The reaction was run to 25% conversion and 80% selectivity
to 11; polar byproducts such as adipamide were left in the
spent MnO2 cake after dilution with toluene and filtration.
Cooling the hot toluene filtrate precipitated anhydrous
5-cyanovaleramide in a high state of purity; recovery and
recycle of unreacted ADN was achieved by concentration
of the mother liquor. Although this was an expeditious way
to obtain pilot-plant quantities of quality 11, the consequences
of disposing of or recycling large quantities of manganese
dioxide provided incentive to find a “greener” process, and
thus enzyme-catalysis was investigated.
Aliphatic nitriles are readily hydrolyzed to the corre-
sponding amides by the nitrile hydratase (EC 4.2.1.84) of a
variety of bacteria and fungi.8 In the present case, we required
sufficient regioselectivity to minimize adipamide formation;
the absence of amidase activity that could result in carboxylic
acid formation was also important. A variety of microbes
were evaluated, and Rhodococcus erythropolis A49 was
initially selected for process development.
R. erythropolis A4 cells were immobilized in calcium
alginate beads prior to use. Hydrolysis reactions were run at
5 °C, as the stability of the enzyme decreased markedly
above this temperature. Batch reactions in 20 mM sodium
butyrate/5 mM CaCl2 buffer were begun as a three-phase
mixture of ADN and a suspension of catalyst beads in 20
mM sodium butyrate/5 mM CaCl2 (pH 7.0) to produce 0.75
M 5-cyanovaleramide. Extending the reaction time once
conversion was nearly complete caused very slow conversion
of 11 to adipamide. Catalyst recycle was accomplished
simply by removing 90% of the solution phase, followed by
the addition of ADN and buffer to the heel. Sixty consecutive
batch reactions converted a total of 2400 lb of adiponitrile
to afford a 9.0 wt % solution of 11 in 93% yield, with a
catalyst productivity (kg/kg) of greater than 1000/1. Isolation
of 11 consisted of distillation of water, dissolution in boiling
methanol, and filtration of insoluble adipamide and salts. The
resulting methanolic solution was suitable for use in the
subsequent step.
enzyme so activated will rapidly lose activity if stored in
the absence of light prior to use. The requirement of light
activation of the cells during harvesting from fermentation
broth added additional cost to catalyst manufacture. With
more extensive screening we identified a more desirable
catalyst. Pseudomonas chlororaphis B23 had been first
isolated and characterized by H. Yamada and co-workers11
and was later used in immobilized form by the Nitto
Chemical Industry Co. (now merged with Mitsubihi Rayon
Co.) for the manufacture of acrylamide from acrylonitrile.12
This highly selective monohydration method now did not
require an activation step and furthermore proved to be
superior in both enzyme stability and productivity to R.
erythropolis A4.
The first commercial-scale production of 11 using the P.
chlororaphis B23/alginate beads converted a total of 12.7
Mt of ADN in 58 consecutive 400-gal batch reactions with
catalyst recycle. Using the same procedures and conditions
as previously defined, now it was possible to produce a final
product concentration of 1.5 M (19 wt %) in the aqueous
buffer, increasing the reaction time from 3.5 to 5.5 h. At
97% conversion of ADN, 13.6 Mt (93%) of 5-cyanovaler-
amide was obtained, now with a productivity (kg/kg) of 3150/
1.
Hofmann Rearrangement and Pinner Cyclization.
Scale-up of the Hofmann rearrangement of 11 presented the
challenge of safely generating and reacting large quantities
of the high-energy N-bromoamide salt. The best methods
we could find in the literature for generating high yields of
methyl carbamates from amides in methanol involved the
use either of very low temperature13 or rapid decomposition.14
We found an improved and more practical procedure was
to form the sodium salt of the N-bromoamide by adding
exactly 1 equiv of bromine to a solution of 12 in methanol
containing slightly more than 2 equiv of sodium methoxide
at 0-5 °C. Accelerated-rate calorimetry (ARC) tests of
methanol solutions thus generated showed that in case
cooling was lost the intermediate would be stable for ca. 24
h before an uncontrollable reaction would occur. The safest
way to conduct the exothermic decomposition to 11 was thus
to feed the cold solution of the intermediate to boiling
methanol, whereby yields of 92-94% could be consistently
achieved on a large scale.
The nitrile hydratase of R. erythropolis A4 is unusual in
that it requires a light-activation step,10 and the microbial
(6) (a) Berther, C. Chem. Ber. 1959, 92, 2616 [basic ion-exchange resin]; (b)
Wiley: R. H.; Morgan, H. S., Jr. J. Org. Chem. 1950, 15, 800 [basic
hydrogen peroxide]; (c) Zil’berman, E. N.; Kulikova, A. E. J. Gen. Chem.
USSR 1959, 29, 1671 [HCl gas/ether]; (d) Witzel, T.; Fuchs, E.; Merger, F.
U.S. Patent 5,347,033, 1994 [Cu catalyst].
The intramolecular Pinner-type reaction of 11 was orig-
inally conducted in diethyl ether to provide crystalline 13;
replacing this hazardous solvent with a more suitable one
such as toluene resulted in the precipitation of 13 as an oil
which trapped HCl, making the material hygroscopic and
difficult to carry into the subsequent step. To induce
(7) Sieja, J. Personal communication.
(8) (a) Holland, H. L. Curr. Opin. Chem. Biol. 1998, 2, 77. (b) Sugai, T.;
Yamazaki, T.; Yokoyama, M.; Ohta, H. Biosci. Biotechnol. Biochem. 1997,
61, 1419. (c) Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin Trans. 1
1997, 3197. (d) Hoenicke-Schmidt, P.; Schneider, M. P. J. Chem. Soc.,
Chem. Commun., 1990, 648. (e) Blakey, A. J.; Williams, E.; O’Reilly, C.
FEMS Microbiol. Lett. 1995, 129, 57. (f) Crosby, J.; Moilliet, J.; Parrat, J.
S.; Turner, N. J. J. Chem. Soc., Perkin Trans. 1 1994, 1679. (g) DeRaadt,
A.; Klempier, N.; Faber, K. T.; Griengl, H. J. Chem. Soc., Perkin Trans. 1
1992, 137. (h) Yokoyama, M.; Sugai, T.; Ohta, H. Tetrahedron: Asymmetry
1993, 4, 1081. (i) Cohen, M. A.; Sawden, J.; Turner, N. J. Tetrahedron
Lett. 1990, 31, 7223.
(9) (a) Bernet, N.; Arnaud, A.; Galzy, P. Biocatalysis 1990, 3, 259. (b)
Ingvorsen, K.; Godtfredsen, S. E.; Tsuchiya, R. T. Ciba Found. Symp. 1988,
140 (Cyanide Compd. Biol.), 16. (c) Andresen, O.; Godtfredsen, S. E. 178106
B1, 1993.
(10) (a) Nagamune, T.; Kurata, H.; Hirata, M.; Honda, J.; Hirata, A.; Endo, I.
Photochem. Photobiol. 1990, 51, 87. (b) Honda, J.; Kandori, H.; Okada,
T.; Nagamune, T.; Shichida, Y.; Sasabe, H.; Endo, I. Biochemistry 1994,
33, 3577.
(11) (a) Nagasawa, T.; Nanba, H.; Ryuno, K.; Takeuchi, K.; Yamada, H. Eur.
J. Biochem. 1987, 162, 691. (b) Yamada, H.; Ryuno, K.; Nagasawa, T.;
Enomoto, K.; Watanabe, I. Agric. Biol. Chem. 1986, 50, 2859. (c) Asano,
Y.; Yasuda, T.; Tani, Y.; Yamada, H. Agric. Biol. Chem. 1982, 46,
1183.
(12) (a) Ashina, Y.; Suto, M. Bioprocess Technol. 1993, 16, 91. (b) Kobayashi,
M.; Nagasawa, T.; Yamada, H. Trends Biotechnol. 1992, 10, 402.
(c) Nagasawa, T.; Ryuno, K.; Yamada, H. Experientia 1989, 45, 1066.
(d) Ryuno, K.; Nagasawa, T.; Yamada, H. Agric. Biol. Chem. 1988, 52,
1813.
(13) Radlick, P.; Brown, L. R. Synthesis 1974, 290.
(14) Murr, B. L.; Lester, C. T. J. Am. Chem. Soc. 1955, 77, 1684.
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