Enhancement of whole cell dioxygenase biotransformations of haloarenes by toxic ionic liquids

Article abstract of DOI:10.1039/c4ra00640b

Accessing chirally pure cis-diols from arenes using micro-organisms over-expressing toluene dioxygenase (TDO) is now well established, but the conversions remain low for the more toxic and volatile substrates. For such arenes, improved production has alre

Full text of DOI:10.1039/c4ra00640b

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Enhancement of whole cell dioxygenase
biotransformations of haloarenes by toxic ionic
Cite this: RSC Adv., 2014, 4, 19916
liquids†
a
b
b
bc
C. C. R. Allen, C. J. Boudet, C. Hardacre and M. E. Migaud*
Accessing chirally pure cis-diols from arenes using micro-organisms over-expressing toluene dioxygenase
TDO) is now well established, but the conversions remain low for the more toxic and volatile substrates. For
(
such arenes, improved production has already been achieved in the presence of hydrophobic non-toxic
ionic liquids (ILs) acting in the form of a reservoir for the arene substrate. Yet, the costs associated with
such ILs require extensive process development to render them viable. Herein, we show that
optimization of the hydrophobic IL's cationic moiety and of the IL's concentration are key to enhanced
conversion yielding between a 2–5 fold yield increase in the conversion of four haloarenes (Ph–X; X ¼ F,
Cl, Br, I). Additionally, we report that hydrophilic imidazolium-based ILs offer opportunities to achieve
similarly high yielding biotransformations, with further improved reaction rates (<6 h), and this at very low
ILs' concentrations (0.0015 VIL/Vaq). We also demonstrate that the increased biotransformations are due
to these ILs being inhibitors of cellular respiration processes and thus favoring the shunting of NADH and
Received 22nd January 2014
Accepted 4th April 2014
DOI: 10.1039/c4ra00640b
www.rsc.org/advances
2
O towards the overexpressed biocatalytic process.
to the biocatalyst at concentrations remaining below the
inhibitory concentration, thereby improving regio-selectivity
Introduction
8
The use of whole cell biocatalysis to obtain chirally-dened ne and/or overall yields. As such, the development of biocatalytic
chemicals and the development of “greener” chemical processes in alternative media has been of growing interest even
processes is becoming common practice for the chemical though the rationale underpinning the observed effects
1,2
9–12
industry. For instance, the versatile production of chirally remains somewhat uncharted.
pure cis-diols from arenes can be achieved in variable yields
Previous studies, including the pioneering work of Cull
3
13
using organisms such as Pseudomonas putida UV4. Such et al. have shown that ILs, including hydrophobic ILs, can be
compounds, which cannot be obtained effectively via any other used to enhance biotransformations with a variety of whole cell
8,14,15
sources, are ideal materials to be used in ne chemical and biocatalysts.
The primary mechanism of enhancement with
4
–6
chiral ligand syntheses. While successful, improvements in hydrophobic substrates is thought to involve increasing
the biotransformation conditions and related processes are substrate availability and, therefore, mass transfer to the
always being sought. To this end, organic solvents or mixed/ enzyme, whilst decreasing free substrate overall concentration
biphasic aqueous solutions/organic media whereby improved to limit cell toxicity. However, other studies have shown that ILs
conversions/yields can be achieved, have been extensively can be strong inhibitors for whole cell metabolic processes. The
8
studied. These have been developed to overcome limitations limited rationale for such broad ranging behaviour has thus
2,15
due to low substrate solubility in aqueous solutions or to limited their widespread application.
7
minimise product inhibition. For example, reactions per-
In an attempt to examine how hydrophobic ionic liquids can
formed in mono- or biphasic conditions for isolated enzymes or be used as suitable agents in the biphasic whole-cell dihydrox-
whole cell systems have been developed to release the substrate ylation of halobenzenes by P. putida UV4, conditions were
identied whereby improved yields and reaction rates were
easily achieved, but for which the biocatalyst was compromised
School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 by these same conditions. The aims were therefore to establish
a
7
BL, Northern Ireland, UK
how the ILs could be used to enhance biotransformation rates
via a two phase system while improving the mass transfer, and
establish the mechanism(s) which was responsible for the
increases in rate observed. This allowed for routes, via inhibi-
tion of cellular biochemistry leading to an indirect enhance-
ment of enzyme activity, to be probed. Therefore, for the rst
b
School of Chemistry and Chemical Engineering, Queen's University of Belfast,
Stranmillis Road, BT9 5AG, Northern Ireland, UK
c
School of Pharmacy, Queen's University of Belfast, 97 Lisburn Road, Belfast, BT9 7BL,
Northern Ireland, UK. E-mail: m.migaud@qub.ac.uk
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra00640b
1
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time, the nature of the IL and its effect on the biocatalyst has nesulfonyl)imide ([C10dmim][NTf
2
]); N-butyl,N-methylpyrro
been related to the overall biotransformation process.
lidinium-bis(triuoromethanesulfonyl)imide ([C mpyrrol][NTf ]),
4 2
N-octyl,N-methylpyrrolidinium-bis(tri-uoromethanesulfonyl)
imide ([C mpyrrol][NTf ]), trihexyl-tetradecyl-phosphonium-
8
2
Results and discussion
Impact of hydrophobic ILs on cis-dihydroxylation
bis(triuoromethanesulfonyl)imide ([P66614][NTf
carbonylmethyl-1,2-dimethyl-imidazolium-bis(triuorometha
nesulfonyl)imide, [C 13OCOCH dmim][(CF SO N]; 3-
2
]); 3-hexoxy-
biotransformations
6
H
2
3
2 2
)
The biocompatibility of secondary phases is an important octoxycarbonylmethyl-1,2-dimethylimidazolium-bis(triuoro-
criterion in the choice of the solvent for the whole cell methanesulfonyl)imide, [C 17OCOCH dmim][(CF SO N], N-
Therefore, an understanding hexoxycarbonyl-methyl-N-methylpyrrolidinium-bis(triuoro-
of the toxicity of the ILs for the biotransforming cell lines is methanesulfonyl)imide, [C 13OCOCH mpyrrol][(CF SO N];
8
H
2
3
2 2
)
16–18
biotransformation processes.
H
)
2 2
6
2
3
crucial. Pfruender et al. have shown that the biocompatibility N-octoxycarbonyl-methyl-N-methylpyrrolidinium-bis(triuoro-
ꢀ
and partition coefficient of four ILs based on the [PF
6
]
and methanesulfonyl)imide, [C
8
H
17OCOCH
2
mpyrrol][(CF
3
SO
2
)
2
N],
ꢀ
17,18
[NTf2] anions,
including [C mim][NTf ], had a major posi- 3-(N-butyl-N-methylcarbamoylmethyl)-1,2-dimethylimidazolium-
4 2
tive impact on the yield of conversion of 4-chloroacetophenone bis(triuoromethanesulfonyl)imide, [C
4
H
9
CH
3 2
NCOCH dmim]-
0
to (R)-1-(4-chlorophenyl)ethanol by Lactobacillus ker compared [(CF
3
SO
2
)
2
N] and N -(N-butyl-N-methylcarbamoylmethyl)-
0
with the addition of organic solvents. The improved yields were N -methylpyrrolidinium-bis(triuoromethanesulfonyl)imide
proposed to be due to the non-destructive effect of the ILs on [C
H CH NCOCH mpyrrol][(CF SO ) N]. The volume ratio of
9 3 2 3 2 2
4
the cell membrane when compared with the organic solvents. the IL:water in the two phase system ranged from 0.0015 to 0.02
21
Additional studies led to the proposal that the formation of (VIL/Vaq) ratio.
small droplets of ILs containing the arene substrate were
In the rst instance, the initial reaction rates of chloroben-
generated and improved the arene's dispersion in solution thus zene by P. putida UV4 in the presence (0.02 (VIL/Vaq)) or absence
overcoming the high viscosity associated with the ILs used. In of ILs were recorded and the amounts of biotransformed
comparison, Stephens and co-workers showed that the same product were measured aer 6 h of reaction (Table 1). Under
non-water miscible IL ([C mim][NTf ]) was toxic to the E. coli these conditions, only [C dmim][NTf ] was found to result in
4 2 8 2
strain MG1655 pDTG601A over-expressing TDO and, therefore, improved yields for the P. putida UV4 catalysed biotransfor-
was unsuitable for the oxygenase-based biotransformations mation compared with the non-IL based system. For the imi-
catalysed by that clone, while tetraalkyl phosphonium and tet- dazolium-based ILs incorporating a side chain below eight
ꢀ
raalkyl ammonium [NTf
2
]
based ILs provided 2.5 fold carbons, the biotransformation rate was reduced which may be
8
enhancement for the biotransformations.
In order to establish how selected ILs enhance the conver- [C
sion of arenes to cis-diols by another ring-hydroxylating dioxy- [NTf
genase expressing organism, the TDO expressing bacterium P. inhibitory effect on the biotransformation process (Table 2).
putida UV4 was chosen. This organism has been shown to have Finally, the effect of the [P66614][NTf ] ILs on the initial rates and
a wide range of activity towards diverse arene substrates and is, overall yields were minimal when compared with the control
due to the IL toxicity (e.g. [C
(d)mim][NTf ]) of the enzyme. At 0.02 (VIL/Vaq), [C
] and the [C mpyrrol][NTf ] demonstrated mainly an
4
(d)mim][NTf
2
]) or inhibition (e.g.
6
2
4
mpyrrol]-
2
8
2
2
19
therefore, a useful industrial catalyst. Investigative whole cell (Table S2 ESI†). These results are in agreement with Stephens
biotransformations were rst conducted with chlorobenzene et al. where the C4 alkyl chain containing ILs proved unsuitable
8
(
Ph–X, where X ¼ Cl), as the biotransformation of chloroben- media to conduct biotransformations. The difference in
zene to cis-1,2-dihydroxy 3-chlorobenzene has been demon- biotransformation proles, in particular with regards to
strated to be a very high yielding biotransformation with this “toxicity” vs. “inhibition”, led to investigating the direct effects
enzyme system.
19,20
of the ILs on the biocatalyst and this as a function of the IL's
The initial rates of the biotransformation of chlorobenzene concentration and of the IL's cation.
to cis-1,2-dihydroxy 3-chlorobenzene with bacterium P. putida
Clearly, ILs incorporating short chain lengths had a detri-
UV4 and the maximum conversion yields for the product were mental effect on the overall biotransformation process (Tables 1
1
quantied by H-NMR. In these studies, a range of ionic liquids and 2). However, the imidazolium-based ILs which incorpo-
employed under biphasic conditions was examined and these rated longer side-chains (n > 6), exhibited faster initial reaction
ILs were: 1-butyl,3-methylimidazolium-bis(triuoromethane- rates and 3-fold increase in yields aer 2 h when compared with
sulfonyl)imide ([C
bis(triuoromethanesulfonyl)imide
methyl,3-octylimidazolium-bis(triuoromethanesulfonyl)imide which shows the reaction-time prole for the conversion of
[C mim][NTf
]), 1-decyl,3-methylimidazolium-bis(triuoro-metha- chlorobenzene in the biphasic system using [C dmim][NTf
nesulfonyl)imide ([C10mim][NTf
]), N-butyl,N-methylmethyl- present in volume ranging between 0 and 0.02 (VIL/Vaq) ratio.
imidazolium-bis(triuoromethanesulfonyl)imide [C
dmim]- From this trend, it is clear that minimal amounts of IL are
NTf2], 1-hexyl,2,3-dimethylimidazolium-bis(tri-uorometha- required to achieve a sizable enhancement in the biotransfor-
4
mim][NTf
2
]), 1-hexyl,3-methylimidazolium- the control experiment where no IL was present.
([C mim][NTf ]), 1- The effect of the volume ratio can be best visualised in Fig. 1,
6
2
(
8
2
8
]
2
2
4
[
nesulfonyl)imide
([C dmim][NTf ]),
1-octyl,2,3-dimethyl- mation with 0.0015 (VIL/Vaq) showing the most signicant
dmim]- increase observed.
2
]), 1-decyl,2,3-dimethylimidazolium-bis(tri-uorometha-
6
2
imidazolium-bis(triuoromethanesulfonyl)imide ([C
NTf
8
[
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Interestingly, when the same volume ratio (0.0015) was used
employing [C mpyrrol][NTf ] a similar enhancement in the
8
2
biotransformation activity was observed as found with
C dmim][NTf ] (Fig. 2). In contrast, little effect was observed on
[
8
2
the initial rates and overall bioconversion using the phospho-
nium based ILs at these low concentrations. This is in stark
contrast to the work of Cornmell et al. who reported >200%
bioconversion enhancement using an E. coli cloned TDO for
8
this phosphonium IL. However, the concentration of ILs used
in the present work is very different from that employed in other
studies with dioxygenases, where a phase ratio greater than 0.20
was used. The different behaviour may, therefore, be linked
with improved mass transfer at high IL loading.
To further examine whether other structural aspects of the
imidazolium and pyrrolidinium cationic part of the ILs which
impacts on their polarity and solubility, play a role in the
observed results, a range of ester and amide modied imida-
zolium and pyrrolidinium-bis(triuoromethanesulfonyl)imide
based ILs were examined at 0.0015 (V /V ). The ester con-
IL aq
taining
17OCOCH
and [C 17OCOCH
side chains on the imidazolium cation resulted in an increase in
rate and activity similar to that found for [C dmim][NTf ] (see
ILs
included
dmim][NTf ],
mpyrrol][NTf
[C H OCOCH dmim][NTf ],
6
13
2
2
[C
8
H
2
2
[C
6
H
13OCOCH
2
mpyrrol][NTf
2
],
8
H
2
2
]. The presence of an ester
8
2
Table S6†) over the same period, with sustained activity with the
longer chain, albeit slightly lower than that of [C dmim][NTf ].
8
2
Surprisingly, the modied pyrrolidinium based ILs displayed
no effect on the biocatalytic process. The homologous amide
chains containing ILs ([C
4
H
9
CH
]) were also compared with
] in the biotransformation of chlorobenzene by
3 2 2
NCOCH dmim][NTf ] and
[C
C
4
8
H
9
CH
3 2 2
NCOCH mpyrrol][NTf
[
dmim][NTf
2
P. putida UV4 (Fig. 3).
Unlike ester side chains, the amide side chain on the imi-
dazolium or pyrrolidinium cations showed little effect on the
biotransformation rates. However, this functionalization,
despite being of similar chain length to the C alkyl parent, did
6
not result in the inhibition of biocatalyst unlike that found in
the case of [C
increase to high conversion over the rst six hours of
biotransformation was observed for [C CH NCOCH dmim]-
NTf ] (Fig. 3). This may be due to the fact that the delivery of
6 2
dmim][NTf ], for example. In this case, a linear
4
H
9
3
2
[
2
substrate in the aqueous solution may have been limited by the
high viscosity of the amide functionalised IL which was greater
that of [C dmim][NTf ]. Alternatively, this IL might be working
8
2
via a different mechanism from the [C
mpyrrol][NTf ] ILs.
8 2
dmim][NTf ] and the
[C
8
2
Impact of hydrophobic ILs on cis-dihydroxylation
biotransformations of Ph–X
To examine whether the biotransformation optimised for
chlorobenzene could also be applied with similar enhanced
conversions to the other haloarenes, these conditions were
applied to uoro-, bromo- and iodo-benzene in the presence of
[
C dmim][NTf ] and [C mim][NTf ] ILs at 0.0015 (V /V ) and
8 2 8 2 IL aq
22,23
compared with the reaction in the absence of the IL.
Table 3
summarises the results of these biotranformations. For all
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Table 2 Biphasic biotransformation of chlorobenzene in the presence of phosphonium and pyrrolidinium based-IL with P. putida UV4 (2.0% (VIL
/
ꢀ1
a
Vaq) IL + substrate) for OD600 ¼ 0.9 or 0.35 g L dcw
Control
[P66614][NTf
2
]
[C
4
pyrrol][NTf
2
]
8 2
[C pyrrol][NTf ]
Conversion @ 6 h [%]
Initial reaction rate [mM h
36.8 (ꢁ0.6)
35.5 (ꢁ0.7)
0.54 (ꢁ0.3)
n.r.
n.r.
15.4 (ꢁ0.0)
0.56 (ꢁ0.0)
ꢀ1
]
0.47 (ꢁ0.01)
a
n.r. ¼ no reaction; calculated values show mean of duplicate samples.
Fig. 1 Effect of volume ratio (VIL/Vaq) on the biotransformation yield
using [C dmim][NTf ] phase while maintaining a constant number of
8
2
moles of chlorobenzene substrate, set at 0.98 mmol.
Fig. 3 Biotransformation of chlorobenzene by P. putida UV4 with
novel-amide-IL phases (imidazolium and pyrrolidinium cations).
Conversion of 0.98 mmol chlorobenzene in buffer (pH 7.2), (:)
without IL and biphasic system volumetric ratio 0.0015 (VIL/Vaq), (-)
[
[
C
C
8
dmim][NTf
2
], (A) [C
4
H
9
CH
3
NCOCH
2
dmim][NTf
2
]
and (C)
ꢀ
1
4
H
9
CH NCOCH
3
2
mpyrrol][NTf
2
]. OD600 ¼ 1.8 or 0.70 g L dcw –
data points show mean of duplicate samples.
To establish whether the improve biotransformation is due
to an improved mass transfer and substrate availability, the
solubility of the arenes in the hydrophobic ILs (Fig. 4a) and their
release into the growth medium over time (e.g. for iodobenzene,
Fig. 4b) were determined. The ILs demonstrated an ability to
host effectively the haloarenes and deliver them to the aqueous
phase in a controlled manner over a 4 h period (Fig. 4b).
The solubility of all the arenes in ILs increased as a function
of the chain length of the cation and its increased lipophilicity.
The overall mass transfer coefficient was then determined for
Fig. 2 Biotransformation of chlorobenzene by P. putida UV4 with
0
[
(
.0015 (VIL/Vaq) [C
NTf ]. Conversion of 0.98 mmol chlorobenzene in a buffer (pH 7.2),
:) without IL and biphasic system volumetric ratio 0.0015 (VIL/Vaq
mim][NTf ], (-) [C dmim][NTf ] and (>) [C mpyrrol]-
8 2 8 2 8
mim][NTf ], [C dmim][NTf ] and [C mpyrrol]-
8 2
each of the arenes in the [C (d)mim][NTf ] (Fig. 5). The data
clearly showed that the arene that was most effectively released
from the ILs was, as expected, the uorobenzene and that
2
)
IL, (B) [C
NTf
8
2
8
ꢀ
2
8
1
[
2
]. OD600 ¼ 1.8 or 0.70 g L dcw – data points show mean of increased lipophilicity of the arene resulted in slower release
duplicate samples.
rates. Importantly, the overall mass transfer of chlorobenzene
from [C dmim][NTf ] ILs (n¼ 6, 8 and 10) to the growth medium
n
2
ꢀ
5
ꢀ1
remained constant at ꢂ4 ꢃ 10
s
and was found to decrease
ꢀ
5
ꢀ1
ꢀ5 ꢀ1
only slightly from 6 ꢃ 10
s
to 4 ꢃ 10
s
on increasing the
substrates, the presence of the ILs enhances the overall yields
and rates of the biotransformations with a large increase found
for uorobenzene. This is important as the high volatility of
chain length (n ¼ 6, 8, 10) for the [C
n
mim][NTf ] ILs.
2
Impact of hydrophilic ILs on cis-dihydroxylation
biotransformations

uorobenzene compared with that of the other halogenated
arenes is a major limiting factor for its implementation in
bioprocesses. Here the effect of [C (d)mim][NTf ] might indi- One proposed mechanism for the IL effect on the biotransfor-
8
2
cate improved mass transfer for the uorobenzene through its mation of arenes using P. putida UV4 is that it is due to a
retention in the growth medium.
measurable storage/release process whereby the controlled
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Table 3 Biotransformation of haloarenes by P. putida UV4 with imidazolium hydrophobic-IL phases. Conversion of 0.98 mmol halobenzene in
ꢀ1
buffer (pH 7.2), OD600 ¼ 1.8 or 0.70 g L dcw – data points show mean of duplicate samples
Fluorobenzene
Chlorobenzene
Bromobenzene
Iodobenzene
Without IL
5.1% ꢁ 0.3%
18.3% ꢁ 1.8%
25.6% ꢁ 0.4%
502
33.4% ꢁ 0.4%
68.0% ꢁ 1.7%
70.0% ꢁ 1.6%
210
36% ꢁ 0.9%
83.5% ꢁ 1.2%
84.3% ꢁ 2%
230
41.9% ꢁ 2.4%
81.2% ꢁ 3.4%
77.6% ꢁ 1%
190
[
[
C
C
8
dmim][NTf
mim][NTf
2
]
]
8
2
%
Improvement
mass transfer of the arenes into the growth medium is
responsible for the observed enhancement. Yet, the extent of
the enhancement on the biotransformations for each of these
arenes does not correlate with simple improved mass transfer
or substrate availability as the overall mass transfer coefficient
remains fairly constant for three of the four haloarenes studied
in [C mim][NTf ] and [C dmim][NTf ] (n¼ 6 and 8) ILs, while
n
2
n
2
the overall yields and rate differ as a function of ILs.
Additionally, the presence of a methyl group at the C(2)
position improves the biotransformation more effectively than
the ILs with a H in the C(2) position on the imidazolium cation
despite the fact that these ILs are also more toxic to the cells
(Fig. 6). The increased solubility of the hydrophobic-IL in water,
which was found to be higher for [C mim][NTf ] than [C dmim]-
8
2
8
[
NTf ] (Table S3†) is therefore unlikely to be solely responsible
2
for the “activation through inhibition” of the biocatalysts or
improved mass transfer. In contrast, [C mpyrrol][NTf ], dis-
playing further reduced aqueous solubility in water (Table S4†)
when compared with [C mim][NTf ] and [C dmim][NTf ], dis-
8
2
8
2
8
2
played an inhibitory inuence on the biocatalyst at high
concentration and enhanced biocatalysis both in terms of rate
and over yields when present at low concentration. Additionally,
the C4 and C6 alkyl chain containing ILs, whilst having greater
solubility in water, thus improving mass transfer and decreased
cellular toxicity compared to the longer alkyl chain containing
ILs, completely suppress the biocatalytic conversion (Tables 1
and 2). These latter ILs are likely, therefore, to inhibit enzyme(s)
Fig. 4 (a) Solubility of haloarenes in hydrophobic ILs as a function of
the imidazolium side chain length; %mole fraction of substrate to IL at
equilibrium after 4 h; (b) delivery of iodoarenes into the growth
medium from a saturated [C
8
dmim][NTf
2
]–iodobenzene solution.
ꢄ
0
.0025 (VIL/Vaq) system shaken at 150 rpm at 30 C over 240 min.
Fig. 6 Viable cell count of P. putida UV4 in the presence of 0.02 (VIL
/
V
aq
)
[C(n)mim][NTf (grey), [C(n)dmim][NTf (blue) and control
2
]
2
]
without IL (Horizontal line). Cells re-suspended in carbon source free
Fig. 5 Overall mass transfer coefficient of haloarenes in hydrophobic
(d)mim][NTf ] ILs.
buffer solution (pH 7.2) and exposed to the IL for 3 h. OD600 ¼ 2 or 0.78
[
C
8
2
ꢀ1
g L dcw – data points show mean of triplicate samples with range.
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of the dihydroxylation pathway which are not critical to cell observed even at high IL concentration (Fig. 7). Additionally,
survival. With increased chain length, the [C mpyrrol][NTf ] cell lysis would lead to a dilution effect with the multi-compo-
8
2
inhibits the biotransformation process when present at high nent dioxygenase complex of several orders of magnitude, as
concentration. However at low concentrations, it appears to demonstrated in various studies where enzyme purication of
enable biotransformation via a mechanism similar to that of dioxygenases is made difficult by this very effect – activity is so
the C8-imidazolium-based ILs. Clearly, mass transfer and low that radiochemical based assays are needed to measure the
22
solubility of ILs in the aqueous phase are not the sole param- activity of dioxygenases in cell-free extracts.
eters responsible for the enhanced biocatalysis in the presence
of the octyl-based ILs.
To further test our hypothesis, the effect of the cation on
oxygen respiration in resting P. putida UV4 cells was probed.
Additionally, two hydrophilic ILs ([C dmim]Br and Cells were presented to an oxygen electrode in the presence or
8
[
C mpyrrol]Br) displayed similar effects on biotransformations absence of the IL cation (Fig. 3a–c in ESI†). In these experi-
8
to that of the corresponding-bis(triuoromethanesulfonyl) ments, the data clearly indicates that the imidazolium cation
imide based ILs at low VIL/Vaq. This indicates that the enhancing affects cellular respiration in a very similar fashion to establish
behaviour of the ILs is likely to stem from the cationic compo- inhibitors of oxidative phosphorylation, i.e. cyanide and azide,
nent (Fig. S3†). As these ILs are fully soluble, any effects due to see ESI† for details. As the concentration of these compounds is
the biphasic nature of the system with the growth medium, for increased oxygen turn-over is clearly inhibited in the presence
example maintaining a reservoir of the substrate, which is of an electron donor (e.g. glucose). Cyanide and azide are known
present with the bis(triuoromethanesulfonyl)imide ILs cannot to inhibit directly the Cytochrome C component of the respi-
be used to explain the effect.
ratory chain. Table 4 compares the kinetic parameters of the two
Therefore, an examination of the cation on cellular established inhibitors with the hydrophilic ILs examined. These
biochemistry was undertaken in order to understand their data suggest that the ILs are at least as effective as azide as
mechanism of action. In this case, the ILs may enhance the respiratory inhibitors, under the same conditions. The C8-pyr-
biotransformation through inhibition of respiratory processes. rolidinium was included in this study as a negative control as it
To test this idea further, the effect of the ILs on the growth of the would not be expected to inhibit respiration from earlier data
cells and on the oxygen dependent respiration was studied.
presented, herein.
In addition to establishing the inhibitory properties of
[
C dmim]Br and comparing them with that of [C mpyrrol]Br,
8
8
Synergy between ILs toxicity and whole cell cis-
dihydroxylation using P. putida UV4
their rate and overall yield enhancement was compared with the
biotransformations conducted in the presence of respiratory
Possible inhibition or toxicity effects of the ILs were initially
evaluated through viable cell count experiments (Fig. 6). When
examined for their toxicity towards the biocatalyst, the ILs
incorporating longer side chains were found to inhibit growth.
This was shown clearly for [C
8
mim][NTf
2
], [C10mim][NTf
2
],
[C
6
dmim][NTf ], [C dmim][NTf ] and [C10dmim][NTf
2
8
2
2
]. The
trend of growth inhibition observed with an increase of the alkyl
chain length was in agreement with the results reported by
24
Docherty et al. Herein, the toxicity of the alkyl chains increased
from hexyl to octyl to decyl on the imidazolium cation. This has
also been observed for pyridinium and imidazolium based ILs
containing octyl chains which have been reported to be more
25
toxic than those possessing butyl and hexyl side chains. Whilst
the octylimidazolium based ILs showed high levels of toxicity to
the cells, importantly, this IL also showed signicant
enhancement to whole cell biotransformations.
25
Bernot et al. have proposed the possibility of lipophilic
interactions of ILs with the organisms' cell membrane. It was, Fig. 7 Visible light microscopy image (ꢃ1000 magnification) showing
8 2
P. putida UV4 cells incubated in the presence of the [C dmim][NTf ] IL
at 0.02 (VIL/Vaq). Cells appear to be unaffected by the presence of the
IL with no obvious damage to the cellular envelope. Similar analysis
therefore, unclear whether the effect of the IL phase on the
bacteria was due to the molecular toxicity associated with the
aqueous solubility of the ILs or the phase toxicity associated
with the biphasic surface, or a combination of both. The
6 2 2
was also performed for [C dmim][NTf ] and [C10dmim][NTf ]. Criti-
cally, the paradoxical observations shown herein can be explained
surprising increase in the conversion rate associated with a from one key hypothesis that the oxidative phosphorylation in the
bacterial cell is specifically inhibited by the imidazolium-based ionic
liquids containing octyl groups. This would mean that the presence of
ILs inhibited growth but at the same time enhanced the availability to
the dioxygenase enzymes of both NADH/NAD(P)H electron donors
dramatic decrease in cell availability could potentially be
explained by a cell lysis resulting in the immediate removal of
mass transfer restrictions leading to a rate increase. Yet, even at
low IL concentrations, molecular toxicity remained a critical
and oxygen-obviously a key component for dihydroxylation. This
parameter, whilst retention of microbe cellular integrity was effect is summarised in Scheme 1.
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enhancements, with for instance 89% overall conversion of
chlorobenzene to the cis-diols aer 8 hours (Table 5). An
optimal conversion of chlorobenzene by P. putida UV4 was
achieved in 99% in a shake ask experiment, indicating that the
application of this technique to a fermenter process with
2
controlled O concentration could possibly increase the
production of cis-diols quite dramatically, both in term of
overall yields and time of reaction. The cumulative effect noted
during the co-inhibition experiment would suggest that the ILs
cations inhibit a different complex than the cyanide and azide
anions. Unfortunately, while the data clearly indicate an effect
of these ILs on the electron chain transport pathway, the
experiments do not provide information on the specic enzy-
matic step or steps which the cations are able to compromise.
In summary, the concept that the use of ILs in biotransfor-
mations is to be limited by their property as reservoir of substrate
and this as long as they remain innocuous to the biocatalyst has
been challenged. While hydrophobic ILs were able to release in
Scheme 1 A model for indirect enhancement of biocatalysis rates
using dioxygenase enzymes in P. putida UV4. Diagram showing the
proposed mechanism of action of the ILs on dioxygenase-mediated
biotransformations: (A) – ILs may have a positive effect on biotrans-
formation yields by enhancement of substrate uptake via diffusion to the growth medium a range of haloarenes in a controlled manner
the enzyme active site across the cell membrane; (B) – our data also and therefore allow for an effective mass transfer between two
suggest that the inhibition of oxidative phosphorylation specifically by
the ILs also enhances biotransformation by increasing the pool of
using the parent hydrophilic ILs acting as a biocatalyst enhancer,
NADH equivalents available for the dioxygenase activity provided by
phases, the outcome of this property did not differ from that of
through inhibition. Here, the low molar concentration of imi-
the co-substrate, and also reducing cellular demand for oxygen.
dazolium bromide salts (i.e. hydrophilic IL) used to achieve high
conversion diminishes the need for its recycling, a key aspect to
most IL-based processes, where the ILs are used in much greater
Table 4 Inhibition coefficient of cell respiration for P. putida UV4. The
data show the apparent K
chain transport of P. putida UV4 by [C
i
obtained for the inhibition of the electron quantities. Finally, it has been demonstrated that the inhibition
8
dmim]Br, KCN and NaN3. of oxygen respiration during biotransformation leads to an
Calculated values show mean of duplicate samples. See Fig. E.1 to E.4
in ESI
improvement in the conversion of substrate in dioxygenase
enzymatic reactions. The effect of this inhibition on rates and
overall yields is of particular importance to biotransformation
reactions where the biotransformed product is unstable over
time, and can potentially be useful to any biotransformation
processes where increased NADH availability to the intracellular
medium results in increased enzymatic reaction rates. Nonethe-
less, it is still unclear where the IL-cation interacts within the
electron transfer chain. Further experiments are required with
pure enzymes from the electron transport chain to determine the
mechanism of inhibition of these cations.
Inhibitors of Pseudomonas
putida UV4
Ki(app) – mmol per g
per dry cell
Sodium azide
Potassium cyanide
6.15 ꢁ 0.02
4.85 ꢁ 0.50
5.99 ꢁ 0.43
15.25 ꢁ 2.1
[
[
C
C
8
dmim]Br
mpyrrol]Br
8
inhibitors. This study aimed to establish whether the known
respiratory inhibitors, i.e. cyanide and azide, had the same
effect as the imidazolium based ILs on the biotransformations
Experimental
(
3
Fig. 8). The results demonstrated that the use of NaN and KCN
The general synthetic procedures and characterisations of the
ionic liquids used in this work can be found in the ESI.† All the
biotransformation processes were conducted under biphasic
conditions.
resulted in improved yields for P. putida UV4 catalysed
biotransformations in a similar fashion to that observed for
[C dmim]Br. These data conrmed that there is very little
8
difference on dioxygenase biotransformations between the
cytotoxic ILs and the known respiratory inhibitors.
General biotransformation monitoring procedure
The higher increase in yields observed in the presence of
KCN than for [C
faster inhibition of cell respiration is achieved with cyanide mations with chlorobenzene. The residual substrate concen-
than with [C dmim]Br, allowing a faster initial conversion of tration of chlorobenzene has not been shown. The duplicate
8
dmim]Br may, therefore, be due to the fact that The experiments involved time-course studies of biotransfor-
8
chlorobenzene to its cis-diol product. Nonetheless, the cation of time-course biotransformation studies were measured by HPLC
specic ILs can be utilised to enhance biotransformation reverse phase. The aliquot of a single date point was sampled at
3
through inhibition of cell respiration as demonstrated in Fig. 8. each time interval. In each experiment 0.5 cm of the
In addition, simultaneous addition of [C
8
dmim]Br (2.4 mM) biotransformation mixture was sampled and centrifuged for 2
and KCN (0.5 mM) to the reaction media yielded synergistic min at high speed in an Eppendorf Microcentrifuge 5415 D. The
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Fig. 8 Comparison between [C
8
dmim]Br and (a) sodium azide and (b) potassium cyanide on the biotransformation of 0.98 mmol of chloro-
8
benzene (pH 7.2) by P. putida UV4, (:) without inhibitor, (,) 2.4 mM [C dmim]Br, (a) (A) 4 mM sodium azide and (b) (C) 3.0 mM potassium
ꢀ1
cyanide. OD600 ¼ 1.8 or 0.70 g L dcw. Data points show mean of duplicate samples.
Table 5 Conversion of chlorobenzene with addition of potassium cyanide to an initial concentration of 2.4 mM [C
OD600 ¼ 1.8 or 0.70 g L dcw. Calculated values show mean of duplicate samples
8
dmim]Br with P. putida UV4.
ꢀ1
ꢀ1
[
C
8
dmim]Br – mM
Potassium cyanide – mM
Conversion of chlorobenzene aer 8 h – %
Initial reaction rate – mM h
2.4
2.4
2.4
2.4
0
72.1 ꢁ 4.5
85.9 ꢁ 0.6
89.1 ꢁ 0.5
81.4 ꢁ 3.7
1.63 ꢁ 0.00
2.64 ꢁ 0.18
2.85 ꢁ 0.11
2.85 ꢁ 0.10
0.25
0.5
1
centrifuged supernatant was diluted by half with ethanol and an oxygenase dependent biocatalysis system and suggested that
+
8
centrifuged at high speed to remove any insoluble material the [C dmim] cation could inhibit a different protein than that
before HPLC reverse phase quantication. A standard sample of inhibited by cyanide or azide. We have presented strong
the biotransformation product chlorobenzene cis-diol was evidence to support our hypothesis proposed where the cations
kindly provided by Professor D. R. Boyd and Dr N. D. Sharma of of hydrophobic-ILs containing an octyl chain improve the
2
the School of Chemistry and Chemical Engineering, Queen's biotransformation of enzymes dependent on O and NADH in
University Belfast. The metabolites obtained throughout the whole cell bio-catalyst. This mechanism of action could be
experiments reported in this work were identied by compar- highly complementary to other modes of biotransformation
ison of HPLC retention times and UV spectra with the standard process optimisation. For example, it has been widely accepted
compounds. HP1100 HPLC System (Agilent Technologies) was that non-toxic hydrophobic IL (e.g. tetraalkyl phosphonium-
used with a C8 column (Supercosil 150 ꢃ 4.6 mm, 5 mm) and UV bis(triuoromethanesulfonyl)imide) can be used to enhance
detection to separate metabolite from substrate and ionic biotransformation processes. If this approach was combined
liquids. Chlorobenzene biotransformations were analysed with a low level of an appropriate respiratory inhibitor, e.g.
3
using a binary gradient solvent system (acetonitrile–H
2
O, 1 cm
cyanide or azide, in the same reaction vessel, one could antici-
ꢀ
1
min ow rate). The column was rst equilibrated at 10% pate a synergistic enhancement effect. Further studies could
CH CN–H O, the run started with a linear gradient and help validate this counter-intuitive possibility.
3
2
increased to 40% CH CN over three minutes and followed by a
3
second linear gradient which increased to 60% CH
3
CN over the
next two minutes. A third gradient increase from 60% to 100%
Acknowledgements
CH
linear gradient decrease from 100% to 10% CH
min. An equilibration time of two minutes at 10% CH
nally applied to stabilise the C8 column for the next run. In
3
CN was applied over 5 min, followed immediately with a
CN during 3
CN was
3
We thank the European Social Funds for its nancial support of
CB. P. putida UV4 was kindly provided by Professor D. R. Boyd of
the School of Chemistry and Chemical Engineering, Queen's
University Belfast, Belfast, N. Ireland. We also thank Professor
Sheila Patrick for assistance with microscopy imaging.
3

these biotransformations only one metabolite was formed and
the same HPLC method was applied.
Conclusions
Notes and references
We have demonstrated that under non-mass transfer condi-
tions, cytotoxic ILs can enhance biotransformation rates with
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