10.1002/ejoc.201901682
European Journal of Organic Chemistry
FULL PAPER
General procedure for one-pot reaction. 2 mL mixture solution (25% v/v
acetonitrile in water) containing 25 mM substrate and 5 mM SAS, was
incubated under aerobic conditions at r.t. and exposed to the light of 8 W
blue LED irradiation for 12 hours (the reaction time could be extended to
ensure no residual alcohols). 20 mL cell culture of ketoreductase
(resuspended in 50 mM sodium phosphate buffer, pH 6.5) with glucose
(250 mM) was added into the photocatalytic mixture and shaken at 30 °C
for overnight. The stereoselectivity and yield was determined by chiral GC
or HPLC.
99 % ee
88 % ee
99 % ee
(R)-3g
(R)-3h
(S)-3i
99(50)% yield
99 % ee
52(12)% yield
91 % ee
n.d.
Scaling-up one-pot reaction catalyzed by photocatalyst and
ketoreductase. The scale-up reaction was performed as follows: SAS (0.2
mmol, 20 mol%) and 1 mmol 1a was dissolved in 40 mL mixture solution
of acetonitrile and water (25% v/v acetonitrile as cosolvent). The reaction
mixture was irradiated with blue LEDs for 20 hours (the reaction time could
be extended to ensure no residual alcohols). 400 mL whole-cell culture of
ketoreductase (resuspended in 50 mM sodium phosphate buffer, pH 6.5)
with glucose (10 mmol) was added into the photocatalytic reaction mixture
and shaken at 30 oC for overnight. The reaction solution was extracted with
ethyl acetate for three times, then the organic phase was dried over
anhydrous sodium sulfate and concentrated in vacuum. The obtained
crude product was further separated and purified by flash column
chromatography.
(S)-3j
n.d.
[a]
The obtained photocatalyst reaction system was added to YtbE whole cell
[b]
culture.
Yields and ee values referred to the YtbE-catalyzed reduction
reactions, which were determined by chiral GC or HPLC, and the total yields of
photo-enzymatic cascade reactions were given in parentheses.
assignment was changed in accordance with the Cahn-Ingold-Prelog priority
rules.
[c]
The R/S
Conclusions
In summary, we have reported an enantiocomplementary C-H
hydroxylation process in a one-pot cascade reaction combining
photocatalysis
and
biocatalysis.
A
series
of
enantiocomplementary chiral alcohols are successfully achieved
from readily available aromatic compounds through simple
process with two types of KREDs in high yields (up to 90%) and
stereoselectivity (up to 99%). This photochemo-enzymatic one-
pot whole-cell C-H hydroxylation reaction is conducted in water
and using O2 as oxidant, and these advantages will contribute to
the synthesis of chiral alcohols, which are highly valuable
intermediates for the production of pharmaceutical drugs and
bioactive compounds.
Acknowledgments
The financial support from National Natural Science Foundation
of China (91956128) and Zhejiang Provincial Natural Science
Foundation (LY19B020014) is gratefully acknowledged.
Keywords: whole-cell biocatalysis • chiral alcohols • C-H bond
hydroxylation • one-pot synthesis • photocatalysis
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Experimental Section
General information. Unless otherwise noted, all reagents were obtained
commercially and used without further purification. The H and 13C NMR
1
spectra were recorded with a Bruker AMX400 MHz spectrometer using
TMS as an internal standard in CDCl3. Yields and stereoselectivity were
determined by chiral GC with Agilent CP-chirasil-Dex CB column or chiral
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Preparation of ketoreductases (KtCR and YtbE).100 μL stored bacteria
was first incubated in 5 mL LB media with Kanamycin (50 μg/mL) and then
shaken at 37 oC for overnight. 5 mL preculture was added to 500 mL fresh
LB medium with 50 μg/mL Kanamycin. The cultures were shaken at 37°C
until OD600 at 0.6 and then cooled at 4°C for 30 min. Isopropyl β-
thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM
to induce ketoreductases expression at 25 oC. Cells were harvested by
centrifugation, and resuspended in 50 mM sodium phosphate buffer ( pH
6.5 for KtCR and YtbE) for reaction.
Preparation of racemic alcohols. The given ketone (2 mmol) was added
into a solution of NaBH4 (5 mmol) in methanol (10 mL) at room
temperature (20oC). The solution was stirred until the complete
disappearance of ketone substrate indicated by TLC. The crude product
was evaporated in vacuo and diluted in dichloromethane (20 mL), and then
washed with water (10 mL). The organic phase was separated and dried
over anhydrous magnesium sulfate, and then evaporated in vacuum.
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