10.1002/adsc.202000120
Advanced Synthesis & Catalysis
dehydrogenase (1-20 µL) to the following assay mixtures
(1 mL final volume):
4a+4b: 1H NMR (400 MHz, CDCl3) δ 7.24 – 7.02 (m, 4H),
4.30 – 3.98 (m, 1H), 3.12 (dd, J = 15.9, 4.5 Hz, 1H), 3.07 –
3.06 (m, 1H), 2.99 (dt, J = 17.0, 5.8 Hz, 1H), 2.92 – 2.84
(m, 1H), 2.80 (dd, J = 16.3, 7.9 Hz, 1H), 2.15 – 2.04 (m,
1H), 1.92 – 1.79 (m, 1H).
HSDH assay: 2.5 mM substrate; 50 mM potassium
phosphate buffer at pH 9.0; 0.20 mM NAD(P)+.
FDH assay: 20 mM substrate; 50 mM potassium phosphate
buffer at pH 7.0; 0.20 mM NAD(P)+.
5a1+5a2: 1H NMR (400 MHz, CDCl3) δ 3.76 (q, J = 2.5
Hz, 1H), 1.92 – 1.74 (m, 2H), 1.74 – 1.55 (m, 4H), 1.56 –
1.43 (m, 3H), 1.44 – 1.16 (m, 5H), 1.15 – 0.85 (m, 3H).
One unit of activity is defined as the enzyme activity that
reduces 1 µmol of NAD(P)+ per min under the assay
conditions described above.
HSDHs optimal temperatures were determined by heating
the assay solution in cuvettes in a water bath in the range
20–90 °C for 10 min before adding the enzyme. Results
were compared to blanks.
1
5b1+5b2: H NMR (400 MHz, CDCl3) δ 3.20 (ddd, J =
10.2, 9.1, 4.3 Hz, 1H), 2.18 – 2.07 (m, J = 6.1, 3.0 Hz, 1H),
2.04 – 1.91 (m, 1H), 1.87 – 1.59 (m, 4H), 1.61 – 1.17 (m,
7H), 1.12 – 0.81 (m, 5H).
Small-scale biotransformations of bile acids
Enzymatic reduction of substrates 1-5 catalyzed by
HSDHs
Oxidation of cholic Acid to 3α,12α-dihydroxy-7-oxo-5β-
cholanoic acid or to 3α,7α-dihydroxy-12-oxo-5β-cholanoic
acid. Cholic acid oxidation reactions were coupled with a
pyruvate/LDH system to regenerate NAD+. Specifically,
reactions were carried out in a 1 mL solution containing 50
mM potassium phosphate buffer, pH 8.0, 10 mM cholic
acid, 50 mM sodium pyruvate, 0.4 mM NAD+, 0.5 U LDH
from rabbit muscle, 2 U of opportune 7α-HSDH (entry 11-
13, Table S1, Supporting Information) to obtain 3α,12α-
dihydroxy-7-oxo-5β-cholanoic acid (Scheme S1, A) or
Ls12α-HSDH to obtain 3α,7α-dihydroxy-12-oxo-5β-
cholanoic acid (Scheme S1, B) at 25°C for 24 h.
Reactions catalyzed by HSDHs were coupled with a
formate/FDH system to regenerate NAD(P)H. For the
initial activity screening the general reaction protocol was
as follows: 76 mM NH4HCO2; 0.2 U mL-1 FDH; 0.4 mM
NAD(P)+; HSDH, 3.4 U mL-1; 12.5 mM substrate; 5% v/v
DMSO; 50 mM potassium phosphate buffer, pH 7.0 (total
volume: 1 mL). Reaction catalyzed by Hh7α-HSDH and
Hh7β-HSDH were performed in the presence and in the
absence of 0.4M NaCl. The mixtures were shaken at 25°C
and 100 rpm for 24 to 48 h and monitored over time via
TLC (eluent CH2Cl2). Reaction conversions and
enantiomeric excesses were evaluated by GC-MS, chiral
GC or chiral HPLC analyses. The absolute configurations
were assigned via analytical comparison with standards
(products 1a and 1b) or via optical rotation measurements
and confrontation with values reported in literature
(products 2a and 2b, (+)-5 and (-)-5, 5b1 and 5b2).[49,50]
Reaction progress was monitored by TLC using
chloroform-methanol-acetic acid, 10:1:0.08, as eluting
system.
Reduction of 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid to
ursocholic acid. 3α,12α-diHydroxy-7-oxo-5β-cholanoic
acid reduction was coupled with a formate/FDH system to
regenerate NADH. Specifically, reactions were performed
in a 1 mL solution containing 50 mM potassium phosphate
buffer, pH 8.0, 10 mM cholic acid, 50 mM NH4HCO2, 0.4
mM NADH, 0.5 U FDH from Candida boidinii, 2 U of
opportune 7β-HSDH (entry 26-29, Table S1, Supporting
Information) (Scheme S1, C) at 25°C for 24 h.
Reaction progress was monitored by TLC using the same
eluting system described for cholic acid oxidation
(chloroform-methanol-acetic acid, 10:1:0.08), as eluting
system.
Reactions on substrates 2 and 5 catalyzed by Cae7β-HSDH
were subsequently scaled up to semi-preparative scale (100
mg, 0.7 mmol in 50 mL total volume) following the
protocol described above. The isolated products were
1
characterized via H- and 13C-NMR, ESI-MS, chiral GC
25
and [α]D (see Supporting information, and analytical
methods).
2a+2b: 1H NMR (400 MHz, DMSO) δ 5.05 (d, J = 5.9 Hz,
1H), 4.04 – 3.89 (m, 2H), 3.64 (t, J = 5.4 Hz, 1H), 1.83 –
1.70 (m, 1H), 1.05 (t, J = 7.1 Hz, 3H), 0.74 (d, J = 6.9 Hz,
3H), 0.69 (d, J = 6.8 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 174.90, 77.35, 77.04, 76.72, 74.99, 61.48, 41.00,
32.14, 18.74, 15.97, 14.23; [M+Na]+: 167.1; conversion:
99.9%; isolated yield: 63.2%; ee (chiral GC): 97.1%;
[α]D22: -2.70°.
Preparation of standard racemate mixtures
The reductions of substrates 1-5 were performed following
a standard protocol of reduction with NaBH4.[56] To a
stirred solution of 0.13 M substrate (1 eq, 100 mg) in
MeOH (5 mL) at 0°C, NaBH4 (1 eq, 13 mg) was added.
When the reaction mixture became clear, it was brought to
rt and stirred for 2-4 h. The reaction was quenched with a
saturated aqueous solution of NH4Cl, then it was extracted
with EtOAc (3x). The organic phase was dried over
Na2SO4, filtrated and the solvent was evaporated under
reduced pressure to yield the desired products (1a+1b,
2a+2b, 3a+3b, 4a+4b, 5a1+5a2+5b1+5b2) in quantitative
yields. The products were characterized via 1H-NMR
analysis and chiral GC or HPLC analyses (see Supporting
information, and analytical methods).
5: 1H NMR (400 MHz, CDCl3) δ 2.41 – 2.25 (m, J = 13.5,
5.0, 1.8 Hz, 2H), 2.10 – 2.00 (m, J = 16.3, 5.9, 2.8 Hz, 1H),
2.00 – 1.85 (m, 2H), 1.85 – 1.59 (m, 5H), 1.49 – 1.30 (m,
2H), 1.30 – 1.09 (m, 4H); 13C NMR (101 MHz, CDCl3) δ
212.72, 77.35, 77.03, 76.71, 55.08, 44.96, 41.80, 34.38,
33.04, 26.48, 25.75, 25.43, 25.10; [M+Na]+: 175.1; ee
(chiral GC): 62.2%; [α]D25: +2,75°.
1
5b1+5b2: H NMR (400 MHz, CDCl3) δ 3.29 – 3.11 (m,
1H), 2.23 – 2.06 (m, J = 6.0, 3.0 Hz, 1H), 2.02 – 1.91 (m,
1H), 1.84 – 1.58 (m, 4H), 1.58 – 1.43 (m, 2H), 1.41 – 1.13
1a+1b: 1H NMR (400 MHz, CDCl3) δ 7.49 – 7.30 (m, 1H), (m, 4H), 1.11 – 0.77 (m, 5H); 13C NMR (101 MHz,
5.20 (s, 1H), 3.79 (s, 1H).
CDCl3) δ 77.33, 77.01, 76.70, 75.04, 50.46, 41.13, 35.82,
33.59, 33.43, 29.00, 26.34, 26.15, 24.03; [M+Na]+: 177.1;
conversion (GC-MS): 35.8%; isolated yield: 15%; ee
(chiral GC): 89.1%; [α]D25: +44.45°.
2a+2b: 1H NMR (400 MHz, CDCl3) δ 4.42 – 4.15 (m, 1H),
4.04 (s, 1H), 2.72 (s, 1H), 2.19 – 2.00 (m, J = 13.7, 6.9, 3.5
Hz, 1H), 1.33 (t, J = 7.1 Hz, 1H), 1.05 (d, J = 6.9 Hz, 1H),
0.89 (d, J = 6.9 Hz, 1H).
Acknowledgements
3a+3b: 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 1H),
7.26 – 7.18 (m, 2H), 7.15 – 7.08 (m, 1H), 4.88 – 4.72 (m,
1H), 3.04 – 2.52 (m, 2H), 2.21 – 1.74 (m, 4H).
We would like to kindly acknowledge Prof. Vladimir I. Tishkov
and Dr. Anastasia A. Pometun (M.V. Lomonosov Moscow State
University) for their support in the preparation of the
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