10.1002/adsc.202000859
Advanced Synthesis & Catalysis
freshly by standard methods. Column chromatography was
performed on Roth silica gel (0.040–0.063 mesh);
analytical thin layer chromatography was performed on
Merck silica gel plates 60 F254 using anisaldehyde staining
or N-(1-naphthyl)-ethylenediamine for detection. All Rf
values are reported for the TLC solvent mixture
and then directly measured by plate reader (SpectraMAX
190; Molecular Devices, Software: SoftMax Pro 6.5.1) at
574 nm. Experimental data were fitted to the Michaelis–
Menten equation by nonlinear regression using Origin
software (v9.1G, OriginLab) to obtain kinetic parameters.
Protein concentration was measured by BCA assay
n-BuOH:acetone:H2O:AcOH
=
35:35:23:7. Organic
(bicinchoninic
acid
assay,
Sigma-Aldrich).
All
solvents were removed under vacuum using a rotary
evaporator, and residues were dissolved in H2O and
lyophilized. NMR spectra were recorded on Bruker
DRX500 spectrometer; chemical shifts are referenced to
HOD, = 4.79 ppm. For quantitative NMR measurements
an accurate manual phase correction and a baseline
correction with Whittaker Smoother method were
performed using MestReNova 11.0.3. High-resolution
mass data were determined on a Bruker Impact II ESI-Q-
TOF spectrometer.
measurements were done in triplicate.
Sialidase analysis. The assay mixture (200 µL) contained
horseradish peroxidase (> 15 U), glucose oxidase from
Aspergillus niger (> 0.5 U), -galactosidase from
Aspergillus oryzae (> 0.6 U) (all enzymes from Sigma-
Aldrich), flavin adenine dinucleotide (0.1 mM), 4-
aminoantipyrine (2.1 mM), vanillic acid (2.1 mM) and the
respective substrate (4.0 mM) in sodium acetate buffer
(50 mM, 5.0 mM CaCl2) at pH 5.5. Reactions were
supplemented by a limiting quantity of neuraminidase
(from Clostridium perfringens, > 2 mU, Sigma-Aldrich; or
from Vibrio cholerae, > 10 mU, New England Biolabs).
All assays were conducted as triplicates in 96-well plates at
35 °C and started by addition of a double-concentrated
substrate solution (for competitive assays containing both
substrates at double concentration).
General procedure for Barbier-type reactions. Aldehyde 6
(1 eq.) was dissolved in H2O : THF = 1 : 1 (v/v; 5 mL per
50 mg). Indium powder (2 eq., 200 mesh) and a catalytic
amount of InCl3 were added with vigorous stirring. The
reaction was started by addition of a THF solution
containing the respective allyl bromide (3 eq.), and the
mixture was placed in an ultrasonic bath at RT. When TLC
showed complete conversion, the reaction mixture was
neutralized with 1 M NaOH, filtered through celite and the
solvents were removed under vacuum. The residue was
purified by silica column chromatography.
Acknowledgements
This work was supported by funds from the State of Hessia.
General deprotection procedure for anomeric methoxy
group. Methyl glycosides were dissolved in H2O. Formic
acid was added and the reaction mixture heated to 80 °C
for 4-5 h. Conversion was checked by NMR analysis,
because deprotection was difficult to follow by TLC, and
the procedure repeated if necessary.
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kresol red (2 μL, 10 mg L-1, 0.026 mM), substrate
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9
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