Asymmetric synthesis of both antipodes of β-hydroxy nitriles and β-Hydroxy carboxylic acids via enzymatic reduction or sequential reduction/hydrolysis

Article abstract of DOI:10.1021/jo802495f

Use of isolated carbonyl reductases in the reduction of aromatic β-ketonitriles have completely eliminated the competing α-ethylation, which is often observed with whole cell biocatalysts. By choosing suitable recombinant carbonyl reductase, the reduction

Full text of DOI:10.1021/jo802495f

Asymmetric Synthesis of Both Antipodes of ꢀ-Hydroxy Nitriles and
ꢀ-Hydroxy Carboxylic Acids via Enzymatic Reduction or Sequential
Reduction/Hydrolysis
Haribabu Ankati,† Dunming Zhu,*,†,‡ Yan Yang,† Edward R. Biehl,† and Ling Hua†,§
Department of Chemistry, Southern Methodist UniVersity, Dallas, Texas 75275,
State Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China, and China Research Center, Genencor,
A Danisco DiVision, Shanghai 200335, China
dzhu@smu.edu
ReceiVed NoVember 07, 2008
Use of isolated carbonyl reductases in the reduction of aromatic ꢀ-ketonitriles have completely eliminated
the competing R-ethylation, which is often observed with whole cell biocatalysts. By choosing suitable
recombinant carbonyl reductase, the reduction of ꢀ-ketonitriles afforded (R)- or (S)-ꢀ-hydroxy nitriles
with excellent optical purity and yield. Subsequently, nitrilase-catalyzed hydrolysis of the obtained optically
pure ꢀ-hydroxy nitriles led to the corresponding ꢀ-hydroxy carboxylic acids in high yields. More
importantly, the sequential enzymatic reduction and hydrolysis could be carried out in “two-step-one-
pot” fashion without the isolation of intermediates ꢀ-hydroxy nitriles, lowering the cost and minimizing
the environmental impact. This allows ready access to both antipodes of chiral ꢀ-hydroxy nitriles and
ꢀ-hydroxy carboxylic acids of pharmaceutical importance with excellent optical purity.
Introduction
inflammatory drugs,1 chiral ꢀ-hydroxy carboxylic acids can be
converted to ꢀ-amino acids,2 ꢀ-lactams,3 and ꢀ-lactones4 and
have been used in the synthesis of pheromones.5 The ꢀ-hydroxy
carboxylic acid moiety has often been found in polyketide
natural products such as amphotericin B,6 tylosin, and rosa-
ramicin7 and the marine natural product hapalosin.8 Therefore,
Optically active ꢀ-hydroxy nitriles and ꢀ-hydroxy carboxylic
acids are key building blocks for the synthesis of a variety of
pharmaceutically important compounds. For example, many
biologically active compounds such as ꢀ-blocker drugs contain
1,3-amino alcohol moieties, which are often prepared via
reduction of ꢀ-hydroxy nitriles. In addition to utilization as anti-
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Chem. 1987, 30, 1837–42.
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* To whom correspondence should be addressed. Tel: 214-768-1609. Fax:
214-768-4089.
† Southern Methodist University.
‡ Tianjin Institute of Industrial Biotechnology.
§ Genencor.
1658 J. Org. Chem. 2009, 74, 1658–1662
10.1021/jo802495f CCC: $40.75 2009 American Chemical Society
Published on Web 01/22/2009

Asymmetric Synthesis of ꢀ-Hydroxy Nitriles and Carboxylic Acids
SCHEME 1. Whole-Cell Reduction of
3-Oxo-3-phenylpropanenitrile Concomitant with
r-Ethylation
The ethyl group is proposed to come from the ethanol produced
by the whole cell metabolism.28,29
Optically active ꢀ-hydroxy carboxylic acids are usually
obtained from their ester or amide derivatives via carefully
controlled hydrolysis. Optically active ꢀ-hydroxy carboxylic acid
esters or amides in turn are prepared by asymmetric acetate aldol
reactions,30-33 Reformatsky reactions,34-37 metal-catalyzed
hydrogenation/transfer hydrogenation,16,38-46 and enzymatic47-50
reductions of ꢀ-keto esters or enzymatic resolution of racemic
acylated ꢀ-hydroxy esters.51 Reduction of ꢀ-keto acids has been
achieved with (-)-diisopinocampheylborane affording enantio-
merically enriched ꢀ-hydroxy carboxylic acids.52,53 Optically
active ꢀ-hydroxy carboxylic acids have also been prepared by
the kinetic resolution of racemic ꢀ-hydroxy carboxylic acid
esters.54,55 Although these methods proved successful to some
extent, improvement in the methodology for the synthesis of
optically pure ꢀ-hydroxy carboxylic acids is still sought after.
development of efficient and environmentally benign method-
ologies for the synthesis of enantiomerically pure ꢀ-hydroxy
nitriles and ꢀ-hydroxy carboxylic acids is of practical importance.
For chiral ꢀ-hydroxy nitriles, asymmetric aldol-type reactions
with acetonitrile,9-13 ꢀ-boration of R,ꢀ-unsaturated nitriles
followed by the oxidation,14 borane reductions,15,16 and transfer
hydrogenation17-19 of ꢀ-ketonitriles and lipase- or nitrilase-
catalyzed resolution of racemic ꢀ-hydroxy nitriles have been
reported.20-23 Although these methods have been successful in
some cases, they suffer from some drawbacks such as require-
ment of low reaction temperature, hazardous reagents, unsat-
isfactory enantiomeric purity, or maximum yield of 50%. For
example, chemoenzymatic dynamic kinetic resolution of racemic
ꢀ-hydroxynitriles using Candida antarctica lipase B and
ruthenium catalyst resulted in acetylated ꢀ-hydroxy nitriles with
36-99% ee with concomitant formation of up to 26% of
ꢀ-ketonitriles, limiting its application.24 An alternative approach
to access of chiral ꢀ-hydroxy nitriles is the biocatalytic reduction
of ꢀ-ketonitriles. However, a competing R-ethylation reaction
(Scheme 1) has been often observed in the bioreduction of
aromatic ꢀ-ketonitriles by whole cell biocatalysts such as bakers’
yeast and fungus CurVularia lunata,25,26 resulting in low
chemical yields of the desired ꢀ-hydroxy nitriles. The ethylated
product has not been completely eliminated even in an E. coli
whole cell system overexpressing yeast carbonyl reductases.27
In the course of exploring the application of enzyme catalysts
in green chemical synthesis, we embraced an approach in which
both antipodes of chiral ꢀ-hydroxy nitriles and ꢀ-hydroxy
carboxylic acids could be obtained with excellent optical purity
and yield via enzymatic reduction or sequential reduction/
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J. Org. Chem. Vol. 74, No. 4, 2009 1659

Ankati et al.
SCHEME 2. Bioreduction of ꢀ-Ketonitriles with Isolated Enzymes
TABLE 1. Enzymatic Reduction of ꢀ-Keto Nitriles with Ymr226c
SCHEME 3. Bioreduction of
4,4-Dimethyl-3-oxopentanenitrile
X
time (h)
yielda (%)
eeb (%)
4-H (1a)
4-F (1b)
2,4-F2 (1c)
4-Cl (1d)
4-Br (1e)
4-CH3 (1f)
4-CN (1g)
4-NO2 (1h)
3-NO2 (1i)
3-CH3O (1j)
4-CH3O (1k)
48
24
24
24
24
48
36
36
36
36
24
83
88
85
81
83
85
90
80
78
75
90
99 (S)
99 (S)c
99 (S)c
99 (S)
99 (S)
99 (S)
95 (S)
99 (S)
99 (S)
95 (S)
99 (S)
The hydrolysis of ꢀ-hydroxy nitriles to ꢀ-hydroxy carboxylic
acids still presents a challenging task in synthetic chemistry,
and careful control of the reaction is required.24 Recently, we
have found that nitrilases from cyanobacterium Synechocystis
sp. strain PCC 6803 (NIT6803) and Bradyrhizobium japonicum
strain USDA110 (bll6402) effectively catalyzed the hydrolysis
of ꢀ-hydroxy nitriles to ꢀ-hydroxy carboxylic acids under mild
conditions.23,57 (R)-ꢀ-Hydroxy nitriles (2a-j) obtained from
CMCR-catalyzed reductions were hydrolyzed with nitrilase
NIT6803 to give (R)-ꢀ-hydroxy carboxylic acids without
racemization.56 Compared to chemical hydrolysis of nitriles,
biocatalytic hydrolysis avoids the strong basic or acidic condi-
tions and elevated reaction temperature that often result in the
undesirable elimination of OH for nitriles with ꢀ-hydroxy group,
yielding unsaturated byproduct.58 In order to obtain (S)-ꢀ-
hydroxy carboxylic acids, the (S)-ꢀ-hydroxy nitriles (2a-k)
obtained from Ymr226c-catalyzed reductions were treated with
nitrilase bll6402 in potassium phosphate buffer (pH 7.2).23 After
the reaction was complete as monitored by TLC, the reaction
mixture was worked up and the product ꢀ-hydroxy carboxylic
acids were isolated and characterized by NMR analysis. The
ee values were measured by chiral HPLC analysis.23 The results
are summarized in Table 2. As shown in Table 2, all aromatic
(S)-ꢀ-hydroxy nitriles (2a-k) were converted to the corre-
sponding (S)-ꢀ- hydroxy carboxylic acids (3a-k) in high yields.
Unfortunately, (R)- and (S)-4,4-dimethyl-3-hydroxypentaneni-
trile could not be hydrolyzed by either nitrilase NIT6803 or
bll6402. Further research for an active nitrilase toward these
nitriles is needed.
a Isolated yield. b The ee value was measured by chiral HPLC
analysis unless indicated otherwise.23 c The ee value was measured by
chiral GC analysis.
hydrolysis, respectively, and the preliminary results on the (R)-
enantiomer were communicated previously.56
Results and Discussion
Bioreduction of ꢀ-ketonitriles has remained until now an
unsolved problem because of a competing R-ethylation reaction
(Scheme 1) observed in this bioreduction with whole cell
biocatalysts, resulting in lower chemical yields of the desired
ꢀ-hydroxy nitriles. As reported in our preliminary communica-
tion, reduction of aromatic ꢀ-ketonitriles (1a-j) with an isolated
carbonyl reductase from Candida magnoliae (CMCR) showed
the absence of the competing R-ethylation (Scheme 2).56 Having
screened several carbonyl reductases obeying Prelog’s rule,
which became available in our laboratory, it has been found
that the isolated alcohol dehydrogenase (Ymr226c) from
Saccharomyces cereVisiae efficiently catalyzed the reduction of
various aromatic ꢀ-ketonitriles bearing an electron-withdrawing
or electron-donating group on the phenyl ring to afford (S)-
enantiomer of the corresponding ꢀ-hydroxy nitriles with high
optical purity (Scheme 2 and Table 1). In all cases, no ethylated
product was observed. As such, use of isolated carbonyl
reductase eliminates the competing ethylation reaction in the
biocatalytic reduction of aromatic ꢀ-ketonitriles, and CMCR
and Ymr226c are valuable enzymes for the preparation of both
enantiomers of ꢀ-hydroxy nitriles.
The reduction of an aliphatic ꢀ-ketonitrile, 4,4-dimethyl-3-
oxo-pentanenitrile, was also achieved by enzymes CMCR and
Ymr226c to give (R)- or (S)-4,4-dimethyl-3-hydroxypentaneni-
trile, respectively (Scheme 3). However, the reduction with
CMCR as catalyst showed moderate enantioselectivity (55%
ee as determined by chiral GC analysis). (R)-4,4-Dimethyl-3-
hydroxypentanenitrile could be obtained with excellent enan-
tiomeric purity via the reduction catalyzed by SSCR, a carbonyl
reductase from red yeast Sporobolomyces salmonicolor AKU
4429.
In order to enhance the efficiency for the synthesis of optically
active ꢀ-hydroxy carboxylic acids from ꢀ-ketonitriles, the
possibility of carrying out the reduction and hydrolysis in a
“two-step-one-pot” fashion without the isolation of intermediates
ꢀ-hydroxy nitriles was explored. A ꢀ-ketonitrile was reduced
using a carbonyl reductase; after the reduction was complete
as monitored by TLC analysis, a nitrilase was added to the
resulting reaction mixture. As expected, the reduction product,
ꢀ-hydroxy nitrile, was hydrolyzed smoothly. Table 3 sum-
marizes the results with the catalyst combination of reductase
CMCR and nitrilase NIT6803. Four tested ꢀ-ketonitriles were
(57) Mukherjee, C.; Zhu, D.; Biehl, E. R.; Hua, L. Eur. J. Org. Chem. 2006,
5238–5242.
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J. E.; Ben-Bassat, A.; Chauhan, S.; Payne, M. S.; Hennessey, S. M.; DiCosimo,
R. AdV. Synth. Catal. 2003, 345, 775–782.
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Org. Lett. 2007, 9, 2561–2563.
1660 J. Org. Chem. Vol. 74, No. 4, 2009

Asymmetric Synthesis of ꢀ-Hydroxy Nitriles and Carboxylic Acids
TABLE 2. Nitrilase-Catalyzed Hydrolysis of ꢀ-Hydroxy Nitriles
nitrilase offers an efficient “two-step-one-pot” process to
synthesize both antipodes of ꢀ-hydroxy carboxylic acids from
the readily available ꢀ-ketonitriles.
Conclusion
Effective asymmetric reduction of ꢀ-ketonitriles has been
successfully achieved with isolated recombinant reductases
CMCR and Ymr226c, affording (R)- and (S)-ꢀ-hydroxy nitriles
with high yield and optical purity. The obtained ꢀ-hydroxy
nitriles have been converted to optically active ꢀ-hydroxy
carboxylic acids via nitrilase-catalyzed hydrolysis in high yields.
This novel two-step enzymatic process can be carried out in a
“two-step-one-pot” fashion without the isolation of intermediates
ꢀ-hydroxy nitriles, offering an effective and environmentally
benign methodology for preparation of optically active ꢀ-hy-
droxy carboxylic acids from readily available ꢀ-ketonitriles.
Therefore, the present study allows ready access to both
enantiomers of chiral ꢀ-hydroxy nitriles and ꢀ-hydroxy car-
boxylic acids with excellent enantiomerically purity, which are
important pharmaceutical intermediates and chiral building
blocks in organic synthesis.
X
time (h)
yielda (%)
eeb (%)
4-H (2a)
4-F (2b)
2,4-F2 (2c)
4-Cl (2d)
4-Br (2e)
4-CH3 (2f)
4-CN (2g)
4-NO2 (2h)
3-NO2 (2i)
3-CH3O (2j)
4-CH3O (2k)
24
24
24
24
24
24
24
24
24
24
24
90
93
91
92
89
91
92
87
88
88
91
99
99
99
99
99
99
95
97
99
95
99
a Isolated yield. b The ee value was determined by chiral HPLC
analysis.23
TABLE 3. “Two-Step-One-Pot” Process Using Reductase CMCR
and Nitrilase NIT6803
Experimental Section
Carbonyl Reductase-Catalyzed Reduction of ꢀ-Keto-
nitriles. The reaction was carried out as follows: D-glucose (400
mg), D-glucose dehydrogenase (10 mg), NADPH (10 mg), and
carbonyl reductase Ymr226c (10 mg) were mixed in a potassium
phosphate buffer (50 mL, 100 mM, pH 7.0). To the mixture
was added a 3-oxo-3-phenylpropanenitrile (1a) solution (170 mg
1.17 mmol) in 5.0 mL of DMSO. The mixture was stirred at
room temperature and monitored by TLC until conversion was
complete. The mixture was extracted with methyl tert-butyl ether.
The organic extract was dried over anhydrous sodium sulfate,
and removal of the solvent gave (S)-3-hydroxy-3-phenylpro-
panenitrile (2a-(S), 143 mg, 83% yield), which was identified
X
timea (h)
yieldb (%)
eec (%)
yieldd (%)
H (1a)
4-F (1b)
4-Cl (1d)
3-CH3O (1j)
60/24
24/24
24/24
24/24
90
90
93
92
99
99
99
98
72
77
77
80
a Reduction time/hydrolysis time. b Isolated yield. c The ee value was
determined by chiral HPLC analysis.23 d Combined yield for sequential
process with isolation of ꢀ-hydroxy nitrile.
23,59-61
1
by comparison of H and 13C NMR with literature data.
TABLE 4. “Two-Step-One-Pot” Process Using Reductase
Ymr226c and Nitrilase bll6402
The absolute configurations of the product ꢀ-hydroxy nitriles
were determined by comparison of the sign of specific rotation
data with those in the literature.18,23
Nitrilase-Catalyzed Hydrolysis of ꢀ-Hydroxy Nitriles. The
reaction was carried out as follows: (S)-3-Hydroxy-3-phenyl-
propanenitrile (2a-(S), 70 mg, 0.48 mmol) was suspended in 50
mL of potassium phosphate buffer (100 mM, pH 7.2). Nitrilase
bll6402 (3 mg) was added to the mixture, which was stirred at
30 °C. The reaction was monitored by TLC. After completion
of the reaction, the reaction mixture was acidified with 2 N HCl,
saturated with NaCl, and extracted with ethyl acetate. The
organic extract was dried over sodium sulfate and evaporated
under reduced pressure to give crude product, which was purified
by preparative TLC. The product (S)-3-hydroxy-3-phenylpro-
panoic acid (3a-(S)) was obtained as a white crystalline solid
X
timea (h)
yieldb (%)
eec (%)
yieldd (%)
H (1a)
2,4-F2 (1c)
4-Cl (1d)
48/24
24/24
24/24
24/24
92
93
95
95
99
99
99
99
75
77
75
82
4-CH3O (1k)
a Reduction time/hydrolysis time. b Isolated yield. c The ee value was
determined by chiral HPLC analysis.23 d Combined yield for sequential
process with isolation of ꢀ-hydroxy nitrile.
1
(71 mg, 90% yield), which was identified by comparison of H
and 13C NMR with literature data.23,62,63 The absolute configura-
tions of the product ꢀ-hydroxy carboxylic acids were determined
by comparison of the sign of specific rotation data with those in
the literature.23,64
converted to the corresponding (R)-ꢀ-hydroxy carboxylic acids
with excellent yields and optical purity.
Four ꢀ-ketonitriles were examined by using reductase
Ymr226c and nitrilase bll6402. As shown in Table 4, these
ꢀ-ketonitriles were reduced and then hydrolyzed in a “two-step-
one-pot” process to afford (S)-ꢀ-hydroxy carboxylic acids with
great than 92% yields and in essentially optically pure form.
As shown in Tables 3 and 4, the product yields in the “two-
step-one-pot” process were higher than the combined yields of
the sequential process with isolation of ꢀ-hydroxy nitrile. The
results demonstrate that suitable combination of reductase and
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Ankati et al.
“Two-Step-One-Pot” Process. The reaction was carried out as
follows: D-glucose (400 mg), D-glucose dehydrogenase (10 mg),
NADPH (10 mg), and carbonyl reductase CMCR (10 mg) were
mixed in a potassium phosphate buffer (50 mL, 100 mM, pH 7.0).
To the mixture was added a 3-oxo-3-phenylpropanenitrile (1a)
solution (150 mg 1.03 mmol) in 5.0 mL of DMSO. The mixture
was stirred at room temperature and monitored by TLC until
conversion was complete. The pH was adjusted to 7.2. Nitrilase
NIT6803 (10 mg) was added to the mixture, which was stirred at
30 °C. The reaction was monitored by TLC. After completion of
the reaction, the reaction mixture was acidified with 2 N HCl,
saturated with NaCl, and extracted with ethylacetate. The organic
extract was dried over sodium sulfate and evaporated under reduced
pressure to give crude product, which was purified by preparative
TLC using hexane/ethyl acetate (80: 20). The product (R)-3-
hydroxy-3-phenylpropanoic acid (3a-(R)) was obtained as white
crystalline solid (149 mg, 90% yield).
Acknowledgment. D.Z. thanks the Chinese Academy of
Sciences for support from the Knowledge Innovation Program
(KSCX 2-YW-G-031). This work is financially supported by
DARPA (HR0011-06-1-0032) and the Robert A. Welch Foun-
dation (N-118).
Supporting Information Available: General experimental
1
1
procedures, H, 13C NMR, and specific rotation data, and H
and 13C NMR spectra of ꢀ-hydroxy nitriles and ꢀ-hydroxy
carboxylic acid. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO802495F
1662 J. Org. Chem. Vol. 74, No. 4, 2009