Biocatalytic Approaches to the Enantiomers of Wieland–Miescher Ketone and its Derivatives

Article abstract of DOI:10.1002/ejoc.202100174

Biocatalytic approaches have been investigated in order to isolate the enantiomers of Wieland–Miescher ketone (1) and of its alcoholic derivatives (cis-2 and trans-3). Specifically, two enzymes from our in-house metagenomic collection of oxidoreductases, IS2-SDR and Dm7α-HSDH, catalyzed the kinetic resolution of the starting racemic ketone 1 or its complete conversion into two diastereomeric products, respectively. Moreover, the kinetic resolution of the racemic cis-alcohol (2) was very efficiently obtained (E?2.000) by lipase PS catalyzed acetylation in dry acetone. All the products were isolated with ee≥95 %. Simple chemical elaborations of some of them allowed to isolate the missing enantiomers.

Full text of DOI:10.1002/ejoc.202100174

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Biocatalytic Approaches to the Enantiomers of Wieland–
Miescher Ketone and its Derivatives
Susanna Bertuletti,[a, b] Ikram Bayout,[a, c] Ivan Bassanini,[a] Erica E. Ferrandi,[a]
Nassima Bouzemi,[c] Daniela Monti,[a] and Sergio Riva*[a]
This manuscript is dedicated to Prof. Franco Cozzi on the occasion of his 70th birthday.
Biocatalytic approaches have been investigated in order to
isolate the enantiomers of Wieland–Miescher ketone (1) and of
its alcoholic derivatives (cis-2 and trans-3). Specifically, two
enzymes from our in-house metagenomic collection of oxidor-
eductases, IS2-SDR and Dm7α-HSDH, catalyzed the kinetic
resolution of the starting racemic ketone 1 or its complete
conversion into two diastereomeric products, respectively.
Moreover, the kinetic resolution of the racemic cis-alcohol (2)
was very efficiently obtained (Effi2.000) by lipase PS catalyzed
acetylation in dry acetone. All the products were isolated with
ee�95%. Simple chemical elaborations of some of them
allowed to isolate the missing enantiomers.
Introduction
en-2(3H)-one) which exist as two couples of cis- and trans-
enantiomers (compounds (4aS, 5S)-2a, (4aR, 5R)-2b and (4aS,
5R)-3a, (4aR, 5S)-3b in Scheme 1). These chiral derivatives,
when obtained as enantiomerically enriched species, are known
to be important building blocks for the synthesis of natural,
bioactive products or compounds of pharmaceutical
interest.[13–17] The presence of an enone group, in fact, easily
allows to further manipulate their molecular skeleton and to
transform them into structurally complex compounds.
From a chemical point of view, the reduction of racemic 1
with common hydride-based reducing agents (e.g., NaBH4), due
to the steric hindrance determined by the adjacent C8a methyl
group, usually gives access only to a racemic mixture of the cis-
alcohols (2a and 2b).[18] The two trans-alcohols 3a and 3b
appear to be accessible only from the cis-isomers via low-
efficient protocols of stereoinversion.[19]
Alternative “bio”-based strategies have been investigated to
build a convenient entry to the enantiomers of 1 and/or to
isolate compounds 2a–b and 3a–b as stereo-enriched species.
On this respect, it deserves to be mentioned the outstanding
results obtained by Lerner, Danishefsky and coworkers, who
developed a catalytic antibody able to catalyze the enantiose-
lective Robinson annulation.[20] The reaction was performed at a
110 mg scale and the S-enantiomer 1a could be isolated in
94% yield and 96% optical purity.
More recently a biocatalyzed approach to the synthesis of 1,
via the Robinson annulation, has been proposed, exploiting the
catalytic promiscuity[21] of a commercial lipase from porcine
pancreas.[22] However, a more detailed investigation of the
protein components of that crude preparation demonstrated
that non-lipase contaminant enzymes, and specifically an α-
amylase, were apparently responsible for the observed trans-
formation. In any case, even under optimized conditions, the
results both in terms of yield and enantiomeric excess of 1 were
significantly lower when compared with a control organo-
catalyzed (with L-proline) reaction.[23]
The so-called Wieland-Miescher ketone (1, Scheme 1) is an
important intermediate widely used in the total synthesis of
complex natural products, predominantly with a terpenoid
structure.[1–3] It can be easily prepared in its racemic form from
2-methylcyclohexane-1,3-dione and methyl vinyl ketone via the
Robinson annulation, a tandem process proposed in 1935
including a Michael addition and an intramolecular aldol
condensation.[4]
The enantiomerically enriched form of
1 was firstly
described in 1971, when two different groups of industrial
chemists independently proposed an asymmetric version of the
Robinson annulation catalyzed by proline.[5–7] Since then, the
enantioselective synthesis of one of the enantiomers of 1 ((8aS)-
1a and (8aR)-1b) has become a model target to evaluate the
performances of new chiral organocatalyst,[8–10] as it has been
recently carefully reviewed.[11,12]
The unselective reduction of the C1 carbonyl moiety of 1
should theoretically afford a complex mixture of stereoisomeric
alcohols (5-hydroxy-4a-methyl-4,4a,5,6,7,8-hexahydronaphthal-
[a] S. Bertuletti, I. Bayout, Dr. I. Bassanini, Dr. E. E. Ferrandi, Dr. D. Monti,
Dr. S. Riva
Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”
Consiglio Nazionale delle Ricerche
Via Mario Bianco 9, Milano, 20131, Italy
E-mail: sergio.riva@scitec.cnr.it
http://www.scitec.cnr.it/personale/bianco-ita/sergio-riva
[b] S. Bertuletti
Pharmaceutical Sciences Department
University of Milan
Via Mangiagalli 25, Milano, 20133, Italy
[c] I. Bayout, Dr. N. Bouzemi
Ecocompatible Asymmetric Catalysis Laboratory (LCAE). Badji Mokhtar
Annaba-University,
BP12, Annaba 23000, Algeria
Part of the “Franco Cozzi’s 70th Birthday” Special Collection.Supporting in-
formation for this article is available on the WWW under https://doi.org/
10.1002/ejoc.202100174
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Scheme 1. (Chemo)enzymatic manipulation of racemic Wieland–Miescher ketone (�1) and of its alcoholic derivatives (�2).
A partial kinetic resolution of racemic 1 was obtained by
submitting this compound to the action of a cyclohexanone
monooxygenase from Acinetobacter calcoaceticus, but the
preferred enantiomer was irreversibly oxidized to the corre-
sponding lactone.[18] Nevertheless, a biocatalyzed synthetic
strategy, exploiting the intrinsic enantioselectivity of enzymes,
might offer a simple and efficient solution to the separation of
the two enantiomers of the easily obtainable racemic 1. To our
surprise, quite few reports can be found in the literature on this
topic so far, most of them describing biotransformations with
microbial whole cells. Stereoselective reduction of racemic 1
was firstly described by Prelog using whole cells of Curvularia
falcata.[24–27] Later on, Sugai described the results obtained with
yeast strains of Torulaspora delbrueckii and Candida
melibiosica,[28,29] and Kodama with baker’s yeast.[30] More recently
Janeczko described the reductive performances of Didymos-
phaeria igniaria and Coryneum betulinium strains.[31]
enriched species. On this respect, Shimizu et al. were able to
obtain the stereoisomeric alcohol derivatives of 1 by conduct-
ing an enzymatic asymmetric hydrolysis (exploiting a library of
hydrolases of different origins)[33] of the acetylated racemates of
alcohols 2 and 3 obtained from 1 according to the above
mentioned protocols.[18,19] Best results were obtained on the cis
racemate 2 using a β-amilase from wheat. On the other hand,
to our surprise again, we could not find examples related to the
use of lipases in organic solvents[34] to catalyze the kinetic
resolution, by esterification, of racemic 2 and 3.
In the following we report the results that we have obtained
investigating the performances of new oxidoreductases and a
well-known lipase, together with a critical comparison with the
literature data.
Results and Discussion
Apparently, no examples of the stereoselective reduction of
1 catalyzed by isolated enzymes have been reported to date.
Therefore, we considered racemic 1 an interesting substrate to
evaluate the performance of our in-house collection of dehy-
drogenases, including new metagenomic and extremophilic
enzymes.[32]
Moreover, complementary biocatalyzed strategies, i.e., the
well-known kinetic resolution of racemic alcohols catalyzed by
hydrolases, deserve to be explored to build alternative entries
to Wieland-Miescher ketone derivatives as enantiomerically
One of the most interesting properties of enzymes, from a
synthetic point of view, is their substrate promiscuity, that is
their ability to transform compounds with chemical structures
significantly different from those of their natural substrates, still
maintaining high levels of stereoselectivity. On this respect, we
recently reported on the performances of a collection of new
hydroxysteroid dehydrogenases (HSDHs), identified either by
genome mining or by metagenomics approaches, in the
reduction of a panel of substrates including, in addition to the
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“natural” hydroxysteroids, α-ketoesters and selected ketones
partially resembling the structural features of steroids.[32]
The results obtained in the reduction of racemic 1 via the
preliminary screening of our panel of dehydrogenases is
summarized in Table 1. Among the 15 (mostly new) enzymes
tested, including 13 HSDHs (7α-HSDHs, 7β-HSDHs, and 12α-
HSDHs) and two short-chain dehydrogenases/reductases (SDRs),
only four accepted 1 as a substrate, two of them with
significantly higher conversion values. The reactions with these
two enzymes, IS2-SDR (a SDR identified from a metagenome
obtained from Icelandic hot spring sediments) and Dm7α-HSDH
(a 7α-hydroxysteroid dehydrogenase identified in the genome
of the extremophilic bacterium Deinococcus marmoris strain
PAMC 26562, a radiation-resistant and psychro- and draught-
tolerant bacterium isolated from an Antarctic rock sample),
were analyzed in more details as their chiral phase HPLC
chromatograms showed two different reaction outcomes. As
reported in Figure 1, while with Dm7α-HSDH a complete
conversion of 1 into two products was obtained, IS2-SDR
catalyzed a kinetic resolution of the starting racemic ketone.
The reactions were scaled-up in order to isolate and
characterize the products, using the ancillary enzyme glucose
dehydrogenase (GDH) to regenerate in situ the needed reduced
cofactor (NADH for Dm7α-HSDH and NADPH for IS2-SDR). As
summarized in the left upper part of Scheme 1, the kinetic
resolution of 1 in the reduction catalyzed by IS2-SDR allowed
Table 1. Screening of dehydrogenases for the reduction of racemic 1.[a]
Figure 1. HPLC chromatograms of [a] racemic 1; [b] Dm7α-HSDH stereo-
selective biocatalyzed reduction of racemic 1; [c] IS2-SDR biocatalyzed
kinetic resolution of racemic 1.
Entry Enzyme[b]
Source[c]
Cofactor Conversion[d]
1
2
Ca7α-HSDH
Clostridium absonum
NADP
(H)
NAD(H)
<1%
Dm7α-
HSDH
Ec7α-HSDH
Deinococcus marmoris
>99%
22
the isolation of the enantiomer 1b with a 96.5% e.e ([α]D
–
3
4
Escherichia coli
NAD(H)
6%
<1%
°
88.7 , c 1.0 in CHCl3), in 32% yield. This result compares well,
both in terms of selectivity and reaction outcome, with those
obtained by Sugai with whole cells of T. delbrueckii,[28] by
Kodama with baker’s yeast,[30] and by Janeczko with D.
igniaria.[31]
Later on this reaction was investigated in more details by
submitting the two separate enantiomers to the catalytic action
of IS2-SDR. It was confirmed that the enantiomer 1a (8aS
configuration) was the preferred one, the relative ratio of the
reaction rates of its reduction vs that of 1b being approximately
3 to 1. Moreover, the IS2-SDR-catalyzed reduction of 1a was
stereoselective and the cis alcoholic enantiomer 2a was the
only product. On the contrary, the slow reduction of 1b gave
predominantly the trans alcohol 3b, accompanied by the cis
diastereoisomer 2b (relative ratio 3b vs 2b:4:1).
Hh7α-HSDH Halomonas halodenitrifi- NAD(H)
cans
5
6
7
8
9
NGI7α-
HSDH
Bsp7β-
HSDH
Norwegian metage-
nome
Bacillus sp.
NAD(H)
NAD(H)
<1%
<1%
<1%
<1%
<1%
Ca7β-HSDH
Clostridium absonum
Collinsella aerofaciens
NADP
(H)
NADP
(H)
Cae7β-
HSDH
Hh7β-HSDH Halomonas halodenitrifi- NAD(H)
cans
10
11
12
Rs7β-HSDH
Sc7β-HSDH
Csp12α-
Rhodobacter sphaeroides NAD(H)
Stanieria cyanosphaera
Clostridium sp.
<1%
<1%
<1%
NAD(H)
NADP
(H)
NAD(H)
NADP
(H)
HSDH
13
14
Ls12α-HSDH Lysinibacillus sphaericus
IS2-SDR
<1%
66%
Icelandic metagenome
15
NGI7-SDR
Norwegian metage-
nome
NAD(H)
12%
The stereospecific reduction of 1 was obtained in the
reaction catalyzed by Dm7α-HSDH, which gave a 1:1 mixture of
two diastereoisomers, whose preparative separation was quite
troublesome and could be achieved by using a Biotage® SP1
system for flash chromatography. The two products proved to
be the cis-alcohol 2a derived from the enantiomer 1a and the
[a] Reaction conditions: 10 mM substrate; 50 mM phosphate buffer pH 7;
5% v/v DMSO; 50 mM glucose; 0.4 mM cofactor; 4 UmLÀ 1 DH; 1 UmLÀ 1
GDH; 0.4 M NaCl (only for entries 4 and 9); 25 C, 100 rpm. [b] The library is
composed both of HSDHs (entries 1–13) and of short chain reductases
(SDRs, entries 14 and 15). [c] The recombinant enzymes were either from
defined chemical sources or coming from metagenomic samples, as
detailed in ref [32]. [d] Conversions were evaluated after 48 h by GC/MS
and, for the entries 2, 3, 14, and 15, also by chiral phase HPLC analyses.
°
trans-alcohol 3b derived from the enantiomer 1b (Scheme 1,
right upper part). Pure 3b (>99% ee, [α]D À 111.6 , c 1.0 in
22
°
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CHCl3) was also obtained by Dm7α-HSDH-catalyzed reduction
of the previously isolated enantiomer 1b (Scheme 1, left upper
part). In terms of stereoselectivity, the production of the trans-
alcohol 3b from the ketone enantiomer 1b is in agreement
with data reported by Sugai with whole cells of C. melibiosica
(lower ee values were obtained)[28] and by Prelog with C.
falcata.[24] A similar outcome but with much lower selectivity
was observed by Janeczko in the biotransformations catalyzed
by strains of D. igniaria and of C. betulinium whole cells.[31]
It is worthy to be reminded that the trans-alcohol 3 is not
obtained using the classical chemical reductive reagents and
only the cis-alcohol 2 could be isolated.[18,35] Moreover, once
again it was possible to observe a significant selectivity of a
HSDH, in this case devoted to the regio- and stereoselective
modification of oxygenated substituent at the position C7 of the
steroid skeleton, with compounds not structurally related to a
steroid. For additional examples of the substrate promiscuity of
HSDHs see ref 33 and 36–41.
The easy availability of the racemic mixture of the cis-
alcohol 2a and 2b prompted us also to evaluate an alternative
biocatalyzed approach, exploiting the well-known enantioselec-
tivity of hydrolases in the acylation of secondary alcohols in
organic solvents.[34] As previously stated, to our surprise,
apparently this substrate has never been considered in previous
investigations, despite the fact that it is clearly a suitable target
to be acylated by lipases, as shown by Franssen and coworkers
with a structurally not so different compound.[42]
Compound 2 was dissolved in dry acetone containing a
large excess (10% v/v) of the acylating agent vinyl acetate and
different lipases were added. The reactions were shaken at
Figure 2. HPLC chromatograms of [a] racemic 2+racemic 4; [b] Lipase PS-
biocatalyzed kinetic resolution of racemic 2 after 72 h; [c] After 120 h.
°
45 C and monitored by TLC and chiral phase HPLC. As shown
in Figure 2 we were pleased to find that the so-called lipase PS,
adsorbed on celite, showed a really significant enantioselectiv-
ity. Actually, the value of enantiomeric ratio “E”, that could be
calculated from the values of degree of conversion and ee of
the residual substrate according to the well-known formula
proposed by Sih years ago,[43] was very high: approximately
2.000. This value makes this biocatalyzed acetylation really close
to an ideal kinetic resolution process that spontaneously stops
after the preferred enantiomer is consumed. And this was
indeed the case, and the two pure cis enantiomers, the alcohol
2a and the acetate 4b, could be isolated and characterized.
The same protocol was applied to the diastereomeric
mixture of alcohols 2a and 3b obtained by the reaction
catalyzed by Dm7α-HSDH, but no conversion was observed
with any of the lipases tested, leaving the above-described
chromatographic protocol as the only way to separate these
two compounds. The hydrolysis of the ester 4b furnished the
pure missing enantiomer of the alcohol, that is 2b, while
oxidation of the alcohol 2a gave an easy and efficient entry to
ketone 1a.
ases, including enzymes with different substrate scope as well
as biological origin. The obtained results further exemplify the
substrate promiscuity of this group of enzymes. Moreover, a
more classical approach based on the kinetic resolution of a
racemic mixture of the cis alcohol (2) catalyzed by lipase PS in
acetone proved to be particularly efficient, very close to an ideal
process in terms of enantioselectivity of the biocatalyst.
The dehydrogenase-catalyzed reactions were performed on
a 50–100 mg scale, whereas the lipase-catalyzed kinetic reso-
lution was obtained starting from 1 g of the substrate. All the
reactions can be easily scaled-up as the DHs are available as
recombinant overexpressed proteins, while lipase PS is a cheap
commercially available enzyme. Specifically, the lipase-catalyzed
reaction has not been optimized in terms of substrate
concentration and substrate/biocatalyst ratio, therefore further
improvements of productivity and solvent consumption are
likely to be easily achieved at will.
The trans enantiomer 3a is presently the only isomer that
could not be produced by an enzyme-catalyzed reaction and
this target will be the object of further future investigations.
Conclusion
Wieland–Miescher ketone (1) has been chosen as a model
synthon to evaluate the selectivity of a panel of dehydrogen-
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IS2-SDR assay: 10 mM methyl benzoylformate; 50 mM potassium
phosphate buffer, pH 8.0; 0.20 mM NADPH.
Experimental Section
General
BmGDH assay: 50 mM glucose; 50 mM potassium phosphate buffer,
pH 7.0; 0.20 mM NAD(P)+.
Wieland-Miescher ketone (8a-methyl-3,4,8,8a-tetrahydronaphtha-
lene-1,6(2H,7H)-dione, purity 98.5%) was purchased from Fluoro-
chem (Hadfield, UK). Amano lipase PS (adsorbed by us on celite)[44]
was from Merck (Darmstadt, Germany). All reagents and solvents
were purchased from Merck and used without further purification,
unless otherwise stated.
One unit (U) is defined as the enzyme activity that reduces/oxidizes
1 μmol of NAD(P)(H) per min under the assay conditions described
above.
Analytical methods
Reactions were monitored via TLC (thin-layer chromatography) on
pre-coated glass plates silica gel 60 with fluorescent indicator UV254
and treated with an oxidizing solution [4-hydroxybenzaldehyde
(6.3 g), H2SO4 (50% v/v in H2O, 40 mL), MeOH (400 mL)].
At scheduled times, reaction samples (50 μL) were extracted with
AcOEt and dried over Na2SO4 to afford 10 mM samples, submitted
to GC-MS analysis, or evaporated, resuspended in i-PrOH to a
10 mM final concentration and analyzed by chiral HPLC.
GC-MS analyses were performed using an Agilent HP-5MS column
(30 m×0.25 mm×0.25 μm) on a Finnigan TRACE DSQ GC/MS instru-
Abbreviations
HSDH: Hydroxysteroid Dehydrogenase; U: enzymatic unit; SDR:
Short Chain Dehydrogenase/Reductase; BmGDH: Glucose Dehydro-
genase from Bacilllus megaterium; IPTG: Isopropyl β-D-1-thiogalac-
topyranoside.
°
ment (ThermoQuest, San Jose, CA). Elution conditions: 60 C, 1 min;
À 1
+10 C min until 300 C; hold 1 min; flow rate: 1.0 mLminÀ 1; inlet
°
°
°
°
temperature: 250 C; ion source temperature: 250 C; MS transfer
°
line temperature: 250 C. Retention times: (1a,1b): 13.7 min;
(2a,2b): 14.6 min; (3a,3b): 14.8 min; (4a,4b): 15.4 min.
Chiral phase HPLC analyses were performed on a Shimadzu LC-
20AD high performance liquid chromatography system equipped
with a Shimadzu SPD-20 A UV detector and a Phenomenex Lux 3u
Cellulose-2 chiral column (250 mm×4.6 mm). HPLC conditions:
injection volume 10 μL; mobile phase: i-PrOH: petroleum ether=
1:1; flow rate: 0.7 mLminÀ 1; detection λ: 254 nm; temperature:
Enzyme Preparation
HSDHs/SDRs expression and purification
Expression and purification of HSDHs, SDRs, and BmGDH were
carried out as previously described.[32]
°
33 C. Retention times: (1a): 22.4 min; (1b): 19.1 min; (2a): 6.9 min;
Expression conditions of Dm7α-HSDH were optimized by expres-
sion trials carried out as follows: E. coli BL21(DE3) harboring the
expression plasmid pETiteDm7aHSDH was inoculated overnight in
50 mL LB medium supplemented with 30 μgmLÀ 1 kanamycin
(LBkan30) and grown at 37 C, 220 rpm. The overnight culture was
subsequently inoculated in 1 L LBkan30 medium and incubated at
(2b): 5.9 min; (3b): 7.3 min; (4a): 16.1 min; (4b): 15.8 min. Prior to
performing HPLC analyses, the molar extinction coefficients of 1, 2
and 4 were determined, to be able to assess conversions while
considering the different molar absorbivity of the molecules under
analysis.
°
The nature of the obtained compounds, which have been
previously characterized in the literature, was confirmed by means
°
37 C, 220 rpm. Protein expression was then induced with 1 mM
IPTG at an OD600 of 0.5–1. The culture was transferred to 30, 25 or
1
of H-NMR, mass spectrometry and optical rotation measurements.
°
17 C with shaking at 220 rpm and grown for 24, 48 or 72 h,
respectively. The cells were harvested by centrifugation at
The NMR spectra were acquired in CDCl3 or in DMSO-d6 at rt on a
Bruker AV 400 MHz spectrometer with a z gradient at 400 MHz for
1H-NMR analysis and 101 MHz for 13C-NMR.
°
6000 rpm for 10 min at 4 C and resuspended in 20 mL lysis buffer
(20 mM potassium phosphate (KP) buffer, pH 7.0, 500 mM NaCl,
20 mM imidazole). After disruption by sonication at 4 C, the cell
extract was centrifuged at 12000 rpm at 4 C for 30 min and clear
°
°
ESI-MS spectra were recorded on a Bruker Esquire 3000 PLUS
instrument (ESI Ion Trap LC/MSn System), equipped with an ESI
source and a quadrupole ion trap detector (QIT). The samples were
dissolved in methanol to 1–2 gLÀ 1 and then directly syringed in the
ESI-MS at 4 μLminÀ 1 rate. The analyses were performed in positive
mode. The acquisition parameters were optimized as such: 4.5 kV
needle voltage, 10 LhÀ 1 N2 flow rate, 40 V cone voltage, trap drive
set to 46, 115.8 V capillary exit, 13000 (m/z) sÀ 1 scan resolution over
lysates were assessed for the presence of soluble protein by SDS-
PAGE (12% T, 2.6% C). Protein purification was performed as
previously described.[32] Cell incubation at 17 C for 72 h after
°
induction resulted in the best expression yields (i.e., 108 mg of
pure Dm7α-HSDH (11.575 U) were obtained after protein purifica-
tion).
°
the 35–900 m/z mass/charge range, source temperature 250 C.
Activity assays
Optical rotations were measured on a Jasco P-2000 polarimeter.
The specific rotation was calculated as the [α]λT=α100/cd, where
α represents the recorded optical rotation, c the analyte concen-
tration (mgmLÀ 1), d the cuvette length (dm). As such, the specific
rotation is expressed as 10À 1 degcmÀ 2 gÀ 1. λ is reported in nm and T
Dehydrogenase activities of HSDHs and BmGDH were determined
spectrophotometrically by measuring the reduction of NAD(P)+ at
340 nm (ɛ: 6.22 mMÀ 1cmÀ 1), while the activity of IS2-SDR was
measured by following the oxidation of NADPH at the same
wavelength. Assays were carried out in polyethylene cuvettes at
room temperature by adding the opportune purified dehydrogen-
ase (1–20 μL) to the following assay mixtures (1 mL final volume):
°
in C. λ corresponds to sodium D line (589 nm), thus the optical
rotation is referred to as [α]D. T, c and the solvent were chosen
according to references reported in literature (see below).
HSDH assay: 2.5 mM substrate (cholic acid for 7α- and 12α-HSDHs,
ursodeoxycholic acid for 7β-HSDHs); 50 mM potassium phosphate
buffer, pH 9.0; 0.20 mM NAD(P)+.
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library of enzymes, the general reaction protocol was as follows:
Determination of the molar absorbivity coefficients of
compounds 1, 2and 4
50 mM glucose; 0.2 UmLÀ 1 GDH; 0.4 mM NAD(P)+; 3.4 UmLÀ 1 HSDH;
10 mM substrate; 5% v/v DMSO; 50 mM potassium phosphate
buffer, pH 7.0 (total volume: 1 mL). Reactions catalyzed by Hh7α-
HSDH and Hh7β-HSDH were performed both in the presence and
The molar extinction coefficients of ketone 1, cis-alcohol 2 and cis-
acetate 4 were determined at 254 nm. For each compound, a
10 mM solution in CH3CN was prepared and it was used as a
mother solution to test the absorption of each molecule in 1 mL
quartz cuvettes in the concentration range of 0.1-25 μM in CH3CN
(where linearity of data was observed). ɛ254 (1): 6508 MÀ 1 cmÀ 1; ɛ254
(2): 5889 MÀ 1 cmÀ 1; ɛ254 (4): 2620 MÀ 1 cmÀ 1 (for more details see
Paragraph 1, Supporting information).
°
in the absence of 0.4 M NaCl. The mixtures were shaken at 25 C
and 100 rpm for 24 to 48 h and monitored over time via TLC
(eluent CH2Cl2 :AcOEt=9:1). Reaction conversions and enantiomer-
ic excesses were evaluated by GC-MS and chiral HPLC analyses. The
isolation of pure products was obtained via extraction with AcOEt
of the reaction mixtures and subsequent flash chromatography on
silica gel 60 (70–320 mesh, Merck, eluent CH2Cl2/AcOEt mixtures).
The absolute configurations of the residual substrate 1b, if any, and
of the products 2a and 3b were assigned via optical rotation
measurements and subsequent confrontation with values reported
in literature.[24–27]
Characterization of commercially available racemic 1
1H NMR (400 MHz, CDCl3) δ 5.87 (d, J=1.8 Hz, 1H), 2.84–2.65 (m,
2H), 2.61–2.39 (m, 4H), 2.30–2.04 (m, 3H), 1.72 (qt, J=13.3, 4.4 Hz,
1H), 1.46 (s, 3H). ESI-MS: [M+Na]+: 201.0.
Enzymatic reduction of 1 catalyzed by IS2-SDR
Synthetic chemistry
Following the aforementioned general protocol on a 100 mg scale
(10 mM substrate, 56 mL reaction volume) and stopping the
reaction at 58% conversion, the following products were isolated
Preparation of standard racemate 2, (�)-4a,5-cis-(5-hydroxy-
4a-methyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one)[18]
after flash chromatography (eluent: CH2Cl2 :AcOEt=9:1). 1b: ee:
22
96.7%; [α]D : À 97.0 (c: 1.0 in CHCl3);[47] isolated yield: 33 mg (32%);
°
The reduction of substrate 1 was performed following a standard
protocol with NaBH4. To a stirred solution of 0.4 M substrate (1 eq,
100 mg) in EtOH (5 mL) at 0 C, an ethanolic suspension (0.4 M) of
2a: ee: 90.7%; isolated yield: 30 mg, (29%). NMR and ms spectra
are in accordance with the proposed structures (see Supplemen-
tary).
°
NaBH4 (1 eq, 13 mg) was added dropwise. After 4 hours, the
reaction was quenched with a saturated aqueous solution of NH4Cl,
then it was extracted with AcOEt (3x). The organic phase was dried
over Na2SO4, filtered and the solvent was evaporated under
reduced pressure to yield a crude extract. A flash chromatography
purification afforded the desired products (2a+2b) in up to 90%
yield. The racemic product 2 was characterized by chiral HPLC (see
analytical methods) and 1H-NMR analyses. 1H-NMR (400 MHz, CDCl3)
δ 5.78 (d, J=1.8 Hz, 1H), 3.42 (dd, J=11.6, 4.3 Hz, 1H), 2.50–2.28 (m,
3H), 2.25–2.13 (m, 2H), 1.94–1.78 (m, 3H), 1.76–1.64 (ddd, J=16.9,
13.3, 4.2 Hz, 1H), 1.48–1.33 (qt, J=13.2, 4.0 Hz, 1H), 1.19 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 206.66, 199.56, 168.43, 125.50, 78.28, 41.63,
34.27, 33.72, 32.03, 30.31, 23.20, 15.27. ESI-MS: [M+Na]+: 203.0.
Enzymatic reductions of 1 and 1b catalyzed by Dm7α-HSDH
Following the general protocol on a 50 mg scale (10 mM substrate,
28 mL reaction volume) and bringing the reaction to full conversion
from racemic 1, the following products were isolated via flash
chromatography on a Biotage SP1 working with 50 mg of crude
mixture and a 10 g silica gel Biotage® cartridge in a gradient of
AcOEt in DCM (10 CV 5% AcOEt; 5 CV from 5% to 15% AcOEt,
10 CV 15% AcOEt).
2a: 20 mg, ee: 95.3%, isolated yield: 40.0%. NMR and ms spectra in
accordance with the proposed structure (see Supplementary).
3b: 25 mg, ee: >99%, isolated yield: 50.0%.1H NMR (400 MHz,
CDCl3) δ 5.87 (br s, 1H), 3.65 (br s, 1H), 2.74–2.35 (m, 4H), 2.35–2.23
(m, 1H), 2.14–1.98 (m, 1H), 1.96–1.66 (m, 3H), 1.57–1.46 (m, 1H), 1.24
Preparation of standard racemate 4, (�)-8a,1-cis-(-8a-methyl-
6-oxo-1,2,3,4,6,7,8,8a-octahydronaphthalen-1-yl acetate)[46]
(s, 3H); 13C NMR (101 MHz, CDCl3) δ 199.41, 167.68, 127.03, 75.33,
The acetylation of racemate substrate 2 was performed according
to a standard acetylation protocol with acetyl chloride. To a stirred
solution of 0.1 M substrate (1 eq, 50 mg) in CH2Cl2 (2.8 mL), pyridine
(1.2 eq, 27 μL) and acetyl chloride (1.2 eq, 24 μL) were added. The
reaction was stirred overnight at room temperature. Then, CH2Cl2
was evaporated and the crude extract was resuspended in
saturated aqueous NaHCO3, then extracted (3×) with AcOEt. The
organic phase was dried over Na2SO4, filtered and the solvent was
evaporated under reduced pressure to give the desired products
22
40.93, 34.02, 31.73, 30.80, 28.71, 21.83, 19.83; ee: >99%; [α]D
:
À 111.6 (c: 1.0 in CHCl3);[28] ESI-MS: [M+Na]+: 203.0.[19]
°
Using the enantiomer 1b as a substrate (30 mg scale), the trans-
alcohol 3b was obtained as sole product following, again, the
general protocol. 3b: yield: 91%; ee: >99%.
Enzymatic acetylation of substrate 2b catalyzed by Amano
lipase PS
1
(4a+4b). H NMR (400 MHz, CDCl3) δ 5.81 (d, J=1.8 Hz, 1H), 4.65
(dd, J=11.8, 4.2 Hz, 1H), 2.44–2.30 (m, 3H), 2.30–2.21 (m, 1H), 2.08
(s, J, 3H), 1.98–1.66 (m, 5H), 1.55–1.41 (m, 1H), 1.27 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 198.82, 170.32, 166.67, 125.80, 79.17, 40.39,
33.98, 33.47, 31.76, 26.87, 22.95, 21.07, 16.64. ESI-MS: [M+H]+:
223.1. ESI-MS: [M+Na]+: 245.0.
Racemic substrate 2 (1040 mg, 1 eq) was dissolved in dry acetone
(80 mM substrate, 70 mL reaction volume). Then, excess vinyl
acetate (10% v/v, 14 eq) and Amano lipase PS on celite (1000 mg,
1% wt, catalytic amount) were added. The reaction was shaken at
°
45 C, 200 rpm for six days. The enzyme was filtered away and the
solvent was evaporated to afford a crude mixture of 2a and 4b.
The two products were separated via flash chromatography (CH2Cl2:
Enzymatic reduction of racemic ketone 1 with HSDHs/SDRs
AcOEt gradient from 9:1 to 7:3) to give the subsequent products:
22
General protocol for the enzymatic reduction: the reactions
catalyzed by HSDHs were coupled with a glucose/GDH system to
regenerate NAD(P)H. For the initial activity screening of the whole
°
2a: 660 mg, 50% isolated yield, ee: 97.3%; [α]D : +173.4 (c: 1.0 in
CHCl3).[33] 4b: 407 mg, 39% isolated yield, ee: >99%; [α]D : À 69.5
22
°
(c: 1.0 in CHCl3).[33]
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Full Papers
doi.org/10.1002/ejoc.202100174
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(0.5 M). Then, a 1 M aqueous solution of NaOH was added dropwise
(0.675 mL, 0.675 mmol, 1.5 eq). The reaction was stirred at room
temperature for 2 h, then quenched with a saturated aqueous
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22
(0.377 mmol, 90.0% yield). [α]D : À 180.5 (c: 1.0 in CHCl3).[28] ee
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Keywords: Biocatalysis · Dehydrogenases · Enzymes · Lipases ·
Wieland-Miescher ketone
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Manuscript received: February 11, 2021
Revised manuscript received: March 15, 2021
Accepted manuscript online: March 16, 2021
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© 2021 Wiley-VCH GmbH
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These are not the final page numbers!

FULL PAPERS
A combination of different enzymatic
activities and chemical protocols
were exploited to access the two
enantiomers of Wieland-Miescher
ketone and three of its four alcohols
derivatives as enantioenriched
species. Specifically, the optimization
of biotransformations of WM-ketone
or of substrates (bio)chemically-
prepared from it, mediated by hy-
droxysteroids dehydrogenases, short
chain alcohol dehydrogenases and
lipases, are described.
S. Bertuletti, I. Bayout, Dr. I. Bassanini,
Dr. E. E. Ferrandi, Dr. N. Bouzemi,
Dr. D. Monti, Dr. S. Riva*
1 – 8
Biocatalytic Approaches to the
Enantiomers of Wieland–Miescher
Ketone and its Derivatives