β-Galactosidase-catalyzed intramolecular transglycosylation

Article abstract of DOI:10.1016/S0040-4039(01)01826-3

A novel transglycosylation strategy for efficient preparation of N-acetyllactosamine (Galβl→4GlcNAc, LacNAc) and sialyl LacNAc was developed using β-galactosidase from Bacillus circulans. In order to minimize the competing hydrolysis by forcing the enzyma

Full text of DOI:10.1016/S0040-4039(01)01826-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8501–8505
b-Galactosidase-catalyzed intramolecular transglycosylation
Shiro Komba† and Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Received 24 August 2001; revised 27 September 2001; accepted 28 September 2001
Abstract—A novel transglycosylation strategy for efficient preparation of N-acetyllactosamine (Galb1“4GlcNAc, LacNAc) and
sialyl LacNAc was developed using b-galactosidase from Bacillus circulans. In order to minimize the competing hydrolysis by
forcing the enzymatic transglycosylation to proceed in an intramolecular manner, a novel substrate 9 carrying the donor
(galactose) and the acceptor (N-acetylglucosamine) components linked via a 2-hydroxy-5-nitro-benzylalcohol derived tether was
prepared. Treatment with b-galactosidase from B. circulans afforded the transglycosylation product 11 in 26% yield. Furthermore,
addition of sialyltransferase and CMP–sialic acid to this system gave sialyl LacNAc 18 in 39% yield. © 2001 Elsevier Science Ltd.
All rights reserved.
N-Acetyllactosamine (Galb1“4GlcNAc, LacNAc) is
widespread as a constituent of glycoconjugates, includ-
ing N- and O-linked glycoproteins,1 glycolipids,2 and
glycosaminoglycans.3
tose) or easily obtainable (aryl b-D-galactoside)
galactosyl donor can be used, and (2) compared to
glycosyltransferases, glycosidases are much more stable,
easier to handle, and generally cheaper. However, in
order to achieve efficient transglycosylation, a large
excess of either glycosyl donor (i.e. lactose) or acceptor
(GlcNAc) is usually required and isolation process thus
tends to be complicated.
It is also important as a precursor of ligands for cell
adhesion molecules, for instance selectin4 and CD22.5
Various approaches toward chemical synthesis of Lac-
NAc have been investigated.6–12 Most typically, it was
prepared from lactose via lactal using Lemieux’s azi-
donitration as the key transformation.13,14 Enzymatic
preparation of LacNAc has also been a subject of
extensive investigation. For instance, galactosyltrans-
ferase-mediated glycosylation of N-acetylglucosamine
Since glycosidases are hydrolyzing enzymes in nature, it
would be difficult to suppress the hydrolysis to an
extent that transglycosylation using an equimolar
amount of glycosyl donor/acceptor can be achieved in a
synthetically useful efficiency. One potential tactic to
alleviate this difficulty would be to make the glycosyl
transfer entropically favorable by connecting the two
components via a tether into a single molecule. On
condition that donor and acceptor portions of the
substrate fit simultaneously into the binding pocket of
the enzyme, transglycosylation may be expected to pro-
ceed in an intramolecular manner and override the
competing hydrolysis (Scheme 1).21
(GlcNAc) with UDP–galactose provides
a highly
efficient access to LacNAc.15 Although several types of
glycosyltransferases are rapidly gaining practical use in
preparative scale oligosaccharide synthesis,16 limitation
has been posed by the cost, availability and the stability
of the enzymes and sugar nucleotides.
Alternatively, galactosidase derived from various
sources, particularly the one derived from Bacillus
circulans, have been used for the preparation of Lac-
NAc, taking advantage of their transglycosylating
activity.17–20 A galactosidase based approach is attrac-
tive due to the following reasons: (1) inexpensive (lac-
In order to examine this possibility, compounds 9 and
10 embracing two sugar residues with an ortho and
meta relationship, respectively, were designed. It can be
conceived that both of them are derivatives of p-nitro-
phenyl b-galactoside, an active and well-explored galac-
tosyl donor. These compounds were prepared from
commercially available 2-hydroxy-5-nitro- (2) (ortho)
and 5-hydroxy-2-nitro- (3) benzyl alcohol (meta) as
depicted in Scheme 2. Thus, Lewis acid-mediated reac-
tions with glucosamine donor 1 afforded 4 and 5.
Subsequently, trichloroacetimidate 622 was used as a
Keywords: galactosidase; intramolecular transglycosylation; N-acetyl-
lactosamine; sialyltransferase.
* Corresponding author. Tel.: +81-48-467-9430; fax: +81-48-462-4680;
e-mail: yukito@postman.riken.go.jp
† Special Postdoctoral Researcher.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01826-3

8502
S. Komba, Y. Ito / Tetrahedron Letters 42 (2001) 8501–8505
HO
HO
OH
HO
HO
O
O
OH
O
O
O
NHAc
AcHN
H
OH
O
HO
HO
O
OH
O
OH
HO
O
O
HO
HO
O
O
OH
O
OH
HO
OH
O
O
O
HO
OH
NHAc
HO
O
O
O
H
O
OH
O
OH
OH
O
H
OH
OH
HO
OH
O
O
O
O
O
O
HO
HO
HO
OH
O
NHAc
NHAc
HO
O
HO
O
HO
O
Scheme 1. b-Galactosidase-mediated intramolecular transglycosylation.
OH
OH
OAc
O
OAc
AcO
AcO
2 (ortho)
O
NO2
O
AcO
AcO
NPhth
HO
OAc
BF3•OEt2
CH2Cl2
r.t.
+
OH
NO2
NPhth
OH
1
4 (ortho) ;84%
5 (meta) ;85%
2h
NO2
3 (meta)
OAc
O
AcO
AcO
O
BzO
BzO
NPhth
OBz
O
4
BF3•OEt2
CH2Cl2
r.t.
OBz
O
NO2
O
or
+
BzO
BzO
OBz
5
BzO
O
CCl
3 1day
6
7 (ortho) ;70%
8 (meta) ;98%
NH
1) 75eq. NH2NH2•H2O
EtOH
OH
O
7
or
8
HO
HO
O
100°C, 8h
NHAc
O
NO2
OH
2) Ac2O
MeOH
6h
O
HO
OH
HO
9 (ortho) ;58%
10 (meta) ;75%
Scheme 2. Preparation of transglycosylation substrates.
was followed by selective N-acetylation to give transg-
lycosylation substrates 9 (ortho) and 10 (meta).
galactosyl donor to glycosylate the phenolic hydroxyl
group to give b-glycosides 7 and 8. Somewhat unex-
pectedly, glycosylation of 4 was accompanied by the
formation of a significant amount of corresponding
a-isomer (a:b=1:3.3). Finally, complete deprotection
Enzymatic transglycosylation was examined using 9 or
10 (8 mM) in 50 mM sodium acetate buffer (pH 5.0) at

S. Komba, Y. Ito / Tetrahedron Letters 42 (2001) 8501–8505
8503
36°C in the presence of galactosidase from B. circulans
(Biolacta, Daiwa Kasei Co., Ltd.) and the reactions
were followed by analytical HPLC.23 As shown in Fig.
1, profiles of two reactions are markedly different.
From 9, the formation of LacNAc derivative 11 (ꢀ)
was dominant over 15 even at the early stage of incuba-
tion (i.e. [9]>[13]). Since rates of intermolecular glycosyl
transfer likely to be similar between 9 and 13 (k1:k2),
this result indicates that the intramolecular pathway is
functional (k3>k1, k2) (Scheme 3). On the other hand,
the overall process from meta-oriented 10 was less
efficient. Formation of LacNAc (12) lagged far behind
Gal2GlcNAc (16) and the direct pathway (10“12)
seems insignificant. Gradual formation of 12 would
most likely to be a result of intermolecular transglyco-
sylation (10“14“12 and/or 10“16“12).
Using the preferred substrate 9, effects of substrate
concentration (entries 1–5), organic solvents (entries
6–12), and buffer pH (entry 13) were examined (Table
1). Contrary to several precedents on enzymatic transg-
lycosylation,24 beneficial effect of organic solvent was
not observed. Preparative scale reaction was performed
at 20 mM concentration (entry 4). Thus, using 50 mg
(94 mmol) of compound 9 incubation with b-galactosi-
dase (430 mU) for 3 h afforded 26% yield (13.1 mg) of
11, the structure of which was rigorously confirmed by
NMR analysis of corresponding acetate 17.25
Since the product 11 should be susceptible to further
digestion by b-galactosidase (to provide 13), its yield
does not necessarily reflect the genuine ratio of
intramolecular transglycosylation and hydrolysis (i.e. k3
versus ka). As a matter of fact, the yield of 11 dropped
to 18% after 4 h incubation (Table 1, entry 5). In order
to minimize the extent of post-transglycosylation
hydrolysis and to gain more precise estimation of the
efficiency of transglycosylation, a-(2“6)-sialyltrans-
ferase (Rat, Recombinant, Spodoptera frugiperda; Cal-
biochem Co., Ltd.)26,27 and CMP–sialic acid were
added to the system to trap 11 as a galactosidase
resistant product 18 (Scheme 4, Figs. 2 and 3). As
expected, smooth formation of sialyl-a-(2“6)-LacNAc
18 (ꢁ) was observed and its amount did not decrease
after prolonged incubation. In a preparative scale run,
substrate 9 was used in 25 mg (47 mmol), that was
incubated in 200 mM HEPES buffer (2.4 ml, pH 7.5) in
the presence of b-galactosidase (100 mU), a-(2“6)-sia-
lyltransferase (60 mU) and CMP–sialic acid (30 mg, 46
mmol). After 17 h, successive purification by Sephadex
LH-20 and preparative HPLC afforded sialyl-a-(2“6)-
LacNAc 18 in 39% yield (15.4 mg).28,29
Figure 1. Time course of b-galactosidase-catalyzed reactions.
(a) Compound 9 (8 mM) was incubated with 130 mU of
b-galactosidase in 700 ml of acetate buffer (50 mM, pH 5.0) at
36°C. (b) Compound 10 (8 mM) was incubated with 390 mU
of b-galactosidase under identical conditions as (a). Aliquots
of the mixture was analyzed by HPLC. Peaks were assigned
as follows based on MALDI-TOF MS analysis. ꢂ Starting
material; (9/10); ꢃ GlcNAc (13/14); ꢀ LacNAc (11/12);
ꢁ Gal2GlcNAc (15/16); × Gal2GlcNAc (regioisomer of 15/
16, structure not confirmed); " Gal3GlcNAc.
OH
OH
O
O
HO
HO
O
HO
HO
O
ka
NHAc
NHAc
HO
O
NO2
OH
NO2
O
HO
HO
OH
9 (ortho)
10 (meta)
13 (ortho)
14 (meta)
k3
k1
k2
OH
O
OH
HO
OH
O
OH
OH
OH
O
kb
O
O
O
O
O
HO
HO
HO
OH
OH
NHAc
NHAc
HO
O
NO2
OH
NO2
O
HO
OH
HO
11 (ortho)
12 (meta)
15 (ortho)
16 (meta)
Scheme 3. Possible pathways leading to transglycosylation products.

8504
S. Komba, Y. Ito / Tetrahedron Letters 42 (2001) 8501–8505
Table 1. Effects of solvent and substrate concentration
Entry
9 (mM)
Enzyme (mU)
Solvent (ml)a
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
8
0.8
0.4
20
20
8
8
8
8
8
86
43
A (0.23)
A (2.3)
A (1.2)
A (4.7)
A (4.7)
B (0.23)
B (0.23)
B (0.23)
C (0.23)
D (0.23)
D (0.23)
D (0.23)
E (0.09)
8
48
144
3
15b
4b
21.5
430
430
130
130
130
130
43
3b
26c
18c
7b
4
24
72
96
24
8
24
72
4.5
14b
14b
2b
9
10
11
12
13
10b
13b
1b
8
8
20
43
43
86
15b
a A 50 mM acetate buffer pH 5.0; B 50% acetonitrile in A; C 60% triethyl phosphate in A; D 20% 2-ethoxyethyl ether in A; E 200 mM HEPES
pH 7.5.
b Based on HPLC relative peak integrations, quantified at 245 nm.
c Isolated yield.
OR
O
RO
RO
OR
O
β-Galactosidase
O
O
RO
OR
NHAc
RO
NO2
Acetate buffer (pH 5.0)
3 h, 26%
R
OH
O
H
11
17
HO
HO
O
Ac
NHAc
O
NO2
OH
O
HO
OH
9 (ortho)
HO
OH
OH
HO
AcHN
COOH
O
CMP-sialic acid
HO
O
O
HO
HO
OH
β-Galactosidase
Sialyl transferase
HEPES (pH 7.5)
17 h, 39%
O
O
O
HO
OH
NHAc
HO
NO2
18
Scheme 4. Synthesis of LacNAc and sialyl LacNAc.
Figure 3. Time course of b-galactosidase/a-sialyltransferase
reaction of 9. Compound 9 (20 mM) was incubated with 86
mU of b-galactosidase and a-(2“6)-sialyltransferase (2.4 mU)
in 95 ml of HEPES (200 mM, pH 7.5) 20 mM MgCl2, 5.3 mM
MnCl2, 20 mM KCl, and CMP–sialic acid (20 mM) at 36°C.
Peaks were assigned as follows based on MALDI-TOF MS
analysis. " Starting material (9); × GlcNAc (13); ꢁ Neu-
AcGalGlcNAc (18); ꢂ Gal2GlcNAc (15).
Figure 2. HPLC profiles of b-galactosidase/a-sialyltransferase
reaction of 9. A: 1.5 h, B: 6.5 h. Retention times of starting
material (9), Gal2GlcNAc (15), NeuAcGalGlcNAc (18) and
GlcNAc (13) are 17.87, 18.34, 22.25 and 22.53 min, respec-
tively.

S. Komba, Y. Ito / Tetrahedron Letters 42 (2001) 8501–8505
8505
In conclusion, a novel method for the preparation of
LacNAc and sialyl LacNAc was developed using b-
galactosidase-mediated intramolecular transglycosyla-
tion.
20. Usui, T.; Kubota, S.; Ohi, H. Carbohydr. Res. 1993, 244,
315–323.
21. For a general discussion on the mechanism of retaining
glycosidases, see: Zechel, D. L.; Withers, S. G. Acc.
Chem. Res. 2000, 33, 11–18.
22. Ivanova, I. A.; Ross, A. J.; Ferguson, M. A. J.; Nikolaev,
A. V. J. Chem. Soc., Perkin Trans. 1 1999, 1743–1753.
23. Kanto chemical Mightysil RP-18 (GP 250-3.0) was used
as the reverse-phase analytical HPLC column. The sol-
vent system used was 100% solvent A (0.1% TFA aq.) for
5 min, then the linear gradient 0–30% of solvent B (90%
CH3CN aq. with 0.1% TFA) for 30 min.
24. (a) Yoon, J. H.; Rhee, J. S. Carbohydr. Res. 2000, 327,
377–383; (b) Usui, T.; Morimoto, S.; Hayakawa, Y.;
Kawaguchi, M.; Murata, T.; Matahira, Y.; Nishida, Y.
Carbohydr. Res. 1996, 285, 29–39; (c) Priya, K.;
Loganathan, D. Tetrahedron 1999, 55, 1119–1128.
25. 1H NMR (400 MHz, CDCl3): l 8.35 (d, 1H, JPh4,Ph5 2.7
Hz, Ph6), 8.23 (dd, 1H, JPh3,Ph4 9.0 Hz, JPh4,Ph6 2.7, Ph4),
5.64 (d, 1H, JNH,2 9.3 Hz, NH), 5.36 (d, 1H, J3,4 3.2 Hz,
H-4Gal), 5.11 (dd, 1H, J1,2 8.1 Hz, J2,3 10.5, H-2Gal),
5.04 (t, 1H, J2,3=J3,4 8.3 Hz, H-3GlcN), 4.98 (dd, 1H,
J2,3 10.5 Hz, J3,4 3.4, H-3Gal), 4.83 (d, 1H, Jgem 13.4 Hz,
PhCH), 4.65 (d, 1H, Jgem 13.4 Hz, PhCH%), 4.55 (dd, 1H,
Jgem 12.2 Hz, J5,6 2.9, H-6Gal), 4.50 (d, 1H, J1,2 7.8 Hz,
H-1Gal), 4.41 (d, 1H, J1,2 7.6 Hz, H-1GlcN), 3.89 (t, 1H,
Acknowledgements
This work was supported by the Special Postdoctoral
Researchers Program at RIKEN. We thank Dr. H.
Koshino and his staff for the NMR measurements, and
Ms. A. Takahashi for technical assistance.
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5,6=J5,6% 7.1 Hz, H-5Gal), 3.81 (t, 1H, J3,4=J4,5 8.5 Hz,
H-4GlcN), 3.59 (m, 1H, H-5GlcN), 2.36, 2.15, 2.14, 2.09,
2.06, 2.05, 2.05, 1.97 (8s, 24H, 7Ac and NAc) ppm.
HRMS (FAB) calcd for C35H44N2O21Na (M+Na+):
851.2334. Found: 851.2403.
26. For the combined use of b-galactosidase and sialyltrans-
ferase, see: Herrmann, G. F.; Ichikawa, Y.; Wandrey, C.;
Gaeta, F. C. A.; Paulson, J. C.; Wong, C. H. Tetrahedron
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28. 1H NMR (500 MHz, D2O): l 8.06 (d, 1H, JPh4,Ph5 3.2 Hz,
Ph6), 8.01 (dd, 1H, JPh3,Ph4 9.2 Hz, JPh4,Ph6 3.2, Ph4), 6.49
(d, 1H, JPh3,Ph4 9.2 Hz, Ph3), 4.75 (d, 1H, Jgem 12.8 Hz,
PhCH), 4.63 (d, 1H, Jgem 13.3 Hz, PhCH%), 4.57 (d, 1H,
J1,2 8.2 Hz, H-1Gal), 4.40 (d, 1H, J1,2 7.8 Hz, H-1GlcN),
2.64 (dd, 1H, J3eq.,4 4.1 Hz, Jgem 11.9, H-3eq. sialic acid),
2.00 and 1.95 (2s, 6H, 2NAc), 1.66 (dd, 1H, J3ax.,4=Jgem
11.9 Hz, H-3ax. sialic acid) ppm; 13C NMR (150 MHz,
D2O, NaOD and MeOH): l 176.4 (CPh2), 175.1 (CNAc
=
O GlcN), 174.8 (CNAc=O sialic acid), 173.7 (C-1 sialic
acid), 133.1 (CPh5), 127.6 (CPh4-H), 127.2 (CPh6-H), 126.5
(CPh1), 119.6 (CPh3-H), 103.8 (C-1 Gal), 100.5 (C-1
GlcN), 100.4 (C-2 sialic acid), 80.2 (C-4 GlcN), 74.8 (C-5
GlcN), 74.0 (C-5 Gal), 73.1 (C-3 Gal), 72.7 (C-3 GlcN),
72.7 (C-6 sialic acid), 71.9 (C-8 sialic acid), 70.8 (C-2
Gal), 68.9 (C-4 Gal), 68.6 (C-7 sialic acid), 68.4 (C-4
sialic acid), 67.0 (CBn-H2), 63.6 (C-6 Gal), 62.8 (C-9 sialic
acid), 60.7 (C-6 GlcN), 55.4 (C-2 GlcN), 52.2 (C-5 sialic
acid), 40.5 (C-3 sialic acid), 22.3 and 22.2 (2CNAc-H3)
ppm.
18. Fang, J. W.; Xie, W. H.; Li, J.; Wang, P. G. Tetrahedron
Lett. 1998, 39, 919–922.
19. Sakai, K.; Katsumi, R.; Ohi, H.; Usui, T.; Ishido, Y. J.
Carbohydr. Chem. 1992, 11, 553–565.
29. Since compound 15 can be a substrate for the sialyltrans-
ferase, degalactosylation of the resultant trisaccharide
might be functional as a minor pathway leading to 18.