Deoxy and deoxyfluoro analogues of acetylated methyl β-D- xylopyranoside - Substrates for acetylxylan esterases

Article abstract of DOI:10.1016/j.carres.2004.06.001

Four modified substrates for acetylxylan esterases, 2-deoxy, 3-deoxy, 2-deoxy-2-fluoro, and 3-deoxy-3-fluoro derivatives of di-O-acetylated methyl β-D-xylopyranoside were synthesized via 2,3-anhydropentopyranoside precursors. Methyl 2,3-anhydro-4-O-benzyl-β-D-ribopyranoside was transformed into methyl 2,3-anhydro-4-O-benzyl-β-D-lyxopyranoside in three steps. The epoxide ring opening of 2,3-anhydropentopyranosides was accomplished either by hydride reduction or hydrofluorination. Methyl β-D-xylopyranoside 2,3,4-tri-O-, 2,4-di-O-, and 3,4-di-O-acetates, and the prepared diacetate analogues were tested as substrates of acetylxylan esterases from Schizophyllum commune and Trichoderma reesei. Measurement of their rate of deacetylation pointed to unique structural requirements of the enzymes for the substrates. The enzymes differed particularly in the requirement for the trans vicinal hydroxy group in the deacetylation at C-2 and C-3 and in the tolerance to the presence of trans vicinal acetyl groups esterifying the OH group at C-2 and C-3.

Full text of DOI:10.1016/j.carres.2004.06.001

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2101–2110
Deoxy and deoxyfluoro analogues of acetylated methyl
b- -xylopyranoside––substrates for acetylxylan esterases
D
ꢀ
ꢀ*
Maria Mastihubova and Peter Biely
Institute of Chemistry, Slovak Academy of Sciences, Dꢀubravskꢀa cesta 9, SK-845 38 Bratislava, Slovakia
Received 13February 2003; received in revised form 4 June 2004; accepted 5 June 2004
Abstract—Four modified substrates for acetylxylan esterases, 2-deoxy, 3-deoxy, 2-deoxy-2-fluoro, and 3-deoxy-3-fluoro derivatives
of di-O-acetylated methyl b-
O-benzyl-b- -ribopyranoside was transformed into methyl 2,3-anhydro-4-O-benzyl-b-
ring opening of 2,3-anhydropentopyranosides was accomplished either by hydride reduction or hydrofluorination. Methyl b-
D
-xylopyranoside were synthesized via 2,3-anhydropentopyranoside precursors. Methyl 2,3-anhydro-4-
D
D
-lyxopyranoside in three steps. The epoxide
D
-
xylopyranoside 2,3,4-tri-O-, 2,4-di-O-, and 3,4-di-O-acetates, and the prepared diacetate analogues were tested as substrates of
acetylxylan esterases from Schizophyllum commune and Trichoderma reesei. Measurement of their rate of deacetylation pointed to
unique structural requirements of the enzymes for the substrates. The enzymes differed particularly in the requirement for the trans
vicinal hydroxy group in the deacetylation at C-2 and C-3and in the tolerance to the presence of trans vicinal acetyl groups
esterifying the OH group at C-2 and C-3.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: 2,3-Anhydropyranosides; Deoxy and deoxyfluoro sugars; Acetylxylan esterase specificity
1. Introduction
XylpbOMe) revealed an interesting regioselectivity of
substrate deacetylation. The enzymes, in general,
deacetylated positions 2 and 3, which are compatible
with their function in biodegradation of hemicellulose.
A unique behavior was observed with AcXE from S.
lividans7 belonging to the carbohydrate esterase (CE)
family 4.8 This esterase deacetylated very slowly 2,3-di-
Microbial acetylxylan esterases (EC 3.1.1.72, AcXEs)
represent a group of carbohydrate esterases with great
potential in biotechnology and carbohydrate chemistry.
They deacetylate partially acetylated 4-O-methyl-D-
glucuronoxylan, the main hardwood hemicellulose, or
its fragments generated upon the action of endo-b-
(1 fi 4)-xylanases. Different enzymes with AcXE activity
have recently been purified from various microorgan-
isms.1;2 The three-dimensional structure of AcXEs from
Penicillium purpurogenum3 and Trichoderma reesei4 has
been established. Both enzymes belong to CE family 5
and are typical serine esterases. Recent studies on the
mode of action of AcXEs from T. reesei5 Schizophyllum
commune6 and Streptomyces lividans7 on fully or par-
O-Ac-
The first deacetylation step was, however, immediately
followed by second one, yielding 4-O-Ac-D-
D
-XylpbOMe and 2,3,4-tri-O-Ac- -XylpbOMe.
D
a
XylpbOMe as the main reaction product. The double
deacetylation became better understood after finding
that 2,4-di-O-Ac and 3,4-di-O-Ac-
deacetylated by several orders of magnitude faster as
compared to 2,3,4-tri-O-Ac- -XylpbOMe and 2,3-di-O-
Ac-
-XylpbOMe.7 Since it was difficult to imagine that
D
-XylpbOMe were
D
D
tially acetylated methyl b-
D
-xylopyranoside
(
D
-
the enzyme has an equal ability to deacetylate positions
2 and 3, we proposed that release of the acetyl group in
these two positions, in case the neighboring hydroxyl at
C-3or C-2 is nonacetylated, involves a common 2,3-
ortho ester intermediate. Such structure is believed to
* Corresponding author. Tel.: +42-12-59410-246; fax: +42-12-59410-
222; e-mail: chemjama@savba.sk
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.06.001

2102
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
take part in the acetyl group migration along the pyra-
noid ring of aldoses.9 A similar ortho acid intermediate
was proposed in partial reactions catalyzed by pepti-
dyltransferases (aminoacyl-tRNA synthases) as a result
of an easy aminoacyl group migration between cis-
related 2- and 3-OH groups of ribose in the terminal
nucleotide.10
tives.12 Each AcXE was found to be unique in terms of
role of the vicinal OH-group in the deacetylation
mechanism.
2. Results and discussion
Verification of the hypothesis that ortho ester inter-
mediates or acetyl migration6;7 or reorientation of a
residue4 can take part in the mechanism of deacetylation
of AcXE from S. lividans requires substrate analogues of
2.1. Synthesis of deoxy and deoxyfluoro analogues of
D
-XylpbOMe diacetates (Chart 1)
Methyl 2,3-anhydro-b-
pared from
-arabinose.13 Benzylation of 1 yielded
methyl 2,3-anhydro-4-O-benzyl-b-
-ribopyranoside (2),14
the precursor for the preparation of methyl 2,3-anhy-
dro-4-O-benzyl-b- -lyxopyranoside (5). The decisive
D-ribopyranoside (1) was pre-
acetylated b-
D
-XylpbOMe in which the formation of an
D
hypothetical 2,3-ortho ester or migration of acetyl group
is ruled out. Such analogues are acetylated derivatives of
2-deoxy-, 3-deoxy-, 2-deoxy-2-fluoro- and 3-deoxy-3-
D
D
fluoro-
D
-XylpbOMe. A preliminary report on the syn-
step involved opening of the epoxide ring of 2 using
10 equiv of sodium acetate in acetic acid at 130 °C,
which gave as major compound methyl 3-O-acetyl-4-O-
thesis of these analogues was already published.11 The
analogues have also been used in a study of the mode of
action of AcXE from S. lividans, a carbohydrate esterase
belonging to family 4.12 Replacement of the OH-group
at C-2 or C-3by hydrogen or fluorine resulted in a net
decrease of the rate of deacetylation at the neighboring
position, pointing an essential role of the vicinal OH-
group in the mechanism of deacetylation by the S. livi-
dans AcXE.12
benzyl-b-D-xylopyranoside (3). The reaction was modi-
fied according to Lugemwa and Denison15 It proceeded
with high regioselectivity since only the 3-O-acetate 3
and the 2-O-acetate 4 of methyl 4-O-benzyl-b-D-xylo-
pyranoside were formed (91% overall yield). Probably
due to the trans-acetyl migration9 from O-3to O-2 due
to both the presence of base and the high reaction
temperature, only 66% of the 3-O-acetate 3 was iso-
lated. In order to increase the yield of 3, we were able
to transform almost quantitatively the undesired 2-O-
acetate 4 to the 3-O-acetate 3 chemoenzymatically.
In this report, we present the full description of the
preparation and characterization of di-O-acetylated 2-
deoxy, 3-deoxy, 2-deoxy-2-fluoro and 3-deoxy-3-fluoro
derivatives of
D
-XylpbOMe, and their use in studies of
the mode of action of two AcXEs belonging to the
carbohydrate esterase families 1 and 5.8 The response of
each enzyme to the replacement of the vicinal OH-group
The C-3hydroxyl group of methyl 4- O-benzyl-b-D-xy-
ꢀ
lopyranoside obtained by Zemplen deacetylation of 4
was acetylated regioselectively with vinyl acetate in
the presence of a catalytic amount of lipase PS-30
(Amano).
in
D
-XylpbOMe diacetates was compared with the
behavior of S. lividans AcXE toward the same deriva-
OCH3
OCH3
O
O
O
R1
OCH3
R2
R3O
R3O
RO
O
R1
R2
R1
R
R2
R3
R1
R2 R3
H
OH OAc Bn
OAc OH Bn
F
OH
H
H
1
2
3
8
Bn
OH
OH
Bn
Bn
4
10
12
OH
OH
H
H
F
H
H
F
6
7
OH Bn
9
F
Bn
H
11
14
15
16
17
18
19
OH
OH
OH
H
O
OCH3
OCH3
H
O
OH
H
F
BnO
OAc
OAc
H
Ac
Ac
Ac
Ac
BnO
O
F
13
5
OAc
OAc
F
Chart 1.

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
2103
2,3-Anhydro-4-O-benzyl-b-
D
-lyxopyranoside 5 was
was hydrofluorinated with Bu4NþH2F3ꢀ/KHF2 under
the SL–PTC conditions, the unsaturated product 13 was
isolated from the reaction mixture in 56% yield. Addi-
obtained in 87% yield after tosylation of 3 and alkaline
treatment of the resulting crude tosylate with sodium
methoxide in methanol. The 4-O-benzyl group of 5
avoids the possibility of equilibrium between the first
tionally, the 3-deoxy-3-fluoro-b-
derivative 12 was obtained in 15% yield whereas the
desired 2-deoxy-2-fluoro-b- -xylopyranoside derivative
D-arabinopyranoside
produced
D
-lyxo-2,3-epoxide and the
D
-arabino-3,4-
D
epoxide formed by epoxide-ring migration. The 1H
NMR spectra established the structure of 5 by the shifts
of H-2 and H-3( d 3.33 and 3.37 ppm) and by the small
coupling constants J1;2; J2;3 and J3;4 (2.9, 3,7 and 1.9 Hz),
typical for 2,3-anhydropentopyranosides.
11 was isolated in 6% yield only by column chromato-
graphy. Alternative treatment of 5 by KHF2 in eth-
ane-1,2-diol at 180 °C resulted in an absence of
regioselectivity (Table 1) due to the high temperature,
but satisfactorily afforded 4-O-benzyl-2-deoxy-2-fluoro-
The prepared 2,3-anhydropentopyranosides 1 and 5
were converted to deoxy and deoxyfluoro analogues of
b-D-xylopyranoside (11) (36%), which is inaccessible by
the DAST method due to the occurrence of 1,2-aglycon
migration.18
The drop in regioselectivity and yields of 2-substituted
xylopyranosides from epoxide 5 through trans-diaxial
methyl b-
selective epoxide ring opening of 1 and 5, either by
reduction or hydrofluorination. The known -ribo-2,3-
D-xylopyranoside 6, 7, 9 and 11 by regio-
D
€
epoxides 1 or 2 could be regioselectively transformed to
3-substituted xylopyranosides by preferential nucleo-
philic attack at C-3as indicated in previous stud-
ies.13b;14;16 There is however no mention about the
existence of side products. As an extension of these
results, we observed that this reaction proceeds regio-
selectively also with small nucleophiles like Hꢀ and Fꢀ
(Table 1). We paid also attention to the detection of
possible side products. Reduction of epoxide 1 by
treatment with LiAlH4 in THF at room temperature
ring opening according to the Furst–Plattner rule may
result from electrostatic and steric repulsions of the
incoming nucleophile as well as adoption of a 5H0
transition state conformation during the reaction due to
the anomeric effect and electrostatic repulsion of the
lone-pair electrons of the pyranoid ring and epoxide ring
oxygens.19 The vicinal coupling constants for H-4, H-5a,
and H-5b of 5 measured in CDCl3 at room temperature
are small (J4;5a 3.1 Hz and J4;5b 2.1 Hz) and we also ob-
served a long-range W-coupling between the equatorial
4
gave exclusively the corresponding 3-deoxy-b-
D
-erythro-
H-3and H-5 ( J3;5a 1.6 Hz).
pentopyranoside 6 in 74% yield after purification from
the starting compound 1. The minor methyl 2-deoxy-2-
Catalytic hydrogenation (10% palladium on charcoal)
removed the benzyl group from 9 and 11 to yield the
glycosides 14 and 15. Finally, the resulting deoxy- and
fluoro-b-
yield together with 70% of the desired 3-deoxy-3-fluoro-
-xylopyranoside (7) when 1 was hydrofluorinated
D-arabinopyranoside (8) was isolated in 6%
deoxyfluoro-b-
to afford the target modified AcXE substrates 16–19.
The structures of all methyl b- -xylopyranoside
D-xylopyranosides were fully O-acetyl-ated
b-
D
with Bu4NþH2F3ꢀ and KHF2 at 130 °C under solid-li-
D
quid PTC17 (Table 1).
The possibility of transformation of the newly
derivatives were proven by 1H, 13C, and 19F NMR
spectroscopy. Confirmation of the position of reduction
and fluorination in the modified pentopyranosides was
based on two-dimensional H–H COSY and HMQC
techniques and analysis of geminal, vicinal and long-
range H–H and F–H coupling constants in one-dimen-
prepared methyl 2,3-anhydro-4-O-benzyl-b-
anoside (5) into the corresponding C-2 modified b-
xylopyranosides was also studied. Reduction under the
same conditions as used for the -ribo-2,3-epoxide 1
afforded -xylo and -arabino products in the propor-
tion 1.8:1 (Table 1). After column chromatography on
silica gel, methyl 4-O-benzyl-2-deoxy-b- -threo-pento-
pyranoside (9) and methyl 4-O-benzyl-3-deoxy-b-
D-lyxopyr-
D
-
D
1
D
D
sional H NMR spectra, respectively. For example, the
2
geminal coupling constants JF;H ranged from 52.0 to
49.5 Hz for xylopyranosides 7, 11, 15, 17, and 19 and for
D
2
D
-
arabinopyranosides 8 and 12 values of JF;H were 48.5
and 48.4 Hz, respectively. The proton broad band
threo-pentopyranoside (10) were obtained in 53% and
29% yield, respectively. However, when the epoxide 5
decoupled 13C NMR spectra of 16–19 showed that the
Table 1. Epoxide ring opening of 1 and 5
Starting material
Reaction conditions
Products
-arabino
Ratio
-xylo/ -arabino
D
-xylo
D
Eliminationa
D
D
1
1
5
5
5
LiAlH4/THF
nBu4NþH2Fꢀ3
LiAlH4/THF
nBu4NþH2Fꢀ3
KHF2
6
1/0
7
9
8
11.7/1
1.8/1
0.4/1a
1:1
10
12
12
11
11
13
aThe elimination reaction product 13 was isolated in 56%.

2104
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
1
couplings over one bond JFC ranged from 183to
188 Hz. For the final di-O-acetates of 2- or 3-modified
b-D-xylopyranoside 16–19, some differences in confor-
4
mation could be deduced. The C1 conformation pre-
3
ponderates for derivatives 17–19 (indicated by the JH;H
values). Compound 16 adopts the 1C4 conformation,
which is reflected also by a W-relationship between
4
equatorial H-3and the equatorial H-5 ( J3b;5b 2.1 Hz).
2.2. Response of AcXEs to replacement of the OH-group
in -XylpbOMe diacetates
D
The rates of deacetylation of the acetylated derivatives
of XylpbOMe by AcXE from S. commune (CE 1) and T.
reesei (CE 5) as determined by chromatograms scanning
are shown in Table 2. The data of an earlier study with
the AcXE from S. lividans12 are included for compara-
tive purpose. AcXEs from families 1 and 5 do not show
Figure 1. Scheme illustrating the main differences in structural
requirements for the substrates of AcXEs belonging to three different
CE families. The substrates are shown in hexagonal projections as
recommended by Stick.29 The target acetyl group marked with arrow
and the trans vicinal group are filling the hypothetical enzyme-sub-
strate binding sites. Note the reverse orientation of the binding of
2,3,4-tri-O-Ac- and 3,4-di-O-acetylated derivatives.
such dramatic changes in the rate of deacetylation of
D-
XylpbOMe diacetates at C-2 and C-3due to replace-
ment of the free OH-group by hydrogen or fluorine
atoms as does S. lividans AcXE (Table 2). S. lividans
AcXE is unable to accommodate in its substrate binding
site two vicinal trans-related acetyl groups simulta-
neously. This is mainly the ability of S. commune AcXE.
T. reesei AcXE showed an intermediate behavior.
mune AcXE is not influenced by the replacement of the
vicinal OH-groups by hydrogen or fluorine so strongly
as in the case of the other two AcXEs. The weaker role
of the trans vicinal group on the rate of deacetylation is
further supported by a relatively high rate and lower
regioselectivity of deacetylation of 2,3,4-tri-O-Ac-
XylpbOMe, 2,4-di-O-Ac-XylpbOMe and, particularly,
3,4-di-O-Ac-XylpbOMe, from which two major mono-
acetates are generated on initial deacetylation.6 The
regioselectivity of deacetylation of these derivatives by
AcXE from S. lividans and T. reesei is very high and
leads in all cases to high yields of 4-O-Ac-XylpbOMe.5;7
The major differences between the three investigated
AcXEs are illustrated by drawing the productive com-
plexes with some of the substrates (Fig. 1).
Data in Table 2 for AcXE from S. commune more or
less confirm our previous results obtained by GLC.5–7
The best substrate for S. commune AcXE was found
again to be 3,4-di-O-Ac-XylpbOMe. This diacetate was
deacetylated approximately 3times faster as compared
to the fully acetylated derivative and 7 times faster than
2,4-di-O-Ac-XylpbOMe. Replacement of the OH-group
at C-2 by hydrogen or fluorine in 3,4-di-O-Ac-
XylpbOMe lowered the rate of deacetylation at C-3by
about 50%. Replacement of the OH-group at C-3by
hydrogen in 2,4-di-O-Ac-XylpbOMe lowered the rate of
deacetylation at C-2 to 18%, while the presence of
fluorine at C-2 increased the rate of deacetylation at C-3
by more than four times in comparison to the rate of
deacetylation of 2,4-di-O-Ac-XylpbOMe. These data
show that deacetylation at C-2 or C-3by the S. com-
AcXE from T. reesei responded to structural changes
in XylpbOMe diacetates with an intermediate behavior
between the enzymes from S. lividans and S. commune.
For T. reesei AcXE, fully acetylated XylpbOMe is 5
times worse substrate than 2,4-di-O-Ac-XylpbOMe and
Table 2.
Derivatives of
D
-XylpbOMe
AcXE S. lividansa CE family 4
AcXE S. commune CE family 1
AcXE T. reesei CE family 5
Specific activity
nmol minꢀ1 mgꢀ1
Rel. rate
Specific activityb
nmol minꢀ1 mgꢀ1
Rel. rate
Specific activityb
nmol minꢀ1 mgꢀ1
Rel. rate
2,3,4-tri-O-Ac
2,4-di-O-Ac
3,4-di-O-Ac
0.044
175
34
1.0
35.1
15.3
113
1.0
3.9
19.6
6.6
1.0
5.0
1.7
3923
771
2.8
0.43
3.22
0.47
1.86
1.42
1.80
3-deoxy-2,4-di-O-Ac (16)
3-deoxy-3-fluoro-2,4-di-O-Ac (17) 1.6
0.0631.4
0.08
65.6
50.0
63.3
0.12
25.4
1.5
36.2
1.2
6.5
2-deoxy-3,4-di-O-Ac (18) 0.055
2-deoxy-2-fluoro-3,4-di-O-Ac (19) 0.16
0.38
2.2
4.7
8.7
aData from Ref. 12.
bAverage values from three determinations.

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
2105
about 2 times worse substrate than 3,4-di-O-Ac-
3. Conclusion
XylpbOMe. Already these data indicate that a free vic-
inal OH-group at positions 2 or 3in the substrates plays
an important role in the mechanism of deacetylation by
the T. reesei enzyme. Deoxygenation at C-3in 2,4-di- O-
Ac-XylpbOMe leads to a 40-fold decrease of the rate of
deacetylation at C-2. The same is partially true also for
3,4-di-O-Ac-XylpbOMe. Only deoxygenation at C-2
leads to a decrease in the deacetylation rate at C-3, but
not fluorination. The hydroxy group seems to behave as
a hydrogen bond acceptor since its absence is not only
reversed by fluorine substitution, but the presence of the
fluorine atom brings about a 30% increase in the de-
acetylation rate at both positions 2 and 3. It should be
noted that the enhancing effect of the fluorine atom on
the rate of deacetylation could also be ascribable to its
electron withdrawing effect, increasing the electron
deficiency on the acetyl group.
S. commune AcXE belonging to carbohydrate esterase
family 1 does not require a free vicinal OH-group for its
action so strongly as AcXE from T. reesei and, partic-
ularly, as AcXE from S. lividans for the action of which
a vicinal OH-group is essential (Fig. 1). The vicinal OH-
group in the deacetylation at C-2 or C-3can be replaced
by fluorine for T. reesei AcXE, but not for S. lividans
AcXE. Consequently, the regioselectivity of deacetyl-
ation of the tested substrates by S. commune AcXE is
lower than with the other two AcXEs. The obvious
tolerance of the S. commune enzyme to an acetyl group
esterifying the vicinal OH-group suggests that this
enzyme is capable to operate on doubly acetylated
The 2- and 3-deoxy and 2- and 3-deoxyfluoro ana-
logues of acetylated derivatives of -XylpbOMe were
synthesized by routes that involves the regioselective
nucleophilic epoxide ring opening of a 2,3-anhydro-
pentopyranoside precursor. The rates of deacetylation
D
of the acetylated derivatives of
D
-XylpbOMe, and their
analogues, by AcXEs from S. commune and T. reesei
were determined and compared with the performance of
AcXE from S. lividans.12 AcXEs are members of three
different carbohydrate esterase families, 1, 5, and 4,
respectively.8;21 In accord with this fact, the enzymes
showed a different behavior with respect to the character
of the functional group trans vicinal to the deacetylated
position 2 or 3. Substitution of the vicinal hydroxyl
group by hydrogen hampered dramatically the deacet-
ylation reaction by AcXEs from S. lividans and T. reesei
but not that by AcXE from S. commune. The vicinal
hydroxy group in the acetylated substrate cannot be
replaced by fluorine in S. lividans AcXE, but this can
occur with T. reesei AcXE. This result is not in favor of
a deacetylation mechanism at C-2 and C-3that would
involve a five-membered ortho ester intermediate,7
which still cannot be excluded in the case of AcXE from
S. lividans. Deacetylation by AcXE from S. commune
does not seem to be influenced significantly by the
character of the functional group on the neighboring
carbon atom. The enzyme tolerates well not only the
absence of a vicinal OH-group, but also the presence of
a vicinal acetoxy group. The overall results also support
the view that studies of positional specificity of AcXEs
may require substrates with a larger aglycon than a
methyl group, an aglycon that would guarantee a proper
orientation of the substrate into the substrate binding
sites of AcXEs. Such substrates could be acetylated 4-
nitrophenyl glycosides that have already been synthe-
sized.9;22
xylopyranosyl
residues
of
acetyl-4-O-methyl-D-
glucuronoxylan more efficiently than AcXEs from T.
reesei and S. lividans. The observed rates of deacetyl-
ation of the tested substrates by S. commune AcXE are
not compatible with our earlier hypothesis introduced
for AcXE from S. lividans12 that deacetylation at C-2
and C-3involves a common five-membered ring ortho
ester intermediate. The dependence of the deacetylation
rate at C-2 and C-3on the presence of a free vicinal OH-
group in AcXEs from S. lividans and T. reesei is seem-
ingly in accord with the hypothesis of the formation of
the five-membered ortho ester intermediate, however,
based on the information on size of the substrate bind-
ing site of T. reesei AcXE4 and Penicillium purpuro-
genum AcXE3 (both CE 5), we are inclined to share the
opinion that the acetylated derivatives of XylpbOMe
and its deoxy and deoxyfluoro analogues are too
small substrates to investigate the positional specificity
of the enzymes.4;20 The enzymes can form productive
complexes with these small substrates in different ori-
4. Experimental
4.1. General methods
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. Micro-
analyses were performed with a Fisons EA 1108 ana-
lyzer. 1H and 13C NMR spectra (internal standard
Me4Si) were recorded with a Bruker AM 300 spec-
trometer. Homonuclear correlation experiment COSY-
1
entations. Nevertheless, the prepared analogues of
D
-
45 and two-dimensional H-13C HMQC technique were
XylpbOMe diacetates enable to obtain insight into the
mechanism of deacetylation, particularly to assess the
role of the free vicinal OH-groups in the deacetylation
mechanism.
used.19 F NMR spectra were recorded with a Varian 300
spectrometer in acetone-d6 and chemical shifts are given
relative to CF3CO2H as external standard. Solvents
were distilled from the appropriate drying agents before

2106
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
use. All reactions were monitored by TLC on Silica Gel
60 (0.25 mm, E. Merck) and spraying the plates with 1%
orcinol in 10% (v/v) EtOH soln of H2SO4 followed by
heating at ca. 200 °C. Column chromatography was
performed on Silica Gel 60 (100–160 mesh). Fully
1), 3.99 (dd, 1H, J4;5a 4.6, J5a;5b 11.8 Hz, H-5a), 3.74 (dt,
1H, J3;4 8.3 Hz, H-3), 3.52 (ddd, 1H, H-4), 3.45 (s, 3H,
OCH3), 3.30 (dd, 1H, J4;5b 8.7, H-5b), 2.71 (d, 1H,
JH–OH;H-3 4.1 Hz, H–OH), 2.11 (s, 3H, COCH3); 13C
NMR (CDCl3): d 170.4 (COCH3), 137.8, 2 · 129.7,
127.8, 2 · 127.7 (C–Ph), 101.5 (C-1), 77.1 (C-4), 73.0 (C-
3), 72.8 (CH2), 72.7 (C-2), 62.7 (C-5), 56.4 (OCH3), 20.8
(COCH3). Anal. Calcd for C15H20O6: C, 60.80; H, 6.80.
Found: C, 61.20; H, 6.88.
acetylated methyl b-
XylpbOMe) was prepared by acetylation of commer-
cially available methyl b- -xylopyranoside (Sigma) with
Ac2O in pyridine. Methyl 2,4-di-O-acetyl-b- -xylopyr-
anoside (2,4-di-O-Ac-XylpbOMe) was a kind gift from
D-xylopyranoside (2,3,4-tri-O-Ac-
D
D
ꢀ
Dr. Jan Hirsch. Methyl 3,4-di-O-acetyl-b-
anoside (3,4-di-O-Ac-XylpbOMe) was prepared by
-xylopyranoside
D
-xylopyr-
4.3. Chemoenzymatic transformation of the 2-O-acetate 4
to the 3-O-acetate 3
enzymatic acetylation of methyl b-
D
with Lipase PS-30 (Amano).23 Pure AcXE from
S. commune was obtained as described previously.6;24
Pure AcXE from T. reesei was a generous gift from Dr.
Maija Tenkanen (University of Helsinki, Finland).
Compound 4 (2.2 g, 7.4 mmol) was dissolved in a 0.7 M
soln of MeONa in MeOH (50 mL) and the reaction was
stirred at room temperature. After 5 h, the mixture was
neutralized with Dowex 50 WX8 (Hþ) resin, filtered,
concentrated, and crystallized from i-PrOH–n-hexane to
4.2. Epoxide ring opening of 2 with AcONa–AcOH
obtain methyl 4-O-benzyl-b-
91%); mp 98–100 °C; lit.14 mp 99–100 °C. The resulting
methyl 4-O-benzyl-b- -xylopyranoside (1.5 g, 5.9 mmol)
D-xylopyranoside (1.72 g,
Sodium acetate (32.8 g, 400 mmol) was dissolved in
AcOH (60 mL) with stirring at 100 °C and methyl
D
was dissolved in MeCN (100 mL); then vinyl acetate
(2.5 mL) and Lipase PS-30 (9 g) were added. The reac-
tion suspension was shaken at 35 °C and 200 rpm for
36 h. At the end of the reaction, the lipase powder was
filtered off, the solvent was evaporated from the filtrate
and the residue was triturated with diethyl ether. The
precipitated white solid was separated by filtration. The
yield after crystallization from EtOAc was 1.61 g (92 %).
All physical and spectral characteristics were the same as
for 3.
2,3-anhydro-4-O-benzyl-b-D
-ribopyranoside14 (9.44 g,
40 mmol) was added. The reaction mixture was stirred
and heated to mild reflux for 3h and then poured into
ice-water and extracted with CHCl3. The organic layer
was washed twice with water, dried (Na2SO4), decolor-
ized with charcoal, filtered, and concentrated to obtain a
crude mixture of two products. The mixture was sus-
pended into diethyl ether and the precipitated white
crystals of 3-acetate 3 (6.16 g, 52%) were filtered. The
concentrated filtrate was chromatographed (8:1 toluene–
acetone) to give 3 (1.68 g, 14%) as a first fraction and the
2-acetate 4 as a second fraction.
4.4. Methyl 2,3-anhydro-4-O-benzyl-b-D-lyxopyranoside
(5)
4.2.1. Methyl 3-O-acetyl-4-O-benzyl-b-
D
-xylopyranoside
The 3-acetate 3 (7.40 g, 25 mmol) was dissolved in pyr-
idine (60 mL) in the presence of a catalytic amount of
DMAP. p-Toluenesulfonyl chloride (9.5 g, 50 mmol) was
added at 0 °C and the mixture was stirred at room
temperature. After 72 h, ice-water (60 mL) was added
and the mixture was extracted with CH2Cl2
(3 · 100 mL). The organic layer was washed with 1 N
HCl, two times with water, dried (Na2SO4), and con-
centrated. The crude product (10.46 g, 23mmol, 93%)
was dissolved in 0.7 M MeONa (92 mL) and stirred
overnight at room temperature. The soln was then
neutralized with 1 M H2SO4 and extracted with CH2Cl2
(3 · 100 mL). The organic layer was washed twice with
water, dried (Na2SO4) and concentrated under dimin-
ished pressure. The crude syrup was purified by column
chromatography (2:1 toluene–EtOAc) to afford epoxide
(3). White solid (7.84, 66%), mp 161–162 °C (from
20
EtOAc); lit.16a mp 162–163 °C; ½aŠ ) 49 (c 1.0, CHCl3);
D
22
D
1
lit.16a ½aŠ )48 (c 1.0, CHCl3); Rf 0.27 (8:1 toluene–
acetone); H NMR (300 MHz, CDCl3): d 7.34–7.28 (m,
5H, H-Ph), 5.05 (t, 1H, J2;3 7.9 Hz, H-3), 4.61 (s, 2H,
CH2), 4.28 (d, 1H, J1;2 6.3Hz, H-1), 4.00 (dd, 1H, J4;5a
4.4, J5a;5b 11.8 Hz, H-5a), 3.56 (dt, 1H, J3;4 8.0, J4;5b
8.6 Hz, H-4), 3.50 (s, 3H, OCH3), 3.44 (ddd, 1H, H-2),
3.37 (dd, 1H, H-5b), 2.80 (d, 1H, JH–OH;H-2 5.2 Hz, H–
OH), 2.08 (s, 3H, COCH3); 13C NMR (CDCl3): d 170.8
(COCH3, 137.6, 2 · 128.5, 128.0, 2 · 127.7 (C–Ph), 103.8
(C-1), 74.6 (C-4), 74.0 (C-3), 72.7 (CH2), 71.3(C-2), 62.4
(C-5), 56.9 (OCH3), 21.0 (COCH3). Anal. Calcd for
C15H20O6: C, 60.80; H, 6.80. Found: C, 61.08; H, 6.90.
4.2.2. Methyl 2-O-acetyl-4-O-benzyl-b-
D
-xylopyranoside
5 (5.13g, 87%) as a colorless oil: ½aŠ20 )74 (c 1.0, CHCl3);
D
20
20
D
1
(4). Colorless oil (2.92 g, 25%), ½aŠ )58 (c 1.0, CHCl3);
lit.23 ½aŠ )77 (c 1.0, CHCl3); Rf 0.50 (3:2 toluene–
D
Rf 0.38 (8:1 toluene–acetone); 1H NMR (300 MHz,
CDCl3): d 7.37–7.31 (m, 5H, H-Ph), 4.77 (dd, 1H, J1;2
6.7, J2;3 8.3Hz, H-2), 4.67 (dd, 2H, CH 2), 4.33 (d, 1H, H-
EtOAc); H NMR (300 MHz, CDCl3): d 7.37–7.29 (m,
5H, H-Ph), 4.93(d, 1H, J1;2 2.9 Hz, H-1), 4.67 (dd, 2H,
CH2), 3.80 (dd, 1H, J4;5a 2.1, J5a;5b 12.1 Hz, H-5a), 3.78–

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
2107
3.76 (m, 1H, H-4), 3.59 (dt, 1H, J4;5b 3.1, J3;5b 1.6 Hz, H-
4.6.1. Methyl 3-deoxy-3-fluoro-b-
D
-xylopyranoside (7).
5b), 3.46 (s, 3H, OCH3), 3.37 (dd, 1H, J2;3 3.7 Hz, H-2),
3.33 (dd, 1H, J3;4 1.9 Hz, H-3); 13C NMR (CDCl3): d
137.6, 2 · 128.6, 128.1, 2 · 127.9 (C–Ph), 94.9 (C-1), 72.0
(CH2), 70.4 (C-4), 58.6 (C-5), 55.8 (OCH3), 51.5 (C-2),
50.5 (C-3). Anal. Calcd for C13H16O4: C, 66.09; H, 6.83.
Found: C, 65.61; H, 7.12.
White solid (0.58 g, 70%), mp 104–105 °C (from MeOH–
20
Et2O or CHCl3); ½aŠ )56 (c 1.0, MeOH); lit.16c mp
D
105 °C; Rf 0.35 (9:1 CHCl3–MeOH); 1H NMR
(300 MHz, CDCl3): d 4.38 (ddd, 1H, J2;3 7.9, J3;4 8.1, J3;F
52.0 Hz, H-3), 4.22 (d, 1H, J1;2 6.9 Hz, H-1), 4.06 (ddd,
1H, J4;5a 5.6, J5a;F 5.8, J5a;5b 11.6 Hz, H-5a), 4.00–3.88 (m,
1H, H-4), 3.61 (ddd, 1H, J2;F 12.9 Hz, H-2), 3.55 (s, 3H,
OCH3), 3.30 (dd, 1H, J4;5b 9.4 Hz, H-5b), 2.58 (br s, 1H,
OH), 2.42 (br s, 1H, OH); 13C NMR (CDCl3): d 103.5
(d, JC-1;F 10.0 Hz, C-1), 95.4 (d, JC-3;F 182.5 Hz, C-3), 71.6
(d, JC-2;F 18.3Hz, C-2), 68.4 (d, JC-4;F 19.1 Hz, C-4), 63.7
(d, JC-5;F 7.4 Hz, C-5), 57.2 (OCH3); 19F NMR: d )115.8
(ddt). Anal. Calcd for C6H11FO4: C, 43.37; H, 6.67; F,
11.43. Found: C, 43.19; H, 6.92; F, 11.25.
4.5. Ring-opening of epoxide 1 by hydride reduction
Methyl 2,3-anhydro-b-D
-ribopyranoside13 (1) (0.73g,
5 mmol) was dissolved in dry THF (10 mL), and the soln
was added dropwise to a stirred suspension of LiAlH4
(0.57 g, 15 mmol) in THF (15 mL) under nitrogen at
0 °C. The reaction mixture was allowed to warm up to
room temperature and stirred for 48 h. Water (15 mL)
was added dropwise upon cooling, the mixture was
filtered through a pad of Celite and the residue was
washed with EtOAc. The filtrate was concentrated to
1/5 vol and extracted with EtOAc. The organic layer was
dried (Na2SO4) and concentrated. There was no evi-
dence for the presence of the 2-deoxy-arabino isomer in
the 1H NMR spectrum of the crude product; only about
5% of starting material was found. The crude product
was purified by chromatography on silica gel using 9:1
CHCl3–MeOH as eluent to afford 6.
4.6.2. Methyl 2-deoxy-2-fluoro-b-D-arabinopyranoside
20
(8). Colorless oil (0.05 g, 6%), ½aŠ )71 (c 1.0, MeOH);
D
Rf 0.43(9:1 CHCl 3–MeOH); 1H NMR (300 MHz, D2O):
d 4.67 (ddt, 1H, J1;2 4.1, J2;F 48.5 Hz, H-2), 4.60 (dd, 1H,
J1;F 2.4 Hz, H-1), 4.11 (ddd, 1H, J2;3 6.8, J3;4 3.5, J3;F
11.1 Hz, H-3), 3.94–3.74 (m, 3H, H-4, H-5a, H-5b), 3.46
(s, 3H, OCH3), 2.63(br s, 2H, 2 · OH); 19F NMR: d
)123.6 (dm). Anal. Calcd for C6H11FO4: C, 43.37; H,
6.67; F, 11.43. Found: C, 43.06; H, 7.02; F, 11.18.
4.7. Ring-opening of epoxide 5 by hydride reduction
Methyl 2,3-anhydro-4-O-benzyl-b-D-lyxopyranoside (5)
4.5.1. Methyl 3-deoxy-b- -erythro-pentopyranoside (6).
D
20
D
(2.13g, 9 mmol) was dissolved in dry THF (18 mL), and
the soln was added dropwise to a stirred suspension of
LiAlH4 (1.14 g, 30 mmol) in THF (30 mL) under N2 at
0 °C. The reaction mixture was allowed to warm up to
room temperature and further stirred for 40 h. Water
(20 mL) was added dropwise with cooling, the mixture
was filtered through a pad of Celite and the residue was
washed with CH2Cl2. The filtrate was concentrated to 1/
4 vol and extracted with CH2Cl2. The organic layer was
dried (Na2SO4) and concentrated under diminished
pressure. The resulting crude material was purified by
column chromatography on silica gel using 2:1 toluene–
Colorless oil (0.55 g, 74%), ½aŠ )106 (c 1.0, MeOH); Rf
1
0.45 (9:1 CHCl3–MeOH); H NMR (300 MHz, D2O): d
4.36 (dd, 1H, J1;2 6.1 Hz, H-1), 3.95 (ddd, 1H, J4;5a 4.1,
J5a;5b 11.3Hz, H-5a), 3.85 (dddd, 1H, J3a;4 4.4, J3b;4 9.1,
J
4;5b 7.8 Hz, H-4), 3.52 (ddd, 1H, J2;3a 4.4, J2;3b 9.1 Hz, H-
2), 3.50 (s, 3H, OCH3), 3.38 (dd, 1H, H-5b), 2.26 (ddt,
1H, J3a;5b 1.8 Hz, H-3a), 1.58 (dt, 1H, J3a;3b 12.8 Hz, H-
3b); 13C NMR (CDCl3): d 101.03(C-1), 66.8 (C-2), 65.4
(C-4), 64.5 (C-5), 55.2 (OCH3), 30.9 (C-3). Anal. Calcd
for C6H12O4: C, 48.64; H, 8.16. Found: C, 48.64; H,
8.14.
EtOAc as eluent to obtain the 2-deoxy-b-
anoside 9 and the 3-deoxy-b-
logue 10.
D
-xylopyr-
D-arabinopyranoside ana-
4.6. Ring opening of epoxide 1 by hydrofluorination
The epoxide 1 (0.73g, 5 mmol), Bu 4NþH2F3ꢀ (1.8 g,
6 mmol) and solid KHF2 (0.94 g, 12 mmol) were stirred
and heated at 130 °C until complete disappearance of
the starting compound (12 h). The brown reaction mix-
ture was cooled, diluted with EtOAc (120 mL) and
washed with a satd aq NaHCO3. After drying the organic
layer with Na2SO4, filtration, and evaporation of the
solvent, the crude residue was purified by chromato-
graphy using 10:1 CHCl3–MeOH as eluent to give syrupy
4.7.1. Methyl 4-O-benzyl-2-deoxy-b- -threo-pentopyr-
D
anoside (9). White crystals (1.26 g, 53%), mp 36 °C
20
(from i-Pr2O–cyclohexane); ½aŠ )132 (c 1.0, CHCl3); Rf
D
0.35 (3:2 toluene–EtOAc); 1H NMR (300 MHz, CDCl3):
d 7.37–7.29 (m, 5H, H-Ph), 4.72 (dd, 1H, J1;2a 3.5, J1;2b
4.2 Hz, H-1), 4.65 (dd, 2H, Jgem 12.0 Hz, CH2), 4.05 (dd,
1H, J4;5a 2.4, J5a;5b 12.6 Hz, H-5a), 3.87 (dt, 1H, J2a;3 3.5,
J2b;3 4.2, J3;4 4.2 Hz, H-3), 3.58 (dd, 1H, J4;5b 3.6 Hz, H-
5b), 3.45–3.36 (m, 1H, H-4), 3.43 (s, 3H, OCH3), 2.27
(dt, 1H, J2a;2b 14.1 Hz, H-2a), 1.78 (dt, 1H, H-2b); 13C
NMR (CDCl3): d 138.2, 128.5, 127.9, 127.8 (C–Ph), 99.4
3-deoxy-3-fluoro-b-D-xylopyranoside 7, which solidified
upon storage, and the 2-deoxy-2-fluoro-b-
pyranoside isomer 8.
D-arabino-

2108
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
(C-1), 76.0 (C-4), 71.6 (CH2), 66.3(C-3), 58.4 (C-5), 55.7
(OCH3), 32.7 (C-2). Anal. Calcd for C13H18O4: C, 65.53;
H, 7.61. Found: C, 65.83; H, 7.64.
4.9. Methyl 4-O-benzyl-3-deoxy-a-L-glycero-pent-3-eno-
pyranoside (13)
The epoxide 5 (1.77 g, 7.5 mmol), Bu4NþH2F3ꢀ (4.52 g,
15 mmol) and KHF2 (2.34 g, 30 mmol) were stirred and
heated for 25 h at 135 °C. The dark reaction mixture was
cooled, diluted with CHCl3 (200 mL), and washed with a
satd aq NaHCO3, and brine. After drying the organic
layer with Na2SO4, filtration and evaporation of the
solvent, the residue was chromatographed on silica gel
using toluene, then 2:1 toluene–EtOAc as eluent to give
successively the elimination product 13 (0.99 g, 56%), the
4.7.2. Methyl 4-O-benzyl-3-deoxy-b- -threo-pentopyr-
D
20
anoside (10). Colorless oil (0.70 g, 29%), ½aŠ )114 (c
D
1.0, CHCl3); Rf 0.27 (3:2 toluene–EtOAc); 1H NMR
(300 MHz, CDCl3): d 7.36–7.26 (m, 5H, H-Ph), 4.65 (d,
1H, J1;2 3.4 Hz, H-1), 4.56 (s, 2H, CH2), 4.02 (ddd, 1H,
J2;3a 4.7 Hz, H-2), 3.70–3.59 (m, 3H, H-4, H-5a, H-5b),
3.45 (s, 3H, OCH3), 2.07 (m, 1H, H-3a), 1.81 (ddd, 1H,
J2;3b 11.0, J3b;4 2.9, J3a;3b 12.4 Hz, H-3b). Anal. Calcd for
C13H18O4: C, 65.53; H, 7.61. Found: C, 65.65; H, 7.92.
3-deoxy-3-fluoro-b-
(0.29 g, 15%) and the 2-deoxy-2-fluoro-b-
D
-arabinopyranoside derivative 12
-xylopyr-
D
4.8. Ring-opening of epoxide 5 by hydrofluorination
anoside derivative 11 (0.11 g, 6%) and 13: mp 54–55 °C
20
(from i-Pr2O–cyclohexane); ½aŠ )127 (c 1.0, CHCl3); Rf
D
Epoxide 5 (1.45 g, 6 mmol) and KHF2 (7 g) in ethane-
1,2-diol (28 mL) was heated for 3h at 180 °C. After
cooling, the suspension was neutralized with a satd aq
NaHCO3 and the neutral soln was extracted with CHCl3
(3 · 70 mL), the extract was dried with Na2SO4, filtered,
and evaporated to dryness under diminished pressure.
The residue was purified by column chromatography
(1:0 fi 1:1 toluene–EtOAc) to give first the 2-deoxy-2-
0.36 (3:2 toluene–EtOAc); 1H NMR (300 MHz, CDCl3):
d 7.37–7.29 (m, 5H, H-Ph), 4.84 (dt, 1H, J2;3 3.4, J3;5a 1.3,
J3;5b 1.3Hz, H-3), 4.78 (dt, 2H, Jgem 15.2 Hz, CH2), 4.66
(d, 1H, J1;2 3.6 Hz, H-1), 4.34–4.28 (m, 1H, H-2), 4.15
(dt, 1H, J5a;CH2 1.4, J5a;5b 15.2 Hz, H-5a), 3.98 (dt, 1H,
J5b;CH2 1.2 Hz, H-5b), 3.54 (s, 3H, OCH3), 2.21 (d, 1H,
J2;OH 9.1 Hz, H–OH); 13C NMR (CDCl3): d 154.2 (C-4),
136.2, 128.5, 128.1, 127.7 (C–Ph), 98.7 (C-1), 94.4 (C-3),
69.2 (CH2), 64.8 (C-2), 60.5 (C-5), 56.2 (OCH3). Anal.
Calcd for C13H16O4: C, 66.09; H, 6.83. Found: C, 66.23;
H, 7.06.
fluoro-b-
D
-xylopyranoside derivative 11 and next the 3-
deoxy-3-fluoro-b-
D
-arabinopyranoside derivative 12.
4.8.1. Methyl 4-O-benzyl-2-deoxy-2-fluoro-b-D-xylopyr-
anoside (11). White crystals (0.54 g, 36%), mp 84–85 °C
4.10. Methyl 2-deoxy-b- -threo-pentopyranoside (14)
D
20
(from i-Pr2O–cyclohexane); ½aŠ )44 (c 1.0, CHCl3); Rf
D
0.51 (3:2 toluene–EtOAc); 1H NMR (300 MHz, CDCl3):
d 7.36–7.31 (m, 5H, Ph), 4.67 (dd, 2H, Jgem 11.8 Hz,
CH2), 4.34 (dd, 1H, J1;2 7.2, J1;F 3.7 Hz, H-1), 4.15 (ddd,
1H, J2;3 8.5, J2;F 50.5 Hz, H-2), 3.94 (ddd, 1H, J4;5a 5.0,
J5a;5b 11.7 Hz, H-5a), 3.82 (ddd, 1H, J3;4 8.4, J3;F 15.2 Hz,
H-3), 3.52 (s, 3H, OCH3), 3.57–3.41 (m, 1H, H-4), 3.23
(dd, 1H, J4;5b 9.7 Hz, H-5b), 3.06 (br s, 1H, OH); 13C
NMR (CDCl3): d 137.7, 128.5, 128.0, 127.8 (C–Ph),
101.5 (d, JC-1;F 23.4 Hz, C-1), 91.4 (d, JC-2;F 185.4 Hz, C-
2), 76.8 (d, JC-4;F 7.0 Hz, C-4), 74.0 (d, JC-3;F 18.4 Hz, C-
3), 73.1 (CH2), 63.3 (C-5), 56.8 (OCH3); 19F NMR: d
)121.0 (dd). Anal. Calcd for C13H17FO4: C, 60.93; H,
6.69; F, 7.41. Found: C, 61.18; H, 6.79; F, 7.28.
Compound 9 (1.20 g, 5 mmol) was dissolved in MeOH
(50 mL) and 10% Pd/C (0.25 g) was added. The reaction
mixture was intensively stirred under H2 overnight.
Filtration through Celite, concentration of the filtrate
and purification by flash chromatography (9:1 CHCl3–
MeOH) gave pure 14 (0.70 g, 95%) as a white solid: mp
82–84 °C (from EtOAc–cyclohexane); lit.25 mp 83–84 °C;
20
15
½aŠ )97 (c 1.0, MeOH); lit.25 ½aŠ )167 (c 1.0, CHCl3);
D
D
Rf 0.25 (9:1 CHCl3–MeOH); 1H NMR (300 MHz,
CD3OD): d 4.45 (dd, 1H, J1;2a 2.4, J1;2b 8.0 Hz, H-1), 3.92
(dd, 1H, J4;5a 4.5, J5a;5b 11.6 Hz, H-5a), 3.53 (ddd, 1H,
J2a;3 4.8, J2b;3 9.9, J3;4 7.7 Hz, H-3), 3.41 (s, 3H, OCH3),
3.39 (ddd, 1H, J4;5b 8.5 Hz, H-4), 3.18 (dd, 1H, H-5b),
2.11 (ddd, 1H, J2a;2b 13.0 Hz, H-2a), 1.47 (ddd, 1H, H-
2b); 13C NMR (CDCl3): d 102.4 (C-1), 72.0 (C-4), 71.5
(C-3), 65.9 (C-5), 56.6 (OCH3), 38.4 (C-2). Anal. Calcd
for C6H12O4: C, 48.64; H, 8.16. Found: C, 48.53; H,
8.28.
4.8.2. Methyl 4-O-benzyl-3-deoxy-3-fluoro-b-D-arabino-
20
pyranoside (12). Colorless oil (0.53g, 35%), ½aŠ )168 (c
D
1.0, CHCl3); Rf 0.30 (3:2 toluene–EtOAc); 1H NMR
(300 MHz, CDCl3): d 7.39–7.28 (m, 5H, Ph), 4.84 (dd,
1H, J1;2 3.8, J1;F 3.9 Hz, H-1), 4.72 (dd, 2H, Jgem 12.2 Hz,
CH2), 4.68 (ddd, 1H, J2;3 9.4, J3;4 3.4, J3;F 48.4 Hz, H-3),
4.22 (dt, 1H, J2;F 10.8 Hz, H-2), 3.93–3.91 (m, 1H, H-4),
3.74 (ddd, 1H, J5a;F 2.6, J4;5a 6.3Hz, H-5a), 3.66 (d, 1H,
J4;5b ꢁ0, J5a;5b 12.5 Hz, H-5b), 3.43 (s, 3H, OCH3), 2.11
(br s, 1H, OH); 19F NMR: d )124.8 (dm). Anal. Calcd
for C13H17FO4: C, 60.93; H, 6.69; F, 7.41. Found: C,
61.14; H, 7.02; F, 7.22.
4.11. Methyl 2-deoxy-2-fluoro-b-D-xylopyranoside (15)
Compound 11 (0.50 g, 2 mmol) was dissolved in MeOH
(20 mL) and 10% Pd/C (0.1 g) was added. The suspen-
sion was hydrogenated as described for 14. Purification
by flash chromatography gave pure 15 (0.31 g, 96%) as
white crystals: mp 105–107 °C (from i-Pr2O–i-PrOH);

M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
2109
20
D
½aŠ )56 (c 1.0, MeOH); Rf 0.26 (9:1 CHCl3–MeOH);
60.8 (d, JC-5;F 5.7 Hz, C-5), 56.6 (OCH3), 20.8
(2COCH3); 19F NMR: d )117.0 (ddd). Anal. Calcd for
C10H15FO6: C, 48.00; H, 6.04; F, 7.59. Found: C, 48.11;
H, 6.12; F, 7.48.
1H NMR (300 MHz, CDCl3): d 4.60 (dd, 1H, J1;2 7.5,
J1;F 2.7 Hz, H-1), 4.11 (ddd, 1H, J2;3 8.2, J2;F 50.9 Hz, H-
2), 3.98 (dd, 1H, J4;5a 5.1, J5a;5b 11.2 Hz, H-5a), 3.74
(ddd, 1H, J3;4 8.7, J3;F 14.8 Hz, H-3), 3.70–3.62 (m, 1H,
H-4), 3.56 (s, 3H, OCH3), 3.35 (dd, 1H, J4;5b 10.4, H-5b);
13C NMR (CD3OD): d 57.1 (OCH3), 66.9 (C-5), 71.0 (d,
JC-4;F 7.6 Hz, C-4), 76.4 (d, JC-3;F 17.4 Hz, C-3), 93.6 (d,
JC-2;F 184.9 Hz, C-2), 103.4 (d, JC-1;F 23.4 Hz, C-1); 19F
NMR: d )121.4 (dd). Anal. Calcd for C6H11FO4: C,
43.37; H, 6.67; F, 11.43. Found: C, 43.68; H, 6.78; F,
11.38.
4.12.3. Methyl 3,4-di-O-acetyl-2-deoxy-b- -threo-pento-
D
pyranoside (18). The reaction of acetylation was run with
14 (0.5 g, 3.4 mmol) and pure 18 was obtained as a
20
D
colorless syrup (0.71 g, 90%): ½aŠ )114 (c 1.0, CHCl3);
20
D
19
D
lit.25 ½aŠ )101 (c 1.0, CHCl3); lit.27 ½aŠ )121.9 (c 1.0,
CHCl3); Rf 0.48 (1:1 toluene–EtOAc); 1H NMR
(400 MHz, CDCl3): d 4.94 (ddd, 1H, J2a;3 4.8, J2b;3 8.4,
J3;4 6.9 Hz, H-3), 4.83 (ddd, 1H, J4;5a 4.0 Hz, J4;5b 6.7 Hz,
H-4), 4.53(dd, 1H, J1;2a 3.2, J1;2b 6.4 Hz, H-1), 4.11 (dd,
1H, J5a;5b 12.2 Hz, H-5a), 3.43 (s, 3H, OCH3), 3.39 (dd,
1H, H-5b), 2.24 (ddd, 1H, H-2a), 2.07 (s, 3H, COCH3),
2.06 (s, 3H, COCH3), 1.76 (ddd, 1H, J2a;2b 13.7 Hz, H-
2b); 13C NMR (CDCl3): d 170.2 (COCH3), 170.1
(COCH3), 99.3(C-1), 69.0 (C-4), 68.4 (C-3), 60.8 (C-5),
56.1 (OCH3), 33.1 (C-2), 21.06 (COCH3), 20.9
(COCH3). Anal. Calcd for C10H16O6: C, 51.72; H, 6.94.
Found: C, 51.65; H, 7.08.
4.12. Acetylation of 6, 7, 14, and 15
Compounds 6, 7, 14 or 15 were dissolved in pyridine
(0.9 mL per mmol glycoside) in the presence of a cata-
lytic amount of DMAP. Ac2O (0.8 mL per mmol gly-
coside) was added with cooling at 0 °C. The mixture was
stirred at room temperature for 48 h. Then the soln was
poured onto crushed ice, stirred for 2 h and extracted
with CH2Cl2 (3 · 10 mL per mmol glycoside). The ex-
tract was washed with water, dried (Na2SO4), and after
concentration and flash chromatography (2:1 toluene–
EtOAc) pure 16, 17, 18 or 19 were obtained.
4.12.4. Methyl 3,4-di-O-acetyl-2-deoxy-2-fluoro-b-D-xy-
lopyranoside (19). The reaction was carried out with 15
(0.25 g, 1.5 mmol) yielding 19 (0.35 g, 93%) after isola-
tion by the usual manner: mp 101–102 °C (from i-Pr2O);
4.12.1. Methyl 2,4-di-O-acetyl-3-deoxy-b-
D
-erythro-
pentopyranoside (16). The acetylation was performed
20
½aŠ )14 (c 1.0, CHCl3); Rf 0.54 (1:1 toluene–EtOAc);
D
with 6 (0.5 g, 3.4 mmol) to give 16 (0.72 g, 91%) as a
1H NMR (300 MHz, CDCl3): d 5.28 (ddd, 1H, J2;3 8.0,
J3;4 8.6, J3;F 13.9 Hz, H-3), 4.92 (ddd, 1H, J4;5a 5.0, J4;5b
8.9 Hz, H-4), 4.48 (dd, 1H, J1;2 6.5, J1;F 5.2 Hz, H-1), 4.26
(ddd, 1H, J2;F 49.5 Hz, H-2), 4.09 (dd, 1H, J5a;5b 11.8 Hz,
H-5a), 3.55 (s, 3H, OCH3), 3.39 (dd, 1H, H-5b), 2.11 (s,
3H, COCH3), 2.06 (s, 3H, COCH3); 13C NMR (CDCl3):
d 169.9 (2 COCH3), 101.3(d, JC-1;F 24.2 Hz, C-1), 88.8
(d, JC-2;F 187.9 Hz, C-2), 71.3(d, JCꢀ3;F 21.4 Hz, C-3),
68.6 (d, JC-4;F 6.0 Hz, C-4), 62.0 (C-5), 56.9 (OCH3), 20.7
(2 COCH3); 19F NMR: d )121.5 (ddd). Anal. Calcd for
C10H15FO6: C, 48.00; H, 6.04; F, 7.59. Found: C, 47.89;
H, 6.22; F, 7.46.
20
colorless syrup: ½aŠ )96 (c 1.0, CHCl3); lit.26 ½aŠ )94 (c
D
D
1.0, CHCl3); Rf 0.59 (1:1 toluene–EtOAc); 1H NMR
(300 MHz, CDCl3): d 4.84–4.80 (m, 1H, H-4), 4.74 (ddd,
1H, J1;2 1.7, J2;3a 4.0, J2;3b 1.2 Hz, H-2), 4.59 (d, 1H, H-1),
3.96 (dd, 1H, J4;5a 2.4, J5a;5b 12.7 Hz, H-5a), 3.68 (dt, 1H,
J4;5b 2.3, J3b;5b 2.1 Hz, H-5b), 2.20 (dt, 1H, J3a;4 4.0, J3a;3b
15.4 Hz, H-3a), 3.43 (s, 3H, OCH3), 2.11 (s, 3H,
COCH3), 2.10 (s, 3H, COCH3), 2.04 (ddt, 1H, J3b;4
1.2 Hz, H-3b); 13C NMR (CDCl3): d 170.4, 170.0 (2
COCH3), 98.1 (C-1), 66.9 (C-2), 65.7 (C-4), 61.2 (C-5),
55.3(OCH ), 26.9 (C-3), 21.2, 21.1 (2 COCH3). Anal.
3
Calcd for C10H16O6: C, 51.72; H, 6.94. Found: C, 51.74;
H, 6.86.
4.13. Deacetylation of 2,3,4-tri-O-Ac-XylpbOMe, 2,4-di-
O-Ac-XylpbOMe, 3,4-di-O-Ac-XylpbOMe, 16–19 with
AcXEs and products analysis
4.12.2. Methyl 2,4-di-O-acetyl-3-deoxy-3-fluoro-b-D-
xylopyranoside (17). The reaction was carried out with
7 (0.55 g, 3.3 mmol) to obtain 17 (0.73g, 89%) as white
Substrate solns (10 mM) in 0.1 M sodium phosphate
buffer (pH 6.0) were incubated with the appropriate
amount of purified AcXEs at 40 °C. Aliquots of the
reaction mixtures were analyzed by TLC. Solns of 2,4-
di-O-Ac-XylpbOMe and 3,4-di-O-Ac-XylpbOMe, which
undergo spontaneous acetyl group migration in aqueous
media, were always prepared freshly in the shortest
possible time before enzyme addition and their enzy-
matic treatment was never longer than 15 min. Reaction
mixtures of all substrates were analyzed by TLC on glass
plates of Silicagel G-60 (E. Merck) in 2:1:0.1 EtOAc–
20
needles: mp 80–81 °C (from i-Pr2O); ½aŠ )73( c 1.0,
D
CHCl3); Rf 0.52 (1:1 toluene–EtOAc); 1H NMR
(300 MHz, CDCl3): d 5.06–4.99 (m, 2H, H-2, H-4), 4.57
(ddd, 1H, J2;3 ¼ J3;4 7.9, J3;F 50.4 Hz, H-3), 4.37 (d, 1H,
J1;2 6.4 Hz, H-1), 4.16 (dt, 1H, J4;5a 4.9, J5a;F 5.0, J5a;5b
12.0 Hz, H-5a), 3.47 (s, 3H, OCH3), 3.32 (dd, 1H, J4;5b
8.2 Hz, H-5b), 2.13(s, H3, COCH 3), 2.11 (s, 3H,
COCH3); 13C NMR (CDCl3): d 169.7, 169.3(2 COCH3),
101.0 (d, JC-1;F 8.2 Hz, C-1), 89.7 (d, JC-3;F 187.9 Hz, C-3),
70.4 (d, JC-2;F 20.6 Hz, C-2), 68.9 (d, JC-4;F 20.7 Hz, C-4),

2110
M. Mastihubovꢀa, P. Biely / Carbohydrate Research 339 (2004) 2101–2110
benzene–2-propanol as described in details elsewhere12
The detected chromatograms28 were scanned in a
reflectance mode and the images were analyzed by
densitometry using Un-Scan-It software (Silk Scientific
Co., Orem, UT, USA). The rate of disappearance of the
substrate, was used to estimate the rate of the first de-
acetylation step. The rate was referred to 1 mg of the
protein, thus specific activities of the enzyme to indi-
vidual substrates were calculated. The number of
products as a result of the first deacetylation step was
evaluated on TLC visually.
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