Transglycosylation reactions with a crude culture filtrate from Thermoascus aurantiacus

Article abstract of DOI:10.1016/S0008-6215(00)00061-6

Some characteristics of regioselectivity and acceptor tolerance in transglycosylation reactions, catalysed by a crude culture filtrate from Thermoascus aurantiacus, were examined by employing methanol and monosaccharides as acceptors. When β-D-mannopyranosyl fluoride was employed as the donor, the anomeric configuration of the newly formed bond was found to depend on the structure of the acceptor used. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00061-6

www.elsevier.nl/locate/carres
Carbohydrate Research 327 (2000) 483–487
Note
Transglycosylation reactions with a crude culture filtrate
from Thermoascus aurantiacus
J o¨ rg Ortner , Martin Albert , Katherine Terler , Walter Steiner , Karl Dax *
a
a
b
b
a,
a
Institute of Organic Chemistry, Technical Uni6ersity Graz, Stremayrgasse 16, A-8010 Graz, Austria
b
Institute of Biotechnology, Technical Uni6ersity Graz, Petersgasse 12, A-8010 Graz, Austria
Received 12 October 1999; received in revised form 7 February 2000; accepted 18 February 2000
Abstract
Some characteristics of regioselectivity and acceptor tolerance in transglycosylation reactions, catalysed by a crude
culture filtrate from Thermoascus aurantiacus, were examined by employing methanol and monosaccharides as
acceptors. When b-D-mannopyranosyl fluoride was employed as the donor, the anomeric configuration of the newly
formed bond was found to depend on the structure of the acceptor used. © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Glycosyl fluorides; Glycosidases; Transglycosylation; b-Mannosidase; Thermoascus aurantiacus Miehe
1
9
1
. Introduction
For this purpose a new F NMR method
8], based on simultaneous monitoring of the
[
Due to the biological significance of
fate of different glycosyl fluorides in the pres-
ence of enzyme mixtures, has been developed.
By applying this technique together with the
known thin-layer chromatography (TLC)
method [9] in a screening program, the ther-
mophilic fungus Thermoascus aurantiacus has
proven to be a source of mannosidases as well
as hydrolysing enzymes towards b-cellobiosyl,
oligosaccharidic structures and glycoconju-
gates [1,2], a broad range of chemical [3,4] and
enzymatic [5–7] methods for glycosidic bond
formation has been developed to date. Ap-
proaches employing glycosidases as cheap and
easy to handle biocatalysts are limited by the
number of glycosidases available, especially
when, as in an ongoing program, formation of
b-lactosyl, a-
D
-galactosyl, and a- and b- -glu-
D
b- -mannosidic bonds or regiospecific trans-
D
cosyl fluorides [8].
glycosylation to secondary OH-groups of the
acceptor is required. In some instances, such
problems have been overcome by utilisation of
enzymes from new sources wherein ex-
tremophilic microorganisms are of special
interest.
2
. Results and discussion
To roughly test the glycosyltransfer poten-
tial of these hydrolases, cellobiose 1 and b- -
D
mannopyranosyl fluoride 3 as donors and
methanol as a simple acceptor were used
*
Corresponding author. Tel.: +43-316-8738244; fax: +
(
Table 1, entries 1 and 2). The methyl gly-
4
3-316-8738740.
E-mail address: dax@orgc.tu-graz.ac.at (K. Dax).
cosides 2 and 4, isolated by chromatography
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00061-6

484
J. Ortner et al. / Carbohydrate Research 327 (2000) 483–487
Table 1 Results of transglycosylation reactions
13
and identified by their C NMR spectra [10],
were formed with overall retention of configu-
ration. The b-configuration in mannopyra-
noside 4 was additionally proven by the
1
magnitude of J
of 160 Hz (versus 170
C-1,H-1
Hz reported for the a anomer) [11].

J. Ortner et al. / Carbohydrate Research 327 (2000) 483–487
485
In a second series of experiments, different
donors together with monosaccharidic accep-
tors were used. In all cases the products were
isolated and characterised as their peracetates.
The respective site of glycosylation as well as
the anomeric configuration of the newly formed
identical reaction conditions (Table 1, entry
8).
Starting from b- -mannopyranosyl fluoride
D
(3), formation of the a-configurated intersac-
charidic linkage in disaccharide 11 is in contrast
to own results which were obtained with
methanol as simple acceptor in the presence of
the same culture filtrate (see Table 1, entry 2).
(Transglycosylation reactions using 3 and b-
glucosidase from Agrobacterium sp. [20] also led
bond was elucidated from diacritic NMR data
3
(
as acylation and glycosylation shift, JH,H and
JC-1,H-1) obtained from the acetylated products
1
and verified by comparison of the NMR-data
of the O-deacetylated ones with those reported
for the proposed structures in the literature.
to b- -mannopyranosides under retention of
D
configuration.) Therefore, control experiments
under otherwise identical conditions but using
Using methyl b-
acceptor and p-nitrophenyl b-
noside (5) as donor, the disaccharide b-
1“6)-b- -Glcp-OMe (6) [12] was formed as
D
-glucopyranoside (2) as
-glucopyra-
-Glcp-
D
D-mannose as donor were undertaken. As no
D
disaccharide was produced, formation of 11 in
the former case by (b-mannosidase catalysed)
hydrolysis of the fluoride followed by (a-man-
nosidase catalysed) reverse hydrolysis could be
(
D
the sole product (Table 1, entry 3). In the
reaction with b-lactosyl fluoride (7) as donor,
a mixture of regioisomers was obtained from
which the main product could be isolated and
1
9
excluded. Furthermore, as TLC and F NMR
monitoring disclosed, anomerisation of the b-
fluoride 3 under the reaction conditions did not
take place, thus excluding anomerisation prior
to (a-mannosidase catalysed) transglycosyla-
tion as a possible reaction path. For further
mechanistic studies, the isolation and charac-
terisation of the individual mannosidases (ei-
ther working with retention or inversion of
configuration) from this multi-enzyme mixture
is in progress.
identified as b-
D
-Galp-(1“4)-b-
D
-Glcp-(1“
4
)-b- -Glcp-OMe (8) [13] containing a new
D
b-(1“4) linkage (Table 1, entry 4). In order to
explore the influence of the anomeric configura-
tion of the acceptor on the regioselectivity, an
analogous experiment with 7 and methyl a- -
D
glucopyranoside (9) was performed, but no
products of transglycosylation could be iso-
lated (Table 1, entry 5). A similar result has
been reported by Kobayashi and co-workers,
when using a cellulase from Trichoderma 6iride
[
14].
Interestingly, transglycosylation with inver-
sion of configuration was observed when b-
mannopyranosyl fluoride (3) as donor together
with methyl a- -mannopyranoside (10) as ac-
ceptor was used and a- -Manp-(1“6)-a-
3. Experimental
D
-
General procedures.—Optical rotations were
measured with a Jasco DIP-360 digital polar-
imeter at 589 nm at ambient temperature.
NMR spectra were recorded at 300.13 or 199.98
D
D
D
-
1
13
Manp-OMe (11) [15–18] was isolated as its
peracetate [18] in low yield (Table 1, entry 6).
The same a-(1“6)-linked disaccharide was
MHz ( H) and 75.47 or 50.29 MHz ( C) from
solutions in CDCl using a Bruker MSL 300
and a Varian Gemini 200 apparatus, respec-
tively. As reference standard Me Si ( H and C
NMR) was used. TLC was performed on Silica
Gel 60 F254 precoated aluminium plates (E.
Merck 5554) with detection by charring after
spraying with vanillin/H SO (1% w/v). For
3
1
13
formed when a-
D-mannopyranosyl fluoride
4
(
12) was applied as donor (Table 1, entry 7).
The a-configuration at both anomeric centres in
disaccharide 11 was additionally proved by
1
JC-1,H-1 of 172.2 and 172.8 Hz, respectively.
2
4
No products of mannosyl transfer could be
column chromatography Silica Gel 60, 230–
400 mesh (E. Merck 9385) was used.
Compounds 2, 5, 9 and 10 were purchased
from Fluka; donors 3, 7 and 12 were prepared
according to literature procedures [8,12].
isolated when b-
together with an 1:1 mixture of 2-deoxy-2-
fluoro-a- -gluco- (13) and -a- -mannopyra-
nosyl fluoride (14) [19] was subjected to
D
-mannopyranosyl fluoride (3)
D
D

486
J. Ortner et al. / Carbohydrate Research 327 (2000) 483–487
The crude culture filtrate used in this study
Methyl 2,3,6-tri-O-acetyl-4-O-[2,3,6-tri-O-
was from the strain Thermoascus aurantiacus
acetyl - 4 - O - (2,3,4,6 - tetra - O - acetyl - i -
galactopyranosyl)-i- -glucopyranosyl]-i-
D
-
-
Miehe, and was prepared according to Ref.
D
D
[
8].
glucopyranoside (peracetate of 8).—b-Lactosyl
Procedure A.—To a soln of the donor (0.20
fluoride (7, 200 mg, 0.58 mmol) and methyl
M) in acetate buffer (pH 5.0, 50 mM) contain-
ing 20 vol% MeOH, the lyophilised culture
filtrate (0.5–1 mg per mg donor) was added
and the mixture was shaken at room tempera-
ture (rt) until all the donor was consumed
b- -glucopyranoside (2, 338 mg, 1.74 mmol)
D
were treated according to procedure B yielding
a mixture of regioisomeric trisaccharides (105
mg, S 19%, colourless oil). By repeated
column chromatography (1:1 cyclohexane–
EtOAc), a clean fraction of the title compound
(
typically overnight). After addition of toluene
2
0
and evaporation of the solvent under dimin-
ished pressure, the products were isolated by
was isolated and characterised; [h] −11° (c
D
0
.15, CH Cl ); R 0.33 (1:2 cyclohexane–
2
2
f
column chromatography (5:1 CHCl –MeOH).
13
3
EtOAc); C NMR (50.29 MHz, CDCl ): l
3
Procedure B.—To a soln of the donor (0.50
1
(
7
7
70.4–169.2 (OAc), 101.5, 101.2 and 100.6
M) and acceptor (1.5 M) in acetate buffer (pH
C-1, 1%, 1¦), 76.6 and 76.1 (C-4, 4%), 73.1, 72.8,
5
(
.0, 50 mM), the lyophilised culture filtrate
2.8, 72.6, 72.0 and 71.7 (C-2, 3, 5, 2%, 3%, 5%),
1.1 and 70.9 (C-3¦, 5¦), 66.7 (C-4¦), 62.4, 61.9
1–2 mg per mg donor) was added and the
reaction mixture was shaken at rt until all the
donor was consumed. After addition of
toluene and evaporation of the solvent under
diminished pressure, the crude product mix-
and 60.9 (C-6, 6%, 6¦), 57.1 (OMe), 21.0–20.6
1
(
(
OAc); H NMR (199.98 MHz, CDCl ): l 5.34
3
bd, 1 H, J3¦,4¦ 3.3 Hz, H-4¦), 5.16 and 5.13 (t
each, 1 H each, J =J =J =J 9.2 Hz,
2
,3
3,4
2%,3%
3%,4%
ture was acetylated in Ac O–pyridine. After
2
H-3, 3%), 5.09 (dd, 1 H, J1¦,2¦ 8.1 Hz, J2¦,3¦ 10.3
Hz, H-2¦), 4.93 (dd, 1 H, H-3¦), 4.88 and 4.83
extractive work-up, the peracetates were iso-
lated by column chromatography (2:1
cyclohexane–EtOAc).
(
4
(
3
3
4
dd each, 1 H each, J =J 7.8 Hz, H-2, 2%),
1
,2
1%,2%
.49 and 4.43 (d each, 1 H each, H-1, 1%), 4.37
Methyl
tetra - O - acetyl - i -
glucopyranoside (peracetate of 6).—p-Nitro-
phenyl b- -glucopyranoside (5, 176 mg, 0.58
mmol) and methyl b- -glucopyranoside (2,
40 mg, 1.75 mmol) were brought to reaction
2,3,4-tri-O-acetyl-6-O-(2,3,4,6-
d, 1 H, H-1¦), 4.0–4.6 (m, 6 H, H-6, 6%, 6¦),
D
- glucopyranosyl) - i -
D-
.85 (bd, 1 H, J
.76 (t each, 1 H each, J =J 9.2 Hz, H-4,
6.6 Hz, H-5¦), 3.78 and
5
¦,6¦
4
,5
4%,5%
D
%), 3.5–3.65 (m, 2 H, H-5, 5%), 3.47 (s, 3 H,
OMe), 2.20–1.95 (m, 30 H, 10 OAc).
D
3
Methyl
tetra - O - acetyl - h -
2,3,4-tri-O-acetyl-6-O-(2,3,4,6-
as described in procedure B yielding the title
compound (95 mg, 25%) as a colourless oil.
D
- mannopyranosyl) - h - -
D
2
0
mannopyranoside (peracetate of 11).—Appli-
[
h] −20° (c 1.35, CHCl ); R 0.42 (1:1 cyclo-
D
3
f
13
cation of procedure B to a mixture of a- or
hexane–EtOAc);
CDCl ): l 170.7–169.3 (OAc), 101.5 and 100.8
C NMR (50.29 MHz,
b- -mannopyranosyl fluoride (12 or 3, 150
D
3
mg, 0.823 mmol) and methyl a- -mannopyra-
D
(
7
C-1, 1%), 73.3 (C-5), 72.8 and 72.8 (C-3, 3%),
2.0 (C-5%), 71.3 and 71.1 (C-2, 2%), 69.2, 68.3
and 68.3 (C-4, 6, 4%), 61.8 (C-6%), 57.1 (OMe),
noside (10, 480 mg, 2.47 mmol) led to the title
compound as a colourless oil (43 or 59 mg, 8
2
0
1
or 11%, respectively). [h] +72° (c 1.10,
2
0.7–20.6 (OAc); H NMR (300.13 MHz,
D
1
3
CHCl ); R 0.26 (1:1 cyclohexane–EtOAc); C
CDCl ): l 5.13 (t, 2 H, J =J =J2%,3%=J3%,4%
3
f
3
2,3
3,4
NMR (50.29 MHz, CDCl ): l 170.6–169.8
9
.5 Hz, H-3, 3%), 4.92 and 4.85 (dd each, 1 H
3
(
(
OAc), 98.4 (JC-1,H-1 172.2 Hz, C-1), 97.5
JC-1,H-1 172.8 Hz, C-1%), 69.5, 69.4, 69.2, 69.1,
69.0, 68.7, 66.6 and 66.0 (C-2, 3, 4, 5, 2%, 3%, 4%,
each, J =J 8.0 Hz, H-2, 2%), 5.00 and 4.82
1
,2
1%,2%
(
t each, 1 H each, J =J
9.5 Hz, H-4, 4%),
4
,5
4%,5%
4
.54 and 4.33 (d each, 1 H each, H-1, 1%), 4.20
(
dd, 1 H, J5%,6%a 4.8 Hz, J6%a,6%b 12.4 Hz, H-6%a),
.05 (bd, 1 H, H-6%b), 3.81 (bd, 1 H, J6a,6b 10.5
Hz, H-6a), 3.67–3.60 (m, 2 H, H-5, 5%), 3.55
dd, 1 H, J5,6b 7.4 Hz, H-6b), 3.44 (s, 3 H,
OMe), 2.1–1.9 (m, 21 H, 7 OAc).
5%), 66.7 (C-6), 62.4 (C-6%), 55.3 (OMe), 20.9–
1
4
20.7 (OAc); H NMR (199.98 MHz, CDCl ): l
3
5.40–5.15 (m, 6 H, H-2, 3, 4, 2%, 3%, 4%), 4.83
and 4.67 (d each, 1 H each, J =J 1.3 Hz,
(
1,2
1%,2%
H-1, 1%), 4.22 (dd, 1 H, J5%,6%a 5.4 Hz, J6%a,6%b

J. Ortner et al. / Carbohydrate Research 327 (2000) 483–487
487
[
6] Y. Ichikawa, G.C. Look, C.-H. Wong, Anal. Biochem.,
1
3
2
2.2 Hz, H-6%a), 4.15–4.0 (m, 2 H, H-5, 6%b),
202 (1992) 215–238.
.90 (ddd, 1 H, J4,5 8.5 Hz, J5,6a 5.8 Hz, J5,6b
[7] L.F. Mackenzie, Q. Wang, R.A.J. Warren, S.G. With-
.4 Hz, H-5), 3.75 (dd, 1 H, J6a,6b 10.8 Hz,
ers, J. Am. Chem. Soc., 120 (1998) 5583–5584.
[
8] M. Albert, W. Repetschnigg, J. Ortner, J. Gomes, B.J.
Paul, C. Illaszewicz, H. Weber, W. Steiner, K. Dax,
Carbohydr. Res., 327 (2000) 395–400.
H-6a), 3.54 (dd, 1 H, H-6b), 3.38 (s, 3 H,
OMe), 2.15–1.9 (m, 21 H, 7 OAc).
[9] A. Holazo, H. Shinoyama, Y. Kamiyama, T. Yasui,
Biosci. Biotech. Biochem., 56 (1992) 822–824.
[
[
10] (a) K. Bock, C. Pedersen, Ad6. Carbohydr. Chem.
Biochem., 41 (1983) 27–65. (b) P.K. Agrawal, Phyto-
chemistry, 31 (1992) 3307–3330.
Acknowledgements
11] K. Bock, C. Pedersen, J. Chem. Soc., Perkin Trans. 2,
We appreciate financial support by the Aus-
trian Fonds zur F o¨ rderung der Wis-
senschaftlichen Forschung, Vienna (Project
(
1974) 293–297.
[12] P.-E. Jannson, L. Kenne, I. Kolare, Carbohydr. Res.,
57 (1994) 163–174.
2
[
13] S. Shoda, T. Kawasaki, K. Obata, S. Kobayashi, Car-
1
1021-OECH). Dr Dean Johnson is thanked
bohydr. Res., 249 (1993) 127–137.
for proof-reading the manuscript.
[14] O. Karthaus, S. Shoda, H. Takano, K. Obata, S.
Kobayashi, J. Chem. Soc., Perkin Trans. 1, (1994)
1
851–1853.
15] T. Ogawa, K. Sasajima, Carbohydr. Res., 97 (1981)
05–227.
[
2
References
[
[
16] K.G. Nilsson, Carbohydr. Res., 167 (1987) 95–103.
17] B.A. Spronk, A. Rivera-Sagredo, J.P. Kamerling,
J.F.G. Vliegenthart, Carbohydr. Res., 273 (1995) 11–26.
[
[
[
1] R.A. Dwek, Chem. Re6., 96 (1996) 683–720.
2] H. Lis, N. Sharon, Eur. J. Biochem., 218 (1993) 1–27.
3] K. Toshima, K. Tatsuta, Chem. Re6., 93 (1993) 1503–
[18] Z. Szurmai, L. Janossy, Z. Szilagyi, K. Vekey, J. Car-
bohydr. Chem., 17 (1998) 417–437.
1
531.
[19] J. Ortner, M. Albert, H. Weber, K. Dax, J. Carbohydr.
Chem., 18 (1999) 297–316.
[20] H. Prade, L.F. Mackenzie, S.G. Withers, Carbohydr.
Res., 305 (1998) 371–381.
[
[
4] G.-J. Boons, Tetrahedron, 52 (1996) 1095–1121.
5] E.J. Toone, E.S. Simon, M.D. Bednarski, G.M.
Whitesides, Tetrahedron, 45 (1989) 5365–5422.
.