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reacting acetonitrile with 3-methylcinnamonitrile. All
unknown starting dinitriles were fully characterized by
their spectra data.
5.3.1. Enzymatic hydrolysis of 3-phenylglutaronitrile 1a.
(3S)-4-Cyano-3-phenylbutanoic acid 2a: reaction time 24
h (with acetone) (67%) [h]2D0 +22 (c 1, CHCl3), e.e. 88%.
Mp: 123–124°C; wmax (KBr)/cm−1 2553–3215 (COOH),
2247 (CN), 1697 (CꢀO); lH (CDCl3) 8.97 (br, 1H),
7.26–7.41 (m, 5H), 3.51 (quin., J 7.0, 1H), 2.94 (dd, J
7.7, 16.7, 1H), 2.85 (dd, J 7.2, 16.8, 1H), 2.76 (d, J 6.6,
2H); lC (CDCl3) 176.5, 139.9, 129.1, 128.0, 126.9,
117.7, 38.5, 37.7, 24.4; m/z (EI) 189 (M+, 15%), 171
(10), 162 (36), 143 (38), 134 (57), 107 (100). Found: C,
69.79; H, 5.89; N, 7.44. C11H11NO2 requires C, 69.83;
H, 5.86; N, 7.40%.
5.2.1. 3-(4-Chlorophenyl)glutaronitrile 1c.16a Yield 52%.
Bp: 179–180°C (1 mmHg); wmax (KBr)/cm−1 2248 (CN);
lH (CDCl3) 7.42 (d, J 8.4, 2H), 7.27 (d, J 8.4, 2H), 3.43
(quin., J 6.9, 1H), 2.86 (d, J 7.0, 4H); lC 136.4, 134.7,
129.7, 128.2, 116.8, 38.0, 23.5; m/z (EI) 206 (6), 204
(M+, 18%), 166 (33), 164 (100). Found: C, 64.35; H,
4.34; N, 13.28. C11H9ClN2 requires: C, 64.59; H, 4.43;
N, 13.69%.
5.2.2. 3-(4-Methylphenyl)glutaronitrile 1d.16a Yield 45%.
Bp: 156–158°C (1 mmHg); wmax (KBr)/cm−1 2244 (CN);
lH (CDCl3) 7.23 (d, J 8.1, 2H), 7.16 (d, J 8.2, 2H), 3.35
(quin., J 6,9, 1H), 2.79 (d, J 6.9, 4H), 2.35 (s, 3H); lC
(CDCl3) 138.4, 134.9, 129.9, 126.5, 117.0, 38.1, 23.4,
20.9; m/z (EI) 184 (M+, 21%), 144 (100). Found: C,
78.61; H, 6.43; N, 15.27. C12H12N2 requires: C, 78.23;
H, 6.56; N, 15.20.
5.3.2. Enzymatic hydrolysis of 3-(4-fluorophenyl) gluta-
ronitrile 1b. (3S)-4-Cyano-3-(4-fluorophenyl)butanoic
acid 2b: reaction time 24 h (with acetone) (16%) [h]D20
+46.7 (c 0.9, CHCl3) e.e. 76%. Mp: 100.5–101.5°C; wmax
(KBr)/cm−1 2632–3072 (COOH), 2255 (CN), 1712
(CꢀO); lH (CDCl3) 8.37 (br, 1H), 7.05–7.28 (m, 4H),
3.51 (quin., J 7.0, 1H), 2.90 (dd, J 7.6, 16.7, 1H), 2.84
(dd, J 7.3, 16.7, 1H), 2.74 (d, J 6.6, 2H); lC (CDCl3)
176.3, 163.9, 160.6, 135.7, 135.6, 128.7, 128.6, 117.5,
116.2, 115.9, 38.6, 37.1, 24.6; m/z (EI) 207 (M+, 10%),
180 (31), 161 (21), 152 (32), 148 (20), 125 (100). Found:
C, 63.48; H, 4.98; N, 6.89. C11H10FNO2 requires: C,
63.76; H, 4.86; N, 6.76%.
5.2.3. 3-(2-Methylphenyl)glutaronitrile 1e.16a Yield 42%.
Bp: 134–136°C (0.5 mmHg); wmax (KBr)/cm−1 2249
(CN); lH (CDCl3) 7.20–7.29 (m, 4H), 3.68 (quin., J 6.9,
1H), 2.76 (d, J 6.9, 4H), 2.39 (S, 3H); lC (CDCl3) 136.3,
135.8, 131.4, 128.5, 127.2, 124.9, 117.3, 33.7, 23.0, 21.2;
m/z (EI) 184 (M+, 13%), 144 (100). Found: C, 77.97; H,
6.64; N, 15.29. C12H12N2 requires: C, 78.23; H, 6.56; N,
15.20%.
5.3.3. Enzymatic hydrolysis of 3-(4-chlorophenyl) gluta-
ronitrile 1c. (3S)-3-(4-Chlorophenyl)-4-cyanobutanoic
acid 2f: reaction time 72 h (with acetone) (25%) [h]D25
+14.3 (c 1.4, CHCl3) e.e. 63%. Mp: 119–120°C; wmax
(KBr)/cm−1 2538–3208 (COOH), 2254 (CN), 1707
(CꢀO); lH (CDCl3) 9.01 (br, 1H), 7.34 (d, J 8.4, 2H),
7.21 (d, J 8.5, 2H), 3.48 (quin., J 7.0, 1H), 2.88 (dd, J
7.5, 16.7, 1H), 2.82 (dd, J 7.4, 16.8, 1H), 2.73 (d, J 6.6,
2H); lC (CDCl3) 176.4, 138.4, 133.9, 129.3, 128.4,
117.4, 38.5, 37.2, 24.4; m/z (EI) 225 (7), 223 (M+, 17%),
198 (16), 196 (46), 179 (8), 177 (23), 170 (12), 168 (35),
166 (7), 164 (21), 143 (32), 141 (100). Found: C, 58.75;
H, 4.69; N, 6.28. C11H10ClNO2 requires: C, 59.07; H,
4.51; N, 6.26%.
5.2.4. 3-(4-Methoxyphenyl)glutaronitrile 1f.16a Yield
48%. Mp: 71–72°C; wmax (KBr)/cm−1 2250.7 (CN); lH
(CDCl3) 7.23 (d, J 8.6, 2H), 6.96 (d, J 8,6, 2H), 3.84 (s,
3H), 3.40 (quin., J 6.9, 1H), 2.84 (d, J 6.9, 4H); lC
(CDCl3) 159.5, 129.7, 127.6, 114.5, 116.9, 55.1, 37.7,
23.5; m/z (EI) 200 (M+, 17%), 160 (100). Found: C,
72.23; H, 5.93; N, 13.82. C12H12N2O requires: C, 71.98;
H, 6.04; N, 13.99%.
5.3. General procedure for the biotransformations of
3-substituted glutaronitriles
To an Erlenmeyer flask (150 mL) with a screw cap was
added Rhodococcus sp. AJ270 cells3 (2 g wet weight)
and potassium phosphate buffer (0.1 M, pH 7.0, 50
mL), and the resting cells were activated at 30°C for 0.5
h with orbital shaking. The 3-substituted glutaronitrile
as a fine powder, or dissolved in organic solvents (3
mL) or in hexane (25 mL) solution, or as a gelation
with b-CD (300 mg) and buffer (Table 1) was added in
one portion to the flask and the mixture was incubated
at 30°C using an orbital shaker (200 rpm). The reac-
tion, monitored by TLC, was quenched after the spe-
cified period of time (see Tables 1 and 2) by removing
the biomass via filtration through a Celite pad. The
resulting aqueous solution was basified to pH 12 with
aqueous NaOH (2 M). Extraction with ethyl acetate
gave, after drying (MgSO4) and removing solvent, the
unconverted nitrile. The aqueous solution was then
acidified using aqueous HCl (2 M) to pH 2 and
extracted with ethyl acetate. Pure 3-substituted 4-
cyanobutanoic acid 2 was obtained after removal of the
solvent.
5.3.4. Enzymic hydrolysis of 3-(4-methylphenyl) gluta-
ronitrile 1d. (3S)-4-Cyano-3-(4-methylphenyl)butanoic
acid 2d: reaction time 24 h (42%) (e.e. 64%); 24 h (25%)
(with acetone) [h]D20 +24 (c 1, CHCl3) e.e. 95%. Mp:
114–115°C; wmax (KBr)/cm−1 2589–3208 (COOH), 2247
(CN), 1700 (CꢀO); lH (CDCl3) 9.26 (br, 1H), 7.20 (s,
4H), 3.50 (quin., J 7.0, 1H), 2.93 (dd, J 7.7, 16.6, 1H),
2.85 (dd, J 7.3, 16.7, 1H), 2.76 (d, J 6.6, 2H), 2.38 (s,
3H); lC (CDCl3) 176.8, 137.7, 137.0, 129.7, 126.8,
117.8, 38.7, 37.4, 24.5, 21.1; m/z (EI) 203 (M+, 19%),
176 (39), 157 (17), 148 (27), 121 (100). Found: C, 71.02;
H, 6.71; N, 6.81. C11H11NO2 requires: C, 70.92; H,
6.45; N, 6.89%.
5.3.5. Enzymatic hydrolysis of 3-(2-methylphenyl) gluta-
ronitrile 1e. (3S)-4-Cyano-3-(4-methylphenyl)butanoic
acid 2e: reaction time 20 h (with acetone) (40%) [h]D25
+3.16 (c 1.25, CHCl3) e.e. 35%. Oil: wmax (KBr)/cm−1
2958–3250 (COOH), 2248 (CN), 1711 (CꢀO); lH
(CDCl3) 7.20–7.26 (m, 4H), 3.81 (quin., J 7.0, 1H), 2.91
(dd, J 7.7, 16.7, 1H), 2.83 (dd, J 7.0, 16.8, 1H), 2.70 (d,