Baeyer–Villiger monooxygenase-catalyzed desymmetrizations of cyclobutanones. Application to the synthesis of valuable spirolactones

Article abstract of DOI:10.1016/j.tet.2015.12.071

A series of γ-butyrolactone derivatives, including some spiranic ones, was obtained through desymmetrization of the corresponding prochiral 3-substituted cyclobutanones via Baeyer–Villiger monooxygenase (BVMO)-catalyzed oxidation. After reaction optimization using several commercial enzymes, both antipodes of various lactones were synthesized in most cases with >90% conversion and >80% enantiomeric excess under mild reaction conditions. In some cases alcohol formation was also observed (up to 40% conversion) as an undesired side reaction due to the presence of alcohol dehydrogenases in these preparations. Selected transformations were achieved on a 100 mg scale showing the possibilities of these oxidative biocatalysts as a new source of highly interesting compounds.

Full text of DOI:10.1016/j.tet.2015.12.071

Accepted Manuscript
Baeyer-Villiger monooxygenase-catalyzed desymmetrizations of cyclobutanones.
Application to the synthesis of valuable spirolactones
María Rodríguez-Mata, Iván Lavandera, Vicente Gotor-Fernández, Vicente Gotor,
Susana García-Cerrada, Javier Mendiola, Óscar de Frutos, Iván Collado
PII:
S0040-4020(15)30319-7
10.1016/j.tet.2015.12.071
TET 27397
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 October 2015
Revised Date: 21 December 2015
Accepted Date: 27 December 2015
Please cite this article as: Rodríguez-Mata M, Lavandera I, Gotor-Fernández V, Gotor V, García-
Cerrada S, Mendiola J, de Frutos Ó, Collado I, Baeyer-Villiger monooxygenase-catalyzed
desymmetrizations of cyclobutanones. Application to the synthesis of valuable spirolactones,
Tetrahedron (2016), doi: 10.1016/j.tet.2015.12.071.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Baeyer-Villiger monooxygenase-catalyzed
desymmetrizations of cyclobutanones.
Application to the synthesis of valuable
spirolactones
Leave this area blank for abstract info.
María Rodríguez-Mata,a Iván Lavandera,a Vicente Gotor-Fernández,a Vicente Gotor,a,* Susana García-
Cerrada,b,* Javier Mendiola,b Óscar de Frutos,b and Iván Colladob
a Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias,
University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain
b Centro de Investigación Lilly S.A., Avda. de la Industria, 30, Alcobendas-Madrid 28108, Spain.

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Baeyer-Villiger monooxygenase-catalyzed desymmetrizations of cyclobutanones.
Application to the synthesis of valuable spirolactones
María Rodríguez-Mata,a Iván Lavandera,a Vicente Gotor-Fernández,a Vicente Gotor,a,* Susana García-
Cerrada,b,* Javier Mendiola,b Óscar de Frutos,b and Iván Colladob
a Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, University of Oviedo, C/Julián Clavería 8, 33006
Oviedo, Spain. e-mail: vgs@uniovi.es; Tel.: +34 985 103448
b Centro de Investigación Lilly S.A., Avda. de la Industria, 30, Alcobendas-Madrid 28108, Spain. e-mail: garcia_susana_maria@lilly.com; Tel.: +34 91 6233614
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of γ-butyrolactone derivatives, including some spiranic ones, was obtained through
desymmetrization of the corresponding prochiral 3-substituted cyclobutanones via Baeyer-
Villiger monooxygenase (BVMO)-catalyzed oxidation. After reaction optimization using several
commercial enzymes, both antipodes of various lactones were synthesized in most cases with
>90% conversion and >80% enantiomeric excess under mild reaction conditions. In some cases
alcohol formation was also observed (up to 40% conversion) as an undesired side reaction due to
the presence of alcohol dehydrogenases in these preparations. Selected transformations were
achieved on a 100 mg scale showing the possibilities of these oxidative biocatalysts as a new
source of highly interesting compounds.
Available online
Keywords:
Asymmetric synthesis
Baeyer-Villiger oxidation
Lactones
2009 Elsevier Ltd. All rights reserved
.
Oxygenases
Spiro compounds
1. Introduction
and alcohol dehydrogenase-mediated stereoselective oxidations
of racemic diols or reductions of keto esters can be cited.11
Alternatively, the use of Baeyer-Villiger monooxygenases
(BVMOs) to achieve the BV oxidation of racemic or prochiral
cyclic ketones has also been studied to afford the enantiopure
products.12 This class of oxidoreductases is a promising family of
biocatalysts able to perform the oxidation of carbonylic
compounds as well as certain heteroatoms with excellent regio-
and/or enantioselectivities, by simply using oxygen as final
electron acceptor while releasing water as the only byproduct.13
Thus, although both kinetic and dynamic kinetic resolutions of
racemic ketones could also be performed, desymmetrization
reactions of prochiral substrates represent one of the most
appealing applications as they allow a theoretical yield of 100%
of enantiopure products. More specifically, the desymmetrization
of 3-substituted cyclobutanones via BVMO-catalyzed oxidation
has been successfully performed to gain access to the
corresponding enantioenriched γ-butyrolactones.14
Enantioenriched γ-butyrolactones are highly valuable
compounds displaying
a
broad biological profile with
applications as flavor and aroma constituents.1 In addition, they
also possess multiple uses as building blocks for polymers and
natural product synthesis. As examples, γ-valerolactone is
recognized as a potential intermediate for the production of
fuels,2 and 4-substituted analogues as precursors of analgesics,3
GABA receptor inhibitors,4 and lignans,5 a family of compounds
with broad range of activities including antiviral and
antineoplastic.
The selective synthesis of spiro compounds is also highly
appealing due to their interesting conformational features and
structural implications on biological systems.6 Hence, the spiro
functionality is known to be present in
a variety of
phytochemicals such alkaloids or terpenoids.7 In particular,
spirolactones are important motifs in medicinal and natural
product chemistry.8 The intrinsic rigidity imparted by the spiro
center, together with the latent lactone functionality, has been
postulated as the source of these beneficial activities.
As part of our ongoing interest related to the application of
biological catalysts to obtain valuable compounds, herein we
show the application of several commercially available BVMOs
in the desymmetrization of prochiral cyclobutanones (Scheme 1),
including also interesting spiro derivatives which, to the best of
our knowledge, have not been previously studied under similar
biocatalytic oxidative conditions. The optimization of the
reaction conditions and the scale-up of the biotransformation
have been considered as well.
Among the different strategies to synthesize enantiopure γ-
lactone derivatives, biocatalytic approaches have rapidly gained
ground due to the excellent selectivities demonstrated by
enzymes under mild reaction conditions.9 Among them,
hydrolase-catalyzed (dynamic) kinetic resolutions starting from
the corresponding racemic lactones or hydroxy ester precursors,10

2
Tetrahedron
Table 1. Activity of different BVMOs towards cyclobutanone
ACCEPTED MANUSCRIPT
(1a).a
Scheme 1. General approach to enantioenriched 4-substituted γ-
butyrolactones based on a BVMO-catalyzed desymmetrization
process.
Entry
Enzyme
c (%)b
88
1
2
3
4
5
6
BVMO-P1-C06
BVMO-P1-D08
BVMO-P3-A10
BVMO-P3-A12
BVMO-P3-C07
CDX-003
98
19
2. Results and discussion
47
Six different Baeyer-Villiger monooxygenases acquired from
Codexis were studied towards the oxidation of cyclohexanone, a
typical substrate for BVMOs. An initial enzyme activity
screening was performed using conditions previously optimized
in our research group,15 which imply a 10 mM substrate
concentration, use of co-solvent (DMSO, 1% v/v), NADPH (0.2
mM), glucose (20 mM), glucose dehydrogenase (GDH, 10 U)
and phosphate buffer 100 mM pH 9. The reactions were initially
carried out at 30 ºC and 250 rpm for 24 h in a 0.5 mL scale. In
order to improve the obtained results, some reaction parameters
were modified. Among them, addition of other prosthetic groups
(FAD or FMN) and variations in the nicotinamide (NADH
instead of NADPH), cofactor regeneration systems (glucose-6-
phosphate/glucose-6-phosphate dehydrogenase or sodium
formate/formate dehydrogenase instead of glucose/glucose
dehydrogenase), buffer pH (7, 8, 9 or 10) or temperature (20, 30
or 40 ºC) can be named, but none of them improved the first
reaction conditions. Remarkably, we noticed the formation of the
corresponding alcohol at higher temperatures, due to the
concomitant presence of alcohol dehydrogenase(s) in these
enzymatic preparations. Interestingly, when different co-solvents
were explored, better results were observed especially in the
presence of methanol. For this reason, together with its lower
boiling point compared to that of dimethylsulfoxide, which
allows an easier elimination from the reaction media, methanol
was the co-solvent selected from now on.
52
2
a Reaction conditions (0.5 mL): Cyclobutanone (10 mM) in phosphate buffer
100 mM pH 9 with MeOH (1% v/v), NADPH (0.2 mM), glucose (20 mM),
GDH (10 U) and BVMO (2 mg) for 24 h at 30 ºC and 250 rpm. b Determined
by GC.
These promising results inspired us to employ these enzymes
on useful compounds, as could be the synthesis of a broad family
of enantioenriched (spiro)lactones starting from the
corresponding cyclic ketones. To start with, cyclobutanone
bearing a phenyl group at position 3 (1b)16 was selected as model
prochiral substrate. Under previously optimized conditions, the
screening of the six BVMOs was thereby performed (Table 2).
To our delight, a total conversion into the corresponding lactone
(2b) was achieved in all cases, also reaching excellent
enantiomeric excess for BVMO-P1-D08 and CDX-003 to obtain
the (R)-enantiomer (entries 3 and 7). Interestingly, BVMO-P3-
C07 showed an opposite selectivity although at lower extent
(83% ee, entry 6). A more detailed study demonstrated that
quantitative conversions were obtained after just 4 h of reaction,
indicating the stability of the final product in the reaction media.
For comparison, other previous reports related to BVMO-
catalyzed oxidations on substrate 1b using purified or
overexpressed enzymes, have been added in Table 2 (entries 8-
19). As can be seen, earlier articles have employed lower
substrate concentrations affording lactone 2b with moderate to
high yields and enantiomeric excess. As can be seen,
cyclohexanone monooxygenases (CHMOs) coming from
different sources14e,f,g,h,j allowed synthesizing the (R)-enantiomer,
while other biocatalysts such as steroid monooxygenase (STMO,
entry 10),14j cyclopentanone monooxygenase (CPMO, entry
14),14g and 4-hydroxyacetophenone monooxygenase (HAPMO,
entry 19),14d afforded preferentially (S)-2b.
Once the reaction parameters had been optimized, our
attention was focused on cyclobutanone (1a) for being the
simplest substrate of the family. It turned to be highly reactive,
affording conversions up to 98% into the corresponding lactone
2a as can be seen in Table 1. Additional experiments were
performed in order to contemplate the possibility of carrying out
the biotransformations under stirring conditions. Hence, the
biooxidation of cyclobutanone was performed on a 30 mg scale
in the presence of BVMO-P1-D-08 using magnetic stirring,
observing also a complete conversion at either 250 or 900 rpm
(see conditions in Supporting Information).

3
Table 2. Oxidation of 3-phenylcyclobutanone (1b) using
ACCEPTED MANUSCRIPT
commercial BVMOs.a
Entry Enzyme
1b concentration (mM) 2b (%)b eeP (%)c
Figure 1. Evolution of lactone 2b formation using different
substrate concentration.
1
----d
10
0
----
2
BVMO-P1-C06 10
BVMO-P1-D08 10
BVMO-P3-A10 10
BVMO-P3-A12 10
BVMO-P3-C07 10
>99
>99
>99
>99
>99
>99
46
11 (R)
98 (R)
7 (S)
To discard the possibility of enzyme inhibition by the
substrate, its addition was studied in two portions every 24 h, but
results were even worse (61%). With the same aim, we also
performed the biotransformations at 10 mM for 24 h employing a
biphasic system using 10% v/v of heptane, observing that the
enzymes were slightly deactivated, reaching high conversions
(81-97%) and maintaining their enantioselectivity. As pH could
change in the medium due to higher concentrations of glucose,
and therefore formation of gluconic acid, we increased the
strength of the buffer up to 200 mM, but a decrease in the
enzyme activity was noticed (19% of 2b). Finally, we considered
the possibility of enzyme deactivation after a certain period of
time. This problem should be avoided by adding fresh biocatalyst
after 24 h, but a complete conversion could not be achieved
either with an initial substrate concentration of 20 mM (84%) or
the BVMO addition in two parts (74%).
3
4
5
53 (S)
83 (S)
97 (R)
59 (R)
91 (R)
76 (S)
83 (R)
93 (R)
98 (R)
37 (S)
62 (R)
96 (R)
87 (R)
50 (R)
92 (S)
6
7
CDX-003
CHMOe
CAMOf
STMOg
CHMOh
CHMOi
CHMOj
CPMOk
CHMOl
CHMOm
CHMOn
CHMOo
HAPMOp
10
5
8
9
5
71
10
11
12
13
14
15
16
17
18
19
5
72
n.f.
3
>90
45
3
73
3
66
In this sense, it was also studied the possibility of enzyme
recycling by means of reusing the aqueous phase after extraction
with heptane, which does not inhibit the biocatalyst at 10 mM
substrate concentration (data not shown), but the conversion in
the second cycle remained below 50%. With these results in
hand, the strategy was extended to a broad panel of 3-substituted
cyclobutanones (Figure 2).
n.f.
n.f.
3
>90
>90
54
3
63
3
12
a Reaction conditions (0.5 mL): 1b in phosphate buffer 100 mM pH 9 with
MeOH (1% v/v), NADPH (0.2 mM), glucose (20 mM), GDH (10 U) and
Some of them were commercially available (1f,g,i-k), while
other were synthesized through conventional chemical protocols
(1c-e,h,l-o, see Supporting Information). Table 3 summarizes the
best results found (see the Supporting Information for complete
data).
b
c
BVMO (2 mg) for 24 h at 30 ºC and 250 rpm. Determined by GC.
d
e
Determined by HPLC. Blank reaction with no enzyme. Cyclohexanone
monooxygenase from A. calcoaceticus; reference 14j.
monooxygenase from C. radicicola; reference 14j. Steroid monooxygenase
from R. rhodochrous; reference 14j. Cyclohexanone monooxygenase from
Xanthobacter sp.; reference 14h. Cyclohexanone monooxygenase from
Cyclohexanone monooxygenase from
Brevibacterium, type 1; reference 14g. Cyclopentanone monooxygenase
f
Cycloalkanone
g
h
Initially, the influence of different substituents in the phenyl
ring at position 3 was analyzed (1c-h). Both R- and S-selective
monooxygenases seemed to act better when they were placed at
the para position, leading to almost complete conversions and
high enantiomeric excess into 2e-h (from 86 to 97%, depending
on both substrate and biocatalyst). The meta-substituted
compound 1d offered worse results in terms of reactivity and the
orto-bromo substrate 1c led to lower conversion and
stereoselectivity values in comparison with the 3-(4’-
bromophenyl)cyclobutanone (1e). For instance, BVMO-P1-D08
and CDX-003 were able to achieve usually very high conversions
and ee into the corresponding (R)-lactones, while BVMO-P3-C07
afforded the best results for para-substituted derivatives in order
to gain access to the S-antipodes. We then compare our results
with previous reports using purified or overexpressed BVMOs,
finding that ketone 1f was formerly studied. Hence, results
obtained with P3-C07 and CDX-003 were comparable in terms
of activity and selectivity with biotransformations using various
CHMOs.14g,h
i
j
Brachymonas; reference 14g.
k
l
from Comamonas; reference 14g. Cyclohexanone monooxygenase from
m
Acinetobacter sp.; reference 14f.
Cyclohexanone monooxygenase from
n
Acinetobacter sp., mutant; reference 14f. Cyclohexanone monooxygenase
from Arthrobacter sp.; reference 14e. o Cyclohexanone monooxygenase from
Rhodococcus; reference 14e. p 4-Hydroxyacetophenone monooxygenase from
P. fluorescens; reference 14d. n.f.: Not found.
Prochiral 3-substituted cyclobutanones turned out to be quite
reactive, most likely related to the liberation of ring-strain upon
rearrangement and ring expansion. So, at this point, in order to
enhance the process efficiency and synthetic applicability, we
attempted to increase the substrate concentration up to 50 mM
(Figure 1). However, in spite of maintaining the
enantioselectivity, we came across to a dramatic decrease in the
enzyme activity as well as the formation of the corresponding
alcohol in appreciable amount (1-10%). Remarkably, a 73% of
lactone 2b was reached when the reaction with CDX-003 at 20
mM was allowed continuing for 48 h.

4
Tetrahedron
ACCEPTED MANUSCRIPT
Figure 2. 3-Substituted cyclobutanones subjected to oxidation with commercial BVMOs.
Table 3. Best results found for BVMO-catalyzed oxidations of prochiral 3-substituted cyclobutanones under optimum conditions.a
(S)-Lactone
BVMO
(R)-Lactone
Substrate
Ketone
Lactone (%)b
eeP (%)c
Ketone
BVMO
Lactone (%)b
eeP (%)c
concentration (mM)
concentration (mM)
1c
1d
1e
1f
10
P3-C07
P1-C06
P3-C07
P3-C07
CHMOd
CHMOf
P3-C07
P3-C07
P1-D08
P1-C06
CHMOe
CHMOi
MO1k
51
87
47
97
94
81
95
86
86
82
54
55
53
74
10
10
10
10
2.2
P1-D08
95 (4)
43
89
98
93
96
87
10
79
CDX-003
P1-D08
10
95 (5)
95 (2)
>90
63
99
10
CDX-003
CHMOe
93
n.f.
2.2
10
47
1g
1h
1ig
1j
94 (4)
99
10
10
10
10
2.4
n.f.
3
CDX-003
CDX-003
P3-C07
P1-D08
CPMOh
CHMOj
HAPMOl
MO2m
89 (11)
98
97
87
77
79
63
83
29
90
32
10
10
>99
>99
26
>99
>99
53
10
2.4
n.f.
n.f.
>90
98
>90
35
n.f.
10
95
1kg
1l
10
10
10
P1-D08
P3-A10
P3-C07
29 (41)n
>99n
98
78
96
94
P1-C06
46 (43)n
1n
1og
10
10
P1-D08
P3-A10
>99
>99
93
97
a Reaction conditions (0.5 mL): 1c-l,n,o in phosphate buffer 100 mM pH 9 with MeOH (1% v/v), NADPH (0.2 mM), glucose (20 mM), GDH (10 U) and BVMO
c
d
(2 mg) for 24 h at 30 ºC and 250 rpm. b Determined by GC analysis (alcohol formation in brackets). Determined by chiral HPLC analysis. Cyclohexanone
e
f
monooxygenase from Xanthobacter sp.; reference 14h. Cyclohexanone monooxygenase from Brevibacterium, type 1; reference 14g. Cyclohexanone
g
h
monooxygenase from Rhodococcus; reference 14g. Change in Cahn-Ingold-Prelog (CIP) priority. Cyclopentanone monooxygenase from Comamonas;
i
j
reference 14g. Cyclohexanone monooxygenase from Acinetobacter sp.; reference 14f. Cyclohexanone monooxygenase from Acinetobacter sp., mutant;
k
l
m
reference 14f. Monooxygenase from P. putida, type 1; reference 14a. 4-Hydroxyacetophenone monooxygenase from P. fluorescens; reference 14d.
Monooxygenase from P. putida, type 2; reference 14a. n Determined by 1H-RMN. n.f.: Not found.
Then, our interest turned into the study of the behavior of
these biocatalysts when a heteroatom such as oxygen or nitrogen,
was introduced at the substituent of the prochiral cyclobutanone.
On the one hand, oxygenated derivatives 2i and 2j could be
obtained with BVMO-P3-C07, P1-C06 or P1-D08 with complete
conversions but with moderate enantiomeric excess [from 54%
for (S)-2j to 82% for (S)-2i]. In this sense, previous reports
concerning BVMO-catalyzed oxidations on substrate 1j have
demonstrated that this substrate is usually converted with
moderate selectivities.14a,d,f,g On the other hand, nitrogenated
derivatives 1k and 1l showed several complications. Both
ketones and lactones revealed solubility problems and also
displayed very low GC signals. Hence, it was necessary to work
at a higher scale in order to study the transformations through 1H-
NMR technique. When testing substrate 1k, apart from a low
conversion degree and enantioselectivity, a huge amount of the
corresponding alcohol was detected, in some cases even higher
than the lactone of interest. However, when compound 1l was
treated under the same conditions, complete conversions were
achieved with all tested enzymes except CDX-003. Remarkably,
(S)-lactone 2l was formed in quantitative conversion and 96% ee
using BVMO-P3-A10.
Additionally, and having in mind its possible synthetic
applications, we tested the transformation over ethyl ester 1m,
but only traces of lactone 2m could be detected due to hydrolysis
of the substrate. We also synthesized the benzyl ester derivative
seeking a higher stability towards hydrolysis, but similar results
were found.
Next, we wondered if the set of Baeyer-Villiger
monooxygenases could transform compounds bearing quaternary
centers. With this aim, after synthesizing ketones 1n and 1o (see
Supporting Information), and subjecting them to enzymatic
oxidation, we came across with unexpected results. On the one
hand substrate 1n, with the 3-position quaternized, could be
transformed into both enantiomers of the corresponding lactone
either with BVMO-P3-C07 [(S)-2n, 92% conversion and 94% ee]
or BVMO-P1-D08 [(R)-2n, >99% conversion and 93% ee]. On

5
the other hand substrate 1o, with the quaternary center in an
adjacent position, in spite of being able to react in a more
enantioselective way (97% ee) towards the formation of the
lactone (R)-2o assisted by BVMO-P3-A10, none of the
biocatalysts in the set catalyzed the formation of the other
enantiomer (S)-2o.
When 1s and 1t were subjected to BVMO-catalyzed
ACCEPTED MANUSCRIPT
transformation in the same conditions, we realized that the effects
of the enzymes were completely different on each substrate,
regarding both conversion and selectivity (Table 4). With respect
to 1s, though in some cases conversions reached were acceptable,
the enantiomeric excess of the lactones remained low-to-high. It
can only be outlined the result with BVMO-P3-C07, which led to
the lactone in 64% conversion and 86% ee with a minimum
formation of the alcohol product. Regarding 1t it is worth to
remark that employing BVMO-P3-A10, an excellent conversion
into lactone 2t with high ee (87%) and only 2% of alcohol, was
achieved.
Once evaluated the efficiency of the enzyme set with this type
of substrates, we decided to explore another family with an
increased degree of complexity, the spiro compounds, with
potential applications in medicinal chemistry (Figure 3).17
Once benzylated ketones 1u and 1v had been synthesized, we
realized the complexity of its chemical Baeyer-Villiger oxidation,
as the product formed under the presence of the peracid was the
N-oxide derivative (see Supporting Information). However, this
reason confers additional value to our method employing
biocatalysis. Thus, when ketone 1u was subjected to enzymatic
Baeyer-Villiger oxidation in the same conditions (Table 4), no
product (neither starting material nor lactone) was detected by
GC with most biocatalysts, probably due to the formation of the
N-oxide, which remained in the aqueous phase during the
extraction protocol. Luckily, BVMO-P3-C07 allowed the access
to lactone 2u with a conversion degree up to 79%, although the
process proceeded with only moderate enantioselectivity (59%
ee). Surprisingly, in spite of the N-oxidative competing reaction,
results obtained with substrate 1v were better, detecting the
lactone formation in all cases (see complete data in the
Supporting Information). Remarkably, BVMO-P3-C07 made
possible the synthesis of lactone 2v after 24 h with 82%
conversion and 86% ee. Furthermore, BVMO-P1-D08 acted with
total stereopreference, even though the side reaction still
remained important.
Figure 3. Family of spiro compounds subjected to oxidation with
commercial BVMOs.
To begin with these unprecedented oxidations, we purchased
the N-Boc protected compounds 1p-r. To study if these spiro
derivatives could be appropriate for our BVMOs, achiral
compound 1p was chosen as model substrate. Thus, employing
the same conditions previously optimized, we found that only
BVMO-P3-C07 could afford 90% of the lactone 2p with a
minimum amount of alcohol formation (5%), while the other
monooxygenases remained below 80% with an alcohol formation
up to 37% (see complete data in Supporting Information). In
order to avoid the generation of such quantity of the reduction
product, we attempted the use of an organic solvent (10% v/v)
such as heptane, driving the reaction in a biphasic system, but the
results were quite similar. The presence of heptane neither
deterred or improved the activity of the enzyme, nor prevented
the alcohol formation (data not shown). Then, we carried out the
study with prochiral N-Boc spiro compounds 1q and 1r.
Surprisingly, several biocatalysts led to more than 85% lactone
formation, while only traces of the corresponding alcohol were
detected (see complete data in Supporting Information).
However, owing to the lack of a chromophore group, the
enantiomeric excess of the final products could not be measured.
For this reason, we decided to introduce other common
protecting groups for amines, such as benzyloxycarbonyl (Cbz,
1s and 1t) and benzyl (Bn, 1u and 1v), which are more suitable
for UV detection (see Supporting Information for detailed
conditions).
Once the general studies came to an end, and aiming at a
practical application, we thought about the possibility of scaling-
up the process together with a reduction in the enzyme loading.
In this sense, we handled 15-times higher scale reactions,
keeping proportions in a linear manner (Figure 4). Initially, under
identical conditions, we verified that the results could be
reproduced in Erlenmeyer flasks thermostated at 30 ºC and 250
rpm. Even when the enzyme loading was reduced by a factor of
two (2 mg/mL), complete conversions were achieved. However,
when only 1 mg of enzyme per mL of reaction media was
employed, it was not enough to transform all starting material
after 24 h but the selectivity remained unaltered.
Table 4. Best results found for BVMO-catalyzed oxidation of spiro compounds under optimum conditions.a
(+)-Lactone
(–)-Lactone
Lactone (%)b
66 (15)
98 (2)
Substrate
BVMO
P3-C07
P3-C07
P3-C07
P3-C07
Lactone (%)b
eeP (%)c
86
BVMO
P1-D08
P3-A10
eeP (%)c
68
1s
1t
64 (3)
36
76
87
1u
1v
79
59
n.f.
82
86
P1-D08
46d
>99
a Reaction conditions (0.5 mL): 1s-v (10 mM) in phosphate buffer 100 mM pH 9 with MeOH (1% v/v), NADPH (0.2 mM), glucose
(20 mM), GDH (10 U) and BVMO (2 mg) for 24 h at 30 ºC and 250 rpm. b Determined by GC analysis (alcohol formation in
brackets). c Determined by chiral HPLC analysis. d Low peak intensity, loss of product in the aqueous phase. n.f.: Not found.

6
Tetrahedron
P3-C07 and BVMO-P1-C06 usually offered the best results,
ACCEPTED MANUSCRIPT
while for getting the (R)-antipodes, BVMO-P1-D08 and CDX-
003 showed the best performances. These transformations show
similar, or even better, performances that previous reports with
these cyclobutanones in terms of activity and selectivity,14 the
possibility to carry out the biotransformations being practical at a
higher substrate concentration.
It is noteworthy that when the ketone substrate was not well
accepted by the BVMO, the corresponding alcohol was detected
at some extent, probably due to the undesired presence of alcohol
dehydrogenases in these preparations. In some cases biphasic
systems were tested to improve conversions, and although
enzyme inactivation was not observed at high extent, results
could not be improved. Finally, efforts were focused on the scale-
up of some of the biocatalytic oxidations, showing that reactions
on a 100 mg scale could be easily carried out reducing the
quantity of enzyme employed at smaller scale to obtain similar
conversion and ee values. This study demonstrates a new
sustainable alternative to produce interesting lactones which are
not easily accessible by other conventional chemical methods.
Figure 4. Optimization of the enzyme loading in 15x-scale
biotransformations of 1b with BVMO-CDX-003.
At this point, several selected Baeyer-Villiger oxidation
reactions were scaled up to 100 mg employing half as much
enzyme as in the screening protocols (Table 5 and Table S22),
demonstrating the applicability of the process as almost complete
conversions were reached for most of the studied substrates, and
the stereoselectivity of the processes remained very similar.
Furthermore, the enantioenriched pure lactones could be easily
recovered after a simple extraction protocol when all starting
material was consumed. In other cases, purification through
column chromatography was necessary.
4. Experimental part
4.1. General procedures
Chemicals and cyclobutanones 1a,f,g,i-k,p-r were purchased
from different commercial sources and were of the highest purity
available. Baeyer-Villiger monooxygenases and glucose
dehydrogenase (GDH-105) were acquired from Codexis. Diethyl
ether and tetrahydrofuran used in chemical transformations were
previously dried over sodium under inert atmosphere using
benzophenone as indicator. Phosphoryl chloride (POCl3) was
distilled under reduced pressure before its utilization. The rest of
reagents and solvents were employed without additional
purification.
Table 5. Scale-up in the lactonization reaction of selected
ketones.a
Substrate
BVMO
P3-C07
CDX-003
P3-C07
P1-D08
P3-C07
P3-A10
Lactone (%)a
81
Alcohol (%)b
eeP (%)c
82 (S)
93 (R)
94 (S)
92 (R)
87 (+)
86 (–)
1b
19
0
97 (90)
87
1n
0
>99 (89)
93 (77)
99 (93)
0
1s
1t
0
Chemical reactions were monitored by analytical TLC,
performed on silica gel 60 F254 plates, and visualized by UV or
revealed in a solution of potassium permanganate (1% KMnO4,
5% K2CO3 and 5% NaOH in water) or p-anisaldehyde (2.5% p-
anisaldehyde, 1% H2SO4 and 1% AcOH in ethanol). Flash
chromatography was performed using silica gel 60 (230-400
mesh). IR spectra were recorded on an infrared Fourier transform
spectrophotometer on NaCl pellets. NMR spectra were recorded
at 300.13 (1H), and 75.5 (13C) MHz. The chemical shifts (δ) are
reported in parts per million (ppm) relative to the solvent residual
signals and the coupling constants (J) in Hertz (Hz). ESI-TOF
mode was used to record high-resolution mass spectra (HRMS).
Gas chromatography (GC) analyses were performed on a
standard gas chromatograph equipped with a FID. HPLC
analyses were performed using a standard HPLC chromatograph
with UV detection. Optical rotations were measured using a
standard polarimeter with a sodium lamp (D) and are reported in
units of 10−1 deg cm2 g−1. Melting points were taken on samples
in open capillary tubes and are uncorrected.
0
a Reaction conditions: 1b,n,s,t (100 mg, 10 mM) in phosphate buffer 100 mM
pH 9 with MeOH (1% v/v), NADPH (0.2 mM), glucose (20 mM), GDH
(1250 U) and BVMO (2 mg/mL) for 24 h at 30 ºC and 250 rpm. b Determined
c
by GC analysis (isolated yields in brackets). Determined by chiral HPLC
analysis.
3. Conclusions
In summary, a series of commercially available BVMOs have
been identified as efficient catalysts for the synthesis of
enantioenriched lactones through desymmetrization of prochiral
3-substituted cyclobutanones. Thus, both enantiomers of several
γ-butyrolactones could be obtained in many cases with high or
excellent conversions and selectivities. These derivatives are
highly important as they have found many applications in
different fields as pharmaceuticals and fuel or polymer
precursors, among others. While enzymatic (dynamic) kinetic
resolutions of racemic lactones or hydroxy ester derivatives have
offered interesting results, the desymmetrization of a prochiral
precursor is an appealing process as it can give access to
quantitative yield of an enantiopure product in the best case.
4.2. General method for the preparation of lactones 2b-t
To an ice-cooled solution of the corresponding cyclobutanone
1b-t (0.25 mmol) in dichloromethane (1.0 mL), m-
chloroperbenzoic acid (MCPBA, 88 mg, 0.50 mmol) was added,
and the resulting mixture stirred for 20 h at room temperature.
Then, more dichloromethane was added (5 mL) and the solution
washed repeatedly with a saturated aqueous solution of sodium
bicarbonate (6×5 mL). The resulting organic phase was dried
over anhydrous sodium sulfate, filtered and the solvent
evaporated under reduced pressure. Purification by column
chromatography on silica gel (10-30% EtOAc/hexane) afforded
Herein, starting from various ketones and after optimization of
the enzymatic reaction conditions, it has been demonstrated that
several enzymatic preparations could be employed to efficiently
synthesize chiral γ-butyrolactones substituted at the 4-position,
comprising aromatic rings, (hetero)alkyl chains, and also spiranic
compounds. In fact, to the best of our knowledge, this is the first
report related to a biocatalytic process to get access to such
heterocyclic derivatives. To obtain the (S)-enantiomers, BVMO-

7
lactones 2b-t in 38-99% yield (83% for 2b; 72% for 2c; 99% for
2d; 85% for 2e; 86% for 2f; 88% for 2g; 44% for 2h; 96% for 2i;
77% for 2m; see below for 2j-l,n-t).18
(NaCl) ν 2977, 2880, 1786, 1692, 1470, 1406, 1366, 1323,
ACCEPTED MANUSCRIPT
1286, 1250, 1165, 1150, 1097, 1023, 1016 cm-1; 1H-NMR
t
(300.13 MHz, CDCl3) δ 1.44 (s, 9H, Bu), 2.76 (s, 2H, H-8), AB
system (δA= 3.94, δB= 3.98, 2JAB= 8.9 Hz, 4H, H-1 and H-3), 4.40
(s, 2H, H-5); 13C-NMR (75.5 MHz, CDCl3): δ 28.4 (3C, C-11),
37.3 (C-4), 39.3 (C-8), 59.0 (C-1 and C-3), 77.4 (C-5), 80.5 (C-
10), 156.0 (NCOO), 174.8 (COO); HRMS (ESI+, m/z): calcd for
C11H17NNaO4 [(M+H)+]: 250.1050, found: 250.1097.
4.2.1. 4-((Benzyloxy)methyl)dihydrofuran-2(3H)-one (2j).
58% yield; yellow oil; IR (NaCl) ν 3061, 2913, 2859, 1777,
1495, 1479, 1454, 1421, 1366, 1266, 1174, 1101, 1023, 736 cm-1;
1H-NMR (300.13 MHz, CDCl3) δ 2.37 (dd, 3J = 6.3 Hz, 2J = 17.7
3
2
Hz, 1H, CHH), 2.61 (dd, J = 8.9 Hz, J = 17.7 Hz, 1H, CHH),
2.77-2.91 (m, 1H, CH), 3.45-3.52 (m, 2H, CHCH2O), 4.18 (dd, 3J
= 5.5 Hz, 2J = 9.2 Hz, 1H, OCHH), 4.40 (dd, 3J = 7.5 Hz, 2J = 9.2
Hz, 1H, OCHH), 4.53 (s, 2H, OCH2Ph), 7.27-7.39 (m, 5H, Ph);
13C-NMR (75.5 MHz, CDCl3): δ 31.3 (CH2), 35.6 (CH), 70.5,
70.9 (OCH2, CHCH2O), 73.5 (OCH2Ph), 127.8 (2 CH Ar), 128.0
(CH Ar), 128.7 (2 CH Ar), 137.8 (Cipso), 176.9 (COO); HRMS
(ESI+, m/z): calcd for C12H15O3 [(M+H)+]: 207.1016, found:
207.1007.
4.2.7.
tert-Butyl
3-oxo-2-oxa-7-azaspiro[4.4]nonane-7-
carboxylate (2q): 71% yield; white solid; mp: 65-67 ºC; IR
(KBr) ν 1782, 1691, 1483, 1463, 1402, 1362, 1307, 1261, 1233,
1172, 1152, 1120, 1088, 1026, 1018, 854, 775 cm-1; H-NMR
1
t
(300.13 MHz, CDCl3) δ 1.45 (s, 9H, Bu), 1.93-1.98 (m, 2H, H-
9), 2.46-2.61 (m, 2H, H-4), 3.37-3.46 (m, 4H, H-6 and H-8), AB
system (δA= 4.16, δB= 4.20, 2JAB= 9.2 Hz, 2H, H-1); 13C-NMR
(75.5 MHz, CDCl3): δ 28.5 (3C, C-12), 34.5, 35.6 (C-9), 38.7 (C-
4), 44.5, 44.8 (C-8), 45.6, 46.5 (C-5), 54.2, 55.0 (C-6), 75.9 (C-
1), 80.1 (C-11), 154.4 (NCOO), 175.4 (COO) (presence of
rotamers); HRMS (ESI+, m/z): calcd for C12H19NNaO4
[(M+Na)+]: 264.1206, found: 264.1201.
4.2.2. Benzyl (5-oxotetrahydrofuran-3-yl)carbamate (2k).
43% yield; white solid; mp: 104-105 ºC; IR (NaCl) ν 3055, 2987,
1786, 1723, 1514, 1422, 1170, 1066, 1016 cm-1; 1H-NMR
3
2
(300.13 MHz, CDCl3) δ 2.43 (dd, J = 2.9 Hz, J = 17.8 Hz, 1H,
CHH), 2.74 (dd, 3J = 7.8 Hz, 2J = 17.8 Hz, 1H, CHH), 4.16-4.19
(m, 1H, OCHH), 4.38-4.49 (m, 2H, OCHH + CH), 5.07 (s, 2H,
4.2.8.
tert-Butyl
3-oxo-2-oxa-7-azaspiro[4.5]decane-7-
carboxylate (2r): 63% yield; colorless oil; IR (NaCl) ν 3059,
3026, 2958, 2921, 1785, 1602, 1496, 1445, 1381, 1080, 764 cm-1;
1H-NMR (300.13 MHz, CDCl3) δ 1.44 (s, 9H, tBu), 1.60-1.65 (m,
2H, H-9), 1.67-1.69 (m, 2H, H-10), AB system
(δA= 2.29, δB= 2.43, 2JAB= 17.6 Hz, 2H, H-4), 3.34-3.40 (m, 4H,
H-6 and H-8), 3.96 (d, J = 9.3 Hz, 1H, H-1), 4.11 (d, J = 9.3
Hz, 1H, H-1); 13C-NMR (75.5 MHz, CDCl3): δ 22.5 (C-9), 28.5
(3C, C-13), 33.8 (C-10), 38.6 (C-4), 40.4 (C-5), 43.5 (C-8), 51.2
(C-8), 75.4 (C-1), 80.4 (C-12), 154.7 (NCOO), 175.9 (COO);
HRMS (ESI+, m/z): calcd for C13H21NNaO4 [(M+Na)+]:
278.1363, found: 278.1360.
3
OCH2Ph), 5.84 (br d, J = 3.6 Hz, 1H, NH), 7.28-7.37 (m, 5H,
Ph); 13C-NMR (75.5 MHz, CDCl3): δ 34.8 (CH2), 48.0 (CH),
67.2 (OCH2Ph), 73.7 (OCH2), 128.2 (2 CH Ar), 128.4 (CH Ar),
128.6 (2 CH Ar), 136.0 (Cipso), 155.9 (NCOO), 175.6 (COO);
HRMS (ESI+, m/z): calcd for C12H14NO4 [(M+H)+]: 236.0917,
found: 236.0897.
2
2
4.2.3. Benzyl ((5-oxotetrahydrofuran-3-yl)methyl)carbamate
(2l). 38% yield; colorless oil; IR (NaCl) ν 3338, 3063, 3033,
2929, 1773, 1701, 1534, 1454, 1417, 1376, 1254, 1174, 1019,
1
738 cm-1; H-NMR (300.13 MHz, CDCl3) δ 2.25-2.33 (m, 1H,
CHH), 2.55-2.64 (m, 1H, CHH), 2.76-2.82 (m, 1H, CH), 3.24-
3.28 (m, 2H, CH2NH), 4.05-4.15 (m, 1H, OCHH), 4.33-4.39 (m,
1H, OCHH), 5.09 (s, 2H, OCH2Ph), 5.21 (br s, 1H, NH), 7.29-
7.42 (m, 5H, Ph); 13C-NMR (75.5 MHz, CDCl3): δ 32.1 (CH2),
36.0 (CH), 43.0 (CH2NH), 67.2 (OCH2Ph), 71.0 (OCH2), 128.3
(2 CH Ar), 128.4 (CH Ar), 128.7 (2 CH Ar), 136.2 (Cipso), 156.8
(NCOO), 176.6 (COO); HRMS (ESI+, m/z): calcd for
C13H15NNaO4 [(M+Na)+]: 272.0893, found: 272.0889.
4.2.9.
Benzyl
3-oxo-2-oxa-7-azaspiro[4.4]nonane-7-
carboxylate (2s): 58% yield; colorless oil; [α]D20= +11.6 (c 1,
CHCl3); IR (NaCl) ν 3054, 2984, 2889, 1782, 1701, 1420, 1361,
1267, 1170, 1109, 1029, 736 cm-1; 1H-NMR (300.13 MHz,
CDCl3) δ 1.97-2.04 (m, 2H, H-9), 2.41-2.63 (m, 2H, H-4), 3.41-
3.58 (m, 4H, H-6 and H-8), 4.11-4.23 (m, 2H, H-1), 5.13 (s, 2H,
H-11), 7.32-7.38 (m, 5H, Ph); 13C-NMR (75.5 MHz, CDCl3): δ
34.5, 35.6 (C-9), 38.6 (C-4), 44.7, 45.1 (C-8), 45.7, 46.6 (C-5),
54.7 (C-6), 67.3 (C-11), 75.8 (C-1), 128.2, 128.3, 128.7 (5C, Ph),
136.5 (Cipso), 154.8 (NCOO), 175.2 (COO) (presence of
rotamers); HRMS (ESI+, m/z): calcd for C15H17NNaO4
[(M+Na)+]: 298.1050, found: 298.1079.
4.2.4. 4-Methyl-4-phenyldihydrofuran-2(3H)-one (2n): 93%
yield; White solid; Mp: 49-51ºC; IR (nujol) 3024, 2969, 2930,
2904, 1773, 1601, 1497, 1305, 1173,1094, 1020, 767 cm−1;1H-
2
NMR (300.13 MHz, CDCl3) δ 1.54 (s, 3H, CH3), 2.68 (dd, J =
4
2
4
16.8, J = 0.4, 1H, CHH), 2.93 (dd, J = 16.8, J = 0.6, 1H,
CHH), 4.39-4.47 (m, 2H, OCH2), 7.18-7.23 (m, 2H, Ph), 7.27-
7.33 (m, 1H, Ph), 7.36-7.42 (m, 2H, Ph); 13C-NMR (75.5 MHz,
CDCl3) δ 28.1 (CH3), 42.1 (CH2), 44.2 (C), 78.5 (OCH2), 125.2
(2CH), 127.3 (CH), 129.1 (2CH), 144.4 (C), 176.2 (CO); HRMS
(ESI+, m/z): calcd for C11H12NaO2 [(M+Na)+]: 199.0730, found:
199.0781.
4.2.10.
Benzyl
3-oxo-2-oxa-7-azaspiro[4.5]decane-7-
carboxylate (2t): 51% yield; pale yellow oil; [α]D20= −32.0 (c 1,
CHCl3); IR (NaCl) ν 2936, 2860, 1777, 1698, 1470, 1433, 1365,
1349, 1255, 1238, 1217, 1176, 1101, 1028, 763, 737 cm-1; H-
1
NMR (300.13 MHz, CDCl3) δ 1.53-1.61 (m, 2H, H-9), 1.67-1.71
(m, 2H, H-10), 2.27-2.45 (m, 2H, H-4), 3.35-3.55 (m, 4H, H-6
and H-8), 4.08-4.15 (m, 2H, H-1), 5.13 (s, 2H, H-12), 7.28-7.40
(m, 5H, Ph); 13C-NMR (75.5 MHz, CDCl3): δ 22.4 (C-9), 33.7
(C-10), 38.6 (C-4), 40.2 (C-5), 44.0 (C-8), 51.2 (C-6), 67.6 (C-
12), 75.2 (C-1), 128.1 (2 CH Ar), 128.3 (CH Ar), 128.7 (2 CH
Ar), 136.5 (Cipso), 155.4 (NCOO), 175.7 (COO); HRMS (ESI+,
m/z): calcd for C16H20NO4 [(M+H)+]: 290.1387, found: 290.1394.
4.2.5. 4-(2-(Benzyloxy)propan-2-yl)dihydrofuran-2(3H)-one
(2o): 75% yield; pale yellow oil; IR (NaCl) ν 3055, 2986, 1775,
1
1422, 1180, 1026, 706 cm-1; H-NMR (300.13 MHz, CDCl3) δ
1.26, 1.27 (2 s, 6H, 2 CH3), 2.44-2.54 (m, 1H, CHH), 2.65-2.77
(m, 3H, CHH + CH), 4.32-4.43 (m, 2H, OCH2), 4.46 (s, 2H,
OCH2Ph), 7.25-7.37 (m, 5H, Ph); 13C-NMR (75.5 MHz, CDCl3):
δ 23.0, 23.3 (2 CH3), 29.7 (CH2), 46.1 (CH), 63.9 (OCH2Ph),
69.2 (OCH2), 74.2 (CMe2), 127.1 (2 CH Ar), 127.5 (CH Ar),
128.5 (2 CH Ar), 139.1 (Cipso), 177.5 (COO); HRMS (ESI+, m/z):
calcd for C14H19O3 [(M+H)+]: 235.1329, found: 235.1329.
4.3. Alternative method for the preparation of lactones 2u
and 2v
Owing to the availability of the free electron pair in the
nitrogen atoms in substrates 1u,v, the treatment with MCPBA led
to their N-oxidation. In order to circumvent this problem, a
partial solution for the synthesis of 2v was found in the use of
4.2.6.
tert-butyl
7-oxo-6-oxa-2-azaspiro[3.4]octane-2-
carboxylate (2p): 57% yield; white solid; mp: 154-155 ºC; IR

8
Tetrahedron
ACCEPTED MANUSCRIPT
Oxone®
(2KHSO5.KHSO4.K2SO4,
potassium
105, 1250 U), NADPH (0.2 mM) and the corresponding Baeyer-
peroxomonosulfate) as oxidizing agent. Unfortunately, 2u could
not be synthesized.
Villiger monooxygenase (2 mg/mL). The suspensions were
placed in Erlenmeyer flasks and shaken at 250 rpm in an orbital
shaker at 30 ºC for 24 h. 1 mL aliquots were taken for conversion
and enantiomeric excess analysis (same treatment previously
described). The reactions were stopped by removing the solid
protein through decantation and extracting the aqueous phase
with diethyl ether (3×50 µL). The organic fractions were
combined, dried over anhydrous sodium sulfate, filtered and
evaporated under reduced pressure affording pure lactones when
total conversion were achieved. When some starting product
remained, an additional purification by column chromatography
in silica gel was needed (50-100% EtOAc/hexane).
Thus, Oxone® (17 mg, 0.06 mmol) was added over a solution
of the ketone 1v (10 mg, 0.04 mmol) in a mixture of acetonitrile
and water (10:1, 0.1 M) and stirred at room temperature for 36 h.
Then, the solvents were evaporated and the product purified by
column chromatography on silica gel (2-5% MeOH/CH2Cl2),
affording lactone 2v in 33% yield.
7-Benzyl-2-oxa-7-azaspiro[4.5]decan-3-one (2v). Yellow oil;
IR (NaCl) ν 3054, 2931, 1774, 1453, 1421, 1377, 1352, 1180,
1158, 1075, 1019, 739 cm-1; H-NMR (300.13 MHz, CDCl3) δ
1
1.25-1.68 (m, 4H, H-9 and H-10), 2.17-2.53 (m, 6H, H-4, H-6
4.4.4. Procedure for the enzyme-catalyzed Baeyer-Villiger
oxidation of cyclobutanone (1a) using magnetic stirring
2
and H-8), 3.44-3.54 (m, 2H, H-11), 4.04 (d, J = 9.1 Hz, 1H, H-
1), 4.20-4.23 (m, 1H, H-1), 7.23-7.31 (m, 5H, Ph); 13C-NMR
(75.5 MHz, CDCl3): δ 22.9 (C-9), 33.8 (C-10), 39.9 (C-4), 40.4
(C-5), 53.5 (C-8), 61.4 (C-6), 63.0 (C-11), 76.7 (C-1), 127.4 (CH
Ar), 128.5 (2 CH Ar), 128.9 (2 CH Ar), 138.3 (Cipso), 176.9
(COO); HRMS (ESI+, m/z): calcd for C15H19NNaO2 [(M+Na)+]:
268.1308, found: 268.1326.
In order to check whether mechanical stirring can influence
the BVMO activity, cyclobutanone (1a, 30 mg, 10 m
dissolved in methanol (428 µL, 1% v/v) and KPi buffer (42.4
mL, 100 m , pH 9.0), containing glucose (154 mg, 20 m ).
Then, NADPH (7.1 mg, 0.2 m ), glucose dehydrogenase (GDH-
M) was
M
M
M
105, 850 U) and BVMO-P1-D08 (86 mg, 2 mg/mL) were
subsequently supplemented. The mixture was placed in a 250 mL
round-bottom flask and shaken at 30 ºC at either 250 or 900 rpm
with a magnetic stir bar for 24 h. After this time, complete
conversions were achieved as demonstrated by GC analysis.
4.4. Enzymatic procedures
4.4.1. General procedure for the BVMO-catalyzed oxidation
of prochiral cyclobutanones 1a-j, m-v
In a typical experiment carried out in 1.5 mL tubes (total
5. Acknowledgments
volume of 500 µL), the substrate 1a-j, m-v (10 m
dissolved in methanol (5 µL, 1% v/v) and KPi buffer (100 m
pH 9.0, 482 µL), containing glucose (20 ), glucose
dehydrogenase (GDH-105, 10 U, from stock solution of 1.275
U/µL), NADPH (0.2 m , from a 20 m stock solution) and the
M) was
M
,
This work was supported by Eli Lilly and Company through the
Lilly Research Award Program (LRAP).
m
M
M
M
Supplementary data
corresponding Baeyer-Villiger monooxygenase (2 mg). The
mixture was shaken at 250 rpm at 30 ºC for 24 h. The reaction
was stopped by extracting with diethyl ether (2×400 µL) and
centrifuged at 13000 rpm in order to separate both phases and
pellet the suspended protein. The organic phases were combined,
dried over anhydrous sodium sulfate and analyzed by GC in
order to determine the conversion values. Then, the solvent in
GC samples was evaporated with a continuous flow of nitrogen,
the residue re-dissolved in a mixture of hexane:ethanol 90:10 and
the new sample filtered and analyzed by HPLC, leading to the
measurement of the enantiomeric excess of the lactones. Control
experiments in the absence of enzyme were performed for all
substrates, not observing any reaction product after similar
periods of time (Tables S1-S19).
General
information,
synthesis
of
cyclobutanones,
characterization data, extensive enzymatic screening results, and
analytical data are provided.
References
(1) (a) M. Seitz, O. Reiser, Curr. Opin. Chem. Biol. 2005, 9, 285-
292. (b) R. R. A. Kitson, A. Millemaggi, R. J. K. Taylor,
Angew. Chem. Int. Ed. 2009, 48, 9426-9451. (c) S. Gil, M.
Parra, P. Rodríguez, J. Segura, Mini-Rev. Org. Chem. 2009,
6, 345-358.
(2) (a) W. R. H. Wright, R. Palkovits, ChemSusChem 2012, 5,
1657-1667. (b) P. Azadi, R. Carrasquillo-Flores, Y. J.
Pagán-Torres, E. I. Gürbüz, R. Farnood, J. A. Dumesic,
Green Chem. 2012, 14, 1573-1576. (c) D. M. Alonso, S. G.
Wettstein, J. A. Dumesic, Green Chem. 2013, 15, 584-595.
4.4.2. Alternative procedure for the BVMO-catalyzed
oxidation of prochiral cyclobutanones 1k and 1l
(3) M. Node, T. Kajimoto, M. Ozeki, Heterocycles 2010, 81,
Owing to the problems that both ketones 1k,l and their
corresponding lactones 2k,l showed when analyzed by GC, as
well as their poor solubility in diethyl ether or ethyl acetate, their
enzymatic transformations were carried out in a bigger scale (×3,
2 mL tubes) but maintaining identical reaction conditions. After
24 h, products were extracted with deuterated chloroform (2×400
µL). The organic phases were dried over anhydrous sodium
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1
sulfate, filtered and conversion values were determined by H-
NMR experiments. Similarly, samples were evaporated and the
residue re-dissolved in a mixture of hexane:ethanol 90:10 for
HPLC analyses (see Tables S20-S21).
4.4.3. Procedure for the BVMO-catalyzed oxidation at 100
mg scale of cyclobutanontes 1b, 1n, 1s and 1t
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M
) were
M
, pH
M

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