8158 J. Agric. Food Chem., Vol. 52, No. 26, 2004
Rey et al.
1
99%; H NMR (DMSO d6) δH 1.78 (s, 3H, CH3-S), 1.56-1.68 (m,
Enzymatic Hydrolysis of (()-2-(1-Ethoxyethoxy)-but-3-enenitrile
10a. Yield after extraction ) 86% of (()-2-(1-ethoxyethoxy)-but-3-
enoic acid 9c: 1H NMR (DMSO d6) δH 1.06 (t, 3H, CH3-CH2-O), 1.19
(d, 3H, CH3-CH), 3.40-3.55 (m, 2H, CH3-CH2-O-), 4.32 (m, 1H, CH2d
CHsCH), 4.75-4.66 (qd, 1H, O-CH-O), 4.95-5.2 (m, 2H, CH2dCHs),
5.78-5.91 (m, 1H, CH2dCH), 12.32 (s, 1H, CO2H); 13C NMR (DMSO
d6) δC 15.94 and 16.06 (CH3-CH2-O-), 20.75 and 21.15 (CH3-CH),
60.24 and 61.10 (CH3-CH2-O-), 78.61 and 78.92 (CH2dCHsCH),
98.31 and 98.87 (O-CH-O), 113.86 and 115.0 (CH2dCHs), 138.25
and 138.56 (CH2dCH), 174.56 (CO2H); IR (CHCl3) cm-1 2500-3500,
1725; mass FAB- (GT), m/e (relative intensity) 173 (100), 43 (35).
Enzymatic Hydrolysis of (()-2-Methoxymethoxy-but-3-enenitrile
11a. Yield after extraction ) 80% in (()-2-methoxymethoxy-but-3-
enoic acid 11c: 1H NMR (DMSO d6) δH 3.30-3.95 (m, 3H, CH3-O-),
4.37 (m, 1H, CH2dCHsCH), 4.62-4.71 (dd, O-CH2-O), 5.07-5.23
(m, 2H, CH2dCHs), 5.91-5.98 (m, 1H, CH2dCH), 11.47 (s, 1H,
CO2H); 13C NMR (DMSO d6) δC 55.58 (CH3-O-), 78.87 (CH2dCHs
CH), 94.62 (O-CH2-O), 114.38 (CH2dCHs), 138.11 (CH2dCH),
173.67 (s, 1H, CO2H); IR (CHCl3) cm-1 2500-3500, 1725; mass FAB-
(GT), m/e (relative intensity) 193 (100).
2H, S-CH2-CH2), 2.26 (t, 2H, S-CH2-CH2), 3.79 (CH (OH)CO2H), 11.74
(s, 1H, CO2H); 13C NMR (DMSO d6) δC 15.38 (CH3-S), 30.08 (CH3-
S-CH2), 34.32 (S-CH2-CH2), 69.34 (CH(OH)CO2H), 176.40 (CO2H);
IR (CHCl3) cm-1 2500-3500, 1725; mass FAB- (NBA), m/e (relative
intensity) 149 (100), 122 (5), 46 (15).
Enzymatic Hydrolysis of 2-Acetoxy-4-methylthiobutanenitrile
(4a). HPLC analysis had indicated that all the starting material had
been converted into two more polar products (water/acetonitrile 80:20
(v/v)/0.05% (v/v) trifluoroacetic acid; λ ) 210 nm; flow ) 1 mL/min;
column: Nucleosil RP C18 (5 µm, 100 Å). A liquid was isolated and
shown by 1H NMR spectroscopy to be a mixture of 2-acetoxy-4-
methylthiobutanoic acid 4c (82%) and 2-hydroxy-4-methylthiobutanoic
acid 3c (6%): 1H NMR (DMSO d6) δH 1.75 (m, 2H, S-CH2-CH2),
1.79 (s, 3H, CH3CO2), 1.84 (s, 3H, CH3-S), 2.28 (t, 2H, CH3-S-CH2),
4.69 (td, 1H, CH (OAc)CN), 11.9 (s, 1H, CO2H); 13C NMR (DMSO
d6) δC 15.24 (CH3-S), 21.17 (CH3-CO), 29.73 (CH3-S-CH2), 30.96 (S-
CH2-CH2), 71.35 (CH(OAc)CN), 170.77 (CH3-CO2), 171.86 (CO2H);
IR (CHCl3) cm-1 2500-3500, 1745, 1720.; mass FAB- (NBA), m/e
(relative intensity) 191 (100), 149 (70), 59 (25).
Enzymatic Hydrolysis of 2-Cyan-O-ethyl Acetate 12a. Yield after
extraction ) 94% of 2-carbetoxyacetic acid 12c: 1H NMR (DMSO
d6) δH 1.19 (t, 3H, CH3-CH2-O), 3.33 (s, 2H, HO2C-CH2-CO2Et), 4.12
(q, 3H, CH3-CH2-O), 10.71 (s, 1H, CO2H); 13C NMR (DMSO d6) δC
14.66 (CH3-CH2-O-), 42.34 (HO2C-CH2-CO2Et), 64.47 (CH3-CH2-O),
167.7 (-CO2-CH2-CH3), 168.9 (CO2H); IR (CHCl3) cm-1 2500-3500,
1745, 1725; mass FAB- (GT), m/e (relative intensity) 131 (65), 104
(40), 87 (100), 41 (25).
Enzymatic Hydrolysis of Malononitrile 13a. Yield after extraction
) 87% of 2-cyanoacetic acid 13c: 1H NMR (DMSO d6) δH 3.57 (s,
2H, -CH2-), 11.57 (s, 1H, CO2H); 13C NMR (DMSO d6) δC 25.46
(CH2-CN), 115.4 (CCN), 165.6 (CO2H); IR (CHCl3) cm-1 2500-
3500, 2240, 1725; mass FAB- (GT), m/e (relative intensity) 84 (100),
46 (60).
Enzymatic Hydrolysis of Adiponitrile 14a. Yield after extraction
) 90% of 5-cyanopentanoic acid 14c: 1H NMR (DMSO d6) δH 1.60
(m, 2H, HO2C-CH2-CH2-), 1.62 (m, 2H, NC-CH2-CH2-), 2.23 (t, 2H,
-CH2-CO2H), 2.51 (t, 2H, -CH2-CN), 12.41 (s, 1H, CO2H); 13C NMR
(DMSO d6) δC 16.92 (NC-CH2-CH2), 24.92 (NC-CH2-CH2), 25.31
(CH2-CH2-CO2H), 34.95 (CH2-CO2H), 121.38 (CN), 176.41 (s, 1H,
CO2H); IR (CHCl3) cm-1 2500-3500, 2245, 1730; mass FAB- (GT):
m/e (relative intensity) 126 (100).
Enzymatic Hydrolysis of Fumaronitrile 15a. Yield after extraction
) 72% in 3-cyanoacrylic acid 15c and 28% of 3-cyanoacrylamide 15b.
3-Cyanoacrylic acid 15c: 1H NMR (DMSO d6) δH 5.84 (d, 1H, HO2Cs
CHdCH-CN), 6.54 (d, 1H, HO2CsCHdCHsCN), 13.10 (s, 1H,
CO2H); 13C NMR (DMSO d6) δC 115.03 (HO2CsCHdCHsCN),
119.09 (CN), 153.22 (HO2CsCHdCHsCN), 167.13 (CO2H); IR
(CHCl3) cm-1 2500-3500, 2225, 1695; mass FAB- (GT), m/e (relative
intensity) 96 (100), 52 (10). 3-Cyanoacrylamide 15b: 1H NMR (DMSO
d6) δH 6.56 (d, 1H, H2NOCsCHdCHsCN), 7.04 (d, 1H, H2NOCs
CHdCHsCN), 7.72-7.99 (d, 2H, CONH2); 13C NMR (DMSO d6) δC
109.54 (H2NOCsCHdCHsCN), 117.95 (CN), 144.95 (H2NOCsCHd
CHsCN), 164.01 (CONH2); IR (CHCl3) cm-1 3100-3500, 2235, 1695;
mass FAB- (GT), m/e (relative intensity) 95 (10).
Enzymatic Hydrolysis of 1,4-Dicyanobut-2-ene 16a. Yield after
extraction ) 82% of 5-cyanopent-3-enoic acid 16c: 1H NMR (DMSO
d6) δH 3.04 (dd, 2H, HO2C-CH2), 3.31 (dd, 2H, CH2-CN), 5.51 (td,
1H, NCsCH2sCHd), 5.82 (td, 1H, H2OCsCH2sCHd), 10.51 (s,
1H, CO2H); 13C NMR (DMSO d6) δC 20.34 (CH2-CN), 38.14 (H2OC-
CH2), 119.44 (CN), 122.21 (NCsCH2sCHd), 128.75 (H2OCsCH2s
CHd), 173.44 (CO2H); IR (CHCl3) cm-1 2500-3500, 2255, 1725; mass
FAB- (GT), m/e (relative intensity) 124 (100), 80 (25), 46 (30).
Enzymatic Hydrolysis of 2-Methylglutaronitrile 17a. Yield after
extraction ) 91% of 4-cyanopentanoic acid 17c: 1H NMR (DMSO
d6) δH 1.23 (d, 3H, CH3-CH), 1.79 (td, 2H, NC-CH(CH3)-CH2-), 2.35
(td, 2H, -CH2-CO2H), 2.84 (m, 2H, -CH-CN), 10.89 (s, 1H, CO2H);
13C NMR (DMSO d6) δC 18.08 (CH3-CH), 24.94 (NC-CH-), 29.33
(CH2-CH2-CO2H), 31.89 (CH2-CO2H), 123.78 (CN), 174.25 (s, 1H,
CO2H); IR (CHCl3) cm-1 2500-3500, 2240, 1735; mass FAB- (GT):
m/e (relative intensity) 126 (100), 54 (5).
Enzymatic Hydrolysis of 2-(1-Ethoxyethoxy)-4-methylthio-
butanenitrile (5a). HPLC analysis had indicated that all the starting
material had been converted into a single more polar product (water/
acetonitrile 95:5 (v/v)/0.05% (v/v) trifluoroacetic acid; λ ) 210 nm;
flow ) 1 mL/min; column: Nucleosil RP C18 (5 µm, 100 Å). A liquid
was isolated (4.54 g, 20.4 mmol) and shown by 1H NMR spectroscopy
to be 2-(1-ethoxyethoxy)-4-methylthiobutanoic acid 5c: yield, 93%;
1H NMR (DMSO d6) δH 1.09 (td, 3H, CH3-CH2-O), 1.18 (dd, 3H,
CH3-CH), 1.72-1.84 (m, 2H, S-CH2-CH2), 2.10 (s, 3H, CH3-S), 2.52
(t, 2H, S-CH2-CH2), 3.51 (m, 2H, O-CH2-CH3), 3.84 (m, 1H, O-CH-
CO2H), 4.72 (qd, 1H, O-CH (CH3)-O), 11.54 (s, 1H, CO2H); 13C NMR
(DMSO d6) δC 15.42 (CH3-S), 16.1 (CH3--CH2-O), 21.32 (CH3-CH),
30.07 (S-CH2-CH2), 33.89 (S-CH2-CH2), 62.00 (CH3-CH2-O), 77.06
(CH(CO2H)-O), 99.49 (O-CH(CH3)-O), 176.42 (CO2H); IR (CHCl3)
cm-1 2500-3500, 1725; mass FAB- (NBA), m/e (relative intensity)
221 (100), 152 (40), 122 (30), 46 (65).
Enzymatic Hydrolysis of 2-Methoxymethoxy-4-methylthio-
butanenitrile (6a). HPLC analysis indicated that all the starting material
had been converted into a single more polar product (water/acetonitrile
80:20 (v/v)/0.05% (v/v) trifluoroacetic acid; λ ) 210 nm; flow ) 1
mL/min; column: Nucleosil RP C18 (5 µm, 100 Å). A liquid was
1
isolated (2.98 g, 15.5 mmol) and shown by H NMR spectroscopy to
1
be 2-methoxymethoxy-4-methylthiobutanoic acid 6c: yield, 70%; H
NMR (DMSO d6) δH 1.96 (m, 2H, S-CH2-CH2), 2.05 (s, 3H, CH3-S-
CH2), 2.59 (t, 2H, S-CH2-CH2), 3.31 (s, 3H, CH3-O), 3.91 (m, 1H,
O-CH-CO2H), 4.60-4.72 (dd, 2H, O-CH2-O), 11.32 (CO2H); 13C NMR
(DMSO d6) δC 15.37 (CH3-S), 30.80 (CH3-S-CH2), 33.60 (S-CH2-CH2)
55.74 (CH3-O), 76.55 (O-CH-CO2H), 95.44 (O-CH2-O), 176.13 (CO2H);
IR (CHCl3) cm-1 2500-3500, 1720; mass FAB- (NBA), m/e (relative
intensity) 193 (70), 149 (55), 115 (20), 46 (100).
Enzymatic Hydrolysis of Isobutanenitrile 7a. Yield in isobutanoic
acid ) 71% 7c: 1H NMR (DMSO d6) δH 1.05 (d, 6H, (CH3)2-CH),
2.40 (h, 1H, (CH3)2-CH), 11.74 (s, 1H, CO2H); 13C NMR (DMSO d6)
δC 19.61 ((CH3)2-CH), 33.90 (CH3)2-CH), 178.7 (s, 1H, CO2H); IR
(CHCl3) cm-1 2500-3500, 1715; mass FAB- (GT), m/e (relative
intensity) 87 (100), 79 (20).
Enzymatic Hydrolysis of Acrylonitrile (8a). Yield in acrylic acid
) 93% 8c: 1H NMR (DMSO d6) δH 5.90-6.13 (m, 2H, CH2dCH),
6.32 (dd, 1H, CH2dCH), 12.43 (s, 1H, CO2H); 13C RMN (DMSO d6)
δC 130.7 (CH2dCH), 131.45 (CH2dCH), 171.16 (CO2H); IR (CHCl3)
cm-1 2500-3500, 1695; mass FAB- (GT), m/e (relative intensity) 71
(70), 53 (20). NMR analysis revealed 0.8% of acrylamide.
Enzymatic Hydrolysis of (()-2-Acetoxy-3-butenenitrile 9a. Yield
after extraction ) 92% of (()-2-acetoxybut-3-enoic acid 9c: NMR
analysis showed 6% of (()-2-hydroxy-but-3-enoic acid; 1H NMR
(DMSO d6) δH 2.01 (s, 3H, CH3-CO2), 4.87-5.25 (m, 3H, CH2dCHs
CH), 5.93-6.01 (m, 1H, CH2dCHsCH), 12.33 (CO2H); 13C NMR
(DMSO d6) δC 21.77 (CH3-CO2), 76.69 (CH2dCHsCH), 115.12
(CH2dCH), 135.59 (CH2dCH), 171.24 (CH3-CO2), 176.86 (CO2H);
IR (CHCl3) cm-1 2500-3500, 1740, 1715; mass FAB- (GT), m/e
(relative intensity) 143 (100), 59 (30).