Multienzymatic stereoselective reduction of tetrasubstituted cyclic enones to halohydrins with three contiguous stereogenic centers

Article abstract of DOI:10.1021/acscatal.0c04097

The asymmetric hydrogenation of conjugated tetrasubstituted alkenes with transition-metal catalysts is a challenging reaction, especially for substrates bearing a halide substituent. We describe a two-step multienzymatic reduction of a series of α-halo β-alkyl tetrasubstituted cyclic enones, affording halohydrins with three contiguous stereogenic centers, in good yield and with a high stereoselectivity. The reduction is catalyzed by a stereospecific ene-reductase (OYE2-3 or NemA) and a highly enantioselective alcohol dehydrogenase (ADH). The use of two enantiodivergent ADHs allows the control of the diastereoselectivity. The absolute stereochemical configurations of the products have been determined from the analysis of single-crystal structures (Flack's parameter). The enantiomeric excess (ee) has been determined by derivatization of the products with (R) Mosher's acid. Lastly, we extended our methodology also to a nonhalogenated substrate: the α-methyl ketoisophorone was reduced by two distinct enantiodivergent ene-reductases (flavin mononucleotide- and F₄₂₀-dependent), affording each enantiomer of the saturated ketone with ee > 98%.

Full text of DOI:10.1021/acscatal.0c04097

pubs.acs.org/acscatalysis
Research Article
Multienzymatic Stereoselective Reduction of Tetrasubstituted Cyclic
Enones to Halohydrins with Three Contiguous Stereogenic Centers
Silvia Venturi, Elisabetta Brenna, Danilo Colombo, Marco W. Fraaije, Francesco G. Gatti,* Piero Macchi,
Daniela Monti, Milos Trajkovic, and Emilio Zamboni
Cite This: ACS Catal. 2020, 10, 13050−13057
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The asymmetric hydrogenation of conjugated
tetrasubstituted alkenes with transition-metal catalysts is a
challenging reaction, especially for substrates bearing a halide
substituent. We describe a two-step multienzymatic reduction of a
series of α-halo β-alkyl tetrasubstituted cyclic enones, affording
halohydrins with three contiguous stereogenic centers, in good
yield and with a high stereoselectivity. The reduction is catalyzed
by a stereospecific ene-reductase (OYE2-3 or NemA) and a highly
enantioselective alcohol dehydrogenase (ADH). The use of two
enantiodivergent ADHs allows the control of the diastereoselec-
tivity. The absolute stereochemical configurations of the products
have been determined from the analysis of single-crystal structures
(Flack’s parameter). The enantiomeric excess (ee) has been
determined by derivatization of the products with (R) Mosher’s acid. Lastly, we extended our methodology also to a nonhalogenated
substrate: the α-methyl ketoisophorone was reduced by two distinct enantiodivergent ene-reductases (flavin mononucleotide- and
F420-dependent), affording each enantiomer of the saturated ketone with ee > 98%.
KEYWORDS: ene-reductases, alcohol dehydrogenases, tetrasubstituted enones, chlorohydrins, biocatalysis
by an Ir complex with a chiral phosphine ligand in the presence
INTRODUCTION
■
of t-BuONa.1 Even though selectivity and yields were high, the
substrate scope was limited by R1 = CO2Et. However, if the R1
substituent is a halogen, it is possible to obtain the halohydrins
III. Especially interesting would be the stereoisomers of III
with the halogen substituent trans to the OH group because
they can be easily converted to α-alkyl epoxides IV, which are
synthons that are more versatile than II. But, to the best of our
knowledge, such a retrosynthesis has never been realized, most
likely because the α-haloenones can undergo dehalogenation at
typical reaction conditions of the transition-metal-catalyzed
hydrogenations.2
The one-pot stereoselective reduction of CO and CC
double bonds of tetrasubstituted enones I is an appealing
transformation since it gives secondary alcohols with three
contiguous stereogenic centers, that is, II (Figure 1a).
Surprisingly, its implementation is very recent. It was
accomplished through an asymmetric hydrogenation catalyzed
In addition, it is well-known that the stereoselective
reduction of tetrasubstituted CC double bonds (either
isolated or conjugated), catalyzed by transition-metal com-
plexes with chiral ligands, is one of the most challenging
reactions of organic chemistry.3 Good conversions are
achievable only by using high H2 pressures and/or high
Received: September 18, 2020
Revised: October 12, 2020
Figure 1. (a) Retrosynthesis of alcohols II, chlorohydrins III, and
epoxides IV. (b) Complete reduction of a α-bromo trisubstituted
enone catalyzed by enzymes.
https://dx.doi.org/10.1021/acscatal.0c04097
© XXXX American Chemical Society
ACS Catal. 2020, 10, 13050−13057
13050

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
temperatures.3b,c Therefore, at harsher experimental condi-
tions, the dehalogenation side reaction should increase.
Completely different is the scenario in the field of
biocatalytic reductions.4 Indeed, enzymes with ene-reductase
(ER) activity5 and enzymes with alcohol dehydrogenase
(ADH) activity6 are able to reduce α-halogenated enones
and ketones at mild reaction conditions, and the dehalogena-
tion is usually not observed. Especially the ERs are becoming
valuable catalysts in organic synthesis, not only for their use in
stereospecific reductions of the CC double bond conjugated
with an electron-withdrawing group (EWGs) but also for their
use in enantioselective carbocyclizations7a,b and coupling with
photocatalysts in multistep processes.7c,d
The combination of ER and ADH activities has proven to be
effective in the reduction of many prochiral enones and enals.
Chiral alcohols with up to two stereogenic centers are usually
obtained in good yield and with high stereoselectivity.8
Recently, we have shown that the multienzymatic reduction
of an α-bromo trisubstituted enone proceeded smoothly,
affording the corresponding bromohydrin9 with high optical
purity (Figure 1b).
On the other hand, it should be noted that although in the
last 2 decades many ERs have been discovered and tested, their
substrate scope is restricted to the reduction of trisubstituted
conjugated alkenes. Indeed, so far just two tetrasubstituted
conjugated alkenes have been reduced.8e,10 This is likely due to
the fact that such substrates are believed to be too sterically
hindered11 to be accepted by most of the ERs.
Nonetheless, in this study we show how ene-reductases can
be used as efficient catalysts for the reduction of sterically
demanding substrates such as the tretrasubstituted conjugated
alkenes 1a−1l (Figure 2). In addition to the canonical flavin
mononucleotide (FMN) cofactor dependent ERs (such as the
OYEs belonging to the Old Yellow Enzyme family), also the
enantiodivergent deazaflavin F420-dependent ERs (FDRs) were
investigated.12 Lastly, we show that ERs coupled with ADHs
reduce α-halo tetrasubstituted cyclic enones, affording the
corresponding halohydrins in good yield and with high
selectivity.
Figure 2. Substrate scope of the CC double-bond reduction of
tetrasubstituted conjugated alkenes.
elimination occurred. The latter intermediates being well
accepted by most ERs, they were promptly reduced (Scheme
1a). Moreover, we do not exclude that the formation of
dehalogenated products9,14 could be due to alternative and
competitive chemical paths based on ER-catalyzed radical
reactions.7b
However, it is a fact that the reduction of the chloro
analogues, that is, 1b and 1l, proceed smoothly, and the side
product formation was almost negligible (for 1l < 5%, by GC-
MS). Thus, in view of coupling the two reductive steps on a
semipreparative scale (4.0 mmol), we concluded that the
chloro derivatives are better substrates than their bromo
analogues because the intermediate ketones are more stable at
our reaction conditions.
Concerning the reduction of the 2-chloro-3-alkyl cyclohex-2-
enones, 1b−1d, the conversion with OYE2 decreased
significantly by increasing the alkyl chain length: from 99%
for R2 = Me (1b) to 26% for R2 = n-Pr (1d). In this regard,
Stewart and co-worker observed a similar trend in the OYE1
(similar ER isolated from Saccharomyces carlsbergensis)-
catalyzed reduction of the alkyl cyclohex-2-enone analogues:
from a quantitative conversion for the 3-methylcyclohex-2-
enone, the yield dropped down to 18% for the substrate with
the n-propyl substituent; all products had (S)-configuration).15
Next, we tested the racemic mixture of enone 1e bearing a
methyl group at the C(5) carbon to see whether the ERs can
reduce preferentially one enantiomer over the other, allowing a
kinetic resolution.14b,16 With NemA the conversion was too
high (70%), whereas OYE2 and OYE3 gave the product in a
yield too low (18% and 7%, respectively). However, in both
cases the diastereomeric excess was insignificant because a
RESULTS AND DISCUSSION
■
At first, we tested the CC double-bond reduction of
substrates 1a−l with a set of different recombinant ERs. OYE2
and OYE3 from Saccharomyces cerevisiae and NemA from
Escherichia coli were used because they are known for their
good performances (selectivity and conversion) with sterically
hindered substrates.8e,10 For regeneration of the reduced
nicotinamide adenine dinucleotide phosphate (NADPH)
cofactor, we used glucose dehydrogenase (GDH) and glucose
as the sacrificial cosubstrate.13 In the Supporting Information
the results of the screening can be found (Table S1 and for the
best results see Table 1).
Most of the substrates were reduced with good conversions
and with a discrete diastereoselectivity (up to de > 99% by GC-
MS), except for the menadione and the cyclopentenone chloro
derivatives, 1h and 1i, respectively, which were transformed
into the corresponding saturated ketones in a very low yield.
For the α-bromo substituted substrates, that is, 1a and 1k, we
obtained mainly the debrominated saturated products instead
of 2a and 2k (Scheme 1a). Especially, for the reduction of 1k,
we detected in the reaction mixture also the presence of the
maleimide intermediate (see Supporting Information). Hence,
probably, as soon as 2a and 2k are formed, a spontaneous HBr
13051
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Table 1. Reduction of α-Halo Tetrasubstituted Conjugated
Table 1. continued
Alkenes
g
mg), and GDH (400 U), 24 h at 30 °C. We observed the
h
i
dehalogenated side product. Product in trace, not isolated. Complex
mixture of diastereoisomers.
Scheme 1. (a) Proposed Reduction/Elimination/Reduction
Reaction Sequence in the ER Catalyzed Reductions of α-
Bromo Substituted Substrates; (b) Example of Cascade
Reduction (Method A); (c) Example of Sequential
Reduction (Method B)
complex distribution of the four possible diastereoisomers was
detected (by GC-MS).
The NemA-catalyzed reduction of the chloro derivative of
isophorone, that is, 1f, gave 2f in a good yield of 72%, whereas
both yeast OYEs failed. Interestingly, the doubly activated
chloro-ketoisophorone 1g was instead reduced with OYE2
affording the ketone 2g together with the levodione side
product (8:2), in an overall conversion of 76% and with a good
de of 92%.
For the enzymatic reduction of the carbonyl group to be
coupled to the ER-catalyzed step, we selected two
commercially available ADHs, EVO270 and EVO440, having
stereoselectivity pro (R) and pro (S), respectively. The
cofactor regeneration (NADPH) for the ER + ADH
multienzymatic process was the same as that applied to the
ER-catalyzed biotransformations. Conversions and chemo-
a
b
Best ER, 24 h at 30 °C, see Table S1. Conversion by GC-MS, not
c
d
isolated. By 1H NMR and/or GC-MS. Reaction conditions for
Method A: substrate (4.0 mmol), i-PrOH as cosolvent (1−2% in
volume), ER (OYE2 or NemA, 10−12 mg), GDH (400 U), EVO270
(30−40 mg), NADP+ (20 mg), glucose (6.0 equiv), and pH 7 KPi
buffer (50 mM, 100 mL), 24 h at 30 °C. Isolated yield after column
chromatography purification. Reaction conditions for Method B: (1)
substrate (4.0 mmol), i-PrOH as cosolvent (1−2% in volume), ER
(OYE2 or NemA, 10−12 mg), GDH (400 U), NADP+ (15 mg),
glucose (4.0 equiv), and pH 7 KPi buffer (50 mM, 100 mL), 12 h at
30 °C, and (2) EVO440 (30−40 mg), glucose (2.0 equiv), NADP+ (5
e
f
13052
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Figure 3. Reduction mechanism and possible binding modes of tetrasubstituted cyclic enones in the ER catalytic site: (a) “flipped” binding for
OYE-type ERs and (b) “normal” binding for FDRs.
selectivity data of the screening are available in the Supporting
Information (Table S3). Interestingly, we found that EVO270
was sufficiently chemoselective to be added to the reaction
mixture together with the ER (Method A: [ER + ADH]
cascade reduction, Scheme 1b) because the carbonyl enone
reduction, affording the allylic alcohol byproduct, was in most
cases negligible. In contrast, EVO440 could be added only
after that most of the starting material was consumed, usually
no earlier than after 12 h (Method B: [(1) ER; (2) ADH]
sequential reduction, Scheme 1c).
The outcomes on the relative stereochemistry gave further
evidence that the OYEs-catalyzed reductions proceed by
formal addition of H2 to the CC double bond with anti
stereospecificity.20 The hydride, from the reduced flavin
cofactor (FMNH2), attacks the enone’s β carbon, forming
the carboanion. The latter is then protonated by the acidic
hydroxyl group of a tyrosine residue, in the transoid position
with respect to the FMNH2, resulting in a two stepwise 1,4-
addition (Figure 3a). This reaction mechanism has been
elucidated for the reduction of conjugated tri- and
disubstituted alkenes by a combination of computations21
and not trivial experiments such as deuterium labeling, rate
constant measures, and enzymatic mutations.22 Instead, using
cyclic tetrasubstituted enones is considerably easier to
After having identified the best ER−ADH combinations, we
repeated the biotransformations on a higher scale (4.0 mmol).
The yield, after column chromatography purification, and the
1
diastereomeric excess (by H NMR or GC-MS) are shown in
Table 1. We found that the simultaneous addition of ER
(OYE2 or NemA) with EVO270 (Method A) shows a cascade
effect because the conversion of the starting material improved
substantially with respect to that achieved using the ER
standalone (Table S1). In this regard, remarkable was the
cascade reduction of 1a with OYE2 and EVO270 (Method A,
Scheme 1b), in which the CO of the reactive intermediate 2a,
as soon as it was formed, was reduced with a reaction rate
sufficiently high to minimize the formation of the side product.
Indeed, the cis,trans-3a bromohydrin was isolated at a yield of
49% and with an excellent de (>99% by 1H NMR). In contrast,
the reduction of the same substrate with Method B gave
mainly the 3-methylcyclohexan-1-ol and the trans,trans-3a
isomer was present just in traces (Table 1).
1
determinate the stereochemical course (by H NMR coupling
constants). Indeed, we could easily demonstrate that NemA
(just 36% sequence identity with OYE2) has the same
stereospecificity of the two OYEs (Figure 3a).
The absolute stereochemical configurations of trans,trans
chlorohydrins 3b and 3c were estimated from the Flack
parameter obtained from the single-crystal X-ray diffraction
model (Figure 4a−c); for more details see the Supporting
Information.23 Unfortunately, for most of the cis,trans series,
no crystals could be obtained. The only exception is 3g, to
which the (4S,5R,6S) configuration was assigned again by X-
ray analysis (Figure 4c).
The reduction of the 4-ketoisophorone to give the (R)-
levodione followed by the regio- and enantioselective
reduction of the less hindered carbonyl group has been the
object of intense research because the (4R,6R)-actinol,
produced by a chemo-enzymatic process,17 is a key precursor
for the synthesis of several carotenoids of industrial
relevance.18 In this regard, it is noteworthy that the 5-chloro
derivatives of actinol, that is, the chlorohydrins trans,trans-3g
and cis,trans-3g, were obtained with our methodology in good
yield and with high optical purity. These findings open new
routes to oxygenated carotenoids such as crustaxanthin or to
apocarotenoids such as the 3,4-dihydroionone.19
The NMR characterization of both diastereoisomers of
alcohols 3 allowed us to assign their relative stereochemical
configurations (trans,trans or cis,trans) simply by measuring the
J coupling constants of the CH−X proton signal (see
Supporting Information).
Figure 4. Molecular geometries determined from single-crystal X-ray
diffraction: (a) (1R,2R,3S)-3b; (b) (1R,2R,3S)-3c; (c) (4S,5R,6S)-3g;
(d) (5R,6R)-2j. All crystals were grown in pentane.
13053
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
a
In a biocatalytic transformation, the absolute stereochemical
configuration of the product depends mainly on how the
substrate is oriented into the active site of the enzyme.
Historically, two possible geometries of binding for OYE-type
ERs have been defined: “flipped” or “normal” binding modes
(Figure 3). Hence, by taking into account the absolute
configurations of chlorohydrins (trans,trans)-3b,3c and (cis,-
trans)-3g, determined from the X-ray crystal structures, we
concluded that the OYE2 and NemA arrange the substrates 1b,
1c, and 1g by a “flipped” binding mode, and that EVO270 and
EVO440 have actually enantioselectivity pro (S) and pro (R),
respectively.
Scheme 2. Stereodivergent Reduction of 2j
Although the high diastereomeric excesses achieved can be
explained only if both reductive steps are highly stereoselective,
we still had some doubts about the optical purity of the
products, especially for the cis,trans diastereoisomers, of which
the absolute configuration was indirectly assigned. Since we
were not able to synthesize the racemic mixtures, necessary for
the setup of the chiral GC or HPLC analysis,24 we opted for
the derivatization of the cis,trans chlorohydrins 3a−3f with the
(S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride af-
fording Moscher’s esters ((R)-MTPA-8a−8f, see Supporting
Information).25
1
Both H and 19F NMR spectra of 8a−8f esters gave further
support to our initial assumption about the high stereo-
selectivity of the multienzymatic process since the diastereo-
meric excess was always high (de > 98%, see Supporting
Information).
Lastly, we show how the bioreductions can be very effective
also with non α-halogenated tetrasusbstituted enones. Thus,
according to our ongoing research program on stereoselective
synthesis of chiral flavors and fragrances, and their olfactory
evaluation,26 we focused our interest on the reduction of the
tobacco flavoring agent α-methylketoisophorone27 1j to give 2j
(Scheme 2).
a
Reaction conditions: (i) H2, Pd/C cat., MeOH, 0 °C to rt, 99%
yield; (ii) DMP, CH2Cl2, 0 °C to rt, yield 99%; (iii) same of (i), −10
°C to rt, 70% yield; (iv) FDR-Mha, FGD, F420H2 cofactor, glucose-6-
phosphate in tris·HCl buffer at 24 °C; (v) NemA, GDH, glucose,
NADP+ cofactor in KPi buffer at 30 °C, 84% yield; (vi) Method A:
the same of (iv) together with EVO270, 90% yield; (vii) Method B:
the same of (iv) followed by the addition of EVO440, 82% yield.
The direct synthesis of cis-2j isomer by syn stereospecific
hydrogenation failed (Pd/C in MeOH at 0 °C)28 since a
nonregioselective reduction of the carbonyl groups occurred as
well (a very complex diastereomeric mixture of the
regioisomeric hydroxyketones 3j and 5 was detected by GC-
MS, Scheme 2). Thus, in the attempt to improve the
chemoselectivity toward the formation of cis-2j, we carried
out the reaction at a lower temperature (−10 °C) and for a
shorter reaction time. However, at these new reaction
conditions, we obtained the allylic alcohol 6 with a quite
substrates I into the FDR enzyme active site (Figure 3b).
Three different FDRs were tested and found to convert
quantitatively 1j with a high selectivity (Table S3). FDR from
Mycobacterium hassicum (FDR-Mha) was used for further
experiments. Reduction of 1j with FDR-Mha produced, when
compared with NemA, the other enantiomer of the trans
diketone, that is, (5S,6S)-2j (αD = +173.5° in CH2Cl2), in a
good yield of 80% and with a high stereoselectivity (de > 99%
and ee > 98% by chiral GC).
1
good regioselectivity (6/7 = 93:7, by H NMR). Between the
Unlike the OYEs, FDRs require the reduced F420H2 cofactor,
which was conveniently regenerated during the biotransforma-
tion by means of a F420-dependent glucose-6-phosphate
dehydrogenase (FGD, from R. jostii), and using an excess of
glucose-6-phosphate as sacrificial cosubstrate.31
Then the optically pure (−)-diketone was submitted to the
carbonyl reduction with each of the two enantiodivergent
ADHs. Differently from the Pd/C-catalyzed hydrogenation,
EVO270 reduced the less hindered carbonyl group, affording
the hydroxyketone (4S,5R,6S)-3j, whereas the reduction with
EVO440 was not regioselective at all since we isolated the diol
(1S,4R,5R,6R)-4. However, for both reductions, yield and
stereoselectivity were more than satisfactory (Scheme 2).
two carbonyl groups, the one that was mostly reactive was, to
our surprise, the one more sterically hindered.29
Finally, cis-2j was obtained by oxidation of the mixture of
hydroxyketones 3j and 5. Although the conversion with the
Dess-Martin periodinane was quantitative, the diastereomeric
excess was quite disappointing (trans/cis = 76:24 by 1H
NMR).
Less problematic was the enzymatic reduction of 1j with
NemA, which afforded trans-2j (X-ray structure shown in
Figure 4d, αD = −168.0° in CH2Cl2) in a high yield of 84% and
with a very good diastereo- and enantioselectivity (de > 98% by
GC-MS of the crude material and ee = 99% by chiral GC after
column chromatography, see Supporting Information).
Recently, deazaflavin cofactor (F420)-dependent ene-reduc-
tases (FDRs) were shown to exhibit opposite stereo-
selectivity12 to that of most FMN-dependent ERs,30 including
NemA. This is explained by a “normal” binding mode of
CONCLUSION
■
In summary, we have shown that ERs can reduce efficiently the
CC double bond of sterically demanding substrates such as
13054
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
the α-chloro tetrasubstitued enones, disubstitued maleimides,
and the α-methyl ketoisophorone, usually with good yield and
high stereoselectivity. In this case, the use of an enzymatic
approach is not just a “green” alternative to the transition-
metal-catalyzed hydrogenations, but it remedies a typical
weakness of the latter in reducing sterically hindered
substrates, especially those bearing halide substituents, which
easily undergo dehalogenation.
In addition, the combination of OYE-type ERs with ADHs,
in a cascade process or in a sequential reduction, allowed the
one-pot stereoselective preparation of α-chlorohydrins/alcohol
with three contiguous stereogenic centers, and a diol with four
stereogenic centers, in good yield and with high stereo-
selectivity.
Lastly, this study also shows the benefit of using the newly
discovered F420-dependent ERs (FDRs), which complement
nicely the available OYE-type ERs by their opposite
enantioselectivity. The access to two distinct enantiodivergent
ERs allowed the synthesis of both enantiomers of a chiral
flavor.
Emilio Zamboni − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.0c04097
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Giuseppe Gatti is kindly acknowledged for his assistance in the
TOPSPIN-DAISY simulations. S.V. and D.C. thank POR-
Lombardia, VIPCAT project (Value Added Innovative
Protocols for Catalytic Transformations-ID 228775), for the
financial support.
REFERENCES
ASSOCIATED CONTENT
■
■
(1) Liu, Y.-T.; Chen, J.-Q.; Li, L.-P.; Shao, X.-Y.; Xie, J.-H.; Zhou,
Q.-L. Asymmetric Hydrogenation of Tetrasubstituted Cyclic Enones
to Chiral Cycloalkanols with Three Contiguous Stereocenters. Org.
Lett. 2017, 19, 3231−3234.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c04097.
(2) For an example of dehalogenation used in natural product
synthesis: Li, J.; Yang, P.; Yao, M.; Deng, Y.; Li, A. Total Synthesis of
Rubriflordilactone A. J. Am. Chem. Soc. 2014, 136, 16477−16480.
(3) (a) Hartwig, J. F. Organotransition Metal Chemistry: From
Bonding to Catalysis; University Science Books: Sausalito, CA, 2009;
Preparation and characterization of starting materials
and products of enzymatic reactions, extensive enzy-
matic screenings, optimization of individual and multi-
enzymatic protocols; copies of GC chiral analyses and
1H and 13C NMR spectra (PDF)
̀
́
pp 575−657. (b) Biosca, M.; Pamies, O.; Dieguez, M. Ir-Biaryl
phosphite-oxazoline catalyst libraries: a breakthrough in the
asymmetric hydrogenation of challenging olefins. Catal. Sci. Technol.
2020, 10, 613−624. (c) Dobbs, D. A.; Vanhessche, K. P. M.; Brazi, E.;
Rautenstrauch, V.; Lenoir, J.-Y.; Genet, J.-P.; Wiles, J.; Bergens, S. H.
Industrial Synthesis of (+)-cis-Methyl Dihydrojasmonate by Enantio-
selective Catalytic Hydrogenation; Identification of the Precatalyst
[Ru((−)-Me-DuPHOS)(H)(η6-1,3,5-cyclooctatriene)](BF4). Angew.
MS and single crystal difraction data (CIF)
AUTHOR INFORMATION
■
Corresponding Author
Francesco G. Gatti − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy; orcid.org/0000-0003-0837-4616;
Email: francesco.gatti@polimi.it
̀
Chem., Int. Ed. 2000, 39, 1992−1995. (d) Verendel, J. J.; Pamies, O.;
́
Dieguez, M.; Andersson, P. G. Asymmetric Hydrogenation of Olefins
Using Chiral Crabtree-type Catalysts: Scope and Limitations. Chem.
Rev. 2014, 114, 2130−2169.
Authors
(4) (a) Faber, K. Biotransformation in Organic Chemistry; Springer:
New York, 2011. (b) Hollmann, F.; Arends, I. W. C. E.; Holtmann, D.
Enzymatic Reductions for the Chemists. Green Chem. 2011, 13,
2285−2314.
Silvia Venturi − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy
(5) (a) Gatti, F. G.; Parmeggiani, F.; Sacchetti, A. Synthetic Methods
for Biological Active Molecules: Exploring the Potential of Bioreductions;
Brenna, E., Ed.;Wiley-VCH: Weinheim, 2013; pp 49−84. (b) Too-
good, H. S.; Scrutton, N. S. Discovery, Characterization, Engineering,
and Applications of Ene-Reductases for Industrial Biocatalysis. ACS
Catal. 2018, 8, 3532−3549. (c) Winkler, C. K.; Faber, K.; Hall, M.
Biocatalytic reduction of activated C = C-bonds and beyond:
emerging trends. Curr. Opin. Chem. Biol. 2018, 43, 97−105.
(6) Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Recent
Developments in Asymmetric Reduction of Ketones with Biocatalysts.
Tetrahedron: Asymmetry 2003, 14, 2659−2681.
(7) (a) Heckenbichler, K.; Schweiger, A.; Brandner, L. A.; Binter, A.;
Toplak, M.; Macheroux, P.; Gruber, K.; Breinbauer, R. Asymmetric
Reductive Carbocyclization Using Engineered Ene Reductases. Angew.
Chem., Int. Ed. 2018, 57, 7240−7244. (b) Black, M. J.; Biegasiewicz,
K. F.; Meichan, A. J.; Oblinsky, D. G.; Kudisch, B.; Scholes, G. D.;
Hyster, T. K. Asymmetric redox-neutral radical cyclization catalysed
by flavin-dependent ‘ene’-reductases. Nat. Chem. 2020, 12, 71−75.
(c) Litman, Z. C.; Wang, Y.; Zhao, H.; Hartwig, J. F. Cooperative
asymmetric reactions combining photocatalysis and enzymatic
Elisabetta Brenna − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy
Danilo Colombo − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy
Marco W. Fraaije − Molecular Enzymology Group, University of
Groningen, Groningen 9747 AG, The Netherlands;
orcid.org/0000-0001-6346-5014
Piero Macchi − Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ”G. Natta”, Politecnico di Milano, Milano
20133, Italy; orcid.org/0000-0001-6292-9825
Daniela Monti − Istituto di Scienze e Tecnologie Chimiche
“Giulio Natta” (SCITEC), C.N.R., Milano 20131, Italy;
orcid.org/0000-0002-3399-7973
Milos Trajkovic − Molecular Enzymology Group, University of
Groningen, Groningen 9747 AG, The Netherlands;
orcid.org/0000-0001-7264-1630
13055
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
catalysis. Nature 2018, 560, 355−359. (d) Nakano, Y.; Black, M. J.;
Meichan, A. J.; Sandoval, B. A.; Chung, M. M.; Biegasiewicz, F.; Zhu,
T.; Hyster, T. K. Photoenzymatic Hydrogenation of Heteroaromatic
Olefins Using ‘Ene’-Reductases with Photoredox Catalysts. Angew.
Chem., Int. Ed. 2020, 59, 10484−10488.
(16) (a) Fronza, G.; Fuganti, C.; Grasselli, P.; Barbeni, M.
Stereochemistry of the Yeast-Mediated Conversion of Delta 2-
Decenolide into Delta Decanolide. Tetrahedron Lett. 1992, 33, 6375−
6378. (b) Issa, I. S.; Toogood, H. S.; Johannissen, L. O.; Raftery, J.;
Scrutton, N. S.; Gardiner, J. M. C3 and C6Modification-Specific OYE
Biotransformations of Synthetic Carvones and Sequential BVMO
Chemoenzymatic Synthesis of Chiral Caprolactones. Chem. - Eur. J.
2019, 25, 2983−2988.
(17) (a) Fukuoka, M.; Hiraga, K.; Skihara, T. Process for producing
Levodione. U.S. Patent 6428991 B1, August 6, 2002. (b) Crameri, Y.;
Puentener, K.; Scalone, M. Process for the Preparation of trans-(R,R)-
actinol. U.S. Patent 6187961 B1, February 13, 2002.
(18) Ernst, H. Recent Advances in Industrial Carotenoid Synthesis.
Pure Appl. Chem. 2002, 74, 2213−2226.
(19) Serra, S.; De Simeis, D. Fungi-Mediated Biotransformation of
the Isomeric Forms of the Apocarotenoids Ionone, Damascone and
Theaspirane. Molecules 2019, 24, 19.
(20) (a) Kohli, R. M.; Massey, V. The Oxidative Half-reaction of
Old Yellow Enzyme The Role of Tyrosine 196. J. Biol. Chem. 1998,
273, 32763−32770. (b) Brenna, E.; Fronza, G.; Fuganti, C.; Gatti, F.
G. Stereochemical Analysis of the Enzymatic Reduction of the Double
Bond of r-and—Substituted Nitrostyrenes and –Ethoxycinnamalde-
hyde through Deuterium Labelling Experiments. Eur. J. Org. Chem.
2010, 2010, 5077−5084.
(21) Lonsdale, R.; Reetz, M. T. Reduction of α,β-Unsaturated
Ketones by Old Yellow Enzymes: Mechanistic Insights from
Quantum Mechanics/Molecular Mechanics Calculations. J. Am.
Chem. Soc. 2015, 137, 14733−14742.
(22) Kohli, R. M.; Massey, V. The Oxidative Half-reaction of Old
Yellow Enzyme The Role of Tyrosine 196. J. Biol. Chem. 1998, 273,
32763−32770.
(8) (a) Korpak, M.; Pietruszka, J. Chemoenzymatic One-Pot
Synthesis of γ-Butyrolactones. Adv. Synth. Catal. 2011, 353, 1420−
1424. (b) Brenna, E.; Monti, D.; Gatti, F. G.; Parmeggiani, F.;
Sacchetti, A. Productivity Enhancement of C = C Bioreductions by
Coupling the in situ Substrate Feeding Product Removal Technology
with Isolated Enzyme. Chem. Commun. 2012, 48, 79−81. (c) Brenna,
E.; Monti, D.; Gatti, F. G.; Parmeggiani, F.; Sacchetti, A. Cascade
Coupling of Ene Reductases with Alcohol Dehydrogenases:
Enantioselective Reduction of Prochiral Unsaturated Aldehydes.
ChemCatChem 2012, 4, 653−659. (d) Brenna, E.; Malpezzi, L.;
Monti, D.; Gatti, F. G.; Parmeggiani, F.; Sacchetti, A. Synthesis of
Robalzotan, Ebalzotan, and Rotigotine Precursors via the Stereo-
selective Multienzymatic Cascade Reduction of α,β-Unsaturated
Aldehydes. J. Org. Chem. 2013, 78, 4811−4822. (e) Classen, T.;
̈
Korpak, M.; Scholzel, M.; Pietruszka, J. Stereoselective Enzyme
Cascades: An Efficient Synthesis of Chiral γ-Butyrolactones. ACS
Catal. 2014, 4, 1321−1331. (f) Brenna, E.; Monti, D.; Gatti, F. G.;
Parmeggiani, F.; Sacchetti, A.; Valoti, J. Substrate-E approach to the
Stereoselective Chemo-multienzymatic Cascade Synthesis of Nic-
otiana tabacum Lactone. J. Mol. Catal. B: Enzym. 2015, 114, 77−85.
(g) Brenna, E.; Crotti, M.; Monti, D.; Gatti, F. G.; Parmeggiani, F.;
Pugliese, A.; Santangelo, S. Multi-enzyme Cascade Synthesis of the
most Odorous Stereoisomers of the Commercial Odorant Muguesia®.
J. Mol. Catal. B: Enzym. 2015, 114, 37−41. (h) Brenna, E.; Crotti, M.;
De Pieri, M.; Gatti, F. G.; Manenti, G.; Monti, D. Chemo-Enzymatic
Oxidative Rearrangement of Tertiary Allylic Alcohols: Synthetic
Application and Integration into a Cascade Process. Adv. Synth. Catal.
2018, 360, 3677−3686.
(23) (a) Flack, H. D.; Bernardinelli, G. The Use of X-ray
Crystallography to Determine Absolute Configuration. Chirality
2008, 20, 681−690. (b) Parsons, S.; Flack, H. D.; Wagner, T. Use
of Intensity Quotients and Differences in Absolute Structure
Refinement. Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater.
2013, B69, 249−259.
(24) A sample of 1b was submitted to the most common
hydrogenation protocols, but in no case had it been possible to
isolate 3b; see Supporting Information.
(9) Brenna, E.; Crotti, M.; Gatti, F. G.; Marinoni, L.; Monti, D.;
Quaiato, S. Exploitation of a Multienzymatic Stereoselective Cascade
Process in the Synthesis of 2-Methyl-3-Substituted Tetrahydrofuran
Precursors. J. Org. Chem. 2017, 82, 2114−2122.
(10) (a) Fronza, G.; Fuganti, C.; Serra, S. Stereochemical Course of
Baker’s Yeast Mediated Reduction of the Tri- and Tetrasubstituted
Double Bonds of Substituted Cinnamaldehydes. Eur. J. Org. Chem.
2009, 2009, 6160−6171. (b) Brenna, E.; Gatti, F. G.; Monti, D.;
Parmeggiani, F.; Serra, S. Stereochemical Outcome of the
Biocatalysed Reduction of Activated Tetrasubstituted Olefins by
Old Yellow Enzymes 1−3. Adv. Synth. Catal. 2012, 354, 105−112.
(11) Dobrijevic, D.; Benhamou, L.; Aliev, A. E.; Mendez-Sanchez,
D.; Dawson, N.; Baud, D.; Tappertzhofen, N.; Moody, T. S.; Orengo,
C. A.; Hailes, H. C.; Ward, J. M. Metagenomic ene-reductases for the
bioreduction of sterically challenging enones. RSC Adv. 2019, 9,
36608−36614.
(25) Dale, J. A.; Mosher, H. S. Nuclear Magnetic Resonance
Enantiomer Regents. Configurational Correlations via Nuclear
Magnetic Resonance Chemical Shifts of Diastereomeric Mandelate,
O-Methylmandelate, and. alpha.-Methoxy-.alpha.-Trifluoromethyl-
phenylacetate (MTPA) Esters. J. Am. Chem. Soc. 1973, 95, 512−519.
(26) Brenna, E.; Fuganti, C.; Gatti, F. G.; Serra, S. Biocatalytic
Methods for the Synthesis of Enantioenriched Odor Active
Compounds. Chem. Rev. 2011, 111, 4036−4072.
(27) Heirich, C.; Elte, S., Preparation of alicyclic diketones, C.H.
U.S. Patent 3923898, December 2, 1975.
(12) Mathew, S.; Trajkovic, M.; Kumar, H.; Nguyen, Q.-T.; Fraaije,
M. W. Enantio- and Regioselective Ene-Reductions using F420H2-
Dependent Enzymes. Chem. Commun. 2018, 54, 11208−11211.
(13) Bechtold, M.; Brenna, E.; Femmer, C.; Gatti, F. G.; Panke, S.;
Parmeggiani, F.; Sacchetti, A. Biotechnological Development of a
Practical Synthesis of Ethyl (S)-2-Ethoxy-3-(p-methoxyphenyl)-
propanoate (EEHP): Over 100-Fold Productivity Increase from
Yeast Whole Cells to Recombinant Isolated Enzymes. Org. Process Res.
Dev. 2012, 16, 269−276.
(28) Weyerstahl, P.; Licha, K. Structure-Odor Correlation, XXIV.
Synthesis and Olfactory Properties of Damascone and Damascenone
Analogs. Liebigs. Ann. 1996, 1996, 809−814.
(29) Hennig, M.; Puntener, K.; Scalone, M. Synthesis of (R)- and
(S)-4-Hydroxyisophorone by Ruthenium-Catalyzed Asymmetric
Transfer Hydrogenation of Ketoisophorone. Tetrahedron: Asymmetry
2000, 11, 1849−1858.
(30) To knowledge of the authors, just another example of ER
stereocomplementary to the OYE’s family has been reported:
Lygidakis, A.; Karuppiah, V.; Hoeven, R.; Ni Cheallaigh, A.; Leys,
D.; Gardiner, J. M.; Toogood, H. S.; Scrutton, N. S. Pinpointing a
Mechanistic Switch Between Ketoreduction and “Ene” Reduction in
Short-Chain Dehydrogenases/Reductases. Angew. Chem., Int. Ed.
2016, 55, 9596−9600.
(31) (a) Nguyen, Q.-T.; Trinco, G.; Binda, C.; Mattevi, A.; Fraaije,
M. W. Discovery and Characterization of an F420-Dependent Glucose-
6-Phosphate Dehydrogenase (Rh-FGD1) from Rhodococcus jostii
RHA1. Appl. Microbiol. Biotechnol. 2017, 101, 2831−2842. (b) Drenth,
J.; Trajkovic, M.; Fraaije, M. W. Chemoenzymatic Synthesis of an
́
(14) (a) Tasnadi, G.; Winkler, C. K.; Clay, D.; Hall, M.; Faber, K.
Reductive Dehalogenation of d-Haloacrylic Ester Derivatives
Mediated by Ene-Reductases. Catal. Sci. Technol. 2012, 2, 1548−
1552. (b) Turrini, N. G.; Hall, M.; Faber, K. Enzymatic Synthesis of
Optically Active Lactones via Asymmetric Bioreduction using Ene-
Reductases from the Old Yellow Enzyme Family. Adv. Synth. Catal.
2015, 357, 1861−1871.
(15) Swiderska, M. A.; Stewart, J. D. Stereoselective Enone
Reductions by Saccharomyces Carlsbergensis Old Yellow Enzyme. J.
Mol. Catal. B: Enzym. 2006, 42, 52−54.
13056
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Unnatural Deazaflavin Cofactor That Can Fuel F420-Dependent
Enzymes. ACS Catal. 2019, 9, 6435−6443.
13057
https://dx.doi.org/10.1021/acscatal.0c04097
ACS Catal. 2020, 10, 13050−13057