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R. Tsunekawa et al. / Molecular Catalysis 444 (2018) 84–89
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Table 2
2.11. Benzyl
Reduction of 2b with carbonyl reductases from Chiralscreen® OH.a
(1S,2S)-2-(4-methylbenzenesulfonyl)oxycyclohexanecarboxylate
(5)
enzyme No.
conv. (%)
isomeric ratio of 1b
1R,2S
1S,2R
1S,2S
1R,2R
To a solution of (1S,2S)-1b (162 mg, 0.691 mmol) in anhydrous
pyridine (2.3 mL) was added p-toluenesulfonyl chloride (663 mg,
3.48 mmol) and DMAP (9.0 mg, 73.6 mol). The mixture was stirred
for 4 h at 70 ◦C. The reaction was quenched by the addition of 2 M
hydrochloric acid and the organic materials were extracted with
AcOEt. The organic layer was washed with brine, dried over anhy-
drous Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (20.0 g). Elution with hex-
ane/AcOEt (30:1) furnished (1S,2S)-5 (233 mg, 86.8%) as yellow oil.
1H NMR (400 MHz, CDCl3): ı 1.17–1.39 (m, 2H), 1.47–1.64 (m, 3H),
1.73–1.76 (m, 1H), 1.93–1.96 (m, 1H), 2.16–2.21 (m, 1H), 2.42 (s,
3H), 2.60 (ddd, J = 3.7, 9.8, 10.0 Hz, 1H), 4.80 (ddd, J = 4.3, 9.8, 9.8 Hz,
1H), 4.89 (d, J = 12.3 Hz, 1H), 4.97 (d, J = 12.3 Hz, 1H), 7.25-7.29 (m,
5H), 7.31–7.37 (m, 2H), 7.74 (d, J = 8.1 Hz, 2H); 13C NMR (125 M Hz,
CDCl3): ı 21.5, 23.3, 23.6, 28.0, 31.7, 48.6, 66.3, 81.3, 127.7 (x2),
127.9 (x2), 128.1, 128.4 (x2), 129.5 (x2), 134.2, 135.6, 144.3 172.4;
IR: 663, 744, 806, 875, 917, 973, 1018, 1099, 1174, 1249, 1355, 1461,
E007
E031
E078
58.2
94.3
54.1
37.2
97.0
96.9
62.8
3.0
3.1
0.0
0.0
0.0
0.0
0.0
0.0
a
Determined by 1H NMR and HPLC analyses. For detail, see Section 2.10.
uct was recovered in a conventional manner. The residue was
purified by silica gel column chromatography (5.0 g). Elution with
hexane/AcOEt (10:1) furnished syn-1b (65.5 mg, 34.7%) and anti-
1b (56.0 mg, 29.7%) as colorless oil. For (1R*,2S*)-1b (syn-isomer):
1H NMR (400 MHz, CDCl3): ı 1.24–1.37 (m, 1H), 1.39–1.51 (m,
2H), 1.63-1.78 (m, 3H), 1.83–1.97 (m, 2H), 2.54 (ddd, J = 2.7,
3.9, 11.2 Hz, 1H), 4.16–4.18 (m, 1H), 5.15 (s, 2H), 7.30–7.39 (m,
5H). HPLC [column, Daicel CHIRALPAK® ID, 0.46 cm x 25 cm;
hexane/i-PrOH = 90:10, flow rate 0.5 mL/min, detected at 220 nm],
tR (min) = 19.2, 20.5. For (1R*,2R*)-1b (anti-isomer): 1H NMR
(400 MHz, CDCl3): ı 1.15-1.43 (m, 4H), 1.69-1.78 (m, 2H), 2.00-
2.09 (m, 2H), 2.32 (ddd, J = 3.7, 10.0, 12.3 Hz, 1H), 3.79 (ddd, J = 4.5,
10.0, 10.0 Hz, 1H), 5.15 (d, J = 12.6 Hz, 1H), 5.18 (d, J = 12.6 Hz,
1H), 7.30–7.39 (m, 5H). HPLC analysis in the same conditions for
syn-isomer, tR (min) = 25.1, 32.4. The relationships between the
retention time in HPLC analysis of each peak and stereoisomer of 1b
was assigned as follows, taking the results in Sections 2.2, 2.6 (ethyl
ester), 2.9, and 2.12 (benzyl ester) into account; 19.2 for (1R,2S)-1b,
20.5 for (1S,2R)-1b, 25.1 for (1S,2S)-1b, 32.4 for (1R,2R)-1b.
1598, 1735, 2360, 2865, 2940 cm−1
.
2.12. Benzyl (1S,2R)-2-hydroxycyclohexanecarboxylate (1b)
To a solution of (1S,2S)-5 (108 mg, 0.278 mmol) in toluene
(0.5 mL) was added tetrabutylammonium nitrite (429 mg,
1.49 mmol). The mixture was stirred for 48 h at 60 ◦C. The reaction
was quenched by the addition of brine and the organic materials
were extracted with AcOEt. The organic layer was washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(10.0 g). Elution with hexane/AcOEt (10:1) furnished a yellow
oil. Further purification with preparative TLC (developed with
hexane/AcOEt = 5:1) yielded pure (1S,2R)-1b (32.5 mg, 49.9%) as
a yellow oil. [␣]D22.1–15.8 (c 1.00, CHCl3). Its 1H NMR spectrum
was identical with that for authentic sample of syn-1b. 13C NMR
(100 M Hz, CDCl3): ı 20.0, 23.8, 24.7, 31.7, 46.7, 66.3, 66.6, 128.0
(x2), 128.3, 128.6 (x2), 135.7, 175.6; IR: 734, 973, 1027, 1126, 1162,
1249, 1450, 1720, 2362, 2937, 3455 cm−1; HRMS (ESI +): m/z calcd.
for C14H18O3Na [(M + Na) + ] 257.1154, found 257.1189. HPLC
analysis under the same conditions in Section 2.8, tR (min) = 20.5
[(1S,2R)-1b, >99.9%].
2.9. Benzyl (1S,2S)-2-hydroxycyclohexanecarboxylate (1b)
The harvested wet cells of W. californica (38.7 g) were re-
suspended with phosphate buffer solution (pH 6.5, 0.1 M, 200 mL).
To the mixture was added a solution of 2b (516 mg, 2.22 mmol) in
DMSO (4.0 mL) and glucose (2.0 g) and the resulting mixture was
vigorously stirred for 19 h at 30 ◦C. After similar workup with the
reduction of 2a, the residue was purified by silica gel column chro-
matography (50.0 g). Elution with hexane/AcOEt (10:1) furnished
(1S,2S)-1b (249 mg, 47.9%). [␣]D
+37.9 (c 1.01, CHCl3). Its 1H
21.8
NMR spectrum was identical with that for authentic sample of anti-
1b. 13C NMR (100 M Hz, CDCl3): ı 24.3, 24.9, 28.1, 33.6, 51.3, 66.3,
70.9, 127.9 (x2), 128.2, 128.6 (x2), 135.8, 175.0; IR: 742, 1012, 1060,
1261, 1448, 1716, 2358, 2858, 2939, 3399 cm−1; HRMS (ESI +): m/z
calcd. for C14H18O3Na [(M + Na)+] 257.1154, found 257.1195. HPLC
analysis under the same conditions in Section 2.6, tR (min) = 24.5
[(1S,2S)-1b, >99.9%].
3. Results and discussion
First, by incubating 2a with ten strains of whole-cell yeasts (Sec-
tion 2.3) as shown in Scheme 1, Torulaspora delbruekii NBRC 10921,
Williopsis californica JCM 3600, and Candida floricola JCM 9439 were
chosen based on the progress of reduction by TLC analysis.
The ratio of stereoisomers was determined by combining
1H NMR analysis of the crude product and HPLC analysis after
derivation to the corresponding benzoate 3a, and the results are
summarized in Table 1. In contrast to the other two strains, W. cali-
fornica furnished anti-isomers as the major product, comparable to
that reported for C. gloeosporoides [22].
We became interested in W. californica, because the com-
bination of chemical transformation and the reduction product
would furnish (1S,2R)-1a as shown in Scheme 1. Then the reduc-
tion at millimolar scale was attempted with W. californica several
times. Although it always exhibited high ee for the anti-isomer
form, the isolated yield of the products and the ratio between
diastereomers often fluctuated. Moreover, when attempting stere-
ochemical inversion at C-2 via the corresponding tosylate 3b,
(1S,2R)-1a was obtained at up to 75% yield, but the reproducibility
2.10. Treatment of 2b with carbonyl reductases in the primary kit
of Chiralscreen® OH
According to the reported procedure [30], oxoester 2b (56.6 mg)
was dissolved in 2-propanol (285 L) and each 50 L of the solution
was added to each reaction vessel of pre-adjusted reaction mixture
of Chiralscreen® OH (E001, E007, E031, E039 and E078) involved
in the primary kit. The mixture was stirred at 30 ◦C for 24 h. Then,
AcOEt was added to each vial and stirred very well. The progress
of the reduction was roughly estimated by a TLC analysis: Rf for
syn-1b: 0.37; anti-1b: 0.27 (developed with hexane/AcOEt = 4:1).
Except for the batch to which E001 and E039 were applied (no
progress), the product was analyzed by HPLC as in Section 2.8,
without any purification. The results were summarized in Table 2.