Strengthening the Combination between Enzymes and Metals in Aqueous Medium: Concurrent Ruthenium-Catalyzed Nitrile Hydration - Asymmetric Ketone Bioreduction

Article abstract of DOI:10.1002/cctc.201801005

A dual ruthenium/ketoreductase catalytic system has been developed for the conversion of β-ketonitriles into optically active β-hydroxyamides through an unprecedented hydration/bioreduction cascade process in aqueous medium working in concurrent mode. The ketoreductase-mediated ketone reduction took place with exquisite stereoselectivity and it was simultaneous to the nitrile hydration promoted by the ruthenium catalyst. The overall transformation occurred: (i) employing commercially and readily available catalytic systems (ii) under mild reaction conditions, (iii) with high degree of conversion and excellent stereoselectivity, and (iv) without the need to isolate intermediates and with high final product yields. This genuine process demonstrates the benefits of combining metal and enzymatic catalysis to tackle the limitations arising from each field.

Full text of DOI:10.1002/cctc.201801005

Full Papers
DOI: 10.1002/cctc.201801005
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Strengthening the Combination between Enzymes and
Metals in Aqueous Medium: Concurrent
Ruthenium-Catalyzed Nitrile Hydration - Asymmetric
Ketone Bioreduction
+ [a]
+ [b]
[a]
[a]
´
´
´
´
´
´
Elisa Liardo , Rebeca Gonzalez-Fernandez , Nicolas Rıos-Lombardıa, Francisco Morıs,
[b]
[b]
[b]
[c]
´
´
´
Joaquın Garcıa-Alvarez, Victorio Cadierno, Pascale Crochet,* Francisca Rebolledo,* and
[a]
´
´
Javier Gonzalez-Sabın*
A dual ruthenium/ketoreductase catalytic system has been
developed for the conversion of b-ketonitriles into optically
active b-hydroxyamides through an unprecedented hydration/
bioreduction cascade process in aqueous medium working in
concurrent mode. The ketoreductase-mediated ketone reduc-
tion took place with exquisite stereoselectivity and it was
simultaneous to the nitrile hydration promoted by the ruthe-
nium catalyst. The overall transformation occurred: (i) employ-
ing commercially and readily available catalytic systems (ii)
under mild reaction conditions, (iii) with high degree of
conversion and excellent stereoselectivity, and (iv) without the
need to isolate intermediates and with high final product yields.
This genuine process demonstrates the benefits of combining
metal and enzymatic catalysis to tackle the limitations arising
from each field.
26 Introduction
and biological catalysts in aqueous media, the natural environ-
ment of enzymes. Although these hybrid systems are still in
their infancy, the wider available pool of water-compatible
metal catalysts[4] along with the advances in protein engineer-
ing to produce more stable enzymes have enabled some
remarkable examples of this kind.[5] Nevertheless, when plan-
ning such a chemo-enzymatic strategy a critical pitfall is the
coexistence of both catalysts leading to: a) reciprocal poisoning,
b) degradation because of additives, cofactors, or cosolvents,
and c) incompatibility of reaction conditions.[6] As a result, most
of these hybrid reactions run sequentially or, alternatively, the
catalysts are site-isolated by compartmentalization or encapsu-
lation techniques.[6c,7] Actually, the number of concurrent
chemoenzymatic processes with concomitant action of metal
and enzyme are still very scarce,[8] and include the pioneering
27
28 The development of one-pot multistep catalytic processes is an
29 emerging trend within the synthetic chemistry to produce high
30 added-value chemicals in a more efficient and sustainable
31 manner.[1] To this end, by mimicking natural biosynthetic
32 pathways in living organisms, chemists have successfully
33 implemented several biocatalytic reactions into non-natural
34 synthetic cascades in both in vivo and in vitro approaches.[2]
35 Similarly, a plethora of synthetic steps mediated by chemical
36 catalysts, mainly transition-metal catalysts, have been coupled
37 in organic media.[3] Now, another turn of the screw in
38 constructing catalytic networks consists of combining chemical
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[a] Dr. E. Liardo,+ Dr. N. Rꢀos-Lombardꢀa, Dr. F. Morꢀs, Dr. J. Gonzꢁlez-Sabꢀn
dynamic kinetic resolution (DKR) processes combining
racemizing metal catalyst with an enzymatic transformation,[8a]
ruthenium-catalyzed olefin metathesis coupled with
a
EntreChem SL
Vivero Ciencias de la Salud
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Santo Domingo de Guzmꢁn 33011 (Spain)
E-mail: jgsabin@entrechem.com
monoamine oxidases and P-450 enzymes,[8c,f] a simultaneous
ruthenium-catalyzed isomerization of allylic alcohols-bioreduc-
tion,[8e] and the oxidation of cyclic amines to lactams by a
monoamine oxidase and CuI/H2O2.[8d]
[b] R. Gonzꢁlez-Fernꢁndez,+ Prof. J. Garcꢀa-ꢂlvarez, Prof. V. Cadierno,
Prof. P. Crochet
Laboratorio de Compuestos Organometꢁlicos y Catꢁlisis (Unidad asociada
al CSIC)
Centro de Innovaciꢃn en Quꢀmica Avanzada (ORFEO-CINQA)
Departamento de Quꢀmica Orgꢁnica e Inorgꢁnica
Universidad de Oviedo
In this context, two different approaches have been recently
reported for converting b-ketonitriles into b-hydroxyamides by
cascade processes in aqueous media combining a nitrile
hydration and a ketone reduction (Scheme 1). In one case, a
dual enzymatic system consisting of a ketoreductase (KRED)
and a nitrile hydratase from whole cells of R. rhodochrous
grown in the presence of DEPA (an inhibitor of amidase activity)
was used.[9] In this asymmetric version, the first step is the KRED
catalyzed dynamic reductive kinetic resolution of the ketone to
a b-hydroxynitrile in very high enantio- and diastereomeric
Oviedo E-33006 (Spain)
E-mail: crochetpascale@uniovi.es
[c] Prof. Dr. F. Rebolledo
Departamento de Quꢀmica Orgꢁnica e Inorgꢁnica
Universidad de Oviedo
Oviedo E-33006 (Spain)
E-mail: frv@uniovi.es
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cctc.201801005
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Scheme 2. Ru(IV) complex 1 catalyzed hydration of benzonitrile (2) in the
reaction medium of KREDs.
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Next, the focus was on the compatibility of both catalytic
systems. Accordingly, two model compounds, namely benzoni-
trile (2) and propiophenone (4) which are substrates for each
catalyst respectively, were put together and subjected to the
simultaneous action of a ketoreductase (KREDÀP1-A04) and the
ruthenium complex 1 in the previous buffer at 200 mM
concentration (Scheme 3). Pleasantly, both hydration and
Scheme 1. One-pot tandem processes for converting b-ketonitriles into b-
hydroxyamides.
20 ratio. As the b-hydroxynitrile is formed, the nitrile hydratase
21 catalyzes the water addition enabling the corresponding b-
22 hydroxyamides with excellent stereoselectivity (route a).[10] On
23 the other hand, a concomitant hydration/transfer hydrogena-
24 tion from b-ketonitriles using a single ruthenium(II) complex
25 (route b) was recently reported.[11]
Scheme 3. Compatibility test for the catalytic systems: hydration of benzoni-
trile (2) and reduction of propiophenone (4) catalyzed by the Ru complex 1
and KREDÀP1-A04, respectively.
26
Despite the merit of these concurrent cascade processes,
27 some issues remained challenging; for route a (enzymatic) the
28 amidase activity was not completely inhibited and the resulting
29 mixture of b-hydroxyamide and b-hydroxyacid required chro-
30 matographic purification which decreased the yield,[12] while for
31 route b (metal-catalyzed) the process was performed as a non-
32 asymmetric version, and suffered from harsh reaction condi-
33 tions (1008C and excess of sodium formate) to achieve carbonyl
34 reduction efficiently. On the light of this background, we
35 envisioned a hybrid catalytic system combining an enzymatic
36 ketone reduction with a ruthenium-catalyzed nitrile hydration,
37 to exploit the synergistic effect of each catalysis area and
38 bypass the drawbacks just mentioned.
reduction reactions occurred with conversions >99% after 24 h
at 408C, and the enantiomeric excess (ee) measured for the
resulting (R)-1-phenylpropan-1-ol (5) was >99%. Seeing as the
perfect coexistence of catalysts and tolerance of their reaction
conditions, these findings reveal a compatibility window for the
two synthetic steps, and pave the way for a new tandem
catalytic process.[16]
Anticipated as an important challenge in the present work
is the level of orthogonality between the two processes.[17]
Specifically, the KRED could reduce not only the starting b-
ketonitrile but also the hypothetical b-ketoamide delivered if
the nitrile hydration by the ruthenium complex takes place first.
As a result, the optical purity of the target b-hydroxyamide
would depend on the stereoselectivity of the KRED towards the
two species. Nevertheless, selectivity could be envisaged if one
of the catalysts was much more reactive than the other, which
remains active towards the formed intermediate. With these
premises, we selected b-ketonitrile 6a and assessed the scope
of the reduction by using a commercial kit of KREDs (Codexꢁ
KRED Screening Kit)[18] and two enzymes overexpressed in E.
coli with opposite stereoselectivity, namely the (R)-selective
ADH from Lactobacillus kefir[19] and the (S)-selective from
Rhodococcus ruber.[20] Likewise, the reduction of the respective
b-ketoamide 6b was also considered. In general, both purified/
overexpressed KREDs displayed higher activity towards 6a and
it was possible to identify biocatalysts which gave rise to both
(R)- and (S)-enantiomers with c>99% and ee>99%. On the
contrary, KREDs showed poor activity towards 6b,[21] and only a
few hits joined high conversion and high enantioselectivity. As
an example, KREDÀP2-H07 showed an excellent activity with
39
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41 Results and Discussion
42
43 The first aspect explored was the identification of a medium
44 that balances the requirements for each catalytic reaction type.
45 While the ruthenium-catalyzed hydration of nitriles is usually
46 performed in pure water,[13] the bioreduction requires an
47 aqueous medium consisting of a phosphate buffer pH 7.0
48 (125 mM KPi, including 1.25 mM MgSO4, and 1.0 mM NADP+)
49 and i-PrOH (10%v/v) for recycling the cofactor and driving the
50 equilibrium towards the product. Thus, after assessing a series
51 of ruthenium(II) and (IV) complexes for the hydration of a
52 typical substrate, benzonitrile (2), in the medium required for
53 the KRED, complex
1 emerged as the best candidate
54 (Scheme 2).[14] The treatment of 2 (200 mM) with 3 mol% of 1,
55 at 408C for 24 h, led to benzamide (3) with >95% of
56 conversion (c).[15]
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6a, but very poor with 6b (Scheme 4). Simultaneously, we also
checked the activity of Ru complex 1 in the hydration of 6a
together from the beginning. As expected, the starting 6a
disappeared but a 79:21 mixture of enantiomerically pure (R)-
6c and (R)-6d was obtained, which shows a high efficiency of
the enzyme and a loss of activity of the metallic catalyst during
the process. Because the hydration effectiveness of the complex
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1 is significantly improved at higher temperatures (60–
1008C),[14] the hydrations of 6a and 6c were tested in buffer
medium at 608C, conditions where KREDs also maintain their
catalytic activity. As in both cases the conversions >99% into
amide were observed, the concurrent approach was designed
by using a biocatalyst displaying matched stereopreference
towards the b-ketonitrile/b-ketoamide pair. Gratifyingly,
KREDÀP2-C11 met these requirements catalyzing the formation
of (R)-6c and (R)-6d with high conversions and ee >99%. After
thorough medium engineering, optimal conditions consisted of
carrying out the reaction at 608C, as well as at 100 mM
substrate concentration. In this way, (R)-6d was finally obtained
from 6a with 97% of conversion and ee>99% with metal and
biocatalyst coexisting throughout the entire process (Table 1,
entry 1). Moreover, although the hydration reaction was para-
metrized under argon atmosphere (see Table S1 in the SI),[11] the
concurrent process yielded identical results without the need
for an inert atmosphere. Likewise, and to assess the possible
Scheme 4. Testing pathways for the tandem bioreduction-nitrile hydration of
b-ketonitrile 6a. All reactions in buffer medium at 408C and 200 mM.
18 and 6c at 408C. While 6c was completely converted into the b-
19 hydroxyamide 6d, compound 6a was recovered mostly
20 unaltered.
21
Accordingly, a preliminary concurrent tandem catalysis was
22 essayed on b-ketonitrile 6a (200 mM) by the combined action
23 of KREDÀP2-H07 and complex 1 (3 mol%) at 408C, working
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Table 1. Orthogonal tandem ruthenium catalyzed nitrile hydration-enzymatic reduction in aqueous medium by combining 1 and KREDs.[a]
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Entry
1
Substrate
Enzyme
P2-C11
t [h]
a [%][b]
b[%][b]
c [%][b]
d [%][b]
>99
ee for d [%][c],[d]
16
–
–
–
>99 (R)
2
3
P2-C11
P2-C11
16
18
3
–
–
9
–
97
74
>99 (R)
>99 (R)
17
4
5
P2-G03
L. kefir
18
24
–
–
–
11
–
2
>99
87
>99 (R)
>99 (R)
6
P2-G03
18
–
–
5
95
>99 (R)
7
P1-B10
18
–
7
10
83
>99 (S)
[a] Reaction conditions: 6a–11 a (100 mM) in KPi buffer 125 mM (1.25 mM MgSO4, 1.0 mM NADP+) pH 7.0, i-PrOH (10% v/v), complex 1 (6 mol%), KRED (100%
w/w, mg of enzyme powder per mg of substrate) and stirring at 608C. For entry 5, 9a (100 mM) in KPi buffer 50 mM (1.0 mM MgCl2, 1.0 mM NADP+) pH 7.0, i-
PrOH (10% v/v), complex 1 (6 mol%), L. kefir (33 U/mg of substrate) and stirring at 608C; [b] Ratio of starting material (a), b-ketoamide (b), b-hydroxynitrile (c),
and b-hydroxyamide (d) was measured by NMR and HPLC; [c] Measured by chiral HPLC; [d] Absolute configuration established as detailed in the SI.
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promiscuous activity of one or either catalyst for the alternative
reaction, blank runs accomplished with b-ketonitrile 6a i) in the
absence of complex 1 and ii) in the absence of KRED, yielded
the b-hydroxynitrile 6c or the b-ketoamide 6b as the sole
product, respectively (c>99%).
towards b-ketonitriles with respect to b-ketoamides. Accord-
ingly, the reduction of b-ketonitriles 6a–11 a was accomplished
in the conditions of the previous screening by choosing
stereoselective KREDs. Once the enzymatic step was complete
at 608C (18–24 h), complex 1 (6 mol%) was added and the
reaction mixture stirred overnight at the same temperature. As
a result, both enantiomers of 6d–11 d were isolated in
essentially pure form (c>99%) and ee>99%. Table 2 collects
some representative examples, including the counterpart of
those missing in Table 1.
Encouraged by the success of this dual catalytic system, we
extended the methodology to b-ketonitriles 7a–11 a (Table 1).
Thereby, by using the appropriate KRED and a higher catalyst
loading (6 mol%),[22] it was possible to reach the (R)-enantiomer
10 of most b-hydroxyamides (6d–10d) as well as (S)-11 d in
11 enantiopure form and high degree of conversion (entries 1–7).
12 Although it was challenging to find KREDs meeting the
13 requirements to prepare the hydroxyamide antipodes, the
14 examples compiled in Table 1 are a valuable proof of concept
15 of the successful simultaneous action of enzymes and metal
16 catalysts. Moreover, it is worth highlighting the excellent
17 outcome of the process in several cases, which allowed us to
18 isolate the resulting b-hydroxyamides with very high yield (92–
19 94% for entries 1, 2, and 4). Particularly, the null or very low
20 activity of the overexpressed enzymes towards b-ketoamides,
21 with the exception of 9b (see Tables S2–S13 in the SI),
22 precluded their use in concurrent processes. Nonetheless, the
23 simultaneous catalytic activity of 1 and Lactobacillus kefir on
24 the b-ketonitrile 9a rendered the target b-hydroxyamide (R)-9d
25 in enantiopure form and conversion close to 90% (entry 5).
Additionally, both cyclic and acyclic b-ketonitriles bearing a
stereogenic carbon (12a–15a) were also considered (Table 3).
The main feature of these compounds relies on the lability of
their chiral center which is prone to racemize in the reaction
medium, hence triggering a dynamic reductive kinetic reso-
lution (DYRKR).[24] This fact along with the proven poor activity
of the KREDs towards the corresponding b-ketoamides 12b–
15b,[18] precluded the option of a concurrent process and
turned our attention towards the sequential protocol. The first
step – KRED reduction – was carried out at 308C applying the
conditions already reported for 13a–15a.[10] Moreover, epimeri-
zation of the intermediate b-hydroxynitrile 12c at 608C was
observed, so the bioreduction step of 12a should also be run
at lower temperature. With these premises, the reduction of
12a, 14a, and 15a were carried out with the selected KRED
(Table 3) at pH 7.0 and 308C and quantitatively afforded the
syn-12c and cis-14c-15c stereoisomers in >99: <1 dr and
>99% ee. To obtain the analogous 13c with high diastereose-
lectivity, the bioreduction of 13a was conducted at pH 5.0.
Finally, after proving that the Ru complex 1 catalyzed the
hydration of 13c more efficiently at pH 5.0 than 7.0, the Ru-
catalyzed second step was carried out at this pH value in all
cases. No epimerization of any chiral center was observed in
the second step, being the optically active a-substituted b-
hydroxyamides 12d–15d isolated with the same dr and ee
26
For those cases with mismatched KRED stereoselectivity
27 towards the b-ketonitrile/b-ketoamide pair or incomplete
28 conversion, a practical solution would be a sequential one-pot/
29 two-step process. Although less elegant than the concurrent
30 one with both steps running simultaneously, the stepwise
31 approach offers essentially the same advantages and low
32 ecological footprint.[23] Actually, the only setting would be the
33 addition of the ruthenium complex 1 once the bioreduction
34 was completed. Concerning the order of the steps, this was
35 established in view of the superior activity displayed by KREDs
36
37
Table 2. Sequential enzymatic reduction-ruthenium catalyzed nitrile hydration in aqueous medium by combining 1 and KREDs.[a]
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Entry
Substrate
KRED
Overall conv.
[%][b]
ee
[%][c],[d]
1
2
3
4
5
6
7
8
9
6a
6a
7a
8a
9a
P3-H12
R. ruber
P3-B03
P3-H12
P1-H08
L. kefir
P1-B10
L. kefir
R. ruber
P2-G03
L. kefir
>99
97
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (R)
>99
>99
>99
>99
>99
80
85
>99
75
9a
10a
10a
10a
11 a
11 a
10
11
[a] Reaction conditions: For purified enzymes, b-ketonitrile (100 mM), KRED (100% w/w), i-PrOH (10% v/v), KPi buffer 125 mM (1.25 mM MgSO4, 1.0 mM
NADP+) and shaking at 250 rpm and 608C; For L. kefir reactions, b-ketonitrile (100 mM), L. kefir (33 U/mg of substrate), i-PrOH (10% v/v), KPi buffer 50 mM
(1.0 mM MgCl2, 1.0 mM NADP+) pH 7.0 and shaking as above; For R. ruber reactions, b-ketonitrile (100 mM), R. ruber (11 U/mg of substrate), i-PrOH (10%v/v),
KPi buffer 50 mM (1.0 mM NAD+) pH 7.0 and shaking as above. After 24 h, complex 1 (6 mol%) was added and the mixture stirred at 608C; [b] Conversion for
the first step was complete in all cases; [c] Measured by chiral HPLC; [d] Absolute configuration established as detailed in the SI.
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Table 3. A sequential one-pot dynamic reductive kinetic resolution
hydration of a-substituted b-ketonitriles 12a–15a.[a]
–
3
4
5
6
7
8
9
Entry
1
Substrate
KRED
Product[b]
dr[c]
ee [%][c]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
P3-H12
(2R,3S)-12d
>99: <1
>99
Scheme 5. Synthesis of (S)-(+)-tembamide and (R)-(À)-aegeline.
Conclusions
2
3
P2-D11
P1-B12
(1S,2S)-13d
(1S,2R)-13d
15:85
80:20
>99
>99
In summary, we have developed a concurrent tandem-type
cascade in aqueous medium which provides enantiopure b-
hydroxyamides from b-ketonitriles by the combination of a
Ru(IV)-catalyzed nitrile hydration with a biocatalytic reduction.
Likewise, a complementary sequential cascade was applied to
b-ketonitriles bearing a chiral center. In this case, the obtained
products exhibited very high diastereo- and enantiomeric
excesses as a result of a selective DYRKR. The genuine mutual
compatibility of catalysts reported herein, a frequent recalci-
trant challenge, is an excellent proof of concept of the practical
value of chemoenzymatic one-pot processes for synthetic
chemists. Although immense advances are being made in areas
such as immobilization and compartmentalization, it is no less
true that metal catalysis and biocatalysis are not antagonistic
areas and can efficiently coexist in a synergic fashion. The key
to the success is probably related with the use of ruthenium(IV)
catalysts, which show a compatibility with enzymes far superior
to that observed with their ruthenium(II) congeners. Finally, the
enantiopure alkaloids (S)-(+)-tembamide and (R)-(À)-aegeline
were efficiently prepared by means of a simple synthetic
sequence in very high yields.
4
5
P1-B12
P1-A04
(1S,2R)-14d
(1R,2S)-14d
>99: <1
>99: <1
>99
>99
6
P1-A04
(1R,2S)-15d
>99: <1
>99
[a] Reaction conditions: 12a–15a (100 mM), KPi buffer 125 mM (1.25 mM
MgSO4, 1.0 mM NADP+) pH 7.0 (for 12a, 14a, and 15a) or pH 5.0 (for 13a),
i-PrOH (10% v/v), KRED (100% w/w) and 30 8C. Once completed the
reduction, addition of 1 (6 mol%) and conc. H3PO4 until pH 5.0, and stirring
at 608C; [b] Absolute configuration established as detailed in the SI; [c]
Diastereomeric ratio (dr) and ee measured by chiral HPLC or GC.
30 (Table 3) than the corresponding b-hydroxynitrile intermedi-
31 ate.[25] As a result, and by means of the two synthetic
32 approaches developed herein (concurrent or sequential mode),
33 the joint action of a ruthenium complex and a ketoreductase
34 allowed to address the gaps of the prior methodologies which
35 were based on enzymatic[10] or metal catalysis alone.[11] Specifi-
36 cally, the reduction step was now smoothly accomplished by
37 the enzyme, leading to optically pure hydroxy-compounds,
38 while the metal complex promoted a highly efficient nitrile
39 hydration enabling the b-hydroxyamides without tedious
40 purification.
Experimental Section
41
By last, to illustrate the usefulness of this methodology the
General Procedure for the Conversion of b-Ketonitriles
6a-11a Into b-Ketoamides 6d-11d in a Concurrent Fashion
42 chemoenzymatic syntheses of the naturally occurring alkaloids
43 (S)-(+)-tembamide (active against HIV)[26] and (R)-(À)-aegeline
44 (hypoglycemic activity) were designed.[27] Starting from 3-(4-
45 methoxyphenyl)-3-oxopropanenitrile (8a), the sequential one-
46 pot reaction of KREDÀP2-C11 or KREDÀP3-H12 and complex 1
47 (6 mol%) gave access to the key enantiopure precursors (R)-8d
48 and (S)-8d, respectively, in very high yields (93%). Then, a
49 Hofmann rearrangement of these b-hydroxyamides with iodo-
50 benzene diacetate in acetonitrile at 50 8C gave the oxazolidi-
51 nones (S)-16 or (R)-16 through an intramolecular attack of the
52 hydroxyl group to the isocyanate intermediate (Scheme 5).
53 Finally, basic hydrolysis of each enantiomer of 16 and
54 subsequent treatment of the resulting b-amino alcohol with
55 the required acid chloride enabled the isolation of (S)-
56 (+)-tembamide[28] or (R)-(À)-aegeline in 77% and 73% overall
57 yield, respectively, and >99% ee in both cases.
In
a 2.0 mL Eppendorf tube, the corresponding b-ketonitrile
(100 mM), KRED (100% w/w; mg of enzyme powder per mg of
substrate), complex 1 (3.0 mol% for 6a, 6.0 mol% for 7a–11a), i-
PrOH (10% v/v) and 125 mM phosphate buffer (also containing
1.25 mM MgSO4 and 1.0 mM NADP+) pH 7.0 were added. The
resulting reaction mixture was shaken at 250 rpm and 608C for 16–
24 h. After this time, the mixture was extracted with ethyl acetate
(2ꢂ500mL), centrifugated (90 s, 13000 rpm), and the combined
organic layers were finally dried over Na2SO4. The degree of
1
conversion was measured by H-NMR. The enantiomeric excess of
the corresponding product was determined by chiral HPLC or GC.
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100–103; c) A. Gꢆmez-Baraibar, C. Reichert, C. Mꢅgge, S. H. Seger, H.
Grçger, R. Kourist, Angew. Chem. Int. Ed. 2016, 55, 14823–14827; Angew.
Chem. 2016, 128, 15043–15047.
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General Procedure for the Conversion of b-Ketonitriles 6a–
11a Into b-Ketoamides 6d-11d in a Sequential Fashion
[8] The only examples of concurrent chemo- and biocatalysis include: a) O.
Verho, J.-E. Bꢇckvall, J. Am. Chem. Soc. 2015, 137, 3996–4009; b) F. G.
Mutti, A. Orthaber, J. H. Schrittwieser, J. G. de Vries, R. Pietschnig, W.
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Bornscheuer, Nat. Catal. 2018, 1, 12–22.
In
a 2.0 mL Eppendorf tube, the corresponding b-ketonitrile
(100 mM), KRED (100% w/w; mg of enzyme powder per mg of
substrate), i-PrOH (10% v/v) and 125 mM phosphate buffer pH 7.0
(also containing 1.25 mM MgSO4, 1.0 mM NADP+) were added. The
resulting reaction mixture was shaken at 250 rpm and 608C for
24 h. After this time, complex 1 (6.0 mol%) was added and the
reaction was left stirring overnight at 608C. Then, the mixture was
extracted with ethyl acetate (2ꢂ500 mL), the organic layers were
separated by centrifugation (90 s, 13000 rpm), combined, and
finally dried over Na2SO4. The degree of conversion was measured
by 1H-NMR and it was complete in all cases. The enantiomeric
excess of the corresponding product was determined by chiral
HPLC or GC.
[9] Commercially available bacterium Rhodococcus rhodochrous IFO 15564
displays both nitrile hydratase/amidase activity. a) H. Kakeya, N. Sakai, T.
Sugai, H. Ohta, Tetrahedron Lett. 1991, 32, 1343–1346; b) R. Morꢉn-
Ramallal, R. Liz, V. Gotor, Org. Lett. 2007, 9, 521–524.
[10] E. Liardo, N. Rꢃos-Lombardꢃa, F. Morꢃs, J. Gonzꢉlez-Sabꢃn, F. Rebolledo,
Org. Lett. 2016, 18, 3366–3369.
[11] R. Gonzꢉlez-Fernꢉndez, P. Crochet, V. Cadierno, Org. Lett. 2016, 18,
6164–6167.
[12] Higher amounts of the amidase inhibitor diethylphosphoramidate
(DEPA) were not effective due to microorganism inactivation.
[13] For a recent review on Ru-catalyzed nitrile hydration, see: R. Garcꢃa-
ꢈlvarez, J. Francos, E. Tomꢉs-Mendivil, P. Crochet, V. Cadierno, J.
Organomet. Chem. 2014, 771, 93–104.
17 Acknowledgements
18
19 We are indebted to the MINECO of Spain (CTQ2013-40591-P and
20 CTQ2016-75986-P) and the Gobierno del Principado de Asturias
21 (Project GRUPIN14-006) for financial support. E. Liardo acknowl-
22 edges funding from the European Union’s Horizon 2020 MSCA
23 ITN-EID program (grant agreement No 634200). The authors also
[14] Three ruthenium complexes, known for their ability to hydrate nitriles
under mild conditions, were considered, i.e.: the bis(allyl)-ruthenium(IV)
complex [RuCl2(h3 :h3-C10H16)(PMe2OH)] (1; C10H16 =2,7-dimethylocta-
2,6-diene-1,8-diyl) and the arene ruthenium(II) derivatives [RuCl2(h6-p-
cymene)(PR2OH)] (R=Me (1’), 4-C6H4F (1’’)): a) E. Tomꢉs-Mendivil, F. J.
Suꢉrez, J. Dꢃez, V. Cadierno, Chem. Commun. 2014, 50, 9661–9664; b) E.
Tomꢉs-Mendivil, V. Cadierno, M. I. Menꢄndez, R. Lꢆpez, Chem. Eur. J.
2015, 21, 16874–16886; c) R. Gonzꢉlez-Fernꢉndez, P. J. Gonzꢉlez-Liste, J.
Borge, P. Crochet, V. Cadierno, Catal. Sci. Technol. 2016, 6, 4398–4409;
d) E. Tomꢉs-Mendivil, J. Francos, R. Gꢆnzalez-Fernꢉndez, P. J. Gonzꢉlez-
Liste, J. Borge, V. Cadierno, Dalton Trans. 2016, 45, 13590–13603; e) R.
Gonzꢉlez-Fernꢉndez, P. Crochet, V. Cadierno, M. I. Menꢄndez, R. Lꢆpez,
Chem. Eur. J. 2017, 23, 15210–15221. A parametric study of the catalytic
activity of these ruthenium complexes in the hydration of 2 is shown in
Table S1 in the SI.
[15] The concentration was lowered from the reported 330 mM (ref. 14a) to
200 mM, to fit the maximum concentration tolerated by KREDs for an
efficient performance.
[16] The stability of the KRED was also evaluated in the presence of growing
percentages of the metal complex 1 (up to 20% mol). As a result, the
enzymatic activity remained unaltered in all the cases.
[17] Orthogonal tandem catalysis is defined as a one-pot sequence of
reactions involving two or more functionally distinct catalytic mecha-
nisms, promoted by two or more different catalysts that are present
from the outset. See: T. L. Lohr, T. J. Marks, Nat. Chem. 2015, 7, 477–482.
[18] The Codexꢁ KRED Screening Kit (Codexis, Reedwood City, USA) contains
24 ketoreductases. For the full panel of enzymatic screenings, see
section 3 in the SI.
[19] A. Weckbecker, W. Hummel, Biocatal. Biotransform. 2006, 24, 380–389.
[20] B. Kosjek, W. Stampfer, M. Pogorevc, W. Goessler, K. Faber, W. Kroutil,
Biotechnol. Bioeng. 2004, 86, 55–62.
¨
24 thank Dr. Martin Schu¨rmann (InnoSyn) and Prof. Harald Groger
25 (Bielefeld University) for the generous gift of the KRED of
26 Rhodococcus ruber and Lactobacillus kefir, respectively.
27
28
29 Conflict of Interest
30
31 The authors declare no conflict of interest.
32
33
Keywords: One-pot processes · concurrent tandem catalysis ·
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metal catalyst · chiral b-hydroxyamides · tembamide
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For clarity, only the keto form is drawn in this work.
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[25] As reported in ref. 10, KREDs displayed marked cis-diastereoselectivity,
enabling both enantiomers of cis-14d and 15d in very high optical
purity. Concerning the five-membered analogue, three of the four
possible stereoisomers of 13d could be accessible.
asymmetric bioreduction and a Pd-catalyzed hydrogenation has been
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d’Alessandro, F. Hollmann, Green Chem. 2013, 15, 3318–3331.
1
2
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[26] M.-J. Cheng, K.-H. Lee, I.-L. Tsai, I.-S. Chen, Bioorg. Med. Chem. 2005, 13,
5915–5920.
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[28] The corresponding (R)-tembamide displaying hypoglycemic activity
(ref. 23), would be also accessible from (S)-8d. A one-pot chemo-
enzymatic synthesis of (S)-tembamide by combining sequentially an
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Prof. Dr. F. Rebolledo*, Dr. J.
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Metals and enzymes unite! A chemo-
enzymatic orthogonal tandem
protocol for the highly enantioselec-
tive synthesis of b-hydroxyamides
from b-ketonitriles in water is
reported, establishing a fruitful coop-
eration between metal catalysts and
enzymes for filling in the gaps from
previous methodologies.
Strengthening the Combination
between Enzymes and Metals in
Aqueous Medium: Concurrent
Ruthenium-Catalyzed Nitrile
Hydration - Asymmetric Ketone
Bioreduction