Stereoselective bioreduction of α-diazo-β-keto esters

Article abstract of DOI:10.3390/molecules25040931

Diazo compounds are versatile reagents in chemical synthesis and biology due to the tunable reactivity of the diazo functionality and its compatibility with living systems. Much effort has been made in recent years to explore their accessibility and synth

Full text of DOI:10.3390/molecules25040931

molecules
Article
Stereoselective Bioreduction of α-diazo-β-keto Esters
1
2
2,
1,
Sergio González-Granda , Taíssa A. Costin , Marcus M. Sá * and Vicente Gotor-Fernández *
1
Organic and Inorganic Chemistry Department, University of Oviedo, Avenida Julián Clavería 8,
3
3006 Oviedo, Spain; sergioglezgranda@gmail.com
2
Chemistry Department, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil;
taissacostin@yahoo.com.br
*
Correspondence: marcus.sa@ufsc.br (M.M.S.); vicgotfer@uniovi.es (V.G.-F.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Paola Vitale
Received: 30 January 2020; Accepted: 14 February 2020; Published: 19 February 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Diazo compounds are versatile reagents in chemical synthesis and biology due to the
tunable reactivity of the diazo functionality and its compatibility with living systems. Much effort
has been made in recent years to explore their accessibility and synthetic potential; however, their
preparation through stereoselective enzymatic asymmetric synthesis has been scarcely reported in
the literature. Alcohol dehydrogenases (ADHs, also called ketoreductases, KREDs) are powerful
redox enzymes able to reduce carbonyl compounds in a highly stereoselective manner. Herein, we
have developed the synthesis and subsequent bioreduction of nine
optically active -diazo- -hydroxy esters with potential applications as chiral building blocks in
chemical synthesis. Therefore, the syntheses of prochiral -diazo- -keto esters bearing different
substitution patterns at the adjacent position of the ketone group (N CH , ClCH , BrCH , CH OCH ,
α-diazo-β-keto esters to give
α
β
α
β
3
2
2
2
3
2
NCSCH , CH , and Ph) and in the alkoxy portion of the ester functionality (Me, Et, and Bn), were
2
3
carried out through the diazo transfer reaction to the corresponding
β-keto esters in good to excellent
yields (81–96%). After performing the chemical reduction of -diazo-
α
β-keto esters with sodium
borohydride and developing robust analytical conditions to monitor the biotransformations, their
bioreductions were exhaustively studied using in-house made Escherichia coli overexpressed and
commercially available KREDs. Remarkably, the corresponding α-diazo-β-hydroxy esters were
obtained in moderate to excellent conversions (60 to >99%) and high selectivities (85 to >99% ee)
◦
after 24 h at 30 C. The best biotransformations in terms of conversion and enantiomeric excess
were successfully scaled up to give the expected chiral alcohols with almost the same activity and
selectivity values observed in the enzyme screening experiments.
Keywords: alcohol dehydrogenases; asymmetric synthesis; bioreduction; diazo compounds; enzymes;
hydroxy esters
1
. Introduction
α
-Diazo carbonyl compounds are widely recognized as versatile reagents in organic synthesis
and chemical biology [ ]. The chemistry of -diazo- -hydroxy carbonyl compounds attracts
particular attention, as these functionalized compounds have been employed in the synthesis of
amino acid analogues and heterocycles of biological relevance [ 11]. The conventional preparation of
-diazo- -hydroxy carbonyl compounds relies on the aldol-type addition of a terminal diazo carbonyl
compound with aldehydes or ketones mediated by a strong base such as the organometallics of lithium,
magnesium, or zinc, DBU, and NaOH [12 16]. Asymmetric versions of the aldol reaction involving
-diazo esters and aldehydes have been reported by several authors [ 17 18], but the best yields and
enantioselectivites were achieved by Trost and co-workers using a dinuclear magnesium complex [19 20].
Although each method possesses its own advantages, the use of basic medium generally requires
1–
5
α
β
6–
α
β
–
α
1
,2
,
,
,
Molecules 2020, 25, 931; doi:10.3390/molecules25040931
www.mdpi.com/journal/molecules

Molecules 2020, 25, 931
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strictly anhydrous conditions and cryogenic temperatures, not mentioning the incompatibility of more
labile functional groups, thereby severely limiting the scope of these synthetic methodologies.
An alternative approach to accessing
α
-diazo-
-diazo- -keto esters, although this strategy has only been
22]. Interestingly, the asymmetric reduction of -diazo- -keto esters to
-diazo- -hydroxy esters has not been described so far, even though the
β-hydroxy esters consists of the chemoselective
carbonyl reduction of the corresponding
α
β
applied occasionally [11
,21
,
α
β
the corresponding chiral
α
β
enzyme-catalyzed reduction of carbonyl compounds to secondary alcohols is one of the most reliable
methods to deliver a wide variety of chiral alcohols under mild conditions [23–28].
The use of enzymes, and particularly alcohol dehydrogenases (ADHs, KREDs), as
sustainable catalysts is a powerful synthetic tool for both academy and industry since they are
non-toxic, biodegradable, and display exquisite chemo, regio, and stereoselectivity in synthetic
transformations [29–32]. In this context, remarkable chemoenzymatic transformations involving diazo
compounds have been described in the past years [33–35]. However, the asymmetric reduction of
ketones to alcohols in the presence of the diazo group had been completely neglected until very
recently [36].
Taking into account the importance of
α-diazo-β-hydroxy esters in synthetic chemistry and
chemical biology, and motivated by the absence of studies dealing with the asymmetric reduction of
α
α
-diazo carbonyl compounds, herein is presented the enantioselective synthesis of -functionalized
γ
-diazo- -hydroxy esters through the bioreduction of readily available -diazo-β-keto esters by
β
α
focusing on the screening of ketoreductases as versatile stereoselective biocatalysts of wide applicability.
2
. Results and Discussion
2
.1. Chemical Synthesis of α-Diazo-β-keto Esters 2a–i and Racemic α-Diazo-β-hydroxy Esters 3a–i
The investigation commenced with the racemic synthesis of representative
α
-diazo-
β-hydroxy
esters 3a through the chemical reduction of selected -diazo- -keto esters 2a–i
–
i
α
β
with different
substitution patterns (Scheme 1) in order to develop adequate analytical methods for the measurement
of conversion and enantiomeric excess of the optically active alcohols obtained from bioreduction
experiments (see below). Firstly, the amine-catalyzed diazo transfer reaction developed by S
á
and cols [37] was the method of choice to prepare diazo compounds
and broad substrate scope. Therefore, the preparation of the starting
2
α
due to its high efficiency
-diazo- -keto esters 2b
β
–i
was readily achieved in 81–96% yield (3–5 mmol scale) through the diazo transfer protocol from
-acetamidobenzenesulfonyl azide (p-ABSA) and the corresponding -keto esters 1b (right side
4
β
–i
of Scheme 1 and Table 1), which are commercially available or can be easily prepared from simple
reagents (see the Experimental section). In the particular case of ethyl 4-azido-2-diazo-3-oxobutanoate
(2a), this versatile building block was obtained in 86% yield following the one-pot procedure from
ethyl 4-chloroacetoacetate (1b) to introduce both the azido and diazo functionalities as previously
developed by Sá and cols (left side of Scheme 1 and Table 1) [37].

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Scheme 1. Chemical synthesis of
esters 3a–i.
α-diazo-β-keto esters 2a–i and their reduction to α-diazo-β-hydroxy
Table 1. Chemical synthesis of α-diazo-β-keto esters 2a–i and α-diazo-β-hydroxy esters 3a–i.
Entry
Compound
R1
N CH
ClCH2
ClCH2
CH3
R2
CH CH
Keto Ester 2 (%) a
Hydroxy Ester 3 (%) a
b
1
2
3
4
5
6
7
8
9
a
b
c
d
e
f
g
h
i
86
96
71
75
69
62
78
85
78
71
77
3
2
3
2
2
b
CH CH
3
CH3
CH3
CH3
90
81
92
95
90
94
88
CH OCH
3
2
C H
CH CH
6
5
3
2
CH3
BrCH2
NCSCH2
C H CH
6 5 2
CH CH
3 2
CH CH
3 2
a
Isolated yields after column chromatography on silica gel. b Compounds 2a and 2b were both obtained from ethyl
-chloroacetoacetate (1b).
4
Next, diazo keto esters 2a
in tetrahydrofuran (THF) as the solvent at 0 C for only 10 min to give the expected
–
i
were chemically reduced by employing sodium borohydride (NaBH )
4
◦
α
-diazo-β-hydroxy
esters 3a–i in moderate to high yields after column chromatography purification (62–85%, Table 1).
With -diazo- -keto esters 2a and racemic -diazo- -hydroxy esters 3a in hand, robust
analytical methods were developed for the determination of the conversion values in the corresponding
bioreduction experiments and the measurements of the optical purity of 3a through the
α
β
–
i
α
β
–i
–
i
selection of HPLC techniques and a series of HPLC chiral columns (see Tables S1–S10 in the
Supplementary Material).
2
.2. Bioreduction of α-Diazo-β-keto Esters 3a–i using Alcohol Dehydrogenases
2
.2.1. Bioreduction of Ethyl 4-Azido-2-diazo-3-oxobutanoate (2a)
Ethyl 4-azido-2-diazo-3-oxobutanoate (2a) was selected as the model substrate to find active
alcohol dehydrogenases (ADHs) that would be able to selectively furnish the chiral alcohol 3a through
the chemoselective bioreduction of the keto group. Therefore, a representative group of in-house made
Escherichia coli overexpressed ADHs was evaluated (Table 2), including the enzymes from Rhodococcus
ruber (ADH-A) [38], Thermoanaerobium species (ADH-T) [39], Lactobacillus brevis (LB-ADH) [40
,41],
Ralstonia species (Ras-ADH) [42 43], Sphingobium yanoikuyae (Sy-ADH), [44 45], and Thermoanaerobacter
,
,
ethanolicus (Tes-ADH) [46]. Unfortunately, poor activities (<10% conversion, entries 1–6) were observed
for all these E. coli overexpressed ADHs, except for the Ras-ADH, which led to the formation of a
complex mixture of unidentified products [47].

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Table 2. Bioreduction of ethyl 4-azido-2-diazo-3-oxobutanoate (2a) using different ADHs.
Alcohol 3a ee (%) b
Entry
ADH/KRED
Conversion (%) a
1
2
3
4
5
6
7
8
9
Ras-ADH
LB-ADH
Sy-ADH
Tes-ADH
ADH-T
n.m.c
<5
6
<5
<5
5
-
-
50
-
-
ADH-A
evo-1.1.200
>99
rac
6
5
KRED-P1-A04
KRED-P1-A12
KRED-P1-B02
KRED-P1-B05
KRED-P1-B10
KRED-P1-B12
KRED-P1-C01
KRED-P1-H08
KRED-P2-B02
KRED-P2-C02
KRED-P2-D03
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
KRED-P2-H07
KRED-P3-B03
KRED-P3-G09
40 (S)
94 (S)
95 (S)
96 (S)
96 (S)
99 (S)
99 (S)
99 (S)
86 (S)
74 (S)
98 (S)
97 (S)
98 (S)
78 (S)
7 (S)
-
49
42
14
64
83
81
86
73
84
77
23
89
38
7
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
1
1
1
1
1
1
1
1
1
2
2
2
2
2
<5
7
76 (R)
a
Conversion values were measured by HPLC (see the experimental section for further details and Supporting
b
Information for the correction factor 1.48 employed after developing calibration curves). Enantiomeric excess
values measured by HPLC. c A complex mixture of several products was observed, including the starting material
2a and the final product 3a (n.m.: not measured).
At this point, we decided to turn our attention to commercially available ADHs, including the
evo-1.1.200 from Evoxx Technologies GmbH (entry 7), which has shown to be a selective enzyme for
the reduction of a wide panel of ketones [48], and a kit from Codexis Inc. including 17 ketoreductases
(KREDs, entries 8–24). Gratefully, it was found that some of the tested enzymes led to conversions
to (S)-3a higher than 80% with excellent selectivity (entries 13, 14, 15 and 20): KRED-P1-B12 (83%
conversion, 99% ee), KRED-P1-C01 (81% conversion, 99% ee), KRED-P1-H08 (86% conversion, 99% ee),
and KRED-P2-D12 (89% conversion, 98% ee). It is worth mentioning that in all cases the alcohol (S)-3a
was formed as the sole product, without the presence of any detectable side product.
2
.2.2. Bioreduction of α-Diazo-β-keto Esters 2b–i
After the successful development of stereoselective bioreduction processes for the keto ester 2a
(summarized in Table 3, entries 1–4), the bioreduction of other α-diazo-β-keto esters 2b–i was also
investigated, revealing distinct reactivity patterns as highlighted in Table 3 (complete screenings have
been included in the Supplementary Material, Tables S11–S18).

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Table 3. Selected examples for the stereoselective bioreduction of α-diazo-β-keto esters 2a–i.
Entry
Substrate
R1
N CH2
R2
CH CH
ADH/KRED
Conversion (%) a
ee 3 (%) a
1
2
3
4
5
6
7
8
9
2a
2a
2a
2a
2b
2b
2b
2b
2c
2c
2c
2c
2c
2d
2d
2e
2e
2f
2f
2f
2f
2g
2g
2g
2h
2h
2h
2i
KRED-P1-B12
KRED-P1-C01
KRED-P1-H08
KRED-P2-D12
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
LB-ADH
KRED-P1-A12
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
LB-ADH
KRED-P1-C01
KRED-P2-B02
KRED-P1-C01
KRED-P2-B02
KRED-P1-B02
KRED-P1-B05
KRED-P2-D11
KRED-P2-G03
KRED-P1-C01
KRED-P2-B02
KRED-P2-C02
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
83
81
86
89
99
>99
99
71
>99
99
99
99
71
60
50
82
98
93
84
67
73
40
60
50
98
99
99
92
99
99
99 (S)
99 (S)
99 (S)
98 (S)
99 (R)
98 (R)
98 (R)
99 (R)
>99 (R)
96 (R)
99 (R)
98 (R)
98 (R)
85 (S)
67 (S)
99 (R)
81 (R)
99 (S)
98 (S)
97 (S)
59 (R)
96 (S)
95 (S)
96 (S)
98 (R)
91 (R)
90 (R)
97 (R)
98 (R)
97 (R)
3
3
2
2
2
2
2
2
2
2
N CH2
CH CH
3
3
N CH2
CH CH
3
3
N CH2
CH CH
3
3
ClCH2
ClCH2
ClCH2
ClCH2
ClCH2
ClCH2
ClCH2
ClCH2
ClCH2
CH3
CH CH
3
CH CH
3
CH CH
3
CH CH
3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CH3
CH OCH2
3
CH OCH2
3
C H
CH CH
CH CH
3
6
5
5
5
5
3
2
2
2
2
C H
6
C H
CH CH
6
3
C H
6
CH CH
3
CH3
CH3
CH3
BrCH2
BrCH2
BrCH2
NCSCH2
NCSCH2
NCSCH2
C H CH
6 5
2
2
2
C H CH
6
5
C H CH
6 5
CH CH
3 2
CH CH
3
2
2
2
2
2
CH CH
3
CH CH
3
2i
2i
CH CH
3
CH CH
3
a
Conversion and enantiomeric excess values measured by HPLC.
Overall, the best results in terms of activity and selectivity were obtained with the commercially
available KREDs. Remarkably, for the -diazo- -keto esters bearing different substitutions at the
-position (Cl for 2b and 2c, Br for 2h, and SCN for 2i), it was found that three of the ketoreductases
α
β
γ
studied (KRED-P2-D11, KRED-P2-D12, and KRED-P2-G03) displayed excellent activity (92 to >99%
conversion) and selectivity values (90 to >99% ee; entries 5–7, 10–12, 25–30) towards the formation of
the corresponding (R)-alcohols.
In-house made ADHs did not perform the reduction of the azido-substituted keto ester 2a
satisfactorily (see Section 2.2.1), but LB-ADH was able to reduce both chloro analogues 2b and 2c to the
corresponding alcohols 3b and 3c with reasonable conversion (71%) and high selectivity (99 and 98% ee,
respectively, entries 8 and 13). At this point, the influence of the alkoxy group of the ester functionality
was also considered, and a simple comparison between ethyl 4-chloro-2-diazo-3-oxobutanoate (2b
,
entries 5–7) and its corresponding methyl ester 2c (entries 10–12) demonstrated that there were not
significant differences among KRED-P2-D11, KRED-P2-D12, and KRED-P2-G03, with the reductions
reaching quantitative conversion and excellent selectivity in all cases.

Molecules 2020, 25, 931
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Lower conversions and selectivity were observed for the other methyl esters studied (2d and 2e),
with better results being obtained through the use of either KRED-P1-C01 (for 2d: 60% conversion and
8
5% ee, entry 14; for 2e: 82% conversion and 99% ee, entry 16) or KRED-P2-B02 (for 2d: 50% conversion
and 67% ee, entry 15; for 2e: 98% conversion and 81% ee, entry 17). Similarly, the reactivity of ethyl
-diazo-3-oxo-3-phenylpropanoate (2f) was also dependent of the conditions studied. For instance,
2
only a 67% conversion to the alcohol (S)-3f was reached with KRED-P2-D11 (97% ee, entry 20), although
higher conversions (93 and 84%) and excellent selectivity (98 and 99% ee) were respectively found with
KRED-P1-B02 (entry 18) and KRED-P1-B05 (entry 19). Interestingly, the use of KRED-P2-G03 led to the
formation of the (R)-enantiomer of 3f instead of the expected (S)-configuration (entry 21), although
in this case the conversion (73%) and the selectivity (59% ee) was not as high as those observed for
the formation of (S)-3f. A similar trend was also noticed when the bioreduction of the benzyl ester 2g
was evaluated, with KRED-P1-C01 displaying a very good selectivity (96% ee) but a low conversion
(40%, entry 22), while both KRED-P2-B02 and KRED-P2-C02 led to higher conversions (60 and 50%,
respectively) maintaining the stereoselection level (95 and 96% ee, entries 23 and 24).
2
.2.3. Absolute Configuration Assignment for the Optically Active Hydroxy Esters 3a–i Obtained
through the Bioreduction Process
The best biotransformations in terms of conversion and enantiomeric excess were successfully
scaled-up to a 0.23 mmol scale, moving the reactions from Eppendorf tubes to Erlenmeyer flasks. The
preparative reductions were carried out with seven (2a
that gave conversions higher than 80% in the optimization studies (see Table 3). In all cases, almost
identical results in terms of activity and selectivity were observed for the production of 3a–c,e,f,h,i.
–c,e,f,h,i) out of the nine α-diazo-β-keto esters
2a–i
Thus, the corresponding optically active
α
-diazo-
β
-hydroxy esters 3a
–
c
,
e
,
f
,
h
,
i
were obtained at 82–99%
◦
conversion (73–96% isolated yield) and 96–99% ee after 24 h at 30 C and 250 rpm (see the Materials
and Methods section and the Supporting Information for further details). In all cases, positive values
were found after measurements of the specific optical rotation values, allowing the direct comparison
20
between the alcohol 3f obtained from the bioreduction of 2f with KRED-P1-B02 ([α] = +19.3 (c 0.42,
D
2
D
0
CHCl , 98% ee)) and the data reported in the literature for (S)-3f [49] ([α] = +21.9 (c 0.42, CHCl3,
3
9
1% ee)).
Additional trends in the behavior of the ADHs were observed after performing the bioreduction
experiments. On the one hand, Sy-ADH and ADH-A, which are considered to act following the Prelog
selectivity, displayed in all cases the opposite stereopreference to anti-Prelog enzymes such as LB-ADH
and evo-1.1.200. On the other hand, the commercially available KREDs led to the same enantiomers
as those observed for LB-ADH and evo-1.1.200. For these reasons, and taking into consideration the
priority changes when assigning the absolute configurations, the synthesis of
S)-3a,d,f,g and (R)-3b,c,e,h,i is here claimed (Scheme of Table 3).
α-diazo-β-hydroxy esters
(
3
. Materials and Methods
3
.1. General Methods
Alcohol dehydrogenases and glucose dehydrogenase (GDH-105) were purchased from Codexis
Inc. (Redwood City, CA, USA), while evo-1.1.200 ADH was acquired from Evoxx technologies
GmbH (Monheim am Rhein, Germany). In-house made ADHs were overexpressed in E. coli:
Rhodococcus ruber (ADH-A), Thermoanaerobacter species (ADH-T), Lactobacillus brevis (LB-ADH),
Ralstonia species (Ras-ADH), Sphingobium yanoikuyae (Sy-ADH), and Thermoanaerobacter ethanolicus
(
Tes-ADH) [50]. d-Glucose, NADPH, NADH,
of chemoenzymatic transformations, were acquired from Sigma-Aldrich (Madrid, Spain) and used as
received. -Keto esters 1h and 1i were chemically synthesized by known methods, exhibiting physical
and spectral data in agreement with those reported in the literature [51,52].
β-keto esters 1, and the other reagents for the development
β

Molecules 2020, 25, 931
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1H, 13C, and DEPT NMR spectra were recorded on a Bruker AV300 MHz spectrometer (Bruker
Co., Faellanden, Switzerland). All chemical shifts ( ) are given in parts per million (ppm) and
δ
referenced to the residual solvent signal as the internal standard (for some experiments the carbon
atom directly linked to the diazo functionality was not detected). IR spectra were recorded on a Jasco
−1
FT/IR-4700 spectrophotometer (Jasco-Spain, Madrid, Spain), and νmax values are given in cm for the
main absorption bands. Melting points were measured in a Stuart apparatus SMP3 (Bibby Sterilin,
Staffordshire, UK) by introducing the samples in open capillary tubes, and the measurements are
uncorrected. High-resolution mass spectra (HRMS) experiments were carried out by electrospray
+
ionization in positive mode (ESI ) using a VG AutoSpec Q high-resolution mass spectrometer (Fision
Instrument, Milford, Massachusetts, USA). Measurement of the optical rotation values was carried
out at 590 nm on an Autopol IV Automatic polarimeter (Rudolph Research Analytical, Hackettstown,
NJ, USA).
High-performance liquid chromatography (HPLC) analyses were performed with an Agilent
1
260 Infinity chromatograph with UV detector at 210 nm (Agilent Technologies, Inc., Wilmington, DE,
USA) using chiral HPLC columns (Chiral Technologies Daicel Group, Illkirch Cedex, France) such as
Chiralcel OJ-H (25 cm 4.6 mm, 5 m particle size) and Chiracel AD-H (25 cm 4.6 mm, 5 m particle
×
µ
×
µ
size) with an isocratic flow of 0.8 mL/min. Thin-layer chromatography (TLC) was conducted with
Merck Silica Gel 60 F254 precoated plates (Merck KGaA, Darmstadt, Germany) and were visualized
with UV, potassium permanganate, and vanillin stain. Column chromatography was performed using
silica gel 60 (230–240 mesh, Merck KGaA, Darmstadt, Germany).
3
.2. Chemical Synthesis of Ethyl 4-Azido-2-diazo-3-oxobutanoate (2a)
Azido diazo ester 2a was prepared according to the one-pot procedure from commercially
available ethyl 4-chloroacetoacetate (1b), as described elsewhere [37]. The product 2a was obtained as
a yellowish oil (86% yield; see Table 1) and its spectral data are in agreement with those reported in the
−
literature [37,53]; R (4:1 hexane/EtOAc): 0.43; IR (neat): 2145, 2105, 1715, and 1665 cm ; H-NMR
f
1 1
(
(
CDCl , 300 MHz)
δ
4.37 (s, 2H), 4.28 (q, J = 7.0 Hz, 2H), and 1.31 ppm (t, J = 7.0 Hz, 3H); 13C-NMR
CDCl , 75 MHz) δ 186.9, 161.3, 62.4, 56.4, and 14.7 ppm.
3
3
3
.3. Chemical Synthesis of α-Diazo-β-keto Esters 2b–i
The synthesis of -diazo- -keto esters 2b was performed following a similar protocol to the one
described in the literature [37]. t-BuNH (5.0 mmol, 525 L) was added dropwise to a solution of the
α
β
–i
µ
2
corresponding β-ketoester 1b–i (5.0 mmol) and 4-acetamidobenzenesulfonyl azide (5.0 mmol, 1.20 g)
◦
in dry THF (10 mL) under inert atmosphere at 25 C. The mixture was stirred at room temperature and
monitored by TLC analysis (4:1 hexane/EtOAc). After complete consumption of the starting material,
the mixture was diluted with CH Cl (20 mL), washed with brine (15 mL), dried over Na SO , filtered,
2
2
2
4
and concentrated under reduced pressure. After complete removal of the solvent, the residue was
triturated with diethyl ether (3 10 mL) and the resulting mixture was again concentrated under
reduced pressure. The final solid residue was repeatedly triturated with hexane (3 10 mL) to separate
×
×
out the insoluble ABSNH by decantation. The resulting mixture was filtered, concentrated under
2
reduced pressure, and purified by column chromatography on silica gel (4:1 hexane/EtOAc), obtaining
the corresponding α-diazo-β-ketoesters 2b–i as oils (81–96% yield; see Table 1).
Ethyl 4-chloro-2-diazo-3-oxobutanoate (2b) [53]: Yellowish oil (96% yield); R (4:1 hexane/EtOAc): 0.48;
f
−1 1
IR (neat): 2142, 1715, and 1670 cm ; H-NMR (CDCl , 300 MHz)
2
δ
4.60 (s, 2H), 4.30 (q, J = 7.1 Hz,
H), and 1.32 ppm (t, J = 7.1 Hz, 3H); 13C-NMR (CDCl , 75 MHz)
δ 184.4, 161.1, 76.2, 62.3, 47.4, and
3
3
1
4.6 ppm.
Methyl 4-chloro-2-diazo-3-oxobutanoate (2c) [54]: Yellowish oil (90% yield); R (4:1 hexane/EtOAc): 0.30;
f
−
1 1
IR (neat) 2144, 1715, and 1679 cm ; H-NMR (CDCl , 300 MHz) δ 4.60 (s, 2H), and 3.85 ppm (s, 3H);
3
13
C-NMR (CDCl , 75 MHz) δ 184.3, 161.6, 76.2, 53.0, and 47.3 ppm.
3

Molecules 2020, 25, 931
8 of 14
Methyl 2-diazo-3-oxobutanoate (2d) [55]: Yellowish oil (81% yield); R (4:1 hexane/EtOAc): 0.41; IR (neat):
f
−
139, 1720, and 1657 cm ; H-NMR (CDCl , 300 MHz) δ 3.84 (s, 3H), and 2.48 ppm (s, 3H); C-NMR
3
1
1
13
2
(
CDCl , 75 MHz) δ 190.1, 161.8, 76.2, 52.2, and 28.2 ppm.
3
Methyl 2-diazo-4-methoxy-3-oxobutanoate (2e) [56]: Yellowish oil (92% yield); R (3:1 hexane/EtOAc): 0.43;
f
−
1 1
IR (neat) 2100, 1731, and 1684 cm ; H-NMR (CDCl , 300 MHz) δ 4.51 (s, 2H), 3.82 (s, 3H), and 3.45
3
ppm (s, 3H); 13C-NMR (CDCl , 75 MHz) δ 189.3, 162.0, 76.2, 75.2, 60.0, and 52.8 ppm.
3
Ethyl 2-diazo-3-oxo-3-phenylpropanoate (2f) [55]: Yellowish oil (95% yield); R (4:1 hexane/EtOAc): 0.51;
f
−
1 1
IR (neat): 2184, 1726, and 1649 cm ; H-NMR (CDCl , 300 MHz)
3
δ
7.61–7.58 (m, 2H), 7.50–7.35 (m,
H), 4.19 (q, J = 7.1 Hz, 2H), and 1.20 ppm (t, J = 7.1 Hz, 3H); C-NMR (CDCl , 75 MHz) 187.4, 161.4,
3
13
δ
3
1
37.5, 132.7, 128.7 (2 × CH), 128.3 (2 × CH), 76.6, 62.0, and 14.6 ppm.
Benzyl 2-diazo-3-oxobutanoate (2g) [55]: Yellowish oil (90% yield); R (4:1 hexane/EtOAc): 0.57; IR (neat):
f
−
1 1
2
3
097, 1737, and 1682 cm ; H-NMR (CDCl , 300 MHz)
δ
7.38 (brs, 5H), 5.27 (s, 2H), and 2.48 ppm (s,
190.0, 161.3, 135.1, 128.7 (2 CH), 128.6, 128.4 (2 CH), 76.4, 67.0,
3
13
H); C-NMR (CDCl , 75 MHz)
δ
×
×
3
and 28.3 ppm.
Ethyl 4-bromo-2-diazo-3-oxobutanoate (2h) [53]: Yellowish oil (94% yield); R (4:1 hexane/EtOAc): 0.46; IR
f
−
neat): 2140, 1715, and 1655 cm ; H-NMR (CDCl , 300 MHz) δ 4.39 (s, 2H), 4.31 (q, J = 7.0 Hz, 2H),
3
1 1
(
and 1.32 ppm (t, J = 7.0 Hz, 3H); 13C-NMR (CDCl , 75 MHz) δ 184.1, 160.8, 62.0, 29.6, and 14.3 ppm.
3
Ethyl 2-diazo-3-oxo-4-thiocyanobutanoate (2i) [53]: Pale orange oil (88% yield); R (4:1 hexane/EtOAc):
f
−
1 1
0
.25; IR (neat): 2145, 1710, and 1655 cm ; H-NMR (CDCl , 300 MHz)
δ
4.37–4.30 (m, 4H), and 1.35
ppm (t, J = 6.0 Hz, 3H); C-NMR (CDCl , 75 MHz) δ 184.1, 161.2, 111.8, 62.8, 41.8, and 14.7 ppm.
3
13
3
3
.4. Chemical Reduction of α-Diazo-β-keto Esters 2a–i using Sodium Borohydride
◦
Sodium borohydride (1.0 mmol, 37.8 mg) was added at 0 C to a solution of the corresponding
α
-diazo-
stirred at room temperature for 30 min and monitored by TLC analysis (4:1 hexane/EtOAc). Next, the
reaction was quenched with an aqueous saturated solution of NH Cl (1 mL); the resulting mixture
β-keto ester 2a-i (1.0 mmol) in dry THF (2 mL) under an inert atmosphere. The mixture was
4
was extracted with EtOAc (3
×
5 mL), and the organic phases were combined and washed with brine
(1
×
15 mL). The resulting organic phase was dried over Na SO , filtered, and concentrated under
2
4
reduced pressure. Finally, the crude product was purified by column chromatography on silica gel (4:1
hexane/EtOAc) to give the corresponding α-diazo-β-hydroxy esters 3a-i as oils (62–85% yield).
Ethyl 4-azido-2-diazo-3-hydroxybutanoate (3a): Yellowish oil (71% yield); R (4:1 hexane/EtOAc): 0.32; IR
f
−1 1
(
neat): 3371, 2102, and 1684 cm ; H-NMR (CDCl , 300 MHz)
δ
4.77 (q, J = 6.1 Hz, 1H), 4.25 (q, J = 9.0
Hz, 2H), 3.55 (d, J = 6.1 Hz, 2H), 2.87 (brs, 1H), and 1.29 ppm (t, J = 9.0 Hz, 3H); C-NMR (CDCl , 75
3
13
3
+
+
+
MHz)
δ 166.1, 65.7, 61.4, 54.3, and 14.4 ppm; HRMS (ESI , m/z): calcd for (C H N NaO ) (M + Na) :
6 9 5 3
2
22.0599, found 222.0598.
Ethyl 4-chloro-2-diazo-3-hydroxybutanoate (3b): Yellowish oil (75% yield); R (4:1 hexane/EtOAc): 0.36; IR
f
−
neat): 3386, 2115, and 1667 cm ; H-NMR (CDCl , 300 MHz) δ 4.77 (t, J = 6.0 Hz, 1H), 4.24 (q, J = 7.1
3
1 1
(
13
Hz, 2H), 3.73 (d, J = 6.0 Hz, 2H), 3.42 (brs, 1H), and 1.28 ppm (t, J = 7.1 Hz, 3H); C-NMR (CDCl , 75
3
+
+
MHz)
δ 165.9, 66.8, 61.3, 46.8, and 14.4 ppm. HRMS (ESI , m/z): calcd for (C H ClN NaO ) (M +
6 9 2 3
+
Na) : 215.0195, found 215.0194.
Methyl 4-chloro-2-diazo-3-hydroxybutanoate (3c): Yellowish oil (69% yield); R (4:1 hexane/EtOAc): 0.19;
f
−
1 1
IR (neat): 3419, 2103, and 1683 cm ; H-NMR (CDCl , 300 MHz)
δ
4.79–4.77 (m, 1H), 3.79 (s, 3H),
166.6, 67.2, 52.6, and 47.2 ppm;
HRMS (ESI , m/z): calcd for (C H ClN NaO ) (M + Na) : 201.0037, found 201.0037.
3
13
3
.77–3.71 (m, 2H), and 3.17 ppm (brs, 1H). C-NMR (CDCl , 75 MHz) δ
3
+
+
+
5
7
2
3
Methyl 2-diazo-3-hydroxybutanoate (3d): Yellowish oil (62% yield); R (4:1 hexane/EtOAc): 0.31; IR (neat):
f
−
392, 2135, and 1652 cm ; H-NMR (CDCl , 300 MHz) δ 4.95–4.90 (m, 1H), 3.79 (s, 3H), 2.58 (brs, 1H),
3
1 1
3

Molecules 2020, 25, 931
9 of 14
and 1.41 ppm (d, J = 6.5 Hz, 3H); 13C-NMR (CDCl , 75 MHz)
δ
165.4, 64.5, 59.5, 54.9, and 19.6 ppm.
3
+
+
+
HRMS (ESI , m/z): calcd for (C H N NaO ) (M + Na) : 167.0421, found 167.0427.
5
8
2
3
Methyl 2-diazo-3-hydroxy-4-methoxybutanoate (3e): Yellowish oil (78% yield); R (3:1 hexane/EtOAc): 0.33;
f
−1 1
IR (neat): 3418, 2100, and 1684 cm ; H-NMR (CDCl , 300 MHz)
δ
4.79 (brs, 1H), 3.79 (s, 3H), 3.69–3.54
m, 2H), 3.42 (s, 3H), and 3.08 ppm (brs, 1H); C-NMR (CDCl , 75 MHz) 166.6, 74.0, 65.0, 59.2, and
2.0 ppm; HRMS (ESI , m/z): calcd for (C H N NaO ) (M + Na) : 197.0537, found 197.0533.
3
13
(
δ
3
+
+
+
5
6
10
2
4
Ethyl 2-diazo-3-hydroxy-3-phenylpropanoate (3f) [49]: Yellowish oil (85% yield); R (4:1 hexane/EtOAc):
f
−
.42; IR (neat): 3419, 2098, and 1665 cm ; H-NMR (CDCl , 300 MHz) δ 7.45–7.30 (m, 5H), 5.91 (s, 1H),
3
1 1
0
4
1
13
.28 (q, J = 6.1 Hz, 2H), 3.10 (brs, 1H), and 1.30 ppm (t, J = 6.1 Hz, 3H); C-NMR (CDCl , 75 MHz)
δ
3
+
66.4, 138.8, 128.8 (2
×
CH), 128.4, 125.7 (2
×
CH), 68.8, 61.2, and 14.5 ppm; HRMS (ESI , m/z): calcd for
+
+
(
C H N NaO ) (M + Na) : 243.0743, found 243.0740.
11
12
2
3
Benzyl 2-diazo-3-hydroxybutanoate (3g): Yellowish oil (78% yield); R (4:1 hexane/EtOAc): 0.44; IR (neat):
f
−
397, 2097, and 1688 cm ; H-NMR (CDCl , 300 MHz) δ 7.37–7.34 (m, 5H), 5.22 (s, 2H), 4.94 (q, J
3
1 1
3
13
=
6.6 Hz, 1H), 2.84 (brs, 1H), and 1.40 ppm (d, J = 6.6 Hz, 3H); C-NMR (CDCl , 75 MHz)
δ
165.5,
CH), 68.1, 62.0, and 21.0 ppm; HRMS (ESI , m/z): calcd for
C H N NaO ) (M + Na) : 243.0740, found 243.0740.
3
+
1
(
36.1, 129.0 (2
×
CH), 128.8, 128.6 (2
×
+
+
11
12
2
3
Ethyl 4-bromo-2-diazo-3-hydroxybutanoate (3h): Yellowish oil (71% yield); R (4:1 hexane/EtOAc): 0.34; IR
f
−
neat): 3402, 2079, and 1695 cm ; H-NMR (CDCl , 300 MHz) δ 4.78 (brs, 1H), 4.25 (q, J = 7.1 Hz, 2H),
3
1 1
(
13
3
.76–3.73 (m, 2H), 3.18 (brs, 1H), and 1.29 ppm (t, J = 7.1 Hz, 3H); C-NMR (CDCl , 75 MHz)
δ
165.7,
3
+
+
+
6
6.6, 61.2, 35.6, and 14.4 ppm; HRMS (ESI , m/z): calcd for (C H BrN NaO ) (M+Na) : 258.9698,
6
9
2
3
found 258.9689.
Ethyl 2-diazo-3-hydroxy-4-thiocyanobutanoate (3i): Yellowish oil (77% yield); R (4:1 hexane/EtOAc): 0.17;
f
−
1 1
IR (neat): 3381, 2138, and 1653 cm ; H-NMR (CDCl , 300 MHz)
δ 4.87 (q, J = 6.5 Hz, 1H), 4.26 (q, J =
.1 Hz, 2H), 3.42 (brs, 1H), 3.34–3.30 (m, 2H), and 1.29 ppm (t, J = 9.1 Hz, 3H); C-NMR (CDCl , 75
MHz) δ 165.6, 111.7, 66.5, 61.5, 38.4, and 14.4 ppm; HRMS (ESI , m/z): calcd for (C H N O S) (M ):
6 9 3 3
3
13
9
3
+
+
+
2
15.0211, found 215.0365.
.5. Bioreduction of α- Diazo-β-keto Esters 2a–i using Sy-ADH, TES-ADH, and ADH-T
3
The selected ADH (12 mg) was added to a 1.5 mL Eppendorf tube containing the corresponding
i
α
-diazo-
β-ketoester 2a
–i
(0.015 mmol), DMSO (56
µ
L), PrOH (30
µL), a 10 mM aqueous solution of
NADPH (60
µ
L), and a 50 mM Tris/HCl pH 7.5 buffer (454
µ
L). Then, the Eppendorf tube was closed and
◦
kept under orbital shaking at 250 rpm at 30 C for 24 h. After this time, the product was extracted with
EtOAc (3
×
0.5 mL) and the combined organic layers were dried over anhydrous Na SO . Finally, the
2 4
solvent was carefully evaporated by bubbling nitrogen gas through it, and an aliquot was taken for the
measurement of conversion and enantiomeric excess values of the corresponding α-diazo-β-hydroxy
ester 3a–i by HPLC.
3
.6. Bioreduction of α- Diazo-β-keto Esters 2a–i using LB-ADH
LB-ADH (12 mg) was added to a 1.5 mL Eppendorf tube containing the corresponding
i
α
-diazo-
β-ketoester 2a
–i
(0.015 mmol), DMSO (56
µ
L), PrOH (30
µL), a 10 mM aqueous solution of
MgCl (60
µ
L), a 10 mM aqueous solution of NADPH (60
µ
L), and a 50 mM Tris/HCl pH 7.5 buffer
2
◦
(
394
4 h. After this time, the product was extracted with EtOAc (3
layers were dried over anhydrous Na SO . Finally, the solvent was carefully evaporated by bubbling
µ
L). Then, the Eppendorf tube was closed and kept under orbital shaking at 250 rpm at 30 C for
2
×
0.5 mL) and the combined organic
2
4
nitrogen gas through it, and an aliquot was taken for the measurement of conversion and enantiomeric
excess values of the corresponding α-diazo-β-hydroxy ester 3a–i by HPLC.

Molecules 2020, 25, 931
10 of 14
3
.7. Bioreduction of α- Diazo-β-keto Esters 2a–i using ADH-A
ADH-A (12 mg) was added to a 1.5 mL Eppendorf tube containing the corresponding
i
α
-diazo-
β
-ketoester 2a
–
i
(0.015 mmol), DMSO (56
µ
L), PrOH (30
µ
L), a 10 mM aqueous solution of
L). Then, the Eppendorf tube was closed and
kept under orbital shaking at 250 rpm at 30 C for 24 h. After this time, the product was extracted with
EtOAc (3 0.5 mL) and the combined organic layers were dried over anhydrous Na SO . Finally, the
solvent was carefully evaporated by bubbling nitrogen gas through it, and an aliquot was taken for the
NADH (60
µL), and a 50 mM Tris/HCl pH 7.5 buffer (454
µ
◦
×
2
4
measurement of conversion and enantiomeric excess values of the corresponding α-diazo-β-hydroxy
ester 3a–i by HPLC.
3
.8. Bioreduction of α- Diazo-β-keto Esters 2a–i using Ras-ADH
Ras-ADH (12 mg) was added to a 1.5 mL Eppendorf tube containing the corresponding
-diazo- -ketoester 2a (0.015 mmol), DMSO (75 L), an aqueous solution of GDH (60 L, 10
L), a 10 mM aqueous solution of NADPH (60 L),
L). Then, the Eppendorf tube was closed and kept under
α
β
–i
µ
µ
U), a 50 mM aqueous solution of d-glucose (60
and a 50 mM Tris/HCl pH 7.5 buffer (345
orbital shaking at 250 rpm at 30 C for 24 h. After this time, the product was extracted with EtOAc (3
µ
µ
µ
◦
×
0.5 mL) and the combined organic layers were dried over anhydrous Na SO . Finally, the solvent
2 4
was carefully evaporated by bubbling nitrogen gas through it, and an aliquot was taken for the
measurement of conversion and enantiomeric excess values of the corresponding α-diazo-β-hydroxy
ester 3a–i by HPLC.
3
.9. Bioreduction of α- Diazo-β-keto Esters 2a–i using evo-1.1.200
Evo-1.1.200 (12 mg) was added to a 1.5 mL Eppendorf tube containing the corresponding
i
α
-diazo-
β-ketoester 2a
–i
(0.015 mmol), DMSO (56
µ
L), PrOH (25
µL), a 10 mM aqueous solution of
MgCl (50
µ
L), a 10 mM aqueous solution of NADH (50
µ
L), and a 50 mM Tris/HCl pH 7.5 buffer
2
◦
(
319
4 h. After this time, the product was extracted with EtOAc (3
layers were dried over anhydrous Na SO . Finally, the solvent was carefully evaporated by bubbling
µ
L). Then, the Eppendorf tube was closed and kept under orbital shaking at 250 rpm at 30 C for
2
×
0.5 mL) and the combined organic
2
4
nitrogen gas through it, and an aliquot was taken for the measurement of conversion and enantiomeric
excess values of the corresponding α-diazo-β-hydroxy ester 3a–i by HPLC.
3
.10. Bioreduction of α- Diazo-β-keto Esters 2a–i using Commercially Available ADHs from Codexis Inc.
The selected commercially available Codexis KRED (1 mg) was added to a 1.5 mL Eppendorf tube
i
containing the corresponding
α
-diazo-
β
-ketoester 2a
–
i
(0.013 mmol), PrOH (95
µ
L), and an aqueous
+
solution with Na PO (128 mM), MgSO (1.7 mM) and NADP (1.1 mM) resulting in pH 7.0 (450
µL).
3
4
4
◦
Then, the Eppendorf tube was closed and kept under orbital shaking at 250 rpm at 30 C for 24 h. After
this time, the product was extracted with EtOAc (3 0.5 mL) and the combined organic layers were
dried over anhydrous Na SO . Finally, the solvent was carefully evaporated by bubbling nitrogen
×
2
4
gas through it, and an aliquot was taken for the measurement of conversion and enantiomeric excess
values of the corresponding α-diazo-β-hydroxy ester 3a–i by HPLC.
3
.11. Semi-preparative Bioreduction of
α- Diazo-β-keto esters 2a–c,e,f,h,i using Commercially Available ADHs
from Codexis Inc.
The selected commercially available Codexis KRED (12 mg) was added to a 25 mL Erlenmeyer
i
flask containing the corresponding
α
-diazo-
β
-ketoester 2a
–c
,e,f
,
h
,
i
(0.23 mmol), PrOH (920
µ
L), and
+
an aqueous solution with Na PO (128 mM), MgSO (1.7 mM), and NADP (1.1 mM), resulting in pH
3
4
4
◦
7
.0 (8.28 mL). Then, the Erlenmeyer flask was closed and kept under orbital shaking at 250 rpm at 30 C
for 24 h. After this time, the product was extracted with EtOAc (3 10 mL), and the combined organic
layers were dried over anhydrous Na SO . Finally, the solvent was carefully evaporated by bubbling
×
2
4

Molecules 2020, 25, 931
11 of 14
nitrogen gas through it and an aliquot was taken for the measurement of conversion and enantiomeric
excess values of the corresponding -diazo- -hydroxy ester 3a by HPLC. At this point, the
α
β
–c,e,f,h,i
resulting reaction crude was purified by column chromatography on silica gel (4:1 hexane:EtOAc) to
give the pure corresponding α-diazo-β-hydroxy esters 3a–c,e,f,h,i as yellowish oils.
20
(
S)-3a: [α] = +5.4 (c 0.1, CHCl , 97% ee) after bioreduction with KRED-P2-D12 (83% conversion, 74%
3
D
isolated yield).
20
(
R)-3b: [α] = +7.5 (c 0.1, CHCl , 96% ee) after bioreduction with KRED-P2-D12 (99% conversion, 86%
3
D
isolated yield).
20
(
R)-3c: [α] = +7.1 (c 0.1, CHCl , 98% ee) after bioreduction with KRED-P2-D12 (99% conversion, 96%
3
D
isolated yield).
20
(
R)-3e: [α] = +9.4 (c 0.1, CHCl , 99% ee) after bioreduction with KRED-P1-C01 (82% conversion, 73%
3
D
isolated yield).
2
0
(
S)-3f: [α] = +19.3 (c 0.42, CHCl , 98% ee) after bioreduction with KRED-P1-B02 (91% conversion,
3
D
8
3% isolated yield).
20
(
R)-3h: [α] = +8.5 (c 0.1, CHCl , 96% ee) after bioreduction with KRED-P2-D11 (97% conversion, 89%
3
D
isolated yield).
2
0
(
R)-3i: [α] = +7.8 (c 0.1, CHCl , 96% ee) after bioreduction with KRED-P2-D12 (97% conversion, 91%
3
D
isolated yield).
4
. Conclusions
A family of
α
-diazo-
β-keto esters 2a–i were chemically synthesized in good to excellent yields
(
81–96%), which were later successfully employed as substrates for bioreduction using a variety of
E. coli overexpressed and commercially available alcohol dehydrogenases to give the corresponding
optically active -diazo- -hydroxy esters 3a . Different structural patterns were considered, including
α
β
–i
the substitution at the adjacent position of the ketone group and in the alkoxy portion of the ester
functionality. For most substrates, in particular, those bearing heteroatoms substituted at the 4-position
(Cl, Br or SCN), the commercial enzymes KRED-P2-D11, KRED-P2-D12, and/or KRED-P2-G03 led
to complete conversions to the corresponding alcohols with excellent selectivities (97–99% ee).
3
Alternatively, KRED-P1-C01 and KRED-P2-B02 were shown to be effective for the less reactive
substrates, while the conversions decreased significantly when considering methyl ketones (up to
60% conversion). On the other hand, the type of the alkyl ester studied was not as relevant for
the bioreduction, with methyl, ethyl, or benzyl esters being appropriate substrates to deliver the
corresponding alcohols with high selectivity (85 to >99% ee) depending on the choice of the enzyme.
Thus, straightforward access to the corresponding optically active α-diazo-β-hydroxy esters 3a-i has
been provided. They were readily obtained in a preparative scale with high conversions and excellent
stereoselectivities, depending on the enzyme selection.
Supplementary Materials: A pdf file containing the structures of
a-i, the development of analytical methods in HPLC for the measurement of conversion and enantiomeric
excess values of the bioreduction processes, extensive enzyme-catalysed screenings, and full characterization of
α-diazo-β-keto esters 2a–i and hydroxy esters
3
1
13
novel compounds by NMR spectra ( H and C-NMR experiments) is available on-line. Figure S1. Structures
of -diazo- -keto esters 2a and the corresponding hydroxy esters 3a-i described in this contribution; Table S1.
Retention times of -diazo- -keto esters 2a and their corresponding alcohols 3a in HPLC analyses; Tables
; Tables S11–S18. Extensive enzyme
α
β
–i
β
α
–
i
–i
S2–S10. Calibrate curves in HPLC for the bioreduction of compounds 2a
–i
screenings for the bioreduction of compounds 2b–i.
Author Contributions: Conceptualization, M.M.S. and V.G.-F.; investigation, S.G.-G. and T.A.C.; writing: original
draft preparation, M.M.S. and V.G.-F.; writing: review and editing, S.G.-G., T.A.C., M.M.S., and V.G.-F.; supervision,
M.M.S. and V.G.-F.; funding acquisition, V.G.-F. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness (MEC) Project
CTQ2016-75752-R and the Asturian Regional Government (FC-GRUPIN-IDI/2018/000181).

Molecules 2020, 25, 931
12 of 14
Acknowledgments: Authors would like to thank Wolfgang Kroutil (University of Graz, Austria) for providing us
with alcohol dehydrogenases overexpressed in E. coli cells and the technical support of the Scientific-Technical
Services of the University of Oviedo. M.M.S. and T.A.C. are grateful to CAPES (Coordenaç
ã
o de Aperfeiçoamento
de Pessoal de N
í
vel Superior, Brazil) and CNPq (Conselho Nacional de Desenvolvimento Cient fico e Tecnol gico,
í
ó
Brazil) for fellowships.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Maas, G. New Syntheses of Diazo Compounds. Angew. Chem. Int. Ed. 2009, 48, 8186–8195. [CrossRef]
PubMed]
Ford, A.; Miel, H.; Ring, A.; Slattery, C.N.; Maguire, A.R.; McKervey, M.A. Modern Organic Synthesis with
α-Diazocarbonyl Compounds. Chem. Rev. 2015, 115, 9981–10080. [CrossRef] [PubMed]
Li, Y.-P.; Li, Z.-Q.; Zhu, S.-F. Recent advances in transition-metal-catalyzed asymmetric reactions of diazo
compounds with electron-rich (hetero) arenes. Tetrahedron Lett. 2018, 59, 2307–2318. [CrossRef]
Xiang, Y.; Wang, C.; Ding, Q.; Peng, Y. Diazo Compounds: Versatile Synthons for the Synthesis of
Nitrogen Heterocycles via Transition Metal-Catalyzed Cascade C–H Activation/Carbene Insertion/Annulation
Reactions. Adv. Synth. Catal. 2019, 361, 919–944. [CrossRef]
[
5
.
.
Empel, C.; Koenlgs, R.M. Sustainable Carbene Transfer Reactions with Iron and Light. Synlett 2019, 30,
1
929–1934. [CrossRef]
Dutta, N.B.; Sharma, S.; Chetry, R.L.; Baishya, G. A Green Protocol for the Synthesis of α-Diazo-β-hydroxy
esters and One-Pot Conversion to -Keto-Esters and Imidazo[1,2-a]pyridine-3-carboxylates. ChemistrySelect
019, 4, 5817–5822. [CrossRef]
6
β
2
7
8
9
1
1
.
.
.
Chen, D.F.; Wu, P.-Y.; Gong, L.-Z. Rhodium/Chiral Urea Relay Catalysis Enables an Enantioselective
Semipinacol Rearrangement/Michael Addition Cascade. Org. Lett. 2013, 15, 3958–3961. [CrossRef]
Bayir, A.; Draghici, C.; Brewer, M. Preparation of Tethered Aldehyde Ynoates and Ynones by Ring
Fragmentation of Cyclic γ-Oxy-β-hydroxy-α-diazo Carbonyls. J. Org. Chem. 2010, 75, 296–302.
Zhang, Y.; Wang, J. Recent development of reactions with α-diazocarbonyl compounds as nucleophiles.
Chem. Commun. 2009, 36, 5350–5361. [CrossRef]
0. Xiao, F.; Liu, Y.; Wang, J. DBU-catalyzed condensation of acyldiazomethanes to aldehydes in water and a
new approach to ethyl β-hydroxy α-arylacrylates. Tetrahedron Lett. 2007, 48, 1147–1149. [CrossRef]
1. Zhdanova, O.V.; Korneev, S.M.; Nikolaev, V.A. Chemistry of Diazocarbonyl Compounds: XVIII. Synthesis
and Spectral Parameters of 1,3-Dialkyl-3-hydroxy-2-diazoketones. Russ. J. Org. Chem. 2004, 40, 316–328.
[CrossRef]
1
1
1
2. Moody, C.J.; Morfitt, C.N. Zinc Derivative of Ethyl Diazoacetate in the Synthesis of α-Diazo-β-hydroxy
Esters. Synthesis 1998, 1039–1042. [CrossRef]
3. Jiang, N.; Wang, J. DBU-promoted condensation of acyldiazomethanes to aldehydes and imines under
catalytic conditions. Tetrahedron Lett. 2002, 43, 1285–1287. [CrossRef]
4. Cuevas-Yañez, E.; Muchowski, J.M.; Cruz-Almanza, R. Metalation of
Grignard reagents. A convenient synthesis of -diazo- -ketoesters and mixed esters of
Tetrahedron Lett. 2004, 45, 2417–2419. [CrossRef]
5. Arai, S.; Hasegawa, K.; Nishida, A. One-pot synthesis of
catalysis and application to the catalytic asymmetric aldol reaction. Tetrahedron Lett. 2004, 45, 1023–1026.
CrossRef]
6. Li, P.; Majireck, M.M.; Korboukh, I.; Weinreb, S.M. A mild, efficient method for the oxidation of
-diazo- -hydroxy esters to -diazo- -ketoesters. Tetrahedron Lett. 2008, 49, 3162–3164. [CrossRef]
PubMed]
7. Hasegawa, K.; Arai, S.; Nishida, A. Synthesis of
α
-diazocarbonyl compounds using
α
β
α
-diazomalonate.
1
1
1
1
α-diazo-β-hydroxyesters under phase-transfer
[
α
β
α
β
[
α
-diazo-β-hydroxyesters through a one-pot protocol by
phase-transfer catalysis: Application to enantioselective aldol-type reaction and diastereoselective synthesis
of α-amino-β-hydroxyester derivatives. Tetrahedron 2006, 62, 1390–1401. [CrossRef]
8. Yao, W.; Wang, J. Direct catalytic asymmetric aldol-type reaction of aldehydes with ethyl diazoacetate. Org.
Lett. 2003, 5, 1527–1530. [CrossRef]

Molecules 2020, 25, 931
13 of 14
1
2
2
9. Trost, B.M.; Malhotra, S.; Fried, B.A. Magnesium-catalyzed asymmetric direct aldol addition of ethyl
diazoacetate to aromatic, aliphatic, and -unsaturated aldehydes. J. Am. Chem. Soc. 2009, 131, 1674–1675.
CrossRef]
α,β
[
0. Trost, B.M.; Malhotra, S.; Koschker, P.; Ellerbrock, P. The development of the enantioselective addition of
ethyl diazoacetate to aldehydes: Asymmetric synthesis of 1,2-diols. J. Am. Chem. Soc. 2012, 134, 2075–2084.
[CrossRef]
1. Pagar, V.V.; Jadhav, A.M.; Liu, R.-S. Gold-Catalyzed Formal [3 + 3] and [4 + 2] Cycloaddition Reactions
of Nitrosobenzenes with Alkenylgold Carbenoids. J. Am. Chem. Soc. 2011, 133, 20728–20731. [CrossRef]
[PubMed]
2
2
2
2
2
2. Davies, H.M.L.; Huby, N.J.S. Enantioselective synthesis of tropanes by reaction of rhodium-stabilized
vinylcarbenoids with pyrroles. Tetrahedron Lett. 1992, 33, 6935–6938. [CrossRef]
3. Applegate, G.A.; Berkowitz, D.B. Exploiting Enzymatic Dynamic Reductive Kinetic Resolution (DYRKR) in
Stereocontrolled Synthesis. Adv. Synth. Catal. 2015, 357, 1619–1632. [CrossRef] [PubMed]
4. Hollmann, F.; Arends, I.W.C.E.; Holtmann, D. Enzymatic reductions for the chemist. Green Chem. 2011, 13,
2
285–2313. [CrossRef]
5. Kallergi, M.; Kalaitzakis, D.; Smonou, I. Enzymatic Total Synthesis of Banana Volatile (S)-2-Pentyl
R)-3-Hydroxyhexanoate. Eur. J. Org. Chem. 2011, 3946–3950. [CrossRef]
6. Cuetos, A.; Rioz-Mart nez, A.; Bisogno, F.R.; Grischek, B.; Lavandera, I.; de Gonzalo, G.; Kroutil, W.;
Gotor, V. Access to Enantiopure -Alkyl- -hydroxy Esters through Dynamic Kinetic Resolutions Employing
(
í
α
β
Purified/Overexpressed Alcohol Dehydrogenases. Adv. Synth. Catal. 2012, 354, 1743–1749. [CrossRef]
7. Zadlo, A.; Schrittwieser, J.H.; Koszelewski, D.; Kroutil, W.; Ostaszewski, R. Enantioselective Reduction
of Ethyl 3-Oxo-5-phenylpentanoate with Whole-Cell Biocatalysts. Eur. J. Org. Chem. 2016, 1007–1011.
2
2
[CrossRef]
8. Barik, R.; Halder, J.; Nanda, S. Biocatalytic dynamic kinetic reductive resolution with ketoreductase from
Klebsiella pneumoniae: The asymmetric synthesis of functionalized tetrahydropyrans. Org. Biomol. Chem.
2
019, 17, 8571–8588. [CrossRef]
9. Sheldon, R.A.; Brady, D. The Limits to Biocatalysis: Pushing the Envelope. Chem. Commun. 2018, 54,
088–6104. [CrossRef]
0. Sheldon, R.A.; Pereira, P.C. Biocatalysis engineering: The big picture. Chem. Soc. Rev. 2017, 46, 2678–2691.
CrossRef]
1. Ferreira-Leit
The Protagonism of Biocatalysis in Green Chemistry and Its Environmental Benefits. Catalysts 2017, 7, 9.
CrossRef]
2. Wohlgemuth, R. Biocatalysis—Key to sustainable industrial chemistry. Curr. Opin. Biotechnol. 2010, 21,
13–724. [CrossRef] [PubMed]
3. Coelho, P.S.; Brustad, E.M.; Kannan, A.; Arnold, F.H. Olefin Cyclopropanation via Carbene Transfer Catalyzed
by Engineered Cytochrome P450 Enzymes. Science 2013, 339, 307–310. [CrossRef] [PubMed]
4. Renata, H.; Lewis, R.D.; Sweredoski, M.J.; Moradian, A.; Hess, S.; Wang, Z.J.; Arnold, F.H. Identification of
Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved
Enzymes. J. Am. Chem. Soc. 2016, 138, 12527–12533. [CrossRef]
2
3
3
6
[
ão, V.S.; Cammarota, M.C.; Aguieiras, E.C.G.; Sá, L.R.V.; Fernandez-Lafuente, R.; Freire, D.M.G.
[
3
3
3
7
3
3
3
3
3
5. Waldman, A.J.; Balskus, E.P. Discovery of a Diazo-Forming Enzyme in Cremeomycin Biosynthesis. J. Org.
Chem. 2018, 83, 7539–7546. [CrossRef]
6. Mittmann, E.; Hu, Y.; Peschke, T.; Rabe, K.S.; Niemeyer, C.M.; Bräse, S. Chemoenzymatic Synthesis of
O-Containing Heterocycles from α-Diazo Esters. ChemCatChem 2019, 11, 5519–5523.
7. Costin, T.A.; Dutra, L.G.; Bortoluzzi, A.J.; Sá, M.M. Amine-mediated synthesis of amides from 1,3-dicarbonyl
compounds through a domino diazo transfer/aminolysis process. Tetrahedron 2017, 73, 4549–4559. [CrossRef]
8. Stampfer, W.; Kosjek, B.; Moitzi, C.; Kroutil, W.; Faber, K. Biocatalytic asymmetric hydrogen transfer. Angew.
Chem. Int. Ed. 2002, 41, 1014–1017. [CrossRef]
9. Peters, J.; Minuth, T.; Kula, M.-R. A novel NADH-dependent carbonyl reductase with an extremely broad
substrate range from Candida parapsilosis: Purification and characterization. Enzyme Microb. Technol. 1993, 11,
9
50–958. [CrossRef]
0. Wolberg, M.; Hummel, W.; Wandrey, C.; Müller, M. Highly Regio- and Enantioselective Reduction of
,5-Dioxocarboxylates. Angew. Chem. Int. Ed. 2000, 39, 4306–4308. [CrossRef]
4
3

Molecules 2020, 25, 931
14 of 14
4
1. Leuchs, S.; Greiner, L. Alcohol Dehydrogenase from Lactobacillus brevis: A Versatile Robust Catalyst for
Enantioselective Transformations. Chem. Biochem. Eng. Q. 2011, 2, 267–281.
4
2. Lavandera, I.; Kern, A.; Ferreira-Silva, B.; Glieder, A.; de Wildeman, S.; Kroutil, W. Stereoselective Bioreduction
of Bulky-Bulky Ketones by a Novel ADH from Ralstonia sp. J. Org. Chem. 2008, 15, 6003–6005. [CrossRef]
[PubMed]
4
3. Man, H.; K e˛ dziora, K.; Kulig, J.; Frank, A.; Lavandera, I.; Gotor-Fernández, V.; Röther, D.; Hart, S.;
Turkenburg, J.P.; Grogan, G. Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp.
Reveal the Molecular Basis for Their Recognition of ‘Bulky-Bulky’ Ketones. Top. Catal. 2014, 5, 356–365.
[CrossRef]
4
4
4
4. Lavandera, I.; Oberdofer, G.; Gross, J.; de Wildeman, S.; Kroutil, W. Stereocomplementary Asymmetric
Reduction of Bulky–Bulky Ketones by Biocatalytic Hydrogen Transfer. Eur. J. Org. Chem. 2008, 2539–2543.
[CrossRef]
5. Lavandera, I.; Kern, A.; Resch, V.; Ferreira-Silva, B.; Glieder, A.; Fabian, W.M.F.; de Wildeman, S.; Kroutil, W.
One-Way Biohydrogen Transfer for Oxidation of sec-Alcohols. Org. Lett. 2008, 11, 2155–2158. [CrossRef]
[PubMed]
6. Heiss, C.; Laivenieks, M.; Zeikus, J.G.; Phillips, R.S. Mutation of cysteine-295 to alanine in secondary alcohol
dehydrogenase from Thermoanaerobacter ethanolicus affects the enantioselectivity and substrate specificity of
ketone reductions. Bioorg. Med. Chem. 2001, 7, 1659–1666. [CrossRef]
4
7. Kroutil, W.; Velikonge, S. Method for the Preparation of Substituted Pyrazines via Enzymatic Catalysis.
Patent No. 3,569,594,A1, 20 11 2019.
4
8. Könst, P.; Merkens, H.; Kara, S.; Kochius, S.; Vogel, A.; Zuhse, R.; Holtmann, D.; Arends, I.W.C.E.; Hollmann, F.
Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase. Angew. Chem. Int. Ed. 2012
1, 9914–9917. [CrossRef]
,
5
4
5
9. Wang, W.; Shen, K.; Hu, X.; Wang, J.; Liu, X.; Feng, X. Highly Enantioselective Synthesis of
α
-Diazo-
β
-hydroxy
Esters Using a Bifunctional Titanium Complex. Synlett 2009, 1655–1658. [CrossRef]
0.
M
é
ndez-S
á
nchez, D.; Mourelle-Insua,
Á
.; Gotor-Fern
á
ndez, V.; Lavandera, I. Synthesis of
α
-Alkyl-
β
-Hydroxy
Amides through Biocatalytic Dynamic Kinetic Resolution Employing Alcohol Dehydrogenases. Adv. Synth.
Catal. 2019, 361, 2706–2712. [CrossRef]
5
5
5
5
5
5
1. Kato, T.; Sato, M.; Kimura, H. Studies on Keten and Its Derivatives. Part 89. Ethyl 4-Substituted Acetoacetates:
Synthesis and Reaction with Diketen. J. Chem. Soc. Perkin Trans. 1 1979, 529–532. [CrossRef]
2. Chandrashaker, V.; Ptaszek, M.; Taniguchi, M.; Lindsey, J.S. Synthesis of diverse acyclic precursors to pyrroles
for studies of prebiotic routes to tetrapyrrole macrocycles. New J. Chem. 2016, 40, 8786–8808. [CrossRef]
3. Padwa, A.; Sá, M.M.; Weingarten, M.D. Metal substituted diazo esters as substrates for cross coupling
reaction. Tetrahedron 1997, 53, 2371–2386. [CrossRef]
4. Bagley, M.C.; Buck, R.T.; Hind, S.L.; Moody, C.J. Synthesis of functionalised oxazoles and bis-oxazoles. J.
Chem. Soc. Perkin Trans. 1 1998, 591–600. [CrossRef]
5. Dar’in, D.; Kantin, G.; Krasavin, M. A ‘sulfonyl-azide-free’ (SAFE) aqueous-phase diazo transfer reaction for
parallel and diversity-oriented synthesis. Chem. Commun. 2019, 55, 5239–5242. [CrossRef] [PubMed]
6. Chen, X.; Xie, Y.; Xiao, X.; Li, G.; Deng, Y.; Jiang, H.; Zeng, W. Rh(III)-catalyzed chelation-assisted
intermolecular carbenoid functionalization of
CrossRef] [PubMed]
α-imino Csp3–H bonds. Chem. Commun. 2015, 51, 15328–15331.
[
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