Chemoenzymic Production of Lactams from Aliphatic α,ω-Dinitriles

Article abstract of DOI:10.1021/jo9804386

Five- and six-membered ring lactams have been prepared by first converting an aliphatic α,ω-dinitrile to an ω-cyanocarboxylic acid ammonium salt, using a microbial cell catalyst having an aliphatic nitrilase activity (Acidovorax facilis 72W, ATCC 55746) or a combination of nitrile hydratase and amidase activities (Comamonas testosteroni 5-MGAM-4D, ATCC 55744). The ω-cyanocarboxylic acid ammonium salt was then directly converted to the corresponding lactam by hydrogenation in aqueous solution, without isolation of the intermediate ω-cyanocarboxylic acid or ω-aminocarboxylic acid. Only one of two possible lactam products was produced from α-alkyl-substituted α,ω-dinitriles, where the nitrilase of A. facilis 72W regioselectively hydrolyzed only the ω-cyano group to produce a single cyanocarboxylic acid ammonium salt in greater than 98% yield.

Full text of DOI:10.1021/jo9804386

4792
J . Org. Chem. 1998, 63, 4792-4801
Ch em oen zym ic P r od u ction of La cta m s fr om Alip h a tic
r,ω-Din itr iles
J ohn E. Gavagan,† Susan K. Fager,† Robert D. Fallon,† Patrick W. Folsom,† Frank E. Herkes,‡
Amy Eisenberg,† Eugenia C. Hann,† and Robert DiCosimo*,†
Central Research and Development Department and DuPont Nylon Intermediates and Specialties, E. I.
du Pont de Nemours & Co., Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328
Received March 9, 1998
Five- and six-membered ring lactams have been prepared by first converting an aliphatic R,ω-
dinitrile to an ω-cyanocarboxylic acid ammonium salt, using a microbial cell catalyst having an
aliphatic nitrilase activity (Acidovorax facilis 72W, ATCC 55746) or a combination of nitrile
hydratase and amidase activities (Comamonas testosteroni 5-MGAM-4D, ATCC 55744). The
ω-cyanocarboxylic acid ammonium salt was then directly converted to the corresponding lactam
by hydrogenation in aqueous solution, without isolation of the intermediate ω-cyanocarboxylic acid
or ω-aminocarboxylic acid. Only one of two possible lactam products was produced from R-alkyl-
substituted R,ω-dinitriles, where the nitrilase of A. facilis 72W regioselectively hydrolyzed only
the ω-cyano group to produce a single cyanocarboxylic acid ammonium salt in greater than 98%
yield.
In tr od u ction
dratase (NHase, EC 4.2.1.84) and amidase (EC 3.5.1.4),
can also be used to convert aliphatic nitriles to the
corresponding carboxylic acid ammonium salts in aque-
ous solution.6 The nitrile is initially converted by the
nitrile hydratase to an amide, which is subsequently
converted by the amidase to the corresponding carboxylic
acid ammonium salt. A wide variety of bacteria possess
a diverse spectrum of nitrile hydratase and amidase
activities, including Rhodococcus,7 Pseudomonas,8 Coryne-
bacterium,9 Brevibacterium,10,11 Bacillus,11 Bacteridium,11
and Micrococcus.11 The fungus Fusarium merismoides
TG-1 has also been used as catalyst for the hydrolysis of
aliphatic nitriles and dinitriles.12 In addition to the
production of carboxylic acids, the conversion of ω-hy-
droxynitriles by a microbial catalyst to lactones in one
step has recently been reported.13
Nitriles are readily converted to the corresponding
carboxylic acids by a variety of chemical methods, but
these methods typically require strongly acidic or basic
reaction conditions and high reaction temperatures, and
also often generate inorganic salts as unwanted byprod-
ucts.1 Enzyme-catalyzed hydrolysis of nitriles to the
corresponding carboxylic acids can be preferable to some
chemical methods, since the enzymatic reactions are
often run at ambient temperature, do not require the use
of strongly acidic or basic reaction conditions, and do not
produce large amounts of undesirable byproducts. An
additional advantage of enzyme-catalyzed hydrolysis of
nitriles is that, for a variety of aliphatic or aromatic
dinitriles, the hydrolysis reaction can be highly regiose-
lective, where only one of the two nitrile groups is
hydrolyzed to the corresponding carboxylic acid am-
monium salt.
The use of aromatic nitrilases for the hydrolysis of
aromatic nitriles to the corresponding carboxylic acid
ammonium salts (with no formation of an amide inter-
mediate) has been well-documented,2 but it is only
recently that aliphatic nitrilases (EC 3.5.5.7) from Rhodo-
coccus rhodochrous K22,3 Comamonas testosteroni Ct,4
and Rhodococcus rhodochrous NCIMB 112165 have been
reported. A combination of two enzymes, nitrile hy-
(5) (a) Gradley, M. L.; Knowles, C. J . Biotechnol. Lett. 1994, 16, 41-
46. (b) Bengis-Garber, C.; Gutman, A. L. Appl. Microbiol. Biotechnol.
1989, 32, 11-16.
(6) Sugai, T.; Yamazaki, T.; Yokoyama, M.; Ohta, H. Biosci. Biotech.
Biochem. 1997, 61, 1419-1427.
(7) (a) Meth-Cohn, O.; Wang, M.-X. J . Chem. Soc., Perkin Trans. 1
1997, 3197-3204. (b) Meth-Cohn, O.; Wang, M.-X. Chem. Commun.
1997, 1041-1042. (c) Ho¨nicke-Schmidt, P.; Schneider, M. P. J . Chem.
Soc., Chem. Commun. 1990, 648-650. (d) Blakey, A. J .; Colby, J .;
Williams, E.; O’Reilly, C. FEMS Microbiol. Lett., 1995, 129, 57-62.
(e) Crosby, J .; Moilliet, J .; Parratt, J . S.; Turner, N. J . J . Chem. Soc.,
Perkin Trans. 1 1994, 13, 1679-1687. (f) De Raadt, A.; Klempier, N.;
Faber, K.t; Griengl, H. J . Chem. Soc., Perkin Trans. 1 1992, 137-140.
(g) Yokoyama, M.; Sugai, T.; Ohta, H. Tetrahedron: Asymmetry 1993,
4, 1081-1084. (h) Cohen, M. A.; Sawden, J .; Turner, N. J . Tetrahedron
Lett. 1990, 31, 7223-7226.
(8) Yamada, H.; Asano, Y.; Tani, Y. J . Ferment. Technol. 1980, 58,
495-500.
(9) (a) Yamamoto, K.; Ueno, Y.; Otsubo, K.; Yamane, H.; Komatsu,
K.-I.; Tani, Y. J . Ferment. Bioengin. 1992, 73, 125-129. (b) Tani, Y.;
Kurihara, M.; Nishise, H.; Yamamoto, K. Agric. Biol. Chem. 1989, 53,
3143-3149.
(10) (a) Moreau, J . L.; Bigey, F.; Azza, S.; Arnaud, A.; Galzy, P.
Biocatalysis 1994, 10, 325-340. (b) Kerridge, A.; Parratt, J . S.; Roberts,
S. M.; Theil, F.; Turner, N. J .; Willetts, A. J . Bioorg. Med. Chem. 1994,
2, 447-455.
(11) Andresen, O.; Godtfredsen, S. E. European Patent 178,106B1,
1993.
† Central Research and Development Department.
‡ DuPont Nylon Intermediates and Specialties.
(1) (a) Larock, R. C. Comprehensive Organic Transformations:
Guide to Functional Group Preparations; VCH: New York, 1989; p
993. (b) Schaefer, F. C. In The Chemistry of the Cyano Group;
Rappoport, Z., Ed.; Interscience: New York, 1970; Chapter 6, pp 256-
262.
(2) (a) Kobayashi, M.; Shimizu, S. FEMS Microbiol. Lett. 1994, 120,
217-234. (b) Kobayashi, M.; Nagasawa, T.; Yamada, H. Eur. J .
Biochem. 1989, 182, 349-356.
(3) (a) Kobayashi, M.; Yanaka, N.; Nagasawa, T.; Yamada, H.
Tetrahedron 1990, 46, 5587-5590. (b) Kobayashi, M.; Yanaka, N.;
Nagasawa, T.; Yamada, H. J . Bacteriology 1990, 172, 4807-4815.
(4) (a) Cerbelaud, E.; Bontoux, M.-C.; Foray, F.; Faucher, D.; Levy-
Schil, S.; Thibaut, D.; Soubrier, F.; Crouzet, J .; Petre, D. Ind. Chem.
Libr. 1996, 8, 189-200. (b) Le´vy-Schil, S.; Soubrier, F.; Crutz-Le Coq,
A.-M., Faucher, D.; Crouzet, J .; Pe´tre, D. Gene 1995, 161, 15-20.
A
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S0022-3263(98)00438-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/25/1998

Chemoenzymic Production of Lactams
J . Org. Chem., Vol. 63, No. 14, 1998 4793
Ta ble 1. A. fa cilis 72W-Ca ta lyzed Hyd r olysis of
Alip h a tic r,ω-Din itr ilesa
Sch em e 1
A method for the preparation of five- and six-membered
ring lactams from aliphatic R,ω-dinitriles has now been
developed (Scheme 1), whereby an aliphatic R,ω-dinitrile
is first converted to an ammonium salt of an ω-cyano-
carboxylic acid in aqueous solution using a microbial
catalyst. The ammonium salt of the ω-cyanocarboxylic
acid is then converted directly to the corresponding
lactam or N-alkyl lactam by hydrogenation in aqueous
solution, without isolation of the intermediate ω-cyano-
carboxylic acid or ω-aminocarboxylic acid. Two new
microbial catalysts have been isolated; the first, Acidovo-
rax facilis 72W, contains a nitrilase which converts an
R,ω-dinitrile directly to a corresponding ω-cyanocarboxyl-
ic acid ammonium salt. The second microbial catalyst,
Comamonas testosteroni 5-MGAM-4D, contains a com-
bination of two enzyme activities, nitrile hydratase and
amidase, where an aliphatic R,ω-dinitrile is initially
converted to an ω-cyanoalkylamide by the nitrile hy-
dratase, and the ω-cyanoalkylamide is subsequently
converted by the amidase to the corresponding ω-cyano-
carboxylic acid ammonium salt. When the aliphatic R,ω-
dinitrile is substituted at the R-carbon atom, the A. facilis
72W nitrilase produces the ω-cyanocarboxylic acid am-
monium salt resulting from hydrolysis of the ω-cyano
group with greater than 98% regioselectivity at 100%
conversion, thereby producing only one of the two possible
lactam products during the subsequent hydrogenation.
a
Reactions were run in phosphate buffer (20 mM, pH 7.0) at
27 °C, using 50 mg (wet cell weight) heat-treated A. facilis 72W/
b
mL of total reaction mixture volume. Concentration of ω-cyano-
carboxylic acid ammonium salt in final product mixture. c Yields
based on HPLC analysis of product mixtures.
normally be observed for a combination of nitrile hy-
dratase and amidase enzymes).
Resu lts
Heat-treated resting cells of Acidovorax facilis 72W
were used as catalyst for the hydrolysis of 2-ethylsucci-
nonitrile (1) to 3-cyanopentanoic acid ammonium salt (3),
2-methylglutaronitrile (2) to 4-cyanopentanoic acid am-
monium salt (4), and 2-methyleneglutaronitrile (5) to
4-cyano-4-pentenoic acid ammonium salt (6) in aqueous
reaction mixtures. At complete conversion of these
dinitriles, a 98-100% yield of only one of two possible
ω-cyanocarboxylic acid ammonium salts was typically
produced (Table 1). The corresponding dicarboxylic acid
diammonium salts were the only observed byproducts.
When the same catalyst was employed for the hydrolysis
of the unsubstituted aliphatic R,ω-dinitriles malononitrile
(7), succinonitrile (8), glutaronitrile (9), or adiponitrile
(10), the corresponding ω-cyanocarboxylic acid ammo-
nium salts of 2-cyanoacetic acid (11), 3-cyanopropionic
acid (12), 4-cyanobutanoic acid (13) and 5-cyanopentanoic
acid (14), respectively, were produced in yields ranging
from less than 50% to 99.7%, depending on the chain
length of the dinitrile (Table 1). The major byproducts
from hydrolysis of 8, 9, and 10 were succinic, glutaric,
and adipic acid diammonium salts, respectively, while
the major byproduct from 7 was 2-cyanoacetamide.
Several of the unsubstituted or R-alkyl-substituted
aliphatic R,ω-dinitriles used for the preparation of ω-cy-
anocarboxylic acid ammonium salts were only moderately
water soluble, and their solubility was dependent on the
temperature of the reaction mixture and the salt con-
centration (buffer and/or ω-cyanocarboxylic acid am-
monium salt) in the aqueous phase. In phosphate buffer
Hyd r olysis of Alip h a tic r,ω-Din itr iles. Acidovorax
facilis 72W (ATCC 55746) was isolated from soil samples
using 2-ethylsuccinonitrile (1) as sole nitrogen source.
When resting whole cells of A. facilis 72W were initially
examined as catalyst for the hydrolysis of the R-alkyl-
R,ω-dinitriles 1 or 2-methylglutaronitrile (2) at 27 °C, a
mixture of products was obtained. Over the course of
the hydrolysis reactions, the corresponding dicarboxylic
acid monoamide ammonium salt and dicarboxylic acid
diammonium salt were produced in addition to the
desired ω-cyanocarboxylic acid ammonium salt. It was
found that heating a suspension of A. facilis 72W in 0.10
M phosphate buffer (pH 7.0) at 50 °C for 1 h deactivated
an endogenous nitrile hydratase activity which catalyzed
the production of the dicarboxylic acid monoamides
(which were subsequently converted by an amidase to
the corresponding dicarboxylic acid), but did not affect
the nitrilase activity present. Heat-treated microbial
cells of A. facilis 72W converted R-alkyl-R,ω-dinitriles to
the corresponding ω-cyanocarboxylic acid ammonium
salts resulting from hydrolysis of the ω-cyano group with
extremely high regioselectivity. The identification of the
enzyme as a nitrilase was based in part on the absence
of formation of any amide intermediates by the heat-
treated cells, and the inability of the heat-treated cells
to convert amides to carboxylic acids (which would
(13) Taylor, S. K.; Chmiel, N. H.; Simons, L. J .; Vyvyan, J . R., J .
Org. Chem. 1996, 61, 9084-9085.

4794 J . Org. Chem., Vol. 63, No. 14, 1998
Gavagan et al.
(20 mM, pH 7.0) at 25 °C, the solubility limits of 1, 2, 5,
and 10 were determined to be ca. 0.30 M (32 g/L), 0.52
M (56 g/L), 0.42 M (45 g/L), and 0.60 M (65 g/L),
respectively. Production of aqueous solutions containing
ω-cyanocarboxylic acid ammonium salts at a concentra-
tion greater than the solubilty limit of the starting R,ω-
dinitrile was accomplished in batch reactions by prepar-
ing a mixture which was initially composed of two
phases: an aqueous phase containing the microbial cell
catalyst and dissolved R,ω-dinitrile, and the undissolved
R,ω-dinitrile. As the reaction progressed, the dinitrile
dissolved into the aqueous phase, and over the course of
the reaction a single-phase aqueous product mixture was
obtained. The highest final concentration of aliphatic
ω-cyanocarboxylic acid ammonium salt generated in a
product mixture at complete conversion of an R,ω-
dinitrile was 2.0 M for 4-cyanopentanoic acid ammonium
salt (254 g/L as the carboxylic acid).
The yields of ω-cyanocarboxylic acid ammonium salts
3, 4, 6, and 12 (Table 1) were independent of the
concentration of dinitrile initially present in the reaction
mixture, and independent of the final product concentra-
tion over a range of concentrations from 0.10 to 2.0 M.
The yields of ω-cyanocarboxylic acid ammonium salts 11
and 13 increased slightly with increasing initial dinitrile
concentration or final product concentration over this
same concentration range; at 0.40 M 7 or 9, the yields of
11 and 13 were only 79% and 85%, respectively (com-
pared to 83% at 0.83 M 11 and 92% at 1.38 M 13; Table
1).
F igu r e 1. Time course for the hydrolysis of 2-methylglu-
taronitrile (2, 0.40 M) in aqueous phosphate buffer (20 mM,
pH 7.0) containing A. facilis 72 PF-15 (50 mg wet cell weight/
mL) at 27 °C: 2-methylglutaronitrile (b), 4-cyanopentanoic
acid ammonium salt (4) (2), 2-methylglutaric acid diammo-
nium salt (0).
Ta ble 2. Com p a r ison of A. fa cilis 72W a n d A. fa cilis
72W P F -15 Micr obia l Cell Ca ta lysts for Hyd r olysis of
2-Meth ylglu ta r on itr ile (2)a
4 + 15
(M)
4
15
catalyst
(% yield)b (% yield)b
A. facilis 72W, not heated
A. facilis 72W, heat-treated
A. facilis 72 PF-15, not heated
A. facilis 72W, heat-treated
A. facilis 72 PF-15, not heated
0.10
0.10
0.10
1.00
1.00
63
99
96
99
99
35
1.0
4.0
1.0
1.0
The wet cell weight of the microbial catalyst used in
hydrolysis reactions typically ranged from 10 g/L to 50
g/L of total reaction mixture volume. The temperature
of the hydrolysis reaction was chosen to optimize both
the reaction rate and the stability of the microbial cell
catalyst and was typically 27 °C, although reactions were
run between 15 °C and 35 °C. Catalyst suspensions were
prepared by dispersing the cells in an aqueous phosphate
buffer which maintained the initial pH of the reaction
between 7.0 and 8.0. After addition of the aliphatic
dinitrile, the pH of the reaction mixture changed only
slightly over the course of the reaction due to the
formation of an ammonium salt of the carboxylic acid;
reactions were run to complete conversion of dinitrile
with no additional pH control.
Two mutants of the A. facilis 72W strain were prepared
(by chemical mutagenesis) which produced only very low
levels of the undesirable nitrile hydratase activity re-
sponsible for the formation of the byproducts obtained
when using unheated A. facilis 72W. These mutant
strains, A. facilis 72 PF-15 (ATCC 55747) and A. facilis
72 PF-17 (ATCC 55745), did not require heat-treatment
of the cells prior to use as catalyst for the hydrolysis of
an aliphatic R,ω-dinitrile to the corresponding ammo-
nium salt of an ω-cyanocarboxylic acid (Figure 1). The
yields of 4 and byproduct 2-methylglutaric acid diam-
monium salt (15) which were produced by the hydrolysis
of 2 with unheated and heat-treated A. facilis 72W, and
with the unheated A. facilis 72 PF-15 mutant strain, are
compared in Table 2; a slight increase in the yield of 4
was observed with increasing substrate/product concen-
tration when using the PF-15 catalyst.
a
Reactions were run in phosphate buffer (20 mM, pH 7.0) at
27 °C, using 50 mg (wet cell weight) heat-treated A. facilis 72W/
mL of total reaction mixture volume. Yields based on HPLC
analysis of product mixtures.
b
Comamonas testosteroni 5-MGAM-4D (ATCC 55744),
contained several nitrile hydratase and amidase activi-
ties, and when initially employed as a microbial catalyst
for the hydrolysis of 10, produced adipic acid diammo-
nium salt (16), adipamide (17) and adipamic acid am-
monium salt (18) as the major reaction products, and only
a minor amount of 14. Heating a suspension of the cells
in phosphate buffer (20 mM, pH 7.0) at 50 °C for 1 h
deactivated an undesirable nitrile hydratase activity to
a significant extent, leaving the microbial catalyst with
a second nitrile hydratase activity which converted 10
to 5-cyanovaleramide (19), and an amidase which con-
verted 19 to 14 (Figure 2). At 100% conversion of 10,
heat-treated C. testosteroni 5-MGAM-4D produced 14 in
yields of from 97% (0.10 M) to 88% (1.25 M), where the
yield of 14 decreased and byproduct 18 increased with
increasing substrate/product concentration.
C. testosteroni 5-MGAM-4D was also examined as
catalyst for the hydrolysis of the unsubstituted aliphatic
dinitriles 7, 8, and 9, and the R-alkyl-substituted dini-
triles 1 and 2. Maximum yields of ω-cyanocarboxylic
acids from 7, 8, and 9 were only 6.1%, 70%, and 2.6%,
respectively, with amides and diamides being produced
as major reaction byproducts from 7 and 8. Very little
(<5%) conversion of 9 was obtained under any reaction
conditions. Hydrolysis of 1 produced 3 in at least a 99%
yield at either 0.10 or 1.0 M final product concentrations.
Hydrolysis of 2 (0.40 M) by C. testosteroni 5-MGAM-4D
produced a mixture of 4 (68% yield) and 2-methyl-4-
cyanobutanoic acid (20, 22.3% yield) as the major reaction
products. Reaction rates slowed significantly at all
A second novel microbial catalyst which was capable
of producing higher yields of 14 from 10 than A. facilis
72W was isolated from soil samples using 2-methylglu-
taramide as sole nitrogen source. This microbial catalyst,

Chemoenzymic Production of Lactams
J . Org. Chem., Vol. 63, No. 14, 1998 4795
Ta ble 3. P r od u ction of La cta m s by Hyd r ogen a tion of
ω-Cya n oca r boxylic Acid Am m on iu m Sa ltsa
F igu r e 2. Time course for the hydrolysis of adiponitrile (10,
0.40 M) in aqueous phosphate buffer (20 mM, pH 7.0) contain-
ing C. testosteroni 5-MGAM-4D (20 mg wet cell weight/mL) at
27 °C: adiponitrile (O), 5-cyanopentanoic acid ammonium salt
(14) (2), 5-cyanopentanamide (b), adipamide (9), adipamic acid
ammonium salt (4), adipic acid diammonium salt (0).
substrate/product concentrations above 0.40 M when
using C. testosteroni 5-MGAM-4D as catalyst, whereas
reaction rates with A. facilis 72 W were unchanged at
concentrations of up to 2.0 M substrate/product in single
batch reactions; this difference may indicate that the
nitrile hydratase of C. testosteroni may be more suscep-
tible to substrate and/or product inhibition than the
nitrilase of A. facilis.
The aqueous product mixtures containing ω-cyanocar-
boxylic acid ammonium salts were used directly for the
production of lactams in subsequent hydrogenation reac-
tions after recovery of the microbial cell catalyst by
centrifugation and/or filtration. The recovered catalyst
still maintained significant activity, and could be reused
in subsequent hydrolysis reactions; in one series of
catalyst recycles, 30 g (dry cell weight) of A. facilis 72W
was used in six consecutive batch reactions to produce
at total of 2.59 kg of 4 at concentrations of up to 2.0 M.
The ω-cyanocarboxylic acids were also readily isolated
from these aqueous product mixtures by acidification of
the product mixture and extraction of the resulting
ω-cyanocarboxylic acid with a suitable organic solvent,
followed by crystallization or distillation (see Experimen-
tal Section).
P r ep a r a tion of La cta m s fr om ω-Cya n oca r boxylic
Acid Am m on iu m Sa lts. Hydrogenations of ω-cyano-
carboxylic acid ammonium salts in aqueous solution were
performed using a Raney nickel catalyst at temperatures
of 70 °C to 180 °C. Ammonium hydroxide was added to
reaction mixtures at a concentration of up to 3.0 M (added
in addition to the ammonium ion concentration already
present as the ammonium salt of the carboxylic acid) to
limit reductive alkylation of the imine intermediate
produced during the hydrogenation by the product ω-ami-
nocarboxylic acid.14 Hydrogenation of 14 under these
reaction conditions proceeded to at least 95% conversion
and produced 6-aminohexanoic acid ammonium salt (21)
as the major product, with less than a 3% yield of
caprolactam (22) being produced (Table 3). Yields of 22
of less than 3% were also obtained when aqueous
solutions prepared by mixing authentic 21 and am-
a
1.0
M ω-cyanocarboxylic acid ammonium salt, 500 psig
b
hydrogen, 2 h reaction time. Chromium-promoted Raney-nickel
catalyst, wt % based on wt of acid. c Yields based on GC analysis
d
of product mixtures. 3 h reaction time.
monium hydroxide were treated under these same hy-
drogenation conditions. The cyclization of 21 (prepared
by the hydrogenation of 14 at 70 °C) in aqueous solution
was also attempted at temperatures greater than 200 °C
by first removing the hydrogenation catalyst, then heat-
ing the ca. 1.0 M solution of 21 at 280 °C for 2 h. An
18% yield of 22 was produced with no added ammonia,
and at 1.0 and 2.0 M added ammonia the yields were
17% and 9.0%, respectively.
Using the same reaction conditions described above for
the hydrogenation of 14, aqueous mixtures of 3 were
directly converted to the corresponding lactam 4-eth-
ylpyrrolidin-2-one (23) in yields of up to 91%, and 4 was
directly converted to 5-methyl-2-piperidone (24) in yields
as high as 96% (Table 3). The yields of 23 and 24 both
increased with increasing concentration of added am-
monium hydroxide. Hydrogenation of aqueous solutions
of 6 resulted in hydrogenation of both the nitrile and
carbon-carbon double bond to produce 24 in up to 85%
yield. Aqueous mixtures of 12 were directly converted
to the corresponding lactam 2-pyrrolidinone (25) in yields
of up to 91%, and 13 was directly converted to 2-piperi-
done (26) in yields as high as 94% (Table 3). Hydrogena-
tion of 11 to 2-azetidinone (a four-membered ring lactam)
was not attempted, as â-alanine (the expected hydroge-
nation product) could not be converted to azetidinone in
the presence of a 3-fold excess of added ammonia at 160
°C, and authentic 2-azetidinone was completely converted
to unidentified products under the hydrogenation condi-
tions tested.
Five-membered ring and six-membered ring lactams
were also produced in high yields when aqueous solutions
of ω-aminocarboxylic acids, the expected initial hydro-
genation products of ω-cyanocarboxylic acids, were heated
with a 3-fold molar excess of added ammonia under the
hydrogenation conditions typically employed (500 psig
hydrogen, 180 °C); quantitative conversion of 4-aminobu-
tanoic acid to 25, and 5-aminopentanoic acid to 26, was
(14) De Bellefon, C.; Fouilloux, P. Catal. Rev. Sci. Eng. 1994, 36,
459-506.

4796 J . Org. Chem., Vol. 63, No. 14, 1998
Gavagan et al.
Sch em e 2
obtained. In contrast, cyclization of 6-aminohexanoic acid
in aqueous solution with 2-fold molar excess of added
ammonium hydroxide at 170 °C produced 22 in 39%
yield; the significantly lower yields of 22 obtained upon
hydrogenation of 14 may have resulted from a low
selectivity for production of 21 from 14 under the reaction
conditions employed.
The addition of an excess of ammonia (as ammonium
hydroxide) to the hydrogenations of ω-cyanocarboxylic
acid ammonium salts in aqueous solution resulted in
some conversion of the starting material to the dicar-
boxylic acid monoamide ammonium salt and/or the
dicarboxylic acid diammonium salt under the reaction
conditions employed, but in very low yields relative to
the production of five-membered ring and six-membered
ring lactams. A second byproduct-forming reaction be-
tween excess ammonia and the product lactam could
have produced an equilibrium mixture of the lactam with
its expected ammonolysis product, an ω-aminocarbox-
amide, but the high yields of five-membered and six-
membered ring lactams attained under the reaction
conditions employed suggest that ammonolysis (or base-
catalyzed hydrolysis) of the lactams was not significant.
The preparation of N-methyllactams from 3 or 4 by the
substitution of methylamine for ammonia in the hydro-
genation reactions was also examined. Addition of from
1 to 4 equiv of methylamine (pKa ) 10.62 for the
protonated amine)15 to an aqueous solution of 4 contain-
ing 1 equiv of ammonium ion (pKa 9.25)16 was expected
to convert a significant amount of ammonium ion to
ammonia (due to the relative differences in pKas of
protonated methylamine and ammonium ions in water).
This ammonia could compete with unprotonated methy-
lamine for reaction with the intermediate imine produced
during hydrogenation of the nitrile, and in the case of 4,
produce a mixture of 5-amino-4-methylpentanoic acid
(27) and 5-(N-methylamino)-4-methylpentanoic acid (28)
ammonium salts (Scheme 2). These intermediates would
subsequently cyclize to produce 24 and 1,5-dimethyl-2-
piperidone (29), respectively.
The relative yields of 24 and 29 produced by hydroge-
nation of 4 (1.0 M) in aqueous solution with added
methylamine were dependent on the choice of catalyst.
Raney nickel and ruthenium/alumina each produced 24
as the major lactam product, even in the presence of 3
equiv of methylamine, while 29 was the major product
when using 5% Pd/carbon, or 4.5% Pd/0.5% Pt/carbon,
as catalyst at the same methylamine concentration
(Table 4). When using 5% Pd/carbon as catalyst, the
yield of 29 increased with increasing concentration of
methylamine. The substitution of 2.0 M methylamine
for ammonia in the hydrogenation of an aqueous solu-
tions of 3 (1.0 M) at 140 °C and using 5% Pd/carbon
catalyst produced 4-ethyl-1-methylpyrrolidin-2-one (30)
and 23 in 69.8% and 20.4% yields, respectively, at 96%
conversion.
hydrogenation catalysts was observed when the hydro-
genation of ω-cyanocarboxylic acid ammonium salt mix-
tures produced via microbial hydrolysis were compared
with the hydrogenation of aqueous solutions of the same
ω-cyanocarboxylic acid which were first isolated from the
hydrolysis product mixtures and purified prior to hydro-
genation as the corresponding ammonium salt. The
lactams or N-methyllactams were readily isolated from
the hydrogenation product mixtures by first filtering the
mixture to recover the hydrogenation catalyst and then
distilling the product directly from the resulting aqueous
filtrate. The ammonia produced during the cyclization
reaction, or added to the reaction mixture, could also be
recovered for recycling by this distillation process, thus
avoiding the generation of undesirable inorganic salts as
waste products. The lactams or N-methyllactams were
alternately recovered by filtering the product mixture to
recover the catalyst, adjusting the filtrate to a pH of ca.
7.0 with concentrated HCl and saturation with sodium
chloride, and extraction of the lactam or N-methyllactam
(batch or continuous extraction) with an appropriate
organic solvent.
Discu ssion
There are currently no nonenzymatic catalysts which
hydrolyze only one nitrile group of an aliphatic R,ω-
dinitrile to a carboxylic acid ammonium salt with high
regioselectively at complete conversion of the dinitrile.
Nonenzymatic hydrolysis of a dinitrile can be run to
incomplete conversion (typically less than 20% conver-
sion) to obtain regioselective production of a monoacid,
but the product must then be separated from unreacted
dinitrile and the dinitrile recycled. Nonenzymatic nitrile
hydrolysis also typically employs a strong acid or base
catalyst at elevated temperatures, and the steps required
Lysis of the microbial cell catalysts during the hydroly-
sis reaction, or contaminants present from the microbial
cell catalyst preparation, could have introduced com-
pounds into the hydrogenation reaction mixture (e.g.,
thiols) which could have poisoned the hydrogenation
catalyst activity. No poisoning or deactivation of the
(15) Lange’s Handbook of Chemistry, 14th ed.; Dean, J . A., Ed.;
McGraw-Hill: NY, 1992; p 8.55.
(16) Ibid., p 8.13.

Chemoenzymic Production of Lactams
J . Org. Chem., Vol. 63, No. 14, 1998 4797
Ta ble 4. Rela tive Yield s of La cta m a n d N-Meth ylla cta m fr om Hyd r ogen a tion of 4-Cya n op en ta n oic Acid Am m on iu m
Sa lt (4)a
entry
temp (°C)
[CH3NH2] (M)
catalystb
conv (%)
29 (% yield)c
24 (% yield)c
1
2
3
4
5
6
7
8
9
160
160
140
160
160
160
160
160
160
180
160
3.0
3.0
3.0
1.0
1.25
1.5
2.0
3.0
4.0
3.0
2.3
Raney-Ni
5% Ru/Al2O3
5% Pd/C
5% Pd/C
5% Pd/C
5% Pd/C
5% Pd/C
5% Pd/C
5% Pd/C
100
100
99
100
100
100
100
100
100
97
19.2
19.0
83.4
53.5
68.3
76.4
81.5
81.7
88.4
73.0
94.0
68.5
63.5
0
0
0
0
0
7.8
1.9
6.6
3.1
10
5% Pd/C
4.5% Pd/0.5% Pt/C
11d
99
a
b
1.0 M 4, 500 psig hydrogen, 2 h reaction time. 5 wt %, based on wt of 4 as acid. c Yields based on GC analysis of product mixtures.
d
1.4 M 4, 3 h reaction time, 2.5 wt % catalyst, based on wt of 4 as acid.
to separate and purify the product from this reaction
mixture can be complicated and costly. The enzyme-
catalyzed hydrolysis of dinitriles described herein can be
run to complete conversion in aqueous solution at ambi-
ent temperature and at neutral pH with no added acid
or base. High yields (83-100%) and product concentra-
tions of up to 25% (weight/volume) have been demon-
strated, and product recovery and catalyst recycle were
simple to perform.
thermal instability, and several nitrile hydratases which
have been employed in the commercial production of
acrylamide from acrylonitrile are relatively unstable in
use at temperatures above 5-15 °C.18 Recently, both
nitrilase and nitrile hydratase activities having improved
thermal stability been identified in the thermophile
Bacillus pallidus Dac521.19
Hydrogenations of ω-cyanocarboxylic acid ammonium
salts in aqueous solution were typically run using an
excess of added ammonia (as ammonium hydroxide) to
prevent reductive alkylation of the nitrile, and the pH of
the reaction mixture was typically between 9 and 10.
Under these reaction conditions, the initial hydrogenation
product was an ammonium salt of an ω-aminocarboxylic
acid. The pKas of the carboxylic acid and the protonated
amine of 21 are 4.373 and 10.804, respectively.20 At a
pH between 9 and 10, greater than 99.9% of the carboxy-
lic acid functionality of 21 would be present as the
ammonium salt, and a significant fraction of the ω-amino
group would also be protonated. Although the pKas of
the carboxylic acid and protonated amine of 4-amino-3-
ethylbutanoic acid (from hydrogenation of 3) or 27 have
not been reported, they are most likely similar to those
of the corresponding acid and amine groups of 21.
Intramolecular cyclization to produce a lactam by reac-
tion of the ω-amino group with the ammonium carboxy-
late in excess aqueous ammonia was not expected under
these reaction conditions.
The nitrilase activity of A. facilis 72 W and 72W PF-
15 microbial cell catalysts was particularly effective at
completely converting several R-alkyl-substituted- and
unsubstituted aliphatic R,ω-dinitriles to the correspond-
ing ω-cyanocarboxylic acids (as their ammonium salts)
at concentrations of up to 2.0 M. When the R,ω-dinitrile
was substituted at the R-position (e.g., 1 and 2, byprod-
ucts of a commercial process for the production of
adiponitrile from butadiene and HCN),17 a ω-cyanocar-
boxylic acid was produced with 99-100% regioselectivity
at 100% conversion. When the R,ω-dinitriles were un-
substituted, the regioselectivity of the A. facilis 72W
nitrilase was dependent on the chain length, with an
optimum regioselectivity observed for hydrolysis of 8. In
the case of the dinitrile 10, where A. facilis 72W produced
the dicarboxylic acid diammonium salt 16 as the major
hydrolysis product, C. testosteroni 5-MGAM-4D utilized
a combination of nitrile hydratase and amidase activities
to produce the desired ω-cyanocarboxylic acid ammonium
salt 14 with high regioselectivity. The nitrile hydratase
of heat-treated C. testosteroni 5-MGAM-4D did not show
the same regioselectivity for the hydrolysis of R-alkyl R,ω-
dinitriles to ω-cyanocarboxylic acid ammonium salts as
was found for A. facilis 72W nitrilase.
Cyclization of ω-aminoaliphatic carboxylic acids or
ω-(N-alkylamino) aliphatic carboxylic acids to produce
lactams have typically been run under reaction conditions
which remove water and/or ammonia.21-23 Five-mem-
bered ring lactams have been reported to be produced in
Both the A. facilis 72 W and C. testosteroni 5-MGAM-
4D microbial cell catalysts contained more than one
enzyme capable of nitrile hydrolysis, which initially
resulted in poor selectivities to the desired ω-cyanocar-
boxylic acids. A simple heat treatment of an aqueous
suspension of the microbial cells prior to their use as
catalyst eliminated the undesirable nitrile hydratase
activity, while leaving the relatively heat-stable nitrilase
of A. facilis 72W, or both a nitrile hydratase and amidase
of C. testosteroni 5-MGAM-4D, unaffected. The stability
of the C. testosteroni 5-MGAM-4D nitrile hydratase to
heating at 50 °C for periods of 1-2 h was unexpected;
mesophilic nitrile hydratases have typically been used
only at ambient temperature or lower because of their
(18) (a) Yamada, H.; Kobayashi, M. Biosci. Biotech. Biochem. 1996,
60, 1391-1400. (b) Nagasawa, T.; Shimizu, H.; Yamada, H. Microbiol.
Biotechnol. 1993, 40, 189-195. (c) Hwang, J .-S.; Chang, H.-N. Kor. J .
Appl. Microbiol. Biotech. 1990, 18, 56-60. (d) Ashina, Y.; Suto, M. In
Industrial Application of Immobilized Biocatalysts; Tanaka, A., Tosa,
T., Kobayashi, T., Eds.; Marcel Dekker: New York, 1993; Chapter 7.
(e) Asano, Y.; Fujishiro, Y. T.; Tani, Y.; Yamada, H. Agric. Biol. Chem.
1982, 46, 1165-1174.
(19) Cramp, R.; Gilmour, M.; Cowan, D. A., Microbiology 1997, 143,
2313-2320.
(20) Lange’s Handbook of Chemistry, Dean, 14th ed., J . A., Ed.;
McGraw-Hill: NY, 1992; p 8.22.
(21) (a) Taylor, E. C.; McKillop, A. J . Am. Chem. Soc. 1965, 87,
1984-1990. (b) Taylor, E. C.; McKillop, A.; Ross, R. E. J . Am. Chem.
Soc. 1965, 87, 1990-1995. (c) Blade´-Font, A. Tetrahedron Lett. 1980,
21, 2443-2446. (c) Demmin, T. R.; Rogic, M. M. US Patent 4,329,498,
1982.
(22) Frank, R. L.; Schmitz, W. R.; Zeidman, B. Org. Synth. 1954, 3,
328-329.
(23) Mares, F.; Sheehan, D. Ind. Eng. Chem. Process Des. Dev. 1978,
17, 9-16.
(17) (a) Tolman, C. A.; Seidel, W. C.; Druliner, J . D.; Domaille, P. J .
Organometallics 1984, 3, 33-38. (b) Seidel, W. C.; Tolman, C. A. Ann.
N. Y. Acad. Sci. 1983, 415, 201-221.

4798 J . Org. Chem., Vol. 63, No. 14, 1998
Gavagan et al.
aqueous solution only under acidic conditions,21c requir-
ing the presence of the protonated carboxylic acid and
not the carboxylate salt. The cyclization of 6-aminohex-
anoic acid to 22 using water or ethanol as solvent has
been extensively studied.23 In water, the cyclization
reaction was reversible, and the percentage of 22 was
reported to increase from 38.7% at 180 °C to 92.2% at
250 °C; higher yields of 22 were obtained in ethanol,
which was attributed to a shift in the equilibrium which
favors the free-acid/free-amine form of 6-aminohexanoic
acid in ethanol, rather than the intramolecular alkylam-
monium carboxylate form which predominates in water.
The processes described above for production of lactams
or N-alkyllactams suffer from one or more of the following
disadvantages: the use of temperatures in excess of 250
°C to obtain high yields of lactams when using water as
a solvent, the removal of water from the reaction mixture
to drive the equilibrium toward lactam formation, the
adjustment of the pH of the reaction mixture to an acidic
value to favor lactam formation, or the use of an organic
solvent in which the starting material is sparingly
soluble. Many of these processes generate undesirable
waste streams, or mixtures of products which are not
easily separated. N-alkyllactams have also been pro-
duced by the direct hydrogenation of aqueous mixtures
containing 2, a primary alkylamine, and a hydrogenation
catalyst;24 a mixture of 1,3- and 1,5-dialkyl-2-piperidones
are produced at lower yields than for the corresponding
process described here.
The preparation of five- and six-membered ring lac-
tams in two steps by the initial enzyme-catalyzed hy-
drolysis of aliphatic R,ω-dinitriles to ω-cyanocarboxylic
acid ammonium salts, followed by the direct hydrogena-
tion of the resulting aqueous product mixture, proceeded
in high yield, with little byproduct or waste stream
production, and with a facile method of product recovery.
This method did not require the isolation of the ω-cy-
anocarboxylic acid ammonium salt from the product
mixture of the hydrolysis reaction prior to the hydroge-
nation step, nor did it require the conversion of the
ω-cyanocarboxylic acid ammonium salt to the free acid
prior to hydrogenation, or isolation of the resulting
ω-aminocarboxylic acid ammonium salt from the hydro-
genation product mixture and conversion of the am-
monium salt to the free carboxylic acid prior to cycliza-
tion. The inability to produce four-membered ring lactams,
and the very low yields of the seven-membered ring
lactam 22 produced by these reaction conditions, may be
due in part to the relatively greater ring strain of four-
and seven-membered rings,25 such that intermolecular
polymerization effectively competes with intramolecular
lactamization.
shaker tubes. The percent recovery of aliphatic R,ω-dinitriles
and the percent yields of the hydrolysis products formed in
nitrile hydrolysis reactions were based on the initial amount
of R,ω-dinitrile present in the reaction mixture, and deter-
mined by HPLC using a refractive index detector and either
a Supelcosil LC-18 DB column (25 cm × 4.6 mm diam) and 10
mM acetic acid/10 mM sodium acetate in 2.5% methanol/water
as mobile phase (for hydrolysis of 1, 2, 5, 9, and 10), or a Bio-
Rad HPX-87H column (30 cm × 7.8 mm diam) and 0.001 N
sulfuric acid as mobile phase at 50 °C (for hydrolysis of 7 and
8). The yields of lactams and N-methyllactams produced by
the hydrogenation of aqueous solutions of ω-cyanocarboxylic
acid ammonium salts were based on the initial concentration
of ω-cyanocarboxylic acid ammonium salt present in the
reaction mixture and determined by gas chromatography using
a DB-1701 capillary column (30 m × 0.53 mm ID, 1 µm film
thickness). Isolated yields of ω-cyanocarboxylic acids and
lactams are unoptimized, and melting points are uncorrected.
Chemical shifts for 1H and 13C NMR spectra are expressed in
parts per million positive values downfield from internal TMS.
Identification of lactam products 25 and 26 was made by
comparison of 1H and 13C NMR spectra to commercially
available samples.
Acidovorax facilis 72W (ATCC 55746), Acidovorax facilis 72
PF-15 (ATCC 55747), Acidovorax facilis 72 PF-17 (ATCC
55745), and Comamonas testosteroni 5-MGAM-4D (ATCC
55744) were stored frozen at -80 °C. Wet cell weights of whole
cell catalysts employed in reactions or assays were obtained
from cell pellets prepared by centrifugation of fermentation
broth or cell suspensions in buffer. Dry cell weights were
determined by lyophilization of wet cells, and the ratio of dry
cell weight to wet cell weight for all cells was typically 0.25.
Assays of the nitrilase activity of A. facilis 72 W (heated as a
10% wet cell weight suspension in 0.10 M phosphate buffer
(pH 7.0) for 1 h at 50 °C), A. facilis 72 PF-15, and A. facilis 72
PF-17 were performed by stirring a suspension of 12.5 mg dry
cell weight/mL in 25 mM phosphate buffer (pH 7.0) and 0.30
M dinitrile substrate at 25 °C and analyzing aliquots removed
at 1, 5, 10 and 15 min for the rate of production of ω-cyano-
carboxylic acid ammonium salt. A unit of nitrilase activity
(IU) was equivalent to 1 µmol/min of production of ω-cyano-
carboxylic acid ammonium salt.
Scr een in g Rea ction s for Micr obia l Hyd r olysis of Ali-
p h a tic r,ω-Din itr iles. In a typical procedure, 0.60 g (wet
cell weight) of frozen A. facilis 72W cells (previously heat-
treated at 50 °C for 1 h) and 12 mL of potassium phosphate
buffer (20 mM, pH 7.0) were added to a 15-mL polypropylene
centrifuge tube. After thawing and suspending the cells, the
resulting suspension was centrifuged and the supernatant
discarded. The resulting cell pellet was resuspended in a total
volume of 12 mL of this same phosphate buffer. Into a second
15-mL centrifuge tube was weighed 0.1081 g (1.00 mmol) of
2, and then 9.89 mL of the A. facilis 72W cell suspension
(containing 0.494 g wet cells) was added and the resulting
suspension mixed on a rotating platform at 27 °C. Samples
(0.300 mL) were withdrawn and centrifuged, and then 0.180
mL of the supernatant was placed in a Millipore Ultrafree-
MC filter unit (10 K MWCO) and mixed with 0.020 mL of an
aqueous solution of 0.750 M N-methylpropionamide (HPLC
internal standard). Sufficient 1.0 M HCl was added to lower
the pH of the sample to ca. 2.5, and the resulting solution was
filtered and analyzed by HPLC. After 1.0 h, the HPLC yields
of 4 and 2-methylglutaric acid were 99.3% and 0.7%, respec-
tively, with 100% conversion of 2.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. All chemicals and hydrogenation
catalysts were obtained from commercial sources and used as
received unless otherwise noted. Hydrogenations were per-
formed in a 300-mL stirred autoclave or in 20-mL glass vial
4-Cya n op en ta n oic Acid .26 Into a 4-L Erlenmeyer flask
equipped with a magnetic stir bar was placed 150 g (wet cell
weight) of frozen A. facilis 72W (previously heat-treated in 20
mM phosphate buffer at pH 7.0 at 50 °C for 1 h) and suspended
in a total volume of 2.5 L of potassium phosphate buffer (20
mM, pH 7.0). With stirring, 129.6 g (136.4 mL, 1.20 mol, 0.400
M) of 2 was added, and the final volume adjusted to 3.00 L
with phosphate buffer. The mixture was stirred at 25 °C, and
samples were withdrawn at regular intervals and analyzed
by HPLC. After 21.5 h, the HPLC yields of 4 and 2-methyl-
glutaric acid diammonium salt were 99.5% and 0.5%, respec-
(24) Kosak, J . R. US Patent 5,449,780, 1995.
(25) (a) Gutman, A. L.; Meyer, E.; Yue, X.; Abell, C. Tetrahedron
Lett. 1992, 33, 3943-3946. (b) Patterson, K. H.; Depree, G. J .; Zender,
J . A.; Morris, P. J . Tetrahedron Lett. 1994, 35, 281-284. (c) Sawada,
H. J . Macromol. Sci., Rev. Macromol. Chem. 1970, 51, 151-173. (d)
Wan, P.; Modro, T. A.; Yates, K. Can. J . Chem. 1980, 58, 2423-2432.
(c) Mandolini, L. J . Am. Chem. Soc. 1978, 100, 550-554. (d) DeTar,
D.; Luthra, N. P. J . Am. Chem. Soc. 1980, 102, 4505-4512.

Chemoenzymic Production of Lactams
J . Org. Chem., Vol. 63, No. 14, 1998 4799
tively, with 100% conversion of 2. The reaction mixture was
centrifuged and the supernatant decanted and filtered using
an Amicon 2.5 L Filter Unit equipped with a YM-10 filter (10K
MWCO). The filtrate was placed in a 4.0 L flask, and the pH
of the solution adjusted to 2.5 with 6 N HCl. The resulting
solution was saturated with sodium chloride, and then 1.0 L
portions of the resulting solution were extracted with 4 × 500
mL of ethyl ether. The combined ether extracts were dried
(MgSO4) and filtered, and the filtrate was concentrated to 1.0
L by rotary evaporation at reduced pressure. To the concen-
trate were then added 1.2 L of hexane and 200 mL of ethyl
ether, and the resulting solution was cooled to -78 °C. The
white crystals which formed were isolated by rapid vacuum
filtration and washed with 300 mL of cold (5 °C) hexane.
Residual solvent was removed under high vacuum (150 mil-
litorr) to yield 120.3 g (79% isolated yield) of 4-cyanopentanoic
pH 7.0). After 1.0 h, the HPLC yield of 12 and succinic acid
diammonium salt were 99.7% and 0.3%, respectively, with
100% conversion of 8. The final concentration of 12 in the
filtered product mixture was 1.31 M. A 200-mL portion of the
filtrate was adjusted to pH 2.5 with 6 N HCl and then
saturated with sodium chloride and extracted with 4 × 200
mL of ethyl ether. The combined organic extracts were dried
(MgSO4) and filtered, and the solvent was removed by rotary
evaporation at reduced pressure. The resulting colorless oil
was dissolved in 150 mL of ethyl ether, 100 mL of hexane was
added, and then the resulting solution cooled to -78 °C. The
resulting white solid which crystallized was isolated by
vacuum filtration, and residual solvent was removed under
high vacuum (150 millitorr) to yield 14.32 g (55% isolated yield)
of 3-cyanopropanoic acid: mp 49.5-51.0 °C; 1H NMR (300
MHz, CDCl3) δ 11.49 (s, 1H), 2.78 (t, J ) 6.8 Hz, 2 H), 2.67 (t,
J ) 6.8, 2 H); 13C NMR (75 MHz, CDCl3) δ 175.7, 118.2, 29.5,
12.5; IR (KBr) 3418-2883 (b), 2981, 2954, 2683, 2251, 1713,
1420, 1345 cm-1; MS (EI) m/z 99 (M+, 11), 82 (15), 54 (100),
45 (24); HRMS calcd for C4H5NO2 (M+) 99.0320, found 99.0320.
4-Cya n obu ta n oic Acid .26 The procedure for the prepara-
tion of 4-cyanopentanoic acid was repeated using 15.0 g of
heat-treated A. facilis 72W cells and 42.78 g (0.450 mol) of 9
in a total volume of 300 mL of potassium phosphate buffer
(20 mM, pH 7.0). After 4.0 h, the HPLC yield of 13 and
glutaric acid diammonium salt were 92.3% and 7.7%, respec-
tively, with 100% conversion of 9. The filtered product mixture
was adjusted to pH 3.5 with 6 N HCl and then saturated with
sodium chloride and extracted with 4 × 300 mL of ethyl ether.
The combined organic extracts were dried (MgSO4) and
filtered, and the solvent was removed by rotary evaporation
at reduced pressure followed by stirring under vacuum (100
millitorr) to yield 35.3 g (62% yield) of 4-cyanobutanoic acid
as a pale yellow oil which solidified upon standing. The solid
was recrystallized from 1:1 ethyl acetate/hexane at 5 °C; mp
39.6-40.2 °C; 1H NMR (300 MHz, CDCl3) δ 11.70 (s, 1 H), 2.56
(t, J ) 7.0 Hz, 2 H), 2.50 (t, J ) 7.0 Hz, 2 H), 2.00 (quintet, J
) 7.0 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 177.8, 118.7, 32.0,
20.3, 16.3; IR (CHCl3) 3600-2800 (b), 3022, 2949, 2671, 2250,
1713, 1428, 1417 cm-1; MS (EI) m/z 114 (MH+, 14), 96 (45),
67 (34), 60 (45), 54 (55), 41 (100); HRMS calcd for C5H8NO2
(MH+) 114.0555, found 114.0528.
1
acid: mp 31.9-32.6 °C; H NMR (300 MHz, CDCl3) δ 10.75
(s, 1 H), 2.84-2.72 (m, 1 H), 2.68-2.50 (m, 2 H), 1.99-1.85
(m, 2 H), 1.36 (d, J ) 7.1 Hz, 3 H); 13C NMR (75 MHz, CDCl3)
δ 178.1, 122.0, 31.1, 28.7, 24.8, 17.8; IR (melt) 3700-2800 (br),
3118, 2986, 2673, 2242, 1713, 1387 cm-1; MS (EI) m/z 128
(MH+, 12), 110 (78), 109 (40), 100 (11), 81 (82), 68 (100); HRMS
calcd for C6H10NO2 (MH+) 128.0712, found 128.0704.
3-Cya n op en ta n oic Acid . The procedure for the prepara-
tion of 4-cyanopentanoic acid was repeated using 161 g of heat-
treated A. facilis 72W cells and 325 g (3.00 mol) of 1 in at total
volume of 2.40 L of potassium phosphate buffer (20 mM, pH
7.0). After 183 h, the HPLC yield of 3 was 100%, with 100%
conversion of 1. The concentration of 3 in the filtered product
mixture was 1.26 M. A 300-mL portion of the filtrate was
adjusted to pH 2.5 with ca. 60 mL of 6 N HCl and then
saturated with sodium chloride and extracted with 4 × 200
mL of ethyl ether. The combined organic extracts were dried
(MgSO4) and filtered, and the solvent was removed by rotary
evaporation at reduced pressure at 28 °C. The resulting
slightly yellow viscous oil was stirred under high vacuum (50
millitorr) to remove residual solvent at 28 °C and then cooled
to -20 °C to produce 3-cyanopentanoic acid as a crystalline
white solid (45.9 g, 96% yield): mp 33.0-34.0 °C; 1H NMR
(300 MHz, CDCl3) δ 11.53 (s, 1 H), 3.02-2.93 (m, 1 H), 2.81-
2.61 (m, 2 H), 1.77-1.65 (m, 2 H), 1.11 (t, J ) 7.5 Hz, 3 H);
13C NMR (75 MHz, CDCl3) δ 175.2, 120.5, 35.8, 28.6, 24.8, 11.1;
IR (neat) 3600-2800 (br), 3200, 2974, 2940, 2882, 2244, 1737,
1463, 1411 cm-1; MS (EI) m/z 128 (MH+, 5), 99 (24), 82 (48),
68 (67), 54 (100); HRMS calcd for C6H10NO2 (MH+) 128.0712,
found 128.0708.
5-Cya n op en ta n oic Acid .28 The procedure for the prepara-
tion of 4-cyanopentanoic acid was repeated using 4.0 g of C.
testosteroni 5-MGAM-4D cells (previously heat-treated at 50
°C for 1 h) and 270 g (2.50 mol) of 10 in a total volume of 2.0
L of potassium phosphate buffer (20 mM, pH 7.0) at 27 °C.
After 63 h, the reaction rate had slowed considerably, so an
additional 10.0 g of the microbial cell catalyst was added to
the mixture. After 86 h, the HPLC yields of 14, adipamic acid
ammonium salt, adipamide, and adipic acid diammonium salt
were 88.2%, 4.7%, 6.6%, and 0%, with 100% conversion of 10.
A 200-mL portion of the filtered product mixture was adjusted
to pH 2.5 with 6 N HCl and then saturated with sodium
chloride and extracted with 4 × 200 mL of ethyl ether. The
combined ether extracts were dried (MgSO4) and filtered, and
the solvent was removed by rotary evaporation at reduced
pressure. Remaining ether was removed by stirring the
colorless liquid at room temperature under high vacuum (60
millitorr) for 5 h to yield 27.32 g (95% isolated yield) of
5-cyanopentanoic acid. The 5-cyanopentanoic acid was then
redistilled under vacuum at 110-112 °C (75 millitorr) without
decomposition: 1H NMR (300 MHz, CDCl3) δ 11.58 (s, 1 H),
2.44-2.35 (m, 4 H), 1.81-1.66 (m, 4 H); 13C NMR (75 MHz,
CDCl3) δ 177.9, 119.0, 32.2, 23.9, 22.9, 16.0; IR (neat) 3600-
2800 (b), 2949, 2878, 2248, 1737, 1709, 1459, 1424 cm-1; MS
(EI) m/z 128 (MH+, 18), 110 (100), 82 (25), 81 (40), 68 (48), 54
(60); HRMS calcd for C6H10NO2 (MH+) 128.0712, found
128.0713.
4-Cya n o-4-p en ten oic Acid . The procedure for the prepa-
ration of 4-cyanopentanoic acid was repeated using 50.0 g of
heat-treated A. facilis 72W cells and 133 g (1.25 mol) of 5 in a
total volume of 1.0 L of potassium phosphate buffer (20 mM,
pH 7.0). After 26 h, the HPLC yield of 6 was 100%, with 100%
conversion of 5. The final concentration of 6 in the filtered
product mixture was 1.298 M. A 100-mL portion of the filtrate
was adjusted to pH 2.7 with 6 N HCl and then saturated with
sodium chloride and extracted with 4 × 100 mL of ethyl ether.
The combined organic extracts were dried (MgSO4) and filtered
and the volume of the combined extracts reduced to 100 mL
by rotary evaporation at reduced pressure at 28 °C. To the
concentrate was added 200 mL of hexane, and the resulting
solution was cooled to -78 °C. The resulting white solid which
crystallized was isolated by rapid vacuum filtration and
washed with 100 mL of cold (5 °C) hexane. Residual solvent
was removed under high vacuum (150 millitorr) to yield 9.80
g (60% isolated yield) of 4-cyano-4-pentenoic acid (stored at
-20 °C): mp 26.5-27.0 °C; 1H NMR (300 MHz, CDCl3) δ 11.69
(s, 1 H), 5.94 (s, 1 H), 5.85 (s, 1 H), 2.69-2.59 (m, 4 H); 13C
NMR (75 MHz, CDCl3) δ 177.4, 131.7, 121.0, 117.9, 31.8, 29.3;
IR (neat) 3700-2800 (br), 3150, 2925, 2225, 1740, 1714, 1624,
1435, 1414 cm-1; MS (EI) m/z 126 (MH+, 8), 108 (48), 79 (100),
66 (8), 53 (32); HRMS calcd for C6H8NO2 (MH+) 126.0555,
found 126.0537.
5-Meth yl-2-p ip er id on e (24).29 Into a 100 mL graduated
cylinder was placed 54.4 mL of an aqueous solution of 4 (1.85
M, 0.100 mol, produced by microbial hydrolysis of 2 and
filtered as described above), 12.9 mL of concentrated am-
monium hydroxide (29.3% NH3, 0.20 mol NH3) was added, and
then the final volume was adjusted to 100 mL with distilled
3-Cya n op r op a n oic Acid .27 The procedure for the prepa-
ration of 4-cyanopentanoic acid was repeated using 20.0 g of
heat-treated A. facilis 72W cells and 101.1 g (1.25 mol) 8 in a
total volume of 1.00 L of potassium phosphate buffer (20 mM,

4800 J . Org. Chem., Vol. 63, No. 14, 1998
Gavagan et al.
water. To the resulting solution was added 0.631 g of
chromium-promoted Raney nickel (Grace Davison Raney 2400
Active Metal Catalyst), and the resulting mixture was charged
to a 300-mL stirred autoclave. After flushing the reactor with
nitrogen, the contents of the reactor were stirred at 1000 rpm
and heated at 160 °C under 500 psig of hydrogen for 3 h. After
cooling to room temperature, analysis of the product mixture
by gas chromatography and HPLC indicated a 96.4% yield of
24, with 100% conversion of 4. The product mixture was
filtered to remove the catalyst, adjusted to pH 6.0 with 6 N
HCl, and saturated with sodium chloride. The resulting
solution was extracted with 5 × 100 mL of dichloromethane,
the combined organic extracts were dried (MgSO4), filtered,
and the solvent was removed by rotary evaporation under
reduced pressure to yield a colorless oil. Removal of remaining
solvent under vacuum (100 millitorr) produced a white solid
which was recrystallized from 150 mL of ethyl ether at -78
°C to yield 6.69 g (59% isolated yield) of 24: mp 55.5-56.2
°C; 1H NMR (500 MHz, CDCl3) δ 7.17 (bs, 1 H), 3.33-3.28 (m,
1 H), 2.94-2.89 (m, 1 H), 2.44-2.31 (m, 2 H), 1.95-1.82 (m, 2
H), 1.52-1.43 (m, 1 H), 1.01 (d, J ) 6.6 Hz, 3 H); 13C NMR (75
MHz, CDCl3) δ 172.5, 49.0, 30.7, 29.0, 28.2, 18.4; IR (KBr)
3247, 3208, 3084, 2953, 2932, 2875, 1674, 1651, 1499, 1466
cm-1; MS (EI) m/z 113 (M+, 100), 98 (6), 84 (8), 70 (31), 56
(40), 42 (25); HRMS calcd for C6H11NO (M+) 113.0841, found
113.0840.
mixture was hydrogenated at 500 psig of hydrogen at 70 °C
for 4.5 h and an additional 5 h at 180 °C. Analysis of the
product mixture by HPLC and gas chromatography indicated
a 91.0% yield of 25, with 100% conversion of 12. An 81 mL
portion of the product mixture was filtered to remove the
catalyst, adjusted to pH 7.0 with 6 N HCl, and saturated with
sodium chloride. The resulting solution was extracted with 4
× 150 mL of dichloromethane, the combined organic extracts
were dried (MgSO4) and filtered, and the solvent was removed
by rotary evaporation under reduced pressure to yield a
colorless liquid. This liquid was distilled and the fraction
boiling at 83.0 °C (125 millitorr) collected to yield 3.31 g of 25
(48% isolated yield).
2-P ip er id on e (26).32 The procedure for the preparation of
24 was repeated using 70.6 mL of an aqueous solution of 13
(1.42 M, 0.100 mol, produced by the enzymatic hydrolysis of 9
and filtered as described above) and 19.4 mL of concentrated
ammonium hydroxide (29.3% NH3, 0.30 mol of NH3) in a total
volume of 100 mL. To the resulting solution was added 1.13
g of chromium-promoted Raney Nickel, and the resulting
mixture was hydrogenated at 500 psig of hydrogen and 70 °C
for 3.5 h. Analysis by gas chromatography indicated a 29.7%
yield of 26, with complete conversion of 13. The temperature
was increased to 180 °C for an additional 2 h to give a 93.5%
yield of 26. An 86 mL portion of the product mixture was
filtered to remove the catalyst, adjusted to pH 7.0 with 6 N
HCl, and saturated with sodium chloride. The resulting
solution was extracted with 4 × 150 mL of dichloromethane,
the combined organic extracts were dried (MgSO4) and filtered,
and the solvent was removed by rotary evaporation under
reduced pressure to yield a colorless liquid. This liquid was
distilled and the fraction boiling at 78.0 °C (120 millitorr)
collected to yield 6.28 g of 26 (74% isolated yield) as a white
crystalline solid: mp 37.7-39.1 °C.
4-Eth ylp yr r olid in -2-on e (23).30 The procedure for the
preparation of 24 was repeated using 12.71 g (0.100 mol) of
3-cyanopentanoic acid and 19.34 mL of concentrated am-
monium hydroxide (29.3% NH3, 0.30 mol of NH3) in a total
volume of 100 mL. To the resulting solution was added 0.636
g of chromium-promoted Raney nickel catalyst, and the
resulting mixture was hydrogenated at 160 °C and 500 psig
hydrogen for 2 h. Analysis of the product mixture by gas
chromatography and HPLC indicated a 92.1% yield of 23, with
100% conversion of 3-cyanopentanoic acid. The product
mixture was filtered to remove the catalyst, adjusted to pH
7.0 with 6 N HCl, and saturated with sodium chloride. The
resulting solution was extracted with 4 × 100 mL of dichlo-
romethane, the combined organic extracts were dried (MgSO4)
and filtered, and the solvent was removed by rotary evapora-
tion under reduced pressure to yield a colorless oil. After
removal of the remaining solvent under vacuum (100 millitorr),
the oil was dissolved in 150 mL of ethyl ether, which was then
cooled to -78 °C. After 1 h, a white crystalline solid was
collected by vacuum filtration to yield a total of 8.96 g (79%
1,5-Dim eth yl-2-p ip er id on e (29).33 The procedure for the
preparation of 24 was repeated using 54.4 mL of an aqueous
solution of 4 (1.85 M, 0.100 mol, produced by microbial
hydrolysis of 2 and filtered as described above) and 25.8 mL
of 40 wt % methylamine (9.31 g methylamine, 0.30 mol) in a
100 mL final volume. To the resulting solution was added
0.636 g of 5% Pd on carbon powder, and the resulting mixture
was hydrogenated for 25.5 h at 70 °C and 500 psig hydrogen.
Analysis by gas chromatography indicated a 38.2% yield of 26,
with 54% of unreacted 4. The temperature was increased to
160 °C and 500 psig hydrogen for 4 h, and analysis of the
product mixture by gas chromatography and HPLC indicated
yields of 72.8% yield of 29, 3.5% of 24, 19.9% of 2-methylglu-
taric acid diammonium salt, and 1.6% 4 remaining. A 61 mL
portion of the product mixture was filtered to remove the
catalyst, adjusted to pH 7.0 with 6 N HCl, and saturated with
sodium chloride. The resulting solution was extracted with 4
× 100 mL of ethyl ether, the combined organic extracts were
dried (MgSO4), and filtered, and the solvent was removed by
rotary evaporation under reduced pressure to yield a colorless
liquid. This liquid was distilled and the fraction boiling at
70.0-71.5 °C (3.5 torr) collected to yield 4.65 g (60% isolated
yield) of 29: 1H NMR (500 MHz, CDCl3) δ 3.28-3.24 (m, 1
H), 2.98-2.94 (m, 1 H), 2.92 (s, 3 H), 2.43-2.28 (m, 2 H), 2.02-
1.95 (m, 1 H), 1.87-1.82 (m, 1 H), 1.51-1.43 (m, 1 H), 1.02 (d,
J ) 6.7 Hz, 3 H); 13C NMR (75 MHz, CDCl3) δ 168.9, 56.3,
34.0, 31.0, 29.0, 28.4, 17.9; IR (neat) 2954, 2929, 2875, 1649,
1504, 1462, 1422 cm-1; MS (EI) m/z 128 (MH+, 76), 127 (M+,
90), 112 (11), 85 (25), 84 (27), 70 (15), 57 (43), 44 (100); HRMS
calcd for C7H14NO (MH+) 128.1076, found 128.1075.
1
isolated yield) of 23: mp 40.5-41.5 °C; H NMR (500 MHz,
CDCl3) δ 7.70 (bs, 1 H), 3.51-3.47 (m, 1 H), 3.03-3.00 (dd, J
) 6.2 and 9.5 Hz, 1 H), 2.45-2.33 (m, 2 H), 2.02-1,97 (dd, J
) 7.3 and 16 Hz, 1 H), 1.52-1.46 (quintet, J ) 7.3 Hz, 2 H),
0.93 (t, J ) 7.3 Hz, 3 H); 13C NMR (75 MHz, CDCl3) δ 178.8,
47.9, 36.6, 36.3, 27.4, 11.7; IR (KBr) 3241, 3122, 2958, 2931,
2900, 2875, 1683, 1664, 1493, 1462 cm-1; MS (EI) m/z 113 (M+,
100), 98 (5), 83 (26), 70 (16), 56 (44), 41 (83); HRMS calcd for
C6H11NO (M+) 113.0840, found 113.0841.
2-P yr r olid in on e (25).31 The procedure for the preparation
of 24 was repeated using 75.8 mL of an aqueous solution of
12 (1.31 M, 0.100 mol, produced by the enzymatic hydrolysis
of 8 and filtered as described above) and 19.4 mL of concen-
trated ammonium hydroxide (29.3% NH3, 0.30 mol of NH3) in
a total volume of 100 mL. To the resulting solution was added
0.99 g of chromium-promoted Raney nickel, and the resulting
(26) (a) Foubelo, F.; Lloret, F.; Yus, M. Tetrahedron 1993, 49, 8465-
8470. (b) Applequist, D. E.; Renken, T. L.; Wheeler, J . W. J . Org. Chem.
1982, 47, 4985-4995.
4-Eth yl-1-m eth ylp yr r olid in -2-on e (30). The procedure
for the preparation of 29 was repeated using 79.4 mL of
aqueous 3 (1.26 M, 0.100 mol, produced by the enzymatic
hydrolysis of 1 and filtered as described above) and 17.2 mL
of 40 wt % methylamine (6.21 g methylamine, 0.200 mol) in a
100 mL final volume. To the resulting solution was added
0.636 g of 5% Pd on carbon powder, and the resulting mixture
(27) Ives, D. J . G.; Sames, K. J . Chem. Soc. 1943, 513-517.
(28) Kataoka, M.; Ohno, M. Bull. Chem. Soc. J pn. 1973, 46, 3474-
3477.
(29) J ackman, L. M.; Webb, R. L.; Yick, H. C. J . Org. Chem. 1982,
47, 1824-1831.
(30) (a) Colonge, J .; Pouchol, J .-M. Bull. Soc. Chim. Fr. 1962, 598-
603. (b) Koelsch, C. F.; Stratton, C. H. J . Am. Chem. Soc. 1944, 66,
1883.
(32) The Aldrich Library of 13C and 1H FT-NMR Spectra, 1st ed.;
Volume 1, spectra no. 1292C.
(33) Moehrle, H.; Class, M. Pharmazie 1988, 43, 749-753.
(31) The Aldrich Library of 13C and 1H FT-NMR Spectra, 1st ed.;
Volume 1, spectra no. 1285B.

Chemoenzymic Production of Lactams
J . Org. Chem., Vol. 63, No. 14, 1998 4801
was hydrogenated for 4 h at 140 °C and 500 psig hydrogen.
Analysis of the product mixture by gas chromatography and
HPLC indicated a 69.8% yield of 30 and a 20.4% yield of 23,
with 100% conversion of 3. An 80 mL portion of the product
mixture was filtered to remove the catalyst, adjusted to pH
7.0 with 6 N HCl, and saturated with sodium chloride. The
resulting solution was extracted with 4 × 100 mL of dichlo-
romethane, the combined organic extracts were dried (MgSO4)
and filtered, and the solvent was removed by rotary evapora-
tion under reduced pressure to yield a colorless liquid. This
liquid was fractionally distilled and the fraction boiling at 100
°C (16 torr) collected (5.15 g, 51% yield). The resulting 30
contained <5% 23 as impurity, and redistillation and collection
of the fraction boiling at 128 °C (40 torr) yielded 3.71 g (37%
isolated yield) of pure 30: 1H NMR (500 MHz, CDCl3) δ 3.52-
3.48 (dd, J ) 9.7 and 7.9 Hz, 1 H), δ 3.05-3.02 (dd, J ) 9.7
and 6.4 Hz, 1 H), 2.80 (s, 3 H), 2.47-2.42 (dd, J ) 8.8 and
16.5 Hz, 1 H), 2.30-2.23 (m, 1 H), 2.00-1.96 (dd, J ) 7.7 and
16.5 Hz, 1 H), 1.51-1.45 (m, 2 H), 0.93 (t, J ) 7.3 Hz, 3 H);
13C NMR (75 MHz, CDCl3) δ 172.4, 53.4, 35.7, 31.7, 27.8, 26.3,
10.3; IR (neat) 2960, 2929, 2876, 2859, 1694, 1501, 1462, 1426
cm-1; MS (EI) m/z 127 (M+, 55), 112 (3), 99 (8), 85 (55), 70 (8),
56 (12), 44 (100); HRMS calcd for C7H13NO (M+) 127.0997,
found 127.0998.
Ack n ow led gm en t. L. Winnie Wagner and co-work-
ers of the DuPont Fermentation Resource Facility
provided supplies of microbial catalysts used in this
work.
Su p p or tin g In for m a tion Ava ila ble: Experimental sec-
tion describing the isolation and growth of Acidovorax facilis
72W (ATCC 55746), Acidovorax facilis 72 PF-15 (ATCC 55747),
Acidovorax facilis 72 PF-17 (ATCC 55745), and Comamonas
testosteroni 5-MGAM-4D (ATCC 55744) (6 pages). This mate-
rial is contained in libraries on microfiche, immediately follows
this article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O9804386