Formation of LacNAc mimetics employing novel donor substrates for enzymatic β1→4 galactosylation

Article abstract of DOI:10.1039/b400916a

The formation of LacNAc mimetics employing novel donor substrates for enzymatic β1→4 galactosylation is discussed. In examining C-6 modified 4-nitrophenyl β-D-galacto-pyranosides as donor structures the β-galactosidase revealed a broad substrate specifici

Full text of DOI:10.1039/b400916a

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Formation of LacNAc mimetics employing novel donor substrates
for enzymatic ꢀ1 4 galactosylation
Saskia Weingarten and Joachim Thiem*
Institute of Organic Chemistry, Hamburg University, Martin-Luther-King-Platz 6, D-20146,
Hamburg, Germany. E-mail: thiem@chemie.uni-hamburg.de; Fax: ϩ49 40 42838 4325;
Tel: ϩ49 40 42838 7068
Received 20th January 2004, Accepted 17th February 2004
First published as an Advance Article on the web 26th February 2004
In examining C-6 modified 4-nitrophenyl ꢀ-D-galacto-
pyranosides as donor structures the ꢀ-galactosidase
(Bacillus circulans) revealed an unexpectedly broad
substrate specificity which allowed successful syntheses
of various disaccharide components.
Previously, two modified galactosides were reported as
donor substrates that are the C-6-oxidized 4-nitrophenyl β--
galacto-hexodialdo-pyranoside (9) and the 4-methylumbelli-
feryl 6-sulfo-β--galactopyranoside. The former could be
successfully utilized for disaccharide synthesis, although the
resulting disaccharide was not isolated, but further reacted
in situ12 and the latter could be converted into the corre-
sponding sulfated disaccharide.13
Based on these findings, the primary alcohol function
in galactosides does not seem to be crucial for recognition.
To verify this assumption further C-6-modified donors were
synthesized and tested as substrates for β-galactosidase (B. cir-
culans) with allyl 2-acetamido-2-deoxy-α--glucopyranoside
(1) as well as 2-acetamido-2-deoxy--glucopyranose (2) as
acceptor molecules (Fig. 2).
The important role of carbohydrates in vital biological
recognition processes has increasingly stimulated efforts in
glycoconjugate research. Due to the complexity of con-
ventional oligosaccharide synthesis, enzymatic methods have
experienced growing interest,1–5 which do not require protection
and deprotection steps and therefore provide rapid access to
natural oligosaccharides.
Unnatural and modified oligosaccharides are needed as
potential drug candidates as well as for the investigation of
glycoconjugate structure–function relationships in biochemical
processes. Exploitation of enzymes for this purpose exhibiting
the usual high regio- and stereospecificity is more difficult.
In this case as certain flexibilities concerning their substrates are
allowed, modification of the glycosyl residue may be possible
before glycosylation. Then as an additional advantage the
enzyme-catalysed reaction can be carried out as the last step.
N-Acetyllactosamine is a common structural element in bio-
logically important glycoconjugates, for example as a typical
terminal sequence of N-linked oligosaccharides, as the core unit
of carbohydrates isolated from human milk6 and as an essential
component of ligands of various lectins.7–9 An appropriate
enzyme for its synthesis is the β-galactosidase from Bacillus
circulans, which regioselectively catalyses the β1 4 linkage
formation between a galactosyl donor and a 2-acetamido-
glycosyl acceptor (Fig. 1). Generally it shows a high trans-
glycosylation rate with extraordinary regioselectivity. Product
yields range from 18 to 66%, depending on the leaving group of
the donor and the utilized acceptor. As donor leaving groups,
glucose, para-nitrophenol and ortho-nitrophenol are used
commonly, others were also investigated.10 The acceptor may
have a glucosyl or a galactosyl α/β-configuration with its
anomeric center either unprotected or protected. An acetamido
group at C-2 is necessary to control the regioselectivity, in
other cases, e.g. with a hydroxyl group at C-2, a mixture of
β 1–3-, β 1–4- and β 1–6- linked regioisomeric disaccharides
is obtained.11
Fig. 2 General reaction with derivatized galactopyranosides.
Both, the pNP β--fucopyranoside (3) and pNP α--arabino-
pyranoside (4) proved to be substrates for the β-galactosidase,
yielding the expected disaccharides allyl β--fucopyranosyl-
(1 4)-2-acetamido-2-deoxy-α--glucopyranoside (5) and
α--arabinopyranosyl-(1 4)-2-acetamido-2-deoxy-α--gluco-
pyranoside (6) in extraordinary high 76% and poorer 13% yield,
respectively. The corresponding reaction with 2-acetamido-
2-deoxy-α--glucopyranose (2) gave β--fucopyranosyl-(1 4)-
2-acetamido-2-deoxy--glucopyranose (7) in 66% yield, but in
case of the arabinopyranoside only traces of a newly formed
compound 8 were detectable (Table 1).
Reaction of the C-6 oxidized donor 4-nitrophenyl β--
galacto-hexodialdo-pyranoside (9) could be confirmed and the
disaccharide allyl β--galacto-hexodialdopyranosyl-(1 4)-
2-acetamido-2-deoxy-α--glucopyranoside (10) isolated in 66%
yield. Treatment of this donor 9 with 2 gave the disaccharide 11
in 19% yield.
The precursor of the aldehyde 9 is the 6,7-olefin structure
12,14 which surprisingly readily could be glycosylated to give
the disaccharides allyl 6,7-dideoxy-β--galacto-hept-6-eno-
pyranosyl-(1 4)-2-acetamido-2-deoxy-α--glucopyranoside
(13) or 6,7-dideoxy-β--galacto-hept-6-enopyranosyl-(1 4)-2-
acetamido-2-deoxy--glucopyranose (14) in 31% and 20%,
respectively.
Reductive amination of aldehyde 9 with the amino acid
taurine led to a novel carbohydrate–amino acid conjugate,15
which however was not accepted by the enzyme.
Fig. 1 Glycosylation catalysed by galactosidase from B. circulans.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 9 6 1 – 9 6 2
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Table 1 Galactoside derivatives employed and LacNAc mimetics obtained
No
X
No
Yield (%)
No
Yield (%)
3
4
CH3
H
CHO
5
6
10
13
16
76
13
66
7
8
11
14
—
66
traces
19
20
—
9
12
15
CH᎐CH2
31
᎐
CH2NH2
∼10a
a After acetylation.
Notes and references
† Enzyme catalysed transgalactosylations to disaccharides 5, 6, 10, 13
and 16 were performed according to the procedure described by Farkas
and Thiem;16 in the case of compound 10 the reaction was performed in
phosphate buffer without acetonitrile as cosolvent.
Enzyme catalysed transgalactosylations with 2-acetamido-2-deoxy-
-glucopyranose (2) as acceptor were performed as follows: 150 µmol
glycopyranosyl donor and 300 mg (9 equivalents) of acceptor 2 were
dissolved in 4 ml of 50 mM acetate buffer pH 5.0 and incubated with
4 U β-galactosidase (B. circulans) for 3 h at 55 ЊC. The reaction was
stopped by heating the solution to 100 ЊC. The crude reaction mixture
was lyophilized. The product was separated from remaining educts and
by-products chromatographically on a biogel P2 column with water as
eluent.
1 K. M. Koeller and C.-H. Wong, Nature, 2001, 409, 232–240.
2 S.-I. Shoda, in Glycoscience, B. O. Fraser-Reid, K. Tatsuta and
J. Thiem, Eds., Springer-Verlag, Berlin, Germany, 2001, vol. 2,
pp. 1465––1496.
Fig. 3 Synthesis of an amine functionalized galactosyl donor.
3 D. J. Vocadlo and S. G. Withers, in Carbohydrates in Chemistry and
Biology B. Ernst, G. W. Hart and P. Sinay, Eds., Wiley-VCH-Verlag
GmbH, Weinheim, Germany, 2000, vol. 2, pp. 723––844.
4 C.-H. Wong, in Enzyme Catalysis in Organic Synthesis, 2nd edn.,
K. Drauz and H. Waldmann, Eds., Wiley-VCH-Verlag GmbH,
Weinheim, Germany, 2002, vol. 2, pp. 609––653.
5 K. Ajisaka and Y. Yamamoto, Trends Glycosci. Glycotechnol., 2002,
14, 1–11.
6 C. Kunz and S. Rudloff, Acta Paediatr., 1993, 82, 903–912.
7 M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Sing-
hal, S.-I. Hakomori and J. C. Paulson, Science, 1990, 250,
1130–1131.
To check the steric requirements, 4-nitrophenyl 6-O-allyl-
β--galactopyranoside, which represents a kind of chain-
elongated analogue of the previously recognized 6,7-olefin 12,
was prepared. However, this donor also was not recognized.
Amine 15 was synthesized via 4-nitrophenyl 6-O-sulfonyl-
β--galactopyranoside and 4-nitrophenyl 6-azido-6-deoxy-β--
galactopyranoside (Fig. 3). Neither the 6-O-mesylate nor the
6-azido-6-deoxy compounds were accepted by the enzyme.
However, to our surprise and pleasure the reaction of amine
15 with acceptor 1 could be achieved and resulted in the form-
ation of allyl 6-amino-6-deoxy-β--galactopyranosyl-(1 4)-
2-acetamido-2-deoxy-α--glucopyranoside (16).
To summarize the results, the synthesis of various LacNAc
mimetics could be achieved employing the β-galactosidase from
Bacillus circulans. Apparently, a certain flexibility in the
enzyme’s substrate specificity can be assigned, which proves
that the primary alcohol function of the donor is not crucial
for recognition by this β-galactosidase. Complete lack of a C-6
group diminishes the reaction yield and substituents at C-6
should not be too bulky. Again, charged functionalities do not
present a hindrance for the enzyme catalyzed formation of
disaccharides.†
8 B. K. Brandley, S. J. Swiedler and P. W. Robbins, Cell, 1990, 63,
861–863.
9 E. L. Berg, J. Magnani, R. A. Warnock, M. K. Robinson and E. C.
Butcher, Biochem. Biophys. Res. Commun., 1992, 184, 1048–1055.
10 A. Vetere, L. Novelli and S. Paoletti, J. Carbohydr. Chem., 1999, 18,
515–521.
11 T. Usui, S. Morimoto, Y. Hayakawa, M. Kawaguchi, T. Murata,
Y. Matahira and Y. Nishida, Carbohydr. Res., 1996, 285, 29–39.
12 T. Kimura, S. Takayama, H. Huang and C.-H. Wong, Angew.
Chem., Int. Ed. Engl., 1996, 35, 2348–2350.
13 T. Murata, M. Kosugi, T. Nakamura, T. Urashima and T. Usui,
Biosci., Biotechnol., Biochem., 2001, 65, 2456–2464.
14 D. A. McManus, U. Grabowska, K. Biggadike, M. I. Bird, S. Davies,
E. N. Vulfson and T. Gallagher, J. Chem. Soc., Perkin Trans. 1, 1999,
295–305.
15 S. Weingarten and J. Thiem, Synlett, 2003, 1052–1054.
16 E. Farkas and J. Thiem, Eur. J. Org. Chem., 1999, 3073–3077.
Support of this work by the Fonds der Chemischen Industrie
is gratefully acknowledged.
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