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E. C. Hann et al. / Tetrahedron 60 (2004) 577–581
C. testosteroni S2B-1 and C. testosteroni S5C were each
isolated and characterized for the first time in this
application. The nitrile hydratase and amidase activities of
C. testosteroni 5-MGAM-4D have previously been reported
as catalysts for the regioselective hydrolysis of aliphatic
a,v-dinitriles to the corresponding v-cyanocarboxylic
acids.14 In that application, C. testosteroni 5-MGAM-4D
exhibited both non-regioselective and regioselective
nitrile hydratase activities, and heating a suspension of
C. testosteroni 5-MGAM-4D at 50 8C for 30–60 min was
required to inactivate the non-regioselective nitrile
hydratase activity of the microbial cell catalyst. For
regioselective production of 1 by hydrolysis of 6, no heat-
treatment of C. testosteroni 5-MGAM-4D cells was
required.
products of 6, 3 and 8 were made by comparison of HPLC
retention times to commercially available samples.
The isolation and growth of A. facilis 72W,14,19 E. coli
SS1001,13 and C. testosteroni 5-MGAM-4D14 has been
reported. C. testosteroni S2B and S5C were isolated from
soil samples using standard enrichment procedures and S12-
N medium (S12 medium with ammonium sulfate replaced
with sodium sulfate),20 then grown aerobically in E2
medium containing 0.2% (w/v) 3-hydroxyvaleronitrile at
30 8C. Cell paste isolated from fermentation was frozen at
280 8C without pre-treatment with glycerol or DMSO. Wet
cell weights (wcw) of microbial catalysts were obtained
from cell pellets prepared by centrifugation of fermentation
broth or cell suspensions in buffer. Dry cell weights (dcw)
were determined by microwave drying of wet cells.
Microbial cell enzyme activity was measured by stirring a
suspension of 8.5–12.5 mg dry cell weight/mL in 25 mM
phosphate buffer (pH 7.0) and 0.14 M substrate at 25 8C.
E. coli SS1001 cells were immobilized in both alginate15
and carrageenan beads,16 and A. facilis 72 W cells were
immobilized in alginate beads,15 using previously reported
procedures.
3. Conclusions
A method has been developed for the facile preparation of 1
from 6, where 6 was first prepared by the facile isomeriza-
tion of commercially-available 3. Although C. testosteroni
microbial cell catalysts had higher specific activities for
hydrolysis of 4, regioselective hydrolysis of 6 by A. facilis
72W nitrilase was preferred over the combination of
nitrile hydratase and amidase activities of C. testosteroni
5-MGAM-4D, S2B-1 or S5C, since the latter catalysts each
produced significant conversion of 5 to the corresponding
amide at complete conversion of 4 to 1, whereas there was
no detectable conversion of 5 by the nitrilase at extended
reaction times. The immobilized-cell nitrilase catalysts were
robust under reaction conditions employing high concen-
tration of 6 and catalyst recycle, and high yields of 1 were
obtained with the added advantages of low temperature and
energy requirements, and low waste production when
compared to chemical methods of nitrile hydrolysis.
4.2. Isomerization of 2-Methyl-3-butenenitrile (3)
A mixture of 3 (50 g, 0.62 mol) and activity I basic alumina
(5 g) was heated with stirring at 85 8C. After 18 h, the
mixture was cooled to ambient temperature and filtered to
yield 49 g (98% isolated yield) of a 72:28 (mole/mole)
mixture of (E)- and (Z)-2-methyl-2-butenenitrile, deter-
mined by analysis of the reaction product by gas
chromatography.
4.3. Isomerization of (Z)-2-pentenitrile (7)
A mixture of 7 (402.7 g, 4.96 mol), triphenylphosphine
(16.0 g, 61.1 mmol), and zinc chloride (8.4 g, 61.6 mmol)
(anhydrous) was heated at 70 8C for 22 h. The resulting
mixture was cooled to ambient temperature, the insoluble
zinc chloride filtered from the mixture, and the mixture
vacuum distilled at 82 8C and 130 Torr to separate the
mixture of (Z)- and (E)-2-pentenitriles from triphenylpho-
sphine and soluble zinc chloride. The resulting distillate was
analyzed by gas chromatography, using authentic samples
of (Z)- and (E)-2-pentenitrile as standards; the ratio of (Z)-
and (E)-isomers in the distillate was 78:22 (mole:mole).
4. Experimental
4.1. General
Chemicals were obtained from commercial sources unless
otherwise noted, and used as received. Isolated yields are
unoptimized and melting points uncorrected. 318 and 418
were each isolated from a mixture of 6 by fractional
distillation under vacuum using a 10-plate Oldershaw
column and a reflux ratio of .10:1. The calculated recovery
of nitriles and yields of the hydrolysis products were based
on initial nitrile concentration, and determined by HPLC
using a refractive index detector and either a Supelcosil
LC-18 DB column (30 cm£4.6 mm dia.) and 10 mM acetic
acid/10 mM sodium acetate in 2.5% methanol/water as
mobile phase (for hydrolysis of 4 and 6), or a Supelcosil
LC-18 DB column (15 cm£4.6 mm dia.) and 10 mM acetic
acid/10 mM sodium acetate in 7.5% methanol/water as
mobile phase (for hydrolysis of 3, 7, and 8). Gas
chromatographic analysis of nitriles was performed on a
J&W Scientific DB1701 column (30 m, 0.53 mm ID, 1 mm
film thickness). Chemical shifts for 1H and 13C NMR
spectra are expressed in parts per million positive values
downfield from internal TMS. Identification of hydrolysis
4.4. General procedure for reactions using
unimmobilized cells
In a typical procedure, an aqueous solution (10.0) mL
containing 0.10 M nitrile, cell paste (0.50 g wcw) and
50 mM potassium phosphate buffer (pH 7.0) was stirred at
35 8C At pre-determined times, a 0.100 mL aliquot of the
reaction was removed and mixed with 0.900 mL of 60 mM
N-ethylacetamide (HPLC external standard) in 1:1 aceto-
nitrile:methanol, the sample centrifuged, and the supernatant
analyzed by HPLC. Results are tabulated in Tables 1 and 3.
4.5. Immobilized-cell catalyst recycle reactions
In a typical procedure, distilled, deionized water (15.0 mL),