C. Chen et al.
Molecular Catalysis 510 (2021) 111670
activity of CpKR (Fig. S2) was determined at 35 ◦C in the following
buffers (100 mM) with different pH: sodium citrate (pH 4.0–6.0), KPB
(pH 6.0–8.0) and Tris–HCl (pH 8.0–9.0). The optimum reaction tem-
perature was determined in KPB (100 mM, pH 6.5) at different tem-
peratures in the range of 20–45 ◦C (Fig. S3). The thermostability of CpKR
(Table S2) was measured by incubating 3.8 mg mLꢀ 1 purified enzyme at
different temperatures (25 ◦C, 30 ◦C, 35 ◦C, 40 ◦C) for proper time fol-
lowed by measuring the residual activity.
2.10. Asymmetric reduction of 3-oxo ester 1a by CpKR
The substrate 1a (1.45 g, 5.97 mmol) dissolved in 3 mL of DMSO
(5%, v/v) was added into the 57 mL potassium phosphate buffer (100
mM, pH 6.0) containing 0.2 mM NADP+, 75 g Lꢀ 1 (3 kU Lꢀ 1) of
lyophilized crude CpKR, 1.25 g of glucose (1.2 equiv.) and 7.5 g Lꢀ 1 (3
kU Lꢀ 1) of lyophilized crude GDH. The pH of the reaction was monitored
and maintained around pH 6.0 by auto-titration of 1 M K2CO3. When the
reaction was completed, the supernatant of reaction mixture was
extracted twice with equal volume of ethyl acetate. The collected
organic phase was washed twice with saturated NaCl solution, dried
over anhydrous Na2SO4 and then evaporated under reduced pressure to
obtain (3S)ꢀ 2a (1.38 g, 94.4% isolated yield). 1H NMR (400 MHz,
CDCl3): δ 7.35–7.30 (m, 2H), 6.94–6.89 (m, 2H), 5.00 (d, J = 8.0 Hz,
1H), 4.36 (d, J = 8.0 Hz, 1H), 3.81 (d, J = 1.3 Hz, 6H); 13C NMR (100
MHz, CDCl3): δ 169.57, 160.08, 130.95, 128.30, 114.11, 75.13, 59.26,
55.43, 53.26.
2.6. Substrate scope
In order to explore the potential synthetic applications of the
enzyme, the specific activities of CpKR towards different keto substrates
were determined using the standard assay method as described above.
More details about enzyme specific activities were presented in
Table S3. The kinetic parameters of some characteristic substrates were
also measured by changing the substrates concentration (0ꢀ 20 mM) in
the presence of 0.2 mM NADPH. The stereoselectivity of CpKR towards
these ketones and ketoesters were measured through reactions, which
were composed of 10 mM compound, 1 U Lꢀ 1 purified CpKR, 2 U Lꢀ 1
lyophilized crude GDH, 15 mM glucose, 0.5 mM NADP+ and 1 mL 100
mM KPB (pH 6.0). After shaking for 12 h at 30 ◦C, 200 µL reaction
mixture was withdrawn and extracted with equal volume of ethyl ace-
tate. The enantiomeric excess (ee) of products was determined by GC or
HPLC (Table S3). The analysis methods for different substrates were also
shown in Table S4. The HPLC and GC chromatograms of substrates were
given in the Supplementary Material.
2.11. Synthesis of (2R,3S)-MPGM
The methanol solution (20 mL) containing reduction products (3S)ꢀ
2a (1.38 g, 5.63 mmol) was added to a 50 mL round bottom flask under
nitrogen atmosphere, and then 6 mL of a methanol solution containing
sodium methoxide (0.32 g, 5.9 mmol) was added dropwise at 0 ◦C. After
reacting at 0 ◦C for 2 h, the reaction was quenched with saturated NH4Cl
[11]. The reaction mixture was extracted with methyl tert-butyl ether.
The extracts were then concentrated in vacuum, giving crude (2R,
3S)-MPGM as a yellow oil. The oily product was purified by silica gel
chromatography (hexanes/EtOAc = 10: 1) to provide pure (2R,
2.7. Structural models of CpKR
3S)-MPGM (0.95 g, 81% isolated yield, 99% ee). [α D25
]
= ꢀ 199∘ (c 1.0,
MeOH) [literature: [α D26
]
= ꢀ 196∘ (c 1.0, MeOH)][10]. 1H NMR (400
The structural model of CpKR was implemented with Modeller 9.17
based on the structure of a ketoreductase (PDB code: 5YWN, with a
sequence identity of 48%). The docking results of the substrates (1a, 1u,
1s) and enzyme were shown in Fig. S4. The active sites, NADPH, and
substrates were shown as salmon sticks, gray sticks, and cyan sticks.
MHz, CDCl3): δ 7.23–7.19 (m, 2H), 6.91–6.87 (m, 2H), 4.05 (d, J = 1.7
Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.51 (d, J = 1.8 Hz, 1H); 13C NMR
(100 MHz, CDCl3): δ 168.96, 160.41, 127.34, 126.85, 114.27, 58.08,
56.68, 55.46, 52.69.
2.8. Synthesis of methyl 3-(4-methoxyphenyl)ꢀ 3-oxopropanoate 1 w
2.12. Synthesis of diastereomeric mixture of 2a by NaBH4
A solution of 4-methoxyacetophenone (3 g, 20 mmol) in toluene (50
mL) was added dropwise to a mixture of sodium hydride (1.44 g, 60
mmol) and dimethyl carbonate (4.53 g, 50 mmol) in toluene (100 mL) at
120 ◦C during 2 h. The reaction mixture was then stirred at 120 ◦C for
another 1 h, quenched with ice cooled saturated NH4Cl, adjusted pH to
neutral. The toluene layer was separated and the aqueous layer was
extracted with ethyl acetate [10]. The combined organics were dried
with anhydrous Na2SO4, concentrated in vacuum, and purified by silica
gel chromatography (hexane/EtOAc = 10:1), affording 1w as yellow
liquid (3.6 g, 87% isolated yield).
The mixture of sodium borohydride (378 mg, 10 mmol) and (S)-
proline (1243 mg, 11 mmol) dissolved in THF (10 mL) was heated to
reflux under nitrogen for
2
h. The methyl 2‑chloro-3-(4-
methoxyphenyl)ꢀ 3-oxopropanoate 1a (535 mg, 2.2 mmol) dissolved in
THF (5 mL) was added at 0 ◦C. The reaction mixture was stirred at 0 ◦C
for 20 h, then 10% aqueous HCl was added to adjust the pH to neutral
[11]. The reaction mixture was concentrated in vacuum, diluted with
water, and extracted with ethyl acetate. After concentrating the extracts,
racemic 2a (214 mg, 40% isolated yield) was obtained as light yellow
liquid. 1H NMR (400 MHz, CDCl3): δ 3.66 (s, 2H), 3.84–3.78 (m, 4H),
4.42 (d, J = 6.7 Hz, 1H), 5.07 (d, J = 6.7 Hz, 1H), 6.93–6.85 (m, 2H),
7.34–7.27 (m, 2H). HPLC condition for racemic 2a: Daicel Chiralcel
OJ-H column, hexane/2-propanol = 70: 30, 0.5 mL/min at 254 nm and
35 ◦C. The retention times of (2R,3R)ꢀ 2a, (2S,3S)ꢀ 2a, (2R,3S)ꢀ 2a and
(2S,3R)ꢀ 2a were the same as described above.
2.9. Synthesis of methyl 2-chloro-3-(4-methoxyphenyl)ꢀ 3-
oxopropanoate 1a
A mixture of methyl 3-(4-methoxyphenyl)ꢀ 3-oxopropanoate 1w (3
g, 14.4 mmol), anhydrous CH2Cl2 (50 mL) was stirred for 15 min at
25 ◦C and then slowly added with CH2Cl2 (50 mL) containing sulfuryl
chloride (2 g, 14.8 mmol) at 1 h. The reaction was heated to reflux for 2
h. The mixture was then allowed to cool down and the solvent was
evaporated [10]. The obtained oily substance was purified by silica gel
chromatography (hexanes/EtOAc=10: 1), affording 1a as light yellow
liquid (3 g, 87.3% isolated yield). 1H NMR (400 MHz, CDCl3): δ 3.82 (s,
3H), 3.89 (s, 3H), 5.60 (s, 1H), 6.99 – 6.95 (m, 2H), 8.01–7.96 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 186.80, 166.14, 164.71, 131.95, 126.23,
114.35, 57.76, 55.77, 53.90. HPLC condition for the 1a: Daicel Chiralcel
OJ-H column, hexane/2-propanol = 70: 30, 0.5 mL/min at 254 nm and
35 ◦C, RT = 33.1 min.
3. Results and discussion
3.1. Enzyme screening
Considering that both two stereochemical isomers of (3S)-alcohols
can be transformed in a convergent mode into the required (2R,3S)-
MPGM [11,12], we only needed to evaluate the exact stereo-specificity
of the tested enzymes toward 3-oxo group of the substrate 1a by
calculating the molar ratio of (3S)ꢀ 2a, regardless of the stereochemical
configuration of (2R)- or (2S)-chlorine isomers. We firstly tested some
alcohol dehydrogenases preserved in our homemade ketoreductase kits
3