Substrate evaluation of rhodococcus erythropolis SET1, a nitrile hydrolysing bacterium, demonstrating dual activity strongly dependent on nitrile sub-structure

Article abstract of DOI:10.1002/ejoc.201403201

Assessment of Rhodococcus erythropolis SET1, a novel nitrile hydrolysing bacterial isolate, has been undertaken with 34 nitriles, 33 chiral and 1 prochiral. These substrates consist primarily of β-hydroxy nitriles with varying alkyl and aryl groups at the β position and containing in several compounds different substituents α to the nitrile. In the case of β-hydroxy nitriles without substitution at the α position, acids were the major products obtained, along with recovered nitrile after biotransformation, as a result of suspected nitrilase activity of the isolate. Unexpectedly, amides were found to be the major hydrolysis product when the β-hydroxy nitriles possessed a vinyl group at this position. To probe this behaviour further, additional related substrates were evaluated containing electron-withdrawing groups at the α position, and amide was also observed upon biotransformation in the presence of SET1. Therefore this novel isolate has also demonstrated NHase activity with nitriles that appears to be substrate-dependent.

Full text of DOI:10.1002/ejoc.201403201

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FULL PAPER
DOI: 10.1002/ejoc.201403201
Substrate Evaluation of Rhodococcus erythropolis SET1, a Nitrile Hydrolysing
Bacterium, Demonstrating Dual Activity Strongly Dependent on Nitrile Sub-
Structure
Tracey M. Coady,[a] Lee V. Coffey,[a] Catherine O’Reilly,[a] and Claire M. Lennon*[a]
Keywords: Biotransformations / Enzyme catalysis / Reaction mechanisms / Nitriles
Assessment of Rhodococcus erythropolis SET1, a novel nitrile
of the isolate. Unexpectedly, amides were found to be the
hydrolysing bacterial isolate, has been undertaken with 34 major hydrolysis product when the β-hydroxy nitriles pos-
nitriles, 33 chiral and 1 prochiral. These substrates consist
primarily of β-hydroxy nitriles with varying alkyl and aryl
sessed a vinyl group at this position. To probe this behaviour
further, additional related substrates were evaluated contain-
groups at the β position and containing in several compounds ing electron-withdrawing groups at the α position, and amide
different substituents α to the nitrile. In the case of β-hydroxy
nitriles without substitution at the α position, acids were the
major products obtained, along with recovered nitrile after
biotransformation, as a result of suspected nitrilase activity
was also observed upon biotransformation in the presence
of SET1. Therefore this novel isolate has also demonstrated
NHase activity with nitriles that appears to be substrate-de-
pendent.
26 subunits.[1] The structure of the nitrilase protein is
thought to be a novel α-β-β-α sandwich fold with a triad of
residues, Glu-Lys-Cys, that is essential for the function of
its active site.[7] The substrate scope of a wide range of puri-
fied nitrilases has been determined, and it has been found
that although most exhibit their highest activity with aro-
matic nitriles,[8] some nitrilases have a preference for ali-
phatic nitriles.[9]
The hydrolysis of nitriles under the influence of the nitril-
ase enzyme does not typically lead to amide as an interme-
diate, however, this has been detected in some cases, in vary-
ing quantities, usually depending on the structure of the
nitrile substrate.[10] For example, the nitrilase from Rhodoc-
occus sp. ATCC 39484 produced amide during the conver-
sion of phenylacetonitrile,[10g] and the two nitrilases AtNit1
and AtNit4 from Arabidopsis thaliana also generated amide
as the product with the quantity and enantioselectivity
found to be strongly dependent on the nature of the substit-
uent at the α position of the nitrile substrate.[10a] Amides
were found to be the major products in ZmNIT2 nitrilase-
catalysed hydrolysis of β-hydroxy nitriles.[10e] In addition,
during the hydrolysis of mandelonitrile in the presence of
PfNLase, the stereochemical configuration of the nitrile was
found to exert a major influence on the product profile.[10b]
The filamentous fungus Aspergillus niger K10 demonstrated
broad specificity towards a range of aromatic and aliphatic
nitriles, and a high amide/acid ratio or amide only was gen-
erated in several cases.[8e,11]
Introduction
It is generally accepted that nature employs two enzy-
matic pathways for nitrile hydrolysis, either by direct con-
version from a nitrile to a carboxylic acid by nitrilase en-
zymes[1] or by the hydration of a nitrile followed by hydroly-
sis of the intermediate amide formed by a combination of
nitrile hydratase/amidase.[2] The pharmaceutical industry
has considerable interest in the use of nitrile metabolising
enzymes for the chemo-, regio- or enantioselective pro-
duction of carboxylic acids and amides from nitriles under
mild conditions.[2,3] In particular, β-hydroxy nitriles, such
as 3-hydroxybutyronitrile, 3-hydroxyglutaronitrile and 3-
hydroxy-3-phenylpropionitriles, can act as sources of key
chiral β-hydroxy carboxylic acids through such hydrolysis
reactions. Under classical acid and base conditions, elimi-
nation reactions can compete with hydrolysis.[4] Alterna-
tively, nitrile biocatalysis can often selectively facilitate this
conversion without affecting other acid- or alkali-labile
functional groups present.[5]
Nitrilases, in particular, are found to be inducible en-
zymes composed of one or two types of subunits of dif-
ferent size and number.[6] Many consist of a single polypep-
tide with a molecular mass of approximately 40 kDa that
aggregates to form the active enzyme. The preferred form
of nitrilase enzymes appears to be a large aggregate of 6–
[a] Pharmaceutical and Molecular Biotechnology Research Centre,
Department of Science, Waterford Institute of Technology,
Waterford, Ireland
We recently reported a high-throughput bacterial isolate
screening strategy that resulted in the identification of a
novel nitrile metabolising strain, Rhodococcus erythropolis
SET1, active towards β-hydroxy nitriles.[12] This organism
E-mail: clennon@wit.ie
www.PMBRC.org
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403201.
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T. M. Coady, L. V. Coffey, C. O’Reilly, C. M. Lennon
FULL PAPER
catalysed the hydrolysis of (Ϯ)-3-hydroxybutyronitrile with was allowed to occur for 24 to 72 h depending on the sub-
remarkably high enantioselectivity to afford (S)-(+)-3-hy- strate under evaluation, periodically monitoring by TLC or
droxybutyric acid in 42% yield and Ͼ99.9% ee. We wished HPLC to determine the completion of the reaction. Product
to study the substrate promiscuity of this isolate with a col- ee values were determined by HPLC employing a chiral col-
lection of related nitriles with diverse structures and hence umn with analysis in triplicate. The absolute configurations
examined the effect of varying the alkyl or aryl group β to of the products and recovered starting materials were as-
the nitrile, the substituent present on the hydroxy function- signed by comparison of our HPLC chromatograms and
ality and the remote proximity of the chiral centre to the the specific optical rotations of the purified compounds
cyano group. The results of these studies are reported with those reported in the literature.[3,5a,14–16]
herein.
The substrate 3-hydroxybutyronitrile [(Ϯ)-1a] was hydro-
lysed with optimum enantioselectivity affording (S)-(+)-3-
hydroxybutyric acid (1b) in 42% yield and Ͼ99.9% ee
(Table 1, entry 1).[12] Despite repeated efforts, unreacted
nitrile could not be recovered from the reaction mixture in
this case. The preferred temperature for this biotransforma-
tion was determined to be 25 °C, above which, although
activity was conserved, there was a sharp decrease in
enantioselectivity (previously reported results along with
additional data provided in the Supporting Information).
This temperature was employed for all other biotransfor-
mations. In contrast to the results for (Ϯ)-1a, significantly
lower ee values were obtained for the acid product of the
β-hydroxyalkane nitriles (Ϯ)-2a (Table 1, entry 2), with de-
gradation of the nitriles (Ϯ)-3a and -4a observed (Table 1,
entries 3 and 4). The desymmetrisation of the prochiral 3-
hydroxyglutaronitrile (5a) was more successful with the acid
(S)-(+)-5b obtained in 67% yield, albeit with a moderate ee
of 32% (Table 1, entry 5).
Results and Discussion
Biotransformations of Racemic β-Hydroxyalkane and β-
Hydroxyaromatic Nitriles (؎)-1a–14a
Applying the conditions previously reported in the litera-
ture, the (Ϯ)-β-hydroxyalkane nitriles 1a–4a were prepared
by condensation of acetonitrile with the required aldehyde
(Figure 1). In addition, β-aromatic derivatives (Ϯ)-6a–10a
[3,13]
were synthesised by NaBH4
reduction of the corre-
sponding commercially available ketone.[5a] The nitrile hy-
drolysing activity of SET1 was induced in M9 minimal me-
dia containing 3-hydroxybutyronitrile as the sole source of
N.[12] By using whole cells of SET1, the biotransformation
It was noted that a longer reaction time of 5–7 d was
required for the hydrolysis of β-hydroxyaromatic nitriles
(Ϯ)-6a–10a than for their β-hydroxyalkane counterparts
(Ϯ)-1a–5a. This can be clearly observed by the difference in
the reaction time of 120 h required for the hydrolysis of 3-
hydroxy-3-phenylpropionitrile [(Ϯ)-6a; Table 1, entry 6]
compared with 24 h for 3-hydroxybutyronitrile [(Ϯ)-1a].
Similarly to biotransformations carried out with these -hy-
droxyaromatic nitriles and other nitrilases, acid was ob-
tained as the product of reaction, it must be noted that
in the case of ZmNit2 amide was formed.[10e] In addition,
considerably lower yields and ee values were obtained with
these β-hydroxyaromatic substrates, with the optimum re-
sult obtained with the p-fluorophenyl β-substituted nitrile
(Ϯ)-8a (Table 1, entry 8), which gave a yield of 42.0% of
acid (S)-(–)-8b with 19.8% ee and a recovery of 35.2% of
nitrile (R)-(+)-8a with an ee of 23.2%. These results corre-
late with some previous reports on nitrilase enzymes, which
also exhibited low enantioselectivity for such β-hydroxyaro-
matic nitriles.[17]
The substituent on the benzene ring of the β-hydroxyaro-
matic nitriles (Ϯ)-6a–10a also appeared to exert some influ-
ence on the enantioselectivity and yield. For example, with
the electron-withdrawing p-fluoro group in (Ϯ)-8a (Table 1,
entry 8), the enantioselectivity of the acid (S)-(–)-8b pro-
duced improved to 19.8% as did the yield to 42% in com-
parison with the results obtained with the unsubstituted
phenyl in (Ϯ)-6a (Table 1, entry 6). The increase in the ee
also held for the (S)-acid generated from the biotransforma-
tion of the p-chloro substituted nitrile (Ϯ)-7a (Table 1, en-
Figure 1. The 34 nitrile substrates evaluated in this study.
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Substrate Evaluation of Rhodococcus erythropolis SET1
Table 1. Results of the biotransformation of racemic β-hydroxyalkane and β-hydroxyaromatic nitriles 1a–10a and docked analogues 11a–
14a.[a]
Entry
Substrate
R1
R2
Time [h]
Recovered nitrile a
Yield [%][b] ee [%][c]
Product acid b
Yield%[b]
ee [%][c]
1[d]
2
3
1a
2a
CH3
H
H
H
H
H
H
H
H
24
48
72
72
48
120
120
120
120
120
168
120
168
120
n.d.
50.1
66.4
72.2
22.3
80.0
74.1
35.2
90.0
55.1
91.3
97.1
96.2
86.1
n.d.
1.4
3.0
1.8
–
42.3
49.1
n.d.
n.d.
67.0
19.3
20.1
42.0
n.d.
28.2
n.d.
n.d.
n.d.
14.0
(S)-(+) Ͼ 99.9
1.20
CH2CH
CH(CH3)2
C(CH3)3
CH2CN
C6H5
4-ClC6H4
4-FC6H4
4-MeC6H4
4-MeOC6H4
C6H5
3a
n.d.
n.d.
4
4a
5[d]
6
5a
(S)-(+)-32.0
(R)-(+)-7.0
(S)-(–)-15.8
(S)-(–)-19.8
n.d.
(R)-(+)-7.4
n.d.
n.d.
n.d.
(R)-(+)-99.9
6a
(S)-(–)-5.0
(R)-(+)-8.7
(R)-(+)-23.2
(S)-(+)-7.4
(S)-(–)-21.5
2.1
3.4
1.2
(S)-(–)-4.2
7
8
9
10
11
12
13
14
7a
8a
9a
H
H
Bn
Allyl
TBDMS
Me
10a
11a
12a
13a
14a
C6H5
C6H5
C6H5
[a] The biotransformations were carried out by incubating the nitrile (10 mmol/L) in a suspension of Rhodococcus erythropolis SET1
(OD600nm = 1) in phosphate buffer (pH 7.0) at 25 °C. [b] Isolated yield. [c] Determined by HPLC analysis using a chiral column (see the
Exp. Sect.). The configuration was determined by comparison of HPLC traces and optical rotations with the literature.[5a,21] n.d.: not
detected. [d] Chiral HPLC analysis performed on the corresponding benzyl ether.
try 7), but an improvement in yield was not observed. In due to the improved fit of the β-aromatic substrate, with
contrast, this trend was not observed with electron-donat- the β-methoxy docking moiety enhancing the chiral re-
ing substituents on the phenyl ring of the β-hydroxyaro- cognition of this substrate by the enzyme, leading to a more
matic nitrile, as with (Ϯ)-9a and (Ϯ)-10a (Table 1, entries 9 enantioselective biotransformation.[20] It appears that the
and 10), although the recovered nitrile (S)-(–)-10a, in par- size of the docking group also has an impact on the ee
ticular, appeared to be enantioenriched after the biotrans- values. For example, when a more sterically demanding
formation with an ee of 21.5% obtained.
group was present, as in the case of protected substrates
Enhanced chiral recognition and improved enantio- (Ϯ)-11a–13a, no acid was detected and racemic starting
selectivity were observed previously for the NHase/amidase nitrile was recovered in almost quantitative yield.
biotransformation employing AJ270 upon the addition of
In summary, for this series of substrates (Ϯ)-1a–14a, the
a removable docking group onto the free hydroxy moiety of presence of an alkyl or aryl substituent at the β-position as
3-hydroxy-3-phenylpropionitrile [(Ϯ)-6a].[18] Although this well as a free or protected β-hydroxy moiety significantly
strategy was employed for NHase/amidase-catalysed hy- influenced the outcome of the biotransformation reaction.
drolysis, the effect of such a docking group in the substrate It is also of note that for all the nitriles (Ϯ)-1a–14a, the acid
during hydrolysis in the presence of nitrilase enzyme was was produced as the reaction product along with remaining
investigated. Thus, to improve the ee of the acid (R)-(+)- nitrile with no amide detected or recovered. This suggests
6b obtained upon biotransformation of the corresponding catalysis of the hydrolysis reaction by a nitrilase enzyme
nitrile and possibly the related analogues discussed above, within the whole cell of SET1.
the protected benzyloxy [(Ϯ)-11a], allyloxy [(Ϯ)-12a], tri-
methylsilyloxy [(Ϯ)-13a] and methoxy [(Ϯ)-14a] derivatives
Biotransformations of Racemic β-Hydroxyalkane and β-
were prepared from (Ϯ)-6a by using previously reported
Hydroxyaromatic α-Methylene Nitriles 15a–28a
procedures.[19]
Introduction of a methyl group, in particular, onto the
To further demonstrate the application of R. erythropolis
free hydroxy of the nitrile in (Ϯ)-14a resulted in a dramatic SET1 in nitrile biotransformations, we examined the bio-
increase in the enantioselectivity upon biotransformation. transformation of a variety of α-methylene β-hydroxy-func-
Although 7% ee of the acid (R)-(+)-6b was obtained from tionalised nitriles. Such synthetically useful Baylis–Hillman
the biotransformation of the unprotected substrate (Ϯ)-6a adducts often demonstrate anti-bacterial, anti-tumour and
in 19% yield (Table 1, entry 6), Ͻ99.9% ee of the acid (R)- antifungal activity.[16b]
(–)-14b was produced, albeit in the slightly lower yield of
The starting nitriles (Ϯ)-15a–28a were prepared by the
14% (Table 1, entry 14). This 14-fold increase in ee may be DABCO-catalysed reaction of acrylonitrile with substituted
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FULL PAPER
aliphatic or aromatic aldehydes.[22] The induction, biotrans- transformation reactions were observed to be complete in
formation and analytical conditions were as reported in the shorter times for β-hydroxyalkane α-methylene nitriles (Ϯ)-
previous section.
15a–17a than for their β-hydroxyaromatic α-methylene ana-
Interestingly, SET1 influenced the hydrolysis of this logues (Ϯ)-18a–25a (48–72 h vs. 120 h). In addition, a lower
series of nitriles, and in all cases under the biotransforma- ee of 42.3% of the amide product (S)-(+)-18c along with
tion conditions amides were produced with good-to-excel- an almost 50% reduction in its yield was observed for the
lent ee values and yields in some cases (Table 2, entries 1, 5 biotransformation of the β-hydroxyaromatic α-methylene
and 10). This is in sharp contrast to the previous behaviour nitrile (Ϯ)-18a (Table 2, entry 4) when compared with its β-
observed with this isolate when acids were the products ob- hydroxyalkane α-methylene analogue (Ϯ)-15a from which
tained from the reaction along with recovered nitrile. This amide (S*)-15c was obtained with 87.8% ee (Table 2, en-
can be exemplified by comparing the results obtained from try 1) and in 43.2% yield. Similar reductions in yield and
the biotransformation of substrates 3-hydroxy-2-methylene- ee were observed for the acid products isolated after the
butanenitrile [(Ϯ)-15a] and 3-hydroxybutyronitrile [(Ϯ)-1a]. biotransformation reactions when comparing within the
In the latter case, the acid (S)-(+)-1b was formed with series the results of α-unsubstituted β-hydroxyalkane and β-
Ͼ99.9% ee in 42% yield from 1a (Table 1, entry 1), whereas hydroxyaromatic nitriles in Table 1.
upon the introduction of the vinyl functionality at the α
It was also observed that higher ee values of the amides
position in (Ϯ)-15a, the amide (S*)-15c was obtained in were obtained when the aromatic ring in the β-hydroxyaro-
43.2% yield and with 87.8% ee (Table 2, entry 1). Unre- matic α-methylene nitrile contained the electron-with-
acted nitrile (R*)-15a was also recovered after biotransfor- drawing F group at the para position of the β-aromatic ring,
mation in 42% yield and with 76.5% ee.
as in (Ϯ)-19a (Table 2, entry 5), and the electron-donating
In addition to amide formation, some of the β-hy- OMe group at the ortho position of the aromatic ring, as in
droxyalkane α-methylene substrates, for example, (Ϯ)-17a, (Ϯ)-24a (Table 2, entry 10), rather than at alternative posi-
proved more stable/tolerant of the reaction media than their tions around the ring. In particular, when such groups were
α-unsubstituted analogues. In the case of (Ϯ)-17a compared present at the meta position of the ring, amides were pro-
with (Ϯ)-3a (Table 2, entry 3 vs. Table 1, entry 3), a signifi- duced with significantly lower ee values [a 10-fold reduction
cant yield of 44% of the amide product was isolated with a in the case of the amide produced from m-fluoro-substi-
moderate ee of 16.1%, whereas no acid product was ob- tuted nitrile (Ϯ)-20a vs. p-fluoro-substituted nitrile (Ϯ)-
tained in the biotransformation of (Ϯ)-3a with reduced re- 19a]. Somewhat surprisingly, the recovered nitrile appeared
covery of the starting nitrile, potentially due to degradation to be more enantioenriched when it was meta-substituted,
by the microbial whole cell.
with nitriles 20a and 23a recovered with 46.0 and 38.9% ee,
As was the case for β-hydroxyalkane nitriles (Ϯ)-1a–5a respectively (Table 2, entries 6 and 9). The p-chloro ana-
when compared with their aromatic counterparts, the bio- logue (Ϯ)-25a did not undergo the biotransformation to
Table 2. Results of the biotransformation of racemic β-hydroxyalkane α-methylene and β-hydroxyaromatic α-methylene nitriles 15a–25a
and docked analogues 26a–28a.[a]
Entry
Substrate
R1
R2
Time [h]
Recovered nitrile a
Product amide c
Yield [%][b]
ee [%][c]
Yield [%][b]
ee [%][c]
1
2
3
4
5
6
7
8
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
CH3
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
48
72
72
42.0
69.3
42.2
72.4
61.3
43.2
69.5
64.6
45.2
82.3
97.0
58.6
60.8
76.3
(R*)-76.5
4.2
43.2
22.0
44.1
22.2
30.2
30.1
26.4
28.5
27.3
12.8
n.d.
27.1
22.0
12.2
(S*)-87.8
(S)-(+)-10.8
(S)-(+)-16.1
(S)-(+)-42.3
(S*)-50.4
(S*)-4.9
(S*)-38.4
(S)-(+)-32.1
(S)-(+)-11.6
(S)-(+)-60.5
n.d.
(S)-(+)-33.0
(S*)-26.4
CH3CH2
CH(CH3)2
C6H5
(R*)-12.0
(R*)-18.7
(R*)-22.5
(R*)-46.0
(R*)-14.3
(R*)-10.8
(R*)-38.9
3.8
2.8
8.2
(R)-10.3
1.5
120
120
120
120
120
120
120
120
120
120
120
4-F-C6H5
3-F-C6H5
2-F-C6H5
4-MeO-C6H5
3-MeO-C6H5
2-MeO-C6H5
4-Cl-C6H5
C6H5
9
10
11
12
13
14
4-F-C6H5
2-MeO-C6H5
(S*)-31.3
[a] The biotransformations were carried out by incubating the substrate (10 mmol/L) in a suspension of Rhodococcus erythropolis SET1
(OD = 1) in phosphate buffer (pH 7.0) at 25 °C. [b] Isolated yield determined by using flash chromatography. [c] Determined by HPLC
analysis using a chiral column. The configurations were determined by comparison of HPLC traces and optical rotations with the
literature when available. Tentative assignments are denoted by the use of *; n.d.: not detected.
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Substrate Evaluation of Rhodococcus erythropolis SET1
any great extent (Table 2, entry 11), with the starting mate- lated α-alkyl-substituted aryl nitriles (Ϯ)-30a–34a to assess
rial recovered in quantitative yield. the effect of alternative groups at the α position on the type
Comparison of the reactions of β-hydroxyaromatic α- and amount of biotransformation product obtained. To cir-
methylene nitriles (Ϯ)-19a–24a with their α-unsubstituted cumvent the degradation of mandelonitrile in aqueous solu-
aromatic analogues (Ϯ)-6a–10a revealed no significant in- tion at pH 7, the biotransformation was carried out at the
crease in yield of the hydrolysed product when the vinyl lower pH of 4.[10f] A slight decrease in the activity of SET1
group was introduced. For example, the yield of amide (S)- was observed at this lower pH, which correlates with the
(+)-18c obtained after purification was 22.2% (Table 2, en- results of pH versus activity studies carried out for 3-hy-
try 4), whereas that of the acid (R)-(+)-6b was 19.3% droxybutryonitrile [(Ϯ)-1a] in the presence of SET1 (the re-
(Table 1, entry 6). However, more significantly, a six-fold in- sults of these studies are presented in the Supporting Infor-
crease in ee was observed for the amide (S)-(+)-18c when mation). In addition, rather than the exclusive formation of
compared with the ee of the acid product (R)-(+)-6b pro- amide or acid, both amide (R)-(–)-29c and acid (S)-(+)-29b
duced in the absence of the vinyl group at the α position. were isolated after the hydrolysis along with recovered ni-
Such increases in ee were also observed for nitriles with p- trile (Table 3, entry 1). It is apparent from the time course
fluoro- and p-methoxy-substituted phenyl rings at the β- of the reaction (data not shown) that both acid and amide
position along with vinyl groups at the α position in com- are formed concurrently and that temporary accumulation
parison with their α-unsubstituted counterparts [nitriles of the amide and subsequent hydrolysis does not occur. The
(Ϯ)-19a vs. (Ϯ)-8a, Table 2 entry 5 vs. Table 1, entry 8 as an S enantiomer of mandelic acid [(S)-(+)-29b] was produced
example].
in an extremely high ee of Ͼ99.9%, albeit in a low yield of
To assess the role of the hydroxy group in the biotrans- 8.2%.
formation of the β-hydroxy α-methylene nitrile substrates, it
In contrast, the absence of the α-hydroxy or another elec-
was protected by methylation as described previously. Here, tron-withdrawing functionality in nitrile (Ϯ)-30a generated
however, the ee of the amide product appeared to decrease only the corresponding carboxylic acid product (S)-(+)-30b
upon protection. The ee of the (S)-amide product obtained with an excellent ee of Ͼ99.9% (Table 3, entry 2) in ad-
decreased from 42.3% (Table 2, entry 4) for the reaction of dition to recovered (R)-nitrile in 40.1% yield and 63.0% ee.
the unprotected nitrile (Ϯ)-18a to 33.0% for the transfor- The substrate (Ϯ)-31a, related to (Ϯ)-30a but differing in
mation of the O-methylated nitrile (Ϯ)-26a (Table 2, en- the size of the alkyl group at the α-position, also provided
try 12). A reduction in the ee of the amide was also ob- only the acid product along with recovered nitrile; however,
served from 50.4% after the biotransformation of nitrile a dramatic decrease in the ee of the acid was observed with
(Ϯ)-19a to 26.4% in the case of the O-methylated (Ϯ)-27a the increase in size of the alkyl substituent (Table 3, entry 3
(Table 2, entry 13). Alhough the configurations of the bio- vs. entry 2, 49.5% vs. 99.9% ee) with only a minor re-
transformation products were assigned as noted previously duction in product yield (19.3 vs. 17.3%). The amide was
by comparison of HPLC and optical rotation data with lit- formed in one case in this series of substrates, (Ϯ)-33a,
erature values, the absolute configurations of several of the which afforded after the biotransformation, along with the
products were assigned tentatively in this series.
acid (S)-(+)-33b with an excellent ee of 96.7%, an equiva-
The unexpected quantities of the amides formed during lent amount of the amide (R)-(–)-33c with a lower ee of
the biotransformations of (Ϯ)-15a–28a in the presence of 45.7% as well as recovered nitrile. The production of amide
SET1 led us to investigate mandelonitrile (Ϯ)-29a and re- in this case may possibly be due to inductive electron-with-
Table 3. Biotransformations of α-alkyl-substituted aryl nitriles.[a]
Entry
Substrate
R1
R2
Recovered nitrile a
Yield [%],[b] ee [%][c]
Product acid b
Product amide c
Yield [%],[b] ee [%][c]
Yield [%],[b] ee [%][c]
1
2
3
4
5
6
29a[d]
30a
31a
32a
33a
34a
OH
H
H
H
H
43.2, (S)-(–)-46.0
40.1, (R)-(+)-63.0
45.0, (R)-(+)-55.5
73.1, 1.2
54.1, (R)-(+)-41.7
89.2, 1.2
8.2, (S)-(+)-99.9
19.3, (S)-(+)-99.9
17.0, (S)-(+)-49.5
n.d.
20.0, (S)-(+)-96.7
n.d.
44.1, (R)-(–)-3.7
n.d.
n.d.
n.d.
20.2, (R)-(–)-45.7
n.d.
CH3
CH3CH2
iPr
CH3
CH3
3-COC6H5
4-iPr
[a] The biotransformations were carried out by incubating the substrate (10 mmol/L) in a suspension of Rhodococcus erythropolis SET1
(OD600nm = 1) in phosphate buffer (pH 7.0) at 25 °C. [b] Isolated yield determined by using flash chromatography. [c] Determined by
HPLC analysis using a chiral column. The configurations were determined by comparison of HPLC traces and optical rotations with
the literature and authentic standards when available; n.d.: not detected. [d] The hydrolysis of mandelonitrile was performed in phosphate
buffer at pH 4.0.
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T. M. Coady, L. V. Coffey, C. O’Reilly, C. M. Lennon
Table 4. Variation and yields of different substrate types as well as ee values obtained from relevant nitriles (n.d.: not detected).
FULL PAPER
Substrate
1a
6a
15a
18a
29a
30a
33a
Acid [%]
ee [%]
Amide [%]
ee [%]
42.2
Ͼ99.9 (S)
n.d.
19.3
7.0 (R)
n.d.
n.d.
n.d.
43.2
n.d.
n.d.
22.2
8.2
Ͼ99.9 (S)
44.1
19.3
Ͼ99.9 (S)
n.d.
20.0
96.7 (S)
20.2
n.d.
n.d.
87.8 (S)
42.3 (S)
3.7 (R)
n.d.
45.7 (R)
drawal at the α position by a m-oxo-substituted aryl ring or
steric effects. A comparison of the acid and amide quantity,
structural variation and ee value is provided for relevant
substrates of the series (Ϯ)-1–34a in Table 4. It can be ob-
served that when there is no substitution or a sterically non-
demanding alkyl group is present at the α position (as in
nitriles 1a, 6a and 30a), the acid is formed with high ee
values in several cases, whereas in the presence of an elec-
tron-withdrawing or conjugating group at this position in
the starting nitrile, amide is formed or a combination of
amide and acid in varying ratios (15a, 18a, 29a and 33a).
Mechanistic Insights into Nitrilase/NHase Behaviour
We suggest that a nitrilase enzyme system is responsible
for the generation of the acid and amide products. It is
thought that a conserved catalytic triad of Glu-Lys-Cys is
present at the active site of nitrilases[23] and that hydrolysis
reactions may take place via the formation of a thioimidate
intermediate produced by attack of the thiol group of the
cysteine residue on the carbon atom of the nitrile, followed
by the addition of a H2O molecule.[8d] To determine
Figure 2. Effect of metal ions on the nitrilase activity of R. erythro-
polis SET1. Reactions were performed for 24 h in potassium phos-
phate (100 mm, pH 7.0) containing 1 mm of inorganic metal ions.
The activities were determined in triplicate by using Nesslers micro-
scale colorimetric assay and are reported relative to that observed
in the absence of metal ions.
whether the catalytic cysteine residue is present in SET1, alternative thiol elimination may occur to form the amide
the biotransformation of 3-hydroxybutyronitrile [(Ϯ)-1a] (via intermediate Ib). If an electron-withdrawing group is
was examined in the presence of a range of inorganic salts present at the α position it may destabilise Ia leading to
that have previously been shown to interact with nitrilase more of the amide-forming aryl intermediate Ib. The rela-
enzymes (Figure 2).[8f,24]
tive quantity of the amide could be linked to the extent of
It can be seen from the data in Figure 2 that the thiol- destabilisation of Ia through inductive effects at the α posi-
complexing reagents of Zn2+, Ag+ and Hg2+ in particular, tion. In addition, in the case of α-methylene β-hydroxy
at a concentration of 1 mm, significantly if not completely nitriles, intramolecular hydrogen bonding of the β-hydroxy
inhibit the activity of SET1. The most pronounced effect with the amino moiety via a six-membered ring may lead
was observed with Hg2+ ions, and this indicates the possible to increased formation of the more favourable intermediate
involvement of a thiol-containing conserved cysteine resi- Ib.[10e] It is also thought that steric interactions could play
due in the catalytic mechanism and points to the presence a significant role by weakening potential interactions be-
of a nitrilase enzyme.
tween the charged N atom and Glu residue in intermediate
When amides have been produced as the products of bio- I, and again this could be a factor in the case of the bio-
transformations employing nitrilase, NHase activity has transformations of nitriles such as 15a, 18a, 29a and 33a.
been postulated to be part of the biocatalytic mechanism.[10]
However, in the hydrolysis of α-methylene β-hydroxy ni-
It has been proposed that upon formation of the thioimid- triles, in particular, enzyme linkage to the unsaturated ni-
ate intermediate, if the N atom of the tetrahedral intermedi- trile by conjugate addition of a thiol residue to a doubly
ate contains a positive charge stabilised by the triad Glu activated Michael-type acceptor may be an alternative al-
residue (Ia, Figure 3), ammonia may be eliminated to form beit less likely mechanism. If this was the case, upon the
acid as the product.[10f] In contrast, if this positive charge addition of the first molecule of water, the relative rates
lies on the amino Lys residue rather than on the N atom, of elimination of the thiol-containing cysteine residue from
6
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Substrate Evaluation of Rhodococcus erythropolis SET1
dependent on the structure of the nitrile. The possible in-
volvement of a cysteine residue in the catalytic cycle has
been demonstrated by the almost complete suppression of
the activity of SET1 in the presence of thiol-complexing
metal agents. Further studies on R. erythropolis SET1 are
underway in our laboratory to gain an understanding of its
enantioselectivity and NHase activity.
Experimental Section
Materials and Methods: All reagents were purchased from commer-
cial sources and used without additional purification. 3-Hydroxy-
butyronitrile (1a), mandelonitrile (29a), 2-phenylpropionitrile
(30a), 2-phenylbutyronitrile (31a) and 3-methyl-2-phenylbutaneni-
trile (32a) were purchased from Sigma–Aldrich. 3-Hydroxypent-
ane-1,5-dinitrile (5a), 2-(3-benzoylphenyl)propanenitrile (33a) and
2-(4-isobutylphenyl)propanenitrile (34a), were purchased from TCI
Chemicals Europe. NMR spectra were recorded with a JEOL ECX
400 MHz spectrometer. The chemical shifts (expressed in ppm) of
1
the H and 13C NMR spectra are referenced to the solvent peaks.
TLC was performed on aluminium-backed sheets with silica gel 60
F254 (Merck). Column chromatography was performed with silica
gel 60 (0.04–0.063 mm, Merck). HPLC analysis was performed
with a Hewlett-Packard 1050 series instrument, LCMS was carried
out with an Agilent technologies 1200 series instrument with an
LC/MSD Trap XCT detector and GC–MS with a Varian 450 GC
coupled with a 220 MS.
Figure 3. Proposed nitrilase mechanism for amide formation
adapted from[10f] and including additional stabilisation by proposed
intermolecular hydrogen bonding.
tetrahedral intermediates obtained restoring unsaturation
could influence the ratio of acid/amide formation. Revers-
ible thiol conjugate addition has been demonstrated to be
possible in doubly activated acrylonitrile substrates.[25] As
the enzyme has not been isolated and the crystal structure
not yet determined, further studies are required to deter-
mine the precise mechanism.
Determination of Substrate Specificity
Preparation of the Starting Nitriles and Their Spectroscopic Data:
The following racemic β-hydroxy nitriles were synthesised by re-
duction (NaBH4, EtOH) of the corresponding ketones, and the
products 6–10a were identified by comparison with NMR data re-
ported in the literature.[5a] The Baylis–Hillman nitriles 15a–26a
were prepared by DABCO-promoted Morita–Baylis–Hillman reac-
tion of acrylonitrile with various aldehydes to produce the corre-
sponding α-methylene β-hydroxy nitriles as previously reported,[22]
and the products were identified by comparison with the corre-
sponding reported NMR data.[22] Substrates 11a, 12a and 14a were
prepared from 3-hydroxy-3-phenylpropionitrile as previously re-
ported by Ma et al.[20] The synthetic procedures and characterisa-
tion data for all such previously prepared compounds are provided
in the Supporting Information.
Conclusions
We have shown that R. erythropolis SET1 catalyses the
hydrolysis of a variety of structural analogues of 3-hydroxy-
butyronitrile to afford both acids and amides in high yields
and ee values along with recovered enantioenriched nitrile
in several cases. This work has also identified key substrate
structural attributes that may influence the activity and
enantioselectivity of the isolate. The isolate appears to
transform β-hydroxyalkane and β-hydroxyaromatic nitriles
with significantly improved yields in the case of β-alkyl sub-
strates. The enantioselectivity may also be improved dra-
matically by incorporating a docking group at the hydroxy
position in β-hydroxyaromatic nitriles. This effect has pre-
(R,S)-2-[(4-Fluorophenyl)(methoxy)methyl]acrylonitrile (27a): Solid
Ag2O (0.646 g, 2.82 mmol, 1 equiv.) and iodomethane (1.59 g,
11.3 mmol, 4 equiv.) were added to 2-[(4-fluorophenyl)(hydroxy)-
methyl]acrylonitrile (0.5 g, 2.82 mmol, 1 equiv.) successively and
the resulting mixture was stirred in the dark overnight. The reac-
tion mixture was diluted with acetone (5 mL) and filtered by grav-
ity. The solvent was removed in vacuo to afford the crude product.
The crude product was subjected to silica gel column chromatog-
viously been reported for the NHase/amidase-catalysed hy- raphy eluting with a mixture of hexane/ethyl acetate (80:20). The
title product was obtained as a colourless oil (0.22 g, 41%). The
enantiomers were separated by HPLC analysis using a Chiralcel
OJ-H column with hexane/propan-2-ol (90:10) containing 0.1%
TFA as the mobile phase at a flow rate of 0.8 mL/min to give to
drolysis of β-hydroxy nitriles rather than nitrilase-catalysed
hydrolysis. Protecting the β-hydroxy moiety with a methyl
group, in particular, led to a significant increase in the ee
in one case and may be a viable strategy in the future for
related substrates and SET1.
We also consistently observed that when an electron-
withdrawing group was present in the nitrile at the α posi-
tion the product bias shifts towards the amide rather than
exclusively the acid. We have proposed a dual nitrilase/
peaks: t1 = 11.0 min, t2 = 11.5 min. IR (KBr disc): ν
= 2227,
˜
max
1
1604, 1224, 953, 840 cm–1. H NMR (400 MHz, CDCl3): δ = 7.51
(td, J = 7.3, 1.8 Hz, 1 H, Ar-H), 7.35–7.31 (m, 1 H, Ar-H), 7.22–
7.20 (m, 1 H, Ar-H), 7.09–7.05 (m, 1 H, Ar-H), 6.05 (s, 1 H,
CH2=C), 5.99 (s, 1 H, CH2=C), 5.13 (s, 1 H, 3-H), 3.42 (s, 3 H,
CH3-O) ppm. 13C NMR (100 MHz, CDCl3): δ = 164.0 (C-1Ј),
NHase activity within the microbial whole cell that is highly 134.1 (C-4Ј), 128.1 (CH2=C), 128.0 (C-2’, C-6Ј), 116.9 (C-2), 116.0
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T. M. Coady, L. V. Coffey, C. O’Reilly, C. M. Lennon
FULL PAPER
(C-1), 115.1 (C-3Ј, C-5Ј), 78.1 (C-3), 57.0 (CH3-O) ppm. MS: m/z 1657, 1625, 862, 816 cm–1. 1H NMR (400 MHz, CDCl3): δ = 7.31–
= 192.2 [MH]+. HRMS (ESI): calcd. for C11H11NOF 192.0825
[MH]+; found 192.0817,.
7.27 (m, 1 H, Ar-H), 7.10 (t, J = 7.8 Hz, 2 H, Ar-H), 6.95 (m, 1
H, Ar-H), 6.25 (br, 1 H), 5.92 (s, 1 H, CH2=C), 5.86 (s, 1 H,
CH2=C), 5.62 (br, 1 H), 5.51 (s, 1 H, 3-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 169.5 (C-1), 161.7 (C-1Ј), 143.9 (C-2), 143.4
(C-4Ј), 130.1 (CH2=C), 122.6 (C-5Ј), 121.7 (C-3Ј), 114.7 (C-6Ј),
113.2 (C-2Ј), 73.9 (C-3) ppm. MS: m/z = 196.1 [MH]+. HRMS
(ESI): calcd. for C10H11NO2F 196.0774 [MH]+; found 196.0773.
(R,S)-2-[Methoxy(2-methoxyphenyl)methyl]acrylonitrile (28a): Solid
Ag2O (0.619 g, 2.6 mmol, 1 equiv.) and iodomethane (1.45 mL,
10.4 mmol, 4 equiv.) were added to 2-[hydroxy(2-methoxyphenyl)-
methyl]acrylonitrile 24a (0.5 g, 2.6 mmol, 1 equiv.) successively and
the resulting mixture was stirred in the dark overnight. The reac-
tion mixture was diluted with acetone (5 mL) and filtered by grav-
ity. The solvent was removed in vacuo to afford the crude product.
The product was subjected to silica gel column chromatography
eluting with a mixture of hexane/ethyl acetate (60:40). The title
product was obtained as a colourless oil (0.20 g, 39% yield). The
enantiomers were separated by HPLC analysis using a Chiralcel
OJ-H column with hexane/propan-2-ol (90:10) containing 0.1%
TFA as the mobile phase at a flow rate of 0.8 mL/min to give two
(S*)-2-[(3-Fluorophenyl)(hydroxy)methyl]acrylamide (20c): The title
product was obtained as a white solid (30.1% yield), m.p. 92.3–
93.0 °C. Chiral HPLC analysis using a Chiralcel OJ-H column with
hexane/propan-2-ol (90:10) containing 0.1% TFA as the mobile
phase at a flow rate of 0.8 mL/min gave two peaks: tminor
=
17.27 min, tmajor = 18.10 min. IR (KBr disc): ν = 3382, 3187,
˜
max
1658, 1625, 1044, 947 cm–1. 1H NMR (400 MHz, CDCl3): δ = 7.31–
7.27 (m, 1 H, Ar-H), 7.10 (t, J = 7.8 Hz, 2 H, Ar-H), 6.95 (td, J =
5.0, 3.2 Hz, 1 H, Ar-H), 6.31 (br, 1 H), 5.92 (s, 1 H, CH2=C), 5.71
(br, 1 H), 5.51 (s, 1 H, CH2=C), 5.50 (s, 1 H, 3-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 169.45 (C-1), 161.67 (C-1Ј), 143.9 (C-2),
143.4 (C-3Ј), 130.1 (C-5Ј), 122.63 (CH2=C), 121.7 (C-4Ј), 114.7 (C-
2Ј), 113.2 (C-6Ј), 73.92 (C-3) ppm. MS: m/z = 196.1 [MH]+. HRMS
(ESI): calcd. for C10H11NO2F 196.0774 [MH]+; found 196.0777.
peaks: t1 = 11.2 min, t2 = 12.2 min. IR (KBr disc): ν
= 2225,
˜
max
1601, 1246, 1090, 747, 668 cm–1. H NMR (400 MHz, CDCl3): δ =
7.45 (d, J = 7.5 Hz, 1 H, Ar-H), 7.29 (t, J = 7.3 Hz, 1 H, Ar-H),
7.01 (t, J = 6.4 Hz, 1 H, Ar-H), 6.88 (d, J = 8.2 Hz, 1 H, Ar-H),
5.96 (d, J = 6.8 Hz, 2 H, CH2=C), 5.20 (s, 1 H, 3-H), 3.82 (s, 3 H,
O-CH3), 3.37 (s, 3 H, CH3-O) ppm. 13C NMR (100 MHz, CDCl3):
δ = 169.1 (C-1Ј), 147.8 (CH2=C), 145.5 (C-5Ј), 143.6 (C-3Ј), 128.3
(C-2Ј), 127.4 (C-2), 127.1 (C-4Ј), 117.8 (C-1), 71.4 (C-3), 57.6,
(CH3-O), 56.1 (CH3-O) ppm. MS: m/z = 204.1 [MH]+. HRMS
(ESI): calcd. for C12H13NO3 203.0946 [M]+; found 203.0955.
1
(S*)-2-[(2-Fluorophenyl)(hydroxy)methyl]acrylamide (21c): The title
product was obtained as a white solid (29 mg, 26.4% yield), m.p.
108.3–109.1 °C. Chiral HPLC analysis using a Chiralcel OJ-H col-
umn with hexane/propan-2-ol (90:10) containing 0.1% TFA as the
mobile phase at a flow rate of 0.8 mL/min gave two peaks: tminor
=
General Procedure for the Biotransformation of Racemic Nitriles:
Rhodococcus erythropolis SET1 was grown in M9 minimal media
by using 3-hydroxybutyronitrile (1a) as the sole nitrogen source.
The culture was then re-suspended in potassium phosphate buffer
(0.1 m, pH 7.0) to give an OD600nm of 1.0. The resting cells were
activated at 25 °C for 30 min through orbital shaking. Racemic ni-
trile was added in one portion to the flask, and the mixture was
incubated at 25 °C by using an orbital shaker (250 rpm). The reac-
tion, monitored by TLC or LCMS, was quenched after a specified
period of time (see Tables 1 and 2 and the Supporting Information)
by removing the biomass through centrifugation. The resulting
aqueous solution was adjusted to pH 12 with aqueous NaOH (2 m)
and extracted with ethyl acetate (3ϫ 120 mL). After drying
(MgSO4) and removal of the solvent under vacuum, the residue
was subjected to silica gel column chromatography using a mixture
of hexane and ethyl acetate as the mobile phase to give amide prod-
uct and the recovered nitrile in some cases. The aqueous phase was
then adjusted to pH 4 with hydrochloric acid (2 m) and extracted
with ethyl acetate (3ϫ 120 mL). After drying (MgSO4) and re-
moval of the solvent under vacuum, the residue was purified by
silica gel column chromatography by employing a mixture of hex-
ane and ethyl acetate as eluent to give the pure acid product. The
structures of all the products were fully characterised by spectro-
scopic analyses. The ee values were determined by HPLC analysis
using a chiral stationary phase (see the Supporting Information).
These and specific optical rotation values were compared with lit-
erature data to assign the stereochemistry. The spectroscopic data
associated with new products are presented in this section. All data
associated with previously reported products are provided in the
Supporting Information.
18.7 min, tmajor = 20.10 min. IR (KBr disc): ν = 3369, 3200,
˜
max
1
1664, 1592, 804, 759 cm–1. H NMR (400 MHz, CDCl3): δ = 7.53
(td, J = 7.6, 1.8, 1.4 Hz, 1 H, Ar-H), 7.24–7.16 (m, 1 H, Ar-H),
7.16 (t, J = 7.6 Hz, 1 H, Ar-H), 6.22 (br, 1 H), 5.86 (s, 1 H,
CH2=C), 5.79 (s, 1 H, CH2=C), 5.54 (br, 1 H), 5.46 (s, 1 H, 3-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.5 (C-1), 142.9 (C-
F), 139.2 (C-2), 129.5 (C-2Ј), 127.9 (C-3Ј), 124.3 (CH2=C), 122.0
(C-5Ј), 115.3 (C-4Ј), 115.1 (C-6Ј), 68.9 (C-3) ppm. MS: m/z = 196.1
[MH]+. HRMS (ESI): calcd. for C10H11NO2F 196.0774 [MH]+;
found 196.0774.
(S*)-2-[(4-Fluorophenyl)(methoxy)methyl]acrylamide (27c): The title
product was obtained as a white solid (19 mg, 22.0% yield), m.p.
101.2–102 °C. Chiral HPLC analysis using a Chiralcel AD-H col-
umn with hexane/propan-2-ol (90:10) containing 0.1% TFA as the
mobile phase at a flow rate of 0.8 mL/min gave two peaks: tminor
=
11.60 min, tmajor = 11.11 min. IR (KBr disc): ν = 3197, 1657,
˜
max
1629, 1101, 861, 822 cm–1 1H NMR (400 MHz, MeOD): δ = 7.38–
7.29 (m, 2 H, Ar-H), 7.16 (t, J = 7.7 Hz, 1 H, Ar-H), 7.06 (t, J =
10.1 Hz, 1 H, Ar-H), 5.92 (s, 1 H, 3-H), 5.51 (d, J = 10.1 Hz, 2
H, CH2=C), 3.28 (s, 3 H, CH3O-CH) ppm. 13C NMR (100 MHz,
MeOD): δ = 171.0 (C-1), 143.6 (C-1Ј), 129.7 (C-2), 129.6 (CH2=C),
128.4 (C-4Ј), 124.0 (C-5Ј, C-3Ј), 119.6 (C-6Ј, C-2Ј), 75.1 (C-3), 56.2
(CH3-O) ppm. GC–MS (EI): retention time = 11.48 min; m/z (%)
= 209 (26.3), 194 (16.1), 178 (46.1), 161 (12.4), 139 (100), 123, 44.
HRMS (ESI): calcd. for C11H13NO2F requires 210.0930 MH+;
found 210.0929.
(S*)-2-[Methoxy(2-methoxyphenyl)methyl]acrylamide (28c): The ti-
tle product was obtained as a white solid (15 mg, 12.2% yield),
m.p. 103.0–104.2 °C. Chiral HPLC analysis using a Chiralcel OJ-
(S)-2-[(4-Fluorophenyl)(hydroxy)methyl]acrylamide (19c): The title H column with hexane/propan-2-ol (90:10) containing 0.1% TFA
product was obtained as a white solid (26 mg, 30.2% yield), m.p.
85.1–85.6 °C. Chiral HPLC analysis using a Chiralcel OJ-H col-
umn with hexane/propan-2-ol (90:10) containing 0.1% TFA as the
as the mobile phase at a flow rate of 0.8 mL/min gave two peaks:
tminor = 17.04 min, tmajor = 20.4 min. IR (KBr disc): ν
= 3187,
˜
max
1662, 1629, 1090, 1064, 884, 852 cm–1. 1H NMR (400 MHz,
CDCl3): δ = 7.36 (dd, J = 7.7, 1.8 Hz, 1 H, 3Ј-H), 7.28 (dd, J =
7.3, 1.8 Hz, 1 H, 5Ј-H), 6.97 (t, J = 7.3 Hz, 1 H, 4Ј-H), 6.91–6.86
mobile phase at a flow rate of 0.8 mL/min gave two peaks: tminor
=
16.09 min, tmajor = 17.20 min. IR (KBr disc): ν = 3378, 3195,
˜
max
8
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Date: 05-01-15 13:53:01
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Substrate Evaluation of Rhodococcus erythropolis SET1
van Rantwijk, A. Stolz, R. A. Sheldon, Tetrahedron: Asym-
(d, J = 8.2 Hz, 1 H, 6Ј-H), 6.82 (br, 1 H), 6.18 (s, 1 H, 3-H), 5.51
(s, 1 H, CH2=C), 5.43 (s, 1 H, CH2=C), 3.82 (s, 3 H, CH3-O-Ar),
3.38 (s, 3 H, CH3-O) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.3
(C-1), 157.2 (C-1Ј), 141.3 (C-2), 130.3 (C-3Ј), 130.1 (C-5Ј), 129.3
(C-2Ј), 127.2 (CH2=C), 124.8 (C-4Ј), 120.8 (C-6Ј), 57.2 (Ar-OCH3),
55.5 (CH3O-C) ppm. LRMS (ES): m/z = 222.1 [MH]+. HRMS
(ESI): calcd. for C12H16NO3 222.1130 [MH]+; found 222.1133.
metry 2006, 17, 320–323; c) F. Effenberger, S. Oßwald, Tetrahe-
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Acknowledgments
The authors would like to thank the Wales–Ireland Network for
Scientific Skills for financial support under the Ireland–Wales
2007–2013 INTERREG 4A programme granted to the depart-
ment. The authors would also like to thank Kilkenny VEC and
Waterford Institute of Technology for additional funding and Da-
mien Reid for contribution to the HBN pH and temperature stud-
ies.
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Received: September 11, 2014
Published Online: ᭿
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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9

Job/Unit: O43201
/KAP1
Date: 05-01-15 13:53:01
Pages: 10
T. M. Coady, L. V. Coffey, C. O’Reilly, C. M. Lennon
FULL PAPER
Enzyme Dual Activity
The nitrile hydrolysing bacterial isolate
Rhodococcus erythropolis SET1 has demon-
strated dual nitrilase/NHase activity de-
pending on the nitrile structure. β-Hydroxy
nitriles unsubstituted α to the nitrile group
gave acids along with recovered nitrile as
a result of nitrilase activity of the isolate,
whereas amides were the major product
from α-vinyl β-hydroxy nitriles due to
NHase activity.
T. M. Coady, L. V. Coffey, C. O’Reilly,
C. M. Lennon* ................................ 1–11
Substrate Evaluation of Rhodococcus
erythropolis SET1, a Nitrile Hydrolysing
Bacterium, Demonstrating Dual Activity
Strongly Dependent on Nitrile Sub-Struc-
ture
Keywords: Biotransformations / Enzyme
catalysis / Reaction mechanisms / Nitriles
10
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0