Advanced Synthesis & Catalysis
10.1002/adsc.201900189
a) Initial experiments at pH 8
Initially, to compare different PfBAL/RADH (Co)-CatIB
preparations, cascade reactions were performed in an
16.2 min; benzyl alcohol: 210 nm, R
210 nm, R = 18.5 min; p-MBA (internal standard):
270 nm, R = 17.1 min.
For HPLC samples originating from PfBAL activity
assays, DMBA and TMBZ were separated by isocratic
t
= 10.2 min; benzoin:
t
t
[
11b]
identical manner as described by Jäger et al.,
0 mM TEA buffer pH 8.0 supplemented with ThDP,
MgSO and CaCl
as described above. NADP+ was
provided at a concentration of 0.3 mM. In all cases RADH
using
5
4
2
elution (50 vol% B for 20 min). DMBA (R
TMBZ (R = 9.4 min) were detected at 215 nm, p-MBA
(internal standard, R = 6.1 min) was detected at 270 nm.
t
= 7.6 min) and
t
-
1
was employed at a fixed volumetric activity of 0.5 U ml ,
corresponding to 3 µM active RADH (Table1/Table S2).
To directly compare cascade performance between
reactions carried out using single CatIBs, Co-CatIBs, and
the corresponding soluble controls, soluble PfBAL, and
single PfBAL-CatIBs were supplied in amounts to yield
t
Quantification of all substrates and products were achieved
by calibration using respective reference compounds (see
ESI Figure S5).
the same volumetric activity (and therefore the same Acknowledgements
activity concentration) as the Co-CatIBs, where the
PfBAL/RADH ratio cannot be adjusted. The employed
catalyst concentrations are listed in Table 1/Table S2.
We thank Stefanie Longerich (Heinrich-Heine-Universität
Düsseldorf) for her help during the initial phases of the study,
Ramona Kloß for the set-up of the HPLC analysis, and Prof.
Wolfgang Kroutil (University of Graz, Austria) for providing the
radh gene. Furthermore, we thank Ursula Mackfeld
(Forschungszentrum Jülich GmbH, IBG-1) for her excellent help
with the work-up of PPD as well as GC and NMR analyses. This
work was funded by the Bioeconomy Science Center (BioSC),
which is financially supported by the Ministry of Culture and
Science of North-Rhine Westphalia within the framework of the
NRW Strategieprojekt BioSC (No. 313/32 3-400-002 13). BioSC
is a research cluster consisting of the universities RWTH Aachen,
Düsseldorf, and Bonn, and the Forschungszentrum Jülich.
b) pH optimization
To evaluate the influence of the buffer pH on the cascade
performance and enzyme stability, the reactions were
additionally carried out at pH 7.5 and pH 9.0. For the
reactions at pH 7.5 TRIS buffer (50 mM) was used, while
the reactions at pH 9.0 were carried out in TEA buffer
(
50 mM). Both buffers were supplemented with ThDP,
as described above. NADP+ was
MgSO and CaCl
2
4
supplied at a concentration of 0.8 mM. In both cases
.5 U ml-1 RADH (3 µM active enzyme) and 2.8 U ml-1
0
PfBAL (~0.6 µM active enzyme) were employed
Table 1/Table S2).
(
c) Optimized reaction cascade at pH 9.0 with DMSO as a References
co-solvent
A final set of experiments was carried out to compare the
CatIBs and Co-CatIBs cascade performance under
optimized conditions. Reactions were carried out in 50 mM
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V. Gotor, Adv. Synth. Catal. 2009, 351, 1842-1848.
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4 2
TEA buffer supplemented with ThDP, MgSO and CaCl
+
as described above. NADP was provided at a final
concentration of 0.8 mM as described above. Reactions
were carried out in the presence of 2.5 vol% DMSO. As
-
1
before a fixed RADH volumetric activity of 0.5 U ml
3 µM active enzyme) was used (Table 1/Table S2), while
different volumetric PfBAL activities were employed
(
-
1
(
2.5 U ml for single TDoT- and 3HAMP-CatIBs (0.5 µM
[
3] a) D. Kihumbu, T. Stillger, W. Hummel, A. Liese,
Tetrahedron-Asymmetr 2002, 13, 1069-1072; b) J. Kulig,
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Romano, F. Molinari, Adv. Synth. Catal. 2011, 353, 2359-
-
1
active enzyme); and variable U ml for the corresponding
Co-CatIBs; see Table 1/Table S2).
HPLC analysis
All HPLC samples were analyzed using a Thermo
Scientific Dionex Ultimate 3000 HPLC system, equipped
with
a
Diode Array detector DAD-3000 (both:
ThermoFisher Scientific, Waltham, MA, USA) and
Chiralpak® IE column (4,6 µm x 250 mm, 5 µm particle;
pre-column: Chiralpak® IE; 4 mm x 10 mm; both Daicel,
2
362.
[
4] a) J. Wachtmeister, A. Jakoblinnert, D. Rother, Org.
Process Res. Dev. 2016, 20, 1744-1753; b) A. Jakoblinnert,
D. Rother, Green Chem. 2014, 16, 3472-3482; c) J. K.
Kulig, Dissertation thesis, Heinrich-Heine Universität
Düsseldorf 2013.
Tokyo, Japan). A binary mobile phase (A: dd H
acetonitrile; flow rate 1 ml min ) was used for separation,
employing two different protocols as described before.
2
O and B:
-
1
[
11b]
For samples originating from cascade reactions, the
following gradient was used: 15 vol% B for 8 min,
[5] B. M. Choudary, N. S. Chowdari, S. Madhi, M. L. Kantam,
3
5 vol% B for 3 min, 60 vol% B for 3 min, and 15 vol% B
for 3 min. Retention times: PPD: 210 nm, R = 11.2 min;
HPP: 245 nm, R = 15.4 min; benzaldehyde: 245 nm, R
J. Org. Chem 2003, 68, 1736-1746.
t
t
t
=
1
0
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