Enantioselective synthesis of (R)-2-arylpropanenitriles catalysed by ene-reductases in aqueous media and in biphasic ionic liquid-water systems

Article abstract of DOI:10.1002/cctc.201402205

The enantioselective reduction of α-methylene nitrile derivatives catalysed by ene-reductases affords the corresponding (R)-2-arylpropanenitriles with high conversion values. The reaction is investigated either in aqueous medium (with an organic cosolvent or by loading the substrate onto hydrophobic resins) or in a biphasic ionic liquid-water system. The use of ionic liquids, herein with isolated ene-reductases, is found to improve the work-up and the substrate recovery method. The synthetic manipulation of the final chiral nitrile derivatives indicates how this biocatalysed method can be exploited for the preparation of a wide range of chiral compounds.

Full text of DOI:10.1002/cctc.201402205

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DOI: 10.1002/cctc.201402205
Enantioselective Synthesis of (R)-2-Arylpropanenitriles
Catalysed by Ene-Reductases in Aqueous Media and in
Biphasic Ionic Liquid–Water Systems
Elisabetta Brenna,*[a] Michele Crotti,[a] Francesco G. Gatti,[a] Alessia Manfredi,[a]
Daniela Monti,[b] Fabio Parmeggiani,*[a] Sara Santangelo,[a] and Davila Zampieri[a]
The enantioselective reduction of a-methylene nitrile deriva-
tives catalysed by ene-reductases affords the corresponding
(R)-2-arylpropanenitriles with high conversion values. The reac-
tion is investigated either in aqueous medium (with an organic
cosolvent or by loading the substrate onto hydrophobic resins)
or in a biphasic ionic liquid–water system. The use of ionic liq-
uids, herein with isolated ene-reductases, is found to improve
the work-up and the substrate recovery method. The synthetic
manipulation of the final chiral nitrile derivatives indicates how
this biocatalysed method can be exploited for the preparation
of a wide range of chiral compounds.
Introduction
A recent perspective,[1] outlined by the medicinal chemistry
subgroup of the American Chemical Society’s Green Chemistry
Institute Pharmaceutical Roundtable, has highlighted the ad-
vantages of using biocatalysed synthetic methods, not only for
the optimisation of sustainable drug manufacturing processes
but also at the medicinal chemistry level. Typical features of
enzyme-mediated transformations are: 1) exquisite chemo-,
regio-, and stereoselectivity, which decreases the number of re-
action steps required and enables high stereoisomeric purity
values; 2) mild reaction conditions, which usually avoid exten-
sive heating and high pressure; 3) high synthetic potential;
and 4) the renewability and biodegradability of enzymes them-
selves.
yeast (BY; Saccharomyces cerevisiae).[2] The enzymes responsible
for this reaction (including those present in BY) are called ene-
reductases (ERs; EC 1.6.99.1), and most of them belong to the
well-known family of old yellow enzymes (OYEs),[3] which are
characterised from a biochemical viewpoint since the 1930s,
but only recently studied in detail for their use in preparative
biocatalysis applications.[4]
These enzymes can reduce suitably substituted alkenes with
high conversion and excellent enantiomeric purities that are
activated towards this bioreduction by one or two electron-
withdrawing groups (EWGs). The effects due to the stereo-
chemistry of the double bond as well as due to the steric and
electronic properties of the activating EWGs and of other sub-
stituents are currently under investigation[5] so as to define the
limits and the potential of this kind of enzyme-mediated
reaction.
A possible approach to promote the use of biocatalysis,
either in the early stage of drug development or during the
optimization of the process route, is to enrich the enzyme tool-
box available to synthetic chemists to perform classical organic
transformations. For example, the stereoselective reduction of
suitably substituted C=C bonds is one of the best methods to
introduce stereogenic centres in the synthesis of chiral com-
pounds. The first approach to the enzyme-mediated variant of
this reduction was based on the exploitation of the resting
cells of various microorganisms, particularly those of baker’s
With this aim, we investigated the capability of ERs to
reduce C=C bonds bearing a nitrile moiety as an EWG, which
affords valuable enantiopure building blocks for synthetic ap-
plications. The relevance of this study lies mainly in the fact
that the nitrile functional group is a pharmacophore in many
biologically active compounds[6] and a versatile precursor of
other functionalities such as carboxylic acids, amides, alde-
hydes, ketones, and amines. A significant interest exists in the
optimisation of highly selective synthetic methods to prepare
or manipulate nitrile derivatives,[7] especially the chiral ones.[8]
Herein, we report on the optimisation of the enzyme-medi-
ated stereoselective reduction of suitably substituted unsatu-
rated nitriles, which represents a novelty in the field of bioca-
talysis because ERs have been rarely reported (see the next
section) to be active in the conversion of unsaturated nitriles
with no other EWGs on the C=C bond.
[a] Prof. E. Brenna, M. Crotti, Dr. F. G. Gatti, A. Manfredi, Dr. F. Parmeggiani,
S. Santangelo, Dr. D. Zampieri
Department of Chemistry, Materials and Chemical Engineering
“Giulio Natta” Politecnico di Milano
Piazza Leonardo Da Vinci 32, 20133 Milano (Italy)
Fax: (+39)02-23993180
E-mail: mariaelisabetta.brenna@polimi.it
fabio.parmeggiani@polimi.it
[b] Dr. D. Monti
Istituto di Chimica del Riconoscimento Molecolare, C.N.R.
Via Mario Bianco 9, 20131 Milano (Italy)
This enzymatic method is also an improvement in organic
synthesis. Despite recent advances in the metal-catalysed ste-
reoselective hydrogenation, the development of effective
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201402205.
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methods in this area for the reductions of unsaturated nitriles
is still a challenge because of their intrinsic low reactivity[9] and
the linearity of the CN group. Owing to their electronic struc-
ture, nitriles prefer end-on coordination to metal ions, which is
not suitable for the hydrogenation of a conjugated double
bond.[10] Only a few reports exist in the literature of this
field,[9,10] and in some cases, an additional functionality, such as
a carbonyl or an alcohol group, was required to enable the co-
ordination of the substrate to the metal of the catalyst.[11] Fur-
thermore, the chemoselection between the olefinic double
bond and the nitrile group in hydrogenation poses additional
problems.
Results and Discussion
Scheme 1. Synthesis and bioreduction of nitriles 1a–k, and synthetic manip-
ulation of the products. Reagents and conditions: a) (CH2O)n, toluene, K2CO3,
nBu4NI; b) ER reduction; c) H2O2, Na2CO3, H2O; d) HCl (37%), reflux; e) diphe-
nylphosphoryl azide, toluene, 808C; then HCl (10%), 608C; f) N-bromosucci-
nimide, 1,8-diazabicyclo[5.4.0]undec-7-ene, MeOH, reflux.
The few data found in the literature indicated that the pres-
ence of the CN moiety as the sole activating group of the
double bond is not enough to promote reduction, except the
a-methylene nitrile derivative 1a. Specifically, BY was shown to
catalyse the conversion of 2-phenyl-2-propenenitrile (1a) into
the R enantiomer of the corresponding reduced product with
high ee and 64% yield in the presence of petroleum ether.[12]
No further investigation was performed on this class of com-
pounds.
involves a [2+2] cycloaddition of the methylenic double bonds
of the two molecules of acrylonitrile.
The reference racemic propanenitriles 2a–k were obtained
either by the hydrogenation of the methylenic parent com-
pound on Pd/C or by alkylation of the starting arylacetonitrile
with sodium hydride and methyl iodide in THF.
In other reports involving isolated ERs of the OYE family,
pentaerythritol tetranitrate reductase and OYE1 were described
to be unable to reduce trans-cinnamonitrile and estrogen-
binding protein EBP1 did not reduce crotonitrile.[13] In 2008,[14]
a range of commercially available NAD(P)H-dependent ERs
were used to reduce various (Z)-p-substituted phenyl buteneni-
triles to the corresponding R enantiomers with good to high
enantioselectivities (70–99% ee). A known pharmaceutical syn-
thon (6-chloro-5-methylspiro[1H-indene-1,4’-piperidine]-3-car-
bonitrile) was also submitted to the same reaction, although
the absolute configuration of the product was not determined.
Two contemporary reports have described the possibility of re-
ducing the C=C bond of a,b-unsaturated nitriles by ERs with
an ester group present as a substituent at the olefinic carbon
atom in the b position.[15,16]
BY and OYE1–3 bioconversions in aqueous systems
The a-methylene nitrile derivatives 1a–k were submitted to
the bioreduction with whole-cells BY in aqueous medium by
adsorbing the substrate on a hydrophobic resin (polystyrene
XAD1180N) to ensure its slow release in the aqueous phase,
which prevented toxic and inhibitory effects on the cells. Paral-
lel experiments were performed with OYE1–3 in aqueous
medium with DMSO as a cosolvent. In view of designing a scal-
able and convenient process, we were not interested in per-
forming the reactions with stoichiometric amounts of the ex-
pensive NADPH cofactor required by the enzyme as a source
of hydride; this problem was circumvented by using glucose
dehydrogenase (GDH) as a regeneration enzyme and glucose
as a cheap cosubstrate. This cofactor regeneration system ena-
bles the use of the cheaper oxidised form of the cofactor
(NADP+) only in a catalytic amount. The results of all the biore-
duction experiments in aqueous systems are compared in
Table 1.
With the aim of exploring the biocatalysed synthesis of
chiral nitriles, we prepared a-methylene nitrile derivatives 1a–
k and submitted them to the biocatalysed reduction. We envis-
aged the possibility of exploiting their enantioselective reduc-
tion to create a benzylic stereogenic centre, bearing a methyl
group in this position, for the preparation of chiral building
blocks to be used in the synthesis of active pharmaceutical
ingredients.
The enantioselectivity of the bioreduction was 99% for most
substrates, by using either BY or isolated ERs. These value de-
creased slightly in the reaction of halogen-substituted deriva-
tives and OYE1–3, except in the case of o-Cl and o-F com-
pounds. Enantioselectivity decreased considerably with the o-
NO2 derivative 1 f, which gave a racemic material upon react-
ing with OYE2 and OYE3, and only modest values of enantiose-
lectivity (50–72%) were obtained with OYE1 and BY.
Synthesis of the substrates
a-Methylene nitrile 1a–k were prepared through the reaction
of the arylacetonitrile with paraformaldehyde, potassium car-
bonate, and tetra-n-butylammonium iodide in toluene at 808C
(Scheme 1).
As for the para-substituted derivatives, the isolation of the
desired compound was made more difficult in some cases be-
cause of the occurrence of a dimerisation process, which likely
Similar conversion values (60–90%) were obtained by using
either BY or OYE1–3 with Ph, p-Me, and m-Cl derivatives 1a, d,
and h. As for other biotransformations, the highest conversion
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to reduce a set of ketones in a [BMIM][PF6]–water
mixture (BMIM=1-butyl-3-methylimidazolium hexa-
fluorophosphate).[22] An increase in conversion (from
44 to 80%) and enantioselectivity (from 62 to 84%)
values was obtained in the reduction of 4-chloroace-
toacetate to (S)-4-chloro-3-hydroxybutanoate in
20 vol% [BMIM][PF6] in the presence of BY.[21]
Table 1. Results of the biocatalysed reductions of substrates 1a–k in aqueous
systems.
Substrate Aryl group
BY
OYE1
c[c] ee[b]
[%] [%]
OYE2
c[c] ee[b]
[%] [%]
OYE3
c[c] ee[b]
[%] [%]
c[a] ee[b]
[%] [%]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
phenyl
o-tolyl
m-tolyl
p-tolyl
1-naphthyl
o-nitrophenyl
o-chlorophenyl
52 99 (R)
95 99 (R)
90 98 (R)
90 95 (R)
82 99 (R)
31 72 (R)
74 99 (R)
65 99 (R)
99 99 (R)
52 99 (R)
91 99 (R)
65 99 (R)
96 50 (R)
96 99 (R)
66 82 (R)
72 99 (R)
52 99 (R)
52 99 (R)
95 99 (R)
49 99 (R)
94 rac
65 99 (R)
17 99 (R)
94 99 (R)
94 99 (R)
Moran et al. described the consecutive reduction
of C=C and C=O bonds of (Z)-3-halo-4-phenyl-3-
buten-2-one mediated by various microorganisms in
the [BMIM][PF6]–water biphasic system, which gave
the corresponding halohydrins with better diastereo-
selectivity and enantioselectivity than did the reduc-
tion in pure water.[23]
5
5
–
rac
18 99 (R)
69 90 (R)
24 99 (R)
66 90 (R)
m-chlorophenyl 65 82 (R)
p-chlorophenyl
o-fluorophenyl
2
–
9
89[d]
5
90[d]
8
88[d]
1j
1k
81 99 (R)[e] 15 99 (R)[e] 15 99 (R)[e] 10 99 (R)[e]
p-fluorophenyl 100 99 (R) 47 88 (R) 47 64 (R) 45 88 (R)
On the basis of these literature data, we decided
to investigate our BY reactions in a biphasic 1:10
[BMIM][PF6]–water system: the experimental results
are presented in Figure 1a and b. The conversion
and ee values of these reactions were generally
higher than those obtained with OYE1–3-mediated
reductions in water with DMSO as a cosolvent and
similar to the values obtained by using BY with the
[a] Conversion values calculated by GC analysis of the crude mixture after 72 h of re-
action; [b] The ee values calculated by GC or HPLC analysis on a chiral stationary
phase; [c] Conversion values calculated by GC analysis of the crude mixture after 24 h
of reaction; [d] The absolute configuration could not be determined because the satu-
rated nitrile derivative could not be isolated; [e] The R configuration was attributed by
analogy to the sign of the optical rotation value of the nitrile derivative.
values were generally obtained with BY and the lowest with
OYE3, except for the m-Me derivative 1c for which approxi-
mately 90% conversion values were obtained with both BY
and OYE3.
use of resin. However, the work-up and product recovery from
the biphasic IL–water system was easier and more effective
than the extraction of the organic products from the resins,
and thus isolation yields increased.
In the case of the o-Cl derivative 1g, we experimented with
the use of the biphasic IL–water system in the biotransforma-
tion with OYE1–3. It has been reported in the literature that
the Lactobacillus brevis alcohol dehydrogenase-catalysed enan-
tioselective reduction of 2-octanone in a biphasic system com-
posed of an aqueous buffer and [BMIM][Tf2N],[24] with the
enzyme and the cofactor dissolved in the aqueous phase and
physically separated from the substrates and products mainly
in the IL phase, showed higher reaction rates than that in the
aqueous buffer/methyl tert-butyl ether biphasic system. For
substrate 1g, we performed the OYE-mediated reductions in
biphasic IL–water systems with either [BMIM][PF6] or [BMIM]-
[Tf2N]. The latter is used in the only literature example describ-
ing the use of ILs with isolated enzymes.[24] The corresponding
experimental data are presented in Figure 1c and d. Notably,
in the case of [BMIM][PF6], the biphasic medium had a benefi-
cial effect on the conversion values obtained in the biocata-
lysed reductions with isolated OYEs, most likely owing to
a better dissolution of the substrate.
BY and OYE1–3 bioconversions in biphasic ionic liquid–
water systems
The low solubility of these arylacrylonitriles in aqueous
medium prompted us to investigate the possibility of perform-
ing the bioreductions with BY in a biphasic system composed
of water and an immiscible ionic liquid (IL).
Over the past decade, a large number of applications of ILs
in biocatalysed reactions (free enzymes and whole cells)[17]
have been discussed in the literature, mostly dealing with the
so-called second generation ILs,[18] that is, those containing
À
weakly coordinating anions, such as PF6 and Tf2À, which are
characterised by high water and air stability. Furthermore, third
generation ILs have been introduced, with structures contain-
ing biodegradable or readily available ions, such as natural
bases, amino acids, and natural carboxylic acids.[18] The evolu-
tion of ILs is represented by deep eutectic solvents and mix-
tures of solid salts (e.g., choline chloride) and hydrogen bond
donors (e.g., urea), which are cheaper and more biodegradable
than ILs and do not require purification in their production
process.[19]
To the best of our knowledge, this study represents the first
application of isolated ERs in the IL–water system; it clearly
demonstrates that these enzymes are compatible with hydro-
phobic ILs and that the method is efficient and advantageous
in terms of yields, selectivity, and work-up practicality.
The set-up of biphasic systems involving immiscible ILs has
been highlighted as a promising research area in whole-cell
biotransformations[20] because the non-invasive effects of hy-
drophobic ILs on cellular membranes make them superior to
many organic solvents. With substrates and products that are
poorly soluble in water, as in our case, the IL can act as an ef-
fective substrate reservoir and in situ extracting agent.[21] Ho-
warth et al. reported the first example of a whole-cell biore-
duction in ILs in which immobilised BY was used as a catalyst
Absolute configurations of the products
The absolute configuration of the Ph, p-Me, 1-naphthyl, and m-
Cl saturated nitriles 2a, d, e, and h has been reported in the
literature(see the Supporting Information). The mild and high
yield conversion of (+)-p-F-2k and (+)-o-Cl-2g into (R)-amides
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Figure 1. Results of the biocatalysed reductions in biphasic IL–water systems (&: [BMIM][PF4], &: [BMIM][Tf2N]) compared with those obtained in aqueous
systems (&): a) conversion and b) ee values for the BY-mediated reductions of substrates 1a–k after 72 h; c) conversion and d) ee values for the ER-mediated
reductions of substrate 1g after 24 h.
3k and g (Scheme 1) was performed through the reaction with
hydrogen peroxide in 10% aqueous solution of Na2CO3. (+)-o-
Me-2b and (+)-m-Me-2c were hydrolysed to afford the corre-
sponding carboxylic acid (R)-4 by heating to reflux for 5 h in
HCl (37%). A two-step method involving first the transforma-
tion of the CN group of (À)-o-NO2-2 f into the carboxylic acid
function and then the reaction with diphenylphosphoryl azide
and triethylamine in toluene at 808C followed by treatment
with diluted HCl at 608C afforded the corresponding (R)-amine
5. (+)-o-Me-2b was converted into the derivative (R)-6 by re-
acting the corresponding amide with N-bromosuccinimide and
1,8-diazabicyclo[5.4.0]undec-7-ene in methanol heated to
reflux.
be used for the diffusion of the nitrile pharmacophore in phar-
maceutical applications. The synthetic versatility of the nitrile
moiety can be exploited for the conversion of arylpropaneni-
triles (R)-2 into amides (R)-3 and amines (R)-5 (Scheme 1)
under the same reaction conditions used for the assignment of
the absolute configuration of some of the reduced products of
this work.
The enantioselective synthesis of (R)-2-arylpropanamides,
such as reparixin (Figure 2),[25] is a relevant achievement in
preparative organic chemistry because these derivatives are
useful in the prevention and treatment of tissue damage due
to the exacerbate recruitment of polymorphonuclear neutro-
Synthetic manipulation of aryl-
propanenitriles
As pointed out in the previous
section, in most cases the enan-
tiomerically enriched arylpropa-
nenitriles had to be manipulated
to determine their absolute con-
figuration by chemical correla-
tion. This process thus demon-
strated the synthetic potential of
these chiral derivatives. Arylpro-
panenitriles (R)-2 themselves can Figure 2. Chiral active pharmaceutical ingredients containing (R)-amide and amine moieties.
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phils (leukocytes) at the inflammatory sites. They are used in
the treatment of psoriasis, ulcerative colitis, glomerulonephritis,
acute respiratory insufficiency, and rheumatoid arthritis.[26]
The preparation of (R)-amines 5 through the Curtius rear-
rangement of carboxylic acids (R)-4 with loss of one carbon
atom and retention of configuration is a valuable method be-
cause of the widespread diffusion of the structural unit of (R)-
2-arylethanamine as the key motif in many drug candidates.[27]
The relevant examples of chiral amino drugs for which the
most bioactive enantiomer is the one showing the R configura-
tion are illustrated in Figure 2. The drug candidate AMG 628[28]
is a transient receptor potential antagonist used for the treat-
ment of chronic pain, and cinacalcet hydrochloride[29] (Sensipar
Amgen) is the first calcimimetic agent approved by the Food
and Drug Administration for the treatment of secondary hyper-
parathyroidism. Other candidates such as calindol,[30]
Calhex 231,[31] NPS R-467,[32] and Amgen pyrazole[33] (Figure 2)
with potent and selective activity on the parathyroid calcium
receptor are under pharmacokinetic studies. A series of small
molecules developed as effective antivirals against severe
acute respiratory syndrome[34] are (R)-2-naphthylethanamine
derivatives.
tional solution to load the substrate onto a hydrophobic resin
or to use biphasic ionic liquid–water systems improves the
work-up method and the recovery of the reduced product.
Ionic liquids were combined with isolated old yellow en-
zymes, which afforded good results in terms of conversion and
enantioselectivity in the case of [BMIM][PF6] (BMIM=1-butyl-3-
methylimidazolium hexafluorophosphate). The ionic liquid
phase represents a good reservoir that makes water-insoluble
products gradually available to the reaction catalysed by these
reducing enzymes.
Experimental Section
Sources of strains and enzymes
Either fresh or freeze-dried commercial BY (S. cerevisiae) was used
for whole-cell biotransformations. OYEs (OYE1 from Saccharomyces
pastorianus and OYE2 and OYE3 from S. cerevisiae) and GDH (from
Bacillus megaterium) were overexpressed in Escherichia coli BL21
(DE3). Detailed methods are reported in the Supporting
Information.
The biological significance of enantiopure (R)-arylethana-
mines prompted us to demonstrate the versatility and the ap-
plicability of our method by converting (R)-arylethanamines
into the corresponding amines, not only the o-NO2 derivative
(R)-2 f (for the configuration assignment) but also phenyl and
1-naphthyl derivatives (R)-2a and (R)-2e. Amines (R)-5a, e, and
f were finally prepared from the arylacetonitrile by using
a four-step sequence in satisfactory yields and high enantio-
meric purity.
OYE-mediated bioreduction in aqueous system
General procedure: The substrate (5 mmol) dissolved in DMSO
(10 mL) was added to a KPi buffer solution (1.0 mL, 50 mm, pH 7.0)
containing glucose (20 mm), NADP+ (0.1 mm), GDH (4 UmLÀ1), and
the required OYE (40 mgmLÀ1). The mixture was incubated for 24 h
in an orbital shaker (160 rpm, 308C). The solution was extracted
with EtOAc (2ꢁ250 mL), centrifuging after each extraction
(15000 g, 1.5 min), and the combined organic solutions were dried
over anhydrous Na2SO4.
Conclusions
BY fermentation in aqueous system
General procedure: A mixture of BY (100 g) and d-glucose (40 g) in
tap water (800 mL) was prepared. After stirring at 308C for 10 min,
the nitrile derivative (3.0 g) adsorbed on a hydrophobic resin (60 g;
polystyrene XAD1180N) was added in one portion. The mixture
was kept under stirring for 72 h at RT and then filtered on a cotton
plug. The collected mass was washed repeatedly with water to
remove most of the cells. The resin was then collected and extract-
ed twice in sequence with acetone (200 mL) and EtOAc (200 mL).
The development of a catalytic stereoselective synthesis of
enantiopure (R)-arylpropanenitriles and the corresponding
amides and arylethanamines through an efficient and environ-
mentally friendly method not involving toxic or hazardous re-
agents and with minimal use of organic solvents offers enor-
mous promise for optimising new routes to prepare a wide
range of pharmaceutical products. The nitrile moiety, which is
necessary to act as an activating electron-withdrawing group
in the bioreduction step, can be subsequently submitted to
valuable chemical manipulations, which gives access to various
derivatives.
1
The organic phase was concentrated to /3 of its volume, washed
with brine, dried over Na2SO4, and evaporated under reduced pres-
sure. The resulting mixture of products was then separated by
using column chromatography in hexane with increasing amounts
of EtOAc.
This enzyme-mediated method gives the possibility of creat-
ing a benzylic stereogenic centre by reduction of a C=C bond
conjugated to a CN moiety, a transformation which is difficult
to accomplish through metal catalysis. In this case, biocata-
lysed reductions represent a complementary activity to metal-
catalysed hydrogenation: 1) the nitrile moiety can establish hy-
drogen bonds in the active site of the enzyme to promote the
substrate binding; 2) the bioreduction is completely chemose-
lective towards the reduction of the C=C bond.
OYE-mediated bioreduction in the biphasic IL–water system
General procedure: The substrate (50 mg) dissolved in [BMIM][PF6]
or [BMIM][Tf2N] (1500 mL) was added to a KPi buffer solution
(15 mL, 50 mm, pH 7.0) containing glucose (4 equiv. with respect
to the substrate, ꢀ250 mg), NADP+ (0.05 mm), GDH (4 UmLÀ1),
and the required OYE (40 mgmLÀ1). The mixture was incubated for
24 h in an orbital shaker (160 rpm, 308C). The separated IL phase
was extracted with iPr2O (3ꢁ5 mL), centrifuging after each extrac-
tion (3000 g, 5 min), and then the combined organic solutions
were dried over Na2SO4 and concentrated under reduced pressure.
In this case, the use of whole-cell systems does not induce
any undesired side reaction and has the advantage of employ-
ing the cofactor regeneration system of the cells. The opera-
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[8] J. Guin, G. Varseev, B. List, J. Am. Chem. Soc. 2013, 135, 2100–2103; J.
Choi, G. C. Fu, J. Am. Chem. Soc. 2012, 134, 9102–9105.
BY fermentation in the biphasic IL–water system
General procedure: The substrate (300 mg) dissolved in [BMIM][PF6]
(6 mL) was added to a mixture of BY (30 g) and d-glucose (10 g) in
tap water (300 mL) at 358C. The mixture was stirred in an orbital
shaker (220 rpm, 308C) for 1–4 days. After separation of the
phases, the aqueous solution was washed with CH2Cl2 and the or-
ganic extract was added to the IL phase to recover all the IL and
products in the organic phase. The latter was dried over Na2SO4
and concentrated under reduced pressure. The residue was
washed with iPr2O (4ꢁ3 mL) to separate the IL, and then the com-
bined ethereal extract was dried over Na2SO4 and concentrated
under reduced pressure. The resulting mixture of products was
then separated by using column chromatography in hexane with
increasing amounts of EtOAc.
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Prof. Neil C. Bruce (Department of Biology, University of York, UK)
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Science and Engineering, ETH Zurich, Basel, Switzerland) are
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Keywords: asymmetric synthesis
enantioselectivity · ionic liquids · nitriles
·
biotransformations
·
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Received: April 4, 2014
Revised: May 22, 2014
Published online on July 8, 2014
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ChemCatChem 2014, 6, 2425 – 2431 2431