Microbial decyanation of 1-benzylpyrrolidine-2,5-dicarbonitrile. Mechanistic investigations

Article abstract of DOI:10.1016/j.molcatb.2010.11.006

Various bacterial and fungal strains were screened for their ability to catalyse the regioselective hydrolysis of 1-benzylpyrrolidine-2,5-dicarbonitrile (1). Among the examined strains, Rhodococcus opacus sp-lma whole cells transformed both isomers of 1 into 1-benzyl-5-cyano-2-pyrrolidinone (2) and N-benzylacetamide (3). These reactions are difficult to achieve chemically and the synthesis of compound 2 did not compete with microbiological catalysis in terms of efficiency and respect for the guidelines of green chemistry. To distinguish between an oxidative or hydrolytic based-mechanism, the origin of the oxygen atom in 2 was investigated by using ¹⁸O₂ and ¹⁸OH₂ coupled with GC-MS analysis. These experiments confirmed that the oxygen atom in 2 came from water and not from molecular oxygen. The reaction is probably initiated by the dehydrogenation of 1 to generate the iminium ion, which could be trapped by a water molecule to form the cyanohydrin. The cyanohydrin intermediate would spontaneously break down to the γ-lactam product 2. Conversion of 1 to 2 by induced rat liver microsomes suggests the involvement of a Cyt P-450-type enzyme. A mechanism that accounts for the formation of 3 is also proposed.

Full text of DOI:10.1016/j.molcatb.2010.11.006

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 211–215
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microbial decyanation of 1-benzylpyrrolidine-2,5-dicarbonitrile.
Mechanistic investigations
a
b
a
c
a,∗
Lucimar Pinheiro , Didier Buisson , Sylvie Cortial , Marcel Delaforge , Jamal Ouazzani
a
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, C.N.R.S, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
Unité Molécules de Communication et Adaptation des Microorganismes FRE3206, Muséum National d’Histoire Naturelle,
b
Département RDDM, CP54 57 rue Cuvier, 75005 Paris, France
c
CEA Saclay, iBiTec-S, SB2SM, URA2096 CNRS, Bat 532, 91191 Gif-sur-Yvette cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2010
Received in revised form 30 October 2010
Accepted 3 November 2010
Available online 11 November 2010
Various bacterial and fungal strains were screened for their ability to catalyse the regioselective hydrol-
ysis of 1-benzylpyrrolidine-2,5-dicarbonitrile (1). Among the examined strains, Rhodococcus opacus
sp-lma whole cells transformed both isomers of 1 into 1-benzyl-5-cyano-2-pyrrolidinone (2) and N-
benzylacetamide (3). These reactions are difficult to achieve chemically and the synthesis of compound
2
did not compete with microbiological catalysis in terms of efficiency and respect for the guidelines
of green chemistry. To distinguish between an oxidative or hydrolytic based-mechanism, the origin of
Keywords:
Rhodococcus opacus
Decyanation
1
8
18
the oxygen atom in 2 was investigated by using O2 and OH2 coupled with GC–MS analysis. These
experiments confirmed that the oxygen atom in 2 came from water and not from molecular oxygen.
The reaction is probably initiated by the dehydrogenation of 1 to generate the iminium ion, which could
be trapped by a water molecule to form the cyanohydrin. The cyanohydrin intermediate would spon-
taneously break down to the ␥-lactam product 2. Conversion of 1 to 2 by induced rat liver microsomes
suggests the involvement of a Cyt P-450-type enzyme. A mechanism that accounts for the formation of
␣
-Aminonitrile
Cyt P-450
3
is also proposed.
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
Nitriles are an important group of chemicals with an industrial
The efficiency and selectivity of these enzymes has driven
organic chemists to consider their use in biocatalysis to enantio-
and regio-selectively access chiral nitriles, carboxylic acids or
amides [9,10].
and environmental significance. They are used as solvents (e.g., ace-
tonitrile), in the fibre and plastic industry (e.g., Nylon and Acrylic)
and as pesticides (e.g., Casoron, Bentrol and Bucril). A number of
nitriles have been reported to be potent carcinogenic, neurotoxic
and environmental pollutants [1,2]. Soil, air and water contami-
nated with nitriles require remediation [3], and bio-remediation
represents a credible alternative to chemical processes.
The enzymatic conversion of nitriles [4] proceeds via different
pathways, as summarised in Fig. 1. Pathway I corresponds to a
direct hydrolysis of a nitrile to the carboxylic acid by a nitrilase
In the search for an environmentally benign and effective
method for a regioselective hydrolysis of 1-benzylpyrrolidine-2,5-
dicarbonitrile (1), we screened various fungi and bacteria. The
results confirmed the capacity of Rhodococcus strains to degrade
nitriles [11] (R. rhodocrous ATCC 21197, R. erythrophilus DCL14
and Rhodococcus opacus sp-lma). This catalysis involves an unusual
enzymatic decyanation mechanism consisting of the conversion
of the cyanopyrrolidine ring to the corresponding imminium ion,
witch leads to the obtained compounds after a series of rearrange-
ment.
[
5]. In II and III, a nitrile is converted by a nitrile hydratase to the
corresponding amide, which is subsequently converted to the car-
boxylic acid by an amidase [6]. The step IV involves the conversion
of an aldehyde to hydroxynitrile, that can be further hydrolysed to
the corresponding hydroxyacid or hydroxyamide by a nitrilase [7].
In V, a nitrogenase catalyses the release of nitrogen to convert the
CN to a CH3 group [8].
2
. Experimental
2.1. Chemicals and analytical techniques
1-Benzylpyrrolidine-2,5-dicarbonitrile (1) was kindly provided
by ORIL-Industrie (Servier-Group, Bolbec, France). Solvents and
compounds were obtained from Carlo Erba, and Sigma–Aldrich
and were of the highest grade required by their use. Formic acid
(98–100%) was obtained from Merck, Germany. Dioxygen 18O2
∗ Corresponding author. Tel.: +33 1 69 82 30 01.
E-mail address: jamal.ouazzani@icsn.cnrs-gif.fr (J. Ouazzani).
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.11.006

2
12
L. Pinheiro et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 211–215
H
H
OH
H
O
C
O
C
I
II
R
R
CN
R
CN
R
R
NH2
NH2
OH
H
I
III
I
V
IV
O
H
H
H
H
R
COOH
R
COOH
R
H
OH
I : Nitrilase, II : Nitrile hydratase, III : Amidase, IV Oxynitrilase, V : Nitrogenase
Fig. 1. Enzymatic conversions of nitriles.
(
99 atom% 18O) was purchased from Cortecnet, France. 18OH (97%)
2.4. Bioconversion conditions
2
was purchased from Cambridge Isotope Laboratories, UK. Bacto-
tryptone, yeast extract and malt extract were purchased from Difco,
Incubations were performed with resting cells. Fresh biomass
(2 g) was added to Erlenmeyer flasks (125 mL) containing 1-
benzylpyrrolidine-2,5-dicarbonitrile (1) (1 mg mL ) and water
1
and soya peptone was obtained from Organotechnie. Proton ( H)
1
3
−1
and carbon ( C) NMR spectra were recorded on Bruker spectrome-
ters, specifically an Avance 300 NMR or 500 NMR (300 and 500 MHz,
respectively).
(25 mL). The substrate was added as a solution in DMF (0.5%,
v/v). Control experiments were also conducted with substrate or
◦
biomass alone. The mixtures were stirred in a rotary shaker (30 C,
1
50 rpm). The progress of the biotransformation was monitored by
2.2. Gas chromatography–mass spectrometry (GC–MS) assays
GC as previously described.
The preparative incubation was conducted in 500 mL of water
containing 1-benzylpyrrolidine-2,5-dicarbonitrile (200 mg) and
Rhodococcus opacus sp-lma biomass grown on LB medium (20 g wet
weight). The reaction was monitored for 5 days. After this time, the
biomass was removed by centrifugation (4500 rpm, 20 min), and
the supernatant was extracted with AcOEt (3× 500 mL). The organic
layers that contained the target compounds were evaporated under
reduced pressure. The residue (160 mg) was purified by flash chro-
matography on silica gel (60% ethyl acetate: 40% heptane) to give
GC–MS data were obtained on a Hewlett-Packard 5890 Series
II gas chromatograph equipped with a 30 m × 0.25 mm × 0.25 ␮m
analytical column (HP-5). The capillary column was directly cou-
pled to a mass spectrometer (HP 5972-MS). The analyses were
performed under the following conditions: (a) the injector and
◦
◦
transfer line were set at 190 C and 280 C, respectively; (b) the
◦
◦
temperature program was set at 90 C (2 min) to 230 C (2 min) at
C min ; (c) the injection volume was 1 ␮L; (d) the split ratio was
:30; (e) the carrier gas flow was 25 cm s He; (f) the mass spectra
◦
−1
8
1
−
1
4
3 mg of 2 and 36 mg of 3.
1
were recorded at 70 eV. Prior to injection, all samples (800 ␮L) were
extracted with ethyl acetate (600 ␮L), agitated on a vortex mixer
and centrifuged at 14,000 rpm for 10 min.The supernatant (300 ␮L)
was diluted with ethyl acetate (800 ␮L), and the solution (200 ␮L)
was transferred to a GC vial and analysed by GC–MS. Under these
-Benzyl-5-cyano-2-pyrrolidinone 2, 1H NMR (CD OD,
3
3
1
7
4
00 MHz) ı 7.40–7.28 (m, 5H), 4.93 (d, J = 15 Hz, 1H), 4.45 (q,
13
H), 4.18 (d, J = 15 Hz, 1H), 2.70–2.26 (m, 4H); C NMR (CD OD,
3
5.5 MHz): ı 176.7, 136.5, 130.2, 130, 129.5, 129.4, 129.3, 118.8,
+
9.3, 46.6, 30.2, 24.6; ESI-MS m/z: 201 [M+H] ; HRMS calcd
•
+
conditions, the m/z values were 184 [M −CN] for 1a and 1b, 173
+
for C12H12N O [(M+H) ] 201.1028, found 201.1031; 2D NMR
2
•
+
•+
[
M
−CN] for 2, and 149 [M ] for 3.
experiments (COSY, HMBC, HSQC) were recorded on 500 MHz and
confirms the structure of 2.
N-Benzylacetamide 3, 1H NMR (CDCl3, 300 MHz,) ı 7.35–7.21
2
.3. Microorganisms and culture conditions
13
(
m, 5H), 4.36 (s, 2H), 1.99 (s, 3H,); C NMR (CDCl , 75 MHz) ı 173.2,
3
1
40.1, 129.7, 129.6, 128.7, 128.6, 128.4, 44.4, 22.5; ESI-MS m/z: 150
◦
Microorganisms were stored (at 4 C) on agar slants containing
+
[
M+H] .
−
1
the media reported below supplemented with agar at 20 g L . A
screen involving 7 fungi and 6 bacteria was conducted. The fungi
examined were Aspergillus ochraceus ATCC 1009, Rhizopus arrhizus
ATCC 11145, Geotrichum candidum CBS 23376, Cunningamella
echinulata NRRL 3655, Curvularia lunata NRRL 2380, Fusarium oxys-
porum AP68MLPPON and Beauvaria bassina ATCC 7159. The fungi
were grown at 27 C on a rotary shaker (200 rpm) in a medium with
the following composition (per litre): 0.5 g KH PO , 1 g K HPO ,
0 g d-glucose, 10 g corn steep, 0.5 g MgSO , 2 g NaNO , 0.5 g
KCl and 0.02 g FeSO . The bacteria investigated were Rhodococcus
rhodocrous ATCC 21197, Rhodococcus erythrophilus DCL14, Pseu-
domonas putida CIP.59.19, Agrobacterium tumefaciens C58, Bacillus
licheniformis CIP.52.71.T, and Rhodococcus opacus sp-lma and were
isolated in our laboratory. Bacteria were cultivated on classical
media (LB and YMS).
The culture media were sterilised at 120 C for 20 min.
The microorganisms were cultivated at 30 C for 72 h in an
orbital shaker (200 rpm). Biomass was recovered by centrifugation
4100 rpm, 30 min, 10 C) and used immediately.
.5. 18O and 18OH2 incubations
2
Rhodococcus opacus sp. was grown in an LB medium (300 mL),
harvested and resuspended in H O (9 mL). The cell suspension
2
◦
(2 mL) was transferred to 10 mL-vials, which were subsequently
2
4
2
4
sealed with a septum. Air was replaced with argon using three
vacuum-purge cycles. The headspace of the vial was then evacu-
ated and replaced with O2 or pure dioxygen enriched with O2
3
4 3
16
18
4
18
(99 atom% O). One of the vials was maintained under an argon
atmosphere. In a second series of experiments, the cell suspen-
sion (1 mL) was transferred to Eppendorf tubes and centrifuged
at 14,000 rpm for 5 min. The supernatant was removed, and the
biomass was transferred to a 5 mL-vial and resuspended in water
enriched with O (97 atom% O). A control experiment was con-
ducted using the same conditions with unlabelled water. Finally,
◦
18
18
◦
1
0 micromoles of substrate were added by an injection of 5 ␮L of a
◦
(
−1
solution in DMF (final concentration 1.15 g L ).

L. Pinheiro et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 211–215
213
Fig. 2. GC–MS data were obtained on a Hewlett-Packard 5890 Series II gas chromatograph equipped with a 30 m × 0.25 mm × 0.25 ␮m analytical column (HP-5) coupled to
a mass spectrometer (HP 5972-MS). Prior to injection, all samples (800 ␮L) were extracted with ethyl acetate (200 ␮L), and then 1 ␮L of the extracted is injected on the GC
column.
A series of experiments were conducted with lyophilised
biomass. For this procedure, 0.1 g of dry biomass was resuspended
in water enriched with 18O (1 mL, 97 atom% O). A solution of 1 in
were concentrated to 100 ␮L, and then 1 ␮L of each was directly
injected onto the GC–MS.
18
−
1
DMF (10 ␮L) was added to reach a final concentration of 1.15 g L
.
3. Results and discussion
A control experiment with unlabelled water was also conducted.
◦
All the experiments were stirred in a rotary shaker (32 C,
00 rpm). The progress of the biotransformation was monitored
Rhodococcus related strains convert the substrate 1 to two
2
derivatives, as observed in the GC chromatograms. After a series
of optimisations investigating the effects of wet biomass weight,
medium (LB, YMS), temperature and pH, the strain Rhodococ-
cus opacus sp-lma, was found to be the most effective and
was able to completely convert the starting substrate in 3 days
by GC–MS at 24, 48 and 96 h. A separate starting sealed vial was
used for each data point to avoid any disturbance due to sample
withdrawal.
2.6. Preparation of rat liver microsomes and microsomal
(Figs. 2 and 3). A preparative incubation was conducted to isolate
incubations
sufficient amounts of the compounds to elucidate their structures.
Thus both trans and cis isomers of 1 were converted to 1-benzyl-
Male Sprague–Dawley rats (175–200 g) were treated with dex-
5
-cyano-2-pyrrolidinone 2 and N-benzylacetamide 3. All products
amethasone (100 mg/kg, intra-peritoneal in corn oil) once per day
for three days. The animals were then killed, and their liver micro-
somes were prepared according to standard methods [11]. The
protein and cytochrome P-450 contents were measured using stan-
dard techniques [12].
were identified and characterised by Mass and NMR spectroscopy
1
13
1
1
(
H and C, H, H-gCOSY, HSQC and gHMBC).
A stock solution of 1 was prepared in DMSO. Compound 1 was
incubated with rat liver microsomes in the presence of NADPH at
R. opacus sp.lma
◦
3
7 C in 1 mL of 50 mM Tris–HCl buffer pH 7.4. The final concen-
N
N
HN
H O, 32 oC, 3 days
O +
O
trations of compound 1 and NADPH were 2.13 ␮M and 4.2 ␮M,
respectively. Microsomes were added to reach a final concentra-
tion of 4 ␮M of cytochrome P-450. The incubations, including all
required controls, were terminated after 2 h by the addition of ethyl
acetate (500 ␮L) and extensive extraction. The organic extracts
N
N
2
N
1
2
3
Fig. 3. Bioconversion products of 1-benzylpyrrolidine-2,5-dicarbonitrile
Rhodococcus opacus sp-lma.
1 by

2
14
L. Pinheiro et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 211–215
(Fig. 5). Although this synthesis offered access to 2, the overall
yield was limited to 12% (instead of 22% in non optimized biocat-
alytic condition), and the reaction catalysts and conditions did not
compete with microbiological catalysis in terms of efficiency and
respect for the guidelines of green chemistry.
To elucidate the mechanism of this reaction and to discrimi-
nate between hydroxylation or deshydrogenation step, we carried
out incubation experiments with 18O2 and OH2 under different
18
18
conditions. The bioconversion and incorporation of the O label
were monitored using GC–MS analysis. Among the various condi-
18
tions screened, only the combination of air and OH2 led to 50%
1
8
18
incorporation of O in 2 and 3. No O incorporation was observed
with 18O , indicating that molecular oxygen is not involved in
2
the reaction. As the oxygen atom in 2 seems to be derived from
Fig. 4. Chemical decyanation routes.
18
OH , the water contained in the wet biomass may account for
2
the incomplete incorporation of the isotopic label. Therefore, an
equivalent incubation was performed with a corresponding quan-
tity of lyophilised biomass. In support of our hypothesis, the use of
lyophilizedbiomass ledto86%and75%of 18Oincorporation in 2 and
3, respectively. Based on these observations, a mechanism of decya-
nation of 1 is proposed in Fig. 6. Dehydrogenation of 1 leads to the
iminium-carbonium derivative. The carbocation could be trapped
by a water molecule to form the cyanohydrin intermediate, that
spontaneously breaks down to the ␥-lactam product 2.
Incubation monitoring showed that compound 3 is formed
simultaneously with and not subsequently to compound 2. The
incubation of 2, isolated from previous experiments, with R. opacus
did not lead to compound 3, nor did it form any other derivatives.
From day 3 to day 7, the concentration of compound 2 remained
constant, whereas compound 3 was further degraded to com-
pounds that were not detected by GC. After one week of incubation,
compound 2 was isolated in almost pure form in 40% yield after
biomass removal and extraction in ethyl acetate.
This unexpected microbial decyanation route encouraged us to
compare it to chemical reactions that achieve the same transforma-
tion. The catalysis observed with R. opacus resembles the oxidative
decyanation of secondary nitriles to ketones by a three-step process
involving the alpha-halogenation of the nitrile derivative followed
by the substitution of the halogen by hydroxide, and the conversion
of the resulting cyanohydrin to the corresponding ketone [12] (bold
arrows in Fig. 4). In our study, this method proved unsuitable for the
halogenation of compound 1. Kyler and Watt [13] also reported the
ineffectiveness of this method and compared five procedures on a
large diversity of nitriles (Fig. 4). The authors concluded that none
of these routes is universal, and they suggested testing all of them
for each specific nitrile compound. Furthermore, these methods
have been established for mononitriles, and there is no expectation
of selectivity in the case of dinitriles. The alternative route (bold
dashed arrows in Fig. 4) was applied to compound 1; however, the
halogenation step was not successful.
Our results obtained with 18OH2 and 18O contrast with the pro-
posed mechanism of degradation of pyrrolidines and piperidines by
microorganisms [20]. The first step leading to the lactam is proba-
bly the formation of an iminium ion rather than monooxgenation.
The formation of the benzyl-acetamide 3 could be due to a rear-
rangement according to Fig. 7.
To obtain more support for the proposed mechanism, we
searched the literature for enzymes that may catalyse the con-
version of the pyrrolidine ring to the iminium derivative. Ho
and Castagnoli [21] reported the mammalian metabolism of ben-
zylpyrrolidines by liver microsome preparations in the presence
and absence of a cyanide ion. In the presence of a cyanide ion, the
authors identified a cyanohydrin derivative by mass spectrometry
Besides this approach, methods for the direct oxidation of alpha-
aminonitriles to the corresponding lactams with peracids [14] or
through electron transfer photoinduced oxidation [15] were inves-
tigated and were effective for acyclic amino-nitriles. We have
screened these approaches using a peracid (mCPBA) or a highly
reactive oxygen species (hydroxyl radical generated by Fenton oxi-
dation); both were unsuccessful and led only to degradation of the
starting material. These results underscore the difficulty in achiev-
ing the conversion of 1 to 2 and the synthetic potential of the
microbial alternative.
After encountering some initial difficulty with accessing com-
pound 2 from 1 through chemical steps, we achieved the synthesis
of 2 by applying previously reported classical reactions [16–19]
H
O
N
3 3 3 2
(CH ) SiCN, BF .OEt
KOH, DMF
Br-Bn
NaBH4, 2N HCl
EtOH
PhSO2H, CaCl2
CH2Cl2, rt
O
O
N
O
N
O
N
CH2Cl2, -78 to rt, 17h
SO2Ph (Ref 19,20)
O
N
(
Ref 17)
(Ref 18)
OEt
(Ref 19, 20)
O
N
2
Fig. 5. Synthesis of the 1-benzyl-5-cyano-2-pyrrolidinone 2, according to reported procedures.
H2O
R. opacus
N
N
N
N+
N
N
N
N
N
N
N
H
O
N
N
N
O
1
Iminium ion
Carbonium ion
2
Cyanohydrin
Fig. 6. Proposed mechanism of microbial decyanation supported by the 18O2 and 18OH2 experiments.

L. Pinheiro et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 211–215
215
In conclusion, Rhodococcus opacus is able to catalyse the con-
version of aminonitrile 1 to the corresponding lactam 2 efficiently.
Access to compound 2, is difficult or impossible to achieve by direct
oxidation, hydrolysis or independent chemical synthesis. Rat liver
microsomes, known to express large quantities of Cyt P-450, are
able to catalyse this reaction. This result suggests that this type of
enzyme, highly represented in Rhodococcus genera, is involved in
the conversion of 1 to the iminium intermediate. This reaction will
be extended to other aminonitriles to further demonstrate its use-
fulness as a “green” alternative to harsh chemical procedures for
accomplishing the same transformation.
18OH
18O
2
N
+
N
N
N
N
HN
18O
N
N
3
Iminium ion
Fig. 7. Proposed mechanism for the formation of compound 3.
Acknowledgments
The authors want to thank Oril-Industrie, Bolbec, France, for
financial support.
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2
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401.
This result strongly supports the involvement of Cyt P-450 type
enzymes in the conversion of compound 1 by Rhodococcus opacus.
This type of enzyme is well-represented in Rhodococcus genera and
accounts for a variety of oxidative reactions [11,26].
25] P. Kremers, Ph. Beaune, T. Cresteil, J. De Graeve, S. Columelli, J.P. Leroux, J. Gielen,
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J. Struct. Biol. 170 (2010) 548.