Enzymatic galactosylation of cello- and chito-oligomers

Article abstract of DOI:10.1080/07328300802030779

Terminal galactosylations of cello- and chito-oligomers are studied employing galactosyltransferase with and without α-lactalbumin. The lactose synthase complex allows just for galactosylation of cellobiose in lower yield but not for higher cello-oligomers. In contrast, the affinity in galactosylation of chito-oligomers increases with higher members to reach the maximum at chitohexaose with only a 30% reduction in transfer rate. In addition to kinetic data, preparative studies with a number of acceptor substrates gave galactosylated oligosaccharides in high yields. (Figure Presented). Copyright Taylor & Francis Group, LLC.

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Enzymatic Galactosylation of Cello‐ and
Chito‐Oligomers
Harald Streicher a , Oliver Scheel a & Joachim Thiem a
a Faculty of Science, Department of Chemistry, University of Hamburg ,
Hamburg, Germany
Published online: 06 May 2008.
To cite this article: Harald Streicher , Oliver Scheel & Joachim Thiem (2008) Enzymatic
Galactosylation of Cello‐ and Chito‐Oligomers, Journal of Carbohydrate Chemistry, 27:1, 41-49, DOI:
10.1080/07328300802030779
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Journal of Carbohydrate Chemistry, 27:41–49, 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300802030779
Enzymatic Galactosylation
of Cello- and Chito-
Oligomers
Harald Streicher, Oliver Scheel, and Joachim Thiem
Faculty of Science, Department of Chemistry, University of Hamburg, Hamburg,
Germany
Terminal galactosylations of cello- and chito-oligomers are studied employing galacto-
syltransferase with and without a-lactalbumin. The lactose synthase complex allows
just for galactosylation of cellobiose in lower yield but not for higher cello-oligomers.
In contrast, the affinity in galactosylation of chito-oligomers increases with higher
members to reach the maximum at chitohexaose with only a 30% reduction in
transfer rate. In addition to kinetic data, preparative studies with a number of
acceptor substrates gave galactosylated oligosaccharides in high yields.
Keywords Enzyme catalysis, Galactosyltransferase, Cello-oligomers, Chito-
oligomers
Received July 12, 2007; accepted January 1, 2008.
Address correspondence to Joachim Thiem, Faculty of Science, Department of
Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, D-20146, Hamburg,
Germany. E-mail: joachim.thiem@chemie.uni-hamburg.de
41

H. Streicher et al.
42
INTRODUCTION
Among some others, galactosyltransferase (GalT, EC 2.4.1.22) is one of the
glycosyltransferases that represents important exceptions from the one-
enzyme-one-linkage hypothesis.[1] Dependent on the presence or absence of
the so-called “specifier protein” a-lactalbumin, it shows two separate
substrate specificities. GalT alone transfers galactose from UDP-Gal onto
N-acetylglucosamine; however, GalT and a-lactalbumin together form
the lactose synthase complex, which in turn transfers galactose from
UDP-Gal onto glucose to give lactose in the mother milk of mammals
(Formula 1).[2–6]
Earlier studies concerning the variability of the acceptor substrates
focused on the use of unnatural substrates to achieve information on kinetics
and perhaps the topology of the active center.[7–15] In following such an
approach, the present study was to check the potential galactosylations of
cello- and chito-oligomers. It can be considered that kinetic data and the
results of preparative transformations should allow for further information
regarding the bioreactions of GalT.
Model compounds for these studies were cello-oligomers 3 obtained by acet-
olysis of cellulose and subsequent deacetylation.[16] Chito-oligomers 4 could be
obtained following an older procedure that could be improved to obtain higher-
molecular-weight derivatives.[17,18]
In the glucose series glucose itself (3a, n ¼ 0) is the genuine natural and
thus an excellent substrate. It was shown previously that cellobiose (3b,
n ¼ 1) could be accepted; however, kinetic data prove it to be a poor substrate.[7]
This is also reflected in the preparative transformation, which results in quite
low yields. Higher cello-oligomers were not hitherto studied, and that is in
focus of this contribution.
In case of N-acetylglucosamine, there were kinetic data for chito-oligomers
up to the tetramer (4, n ¼ 3).[7] The affinities reported increase in going from
GlcNAc to (GlcNAc)2; however, for (GlcNAc)3 and (GlcNAc)4, they decreased
again. A preparative galactosylation of chitobiose (4, n ¼ 1) was reported pre-
viously to give Galß1-4GlcNAcß1-4GlcNAc (6, n ¼ 1) in 18% yield.[19] This
work was further to focus on galactosylation of higher chito-oligosaccharides
employing GalT.
Formula 1: Galactosylation of glucose and N-acetylglucosamine.

Galactosylation of Cello- and Chito-Oligomers
43
RESULTS AND DISCUSSION
Kinetic studies of cello-oligomers made use of the transformations as depicted
in Scheme 1. Employing UDP-Gal (2), galactosylation was catalyzed by galac-
tosyltransferase (GalT) of the substrates 3 or 4, respectively. The resulting
UDP was phosphorylated with pyruvate kinase (PK) and phosphoenol
pyruvate (PEP) to give UTP and pyruvate. Pyruvate was reduced with
NADH and lactate dehydrogenase (LDH) to give (S)-lactate and NADþ. This
well-known approach allows for easy photometric assay of the decreasing
extinction due to the decreasing concentration of NADH at 340 nm.[20]
For the genuine substrate glucose (3a, R ¼ OH, n ¼ 0) and for the unna-
tural substrate cellobiose (3b, R ¼ OH, n ¼ 1), the data from the literature
could be confirmed. Thus, glucose showed a Km of 5 mM (ref. 12, Km ¼ 5 mM)
and cellobiose Km ¼ 826 mM (ref. 7, Km ¼ 833 mM). Tests with higher cello-
oligomers (cellotriose 3c, R ¼ OH, n ¼ 2; cellotetraose 3d, R ¼ OH, n ¼ 3;
cellopentaose 3e, R ¼ OH, n ¼ 4) gave no products and thus evidence that
none of these could function as substrates for GalT. Thus, the affinity from
the mono- to the disaccharide decreases drastically, and higher gluco-oligomers
of the ß,1-4-linked type are not recognized by the enzyme.
Correspondingly chito-oligomers (4a–g, R ¼ NHAc, n ¼ 0–6) were studied
(Sch. 1) in 0.1 M aqueous solutions, but due to low solubility, the higher chito-oli-
gomers had to be measured in 0.02 M aqueous solutions. Whereas in such cases
the Km values may be compared, the vmax values cannot.[20] Thus, both the
pentamer (4e, n ¼ 3) and the hexamer (4f, n ¼ 5) were measured at two concen-
trations, and this allowed for correlation of the data for the heptamer (4g, n ¼ 6).
Scheme 1: Galactosyltransferase (GalT)-catalyzed galactosylation of cello- and chito-
oligomers. PK, pyruvate kinase; LDH, lactate dehydrogenase.

H. Streicher et al.
44
As depicted in Figure 1 (cf. also Table 1), the Km values decrease steadily in
going form GlcNAc (4a, n ¼ 0: 3.6 mM) to chitohexaose (4f, n ¼ 5) and chito-
heptaose (4 g, n ¼ 6), reaching the limiting value of Km ¼ 0.2 mM and thus
the highest affinity. Figure 1 (cf. Table 1) shows the relative rate of the galac-
tose transfer on to the chito-oligomers. Apparently, there is some decrease in
going from GlcNAc (4a, n ¼ 0) to chitotetraose (4d, n ¼ 3) of about 70%, after
which the rate remains constant. Overall, the affinity in going to higher
chito-oligomers increased drastically, whereas there is a less substantial
effect on the transfer rate.
For preparative use direct application of UDP-Gal (2) is too costly, and thus
cheaper UDP-Glc (1) is employed and transformed in situ into 2 with UDP-
glucose-4-epimerase. As observed in the kinetic studies, chitotriose (4c) can
be used in 20 mM concentration; however, for the higher chito-oligomers this
would result in substrate saturation, and thus chitotetraose (4d) and chitopen-
taose (4e) were employed in 5 mM concentrations. The literature data for for-
mation of the trisaccharide galactosylchitobiose (6b) was 60%,[21] and here
we observed a slight increase in the formation of the tetrasaccharide galacto-
sylchitotriose (6c) to 63% yield after 3 d of incubation and workup. Alternative
syntheses of 6b from chitobiose (4b) as well as of 6c from chitotriose (4c) and
p-nitrophenyl ß-D-galactopyranoside by transgalactosylation employing ß-
[22]
galactosidase (B. circulans) in 10%
and 27%,[23] respectively, were
Figure 1: Michaelis constants (Km, A) and relative rates (vrel,O) of chitooligomer acceptor
substrates in GalT-catalyzed galactosylation.

Galactosylation of Cello- and Chito-Oligomers
45
Table 1: Kinetic data of (GlcNAc)n acceptor substrates in galactosylation with GalT.
Chito-oligomer
substrates
vmax
Km [mM]
(mmol min21
)
vrel (%)
GlcNAc 4a
3.6
2.5
1.2
0.7
0.6
0.2
0.2
7.5
6.5
6.3
5.3
5.0
5.2
5.3
100
87
84
71
67
69
71
(GlcNAc)2 4b
(GlcNAc)3 4c
(GlcNAc)4 4d
(GlcNAc)5 4e
(GlcNAc)6 4f
(GlcNAc)7 4g
reported. For galactosylations of chitotetraose (4d) and chitopentaose (4e), a
considerably improved transfer and yield was obtained after 2 d of incubation
and workup to give the pentasaccharide galactosylchitotetraose (6d) in 81%
yield and the hexasaccharide galactosylchitopentaose (6e) in 98% yield. As
obvious from these data, the slight decrease in reaction rate is of little influence
regarding a preparative approach. In particular, generally a high transfer
could be observed for oligomers larger than the tetrasaccharide.
SUMMARY
These findings underline the anticipated biological function of galactosyltrans-
ferase. In mammals and the presence of a-lactalbumin, the lactose synthase
complex is formed and suited to accept and galactosylate glucose as the only
substrate to give lactose (5b). A minor deviation allows for the acceptance of
the unnatural substrate cellobiose (3b), which is still galactosylated in 23%
yield to give the trisaccharide galactosylcellobiose (5b)[19]; however, higher
cello-oligomers (3c, 3d, 3e) did not show any transfer.
In contrast, the GalT-catalyzed galactosylation of
a
terminal
N-acetylglucosamine unit did not require any copeptide and improved drasti-
cally with the increasing oligosaccharide chain as indicated by the improving
affinity, and this again reflects in the preparative yields. It would be interesting
to look into the acceptor properties of pure malto-oligomers and terminally
N-acetylglucosaminylated oligosaccharides resembling chito-oligomers, and
studies in this direction are in progress.
EXPERIMENTAL
General Remarks
Commercially available starting materials were used without further puri-
fication. Solvents were dried according to standard methods. Purifications of

H. Streicher et al.
46
the products were carried out by column chromatography using Biogel P2 or
P4. The enzymatic reactions were incubated in a Thermomixer Comfort
(Merck) at 600 rpm. The nuclear magnetic resonance spectra were recorded
on Bruker AM-300 or AMX-400. All chemical shifts are quoted in ppm down-
field from TMS or referred to the characteristic signal of HDO in D2O
(4.63 ppm). Mass spectra were recorded on Bruker MALDI-Tof Biflex III.
GalT from bovine milk (EC 2.4.1.22), UDP-galactose-4-epimerase (EC
5.1.3.2), PK (EC 2.7.1.40), LDH (EC 1.1.1.27), UDP-Glc, and UDP-Gal were
from Sigma.
Kinetic Data
The maximum reaction rate (vmax) and the Michaelis constant (Km) were
determined for the acceptor substrates cello-oligomers 3a–3e (R ¼ OH,
n ¼ 0–4) and chito-oligomers 4a–4g (R ¼ NHAc, n ¼ 0–6). In a total volume
of 1 mL the Tris-HCl buffer (100 mM, pH 7.5) containing MgCl2 (10 mM),
MnCl2 (10 mM), and KCl (37 mM) and NADH (30 mM), PEP (100 mM), UDP-
Gal (20 mM), and a-lactalbumin (0.1 mg/mL) were incubated with PK (10U),
LDH (10U), GalT (0.1U), and the Glcn acceptor substrates (3a–3e) in 0.1
aqueous solution. In case of the GlcNAc acceptor substrates (4a–4g),
a-lactalbumin was omitted. The decreasing extinction was followed for 5 to
10 min at 340 nm and the linear part of the graph analyzed employing the
Lineweaver-Burk and also the Hanes approach.
General Preparation (GP)
The acceptor substrate and UDP-Glc (1) were dissolved in degassed cacody-
late buffer (100 mM, pH 7.5) containing MnCl2 (5 mM). UDP-glucose-4-epimer-
ase and galactosyltransferase were added and the mixture incubated at 308C.
Every 24 h the pH was adjusted to 7.5; the progress was checked by the UDP
assay. After termination proteins were removed by centrifugation and the
residue chromatographed on Dowex 2 ꢀ 8 (Cl2) ion exchange resin. Elution
with doubly distilled water (250 mL) left charged material on the column and
gave the products. After concentration, purification was by gel chromatography
(Biogel P2 and P4) and final freeze drying.
b-D-Galactopyranosyl-(1-4)-ß-D-glucopyranosyl-(1-4)-D-glucose (5b)
Following the GP cellobiose (3b, 69 mg, 200 mmol, 20 mM) was mixed with
UDP-Glc (1, 131 mg, 200 mmol, 20 mM), a-lactalbumin (5 mg), UDP-Glc-4-epi-
merase (2U, twice), and Gal T (1U, twice) in buffer (10 mL) and incubated for
5 d; the second enzyme charge was added on day 3. Workup and purification
1
on Biogel P2 gave 5b (23 mg, 23%) as colorless amorphous material. H NMR

Galactosylation of Cello- and Chito-Oligomers
47
(D2O): d ¼ 5.23 (d, J1,2 ¼ 3.9 Hz, 1H, H-1a), 4.67 (d, J1,2 ¼ 7.9 Hz, 1H, H-1ß),
4.55 (d, J1 ,2 ¼ 7.9 Hz, 1H, H-10, 4.46 (d, J1 ,2 ¼ 7.9 Hz, 1H, H-100).
A corresponding preparative size approach for galactosylation of cellotriose
(3c, 100 mg, 198 mmol, 20 mM) did not give any compound 5c.
0
0
00 00
ß-D-Galactopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-
acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-D-
glucose (6c)
Following the GP tri-N-acetylchitotriose (4c, 60 mg, 96 mmol, 20 mM) was
mixed with UDP-Glc (1, 63 mg, 96 mmol, 20 mM), and UDP-Glc-4-epimerase
(4U) and GalT (2U) and were incubated in buffer (4.8 mL) for 3 d. Workup
and purification on Biogel P2 gave tetrasaccharide 6c (48 mg, 63%) as colorless
amorphous material; [a]D20: –1088 (c 0.08, H2O) [Lit.:[8] –1148 (c 0.1, H2O)]; 1H
NMR (D2O): d ¼ 5.21 (d, J1,2 ¼ 2.6 Hz, 1H, H-1a), 4.71 (d, J1,2 ¼ 8.3 Hz, 1H,
H-1ß), 4.62 (each d, J1 ,2 ¼ J1 ,2 ¼ 7.8 Hz, 2H, H-10 and H-100), 4.84
0
0
00 00
000;,2000
000,4000
000,5000
(d, J1
¼ 7.8 Hz, 1H, H-1000;), 3.95 (dd, J3
¼ 3.3 Hz, J4
¼ 1.1 Hz, 1H,
H-4000), 3.60 (dd, J1
¼ 7.8 Hz, J2
¼ 9.9 Hz, 1H, H-2000), 2.08 (each s,
000;,2000
000,30000
6H, CH3), 2.06 (s, 3H, CH3). C30H51N3O21: 789.30; FAB-MS m/z 790
[M þ H]þ, 812 [M þ Na]þ.
ß-D-Galactopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-
acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-
glucopyranosyl-(1-4)-2-acetamido-2-deoxy-glucose (6d)
Following the GP tetra-N-acetylchitotetraose (4d, 100 mg, 120 mmol,
5 mM) was mixed with UDP-Glc (1, 79 mg, 120 mmol, 5 mM), UDP-Glc-4-epi-
merase (4U), and GalT (2U) and incubated in buffer (24 mL) for 2 d. Workup
and purification on Biogel P4 gave the pentasaccharide 6d (97 mg, 81%) as a
white amorphous solid; C38H64N4O26: 992.95; FAB-MS m/z 994 [M þ H]þ,
1016 [M þ Na]þ, 1032 [M þ K]þ.
ß-D-Galactopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-
acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-
glucopyranosyl-(1-4)-2-acetamido-2-deoxy-ß-D-glucopyranosyl-(1-4)-2-
acetamido-2-deoxy-ß-D-glucose (6e)
Following the GP penta-N-acetylchitopentaose (4d, 100 mg, 97 mmol, 5 mM)
was mixed with UDP-Glc (1, 64 mg, 97 mmol, 5 mM), UDP-Glc-4-eimerase (4U),
and GalT (2U) and incubated in buffer (19.4 mL) for 2 d. Workup and purification
on Biogel P2 gave the hexasaccharide 6e (114 mg, 98%) as a colorless amorphous
material; C46H77N5O31: 1195.45; FAB-MS m/z 1218 [M þ Na]þ.

H. Streicher et al.
48
ACKNOWLEDGMENTS
Support of this work by the Deutsche Forschungsgemeinschaft and the Fonds
der Chemischen Industrie is gratefully acknowledged.
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