Preparation of chiral β-hydroxytriazoles in one-pot chemoenzymatic bioprocesses catalyzed by Rhodotorula mucilaginosa

Article abstract of DOI:10.1016/j.procbio.2018.12.011

Chemoenzymatic strategies for the preparation of enantiopure β-hydroxytriazoles were designed. These and other related compounds are particularly relevant because of their antitubercular bioactivities and as β-adrenergic receptor blockers. The ability of

Full text of DOI:10.1016/j.procbio.2018.12.011

ProcessBiochemistryxxx(xxxx)xxx–xxx
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Preparation of chiral β-hydroxytriazoles in one-pot chemoenzymatic
bioprocesses catalyzed by Rhodotorula mucilaginosa
Celeste Aguirre-Pranzonia, Rodrigo D. Tossoa, Fabricio R. Bisognob, Marcela Kurina-Sanza,⁎
,
Alejandro A. Ordena,⁎
a INTEQUI-CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, Almirante Brown 1455, D5700HGC, San Luis, Argentina
b INFIQC-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000, Córdoba,
Argentina
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chemoenzymatic strategies for the preparation of enantiopure β-hydroxytriazoles were designed. These and
other related compounds are particularly relevant because of their antitubercular bioactivities and as β-adre-
nergic receptor blockers. The ability of Rhodotorula mucilaginosa LSL to stereoselectively reduce prochiral ke-
tones coupled to copper(I)-catalyzed azide-alkyne cycloaddition was exploited. The reactions were performed in
aqueous medium and at room temperature. R. mucilaginosa LSL offered the advantage of internal redox cofactor
recycling. Notably, the biocatalyst was compatible with all the chemicals required namely sodium azide, copper
sulfate and alkynes and showed a broad substrate scope reducing small-bulky and bulky-bulky ketones stereo-
selectively. Considering this, two one-pot processes were assessed to synthesize enantiopure (R)-β-hydro-
xytriazoles. A one-pot, three-step sequential transformation allowed obtaining enantiopure products up to 65%
yield starting from α-chloro arylketones. On the other hand, with α-bromo arylketones, a one-pot cascade
process furnished the same products in ca 80% isolated yield.
(R)-β-hydroxytriazoles
Divergent-convergent process
Biocatalysis
Bulky-bulky ketones
Bioreduction
Stereoselectivity
1. Introduction
the screening of new KRED possessing the ability of accommodating
bulky substrates is still necessary for the expansion of the enzymatic
The combination of sustainable tools with efficient processes is
continuously searched [1,2]. Particularly, one-pot procedures minimize
time, number of unit operations and costs of complex molecule synth-
esis. On the other hand, biocatalysts have gained place as promising
tools in organic chemistry. A good example is the industrial synthesis of
atorvastatin using a ketoreductase (KRED) and halohydrin dehalo-
genase (HHdH) in its synthetic route [3] Some biocatalysts have been
tailored by protein engineering and designed for the synthesis of
bioactive compounds [4,5].
The asymmetric reduction of prochiral ketones is a good strategy to
obtain enantiopure sec-alcohols, which can be used as building blocks
for the synthesis of chiral pharmaceutical intermediates and fine che-
micals. For this purpose, bioreductions mediated by ketoreductases
(KRED) are widely used. There is a huge number of KRED described in
literature that shows high stereoselectivity with a broad substrate scope
[6] although most of them are limited to ketones bearing small-bulky
substituents [7]. So far, only a few biocatalysts having the ability of
reducing bulky-bulky ketones have been reported [8–11]. Nevertheless,
toolbox.
Azole compounds have been extensively studied as potential targets
for drug discovery since they possess a broad range of biological
properties, including antimicrobial, antiviral, antiepileptic, anti-HIV
[12]. In particular, 1,2,3-triazoles are active against tuberculosis
[13–15]. Besides, β-hydroxytriazole derivatives, specifically the R en-
antiomers, have been reported as potential β-adrenergic receptor
blockers [16,17].
One of the most practical and reliable chemical methods to syn-
thesize 1H-1,2,3-triazoles is the copper(I)-catalyzed azide-alkyne cy-
cloaddition (CuAAC) under mild conditions via click chemistry [18].
Interesting examples for the preparation of enantiopure β-hydro-
xytriazoles derivatives by chemoenzymatic approaches can be surveyed
in the literature, being ADHs the most employed enzymes in these
multi-step procedures. In all these strategies, the stereocenter was de-
fined before the CuAAC reaction [19–24]. In a recent report, a bior-
eduction of a β-ketotriazole was performed by a strain of Penicillium
citrinum affording the corresponding (S)-β-hydroxytriazole in high
⁎ Corresponding authors.
E-mail addresses: pranzonica@gmail.com (C. Aguirre-Pranzoni), rdtosso@unsl.edu.ar (R.D. Tosso), fbisogno@fcq.unc.edu.ar (F.R. Bisogno),
marcelakurina@gmail.com (M. Kurina-Sanz), aaorden@unsl.edu.ar (A.A. Orden).
https://doi.org/10.1016/j.procbio.2018.12.011
Received 10 September 2018; Received in revised form 4 December 2018; Accepted 10 December 2018
1359-5113/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Aguirre-Pranzoni,C.,ProcessBiochemistry,https://doi.org/10.1016/j.procbio.2018.12.011

C. Aguirre-Pranzoni et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
Fig. 1. Chemoenzymatic synthesis of (R)-β-hydroxytriazoles.
Fig. 2. Enantioselective bioreduction of β-ketotriazoles with
whole cells of R. mucilaginosa LSL.
Table 1
Stereoselective reduction of azidoketones and β-ketotriazoles with R. mucilaginosa LSL.
Entry
Substratea
Product
Conversion [%]b
99
ee [%]
> 97c
Isolated yield (%)
69
1
5a
(R)-5b
(R)-6b
(R)-7b
2
3
4
6a
7a
8a
99
99
99
> 97c
71
65
70
98d
> 99d
(R)-8b
(R)-9b
5
6
9a
99
99
> 99
> 99
58
62
10a
(R)-10b
eafter purification by column chromatography.
a
Substrate concentration: 2 mM. Reaction conditions: R. mucilaginosa LSL resting cells (1.5 g) in buffer phosphate pH 6.5 (30 mL), 24 h at room temperature.
conversion determined by GC-FID.
ee determined by chiral GC-FID of the acetylated samples.
ee determined by chiral HPLC.
b
c
d
Fig. 3. Chemoenzymatic one-pot, three-step sequential pro-
cedure to obtain (R)-β-hydroxytriazoles.
enantiomeric excess (ee) values [25].
arylketones [26]. In the present work, we took advantage of the ro-
bustness and broad substrate scope of R. mucilaginosa LSL to design
chemoenzymatic processes with the aim of preparing (R)-β-hydro-
xytriazoles.
Previously, we reported a novel wild type biocatalyst, Rhodotorula
mucilaginosa LSL, isolated from a soil sample of a landfarming site,
capable of carrying out the stereoselective reduction of a series of
2

C. Aguirre-Pranzoni et al.
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Fig. 4. Schematic representation of the reactions involved in the divergent-convergent one-pot process for the synthesis of (R)-β-hydroxytriazoles.
2. Results and discussion
forming reagents. Once the β-ketotriazoles were formed, R. mucilagi-
nosa LSL lyophilized cells were added and incubated under non-sterile
conditions giving the complete and stereoselective reduction of the
bulky-bulky substrates after 24 h (Fig. 3). Notably, the copper species
present in the reaction medium did not affect the enzymatic machinery
responsible of catalyzing the ketones reduction and the redox cofactor
regeneration. Afterwards, only one purification procedure was neces-
sary to yield the (R)-β-hydroxytriazoles with a slight improvement in
the isolated yield (see Table S3, SI).
In a first approach, we synthesized (R)-β-hydroxytriazoles (7b-10b)
with excellent ee values in a three-step sequential procedure starting
from the commercially available 2-halo-1-phenylethanones (1a-4a).
Thus, these compounds were treated with sodium azide and then the α-
azidoketones (2 mM) were stereoselectively reduced to (R)-2-azido-1-
arylethanols (5b and 6b) by whole cells of wild-type R. mucilaginosa
LSL. The biocatalyst was used either as resting or lyophilized cells in
phosphate buffer (30 mL, pH 6.5) and sucrose (1.2 g) at room tem-
perature, without the addition of external redox cofactors, showing
exquisite stereoselectivity, as previously reported [26]. Then, the pur-
ified azidoarylethanols (R)-5b and (R)-6b were subjected to CuAAC
with either phenylacetylene or propargyl alcohol catalyzed by in situ
formed Cu(I) [Cu(II)/ascorbate system] in a mixture of water-di-
methylsulfoxide (DMSO) at room temperature (Fig. 1). In every case,
the products were recovered in ca 40% final isolated yield (see Table
S3, SI).
Alternatively, we assessed the bioreduction of the β-ketotriazoles
7a-10a assuming that these bulky-bulky ketones could be accepted as
substrates for the KRED of R. mucilaginosa LSL. A complete conversion
to the corresponding (R)-β-hydroxytriazoles in 24 h was achieved and
the stereoselectivity was excellent with ee values higher than 98%
(Fig. 2). Taking into account the instability of the β-hydroxyazides [27]
and based on these results, we decided to invert the last two steps of the
synthetic route. Following this methodology, the overall yield increased
reaching 60% of the isolated products (see Table S3, SI). In this manner,
it was demonstrated that R. mucilaginosa LSL accepts α-azidoketones
5a-6a (small-bulky substrates) and β-ketotriazoles 7a-10a (bulky-bulky
substrates) (Table 1) with the same stereopreference (R).
On the other hand, we tried to develop a one-pot cascade strategy
starting from α-chloroketones and all the chemicals and the lyophilized
biocatalyst. In this process, chlorine was substituted by the azide nu-
cleophile in SN2 reaction and, in competition the α-chloroketones were
bioreduced to the corresponding chlorohydrins. This divergence furn-
ished two different products resulting in a low conversion to the desired
products (data not shown). Alternatively, we envisaged the use of α-
bromoketones in aqueous medium at pH 4.5. This reaction condition
was selected to avoid the formation of by-products such as epoxides, as
previously reported [26]. Under this condition, bromine was sub-
stituted by the azide nucleophile in SN2 reaction and, in parallel a small
amount of the α-bromoketones were bioreduced to the corresponding
bromohydrins (R)-3b and (R)-4b. Since bromide is a better leaving
group than chloride, the bromohydrins were also reactive in SN2 re-
action and were transformed to the β-hydroxyazides. The formation of
all these intermediates was confirmed by TLC and GC-FID (data not
shown). Consequently, these products converged efficiently to the
synthesis of the (R)-β-hydroxytriazoles 7b-10b (Fig. 4) in up to 80%
isolated yield (see Table S3, SI). This increase in the yield could be
attributable to i) the immediate consumption of the unstable azide
compounds as soon as they are formed, ii) the complete substitution of
the bromine by the azide even in the bromohydrin intermediate, iii) the
ability of R. mucilaginosa LSL to reduce the bromoketones, the azido-
ketones as well as the bulky-bulky β-ketotriazoles, and iv) the avoid-
ance of intermediates isolation steps.
Encouraged by these results and in order to improve the yields of
the (R)-β-hydroxytriazoles, we designed one-pot strategies. On the one
hand, by starting from α-chloroketones, a one-pot, three-step sequential
procedure was developed. In the first step, both compounds 5a and 6a
were obtained by azidation of α-chloroacetophenones. Immediately
after completion of the reaction, the azides were subjected to CuAAC
reaction by adding the corresponding alkynes and the Cu(I) in situ
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3. Conclusions
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Funding
This work was supported by grants from UNSL (PROICO 2-1716),
CONICET (PIP 1122015 0100090) and ANPCyT (PICT 2014 0654);
C.A.P. and R.D.T. are postdoctoral CONICET fellows; M.K.S, A.A.O and
F.R.B. are members of the Research Career of CONICET.
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Authors thank Lic. Mónica Ferrari for technical assistance, Prof.
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