Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009

Article abstract of DOI:10.1016/j.tetlet.2009.12.094

A study has been carried out into the biocatalytic hydration of nitriles using the nitrile hydratase enzyme from Rhodopseudomonas palustris CGA009. It has been shown that this nitrile hydratase can hydrate aliphatic, aromatic and heterocyclic nitriles under very mild conditions, in mixtures of pH 7 buffer and a range of organic solvents, often with excellent chemoselectivity. The major determinant of hydration occurring is the degree of steric hindrance around the nitrile moiety and/or size of the substrates.

Full text of DOI:10.1016/j.tetlet.2009.12.094

Tetrahedron Letters 51 (2010) 1639–1641
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Tetrahedron Letters
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Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from
Rhodopseudomonas palustris CGA009
a
Gary W. Black a, Thomas Gregson b, Christopher B. McPake a, Justin J. Perry a, , Meng Zhang
*
a School of Applied Sciences, Northumbria University, Newcastle Upon Tyne NE1 8ST, UK
b Isotope Chemistry and Metabolite Synthesis, sanofi-aventis, Alnwick Research Centre, Willowburn Avenue, Alnwick NE66 2JH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A study has been carried out into the biocatalytic hydration of nitriles using the nitrile hydratase enzyme
from Rhodopseudomonas palustris CGA009. It has been shown that this nitrile hydratase can hydrate ali-
phatic, aromatic and heterocyclic nitriles under very mild conditions, in mixtures of pH 7 buffer and a
range of organic solvents, often with excellent chemoselectivity. The major determinant of hydration
occurring is the degree of steric hindrance around the nitrile moiety and/or size of the substrates.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 7 September 2009
Revised 8 December 2009
Accepted 16 December 2009
Available online 24 December 2009
Keywords:
Nitrile hydratase
Nitrile hydration
Solvent stability
1. Introduction
(I)
(II)
NHase
Amidase
Traditional abiotic conditions which are used for nitrile hydra-
tion to primary amides (I) are generally highly acidic or basic,
and require high temperatures. These forcing conditions can often
cause the amides to undergo hydrolysis of the C–N bond to give the
corresponding carboxylic acid (II) (Scheme 1), as well as being
incompatible with many other functional groups.1
R
CONH2
R
CO2H
R
CN
H2O
NH3
H2O
Scheme 1.
it has been proposed that the bottleneck en route to the binding
site is only 7 Å, which could limit the range of possible substrates
and make NHases very sensitive to steric factors.9 Third, aqueous
media would appear to limit their applicability in the hydration
of inherently hydrophobic nitriles, though organic co-solvents
can be used to aid substrate availability to the enzyme.10
The nitrile hydratase from Rhodopseudomonas palustris CGA009
can be determined from primary sequence data as being a cobalt-
centred NHase from the presence of the amino acid sequence
VCTLCSC in the active site on the alpha subunit. It has been over-
expressed in Escherichia coli BL21(DE3) giving a microbial source
which is free of competing nitrilase and nitrile hydratase activities.
Here we report on its performance and regioselectivity during
hydration of nitriles under mild conditions, and probe its limita-
tions with regards to stability, steric demand of substrates and
the use of a mixed aqueous/organic media.
Nitrile hydratases (NHases) are a class of metalloenzyme which
selectively hydrate nitriles into the corresponding primary amide.
They are commonly found in prokaryotes which also exhibit nitri-
lase (nitrile to carboxylic acid hydrolysis) and amidase (primary
amide to carboxylic acid hydrolysis) activities, and are part of
nitrogen catabolism. They are two-subunit enzymes and the metal
anchored in their active site can be either a non-haem iron (Fe-type
NHase) or a non-corrinoid cobalt (Co-type NHase).2
Hydration of nitriles using an NHase however, can be carried
out in aqueous media, without using metal catalysts and at ambi-
ent temperatures making a biocatalytic approach a significantly
‘greener’ option.3 A further advantage to a biocatalytic approach
is the inherent regio- and stereoselectivity that hydration via an
NHase has been shown to exhibit.4–7
Despite these advantages there are three major limitations
which have hindered the more widespread use of NHases as puri-
fied enzymes in synthesis. First, they have been described as rela-
tively unstable and hence problematic to work with.8 Second,
NHases have a highly conserved three-dimensional structure, and
1.1. Effect of organic co-solvents on enzyme performance
As one of the drawbacks of using enzymatic methods for hydra-
tion of nitriles is the relative insolubility of the substrate in pH 7
buffer, conversion of 4-hydroxybenzonitrile 1 in a range of mixed
* Corresponding author.
E-mail address: justin.perry@unn.ac.uk (J.J. Perry).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.094

1640
G. W. Black et al. / Tetrahedron Letters 51 (2010) 1639–1641
Table 2
aqueous/organic media was investigated using the NHase as a cell-
free extract (CFE). This substrate was chosen because at the con-
centration used in this experiment, it was fully soluble in pH 7 buf-
fer and all the mixed media investigated. It can be seen from the
results that the NHase was very robust to significant proportions
of DMSO and methanol (Table 1). This robustness compares very
favourably with a cell-free extract of the Fe-type NHase from Rho-
dococcus erythropolis AJ270, which under the same conditions
showed negligible tolerance for methanol as a co-solvent and could
only tolerate a 20% v/v mixture with DMSO before performance de-
creased significantly. In all cases, the only reaction outcome was
hydration and a hydrolysis product was not observed by any ana-
lytical technique.
NHase-mediated hydration of nitriles containing a benzene or heterocyclic moietya
0.25 mmol, 17 h, 30 oC
O
R
N
phosphate buffer pH 7.0 (3.75 ml)
DMSO (1.25 ml)
R
NH
2
NHase (2.0 µl, PE)
3
4
Entry
1
Substrate
% Conversion 4
>99.9
HO
1
1.2. Hydration of heterocyclic nitriles
2
3
4
>99.9
28
Having identified that this enzyme was able to operate in mixed
aqueous/organic media as a CFE, the solvent conditions of 3:1 pH 7
phosphate buffer/DMSO were used with a purified enzyme (PE)
preparation, and complete hydration of 1 was again observed.11
A panel of nitriles 3a–h containing either a benzene or heterocyclic
moiety was subjected to hydration using this NHase, with all ex-
cept 3b and 3e showing complete reaction with no remaining
starting material present as indicated by TLC or GC–MS (Table 2).
For reasons which may be related to mechanism and the shape
of the active site, both compounds which hydrate poorly contained
a methylene linker between the nitrile and the aromatic groups. It
is interesting to note that this Co-type NHase is unaffected by a 2-
pyridyl substitution pattern, unlike the Fe-type R. erythropolis
AJ270 NHase.9
3a
3b
3c
>99.9
5
6
7
>99.9
89
S
3d
S
3e
1.3. Hydration of substituted benzonitriles
To examine the sensitivity of this enzyme to steric crowding
around the nitrile, and its chemoselectivity, PE NHase was used
to hydrate a range of substituted benzonitriles with various de-
grees of steric hindrance offered by groups at the ortho and para
positions. The results show that this enzyme is very sensitive to
the steric bulk of ortho substitution and that even a methyl group
ortho to the nitrile in 5c is enough to decrease the percentage con-
version. The ortho-substituted bromine in 5d also inhibits hydra-
tion although ortho fluorine and ortho chlorine substitution (5a
and 5b) does not. Para-substituted benzonitriles 5h–p were hy-
drated with very high conversion although the bulky ester groups
in 5o–p do not lead to almost quantitative conversion as with other
substituents (Table 3).
>99.9
O
3f
8
9
>99.9
>99.9
N
3g
N
3h
a
Conversion was determined by correlation of the appropriate signals on GC–MS.
The quantitative conversion of 2,6-difluorobenzonitrile 6 to 2,6-
difluorobenzamide 7 (Scheme 2) could also be achieved without
Table 3
NHase-mediated hydration of substituted benzonitrilesa
O
Table 1
0.25 mmol, 17 h, 30 oC
CN
Effect of different organic co-solvents at different v/v percentages on the activity of
the NHase towards 4-hydroxybenzonitrilea
NH2
X
phosphate buffer pH 7.0 (3.75 ml)
MeOH (1.25 ml)
X
O
0.25 mmol, 24 h, 30 o
C
NHase (PE, 0.2
CN
µl)
NH2
phosphate buffer (pH 7.0) /
organic solvent mixture (5.0 ml total)
NHase (0.5 ml CFE)
4
5
HO
HO
Entry
X
ortho
% Conversion
para
% Conversion
1
2
1
2
3
4
5
6
7
8
9
F
Cl
Me
Br
5a
5b
5c
5d
5e
5f
—
—
5g
>99.9
95
17
5
5
3
—
—
0
5h
5i
5j
5k
5l
5m
5n
5o
5p
>99.9
>99.9
>99.9
>99
>99.9
>99
>99
98
95
Entry
% Co-solvent (v/v)
% Conversion (DMSO)
% Conversion (MeOH)
1
2
3
4
5
6
0
>99.9
>99.9
>99.9
>99.9
>99.9
4
>99.9
>99.9
>99.9
>99.9
35
10
20
30
40
50
CF3
OMe
COMe
CO2Me
CO2Et
2
a
a
Conversion was determined by correlation of the appropriate signals on GC–MS.
Conversion was determined by correlation of the appropriate signals on GC–MS.

G. W. Black et al. / Tetrahedron Letters 51 (2010) 1639–1641
1641
F
F
O
F
2. General procedure for the biotransformation
0.25 mmol, 17 h, 30 oC
CN
F
NH2
phosphate buffer pH 7.0 (3.75 ml)
DMSO (1.25 ml)
NHase (PE, 0.2 µl)
The nitrile substrate was added to a mixture of organic co-sol-
vent (as detailed in Tables 1–3) and 100 mM potassium phosphate
buffer, pH 7.0 (giving a total reaction volume of 5.0 ml), and the en-
zyme (as CFE or PE as detailed below) was added. The solution was
mixed using a standard laboratory magnetic stirrer for 17–24 h at
30 °C whilst following the progress of the reaction by TLC. The
reaction medium was extracted with EtOAc (3 Â 5.0 ml) and the
organic extract dried with MgSO4 and analysed by GC–MS. Com-
pound identities were established by comparison with published
NMR, IR and melting point data.
6
7
Scheme 2.
detectable amounts of products resulting from either cleavage of
the amide bond or nucleophilic substitution of a fluoride. High-
lighting the sensitivity of this enzyme to steric crowding in the
ortho position, the equivalent dichloro-compound showed less
than 5% hydration under similar conditions.
Acknowledgements
We acknowledge the EPSRC National Mass Spectrometry Ser-
vice Centre, Swansea, for providing mass spectrometric data, Mr.
Edwin Ludkin for helping with GC–MS analysis, and ESPRC for
funding a MRes studentship (C.B.M.).
1.4. Hydration of larger aromatic nitriles
One of the acknowledged limitations of NHase methodologies
in synthesis is the sensitivity of these enzymes to the overall
dimensions of the substrate.9 Under the reaction conditions used
for the compounds in Table 3, this NHase was unable to hydrate
the nitrile functionality in diphenylacetonitrile 8, a lack of reactiv-
ity which can be ascribed to its steric bulk, as it was recovered from
the reaction mixture, quantitatively.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.12.094.
CN
References and notes
1. Rappoport, Z. The Chemistry of the Cyano Group, 1st ed.; Wiley Interscience,
1970.
2. Kobayashi, M.; Shimizu, S. Curr. Opin. Chem. Biol. 2000, 4, 95.
3. Asano, Y.; Tani, Y.; Yamada, H. Agric. Biol. Chem. 1980, 44, 2251.
4. Gavagan, J. E.; Fager, S. K.; Fallon, R. D.; Folsom, P. W.; Herkes, F. E.; Eisenberg,
A.; Hann, E. C.; DiCosimo, R. J. Org. Chem. 1998, 63, 4792.
5. Ewert, C.; Lutz-Wahl, S.; Fischer, L. Tetrahedron: Asymmetry 2008, 2573, 19.
6. Wang, M.-X. Top. Catal. 2005, 35, 117.
7. Wang, M.-X. Chimia 2009, 63, 331.
8. van Pelt, S.; Quignard, S.; Kubác, D.; Sorokin, D. Y.; van Rantwijk, F.; Sheldon, R.
A. Green Chem. 2008, 10, 395.
9. Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin Trans. 1 1997, 1099.
10. Prepechalová, I.; Martínková, L.; Stolz, A.; Ovesná, M.; Bezouska, K.; Kopecky, J.;
Kren, V. Appl. Microbiol. Biotechnol. 2001, 55, 150.
11. Both CFE and PE of this Co-type NHase are stable with this proportion of either
DMSO or MeOH, and we believe, can be used interchangeably in these
transformations.
8
It has been shown that NHase enzyme CGA009 is capable of
hydrating a wide variety of nitriles (aryl, alkyl and heterocyclic),
under very mild reaction conditions, and in a mixed aqueous/or-
ganic media which solubilise compounds that are relatively hydro-
phobic. Whilst its use is limited to nitriles with low steric demands
and to smaller substrates, it shows good stability and good conver-
sions and should prove a useful biocatalyst. Studies are currently in
progress on the enantiospecificity of this enzyme with chiral
nitriles.