Regio- and stereoselective synthesis of chiral nitrilolactones using Baeyer–Villiger monooxygenases

Article abstract of DOI:10.1016/j.tet.2015.12.047

This work describes the regio- and enantioselective synthesis of nitrile-containing chiral lactones from easily accessible ketone precursors using Baeyer–Villiger monooxygenases. These biocatalysts controlled the distribution of regioisomers much more tightly than commonly used stoichiometric reagents, additionally with good to excellent optical purity of products. A surprising case of strong stereoelectronic control was also observed. We tested a library of 14 catalysts using five-to eight-membered cyclic ketones with two different tether lengths to the nitrile group. In all but the largest series we found suitable wild-type enzymes for preparative scale synthesis of the target compounds. The diverse possibilities to further functionalize lactones and nitriles make this method interesting for the generation of chiral building blocks.

Full text of DOI:10.1016/j.tet.2015.12.047

Accepted Manuscript
Regio- and stereoselective synthesis of chiral nitrilolactones using Baeyer-Villiger
monooxygenases
Michael J. Fink, Radka Snajdrova, Alexander Winninger, Marko D. Mihovilovic
PII:
S0040-4020(15)30295-7
10.1016/j.tet.2015.12.047
TET 27373
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 23 November 2015
Revised Date: 9 December 2015
Accepted Date: 22 December 2015
Please cite this article as: Fink MJ, Snajdrova R, Winninger A, Mihovilovic MD, Regio- and
stereoselective synthesis of chiral nitrilolactones using Baeyer-Villiger monooxygenases, Tetrahedron
(2016), doi: 10.1016/j.tet.2015.12.047.
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Regio- and stereoselective synthesis of chiral
nitrilolactones using Baeyer-Villiger
monooxygenases
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Michael J. Fink, Radka Snajdrova, Alexander Winninger and Marko D. Mihovilovic
Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163, 1060 Vienna, Austria

1
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Tetrahedron
journal homepage: www.elsevier.com
Regio- and stereoselective synthesis of chiral nitrilolactones using Baeyer-Villiger
monooxygenases
Michael J. Fink a,b , Radka Snajdrovaa,c, Alexander Winningera and Marko D. Mihovilovica
aInstitute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163, 1060 Vienna, Austria.
bPresent affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, MA 02138, United States of America.
cPresent affiliation: Medicines Research Centre, GlaxoSmithKline, Gunnel’s Wood Road, Stevenage, Herts SG1 2NY, United Kingdom.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
This work describes the regio- and enantioselective synthesis of nitrile-containing chiral lactones
from easily accessible ketone precursors using Baeyer-Villiger monooxygenases. These
biocatalysts controlled the distribution of regioisomers much more tightly than commonly used
stoichiometric reagents, additionally with good to excellent optical purity of products. A
surprising case of strong stereoelectronic control was also observed. We tested a library of 14
catalysts using five- to eight-membered cyclic ketones with two different tether lengths to the
nitrile group. In all but the largest series we found suitable wild-type enzymes for preparative
scale synthesis of the target compounds. The diverse possibilities to further functionalize
lactones and nitriles make this method interesting for the generation of chiral building blocks.
Available online
Keywords:
Biocatalysis
BVMO
Redox biotransformation
Chiral building block
Stereoelectronic effects
2009 Elsevier Ltd. All rights reserved
.
Corresponding author. E-mail: michael.j.fink@tuwien.ac.at

2
1. Introduction
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The widespread use of biocatalysts in organic-synthetic research
still much depends on their ease of use, the predictability of
substrate acceptance, and to a lesser extent on the availability of
stereocomplementary enzymes in asymmetric synthesis. Whereas
industrial chemists have long embraced the potential of
biocatalysis and have created productive overlaps of
biotechnology, biocatalysis and organic synthesis in the production
of bulk and fine chemicals, as well as marketed pharmaceuticals1
— scientists working in exploratory research often shun the option
of enzymatic catalysis for its alleged higher initial effort to reach a
set goal of selectivity, or scale. Stronger innovation and
development is thus needed to bring these powerful methods closer
together.2,3
We envision two main areas for improvement to facilitate the
broader application of enzymes in organic synthesis: catalyst
formulation and stability, and development as well as
demonstration of reliable use for certain reactions. The first issue is
critical to the user, but given the inherent complexity of enzymes
the solutions to this problem are usually more elaborate than e.g.
simple exclusion of oxygen and moisture for certain metal
catalysts. Tremendous effort is invested on this topic.4-6 The second
area addresses the organic chemist’s desire to have rules to predict
selectivity, or more generally, reaction outcome (cf. Prelog’s rule7
for alcohol dehydrogenases or Kazlauskas’s rule8 for lipases).
Alternatively, clear superiority over other catalysts or reagents also
promotes usage (e.g. Merck & Co.’s department of research and
development has recently restricted their screening of carbonyl
reduction reactions to enzymes only).9 With this work we want to
contribute in this way for the class of Baeyer-Villiger
monooxygenases (BVMOs).
The first BVMOs were discovered more than 40 years ago; they
are so far indisputably the best catalysts for asymmetric Baeyer-
Villiger oxidations (BVOx).10-12 Many enzymes of this class have
been characterized in great detail from both biochemical and
synthetic perspectives, resulting in knowledge of multiple hundred
accepted substrates.13 Still, most synthetic studies focused on a
holistic description of an enzyme’s substrate profile using model
compounds, or showed the performance of particular BVMOs in
the synthesis of certain molecules of interest. This continued
attention and the emergence of such publications actually
illustrates the lack of a general rule for reactivity and selectivity.
This study aims to establish BVMOs as excellent catalysts for
the synthesis of nitrile-containing lactones as chiral building blocks
derived from their respective nitriloketones. These starting
materials are themselves easily accessible from cheap bulk
chemicals. The combination of three functional groups
(carboxylate, secondary alcohol and nitrile) with strongly distinct
reactivity patterns provides a plethora of possible reactions. Our
investigations were based on the discovery of this activity by
Alphand and co-workers on two cyclohexanone substrates.14 We
expanded the scope to cyclopentanones, –heptanones and –
octanones, identified general trends and independently
corroborated the assignment of absolute configuration, all in order
to facilitate the planning of synthetic strategies using the products
described herein.

3
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Scheme 1. Synthesis of racemic ketones 1a–8a via Stork enamine synthesis and their biocatalytic Baeyer-Villiger oxidation towards regioisomeric normal
lactones 1b-6b and abnormal lactones 1c-6c. Reagents and conditions: a) 1. pyrrolidine, benzene or toluene, Dean-Stark trap, 2. chloroacetonitrile, dioxane, then
H2O; b) 1. as in a), 2. acrylonitrile, dioxane, then H2O. NADPH: nicotinamide adenine dinucleotide, reduced form.
E>200). CDMO performed sluggishly, and CPDMO gave results
similar to the CHMO-group’s. In total, we detected both the
remaining ketone enantiomer (–)-1a and the normal lactone (–)-
2. Results and Discussion
We synthesized eight racemic ketone substrates 1a–8a via
Stork enamine reactions (Scheme 1).15 First, the respective
1b with excellent optical purity, but its regioisomer 1c only with
unsubstituted cycloketones were condensed with pyrrolidine as
poor optical enrichment.
benzene or toluene solutions in a Dean-Stark trap apparatus. We
In strong contrast to the above results, all CHMO-types except
CHMOBrevi plus CDMO and CPDMO converted the
used the resulting enamines without further purification for
alkylation with chloroacetonitrile (for compounds 1a, 3a, 5a and
7a) or for Michael addition to acrylonitrile (for compounds 2a,
4a, 6a and 8a). All reactions and subsequent purifications
proceeded smoothly; the conjugate additions generally gave
higher yields (up to 88% after vacuum distillation, ketone 4a).
,
1
enantiomers of cyclohexanone 3a with almost perfect
stereospecificity to the regioisomers 3b and 3c (Figure 1b), as
suggested by the initial finding using CHMOAcineto.
14 The CPMO-
types had negligible activity, and CHMOBrevi 1 produced only 3b.
Interestingly, in addition to the high specificity, we observed a
pronounced difference in rate constants for the ketone
enantiomers. Thus at first one enantiomer was preferentially
depleted, before the remaining one was converted via a different
transition state mechanism (stereoelectronic control).16,17 This
was a peculiar finding from a kinetic and structural aspect.
In preparation for the biocatalyst screening reactions, all
expected lactone products were synthesized by BVOx using
meta-chloroperbenzoic
acid
(mCPBA)
for
analytical
characterization and GC method development (data not shown).
We selected 14 BVMOs for the evaluation: three linear ketone-
converting enzymes (phenylacetone monooxygenase, PAMO,
from Thermobifida fusca, p-hydroxyacetophenone MO,
HAPMO, from Pseudomonas fluorescens, a steroid MO, STMO,
from Rhodococcus rhodochrous) and 11 cycloketone-converting
enzymes, which are further sub-grouped in seven cyclohexanone
MO-type (CHMOs from Acinetobacter calcoaceticus,
Cycloheptanone 5a was the first substrate in this library to
illustrate the known limitations in ring size for substituted
cycloketones in BVMO-catalyzed reactions (Figure 2a).18,17,19
Only six enzymes could accommodate this ring in their active
site, and in all these biotransformations we only detected the
formation of normal lactone 5b. The large-ring converting
BVMOs CDMO19,20 and CPDMO17,18,21 were already previously
described as not proficient at oxidizing bulky and/or substituted
medium to large ring ketones. Neither did CPMO-types
measurably oxidize 5a. Mainly CHMO-type enzymes were active
in kinetic resolution of this substrate with moderate selectivity.
We measured the best result using CHMORhodo 2 (E = 11 at 39%
relative conversion). In general, the reaction rate was also much
lower than for the smaller ring sizes we tested before. As we
expected after these findings, we could not detect any product
formation with both cyclooctanone derivates 7a and 8a with any
of the 15 tested BVMOs.
Arthrobacter
sp.
BP2,
Brachymonas
petroleovorans,
Brevibacterium epidermis, R. ruber, Xanthobactersp. ZL5), two
cyclopentanone MO-type (CPMO from Comamonas sp. NCIMB
9872 and CHMO from B. epidermis) and two large ring-
converting BVMOs (cyclododecanone MO, CDMO, from R.
ruber and a Pseudomonad cyclopentadecanone MO, CPDMO).
The Supplementary Data contains detailed information and
references. All enzymes were tested as heterologous whole-cell
over-expression systems in Escherichia coli BL21(DE3) or
TOP10, which harbored standard vectors with a T7 promotor for
expression control (except PAMO, HAPMO, STMO. and
CPDMO: ara regulon). The reactions were carried out in multi-
well plates with 1 mL initial volume for up to 24 h, starting in the
early exponential growth phase of the bacteria. Samples were
withdrawn after 4, 8 and 24 h, extracted with EtOAc, and
analyzed the extracts on chiral-phase GC-FID. Additional
All active wild-type BVMOs in our test set converted the
three remaining substrates 2a, 4a and 6a to the respective normal
lactones, exclusively, in kinetic resolution reactions. It was
reported that stoichiometric reagents as mCPBA or
trifluoroperacetic acid favored the nucleophilicity-driven
rearrangement, but also produced substantial amounts of the
abnormal product from 4a (6–20%).14 We could corroborate this
trend with ketones 2a and 6a qualitatively during the synthesis of
racemic reference samples (unquantified reactions, data not
shown). We further hypothesized that the virtually absolute
regioselectivity of BVMOs with these substrates was promoted
by the lower inductive effect of the nitrile group on the α-
carbonyl carbon atom over three σ-bonds in ketones 2a, 4a and
6a vs. two σ-bonds in 1a, 3a. Cycloheptanone 5a was too poor a
substrate to possibly be taken into consideration for this trend.
reactions
containing
acetophenone
(PAMO),
p-
hydroxyacetophenone
(HAPMO), 3-benzylcyclobutanone
(STMO) and rac-bicyclo[3.2.0]hept-2-en-6-one (all other
BVMOs) were performed and analyzed as positive controls for
enzyme activity (data not shown).
2.1. Results from screening reactions on analytical scale
Already with the first substrate, cyclopentanone 1a, we found
a strongly different trend than expected from the known results14
with CHMOAcineto and the higher homolog ketone 3a (1:1 ratio of
regioisomers). All active enzymes preferentially catalyzed the
formation of the levorotatory normal lactone (–)-1b (Figure 1a);
even at full conversion of the starting material, we only found
<20% of abnormal lactone 3c in any of the reactions. CHMO-
type BVMOs were moderately enantioselective, with ee-values
ranging from 50–90% (selectivity factor E up to 33). Excellent
selectivity was achieved with CPMO-types (up to >99% ee,
All cycloketone-converting enzymes accepted cyclopentanone
2a as a substrate (Figure 2b) and produced (R)-2b exclusively.
We measured comparable reaction rates throughout the catalysts
(40–50% conversion after 4 h), except for CDMO (only 36%
after 24 h). CPMO-types, CPDMO and CDMO could not

4
discriminate between ketone enantiomers, but most CHMO-types
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showed excellent enantioselectivity. CHMOBrevi 1 scored the best
and preparatively highly useful result (E>200 at 20%, E = 79 at
38% conversion).

5
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Figure 1. Analytical screening data for BVMO-catalyzed reactions with the partial formation of abnormal lactones c (from ketones 1a and 3b). The enzymes
are grouped by phylogenetic clusters (cf. ref. 22). For each enzyme, the top bar value was determined after 4 h, the bottom bar value after 8 h. All data is color-
coded: light grey for normal lactones, dark grey for abnormal lactones. Left boxes: relative conversion of racemic ketone to regioisomeric products. Center
boxes: enantiomeric excess of normal lactone. Right boxes: enantiomeric excess of abnormal lactone.
Figure 2. Analytical screening data for BVMO-catalyzed reactions towards normal lactones b only (from ketones 5a, 2a, 4a and 6a). The enzymes are
grouped by phylogenetic clusters (cf. ref. 22). Left boxes: relative conversion of racemic ketone to normal lactone. Right boxes: selectivity factor for the kinetic
resolution, calculated according to Sih’s equation E = [ ln(1 – eeS) – ln(1 + eeS/eeP) ] / [ ln(1 + eeS) – ln(1 + eeS/eeP) ] from ee of substrate (eeS) and ee of product
(eeP) values. a: values after 24 h only. b, c: values after 4 h and 8 h. d: values after 8h and 24 h.

6
Again, all cycloketone-converting catalysts efficiently
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oxidized cyclohexanone 4a to normal lactone 4b (Figure 2c).
CHMO-types and CDMO catalyzed the formation of (R)-4b with
excellent selectivity (E>200), whereas the CPMO-types showed
poor, but enantiodivergent selectivity. CPDMO had no apparent
preference for the ketone enantiomers over the course of the
reaction. We observed a similar reactivity and selectivity pattern
Scheme 3. Reduction of the enantiomerically enriched ketone (–)-2a to
the previously characterized trans-configured alcohol 1S,2R-2d.
for cycloheptanone 6a. CHMO-types save CHMOBrevi were
1
This goal was achieved by reducing enantioenriched (–)-2a,
obtained by kinetic resolution using CHMOBrevi 1, to the
corresponding diastereomeric cyclopentanols using sodium
borohydride, and subsequent separation of the diastereomers by
careful column chromatography. Despite the massive loss in
chiral information due to the strongly basic reaction conditions
(ee2a = 88%, ee2d<30%), we were able to measure the sign of
optical rotation of the trans-isomer. By comparison to the
published value for this compound29 we safely assigned the S-
configuration to (–)-2a, as the main topic of the cited study was
the assignment of absolute configurations to mono- and
polycyclic lactones similar to our compounds. Our independent
pathway thus corroborated the previous assignment, and followed
the general trend in BVMO-catalyzed oxidations of 2-substituted
cycloketones: the preferential oxidation of R-ketones.19
active and highly selective for the formation of (–)-6b, and
CPDMO produced racemic 6b. In this case, we could not
measure any activity with CPMO-types and CDMO.
Scheme 2. Formation of abnormal lactone 4c using variant F156L of
CPMOComa
.
Inspired by results from a concurrent BVMO project,23 we
found a surprising new activity when screening a library of
CPMO variants24-26 with ketone 4a. Not only did stereoselectivity
improve (although not better than CHMOBrevi 2), but the F156L
variant also preferentially produced the abnormal lactone (–)-4c
(approx. ratio 3:7 4b/4c; Scheme 2), unfortunately with mediocre
enantioselectivity (45% ee). A double mutant, F156H-G157L,
performed with better selectivity (75% ee, 1:1 4b/4c), but at a
very low rate (see Supplementary Data). This collection of
mutants had already previously been generated with the aim of
improving the selectivity and/or of extending the substrate
acceptability of wild-type CPMO.24-26 Four active site positions
were varied (F156, G157, G449, F450), to modulate available
space and hydrogen bonding properties specifically without
randomization, including four double mutants. These results
strongly suggest that migratory preference can change drastically
with rather small perturbations in catalyst structure; only recently
a similar effect was demonstrated on another BVMO in a
mutagenesis study addressing regioselectivity.27
2.3. Exemplary preparative biotransformations
We performed preparative biotransformations with substrates
1a–6a to confirm the results we obtained in the screening
reactions, and to demonstrate the usefulness of the method in the
synthesis of chiral nitrilolactone building blocks (multi-100 mg
scale). The reactions were linearly scaled up from the 1 mL scale
in standard shake flasks and without pH control. We isolated
most products in high to excellent optical purity, and with
reasonably good yields for kinetic resolution reactions (Table 1).
Table 1. Summary of preparative biotransformation results.
Entry
Ketone
1a
Enzyme
Product 1
Product 2
In summary, we found that CHMO-type BVMOs perform best
with nitriloketones. As often, CPMO-types acted as
complementary catalysts, either in terms of activity or selectivity.
The large ring-converting BVMOs CPDMO and CDMO were
not particularly useful in this substrate class. This feature was
surprising, given the otherwise wide applicability of CDMO.19
The linear ketone-converting BVMOs in our test set completely
failed to accept any of the substrates with a single exception:
HAPMO converted ketone 2a enantiodivergently to (+)-2b, but
with poor selectivity (E<10; see Supplementary Data).
(–)-1a
46%, 58% ee
(–)-(S)-2a
40%, 98% ee
(–)-1b
41%, 97% ee
(–)-(R)-2b
31%, >99% ee
1
2
3
4
5
6
7
8
CHMOBrevi 1
CHMOBrevi 1
0.3 g scale
2a
CHMOBrevi 1
1.2 g scalea
(–)-(S)-2a
47%, 88% ee
(–)-3b
19%, >99% ee
(–)-(R)-2b
33%, 97% ee
(–)-3c
34%, >99% ee
3a
4a
CHMOBrachy
CHMOArthro
CPMOF156L
CHMORhodo 2
CHMOXantho
(+)-(S)-4a
38%, 97% ee
(+)-(S)-4b
18%, 96% ee
(–)-5a
41%, 68% ee
(–)-(R)-4b
39%, 98% ee
(–)-4c
47%, 59% ee
(+)-5b
10%, 66% ee
2.2. Corroboration of the assignment of absolute configuration
5a
6a
In their initial publication14 Berezina et al. assigned the
(–)-6a
43%, 99%
(–)-6b
42%, 95%
absolute configuration of (+)-2a by comparison of the sign of
28
a The reaction was performed in a small bench-top bioreactor with pH and
aeration control.
optical rotation to a published value. The authors of the cited
reference had based their assignment on the reaction mechanism
of the alkylation of cyclohexanone (R)-prolinol enamine with
acrylonitrile. This assignment was unfortunately never cross-
validated or confirmed using unambiguous methods, e.g. single-
crystal XRD. We opted to obtain a second reference point for the
assignment of absolute configuration in this series in order to
provide higher fidelity in stereochemical planning. After all, this
method should serve as a means to generate chiral building
blocks for more elaborate synthetic targets.
We observed substantial racemization of the enantiomerically
enriched cyclopentanones and –hexanones upon purification on
regular silica, but not of the cycloheptanones (see Experimental
Section). We did not further investigate or optimize this issue,
but are convinced that other more neutral chromatographic
stationary phases would alleviate the problem.
We performed the transformations of ketone 2a on different
scales and reaction vessels (Table 1, entries 2 and 3), and found
that the controlled environment of a bioreactor improved the
results only slightly in yield. In any case, it was convenient and
easy to quench the reaction after such time that both starting

7
material and product could be isolated in highly enantio-enriched
form (Figure 3).
resolution mass spectrometry was performed on a Thermo
ACCEPTED MANUSCRIPT
Scientific LTQ Orbitrap XL hybrid FT-MS, equipped with a
Thermo Fischer Exactive Plus Orbitrap (LC-ESI+) and a
Shimadzu IT-TOF Mass Spectrometer.
4.2. General — Cultivation of E. coli
Bacterial cultures were incubated in baffled Erlenmeyer flasks
in orbital shakers (InforsHT Multitron 2 Standard) at 200 rpm.
All materials and biotransformation media were sterilized by
autoclaving at 121 °C for 20 min. Various aqueous stock
solutions were sterilized by filtration through 0.20 µm syringe
filters. Agar plates were prepared with LB medium (10 g L-1
bacto-peptone, 5 g L-1 yeast extract, 10 g L-1 NaCl) supplemented
by 1.5% w/v Agar Agar.
4.3. General procedure for the alkylation of cycloketones via the
corresponding pyrrolidine enamines
Following the procedures of Stork,15 the cycloketone and
pyrrolidine (1.1 equiv.) were dissolved in benzene or toluene
(300 mL mol-1 ketone; toluene for n>1) and refluxed for 5–10 h
(cyclopentanone and –hexanone) or 24–48 h (cycloheptanone and
–octanone). Excess pyrrolidine and solvent were removed under
reduced pressure and the yellow to brown oily residue was
directly used for further reactions. For medium-sized rings a
small amount of p-toluenesulfonic acid was added (n>1).
Figure 3. Time course of preparative kinetic resolutions; ee values were
deteremined by chiral-phase GC; conversion values were calculated from ee
data according to Sih’s equations.30 Top: ketone 2a using CHMOBrevi 1
.
Bottom: ketone 4a using CHMOArhtro
.
3. Conclusion
In this work we demonstrated the potential of BVMOs in the
synthesis of chiral nitrilolactones. The respective starting
materials were easily accessible from cheap starting materials
(cycloketones, pyrrolidine, chloroacetonitrile or acrylonitrile).
We identified suitable biocatalysts to generate 6-, 7- and 8-
membered lactones with excellent optical purity and good yields
on the sub-gram to gram-scale. In total, CHMO-type BVMOs
performed best, with CPMO-types serving as a useful extension
for different selectivity. Large ring-converting and linear ketone-
converting BVMOs were found unsuitable for this substrate
class. In most cases, the nucleophilicity-goverened, normal
lactones were formed with high selectivity. We also observed and
interpreted cases which deviated from this trend. We are
convinced that the extensive analytical screening reaction data
and the exemplary preparative biotransformations we presented
are sufficient to make use of this method for the synthesis of such
chiral building blocks. Their lactone and nitrile moieties should
provide ample opportunity for various synthetic strategies.
Next, the enamine (typically 5–20 g) was dissolved in
anhydrous dioxane (400 mL mol-1) under an argon atmosphere.
Then the electrophile (1.5 equiv.; chloroacetonitrile for
compounds 1, 3, 5 and 7, or acrylonitrile for compounds 2, 4, 6
and 8) was added in one shot using a syringe. Occasionally, more
electrophile was added in case of stagnant reactions. The mixture
was refluxed for 10–48 h until complete conversion of the
enamine could be detected by GC-MS. Subsequently, water
(200 mL mol-1) was added and the biphasic mixture was heated
to reflux for 30–60 min. Dioxane was then removed by rotary
evaporation and the residue was partitioned between EtOAc
(2 mL mmol-1) and 2N HCl (0.5 mL mmol-1). The aqueous phase
was extracted with EtOAc (4 x same volume as before) and the
pooled extracts were dried over sodium sulfate and concentrated
under reduced pressure. Purification of the brown crude oils was
performed on MPLC or via distillation.
4. Experimental
4.4. 2-(2-Oxocyclopentyl)acetonitrile 1a
Following the general procedure in section 4.3 with
cyclopentanone pyrrolidine enamine (9.0 g, 66 mmol) and
chloroacetonitrile and purification via chromatography
(LP/EtOAc 4:1) yielded the title ketone as a yellow oil (1.1 g,
14%). The spectral data was in agreement with previous
literature.31 1H-NMR: δ 1.61-1.89 (m, 2H), 2.00-2.20 (m, 2H),
2.27-2.47 (m, 4H), 2.59-2.72 (m, 1H). 13C-NMR: δ 17.1 (t), 20.1
(t), 28.8 (t), 37.0 (t), 45.3 (d), 118.0 (s), 216.2 (s).
4.1. General — Chemistry
Unless noted otherwise, all reagents were purchased from
commercial suppliers and used without further purification.
Anhydrous dioxane, THF and toluene was obatained by passing
the pre-distilled solvents over Al2O3 columns (PURESOLV,
Innovative Technology). Chromatography solvents were distilled
prior to use. Column chromatography was performed on a Büchi
Sepacore Flash System using silica gel from Merck (40-63 µm)
using the indicated solvent mixtures. NMR spectra were recorded
from CDCl3 solutions on a Bruker AC 200 (200 MHz 1H
resonance) spectrometer and chemical shifts are reported in ppm
relative to tetramethylsilane. Reaction progress of non-
biocatalytic reactions was determined using GC-MS(EI) (Thermo
Finnigan Focus GC / DSQ II, capillary column BGB5 (30m x
0.32 mm ID). Enzyme screening data and enantiomeric excess of
products was determined via GC-FID using a BGB175 (30 m x
0.25 mm ID, 0.25 µm film) or BGB173 (30 m x 0.25 mm ID,
0.25 µm film) column on a ThermoQuest Trace GC 2000 and a
ThermoFocus GC. Specific rotation was measured on an Anton
Paar MCP500 polarimeter at the specified conditions. High
4.5. 3-(2-Oxocyclopentyl)propanenitrile 2a
Following the general procedure in section 4.3 with
cyclopentanone pyrrolidine enamine (7.5 g, 55 mmol) and
acrylonitrile and purification via vacuum distillation yielded the
title ketone as a pale yellow oil (4.1 g, 54%). b.p.: 123–124 °C at
1
5 mbar. H-NMR: δ 1.37–1.84 (m, 3H), 1.87–2.37 (m, 6H), 2.47
(dt, 2H, J1 = 3.6 Hz, J2 = 7.2 Hz). 13C-NMR: δ 15.4 (t), 20.4 (t),
25.5 (t), 29.3 (t), 37.7 (t), 47.5 (d), 119.4 (s), 219.4 (s).
4.6. 2-(2-Oxocyclohexyl)acetonitrile 3a
Following the general procedure in section 4.3 with
cyclohexanone pyrrolidine enamine (15.1 g, 100 mmol) and

8
chloroacetonitrile and purification via chromatography
(LP/EtOAc 4:1) yielded the title ketone as a dark yellow oil (4.7
g, 34%). The spectral data was in agreement with previous
literature.32 1H-NMR: δ 1.36-2.72 (m, 11H). 13C-NMR: δ 17.9 (t),
24.9 (t), 27.6 (t), 33.4 (t), 41.6 (t), 46.8 (d), 118.6 (s), 208.8 (s).
under the same conditions until an optical density of 0.2–0.6
ACCEPTED MANUSCRIPT
was reached. Inducing agent (see Supplementary Data) and β-
cyclodextrin (1 equiv.) were added, the mixture was thoroughly
mixed and split in 1.0 mL aliquots into 24-well plates. Substrates
were added as 0.8 M solutions in dioxane to a final concentration
of 4 mM. The plates were sealed with adhesive film and
incubated at the appropriate temperature (see Supplementary
Data) in an orbital shaker for up to 24 h. Analytical samples were
prepared by extraction of 0.5 mL of biotransformation culture
with 1.0 mL EtOAc (supplemented with 1 mM methyl benzoate
as internal standard) after centrifugal separation of the cells
(approx. 15 kRCF, 1 min, rt).
4.7. 3-(2-Oxocyclohexyl)propanenitrile 4a
Following the general procedure in section 4.3 with
cyclohexanone pyrrolidine enamine (454 g, 3 mol) and
acrylonitrile and purification via vacuum distillation yielded the
title ketone as a colorless liquid (400 g, 88%). b.p.: 144-146 °C at
10 mbar. The spectral data was in agreement with previous
literature.33 1H-NMR: δ 1.16–2.12 (m, 8H), 2.18–2.49 (m, 5H).
13C-NMR: δ 15.0 (t), 25.0 (t), 25.5 (t), 27.8 (t), 34.1 (t), 42.1 (t),
48.7 (d), 119.7 (s), 211.7 (s).
4.13. General procedure for preparative biotransformations in
shake flasks
LB growth medium was inoculated, induced, and charged
with additives as described in section 4.12. The substrate was
then added directly to the shake flask as approx. 10% (w/v)
solution in dioxane. Incubation at the appropriate temperature
was carried out until the desired degree of conversion was
determined via GC control. The aqueous solution was then
centrifuged (17 kRCF, 15 min, 4 °C) and the supernatant was
extracted with EtOAc (5x equal volume). The pooled organic
layers were washed with brine, dried over sodium sulfate and
concentrated. The crude compounds were purified by
chromatography on silica using LP/EtOAc mixtures.
4.8. 2-(2-Oxocycloheptyl)acetonitrile 5a
Following the general procedure in section 4.3 with
cycloheptanone pyrrolidine enamine (4.56 g, 28 mmol) and
chloroacetonitrile and purification via chromatography
(LP/EtOAc 5:1) yielded the title ketone as a yellow oil (1.28 g,
31%). The spectral data was in agreement with previous
literature.34,35 1H-NMR: δ 1.23-2.03 (m, 8H), 2.34-2.70 (m, 3H),
2.87-3.00 (m, 1H). 13C-NMR: δ = 19.7 (t), 23.6 (t), 28.9 (t), 29.2
(t), 30.7 (t), 43.1 (t), 48.1 (d), 119.0 (s), 211.6 (s).
4.9. 3-(2-Oxocycloheptyl)propanenitrile 6a
4.14. (–)-2-(2-Oxocyclopentyl)acetonitrile (–)-1a
Following the general procedure in section 4.3 with
cycloheptanone pyrrolidine enamine (13.6 g, 82 mmol) and
acrylonitrile and purification via chromatography (LP/EtOAc
10:1) yielded the title ketone as a pale yellow oil (5.5 g, 40%).
The spectral data was in agreement with previous literature.36 1H-
NMR: δ 1.17–2.12 (m, 10H), 2.21–2.58 (m, 4H), 2.64–2.78 (m,
1H). 13C-NMR: δ 15.2 (t, C-2), 23.7 (t), 27.5 (t), 28.8 (t), 29.0 (t),
31.6 (t), 43.4 (t), 49.9 (d), 119.6 (s), 214.3 (s).
Following the general procedure in section 4.13 with racemic
ketone 1a (100 mg, 812 µmol, 4 mM) and CHMOBrevi 1, after
10.5 h reaction time and subsequent purification (LP/Et2O 1:3),
the title compound was obtained as a yellow oil (46 mg, 46%).
NMR data: see 1a. ee (GC) = 58%. αD = –115.4 (c 0.54,
CHCl3).
25
4.15. (–)-2-(6-oxotetrahydro-2H-pyran-2-yl)acetonitrile (–)-1b
Following the general procedure in section 4.13 with racemic
ketone 1a (100 mg, 812 µmol, 4 mM) and CHMOBrevi 1, after
10.5 h reaction time and subsequent purification (LP/Et2O 1:3),
the title compound was obtained as a yellow oil (46 mg, 41%).
1H-NMR: δ 1.58–2.13 (m, 4H), 2.35–2.79 (m, 4H), 4.46–4.62
(m, 1H). 13C-NMR: δ 18.2 (t), 24.6 (t), 27.0 (t), 29.1 (t), 74.8 (d),
115.9 (s), 169.9 (s). ee (GC) = 97%. αD25 = –4.0 (c 0.70, CHCl3).
ESI-FT-MS m/z [M+H]+ calc. 140.07061, found 140.07039, ∆
1.54 ppm.
4.10. 2-(2-Oxocyclooctyl)acetonitrile 7a
Following the general procedure in section 4.3 with
cyclooctanone pyrrolidine enamine (4.22 g, 24 mmol) and
chloroacetonitrile and purification via chromatography
(LP/EtOAc 10:1) yielded the title ketone as a pale yellow oil
(2.05 g, 53%). The spectral data was in agreement with previous
literature.37 1H-NMR: δ 2.57-3.10 (m, 2H, CH2-CN), 1.35-2.5 (m,
13H). 13C-NMR: δ 17.0 (t), 22.6 (t), 22.9 (t), 23.9 (t), 25.7 (t),
29.6 (t), 40.5 (t), 44.7 (d), 117.3 (s), 213.8 (s).
4.16. (–)-(S)-3-(2-Oxocyclopentyl)propionitrile (–)-(S)-2a
4.11. 3-(2-Oxocyclooctyl)propanenitrile 8a
Following the general procedure in section 4.13 with racemic
ketone 2a (300 mg, 2.19 mmol, 4 mM) and CHMOBrevi 1, after
10.5 h reaction time and subsequent purification (LP/EtOAc 2:1),
the title compound was obtained as a colorless oil (121 mg,
40%). NMR data: see 2a. ee (GC) = 98% before purification,
85% after purification. αD25 = –100.8 (c 2.42, CHCl3).
Following the general procedure in section 4.3 with
cyclooctanone pyrrolidine enamine (16.0 g, 89 mmol) and
acrylonitrile and purification via chromatography (LP/EtOAc
10:1) yielded the title ketone as a pale yellow oil (4.0 g, 25%).
The spectral data was in agreement with previous literature.36 1H-
NMR: δ 1.00-2.53 (m, 16H), 2.72-2.86 (m, 1H). 13C-NMR:
δ 15.4 (t), 24.6 (t), 24.9 (t), 25.0 (t), 27.2 (t), 27.5 (t), 32.9 (t),
42.9 (t), 48.3 (d), 119.4 (s), 218.4 (s).
4.17. (–)-(R)-3-(6-Oxotetrahydro-2H-pyran-2-yl)propionitrile (–
)-(R)-2b
Following the general procedure in section 4.13 with racemic
ketone 2a (300 mg, 2.19 µmol, 4 mM) and CHMOBrevi 1, after
10.5 h reaction time and subsequent purification (LP/EtOAc 1:1),
the title compound was obtained as a yellow oil (104 mg, 31%).
1H-NMR: δ 1.40–1.66 (m, 1H), 1.71–2.01 (m, 5H), 2.32–2.66
(m, 4H), 4.27–4.43 (m, 1H). 13C-NMR: δ 13.2 (t), 18.3 (t), 27.5
(t), 29.2 (t), 31.4 (t), 77.9 (d), 119.0 (s), 171.1 (s). ee (GC) >99%
before and after purification. αD25 = -132.9 (c 0.38, CHCl3). ESI-
4.12. General procedure for substrate acceptance and
biocatalyst performance screening on analytical scale
A baffled Erlenmeyer flask was charged with LB medium
with appropriate antibiotics supplement (10 mL), inoculated with
a bacterial single colony from an Agar plate and incubated at 37
°C in an orbital shaker o/n. The biotransformation medium,
supplemented with appropriate antibiotics, was then inoculated
with 2% v/v of the preculture and incubated for approx. 1–2 h

9
FT-MS: m/z [M+H]+ calc. 154.08626, found 154.08601, ∆ 1.59
ppm.
4.22. (−)-(R)-3-(7-Oxooxepan-2-yl)propionitrile (–)-(R)-4b
ACCEPTED MANUSCRIPT
Following the general procedure in section 4.13 with racemic
ketone 4a (300 mg, 1.98 mmol, 4 mM) and CHMOArthro, after 5 h
reaction time and subsequent purification (LP/EtOAc 2:1), the
title compound was obtained as a yellow oil (131 mg, 39%). The
spectral data was in agreement with previous literature.38 1H-
NMR: δ 1.38–1.68 (m, 3H), 1.74–2.02 (m, 5H), 2.43–2.59 (m,
4H), 4.25–4.35 (m, 1H). 13C-NMR: δ 13.4 (t), 22.6 (t), 27.8 (t),
31.7 (t), 34.2 (t), 34.6 (t), 77.5 (d), 119.0 (s), 174.8 (s). ee (GC) =
98%. αD25 = –100.5 (c 2.62, CHCl3).
4.18. 3-(2-Hydroxycyclopropyl)propanenitriles 1R,2S-2d and
1S,2S-2d
NaBH4 (248 mg, 6.56 mmol, 5 equiv.) was suspended in
anhydrous THF (20 mL) under an argon atmosphere. Then a
solution of ketone (–)-2a in THF (10 mL) was added at 0–5 °C.
The mixture was stirred at this temperature for 2 h, but no
conversion of starting material was detected. The cooling bath
was removed and stirring was continued for 2 d. More NaBH4
was added (5 equiv.) and stirring was continued (24 h). After 3 d
total reaction time 50% conversion was reached. Another 10
equiv. NaBH4 and THF (20 mL) were added and stirring was
continued at rt. After 4 d the reaction mixture was poured on 2N
HCl (150 mL) and extracted with Et2O (3 x 100 mL). The pooled
extracts were dried over sodium sulfate and concentrated under
reduced pressure. Careful column chromatography (50 g silica,
LP/Et2O 1:2) separated both diastereomers as colorless oils. The
spectral data of the trans isomer was in agreement with previous
4.23. (+)-(S)-3-(7-Oxooxepan-2-yl)propionitrile (+)-(S)-4b
Following the general procedure in section 4.13 with racemic
ketone 4a (265 mg, 1.75 mmol, 7 mM) and CPMOComa-F156L after
22 h reaction time and subsequent purification (LP/Et2O 3:1), the
title compound was obtained as a colorless solid (52 mg, 18%).
m.p.: 48–54 °C. ee (GC) = 96%. αD25 = +56.5 (c 1.04, CHCl3).
4.24. (–)-3-(2-Oxooxepan-3-yl)propanenitrile (–)-4c
1
Following the general procedure in section 4.13 with racemic
ketone 4a (265 mg, 1.75 mmol, 7 mM) and CPMOComa-F156L after
22 h reaction time and subsequent purification (LP/Et2O 3:1), the
title compound was obtained as a colorless solid (139 mg, 47%).
literature.29 1R,2S-2d (48 mg, 26%): H-NMR: δ 1.07–1.25 (m,
1H), 1.45–2.02 (m, 9H), 2.39–2.47 (m, 2H), 3.80 (q, 1H, J = 6.1
Hz). 13C-NMR δ 16.1 (t), 21.5 (t), 29.6 (t, 2C), 34.9 (t), 47.7 (d),
78.9 (d), 120.1 (s). αD20 = –4.0 (c 0.96, CHCl3); Lit.: αD25 = –38.9
(c 7.5, CHCl3).29 d.r. (GC) = 2:98 cis/trans. ee (GC) = 34% ee.
1
m.p.: 62–66 °C. H-NMR δ 1.46-1.80 (m, 5H), 1.83–2.03 (m,
1
2H), 2.04–2.22 (m, 1H), 2.48 (dd, 2H, J1 = 7.6 Hz, J2 = 6.4 Hz),
2.63-2.85 (m, 1H), 4.17-4.34 (m, 2H). 13C-NMR δ 15.1 (t), 27.9
(t), 28.5 (t), 28.7 (t), 30.1 (t), 41.3 (d), 68.6 (t), 119.4 (s), 176.0
1S,2S-2d (15 mg, 8%): H-NMR: δ 1.15–2.04 (m, 10H), 2.31–
2.56 (m, 2H), 4.21 (br s, 1H). 13C-NMR: δ 16.3 (t), 21.8 (t), 25.2
20
(t), 28.6 (t), 35.6 (t), 44.4 (d), 73.9 (d), 120.3 (s). αD = +4.9 (c
25
(s). ee (GC) = 59%. αD = –9.3 (c 1.39, CHCl3). ESI-FT-MS:
0.1, CHCl3). d.r. (GC) = >99:1 cis/trans. ee (GC) = approx. 13%
(no baseline separation).
m/z [M+H]+ calc. 168.10245, found 168.1018, ∆ 3.89 ppm.
4.25. (–)-2-(2-Oxocycloheptyl)acetonitrile (–)-5a
4.19. (–)-2-(7-Oxooxepan-2-yl)acetonitrile (–)-3b
Following the general procedure in section 4.13 with racemic
ketone 5a (100 mg, 661 µmol, 4 mM) and CHMORhodo 2, after 24
h reaction time and subsequent purification (LP/EtOAc 3:1), the
title compound was obtained as a colorless oil (41 mg, 41%).
NMR data: see 5a. ee (GC) = 68% before purification, 66% after
purification. αD25 = –58.8 (c 0.82, CHCl3).
Following the general procedure in section 4.13 with racemic
ketone 3a (300 mg, 2.19 mmol, 4 mM) and CHMOBrachy, after 4 h
reaction time and subsequent purification (LP/EtOAc 1:1), the
title compound was obtained as a colorless oil (64 mg, 19%). The
spectral data was in agreement with previous literature.14 1H-
NMR: δ 1.38–1.82 (m, 3H), 1.88–2.13 (m, 3H), 2.50–2.83 (m,
4H), 4.48–4.59 (m, 1H). 13C-NMR: δ 22.5 (t), 25.2 (t), 27.8 (t),
33.9 (t), 34.7 (t), 74.9 (d), 116.4 (s), 173.7 (s). ee (GC) >99%.
αD25 = –22.6 (c 1.28, CHCl3).
4.26. (+)-2-(8-Oxooxocan-2-yl)acetonitrile (+)-5b
Following the general procedure in section 4.13 with racemic
ketone 5a (100 mg, 661 µmol, 4 mM) and CHMORhodo 2, after 24
h reaction time and subsequent purification (LP/EtOAc 3:1), the
4.20. (–)-2-(2-oxooxepan-3-yl)acetonitrile (–)-3c
1
title compound was obtained as a yellow oil (11 mg, 10%). H-
Following the general procedure in section 4.13 with racemic
ketone 3a (300 mg, 2.19 mmol, 4 mM) and CHMOBrachy, after 4 h
reaction time and subsequent purification (LP/EtOAc 1:1), the
title compound was obtained as a colorless solid (115 mg, 34%).
The spectral data was in agreement with previous literature.14 1H-
NMR: δ 1.44–1.80 (m, 3H), 1.82–2.11 (m, 3H), 2.49 (dd, 1H, J1
= 16.8 Hz, J2 = 7.4 Hz), 2.67 (dd, 1H, J1 = 16.9 Hz, J2 = 5.8 Hz),
2.92–3.07 (m, 1H), 4.14–4.37 (m, 2H). 13C-NMR: δ 21.0 (t), 27.7
(t), 28.4 (t), 29.1 (t), 39.9 (d), 68.9 (t), 118.2 (s), 174.6 (s). ee
(GC) >99%. αD25 = –27.0 (c 2.30, CHCl3).
NMR: δ 1.47–1.94 (m, 8H), 2.44–2.54 (m, 2H), 2.67 (dd, 1H,
J1 = 16.8 Hz, J2 = 5.8 Hz), 2.74 (dd, 1H, J1 = 16.9 Hz, J2 = 6.3
Hz), 4.87–5.00 (m, 1H). 13C-NMR: δ 24.0 (t), 24.4 (t), 26.6 (t),
29.4 (t), 33.1 (t), 36.9 (t), 73.3 (d), 116.5 (s), 175.8 (s). ee (GC) =
25
66% before and after purification. αD = +31.5 (c 0.22, CHCl3).
ESI-FT-MS: m/z [M+H]+ calc. 168.10191, found 168.10170, ∆
1.22 ppm.
4.27. (−)-3-(2-Oxocycloheptyl)propionitrile (–)-6a
Following the general procedure in section 4.13 with racemic
ketone 6a (200 mg, 1.21 mol, 4 mM) and CHMOXantho, after 19 h
reaction time and subsequent purification (LP/EtOAc 4:1), the
title compound was obtained as a colorless oil (85 mg, 43%).
NMR data: see 6a. ee (GC) = 99% after purification. αD25 = -25.9
(c 1.70, CHCl3).
4.21. (+)-(S)-3-(2-Oxocyclohexyl)propionitrile (+)-(S)-4a
Following the general procedure in section 4.13 with racemic
ketone 4a (300 mg, 1.98 mmol, 4 mM) and CHMOArthro, after 5 h
reaction time and subsequent purification (LP/EtOAc 6:1), the
title compound was obtained as a pale yellow oil (113 mg, 38%).
NMR data: see 4a. ee (GC) = 97% before purification, 80% after
purification. αD25 = +24.6 (c 2.26, CHCl3).
4.28. (−)-3-(8-oxooxocan-2-yl)propionitrile (–)-6b
Following the general procedure in section 4.13 with racemic
ketone 6a (200 mg, 1.21 mmol, 4 mM) and CHMOXantho, after
19 h reaction time and subsequent purification (LP/EtOAc 2:1),
the title compound was obtained as a yellow oil (91 mg, 42%).

10
1H-NMR: δ 1.38–2.04 (m, 10H), 2.39–2.63 (m, 4H), 4.60–4.74
(m, 1H). 13C-NMR: δ = 13.8 (t), 23.7 (t), 26.1 (t), 28.8 (t), 31.0
(t), 32.3 (t), 36. 8 (t), 76.2 (d), 119.1 (s), 176.4 (s). ee (GC) =
95% after purification. αD25 = -26.4 (c 1.82, CHCl3). ESI-FT-MS:
m/z [M+H]+ calc. 182.11756, found 182.11728, ∆ 1.51 ppm.
31. Chen, Y.; Ho, D. M.; Lee, C. J. Am. Chem. Soc. 2005, 127, 12184.
ACCEPTED MANUSCRIPT
32. Miura, K.; Fujisawa, N.; Saito, H.; Wang, D.; Hosomi, A. Org. Lett.
2001, 3, 2591.
33. Cren, S.; Schar, P.; Renaud, P.; Schenk, K. J. Org. Chem. 2009, 74,
2942
34. Asselin, A. A.; Humber, L. G.; Dobson, T. A., American Home
Products Corp., USA, US4057559A, 1977; CAN 1978:120981.
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Supplementary Data
36. Hickmott, P. W.; Jutle, K. K.; Pienar, D. H. J. Chem. Soc., Perkin Trans.
1 1990, 2399.
37. Schlichting, O.; Scheuerer, G., BASF AG, Germany, US2882292, 1959;
CAN 1959:121615.
38. Cotarca, L.; Delogu, P.; Maggioni, P.; Nardelli, A.; Bianchini, R.;
Sguassero, S. Synthesis 1997, 328.
A document containing NMR spectra of the lactones isolated
from preparative transformations, numerical data from all
screening reactions and detailed biocatalyst and E. coli strain
information can be downloaded from the publisher’s website.
Acknowledgements
M.J.F. was funded within the EU-FP7 program Oxygreen
(grant # 212281). A.W. received a graduate student fellowship
within the DOC-program by the Austrian Academy of Sciences.
The project was supported by the Austrian Science Fund (FWF)
(project # I19-B10).
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