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doi.org/10.1002/ejoc.202100174
library of enzymes, the general reaction protocol was as follows:
Determination of the molar absorbivity coefficients of
compounds 1, 2and 4
50 mM glucose; 0.2 UmLÀ 1 GDH; 0.4 mM NAD(P)+; 3.4 UmLÀ 1 HSDH;
10 mM substrate; 5% v/v DMSO; 50 mM potassium phosphate
buffer, pH 7.0 (total volume: 1 mL). Reactions catalyzed by Hh7α-
HSDH and Hh7β-HSDH were performed both in the presence and
The molar extinction coefficients of ketone 1, cis-alcohol 2 and cis-
acetate 4 were determined at 254 nm. For each compound, a
10 mM solution in CH3CN was prepared and it was used as a
mother solution to test the absorption of each molecule in 1 mL
quartz cuvettes in the concentration range of 0.1-25 μM in CH3CN
(where linearity of data was observed). ɛ254 (1): 6508 MÀ 1 cmÀ 1; ɛ254
(2): 5889 MÀ 1 cmÀ 1; ɛ254 (4): 2620 MÀ 1 cmÀ 1 (for more details see
Paragraph 1, Supporting information).
°
in the absence of 0.4 M NaCl. The mixtures were shaken at 25 C
and 100 rpm for 24 to 48 h and monitored over time via TLC
(eluent CH2Cl2 :AcOEt=9:1). Reaction conversions and enantiomer-
ic excesses were evaluated by GC-MS and chiral HPLC analyses. The
isolation of pure products was obtained via extraction with AcOEt
of the reaction mixtures and subsequent flash chromatography on
silica gel 60 (70–320 mesh, Merck, eluent CH2Cl2/AcOEt mixtures).
The absolute configurations of the residual substrate 1b, if any, and
of the products 2a and 3b were assigned via optical rotation
measurements and subsequent confrontation with values reported
in literature.[24–27]
Characterization of commercially available racemic 1
1H NMR (400 MHz, CDCl3) δ 5.87 (d, J=1.8 Hz, 1H), 2.84–2.65 (m,
2H), 2.61–2.39 (m, 4H), 2.30–2.04 (m, 3H), 1.72 (qt, J=13.3, 4.4 Hz,
1H), 1.46 (s, 3H). ESI-MS: [M+Na]+: 201.0.
Enzymatic reduction of 1 catalyzed by IS2-SDR
Synthetic chemistry
Following the aforementioned general protocol on a 100 mg scale
(10 mM substrate, 56 mL reaction volume) and stopping the
reaction at 58% conversion, the following products were isolated
Preparation of standard racemate 2, (�)-4a,5-cis-(5-hydroxy-
4a-methyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one)[18]
after flash chromatography (eluent: CH2Cl2 :AcOEt=9:1). 1b: ee:
22
96.7%; [α]D : À 97.0 (c: 1.0 in CHCl3);[47] isolated yield: 33 mg (32%);
°
The reduction of substrate 1 was performed following a standard
protocol with NaBH4. To a stirred solution of 0.4 M substrate (1 eq,
100 mg) in EtOH (5 mL) at 0 C, an ethanolic suspension (0.4 M) of
2a: ee: 90.7%; isolated yield: 30 mg, (29%). NMR and ms spectra
are in accordance with the proposed structures (see Supplemen-
tary).
°
NaBH4 (1 eq, 13 mg) was added dropwise. After 4 hours, the
reaction was quenched with a saturated aqueous solution of NH4Cl,
then it was extracted with AcOEt (3x). The organic phase was dried
over Na2SO4, filtered and the solvent was evaporated under
reduced pressure to yield a crude extract. A flash chromatography
purification afforded the desired products (2a+2b) in up to 90%
yield. The racemic product 2 was characterized by chiral HPLC (see
analytical methods) and 1H-NMR analyses. 1H-NMR (400 MHz, CDCl3)
δ 5.78 (d, J=1.8 Hz, 1H), 3.42 (dd, J=11.6, 4.3 Hz, 1H), 2.50–2.28 (m,
3H), 2.25–2.13 (m, 2H), 1.94–1.78 (m, 3H), 1.76–1.64 (ddd, J=16.9,
13.3, 4.2 Hz, 1H), 1.48–1.33 (qt, J=13.2, 4.0 Hz, 1H), 1.19 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 206.66, 199.56, 168.43, 125.50, 78.28, 41.63,
34.27, 33.72, 32.03, 30.31, 23.20, 15.27. ESI-MS: [M+Na]+: 203.0.
Enzymatic reductions of 1 and 1b catalyzed by Dm7α-HSDH
Following the general protocol on a 50 mg scale (10 mM substrate,
28 mL reaction volume) and bringing the reaction to full conversion
from racemic 1, the following products were isolated via flash
chromatography on a Biotage SP1 working with 50 mg of crude
mixture and a 10 g silica gel Biotage® cartridge in a gradient of
AcOEt in DCM (10 CV 5% AcOEt; 5 CV from 5% to 15% AcOEt,
10 CV 15% AcOEt).
2a: 20 mg, ee: 95.3%, isolated yield: 40.0%. NMR and ms spectra in
accordance with the proposed structure (see Supplementary).
3b: 25 mg, ee: >99%, isolated yield: 50.0%.1H NMR (400 MHz,
CDCl3) δ 5.87 (br s, 1H), 3.65 (br s, 1H), 2.74–2.35 (m, 4H), 2.35–2.23
(m, 1H), 2.14–1.98 (m, 1H), 1.96–1.66 (m, 3H), 1.57–1.46 (m, 1H), 1.24
Preparation of standard racemate 4, (�)-8a,1-cis-(-8a-methyl-
6-oxo-1,2,3,4,6,7,8,8a-octahydronaphthalen-1-yl acetate)[46]
(s, 3H); 13C NMR (101 MHz, CDCl3) δ 199.41, 167.68, 127.03, 75.33,
The acetylation of racemate substrate 2 was performed according
to a standard acetylation protocol with acetyl chloride. To a stirred
solution of 0.1 M substrate (1 eq, 50 mg) in CH2Cl2 (2.8 mL), pyridine
(1.2 eq, 27 μL) and acetyl chloride (1.2 eq, 24 μL) were added. The
reaction was stirred overnight at room temperature. Then, CH2Cl2
was evaporated and the crude extract was resuspended in
saturated aqueous NaHCO3, then extracted (3×) with AcOEt. The
organic phase was dried over Na2SO4, filtered and the solvent was
evaporated under reduced pressure to give the desired products
22
40.93, 34.02, 31.73, 30.80, 28.71, 21.83, 19.83; ee: >99%; [α]D
:
À 111.6 (c: 1.0 in CHCl3);[28] ESI-MS: [M+Na]+: 203.0.[19]
°
Using the enantiomer 1b as a substrate (30 mg scale), the trans-
alcohol 3b was obtained as sole product following, again, the
general protocol. 3b: yield: 91%; ee: >99%.
Enzymatic acetylation of substrate 2b catalyzed by Amano
lipase PS
1
(4a+4b). H NMR (400 MHz, CDCl3) δ 5.81 (d, J=1.8 Hz, 1H), 4.65
(dd, J=11.8, 4.2 Hz, 1H), 2.44–2.30 (m, 3H), 2.30–2.21 (m, 1H), 2.08
(s, J, 3H), 1.98–1.66 (m, 5H), 1.55–1.41 (m, 1H), 1.27 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 198.82, 170.32, 166.67, 125.80, 79.17, 40.39,
33.98, 33.47, 31.76, 26.87, 22.95, 21.07, 16.64. ESI-MS: [M+H]+:
223.1. ESI-MS: [M+Na]+: 245.0.
Racemic substrate 2 (1040 mg, 1 eq) was dissolved in dry acetone
(80 mM substrate, 70 mL reaction volume). Then, excess vinyl
acetate (10% v/v, 14 eq) and Amano lipase PS on celite (1000 mg,
1% wt, catalytic amount) were added. The reaction was shaken at
°
45 C, 200 rpm for six days. The enzyme was filtered away and the
solvent was evaporated to afford a crude mixture of 2a and 4b.
The two products were separated via flash chromatography (CH2Cl2:
Enzymatic reduction of racemic ketone 1 with HSDHs/SDRs
AcOEt gradient from 9:1 to 7:3) to give the subsequent products:
22
General protocol for the enzymatic reduction: the reactions
catalyzed by HSDHs were coupled with a glucose/GDH system to
regenerate NAD(P)H. For the initial activity screening of the whole
°
2a: 660 mg, 50% isolated yield, ee: 97.3%; [α]D : +173.4 (c: 1.0 in
CHCl3).[33] 4b: 407 mg, 39% isolated yield, ee: >99%; [α]D : À 69.5
22
°
(c: 1.0 in CHCl3).[33]
Eur. J. Org. Chem. 2021, 1–8
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