Rhodococcus rhodochrous IFO 15564-mediated hydrolysis of alicyclic nitriles and amides: Stereoselectivity and use for kinetic resolution and asymmetrization

Article abstract of DOI:10.1016/S0957-4166(98)00084-6

The stereocourse and the selectivity of the hydrolysis of alicyclic mono- and dinitriles and amides mediated by Rhodococcus rhodochrous IFO · 15564 has been examined. The stereochemistry of the substrates, as well as the nature of substituents and presence of double bonds in alicyclic rings greatly affected the rate of hydrolysis by nitrile hydratase and amidase. The rate difference between enantiomers or enantiotopic groups, in some cases, enabled kinetic resolution or asymmetrization. The highest enantioselectivity of amidase was observed in the hydrolysis of 5-benzoyloxy-3-cyclohexene-1- carboxamide (E>200), and both enantiomers of methyl 5-hydroxy-3-cyclohexene- 1-carboxylate thus became readily available.

Full text of DOI:10.1016/S0957-4166(98)00084-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1097–1102
Rhodococcus rhodochrous IFO 15564-mediated hydrolysis of
alicyclic nitriles and amides: stereoselectivity and use for kinetic
resolution and asymmetrization
Kaori Matoishi, Akio Sano, Nori Imai, Takahiro Yamazaki, Masahiro Yokoyama,
Takeshi Sugai ∗ and Hiromichi Ohta
Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan
Received 29 January 1998; accepted 23 February 1998
Abstract
The stereocourse and the selectivity of the hydrolysis of alicyclic mono- and dinitriles and amides mediated
by Rhodococcus rhodochrous IFO 15564 has been examined. The stereochemistry of the substrates, as well as the
nature of substituents and presence of double bonds in alicyclic rings greatly affected the rate of hydrolysis by nitrile
hydratase and amidase. The rate difference between enantiomers or enantiotopic groups, in some cases, enabled
kinetic resolution or asymmetrization. The highest enantioselectivity of amidase was observed in the hydrolysis of
5-benzoyloxy-3-cyclohexene-1-carboxamide (E>200), and both enantiomers of methyl 5-hydroxy-3-cyclohexene-
1-carboxylate thus became readily available. © 1998 Elsevier Science Ltd. All rights reserved.
The importance of nitriles in synthetic organic chemistry prompted the elaboration of functional group
transformation under mild conditions, i.e., biocatalytic hydrolysis.1 Many attempts have been devoted to
clarify the regio- and stereoselectivity for aromatic substrates, however, few reports of alicyclic substrates
have appeared so far.2 We became interested in the hydrolysis of alicyclic mono- and dinitriles and
amides mediated by Rhodococcus rhodochrous IFO 15564,3 and the stereocourse and the selectivity on
the substrates was examined.
Table 1 summarizes our results and some comments on them are described below. Firstly, the detection
of amides clearly supports that the hydrolysis by this microorganism proceeds in two steps; the hydration
by nitrile hydratase and the subsequent hydrolysis of the intermediate by amidase. With regard to the
first step, the hydration of cyano groups in the substrates 1 (entry 1), 9 (entry 5), and 14 (entry 7) with a
C_C double bond was faster than the corresponding saturated ones 3 (entry 2), 12 (entry 6), 17 (entry 9),
respectively, and these results suggested a certain interaction between double bonds and nitrile hydratase.
This effect, however, became smaller, between 5 (entry 3) and 7 (entry 4), and between 19 (entry 10) and
24 (entry 15).
∗
Corresponding author. Fax: +81 45 563 5967; e-mail: sugai@chem.keio.ac.jp
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00084-6

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Table 1.

K. Matoishi et al. / Tetrahedron: Asymmetry 9 (1998) 1097–1102
1099
Incubation of meso-dinitrile 19 (entry 10) afforded enantiomerically enriched amide-carboxylate 20
and meso-dicarboxylate 21. Starting from meso-diamide 22 (entry 12), which was revealed as the
intermediate by a shorter-period incubation (entry 11), the same result was obtained. From these results,
the hydrolysis of the amide worked in an enantioselective manner. This was further supported by an
independent experiment of the kinetic resolution of (ꢀ)-20 (entry 13). In contrast, as the incubation
of (ꢀ)-23 (entry 14), a potential precursor of (ꢀ)-20 afforded almost the same result compared with
that from (ꢀ)-20, nitrile hydratase had nearly no enantioselectivity. Indeed, nitrile hydratase of this
microorganism showed rather low enantioselectivity to the series of the substrates in this study (E=1.0 to
6.7).4
Both an unsaturated (19, entry 10) and a saturated (24, entry 15) dinitrile were hydrolyzed in a
similar manner. Thus, in contrast to nitrile hydratase, the rate of hydrolysis by amidase was greatly
affected by the nature and position of substituents on alicyclic rings, rather than the double bonds. The
carbamoyl and carboxyl group placed next to (α) the amide group, which is susceptible to hydrolysis, in
a counter-clockwise orientation in meso-22 and (1R,6S)-20, retards the hydrolysis of the amide group
by amidase (Scheme 1). The result brought about the accumulation of (1R,6S)-20 and meso-21. By

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K. Matoishi et al. / Tetrahedron: Asymmetry 9 (1998) 1097–1102
protecting carboxylic acids as methoxycarbonyl groups, such an effect was more strongly expressed
and the hydrolysis with amidase did not proceed (5 and 7, in entry 3 and 4).
Scheme 1.
We then became interested in the effect of the substituent at the β-position. Smaller substituents such
as hydroxyl groups (30, entry 17) showed no effect. To our surprise, an esterification with a benzoyl group
(32, entry 18) dramatically changed the situation. The introduction of a bulky and lipophilic group8 at
the β-position in a clockwise orientation was found to have a retarding effect as in Scheme 2. In this
case, nitrile hydratase showed a slight preference for the (1R,5R)-enantiomer (E 1.6), while amidase had
a reverse selectivity:9 (1S,5S)-33 was hydrolyzed with a high selectivity (E 30). Figure 1 summarizes
the rate-decreasing substituents on the cyclohex(a/e)ne ring to amidase, which should elucidate the
stereostructure of the active site of amidase (cf. Deigner et al.).10
Scheme 2.
When the substrate concentration of 32 was raised to 0.5% (entry 18 versus 19), the reaction became
slow and the recovery of the starting material (1S,5S)-32 (33% e.e.) as well as (1S,5S)-acid 34 with an
enhanced e.e. (97%) was achieved. This result is explained if an enantioselective inhibition caused by
the final products brought about an enhancement of the apparent E value (>200) of the amidase. By
taking advantage of the high enantioselectivity, a preparative method for the both enantiomers of methyl
Fig. 1.

K. Matoishi et al. / Tetrahedron: Asymmetry 9 (1998) 1097–1102
1101
5-hydroxy-3-cyclohexene-1-carboxylate 3511 was developed as in Scheme 3.12,13 Alkaline treatment of
(1R,5R)-33 and (1S,5S)-34 led to deprotection of the benzoyloxy group, and (1R,5R)-30 and (1S,5S)-
35 (as free carboxylic acid) were produced respectively. Due to a beneficial loss of enantioselectivity
by substituting the benzoyloxy group for the hydroxyl group, the hydrolysis of amide in (1R,5R)-30
proceeded very smoothly under mild conditions (entry 17). In this manner, the enantiomer (1R,5R)-35
could be obtained in a highly enantiomerically enriched state (97% e.e.).
Scheme 3.
References
1. Review: Crosby, J.; Moilliet, J.; Parratt, J. S.; Turner, N. J. J. Chem. Soc., Perkin Trans 1 1994, 1679–1687. Sugai, T.;
Yamazaki, T.; Yokoyama, M.; Ohta, H. Biosci. Biotech. Biochem. 1997, 61, 1419–1427. Recent examples: Hayashi, T.;
Yamamoto, K.; Matsuo, A.; Otsubo, K.; Muramatsu, S.; Matsuda, A.; Komatsu, K. J. Ferment. Bioeng. 1997, 83, 139–145.
Eichhorn, E.; Roduit, J.-P.; Shaw, N.; Heinzmann, K.; Kiener, A. Tetrahedron: Asymmetry 1997, 8, 2533–2536. Meth-Cohn,
O.; Wang, M.-X. Chem. Commun. 1997, 1041–1042.
2. Nishise, H.; Kurihara, M.; Tani, Y. Agric. Biol. Chem. 1987, 51, 2613–2616. Tani, Y.; Kurihara, M.; Nishise, H.; Yamamoto,
K. Agric. Biol. Chem. 1989, 53, 3143–3149. Yamamoto, K.; Ueno, Y.; Otsubo, K.; Yamane, H.; Komatsu, K.; Tani, Y. J.
Ferment Bioeng. 1992, 73, 125–129. Robins, K.; Gilligan, T. Eur. Pat. Appl. EP 502,525 (CA. 118: 37548r); Zimmermann,
T.; Robins, K.; Birch, O. M.; Boehlen, E. Eur. Pat. Appl. EP 524,604 (CA. 118: 211426m).
3. Sugai, T.; Yokoyama, M.; Yamazaki, T.; Ohta, H. Chem. Lett. 1997, 797–798.
4. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104, 7294–7299. In the case of meso-substrates,
the e.e. of the products is considered to reflect the relative reaction rate between enantiotopic groups.
20
5. A methyl ester of (1R,6S)-20: m.p. 115–116°C; [α]D −6.0 (c=2.22, CHCl3); IR (nujol) νmax 3350, 1720, 1660, 1610,
1
1200, 1030 cm−1; H NMR δ 2.1–3.3 (m, 6H), 3.70 (s, 3H), 5.6–5.9 (m, 2H), 6.0 (br., 2H); found: C, 58.60; H, 6.77;
N, 7.54. Calc. for C9H13NO3: C, 59.00; H, 7.15; N, 7.65%. HPLC analysis of the corresponding benzyl ester: Chiralcel
OJ, hexane:i-PrOH (9:1); 0.3 mL/min, tR (min): 74.4 (1S,6R), 81.0 (1R,6S). The absolute configuration was confirmed by
preparing an authentic sample from a known half ester (1R,6S)-36: see Kobayashi, S.; Kamiyama, K.; Iimori, T.; Ohno, M.
Tetrahedron Lett. 1984, 25, 2557–2560.
6. Entry 2: HPLC of 37, Chiralcel OJ, hexane–i-PrOH (50:1); 0.5 mL/min, tR (min): 43.4 (1S,2R,3S,4R), 48.2 (1R,2S,3R,4S).
See Murata, M.; Uchida, H.; Achiwa, K. Chem. Pharm. Bull. 1992, 40, 2610–2613. Entry 3: the absolute configuration
and e.e. was related to that in entry 2 by hydrogenation of the product. Entry 4: the absolute configuration and e.e. was
related to that in entry 2. Entry 7: HPLC of 38, Develosil, CHCl3:EtOAc (25:1); 0.55 mL/min, tR (min): 12.1 (1S,6R), 14.0
(1R,6S). See Nagao, Y.; Hagiwara, Y.; Kumagai, T.; Ochiai, M.; Inoue, T.; Hashimoto, K.; Fujita, E. J. Org. Chem. 1986,
51, 2391–2393. Nagao, Y.; Kume, M.; Wakabayashi, R. C.; Nakamura, T.; Ochiai, M. Chem. Lett. 1989, 239–242. Entry 9:
HPLC of 39, Develosil, CHCl3:EtOAc (25:1); 0.55 mL/min, tR (min): 12.8 (1S,2R), 14.0 (1R,2S). Entry 15: HPLC analysis
of the corresponding benzyl ester, Chiralcel OJ, hexane:i-PrOH (50:1); 1.0 mL/min, tR (min): 15.3 (1S,2R), 17.4 (1R,2S).
The absolute configuration was related to that in entries 10–14 by hydrogenation of the product (Fig. 2).
22
7. (1S,5S)-32: [α]D −30.5 (c=0.93, CHCl3), Chiralcel OJ, hexane:i-PrOH (9:1); 0.5 mL/min, tR (min): 48.3 (1R,5R), 64.4
22
(1S,5S). An authentic (1R,5R)-32 from (1R,5R)-33: [α]D +112.8 (c=0.90, CHCl3). (1R,5R)-33: m.p. 159.0–159.5°C;
22
[α]D +73.3 (c=0.94, CHCl3); IR (KBr) νmax 3440, 1710, 1660, 1620, 1600, 1450, 1320, 1285, 1275, 1250, 715 cm−1
;
1H NMR δ 1.94 (dt, J=9.5, 12.5 Hz, 1H), 2.28–2.47 (m, 3H), 2.65–2.73 (m, 1H), 5.66–5.79 (m, 3H), 5.76–5.79 (m, 1H),
5.91–5.96 (m, 1H), 7.41–7.45 (m, 2H), 7.53–7.57 (m, 1H), 8.02–8.05 (m, 2H); found: C, 68.75; H, 6.28; N, 5.79. Calc. for
C14H15NO3: C, 68.56; H, 6.16; N, 5.71%. HPLC: Chiralcel OJ, hexane:i-PrOH (5:1); 0.5 mL/min, tR (min): 19.1 (1R,5R),
22
22.4 (1S,5S). A methyl ester of (1S,5S)-34: [α]D −86.4 (c=1.25, CHCl3); IR (film) νmax 3030, 2950, 1720, 1595, 1580,
1450, 1270, 715 cm−1; 1H NMR δ 1.96 (ddd, J=8.9, 11.9, 12.5 Hz, 1H), 2.34–2.53 (m, 3H), 2.76–2.87 (m, 1H), 3.67 (s,
3H), 5.61–5.68 (m, 1H), 5.75–5.81 (m, 1H), 5.91–5.98 (m, 1H), 7.40–7.56 (m, 3H), 8.02–8.06 (m, 2H); found: C, 69.00;

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K. Matoishi et al. / Tetrahedron: Asymmetry 9 (1998) 1097–1102
Fig. 2.
H, 6.31. Calc. for C15H16O4: C, 69.22; H, 6.20%. HPLC: Chiralcel OJ, hexane:i-PrOH (50:1); 0.5 mL/min, tR (min): 25.8
(1R,5R), 31.7 (1S,5S). The absolute configurations of 33 and 34 were determined at the stage of 35, respectively.
8. The hydrolysis had worked well with the substrates bearing benzoyloxy groups: Kakeya, H.; Sakai, N.; Sano, A.; Yokoyama,
M.; Sugai, T.; Ohta, H. Chem. Lett. 1991, 1823–1824.; Yokoyama, M.; Imai, N.; Sugai, T.; Ohta, H. J. Mol. Catal. Sect.
B enzymatic 1996, 1, 135–141. In a certain case, however, the hydrolysis became very slow by introducing a too bulky
protecting group such as TBDMS: Sugai, T.; Katoh, O.; Ohta, H. Tetrahedron 1995, 51, 11987–11988.
9. The preference of same enantiomer and/or enantiotopic group by nitrile hydratase and amidase was observed in entry 2, 7
and 9. Related example: Beard, T.; Cohen, M. A.; Parratt, J. S.; Turner, N. J.; Crosby, J.; Moilliet, J. Tetrahedron: Asymmetry
1993, 4, 1085–1104.
10. Deigner, H. P.; Blencowe, C.; Freyberg, C. E. J. Mol. Catal. B: Enz. 1996, 1, 61–70.
11. Smith III A. B.; Hale, K. J.; Laakso, L. M.; Chen, K.; Riéra, A. Tetrahedron Lett. 1989, 30, 6963–6966. Schreiber, S. L.;
Smith, D. B. J. Org. Chem. 1989, 54, 9–10. Kuwahara, S.; Mori, K. Tetrahedron 1990, 46, 8075–8082. Trost, B. M.; Kondo,
Y. Tetrahedron Lett. 1991, 32, 1613–1616. Enzymatic preparation of related compounds: Gu, R.-L.; Sih, C. J. Tetrahedron
Lett. 1990, 31, 3287–3290. Allen, J. V.; Williams, J. M. J. Tetrahedron Lett. 1996, 37, 1859–1862.
12. Marshall, J. A.; Xie, S. J. Org. Chem. 1995, 60, 7230–7237.
22
24
13. (1R,5R)-35: [α]D −5.9 (c=0.39, CHCl3) [Ref.12 [α]D −4.6 (c=2.25, CHCl3)]; IR (film) νmax 3430, 3040, 2960, 1735,
1440, 1380, 1205, 1175, 1050, 1005, 920 cm−1; 1H NMR δ 1.69–1.80 (ddd, J=7.9, 10.6, 12.9 Hz, 1H), 2.25–2.33 (m, 3H),
2.67–2.78 (m, 1H), 3.70 (s, 3H), 4.26–4.32 (m, 1H), 5.71–5.81 (m, 2H); 13C NMR δ 175.6, 130.8, 126.8, 65.9, 51.9, 37.7,
34.1, 27.3. The NMR spectra were identical with those reported previously.12 Found: C, 61.58; H, 7.92. Calc. for C8H12O3:
C, 61.52; H, 7.74%. (1S,5S)-35: [α]D24 +5.5 (c=0.45, CHCl3); IR and NMR spectra were identical with those of (1R,5R)-35.