Facile one-step syntheses of modified O-glycoprotein Galβ1-3GalNAc structures by transglycosylation employing three β-galactosidases from bovine testes, Xanthomonas manihotis, and Bacillus circulans

Article abstract of DOI:10.1080/07328300500208131

Natural O-glycoproteins such as the Thomsen-Friedenreich antigen or gangliosides contain the motif Galβ1-3GalNAc as an important disaccharide with significant biologic activity. The arrangement of spatial functionalities in this structure are of particular interest with regard to the development of potential leads en route to pharmaceuticals. Therefore, it was desired to obtain access to a range of modified derivatives of the aforementioned motif paying particular attention to introducing specific deoxy functions instead of hydroxyl groups. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1080/07328300500208131

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Facile One‐Step Syntheses of Modified
O‐Glycoprotein Galβ1‐3GalNAc
Structures by Transglycosylation
Employing Three β‐Galactosidases from
Bovine Testes, Xanthomonas manihotis,
and Bacillus circulans
a
a
Lars Kröger & Joachim Thiem
a
Institute of Organic Chemistry , University of Hamburg , Hamburg,
Germany
Published online: 20 Aug 2006.
To cite this article: Lars Kröger & Joachim Thiem (2005) Facile One‐Step Syntheses of Modified
O‐Glycoprotein Galβ1‐3GalNAc Structures by Transglycosylation Employing Three β‐Galactosidases
from Bovine Testes, Xanthomonas manihotis, and Bacillus circulans , Journal of Carbohydrate
Chemistry, 24:7, 717-722
To link to this article: http://dx.doi.org/10.1080/07328300500208131
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Journal of Carbohydrate Chemistry, 24:717–722, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300500208131
Facile One-Step Syntheses
of Modified O -Glycoprotein
Galb1-3GalNAc Structures
by Transglycosylation
Employing Three
b-Galactosidases from
Bovine Testes,
Xanthomonas manihotis,
and Bacillus circulans
Lars Kr o¨ ger and Joachim Thiem
Institute of Organic Chemistry, University of Hamburg, Hamburg, Germany
Natural O-glycoproteins such as the Thomsen-Friedenreich antigen or gangliosides
contain the motif Galb1-3GalNAc as an important disaccharide with significant
biologic activity. The arrangement of spatial functionalities in this structure are of
particular interest with regard to the development of potential leads en route to phar-
maceuticals. Therefore, it was desired to obtain access to a range of modified derivatives
of the aforementioned motif paying particular attention to introducing specific deoxy
functions instead of hydroxyl groups.
Keywords Chemoenzymatic glycosylations, b-Galactosidases, T Antigen
Received January 24, 2005; accepted March 7, 2005.
Address correspondence to Joachim Thiem, Institute of Organic Chemistry, University
of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. E-mail: thiem@
chemie.uni-hamburg.de
This paper is dedicated to Prof. Rosa M. de Lederkremer, Buenos Aires, on the occasion
of her 70th anniversary in recognition of her substantial contributions to carbohydrate
chemistry.
717

L. Kr o¨ ger and J. Thiem
7
18
Compared to the well-elaborated classical glycosylation procedures, the in vitro
application of enzymes excels in absolute stereospecificity, mild reaction
[1]
conditions, and the absence of protection-deprotection steps. Glycosidases are
enzymes that normally catalyze the cleavage of glycoside bonds, as opposed to
synthesizing glycosyltransferases. Instead of transferring an enzyme-bound
glycosyl residue to water, the b-galactosidases can transfer it to a different
acceptor. By utilizing novel deoxy acceptor structures in enzymatic glycosylation
reactions with different affinities for the employed enzymes, we anticipated
obtaining additional information about hydrogen bonding of the substrate in the
active site of the enzymes. With this knowledge a map of hydrogen donor and
acceptor interactions may be envisaged, thus allowing prediction of binding affi-
nities and enabling a more systematic application of biocatalysts. In this project,
[
2]
we decided to employ three different b-galactosidases from bovine testes,
[3]
[4]
Xanthomonas manihotis, and Bacillus circulans (recomb., bgaC-gene).
suitable donor for b-galactosidase-catalyzed glycosylations is
p-nitrophenyl b-D-galactopyranoside (pNP-b-D-Galp), releasing p-nitrophenol
A
(
pNP-OH). Its incubation with allyl 2-acetamido-2-deoxy-b-D-galactopyranoside
[5]
1
2
6
in the presence of b-galactosidase from bovine testes gave allyl
-acetamido-2-deoxy-3-O-(b-D-galactopyranosyl)-a-D-galactopyranoside in
7% yield as the single product (Table 1); no formation of regioisomers was obser-
9
a
ved. This synthesis of 9 is superior to all previously published classical organic
methods with regard to conditions employed, efficiency, and total yield.
[6]
[
7]
The regioselective gaiactosylation of methyl glycoside 2 employing the
b-galactosidase from bovine testes was quite successful as well, whereas the
yield was slightly lower in case of the less water-soluble acceptors benzyl and
[
8]
[9]
2
-(trimethylsilyl)-ethyl glycosides 3
and 4,
respectively. The crude
reaction mixture resulting from the treatment of benzyl 2-acetamido-2-
deoxy-b-D-galactopyranoside 3 in the presence of pNP-b-D-Galp had a very
low solubility in aqueous buffers, preventing the use of size exclusion
chromatography during workup. Hence, after the reaction went to completion,
p-nitrophenol was extracted, and the crude mixture was acetylated and
purified.
On removal of the anomeric hydroxyl group such as in 2-acetamido-1,5-
[
9]
anhydro-2-deoxy-D-galactitol 5, no effect on the recognition by the enzyme
from bovine testes was observed compared to the methyl or allyl glycosides.
Apparently, the absence of the 2-acetamido group in 2-deoxy glycoside 6
a
Donor pNP-b-D-Galp and 10 equivalents of the acceptor were dissolved in McIlvain
buffer (50 mM, pH 4.3) and warmed to 378C. b-Galactosidase from bovine testes (4.7
U/mmol) was added, and the mixture was incubated for 50 hr at 378C. The reaction
was stopped by heating to 908C for 5 min, followed by extraction of liberated pNP-
OH. Purification was achieved by size exclusion chromatography on Biogel P-2
columns and filtration through mixed-bed ion exchange resin.

Facile One-Step Synthesis by Transglycosylation
Table 1: Enzymatic galactosylation of GalNAc derivatives.
719
d
13CC-3
a
3
Acceptor
1
R
Product [ppm]
J1
0
0
[Hz]
Enzyme
Yield [%]
,2
OAll
9
10
11
12
13
77.64
77.70
73.78
77.41
80.22
7.6
7.9
7.6
7.6
7.6
Bovine testes
X. manihotis
B. circulans
Bovine testes
X. manihotis
B. circulans
Bovine testes
X. manihotis
B. circulans
Bovine testes
X. manihotis
B. circulans
Bovine testes
X. manihotis
B. circulans
67
21
34
66
24
33
41
16
38
47
19
35
70
19
31
2
3
4
5
OMe
OBn
OSE
H
b
b
b
a
Assigned by HMBC-NMR experiments.
b
Isolated as the corresponding peracetylated product.
prevents an optimal binding into the active site of the enzyme, giving lower
yields of the disaccharide product 14 (Sch. 1). In contrast, 1,5-anhydro-2-
[
10]
deoxy-D-lyxo-hexitol 7
was glycosylated by b-galactosidase from bovine
testes in high yields. Besides, the chemoenzymatic glycosylation of allyl 2-
aectamido-2,4-dideoxy-a-D-xylo-hexopyranoside 8 was accomplished regiose-
lectively at position 3 in particularly high yields.
On treatment of the acceptor glycosides 1–5 with b-galactosidases from
b
c
X. manihotis (Xm) or B. circulans (Bc) the formation of a second disaccharide
product identified as being the corresponding b1-6-regioisomer was observed.
In the first case (Xm), the ratio was approximately 10 : 1, but in the second case
b
Donor pNP-b-D-Galp, 3 equivalents acceptor, b-galactosidase from Xanthomonas man-
ihotis (230 U/mmol), and some BSA were dissolved in sodium acetate buffer (100 mM,
pH 5.5. 10% MeCN) and incubated for 50 hr at 378C. Purification see note a.
c
Donor pNP-b-D-Galp, 3 equivalents acceptor, and b-galactosidase from Bacillus circu-
lans (bgaC-gene, 5.9 U/mmol) were treated in potassium phosphate buffer (100 mM, pH
6
.0. 20% DMF) for 4 1/2 hr at 378C. Purification see note a.

L. Kr o¨ ger and J. Thiem
7
20
Scheme 1: Enzymatic galactosylation of deoxy derivatives. d13C C-3 (ppm) 14: 73.94, 15:
3
7
7.51; 16: 75.59; .J
1
0
,2
0
(Hz) 14: 7.6, 15: 7.9, 16: 7.9.
(Bc) the unwanted regioisomer was formed in considerable amounts giving a ratio
of 3 : 2. To facilitate purification of the desired products, in situ hydrolysis, intro-
[
11]d
duced by Larsson et al.,
of the unwanted disaccharide was applied. The crude
reaction mixtures were diluted and treated with b-galactosidase from Escheri-
chia coli, which has a high specificity for hydrolyzing b1-6-glycosidic linkages,
thus leaving the desired disaccharide linkage intact. In this way, a nonspecific
transglycosylating enzyme can be used for chemoenzymatic syntheses by employ-
ing it in conjunction with a highly specific hydrolyzing enzyme. However, due to a
lower activity and regiospecificity, the yields are reduced compared to the results
with b-galactosidase from bovine testes.
A decrease in yields was observed for all acceptor substrates lacking a
2-acetamido function. This applies to the enzyme from X. manihotis and was
even more pronounced for that from B. circulans. Again, the accompanying
b1-6-regioisomers were hydrolyzed by b-galactosidase from E. coli. In
contrast, the 4-deoxy derivative 8 was galactosylated only at position 3 and
no formation of any regioisomers was observed.
The respective 6-deoxy derivative allyl 2-acetamido-2-deoxy-a-D-fucopyra-
[
12]
noside
was also studied but apparently was not recognized by any of the
d
After denaturation of the respective transglycosylating b-galactosidase, the reaction
mixture was diluted 10-fold with sodium phosphate buffer (50 mM, pH 7.0. 1 mM
MgCl
2
). The b-galactosidasc from Escherichia coli was added and the mixture heated
to 378C for 4 hr. Workup see note a.

Facile One-Step Synthesis by Transglycosylation
721
employed enzymes; the hydroxyl group at position 6 seems to be essential for
binding to all b-galactosidases tested.
The b-galactosidases from bovine testes, X. manihotis, and B. circulans all
show a useful tolerance toward modifications of the acceptor structure, giving
access to derivatives of the Galb1-3-GalNAc motif in moderate to convincing
yields. Further modifications will be tested to ascertain the binding require-
ments for the different enzymes, enabling more accurate predictions of
reactions.
ACKNOWLEDGMENTS
Support of this work by the Deutsche Forschungsgemeinschaft (SFB 470) and
the Fonds der Chemischen Industrie is gratefully acknowledged.
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