FULL PAPERS
Module Systems for the Synthesis of Uridine 5’-Diphospho-a-d-glucuronic Acid
Synthesis of Compound 4, GlcA(b1-3)Gal(b1-
)GlcNAc(b1-linker-t-Boc, by the SuSy/UGDH/
GlcAT-Enzyme Module System
Acknowledgements
4
The authors acknowledge financial support by the Federal
Ministery for Education and Research (BMBF) within the
program “Basistechnologien” for the project “The Golgi
Glycan Factory” (FKZ031A162). The authors acknowledge
support by the DWI Leibniz-Institut für Interaktive Materia-
lien e.V. , RWTH Aachen University, Germany, for carrying
The synthesis of product 4 (1 mL/5 mL batch) was per-
+
formed in buffer A with 0.5 mM NAD , 0.2 mM UDP,
3
00 mM sucrose, 5 mM acceptor substrate 3, 5 mM MnCl2,
0
.25 U/mL SuSy1, 0.5 U/mL His UGDH, 0.5/1.0 U/mL
6
His NOX and 0.5/1.5 U/mL His catGlcAT-P for 24 h at
1
6
6
out the H NMR measurement and thank M.Sc. Ruben R.
308C. The 5 mL batch was performed in a 25-mL flask
Rosencrantz for performing NMR analysis. Furthermore the
authors thank M.Sc. Vera Werthmann for characterization
work on GlcAT-P and the DECHEMA Max Buchner Foun-
dation for financial support (L. Engels). We thank Prof. Dr.
Vladimír K rˇ en (Institute of Microbiology, Academy of Scien-
ces of the Czech Republic Center of Biocatalysis and Bio-
transformation) for providing the starting substrate GlcNAc-
linker-t-Boc.
under gentle (20 rpm) stirring. At different time points ali-
quots were analyzed with RP-HPLC. Experimental condi-
[15a]
tions for product isolation are described by Rech et al.
Product 4 was obtained with an overall yield of 48% (9 mg,
1
1
2 mmol) and characterized by ESI-MS and H NMR spec-
trometry.
Control Experiment of the Cleavage of the Acceptor
Substrate Gal(b1-4)GlcNAc(b1-linker-t-Boc with RP-
HPLC Analysis
References
The experiment was carried out by incubating 0.5 U
His UGDH, 0.5 U His NOX, 0.5 U His catGlcAT-P and
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6
6
6
0
5
.5 U SuSy1 with 2 mM Gal(b1-4)GlcNAc(b1-linker-t-Boc in
0 mM Tris-HCl (pH 7.8) for 24 h at 308C, respectively. A
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Determination of b-Galactosidase Side Activity in the
Purified Protein Pool of Recombinant His UGDH
6
For quantification of the b-galactosidase side activity in the
purified protein pool of recombinant His UGDH 10 mL
6
His UGDH were added to 90 mL of 4 mM pNPG in 50 mM
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6
citrate-Na HPO buffer (pH 6.0) for 24 h at 308C. Samples
2
4
were taken at different time points and reaction was stopped
by the addition of 200 mL 0.2M Na CO . The released pNP
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2
3
was determined with a spectrophotometer at 405 nm using
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enzyme which produces 1 mmol pNP per min under standard
assay conditions. As negative control 10 mL of Milli Q H O
2
instead of the enzyme was added to the assay solution.
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Mass Spectrometry
To confirm the integrity of products 2 and 4 they were ana-
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ESI ionization, probe temperature: 4008C; needle voltage:
[
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4.5 kV; cone voltage: 100 V. 10 mL of a 0.1 mM sample solu-
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tion were concentrated on a Multospher 120 RP 18 HP-3 m
À1
(
602 mm), and applied at a flow rate of 0.2 mLmin using
50% acetonitrile in water as a mobile phase.
[
NMR Spectroscopy
1
H NMR spectra were measured on a Bruker AV 400 Ultra
2
002, 131, 337–347.
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Shield spectrometer in D O (400 MHz; 308C). Chemical
2
[
[
shifts are given in the d scale and coupling constants in Hz.
2
Adv. Synth. Catal. 2015, 357, 1751 – 1762
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1761