Mild Chemoenzymatic Oxidation of Allylic sec-Alcohols. Application to Biocatalytic Stereoselective Redox Isomerizations

Article abstract of DOI:10.1021/acscatal.7b03293

The design of catalytic oxidative methodologies in aqueous medium under mild reaction conditions and using molecular oxygen as final electron acceptor represents a suitable alternative to the traditional oxidative transformations. These methods are especially relevant if other functionalities that can be oxidized are present within the same molecule, as in the case of allylic alcohols. Herein we apply a simple chemoenzymatic system composed of the laccase from Trametes versicolor and 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) to oxidize a series of racemic allylic sec-alcohols into the corresponding α,β-unsaturated ketones. Afterward, these compounds react with different commercially available ene-reductases to afford the corresponding saturated ketones. Remarkably, in the case of trisubstituted alkenes, the bioreduction reaction occurred with high stereoselectivity. Overall, a bienzymatic one-pot two-step sequential strategy has been described with respect to the synthesis of saturated ketones starting from racemic allylic alcohols, thus resembling the metal-catalyzed redox isomerizations of these derivatives that have been previously reported in the literature.

Full text of DOI:10.1021/acscatal.7b03293

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Article
Mild chemoenzymatic oxidation of allylic sec-alcohols.
Application to biocatalytic stereoselective redox isomerizations
Lia Martinez Montero, Vicente Gotor, Vicente Gotor-Fernandez, and Ivan Lavandera
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03293 • Publication Date (Web): 02 Feb 2018
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ACS Catalysis
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Mild Chemoenzymatic Oxidation of Allylic secꢀ
Alcohols. Application to Biocatalytic Stereoselective
Redox Isomerizations
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Lía MartínezꢀMontero,† Vicente Gotor,† Vicente GotorꢀFernández,†,* and Iván Lavandera†,*
† Organic and Inorganic Chemistry Department, University of Oviedo, Avenida Julián Clavería s/n,
33006 Oviedo, Spain.
Corresponding Authors
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*Eꢀmail for V.G.ꢀF.: vicgotfer@uniovi.es., *Eꢀmail for I.L.: lavanderaivan@uniovi.es., Phone: +34 98
5103454; +34 98 5103452. Fax: +34 98 5103446
Abstract. The design of catalytic oxidative methodologies in aqueous medium under mild reaction
conditions and using molecular oxygen as final electron acceptor represents a suitable alternative to the
traditional oxidative transformations. These methods are especially relevant if other functionalities that
can be oxidized are present within the same molecule, as in the case of allylic alcohols. Herein we apply
a simple chemoenzymatic system composed by the laccase from Trametes versicolor and 2,2,6,6ꢀ
tetramethylpiperidinyloxy radical (TEMPO) to oxidize a series of racemic allylic secꢀalcohols into the
corresponding α,βꢀunsaturated ketones. Afterwards, these compounds react with different commercially
available eneꢀreductases to afford the corresponding saturated ketones. Remarkably, in the case of
trisubstituted alkenes, the bioreduction reaction occurred with high stereoselectivity. Overall, a
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bienzymatic oneꢀpot twoꢀstep sequential strategy has been described towards the synthesis of saturated
ketones starting from racemic allylic alcohols, thus resembling the metalꢀcatalyzed redox isomerizations
of these derivatives that have been previously reported in the literature.
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Keywords: Laccases/ Eneꢀreductases/ Oxidation/ Allylic alcohols/ Redox isomerization
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Introduction
Oxidative transformations are one of the pivotal reactions in Organic Chemistry due to the synthetic
relevance of the oxidized products (e.g., carbonylic or carboxylic compounds) as reactive intermediates
for subsequent modifications. Among the different approaches, the oxidation of alcohols into aldehydes
or ketones is one of the most employed transformations on industry.1 While traditionally these methods
have been mediated by stoichiometric amounts of one or more oxidants in organic solvents, the
tendency during the last years has been the development of catalytic oxidative methodologies2 in
aqueous medium3 under mild reaction conditions and using, if possible, molecular oxygen as final
electron acceptor.
These protocols are especially appealing when several functionalities susceptible of being oxidized are
present within the target molecule, as in the case of allylic alcohols, where the C=C double bond can
also react depending on the reaction conditions. Thus, among the different catalytic methodologies
recently reported, the use of chromium(III) complexes,4 palladium nanoclusters,5 gold nanoparticles,6
platinum black,7 silver exchanged molybdovanado phosphoric acid,8 iron(III) chloride complexes,9
palladium on silica,10 ironꢀpicolinate complexes,11 potassium osmium(VI) oxide,12 and palladium on
alumina,13 can be mentioned. However, these processes usually suffer from different drawbacks such as
the formation of undesired byꢀproducts as a consequence of the presence of the metal or a peroxide coꢀ
oxidant, and the use of high temperatures and organic solvents. In the last few years, stable radical
species such as 2,2,6,6ꢀtetramethylpiperidinyloxy (TEMPO) have appeared as interesting alternatives in
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ACS Catalysis
combination with different metals to achieve aerobic oxidations.14 In this context, several strategies
have been disclosed for the oxidation of primary and secondary alcohols using TEMPO in combination
with iron(III),15 copper(I),16 or [bis(acetoxy)iodo]benzene in the presence of bromide ions.17
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Allylic alcohols can act as substrates in a redox (neutral) isomerization process to form saturated
carbonylic derivatives.18 This valuable reaction allows the synthesis of versatile compounds from
readily accessible substrates. Among the different described methodologies, the use of transition metals
is the most widely employed.19 Thus, ruthenium,20 rhodium,21 iridium,21b,22 or palladium23 complexes
have been successfully demonstrated as efficient catalysts for this reaction, usually working at high
temperatures in organic solvents, leading in some cases to low chemoselectivities. Recently, efforts have
been made to develop metalꢀfree24 transformations, or to use water as reaction medium.25 In order to
accomplish a stereoselective version of this reaction with secondary allylic alcohols, kinetic resolutions
with metallic complexes26 or stereospecific isomerizations of enantioenriched substrates to the
corresponding βꢀsubstituted ketones have been described.20c,24b Very recently, Zhao et al. have reported
the first example of a catalytic enantioselective isomerization of racemic secondary allylic alcohols for
the synthesis of ketones with an αꢀtertiary stereocenter up to 90% ee using a rhodium catalyst.27
With these precedents in mind and based on our previous experience in the use of the laccase from
Trametes versicolor (LTv)‒TEMPO catalytic system to perform the oxidation of benzylic secondary
alcohols,28 we next turned our attention in the possibility of extending this simple and mild protocol to
oxidize allylic secondary alcohols (Scheme 1a).29 The exploitation of laccases, blue multicopper
oxidases,30 in combination with a chemical coꢀoxidant allows the design of oxidative transformations in
aqueous media at the expense of the reduction of oxygen, releasing water as the only byꢀproduct. After
the optimization of this oxidation, it was envisaged that the formed α,βꢀunsaturated ketones could act as
suitable substrates for eneꢀreductases (EREDs),31 enzymes capable of reducing C=C double bonds
conjugated to electronꢀwithdrawing groups in a stereoselective fashion, yielding the corresponding
saturated ketones. Thus, depending on the alkene substitution, enantioenriched αꢀ or βꢀsubstituted
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carbonylic compounds can be obtained in a oneꢀpot twoꢀstep strategy (Scheme 1b). This sequential
route resembles the metalꢀcatalyzed redox isomerization of allylic alcohols, and provides a biocatalytic
alternative under very mild conditions. Up to now, very few enzymatic examples of this reaction have
been proposed. Hence, Bruce et al.32 and Hollmann et al.33 have combined the use of an alcohol
dehydrogenase (ADH) and an ERED. This redoxꢀneutral approach is very straightforward, as it allows the
internal recycling of the nicotinamide cofactor, necessary in both steps, thus not requiring an external sacrificial
coenzyme regeneration system. On the contrary, they had to carefully deal with the undesired bioreduction
of the final saturated ketone due to the presence of the ADH. Herein, this problem is overcome as the
final product cannot act as substrate for the laccase‒TEMPO system. On the other hand, Brenna and coꢀ
workers have very recently published a similar methodology to obtain a series of cyclic βꢀhydroxy
esters from the corresponding cyclic derivatives with high yields and ee values via P450ꢀmediated
hydroxylation, followed by oxidation of the allylic alcohol catalyzed by the laccase from Amano M120
and the TEMPO+·BF4‒ salt, and finally an EREDꢀmediated bioreduction.34
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Scheme 1. a) Laccase‒TEMPO catalytic system applied to the oxidation of racemic secondary allylic
alcohols. b) Biocatalytic (stereoselective) redox isomerization of racemic allylic secꢀalcohols via
sequential oneꢀpot twoꢀstep strategy using the laccase‒TEMPO system and an eneꢀreductase.
Results and Discussion
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ACS Catalysis
As a first step, the oxidation of allylic alcohols using LTv‒TEMPO was subjected to study and
optimization. We used commercially available racemic octꢀ3ꢀenꢀ2ꢀol [(±)ꢀ1a, Table 1] as model
substrate, and based on our previous experience with this methodology,28 some parameters were already
considered as optimal [citrate buffer 50 mM pH 5 as buffer medium, TEMPO concentration (33 mol%),
30 ºC and magnetic stirring in an openꢀtoꢀair vessel]. Other factors were optimized, as they were
anticipated as key for the correct reaction outcome. Thus, substrate concentration and the presence of
methyl tertꢀbutyl ether (MTBE) as organic coꢀsolvent were further investigated (see Supporting
Information, Tables S1 and S2). Satisfyingly, it was observed that after 16 h (±)ꢀ1a could be
quantitatively oxidized up to 50 mM substrate concentration into the α,βꢀunsaturated ketone 1b.
Likewise, it was determined that the use of MTBE and an oxygenꢀsaturated buffer did not deter the
chemoenzymatic activity, although their presence was not necessary to accomplish full conversion.
Furthermore, under these mild reaction conditions, byꢀproducts coming from the alkene oxidation were
not detected.
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With these promising results in our hands, we decided to study the scope of this transformation.
Therefore, a set of racemic allylic secꢀalcohols [(±)ꢀ1aꢀ12a, Table 1] were purchased or synthesized
following simple described methodologies (see Supporting Information for more details).35 Also, a
natural compound such as carveol [(5R)ꢀ13a] was studied as suitable substrate.
Substrates were then treated under the conditions previously optimized for alcohol (±)ꢀ1a, obtaining a
complete conversion towards the corresponding α,βꢀunsaturated ketones (40 mM) after 16 h at 30 ºC in
an openꢀtoꢀair vessel under magnetic stirring (Table 1). It is important to remark that byꢀproducts were
not detected, and that both laccase and TEMPO were necessary to perform the oxidations. In the case of
derivatives (±)ꢀ8aꢀ12a, full conversions were not achieved working in buffer medium, as a consequence
of the low solubility presented by these compounds (data not shown). Satisfyingly, quantitative
conversions were attained by the simple addition of MTBE (20% v/v), that helped to dissolve the
starting material (entries 8ꢀ12). During the reaction progress, this organic solvent was eliminated via
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evaporation. At this point (5R)ꢀcarveol [(5R)ꢀ13a], which was present as a mixture of isomers cis:trans
at a molar proportion 46:54, deserves special mention. When the reaction was performed in buffer under
the standard conditions, it was observed that while the cis isomer was completely oxidized to carvone,
35% of the trans diastereoisomer still remained in the reaction mixture (entry 13). Therefore, the
reoxidation of the crude for additional 16 h adding LTv and TEMPO was necessary in order to almost
obtain complete conversion (entry 14). This result is in accordance with a previous study reported by
Knochel and coꢀworkers, which showed that TEMPOꢀcatalyzed oxidations of substituted cyclohexanols
are highly dependent on the axialꢀequatorial position of the alcohol moiety.36
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Table 1. Oxidation of racemic secondary allylic alcohols (±)ꢀ1aꢀ12a and (5R)ꢀ13a with the
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LTv‒TEMPO system.a
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R3 OH
R1
R3
O
LTv TEMPO
R4
R4
R1
Buffer (+ MTBE)
30 ºC, O2
R2
R2
1b-13b
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(±)-1a-12a, (5R)-13a
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OH
OH
(±)-2a, R1= H, R2= H
(±)-3a, R1= Me, R2= H
(±)-4a, R1= H, R2= Me
R1
R2
OH
(±)-1a
(±)-5a
(±)-7a, R1= H, R2= H
OH
(±)-8a, R1= H, R2= m-Br
(±)-9a, R1= H, R2= p-Cl
(±)-10a, R1= H, R2= p-Br
(±)-11a, R1= H, R2= p-NO2
(±)-12a, R1= Me, R2= H
OH
OH
R1
R2
(±)-6a
(5R)-13a
Entry
1
Substrate
(±)ꢀ1a
(±)ꢀ2a
(±)ꢀ3a
(±)ꢀ4a
(±)ꢀ5a
(±)ꢀ6a
(±)ꢀ7a
(±)ꢀ8a
t (h)
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32
MTBE (20% v/v)
c (%)b
No
No
No
No
No
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
65d
2
3
4
5
6
7
8
9
10
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12
13c
14e
No
No
Yes
Yes
Yes
Yes
Yes
No
(±)ꢀ9a
(±)ꢀ10a
(±)ꢀ11a
(±)ꢀ12a
(5R)ꢀ13a
(5R)ꢀ13a
No
98
a
b
For reaction conditions, see the Experimental Section. Determined by GC (see
c
Supporting Information). Starting from a 46:54 cis:trans mixture, determined by
1HꢀNMR. The cis isomer completely reacted. After 16 h, the reaction mixture
d
e
was retreated with LTv‒TEMPO for further 16 h.
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Once developed a simple and mild method to oxidize racemic secondary allylic alcohols to the
corresponding α,βꢀunsaturated ketones, next we envisioned the possibility of coupling this oxidative
process with the (stereoꢀ and) chemoselective EREDꢀcatalyzed bioreduction of the C=C double bond to
obtain saturated ketones (Scheme 1b). Overall, this sequential protocol would provide a biocatalytic
alternative to the transition metalꢀcatalyzed redox isomerizations of allylic alcohols. Furthermore, due to
the excellent selectivity usually shown by these enzymes, the synthesis of αꢀ and/or βꢀsubstituted
derivatives in high ee could also be feasible.
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Hence, a kit of seven commercially available EREDs from Codexis Inc. was used with ketones 1bꢀ13b
(40 mM) under the optimal conditions described by the company to perform the C=C double bond
bioreductions, using as medium phosphate buffer 100 mM pH 7. Glucose (80 mM) and glucose
dehydrogenase (GDH, 20 U/mL) were utilized for the recycling of the nicotinamide cofactor (NADP+,
0.6 mM) necessary in the bioreduction. The reactions were performed at 30 ºC for 24 h under orbital
shaking (250 rpm). Satisfyingly, excellent conversions and very high selectivities were reached for all
the tested substrates. Among them, EREDꢀ103, EREDꢀP1ꢀA04, and EREDꢀP1ꢀE01 usually displayed
the best results (for complete set of data, see Tables S3ꢀS14 in the Supporting Information). Due to
difficulties in the isolation of ketone 6b after the laccaseꢀcatalyzed oxidations, we were unable to
develop the bioreduction study.
Due to the fact that the laccase‒TEMPO system works in citrate buffer 50 mM pH 5, EREDꢀcatalyzed
bioreductions were also studied under these reaction conditions, seeking for the possibility of
developing both steps in a ‘oneꢀpot’ fashion. Gladly, as can be seen in Table 2, excellent results were
achieved in all cases. Even for substrate 3b (entry 3), the reduction proceeded with higher selectivity
than at pH 7, probably due to a slower racemization rate of the final product (R)ꢀ3c. Especially EREDꢀ
P1ꢀE01 and EREDꢀP1ꢀA04 showed excellent conversions and high selectivities for the majority of the
tested substrates.
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Table 2. EREDꢀcatalyzed bioreductions of α,βꢀunsaturated ketones 1bꢀ13b.a
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Entry
1
Substrate
1b
ERED
110
c (%)b
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
ee or de (%)c
n.a.
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P1ꢀA04
P1ꢀE01
P1ꢀE01
P1ꢀA04
P1ꢀE01
103
n.a.
2b
3
87 (R)d
>99 (S)
n.a.
3b
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4b
5
5b
6
n.a.
7b
7
n.a.
8b
8
P1ꢀA04
P1ꢀE01
P1ꢀE01
P1ꢀE01
P1ꢀE01
n.a.
9b
9
n.a.
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12b
(5R)ꢀ13b
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12
n.a.
92 (S)
90 (2R,5R)
a For reaction conditions, see the Experimental Section. b Determined by GC (see Supporting Information).
Determined by achiral or chiral GC or HPLC (see Supporting Information). At pH 7 the enantiomeric
excess of (R)ꢀ3c was 82%. n.a.: not applicable as these substrates are not chiral.
c
d
Once both steps were optimized separately, we attempted to perform them simultaneously in oneꢀpot
since both enzymes could work at the same pH. Hence, the EREDꢀcatalyzed bioreductions were tried
under magnetic stirring, as we have reported that the alcohol oxidations work better under these
conditions.28b We studied the reduction of 1b as model substrate with EREDꢀ110 at pH 5, and
satisfyingly it was observed that quantitative conversion into ketone 1c was achieved after just 6 h (see
Supporting Information). Therefore, encouraged by this promising result, we applied the whole cascade
starting from allylic alcohol (±)ꢀ1a with all necessary reagents and catalysts (LTv‒TEMPO, EREDꢀ110,
NADP+, glucose and GDH) in citrate buffer 50 mM pH 5 in an openꢀtoꢀair vessel (see Supporting
Information). After 16 h we observed complete conversion into unsaturated ketone 1b, but the final
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product 1c was not detected. Consequently, it was obvious that the oxidative step was interfering with
the correct outcome of the bioreduction reaction.
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In order to investigate this problem, the EREDꢀ110ꢀcatalyzed reduction of 1b was performed in an open
vessel, compulsory for the oxidation reaction to avoid oxygen limitation. Under these conditions, only
41% of the saturated ketone 1c was attained after 16 h. Although this effect clearly diminished the
activity of the enzyme,37 still some conversion was observed, unlike the twoꢀstep protocol. Therefore,
another effect should deter the bioreduction process. In fact, when we repeated the cascade starting from
(±)ꢀ1a in a closed tube, formation of 1c was not observed, and the reaction stopped at the intermediate
stage again. It was suspected that TEMPO could inhibit the reduction, therefore, a series of experiments
were performed to study in more detail its effect (data not shown), observing that TEMPO was not
compatible with the glucose‒GDH system required for the NADPH recycling. It has been previously
described that the laccase‒TEMPO system can oxidize monoꢀ and disaccharides at the primary
position,38 thus interfering with the GDH activity. Other NADPꢀrecycling systems such as glucoseꢀ6ꢀ
phosphate‒glucoseꢀ6ꢀphosphate dehydrogenase (Gꢀ6ꢀPꢀDH) or isocitrate‒isocitrate dehydrogenase
(IDH) were also attempted, including the use of stoichiometric amounts of a cofactor mimic such as 1ꢀ
benzylꢀ1,4ꢀdihydronicotinamide,39 but TEMPO hampered the bioreduction process as well.
Due to these incompatibilities, we decided to develop a oneꢀpot twoꢀstep sequential strategy. To check
whether we could directly add the reagents and catalysts of the second reaction right after the oxidation,
a simple experiment was setꢀup. Racemic allylic alcohol (±)ꢀ2a was supplied after incubation of the
laccase‒TEMPO system for 16 h at 30 ºC, observing that after 6 h of reaction, ketone 2b was only
formed in 20% conversion. Compared to the standard reaction (99% conversion after 6 h), it remained
clear that TEMPO lost most of its activity. Hence, after the oxidation of the racemic allylic alcohols
with the LTv‒TEMPO system, glucose, GDH, NADP+, and the corresponding ERED were added to
(stereoꢀ and) chemoselectively reduce the C=C double bond.40 The results obtained are shown in Table
3.
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Table 3. Bienzymatic oneꢀpot twoꢀstep sequential redox isomerization of racemic allylic alcohols.a
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Entry
Substrate
(mM)
ERED
Ratiob
1bꢀ13b
(%)
<1
ee or de
1cꢀ13c
(%)c
1aꢀ13a
(%)
<1
1cꢀ13c
(%)
>99
>99
>99
99
1
2
(±)ꢀ1a (40)
(±)ꢀ2a (40)
(±)ꢀ3a (40)
(±)ꢀ4a (40)
(±)ꢀ5a (40)
(±)ꢀ6a (40)
(±)ꢀ7a (30)
(±)ꢀ8a (30)
(±)ꢀ9a (40)
(±)ꢀ10a (30)
(±)ꢀ11a (30)
(±)ꢀ12a (30)
(5R)ꢀ13a (40)
110
n.a.
P1ꢀA04
P1ꢀE01
P1ꢀE01
P1ꢀA04
P1ꢀE01
P1ꢀE01
103
<1
<1
n.a.
3
<1
<1
88 (R)
>99 (S)
n.a.
4
<1
1
5
<1
<1
>99
>99
>99
>99
>99
99
6
<1
<1
n.a.
7
<1
<1
n.a.
8d
9d
10d
11d
12d
13e
<1
<1
n.a.
P1ꢀA04
P1ꢀE01
P1ꢀE01
P1ꢀE01
110
<1
<1
n.a.
<1
1
n.a.
<1
<1
>99
>99
87
n.a.
<1
<1
92 (S)
2
11
95 (2R,5R)
a For reaction conditions, see the Experimental Section. b Determined by GC (see Supporting Information).
c
d
Determined by achiral or chiral GC or HPLC (see Supporting Information). 20% v/v of MTBE was
added to the reaction mixture in the first step to enhance substrate solubilization. e The oxidation step took
32 h, as described in Table 1. n.a.: not applicable as these substrates are not chiral.
Thus, substrates (±)ꢀ1aꢀ12a and (5R)ꢀ13a (30ꢀ40 mM) were quantitatively oxidized to the
corresponding α,βꢀunsaturated ketones after being treated with the laccase‒TEMPO system during 16 h
under the optimized conditions shown in Table 1 [except for alcohol (5R)ꢀ13a, 32 h]. Afterwards, the
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bioreduction of these intermediates was achieved after 6 h following the conditions previously described
in Table 2. For compounds (±)ꢀ8aꢀ12a, as previously mentioned, MTBE (20% v/v) was necessary to
ensure substrate solubilization. Satisfyingly, for most examples complete conversions into the saturated
ketones were attained, detecting neither the starting material nor the unsaturated ketone intermediates.
Moreover, chiral ketones 3c, 4c, 12c, and 13c were accessed with similar selectivities than those shown
in Table 2, ranging from excellent for βꢀsubstituted ketone (S)ꢀ4c to very high for αꢀsubstituted ketones
(R)ꢀ3c, (S)ꢀ12c, and (2R,5R)ꢀ13c. Globally, these examples can be considered as enzymatic
stereoselective redox isomerizations of allylic alcohols. Compared to the methodology previously
described combining an ADH and an ERED,33 this system presents some advantages. In the
ADH‒ERED protocol, after reaction optimization, (±)ꢀ2a (10 mM) was transformed into ketone 2c in
good chemoselectivity (>90%), although a small quantity of the saturated alcohol was still detected.
Herein substrate concentrations were higher and selectivities were perfect in all cases.
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Finally, to demonstrate the applicability of this strategy, preparative biotransformations (50ꢀmg scale)
were carried out under the optimized reaction conditions for alcohols (±)ꢀ4a and (±)ꢀ6a (at 40 mM) and
(±)ꢀ12a (at 30 mM). In these cases, longer reaction times were required for the bioreduction step (24 h)
to achieve quantitative conversions into the final ketones, that were isolated in good to excellent yields
and optical purities after extraction, (S)ꢀ4c (69% yield, >99% ee), 6c (85% yield), and (S)ꢀ12c (87%
yield, 91% ee).
Conclusions
The stereoselective redox isomerization of allylic alcohols into enantioenriched chiral ketones has been
scarcely proposed via metalꢀcatalyzed transformations. Herein we show a biocatalytic counterpart
merging the action of two different oxidoreductases: a laccase from Trametes versicolor, used to recycle
the chemical oxidant TEMPO which catalyzes the oxidation of a series of allylic alcohols into the
corresponding α,βꢀunsaturated ketones; and an eneꢀreductase, enzyme responsible of the chemoꢀ and
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stereoselective reduction of the C=C double bond of these intermediates, giving access to the final
saturated ketones.
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The oxidative method is especially convenient as it can be performed at room temperature in aqueous
medium and uses oxygen as final electron acceptor, releasing water as the sole coproduct. In addition,
the bioreduction of the α,βꢀunsaturated ketones could be carried out under identical reaction conditions
to those used in the biooxidation step, aiming for a oneꢀpot process. However, incompatibilities between
TEMPO and the cofactor recycling system used in the EREDꢀcatalyzed transformation hampered the
development of this process in a cascade manner, and consequently both enzymatic steps were
combined in sequential oneꢀpot twoꢀstep approach. Under these conditions, different allylic alcohols
were quantitatively transformed into the corresponding saturated ketones, obtaining in some cases chiral
carbonylic derivatives with high enantioꢀ or diastereomeric excess (>88%), even at preparative scale.
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Experimental Section
General methods
NMR spectra were recorded on a 300 MHz spectrometer. Gas chromatography (GC) analyses were
performed on standard GC chromatographs equipped with a FID detector. Thinꢀlayer chromatography
(TLC) was conducted with silica gel precoated plates and visualized with UV and potassium
permanganate stain. Column chromatography was performed using silica gel (230ꢀ400 mesh).
General protocol for the oxidation of allylic alcohols with the LTv‒TEMPO system. In an openꢀ
toꢀair test tube, TEMPO (4.1 mg, 33 mol%) was added to a solution of the racemic allylic alcohol (±)ꢀ
1aꢀ12a or (5R)ꢀ13a (0.08 mmol, 40 mM) in citrate buffer 50 mM pH 5 (2 mL). For substrates (±)ꢀ8aꢀ
12a, MTBE (400 ꢁL) was also added for solubility reasons. The reaction mixture was stirred for a few
minutes to dissolve all the reagents and then the laccase from Trametes versicolor was added (5 U). The
reaction was maintained under magnetic stirring at 30 ºC for 16 h. After this time, the reaction crude
was extracted with EtOAc (2 x 2 mL). The organic layers were combined, dried over Na2SO4, and an
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aliquot was taken for determination of conversion values by GC analysis (see Table 1). In the case of
(5R)ꢀ13a, after the first 16 h, TEMPO (4.1 mg, 33 mol%) and laccase from Trametes versicolor (5 U)
were added again. The reaction was stirred under the same conditions for additional 16 h and then was
treated as explained above.
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General procedure for the (stereoselective) reduction of α,βꢀunsaturated ketones catalyzed by
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EREDs. In a Falcon tube, a stock solution containing Dꢀ(+)ꢀglucose (144 mg, 0.8 mmol), GDHꢀ105 (20
mg), and NADP+ (5 mg, 6 µmol) in 9 mL of phosphate buffer 100 mM pH 7 or citrate buffer 50 mM pH
5 was prepared. The resulting mixture was stirred for a few minutes, and then 1 mL was added in a 2ꢀ
mL Eppendorf vial containing the corresponding ERED (10 mg). Then, substrate 1bꢀ13b (final
concentration: 40 mM) was added. The reaction was shaken at 30 ºC and 250 rpm for 24 h, and then
extracted with EtOAc (2 x 0.5 mL). The organic layers were combined and dried over Na2SO4.
Conversions and enantiomeric excess were determined by GC or HPLC (see Table 2).
General procedure for the sequential oneꢀpot twoꢀstep synthesis of (chiral) saturated ketones
starting from racemic allylic alcohols. In an openꢀtoꢀair test tube, TEMPO (4.1 mg, 33 mol%) was
added to a solution of the racemic allylic alcohol (±)ꢀ1aꢀ12a or (5R)ꢀ13a (0.08 mmol; 40 mM in the
buffer medium for substrates (±)ꢀ1aꢀ6a, (±)ꢀ9a and (5R)ꢀ13a; 30 mM in the buffer medium for
substrates (±)ꢀ7a, (±)ꢀ8a, (±)ꢀ10aꢀ12a) in citrate buffer 50 mM pH 5 (for substrates (±)ꢀ1aꢀ7a and (5R)ꢀ
13a), or in a biphasic mixture of citrate buffer 50 mM pH 5 and MTBE (20% v/v) (for substrates (±)ꢀ8aꢀ
12a). The reaction mixture was stirred for a few minutes to dissolve all the reagents, and then the
laccase from Trametes versicolor (5 U) was added. The reaction was maintained under magnetic stirring
for 16 h at 30 ºC, observing the complete evaporation of MTBE when it was used. To the resulting
reaction crude, D
ꢀ(+)ꢀglucose (29 mg, 0.16 mmol), GDHꢀ105 (20 U/mL), NADP+ (0.6 mM), and the
corresponding ERED (20 mg) were added. The test tube was closed and the reaction was maintained
under magnetic stirring at 30 ºC for 6 h, and then extracted with EtOAc (2 x 2 mL). The organic layers
were combined, dried over Na2SO4 and centrifuged to obtain the supernatant. Conversion (and
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enantiomeric excess) values into the corresponding saturated ketones 1cꢀ13c were determined by GC or
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HPLC analysis (see Table 3).
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Supporting Information. The Supporting Information is available free of charge on the ACS
Publications website at DOI: xxxxx.
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Chemical synthesis and characterization of starting materials for enzymatic reaction, extensive ERED
screenings, optimization of individual and multienzymatic protocols, analytics, copies of HPLC chiral
analyses, and 1H and 13C NMR spectra for described organic compounds.
Acknowledgments
Dedicated to Professor Miguel Yus in occasion on his 70th birthday.
Financial support from MICINN (Project CTQ2013ꢀ44153ꢀP and CTQ2016ꢀ75752ꢀR) and the Regional
Government from the Principado de Asturias (Project FCꢀ15ꢀGRUPIN14ꢀ002) are gratefully
acknowledged. L.M.ꢀM. thanks the Regional Government from the Principado de Asturias for her
predoctoral fellowship Severo Ochoa.
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[RuClCp(PTA)2],
[RuClCp(HPTA)2]Cl2·2H2O, [RuCpꢀ(DMSOꢀκS)(PTA)2]Cl, [RuCp(DMSOꢀκS)(PTA)2](OSO2CF3)
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glucose, and GDH were supplied. In our case this was not necessary.
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