3
4
C. Kamerke et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 27–34
phosphate, trisaccharide phosphate). In addition the sensitivity
is greatly enhanced, so the UDP-tetrasaccharide could clearly be
identified migrating in front of the UDP-trisaccharide. It is likely,
that the separation selectivity changes are due to the change of
the buffer system and the coupling to a sheath liquid interface
means a high degree of freedom to combine CE-devices to mass
spectrometers.
We conclude that enzyme activation and deactivation by MWI
during in the course of the enzymatic reaction could be exploited
for the synthesis of other condensation products using hydrolases.
Work is in progress to exploit this strategy for the synthesis of a
range of glycoconjugates with glycosidases in our group.
[
28], clearly visible for the presence of multiple signals for iso-
baric compounds: UDP-disaccharide derived from UDP-Glc gives
rise to a partially separated triple peak (Fig. 7B), indicating three
different isomers. Those are expected to be Gal(1–4)Glc␣1-UDP,
Gal(1–6)Glc␣1-UDP and also Gal(1–3)Glc␣1-UDP, as products
with UDP-Glc as acceptor substrate. Multiple signals are also vis-
ible for the higher homologues, though the resolution of isobaric
regioisomers is lower due to shorter migration times.
Also for the N-acetylated counterparts, multiple signals are
present: UDP-disaccharide derived from UDP-GlcNAc and the
corresponding trisaccharide show double signals in CE-MS
Acknowledgements
C.K. and L.E. acknowledge financial support by the DFG within
the International Research Training Group 1628 “Selectivity in
Chemo- and Biocatalysis - SeleCa” and by the excellence initiative
of the German federal and state governments through ERS@RWTH
Aachen University. We thank Wolfgang Gloy (Institute of Biotech-
nology, RWTH Aachen University) for assistance in HPAEC-PAD
analysis. M.P. and C.H. acknowledge financial support by Helmholtz
Initiative and Networking Fund.
(
Fig. 8B), indicating two regioisomers. These can be ascribed to
Gal(1–4)GlcNAc␣UDP (UDP-LacNAc) and to a lower extent for the
Gal(1–6)GlcNAc␣UDP isomer corresponding to the specificity of
the enzyme derived from hydrolysis experiments (see supporting
information on Section 1.2). The 1–3 regioisomer is not formed,
probably because of sterical hindrance through the N-acetyl group
of UDP-GlcNAc.
The regioselectivity for 1–4- but not 1–3-glycosidic link-
ages with UDP-GlcNAc as acceptor is consistent with our previous
results [8] where we analysed the regioisomers of the UDP-
oligosaccharides synthesised with commercial -galactosidase
from B. circulans extensively by NMR. Here, by direct mass anal-
ysis out of the reaction mixture and the analysis of the enzyme’s
regioselectivity by specific hydrolysis of disaccharides with defined
glycosidic bonds the product structure can easily be proofed avoid-
ing laborious isolation of single substances.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2012.04.003.
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