½aꢀ À49.8°
(c 1.0, CHCl3), lit.22 158–159 °C (EtOAc–hexane). H
1
NMR (300 MHz, CDCl3): d 8.22 (d, 2H, J 9.2 Hz, H-30, H-50),
7.09 (d, 2H, J 9.2 Hz, H-20, H-60), 5.29 (d, 1H, J1;2 5.7 Hz,
H-1), 5.20 (dd, 1H, J1;2 5.7 and J2;3 7.5 Hz, H-2), 5.02 (t,
0
acetyl group along the flexible b-
D
-xylopyranoside ring
1H, J3;4 7.2 and J2;3 7.3Hz, H-3), 4.18 (dd, 1H, J4;5 4.3and
J5;5 12.0 Hz, H-50), 3.90 (dt, 1H, J4;5 4.4, J3;4 7.0 and J4;5
7.1 Hz, H-4), 3.60 (dd, 1H, J4;5 7.2 and J5;5 12.0 Hz, H-5),
0
0
in buffered aqueous media were characterised by rate
constants. The results are important for studies of the
substrate structure requirements, regioselectivity and
mechanism of action of various carbohydrate esterases.
0
2.16 (s, 3H, COCH3), 2.11 (s, 3H, COCH3). 13C NMR
(75 MHz, CDCl3): d 171.0 (COCH3), 169.2 (COCH3),
161.1, 143.0, 2 · 125.8, 2 · 116.5 (C–Ar), 97.6 (C-1), 73.7
(C-3), 69.4 (C-2), 67.7 (C-4), 64.5 (C-5), 20.8 (COCH3),
20.7 (COCH3). Anal. Calcd for C15H17NO9: C, 50.71; H,
4.82; N, 3.94. Found: C, 50.42; H, 5.16; N, 3.69.
4. Experimental
4.1. General methods
4.3. The HPLC course monitoring of mono-O-acetylation
LPS-30 was a kind gift from Amano (Japan). NPh-b-
Xylp was purchased from Lachema (Brno, Czech
Republic). Per-O-acetylated 4-nitrophenyl b- -xylopyr-
D
-
and subsequent di-O-acetylation of NPh-b- -Xylp in
different organic solvents
D
D
anoside was prepared by conventional acetylation using
Ac2O in pyridine and after isolation crystallised from
ethanol. Solvents were distilled from the appropriate
drying agents before use. All reactions were monitored
by TLC on Silica Gel 60 (0.25 mm, E. Merck, Darms-
tadt, Germany). Compounds were detected with 1%
orcinol in 10% (v/v) EtOH soln of H2SO4 at ca. 200 °C.
HPLC chromatographic analysis was carried out on a
chromatograph equipped with a silica gel column (Bio-
spher Si 100 5 lm). Each sample was injected through a
20 lL loop and the column was eluted with hexane–
EtOAc at a rate of 1.0 mL/min. The elution of acetates
NPh-b-D-Xylp (27.1 mg, 0.1 mmol) was dissolved in a
ꢁ
selected organic solvent (954 lL), then MS 4 A (250 mg),
vinyl acetate (46 lL, 5 equiv) and 270 mg LPS-30 were
added. The reaction mixtures were shaken at 40 °C at
200 rpm. Aliquots (20 lL) were withdrawn at different
time intervals and filtered (Durapore filter, 0.45 lm).
The filtered solution was analysed by HPLC. The elu-
tion times in 2:3hexane–EtOAc, were 3.8 min for NPh-
2,3,4-tri-O-Ac-b-
7.8 min for 2, 10.2 min for 5, 17.5 min for 6, 20.5 min for
4 and 48.9 min for NPh-b- -Xylp. The amount of each
D-Xylp, 4.3min for 3, 5.6 min for 1,
D
acetate was calculated from the integration of chro-
matogram peak areas.
of NPh-b-D-Xylp was monitored with a calibrated UV
detector at 305 nm. Column chromatography was per-
formed on Silica Gel 60 (100–160 lm) in various solvent
systems. Melting points were determined on a Kofler
hot-stage and are uncorrected. Optical rotations were
measured with a Perkin–Elmer 241 polarimeter at 20 °C.
1H NMR spectra were recorded at 300 MHz with Bru-
ker AM 300 (Me4Si as internal standard). Homonuclear
correlation with 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.
4.4. Preparative di-O-acetylation of 4-nitrophenyl b-D-
xylopyranoside catalysed by LPS-30
NPh-b-D-Xylp (1 g, 3.7 mmol) was suspended in toluene
ꢁ
(300 mL). Molecular sieves (4 A, 15 g), vinyl acetate
(3.4 mL, 37 mmol, 10 equiv) and LPS-30 (9 g) were
added. The reaction mixture was shaken at 40 °C and
200 rpm for 72 h. The reaction was stopped by filtrating
off the lipase. The filtrate was concentrated and the
residue fractionated by column chromatography (2:1
toluene–EtOAc) to give 3 as first eluted and 2 as next
eluted compound.
4.2. 4-Nitrophenyl 2,3-di-O-acetyl-b-
D
-xylopyranoside (1)
-xylopyranoside
(1 g, 2.5 mmol) was dissolved in toluene (100 mL), then n-
4.4.1. 4-Nitrophenyl 2,4-di-O-acetyl-b-D-xylopyranoside
4-Nitrophenyl 2,3,4-tri-O-acetyl-b-
D
(2). White solid (0.33 g, 25%), mp 153–155 °C (toluene),
20
½aꢀ À66.2° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3):
D
BuOH (0.8 mL) was added followed by addition of LPS-
d 8.21 (dt, 2H, J 2.1, 3.3 and 9.2 Hz, H-30, H-50), 7.10 (dt,

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
1359
2H, J 2.1, 3.3 and 9.2 Hz, H-20, H-60), 5.30 (d, 1H, J1;2
5.7 Hz, H-1), 5.14 (dd, 1H, J1;2 5.7 and J2;4 7.8 Hz, H-2),
for C13H15NO8: C, 49.84; H, 4.83, N, 4.47. Found: C,
49.65; H, 5.02; N, 4.29.
0
4.92 (ddt, 1H, J4;5 4.4, J4;5 6.8 and J3;4 6.8 Hz, H-4), 4.23
(dd, 1H, J4;5 4.4 and J5;5 12.3Hz, H-5 0), 3.93 (m, 1H, H-
4.5.2. 4-Nitrophenyl 3-O-acetyl-b-D-xylopyranoside (5).
0
0
0
3), 3.59 (dd, 1H, J4;5 6.6 and J5;5 12.3Hz, H-5), 2.82 (d,
White solid (0.19 g, 16%), mp 160–162 °C (CH2Cl2–
20
1H, J3;OH 5.4 Hz, OH), 2.15 (s, 6H, 2 · COCH3). 13C
NMR (75 MHz, CDCl3): d 170.7 (COCH3), 170.1
(COCH3), 161.1, 143.1, 2 · 125.8, 2 · 116.6 (C–Ar), 97.9
(C-1), 71.9, 71.3, 70.9 (C-2, C-3, C-4), 64.6 (C-5), 20.9
(2 · COCH3). Anal. Calcd for C15H17NO9: C, 50.71; H,
4.82, N, 3.94. Found: C, 50.61; H, 5.03; N, 3.83.
cyclohexane), ½aꢀ À36.1° (c 1.0, MeOH). 1H NMR
D
(300 MHz, CDCl3): d 8.21 (dt, 2H, J 2.1, 3.3 and 9.3 Hz,
H-30, H-50), 7.12 (dt, 2H, J 2.1, 3.3 and 9.3 Hz, H-20, H-
60), 5.26 (d, 1H, J1;2 5.4 Hz, H-1), 4.94 (t, 1H, J3;4 7.1 and
0
0
J2;3 7.1 Hz, H-3), 4.14 (dd, 1H, J4;5 4.1 and J5;5 12.1 Hz,
H-50), 3.86–3.93 (m, 2H, H-2, H-4), 3.58 (dd, 1H, J4;5 7.1
0
and J5;5 12.1 Hz, H-5), 2.92 (d, 1H, J 5.5 Hz, OH), 2.80
4.4.2. 4-Nitrophenyl 3,4-di-O-acetyl-b-
D
-xylopyranoside
(d, 1H, J 5.1 Hz, OH), 2.22 (s, 3H, COCH3). 13C NMR
(75 MHz, CDCl3): d 171.9 (COCH3), 161.3, 142.9,
2 · 125.9, 2 · 116.5 (C–Ar), 99.7 (C-1), 75.3(C-3), 69.7,
67.9 (C-2, C-4), 64.4 (C-5), 21.0 (COCH3). Anal. Calcd
for C13H15NO8: C, 49.84; H, 4.83, N, 4.47. Found: C,
49.91; H, 4.98; N, 4.37.
(3). White solid (0.81 g, 62%), mp 151–153 °C (EtOH),
20
½aꢀ À83.0° (c 1.0, CHCl3). 1H NMR (300 MHz,
D
CDCl3): d 8.22 (d, 2H, J 9.1 Hz, H-30, H-50), 7.11 (d, 2H,
J 9.1 Hz, H-20, H-60), 5.23(d, 1H, J1;2 5.8 Hz, H-1), 5.16
0
(t, 1H, J2;3 7.7 and J3;4 7.7 Hz, H-3), 5.01 (ddt, 1H, J4;5
0
4.6, J4;5 7.3and J3;4 7.4 Hz, H-4), 4.18 (dd, 1H, J4;5 4.5
and J5;5 12.1 Hz, H-50), 3.90 (br dt, 1H, J2;OH 5.8, J1;2 5.9
4.5.3. 4-Nitrophenyl 4-O-acetyl-b-D-xylopyranoside (6).
0
0
and J2;3 7.3Hz, H-2), 3.57 (dd, 1H, J4;5 7.4 and J5;5
White solid (0.21 g, 18%), mp 142–143 °C (EtOAc–
20
diisopropyl ether), ½aꢀ À83.7° (c 1.0, MeOH). 1H NMR
12.1 Hz, H-5), 2.79 (d, 1H, J2;OH 5.8 Hz, OH), 2.16 (s,
3H, COCH3), 2.11 (s, 3H, COCH 3). 13C NMR (75 MHz
CDCl3): d 170.6 (COCH3), 169.7 (COCH3), 161.2, 143.0,
2 · 125.8, 2 · 116.5 (C–Ar), 99.9 (C-1), 72.2 (C-3), 70.3
(C-2), 68.4 (C-4), 62.0 (C-5), 20.9 (COCH3), 20.8
(COCH3). Anal. Calcd for C15H17NO9: C, 50.71; H,
4.82, N, 3.94. Found: C, 50.64; H, 4.89; N, 3.89.
D
(300 MHz, CDCl3): d 8.21 (dt, 2H, J 2.1, 3.3 and 9.3 Hz,
H-30, H-50), 7.13(dt, 2H, J 2.1, 3.3 and 9.3 Hz, H-20,
H-60), 5.16 (d, 1H, J1;2 6.8 Hz, H-4), 4.90 (dt, 1H, J4;5
0
0
4.7, J3;4 7.7 and J4;5 7.5 Hz, H-4), 4.19 (dd, 1H, J4;5 4.7
and J5;5 12.2 Hz, H-50), 3.82–3.88 (m, 2H, H-2, H-3),
0
0
3.54 (dd, 1H, J4;5 7.4 and J5;5 12.2 Hz, H-5), 2.16 (s, 3H,
COCH3). 13C NMR (CDCl3): d 170.7 (COCH3), 161.3,
143.0, 2 · 125.8, 2 · 116.6 (C–Ar), 100.1 (C-1), 72.8
(C-4), 72.5, 71.7 (C-2, C-3), 62.4 (C-5), 20.9 (COCH3).
Anal. Calcd for C13H15NO8: C, 49.84; H, 4.83, N, 4.47.
Found: C, 49.69; H, 5.14; N, 4.31.
4.5. Preparative mono-O-acetylation of 4-nitrophenyl
b-D-xylopyranoside catalysed by LPS-30
Vinyl acetate (1.7 mL, 0.0185 mmol, 5 equiv), LPS-30
ꢁ
(5 g) and 4 A molecular sieves (8 g) were added to a soln
of NPh-b-
D
-Xylp (1 g, 3.7 mmol) in t-BuOH (200 mL).
4.6. The HPLC kinetic study of acetyl migration on
The reaction mixture was shaken at 40 °C and 200 rpm
for 40 h. The reaction was stopped by filtrating off
lipase. The filtrate was concentrated and the residue was
fractionated by column chromatography (2:1 fi 1:1 tol-
uene–EtOAc) to give first diacetates 2 and 3 (0.29 g,
22%), then monoacetate 5. The second eluted mono-
acetate was 4 and the last eluted was 6.
NPh-b- -Xylp acetates in phosphate buffer
D
Acetates 1–6 (0.002 mmol) were dissolved in Me2SO
(80 lL) and mixed with 0.1 M sodium phosphate buffer
(1920 lL) of appropriate pH and preheated to 40 °C.
The reaction mixtures were incubated at 40 °C. Aliquots
(50 lL) were withdrawn at time intervals and directly
injected to the HPLC system using a silica gel column
eluted with 1:2 hexane–EtOAc. Elution times were
3.6 min for 3, 4.3min for 1, 5.6 min for 2, 6.6 min for 5,
4.5.1. 4-Nitrophenyl 2-O-acetyl-b-D-xylopyranoside (4).
White solid (0.22 g, 19%), mp 178–180 °C (EtOAc–tol-
20
D
1
uene), ½aꢀ À7.9° (c 1.0, MeOH). H NMR (300 MHz,
11.4 min for 6, 14.6 min for 4 and 30.8 min for NPh-b-D-
Xylp. The data obtained by integration of the peak areas
were used to calculate the rate constants of AcM.
CDCl3): d 8.21 (dt, 2H, J 2.1, 3.2 and 9.2 Hz, H-30,
H-50), 7.10 (dt, 2H, J 2.1, 3.2 and 9.2 Hz, H-20, H-60),
5.21 (d, 1H, J1;2 6.2 Hz, H-1), 5.05 (dd, 1H, J1;2 6.3and
0
0
J2;3 7.9 Hz, H-2), 4.16 (dd, 1H, J4;5 4.5 and J5;5 11.8 Hz,
4.7. Test for AcM in the presence of silica gel
H-50), 3.85 (dt, J4;5 4.5, J3;4 7.8 and J4;5 8.0 Hz, 1H, H-4),
0
3.75 (t, 1H, J3;4 7.8 and J2;3 7.7 Hz, H-3), 3.52 (dd, 1H,
Samples of diacetates 1–3 (0.7 mg) dissolved in EtOAc
(2 mL) were shaken at 30 °C in the presence of 210 mg
silica gel (Lachema, 100–160 lm). No traces of AcM
products were observed in the filtrates of the mixtures
within 3days when monitored by HPLC.
J4;5 8.2 and J5;5 11.8 Hz, H-5), 2.16 (s, 3H, COCH3). 13
C
0
NMR (75 MHz, CDCl3): d 170.5 (COCH3), 161.1, 143.1,
2 · 125.8, 2 · 116.5 (C–Ar), 98.2 (C-1), 74.0 (C-2), 72.7
(C-3), 69.6 (C-4), 64.6 (C-5), 20.9 (COCH3), Anal. Calcd

1360
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 1353–1360
ꢀ
^ ꢀ
7. Biely, P.; Mastihubova, M.; Cote, G. L.; Green, R. V.
Biochim. Biophys. Acta 2003, 1622, 82–88.
Acknowledgements
8. (a) Chen, H.-P.; Hsiao, K.-F.; Wu, S.-H.; Wang, K.-T.
Biotechnol. Lett. 1995, 17, 305–308; (b) Bastida, A.;
This work was supported by a grant from the Slovak
Grant Agency for Science VEGA 2/3079/23. The au-
ꢀ
Fernandez-Lafuente, R.; Fernandez-Lorente, G.; Guisan,
ꢂ
ꢀ
thors thank A. Karovicova, J. Tonka and K. Paule for
NMR measurements, optical rotations and microanal-
yses. We also thank G. Greif and V. Puchart for help
with graphical analysis and S. Kozmon for computa-
J. M.; Pagani, G.; Terreni, M. Bioorg. Med. Chem. Lett.
1999, 9, 633–636.
9. (a) Horrobin, T.; Tran, Ch. H.; Crout, D. J. Chem. Soc.,
Perkin Trans. 1 1998, 1069–1080; (b) Terreni, M.; Salvetti,
ꢀ
R.; Linati, L.; Fernandez-Lafuente, R.; Fernandez-
tional visualisation of X-ray data of PNP-b-D-Xylp
Lorente, G.; Bastida, A.; Guisan, J. M. Carbohydr. Res.
2002, 337, 1615–1621.
published by Le Questel et al.21
10. Hsiao, K.-F.; Yang, F.-L.; Wu, S.-H.; Wang, K.-T.
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