An enzyme library approach to biocatalysis: Development of nitrilases for enantioselective production of carboxylic acid derivatives

Article abstract of DOI:10.1021/ja0259842

The discovery, from Nature, of a large and diverse set of nitrilases is reported. The utility of this nitrilase library for identifying enzymes that catalyze efficient production of valuable hydroxy carboxylic acid derivatives is demonstrated. Unprecedented enantioselectivity and substrate scope are highlighted for three newly discovered and distinct nitrilases. For example, a wide array of (R)-mandelic acid derivatives and analogues were produced with high rates, yields, and enantiomeric excesses (95-99% ee). We also have found nitrilases that provide direct access to (S)-phenyllactic acid and other aryllactic acid derivatives, again with high yields and enantioselectivities. Finally, different nitrilases have been discovered that catalyze enantiotopic hydrolysis of 3-hydroxyglutaronitrile to afford either enantiomer of 4-cyano-3-hydroxybutyric acid with high enantiomeric excesses (>95% ee). The first enzymes are reported that effect this transformation to furnish the (R)-4-cyano-3-hydroxybutyric acid which is a precursor to the blockbuster drug Lipitor. Copyright

Full text of DOI:10.1021/ja0259842

Published on Web 07/10/2002
An Enzyme Library Approach to Biocatalysis: Development of Nitrilases for
Enantioselective Production of Carboxylic Acid Derivatives
Grace DeSantis,* Zuolin Zhu, William A. Greenberg, Kelvin Wong, Jenny Chaplin, Sarah R. Hanson,
Bob Farwell, Lawrence W. Nicholson,1 Cynthia L. Rand,1 David P. Weiner, Dan E. Robertson, and
Mark J. Burk*
DiVersa Corporation, 4955 Directors Place, San Diego, California 92121
Received February 20, 2002
Biocatalytic processes can offer unique advantages in transfor-
mations that are challenging to accomplish through conventional
chemical methods.2 Nitrilases (EC 3.5.5.1) catalyze the mild
hydrolytic conversion of organonitriles directly to the corresponding
carboxylic acids.3 Fewer than 15 microbially derived nitrilases have
been characterized and reported to date.3,4 Several nitrilases
previously have been explored for the preparation of single-
enantiomer carboxylic acids, although little progress has been made
in the development of nitrilases as viable synthetic tools. We now
report the discovery of a large and diverse set of nitrilases and
herein demonstrate the utility of this nitrilase library for identifying
enzymes that catalyze efficient enantioselective production of
valuable hydroxy carboxylic acid derivatives.
to mandelic acid. Preliminary results revealed that 27 enzymes
afforded mandelic acid in >90% ee. One enzyme (nitrilase I) was
studied in greater detail and was found to be very active for
hydrolysis of mandelonitrile. Under standard conditions using 25
mM 3a, and 0.12 mg/mL enzyme in 10% MeOH (v/v) 0.1 M
phosphate buffer at 37 °C and pH 8, (R)-mandelic acid was formed
quantitatively within 10 min and with 98% ee. To confirm synthetic
utility, the reaction was performed using 1.0 g of 3a (50 mM) and
9 mg of nitrilase (0.06 mg/mL nitrilase I); after 3 h (R)-mandelic
acid was isolated in high yield (0.93 g, 86%) and with 98% ee.
In an effort to access the most diversified range of enzymes that
can be found in Nature, we create large genomic libraries by
extracting DNA directly from environmental samples that have been
collected from varying global habitats.5 We have established a
variety of methods to identify novel activities through screening
these libraries.6 Using this approach, we now have discovered and
characterized over 200 new nitrilases.7 All nitrilases were defined
as unique at the sequence level and were shown to possess the
conserved catalytic triad Glu-Lys-Cys which is characteristic for
this enzyme class.8 Each nitrilase in our library was overexpressed
and stored as a lyophilized cell lysate to facilitate rapid evaluation
of the library for particular biocatalytic functions.
Our initial investigations focused upon the efficacy of nitrilases
for production of R-hydroxy acids 2 through hydrolysis of cyano-
hydrins 1. Cyanohydrins are well-documented to racemize under
basic conditions through reversible loss of HCN.9 Thus, a dynamic
kinetic resolution (DKR) process is possible whereby an enzyme
selectively hydrolyzes only one enantiomer of 1, affording 2 in
100% theoretical yield and with high levels of enantiomeric purity.
Table 1. Nitrilase I-Catalyzed Production of Mandelic Acid
Derivatives and Analogues 4 under DKR Conditionsa
c
entry
Ar in 4
specific activityb
TOF
%eed
1
2
3
4
5
6
7
8
9
C6H5
50
3
10
9
6
3
21
5
5
33
30
28
98
97
96
95
98
99
99
95
98
97
95
2-Cl-C6H5
2-Br-C6H5
2-Me-C6H5
3-Cl-C6H5
3-Br-C6H5
4-F-C6H5
1-naphthyl
2-naphthyl
3-pyridyl
1.7
5.6
5.1
3.4
1.7
11.8
2.8
2.8
18.6
16.8
10
11
3-thienyl
a Reactions were conducted under standard conditions (see text). Reaction
time for complete conversion to 4 was 1-3 h. Entries 8-9 were conducted
at pH 9 and 5 mM substrate concentration. b Specific activities were mea-
sured at 5 min transformation timepoints and are expressed as µmol mg-1
min-1 c TOF ) turnover frequency, mol product/mol catalyst/s. d Enantio-
.
selectivites were determined by chiral HPLC analysis. Hydroxy acids were
isolated, and absolute configurations were determined to be (R) in all cases.
We next explored the substrate scope of nitrilase I. As shown in
Table 1, a broad range of mandelic acid derivatives as well as
aromatic and heteroaromatic analogues (4) may be prepared through
this method. Nitrilase I tolerates aromatic ring substituents in the
ortho-, meta-, and para-positions of mandelonitrile derivatives, and
products of type 4 were produced with high enantioselectivities.
Other larger aromatic groups such as 1-naphthyl and 2-naphthyl
also are accommodated within the active site, again affording the
acids 4 with high selectivity (Table 1, entries 8-9). Finally,
3-pyridyl and 3-thienyl analogues of mandelic acid were prepared
readily using this process (Table 1, entries 10-11). To our
knowledge, this is the first reported demonstration of a nitrilase
that affords a range of mandelic acid derivatives and heteroaromatic
One important application of nitrilases involves commercial
production of (R)-mandelic acid from mandelonitrile.4d,10 Mandelic
acid and derivatives find broad use as intermediates and resolving
agents for the production of many pharmaceutical and agricultural
products.11 However, the few known nitrilases derived from cultured
organisms have not been found useful for efficient and selective
hydrolysis of analogous substrates.
We first screened our nitrilase library for activity and enanti-
oselectivity in the hydrolysis of mandelonitrile (3a, Ar ) phenyl)
* To whom correspondence should be addressed. E-mail: gdesantis@diversa.com;
mburk@diversa.com.
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10.1021/ja0259842 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
analogues of type 4. High activity on the more sterically encum-
bered ortho-substituted and 1-naphthyl derivatives is particularly
noteworthy.
nitrilases, this process was operated on a 1.0 g scale (240 mM 7,
30 mg enzyme, 22 °C, pH 7) and after 22 h, (R)-8 was isolated in
98% yield and 95% ee. Interestingly, the same screening program
also identified 22 nitrilases that afford the opposite enantiomer (S)-8
with 90-98% ee. Thus, our extensive screen of biodiversity has
uncovered enzymes that provide ready access to either enantiomer
of the intermediate 8 with high enantioselectivities. Our discovery
of the first enzymes that furnish (R)-8 underscores the advantage
of having access to a large and diverse library of nitrilases.
By plumbing our environmental genomic libraries created from
uncultured DNA, we have discovered a large array of novel
nitrilases. This study has revealed nitrilases that furnish mandelic
and aryllactic acid derivatives, as well as either enantiomer of
4-cyano-3-hydroxybutyric acid in high yield and enantiomeric
excess. A more detailed survey of substrate scope associated with
our nitrilase library is underway and will be reported in due course.
We next examined the preparation of aryllactic acid derivatives
6 through hydrolysis of the corresponding cyanohydrins 5. Phenyl-
lactic acid and derivatives serve as versatile building blocks for
the preparation of numerous biologically active compounds.11 Upon
screening our nitrilase library against the parent cyanohydrin 5a
(Ar ) phenyl), we found several enzymes that provided 6a with
high enantiomeric excess. One enzyme, nitrilase II, was further
characterized. A reaction using 2 mg of nitrilase (0.016 mg/mL
nitrilase II) allowed complete transformation of 1.0 g of 5a (50
mM) within 6 h and afforded (S)-phenyllactic acid (6a), which was
isolated in high yield (0.95 g, 84%) and with 96% ee. The highest
enantioselectivity previously reported for biocatalytic conversion
of 5 to 6 was 75% ee achieved through a whole cell transformation
using a Pseudomonas strain.12
Acknowledgment. We thank B. Morgan, J. McQuaid, K.
Keegan, I. Saliba, and N. Farid for assisting with preliminary
enzyme screening, and P. Kretz, E. Chi, A. Milan, M. Miller, D.
Wyborski, and I. Andruszkiewicz for enzyme discovery. T. Rich-
ardson, M. Podar, and L. Waters are gratefully acknowledged for
bioinformatic analysis. We thank D. Baptista, J. Gemsch, and L.
Bibbs for sequencing, R. Melkus and X. Tan for subcloning, P.
Chen for analytical assistance, and A. Flordeliza, D. DiMasi and
A. Vasavada for expression. We thank K. Avery (Dow) for
discussion on substrate synthesis. Special thanks goes to Mark
Madden for providing initial inspiration for the nitrilase program,
and to Jay Short for valuable discussions and support.
Table 2. Nitrilase II-Catalyzed Production of Aryllactic Acid
Derivatives and Analogues 6 under DKR Conditionsa
c
entry
Ar in 6
specific activityb
TOF
%eed
1
2
3
4
5
6
7
8
9
C6H5
25
160
121
155
21
22
64
10.5
11.6
3.4
2.3
16
100
76
97
13
14
40
6.6
96
95
95
91
95
99
96
99
97
96
97
2-Me-C6H5
2-Br-C6H5
2-F-C6H5
3-Me-C6H5
3-F-C6H5
1-naphthyl
2-pyridyl
3-pyridyl
2-thienyl
Supporting Information Available: Materials and methods and
amino acid sequence (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
7.2
2.1
1.4
10
11
References
3-thienyl
(1) Address: Dow Chemical Co., Midland, MI.
a Reaction conditions as in Table 1, except 0.016 mg/mL nitrilase
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Ortho and meta substituents appear to be tolerated well by
nitrilase II, with ortho substituted derivatives surprisingly being
converted with higher rates relative to the parent substrate 5a. Novel
heteroaromatic derivatives, such as 2-pyridyl-, 3-pyridyl, 2-thienyl-,
and 3-thienyllactic acids, were prepared with high conversions and
enantioselectivities (Table 2, entries 8-11). Unexpectedly, para
substituents greatly lowered the rates of these reactions, with full
conversion taking over two weeks under these conditions (data not
shown).
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of the readily available prochiral substrate 3-hydroxyglutaronitrile
(7)13 to afford hydroxy acid (R)-8 which, once esterified to (R)-9,
is an intermediate used in the manufacture of the cholesterol-
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We screened our nitrilase library and have discovered four unique
enzymes that provided the required product (R)-8 with high
conversion (>95%) and >90% ee. Using one of the (R)-specific
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