One-Pot Enzymatic-Chemical Cascade Route for Synthesizing Aromatic α-Hydroxy Ketones

Article abstract of DOI:10.1021/acscatal.0c04961

2-Hydroxyacetophenone (2-HAP) is an important building block for the production of a series of natural products and pharmaceuticals; however, there is no safe, efficient, and economical method for 2-HAP synthesis. Here, a one-pot enzymatic-chemical cascade route was designed for synthesizing 2-HAP based on retrosynthetic analysis. First, a spontaneous proton-transfer reaction was designed using a computational simulation that enabled 2-HAP synthesis from the isomer 2-hydroxy-2-phenylacetaldehyde. A route for 2-hydroxy-2-phenylacetaldehyde synthesis was then constructed by introducing the unnatural substrate glyoxylic acid into a C-C ligation reaction catalyzed by Candida tropicalis pyruvate decarboxylase. Assembly and optimization of this enzymatic-chemical cascade route resulted in a final yield of 92.7%. Furthermore, stereospecific carbonyl reductases were introduced to construct a synthetic application platform that enabled further transformation of 2-HAP into (S)- and (R)-1-phenyl-1,2-ethanediol. This method of cascading spontaneous chemical and enzymatic reactions to synthesize chemicals offers insight into avenues for synthesizing other valuable chemicals.

Full text of DOI:10.1021/acscatal.0c04961

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Research Article
One-Pot Enzymatic−Chemical Cascade Route for Synthesizing
Aromatic α‑Hydroxy Ketones
Lei Wang, Wei Song, Binju Wang, Yan Zhang, Xin Xu, Jing Wu, Cong Gao, Jia Liu, Xiulai Chen,
Jinghua Chen, and Liming Liu*
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ABSTRACT: 2-Hydroxyacetophenone (2-HAP) is an important
building block for the production of a series of natural products and
pharmaceuticals; however, there is no safe, efficient, and economical
method for 2-HAP synthesis. Here, a one-pot enzymatic-chemical
cascade route was designed for synthesizing 2-HAP based on
retrosynthetic analysis. First, a spontaneous proton-transfer reaction
was designed using a computational simulation that enabled 2-HAP
synthesis from the isomer 2-hydroxy-2-phenylacetaldehyde. A route
for 2-hydroxy-2-phenylacetaldehyde synthesis was then constructed
by introducing the unnatural substrate glyoxylic acid into a C−C
ligation reaction catalyzed by Candida tropicalis pyruvate decarbox-
ylase. Assembly and optimization of this enzymatic−chemical
cascade route resulted in a final yield of 92.7%. Furthermore,
stereospecific carbonyl reductases were introduced to construct a synthetic application platform that enabled further transformation
of 2-HAP into (S)- and (R)-1-phenyl-1,2-ethanediol. This method of cascading spontaneous chemical and enzymatic reactions to
synthesize chemicals offers insight into avenues for synthesizing other valuable chemicals.
KEYWORDS: 2-hydroxyacetophenone, spontaneous chemical reaction, pyruvate decarboxylase, glyoxylic acid, (S)-1-phenyl-1, 2-ethanediol
oxidation of α-aryl vicinal diols catalyzed by alcohol
dehydrogenases1 and C−C bond-formation reaction between
benzaldehyde and formaldehyde catalyzed by benzaldehyde
lyases.14 However, the limiting factor of the former method is
the high price of the starting substrate 1-phenylethane-1,2-diol,
whereas that of the latter is the toxicity of the substrate
formaldehyde and production of byproduct benzoin. There-
fore, it remains necessary to develop a safer and more
economical method for efficient synthesis of 2-HAP.
In this study, an enzymatic−chemical cascade route for 2-
HAP synthesis was designed based on the catalytic mechanism
of pyruvate decarboxylase (PDC)15 and hybrid cluster-
continuum (HCC) model calculation. The route for 2-HAP
synthesis was constructed using benzaldehyde and glyoxylic
acid as substrates by introducing PDC and a spontaneous
proton-transfer reaction. Additionally, a route for synthesizing
the optically pure compounds (S)- and (R)-1-phenyl-1,2-
INTRODUCTION
■
α-Hydroxy ketones are important compounds used to
synthesize a series of natural products and pharmaceuticals1−3
in the field of organic chemistry. Among these compounds, 2-
hydroxyacetophenone (2-HAP) is a promising and cost-
effective synthetic intermediate widely used as a building
block for the synthesis of valuable larger molecules, such as α-
ketoesters, syn-amino alcohols, and antitumor Schiff bases.2,4−6
Additionally, 2-HAP is used to prepare optically pure
chemicals, such as (R)- and (S)-1-phenyl-1,2-ethanediol,
which can be used as medicine intermediates and liquid
crystal materials, respectively.7,8
2-HAP can be produced using chemical and biocatalytic
methods. The chemical synthesis methods mainly include
photocatalytic oxidation of phenethyl alcohol, photocatalytic
decarboxylative carbonylation of carboxylic acid,9,10 oxidation
of phenylglycol or terminal olefin by metal and nanometal
catalysis,11,12 and/or dehalogenation of bromoacetophenone
catalyzed by microwave radiation.13 Unfortunately, chemical
methods include disadvantages such as harsh reaction
conditions, high-priced substrates and catalysts, and technical
challenges (such as catalyst stability and heavy metal recovery).
By contrast, biocatalysis enables 2-HAP synthesis using a
highly efficient and environmentally friendly approach.
Currently, enzymatic synthesis of 2-HAP mainly include
Received: November 13, 2020
Revised: February 5, 2021
Published: February 16, 2021
https://dx.doi.org/10.1021/acscatal.0c04961
© 2021 American Chemical Society
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Figure 1. Retrosynthetic analysis of 2-HAP.
Figure 2. BMK/6-311++G(d,p) relative energies (in kcal/mol) for 4a → 5a conversion in the reaction solution. The relative energies are given as
electronic energy first and then free energy (in parentheses) and shown along with B3LYP/6-31G(d)-optimized key species along the reaction
pathway. (a) BMK/6-311++G(d,p) relative energies (in kcal/mol) for 4a → 5a conversion in aqueous solution. (b) BMK/6-311++G(d,p) relative
energies (in kcal/mol) for the acid-catalyzed 4a → 5a conversion in aqueous solution. (c) B3LYP/B1-optimized structures of 4a and 5a. The
calculated relative Gibbs energies (in kcal/mol) at the BMK/B2 level shown below the structures. (d) BMK/6-311++G(d,p) relative energies (in
kcal/mol) for the Mg2+-catalyzed 4a → 5a conversion in aqueous solution.
ethanediol was generated by cascading stereospecific carbonyl
reductases.
glyoxylic acid and benzaldehyde for the synthesis of 4a,
followed by 4a conversion to 2-HAP (5a) by a proton-transfer
reaction.
Exploring the Spontaneous Reaction of 4a to 5a.
During this cascade reaction, the intermediate product 4a is
unstable and difficult to obtain, making it experimentally
difficult to prove that 4a can convert into 5a. Therefore, the
relative Gibbs energy of 4a and 5a was calculated, revealing
that the relative energy of 5a was lower than that of 4a,
theoretically indicating that 4a can be spontaneous converted
to 5a (Figure 2c). This spontaneous reaction was then
explored using an HCC model calculation under water-, acid-,
and Mg2+-catalyzed conditions, respectively, because this
reaction occurs in an enzymatic reaction environment that
includes water molecules, H+, and Mg2+.15 Under water-
catalyzed conditions (Figure 2a), transformations from 4a to
5a comprised two keto−enol tautomerization steps, where the
proton was first transferred from C2−H to O1 (RC1 → IC1b),
followed by transfer from O2−H to C1 (IC1b′ → PC1);
therefore, the rate-determining step involved deprotonation at
RESULTS
■
Design of an Enzymatic−Chemical Cascade Reaction
to Synthesize 2-HAP (5a). 2-HAP (5a) is an α-hydroxy
ketone compound that contains a carbonyl group and a
hydroxyl group and can be synthesized from its isomer 2-
hydroxy-2-phenylacetaldehyde (4a) through keto−enol tauto-
merization via proton transfer. As shown in Figure 1, a
chemical route was first designed to synthesize 2-HAP from 4a.
4a has a special sec-α-hydroxy ketone moiety that allows it to
be synthesized from simple building blocks, such as
benzaldehyde and formaldehyde, through the C−C bond-
formation reaction catalyzed by aldolase. Moreover, 4a can also
be synthesized from benzaldehyde and glyoxylic acid via PDCs
catalysis. Given the toxicity of formaldehyde, the route
catalyzed by PDCs was second designed. Therefore, the
enzymatic−chemical cascade reaction shown in Figure 1
involves a PDC-catalyzed C−C carboligation reaction with
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Figure 3. Natural reaction catalyzed by PDC and the proposed enzymatic−chemical cascade reaction.
Table 1. Summary of Different Pyruvate Decarboxylases Tested for 2-HAP Formation
a
entry
pyruvate decarboxylase
stains
yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
CtPDC1
CtPDC2
CtPDC3
ScPDC
Candida tropicalis 1798
Candida tropicalis 1798
Candida tropicalis 1798
Saccharomyces cerevisiae BY4741
Candida glabrata
Candida glabrata
Candida glabrata
Candida utiliz 1767
Candida utiliz 1767
69.4
n.d.
n.d.
47.4
41.8
n.d.
45.5
30.6
17.9
53.1
48.1
39.4
n.d.
7.7
CgPDC1
CgPDC2
CgPDC3
CuPDC1
CuPDC2
CuPDC3
CuPDC4
SpPDC1
SpPDC2
SpPDC3
SpPDC4
KmPDC
PpPDC
Candida utiliz 1767
Candida utiliz 1767
Schizosaccharomyces pombe 1056
Schizosaccharomyces pombe 1056
Schizosaccharomyces pombe 1056
Schizosaccharomyces pombe 1056
Kluyveromyces marxiamus 1056
Pichia pastoris GS115
n.d.
44.6
2.7
a
Reactions were performed in 1 mL system with E. coli cells expressing the respective pyruvate decarboxylase, the screening conditions: 2 M MOPS
buffer, pH 6.5, 5 mM MgSO4, 0.5% DMSO, 5 mM thiamine diphosphate (ThDP), 5 mM benzaldehyde, 20 mM glyoxylic acid, 30 g/L whole cells,
30 °C, 200 rpm, and 24 h. HPLC analytics. Results are the average of duplicate reaction. n.d. not detectable.
C2 (TS 1a) or protonation at C1 (TS 1d). Under acid-
catalyzed conditions (Figure 2b), the deprotonation reaction
occurred at C2, which was also the rate-determining step.
Under Mg2+-catalyzed conditions (Figure 2d), the reactions
involved an H-shift from C2 to C1, which differed from both
the water- and acid-catalyzed reactions. Furthermore, the
Gibbs energy barriers of the water-, acid-, and Mg2+-specific
conversions were 25.2, 23.1, and 23.3 kcal/mol, respectively,
suggesting the acid-catalyzed reaction as the most favorable.
Construction of the Enzymatic Reaction for 4a
Synthesis. As shown in Figure 3, (R)-phenylacetylmethanol
(3)16 can be produced from benzaldehyde and pyruvate by
PDC catalysis. Following this reaction mechanism, 17 PDCs
were selected to catalyze the carboligation reactions of
benzaldehyde (1a) with glyoxylic acid (2) for efficient
synthesis of 4a. These PDCs were overexpressed in Escherichia
coli and then used as whole-cell catalysts to synthesize 4a
(because 4a can spontaneously convert to 2-HAP, 2-HAP was
detected as the target product) (Table 1). We found that the
PDC from Candida tropicalis 1798 (CtPDC1) exhibited the
highest yield (69.4%) and this PDC was subsequently chosen
as the catalyst for further experiments.
On the basis of the catalytic mechanism of PDC on natural
substrates (Figure 4a), a putative formation mechanism of 4a
from 1a with 2 was proposed (Figure 4b). First, the cofactor
undergoes deprotonation to form a reactive ylide form,
followed by nucleophilic attack by the ThDP C2 carbanion
of the donor substrate 2 (Figure 2b, shown in blue) to produce
a highly reactive intermediate (LThDPa). Second, the resonant
carbanion/enamine forms of the postdecarboxylation inter-
mediate 2-hydroxyethyl-ThDP (HEThDP−a) are produced by
LThDPa decarboxylate. Subsequently, the “acceptor” species
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Figure 4. Mechanism of the natural reaction catalyzed by PDCs and the putative formation mechanism of4a. (a) The reaction mechanism
involving natural substrates catalyzed by PDC.18 The catalytic process of PDC can be subdivided into six elementary steps: (1) the cofactor is
deprotonated to form a reactive ylide; (2) the ThDP C2 carbanion attacks the donor substrate carbonyl in a nucleophilic manner to yield the
tetrahedral predecarboxylation intermediate 2-lactyl-ThDP (LThDP); (3) decarboxylation of LThDP generates the resonant carbanion/enamine
forms of the postdecarboxylation intermediate 2-hydroxyethyl-ThDP (HEThDP−); (4) if the second substrate is H+, protonation at C2α of
HEThDP− yields its conjugate acid HEThDP; (5) the C2α−OH group of HEThDP is ionized, and cleavage of its C2−C2α bond leads to
liberation of the acetaldehyde product; and (6) if the second substrate is 1a, the HEThDP− carbanion attacks the acceptor 1a carbonyl group in a
nucleophilic manner to yield (R)-PAC. (b) The putative formation mechanism of 4a.
Figure 5. UV−vis spectra of 1a reacting with the natural substrate pyruvic acid and the unnatural substrate 2. (a) UV−vis spectra of reaction with
the natural substrate pyruvic acid over the course of 2 h. (b) UV−vis spectra of reaction with the unnatural substrate 2 over the course of 2 h.
Spectra were collected on a Shimadzu UV2550 spectrophotometer in a quartz cuvette with a 1 cm path length at 30 °C. Samples were prepared in a
total volume of 1 mL with 10 μM of purified enzyme (with 1a 1 mM, 2 15 mM, Mg2+ 5 mM, 0.5% DMSO and ThDP 1 mM added, as needed) in
2.5 M MOPS buffer (pH 6.5).
1a (Figure 4b, shown in red) is attacked by the activated
aldehyde intermediate (HEThDP−a) and directly generates
product 4a. To confirm the formation mechanism of 4a, the
reaction-process curve was generated using UV−vis spectros-
copy by first incubating 10 μM CtPDC1 with 1 mM 1a in 2.5
M MOPS buffer (Mg2+ 5 mM, 0.5% DMSO and ThDP 1
mM). At this point, the enzyme is still in the resting state17
(Figure 5a and 5b, dotted lines), because when 1a as the only
substrate, there was no catalytic reaction occurred (Figure S1
of Supporting Information, SI). After addition of 15 mM
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Figure 6. Putative mechanism and verification of donor−acceptor exchange caused by CtPDC1. (a) Speculative mechanism of donor−acceptor
exchange catalyzed by CtPDC1. The cofactor ThDP is deprotonated to form a reactive ylide, followed by nucleophilic attack by the ThDP C2
carbanion of the donor 1a (shown in blue) to yield intermediate A, which attacks an “acceptor” species formaldehyde (shown in red) formed by
decarboxylation of 2 to produce the product 5a. (b) Verification of the donor−acceptor exchange mechanism via CtPDC1 catalysis.
pyruvic acid or 2, no significant change in the curve was
observed immediately; however, the absorbance at between
240 and 260 nm gradually decreased in 2 h (Figure 5). The
trends of the spectral curves associated with the natural (Figure
5a) and unnatural (Figure 5b) reactions were determined as
being the same, indicating similar reaction processes and
transition states. This suggested that our proposed formation
mechanism of 4a from 1a with 2 was correct.
Assembly and Optimization of Chemoenzymatic
Synthesis of 2-HAP. With 1a and 1b as substrate, the
product 5a and 5b were produced by the enzymatic-chemical
cascade reaction of CtPDC1-catalyzed C−C carboligation
reaction and spontaneous proton transfer. The relative
molecular mass of 5a and 5b were 136.08 and 151.0124
(Figure S2), respectively, detected by GC−MS and LC−MS.
The products were then separated and purified using
preparative high-performance liquid chromatography and
determined by 1H and 13C NMR spectroscopy, which revealed
a CH2-absorption peak for product 5 rather than a CH-
absorption peak for product 4 (Figures S3a and S4a). These
results demonstrated that the enzymatic−chemical cascade
reaction successfully synthesized 5a and 5b.
CtPDC1. The first way is to detect the presence of
intermediate A by UV−vis spectroscopy and fluorescence
spectroscopy (Figure 6a); however, given the speed of the
reaction process, it was difficult to capture intermediate A. The
second way is to determine whether 1a becomes the donor
substrate (Figure 6b). In theory, if 1a is the donor substrate, it
will react with the acceptor substrate 1a to produce product B
via C−C carboligation. In fact, in the reaction described, 1a
was the only substrate, and no product B was detected (Figure
S1). Because 1a was not used as the donor substrate, formation
of 5a was not a result of enzyme−catalyzed donor−acceptor
exchange.
The transformation conditions of the enzymatic−chemical
cascade reaction were then optimized (Figure 7), resulting in
the following: 1a 4.5 mM, 2 15 mM, Mg2+ 5 mM, ThDP 1
mM, 0.5% DMSO, MOPS buffer 2.5 M, reaction temperature
30 °C, pH 6.5, and a 3.5-h reaction duration. The optimal
conditions increased the titer of 5a to 3.7 mM with a 82.7%
yield (Figure 7). To further increase the production efficiency
of 5a, a homodimer structural model of CtPDC1 in complex
with two cofactors (ThDP) and two Mg2+ was generated via
homologous modeling using Kluyveromyces lactis PDC (PDB
ID: 2VJY; 70% identity) as the template. Docking of substrate
2 and natural substrate 2* to the allosteric region of the model
There are two ways to verify whether the formation of 5a
was caused by donor−acceptor exchange catalyzed by
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Figure 7. Optimization of transformation conditions of the enzymatic−chemical cascade reaction. Optimization of (a) 2 concentration, (b) Mg2+
concentration, (c) thiamine diphosphate (ThDP) concentration, (d) the reaction temperature, (e) reaction pH, and (f) reaction time. Initial
conditions: 1a 4.5 mM, 2 20 mM, Mg2+ 5 mM, ThDP 5 mM, 0.5% DMSO, MOPS buffer 2.5 M, reaction temperature 30 °C, pH 6.5, and a 24-h
reaction time.
Figure 8. Docking of (a) pyruvic acid and (b) 2 substrates into the allosteric region of CtPDC1.
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of CtPDC1 resulted in substrates in approximately the same
orientation (Figure 8); therefore, residues H94, C223, A289,
312H, and 313S, all of which showed interactions with the
substrate in the allosteric region, were selected for site-directed
saturation mutation. Among these mutations, CtPDC1C223E
resulted in an increased yield of 92.7%, 10% higher than that of
CtPDC1WT. This was a result of increases and decreases in kcat
and Km (2) for CtPDC1C223E of 1.7 and −0.4, respectively,
relative to CtPDC1WT (Table 2), suggesting increased affinity
for the substrate.19
meta-substituted benzaldehyde as substrates and catalyzes the
synthesis of the corresponding 2-HAP derivatives, though the
yields are relatively low (0.2−23.3%). (2) The electron-
withdrawing or donating substituents at the para position
(Table 3 and 5b, e, h, k, and n) result in relatively high yields
relative to substrates of ortho- or meta-substituents. Among
para-substituents, electron-withdrawing substituents (5e, h,
and k) are more reactive than electron-donating ones (5b and
n). (3) In general, the yields from substrates of meta-
substituents were higher than those of substrates of ortho-
substituents. In conclusion, the high yields from electron-
withdrawing para-substituents might be due to the attraction of
the electron-withdrawing groups on the benzene ring, resulting
in less charge on α-C, which is favorable for the nucleophilic
addition reaction.
Table 2. Biochemical Characterization of CtPDC1WT and
Mutants
Km (2)
[mM]
k
cat/Km (2)
yield
[%]
enzyme
kcat [s−1
]
[mM−1 s−1
]
Constructing the Cascade Reaction to Produce (S)-
and (R)-1-Phenyl-1,2-ethanediol. CtPDC1C223E can con-
vert 1a and 2 to 2-HAP; therefore, the ability of 2-HAP to be
used as a substrate to synthesize other higher-value products
was determined. A cascade reaction was then designed with
PDC, carbonyl reductase (SCR or RCR), and glucose
dehydrogenase (GDH) to produce optically pure (S)- and
(R)-1-phenyl-1,2-ethanediol [(S)- or (R)-PED] using 1a and 2
as substrates (Figure 9). Additionally, based on the literature
CtPDC1WT
6.9 0.22
6.5 0.16
96.4 0.14
98.1 0.12
14.0
15.1
82.7
92.7
CtPDC1C223E
Since the property and position of any substituent in a
benzene ring affect the nucleophilic addition reaction, the
effects of the electron-withdrawing and donating substituents
on reaction yield were investigated (Table 3). It was found that
(1) CtPDC1C223E accepts various forms of ortho-, para-, or
a
Table 3. Substrate Scope of C−C Carboligation Reaction Catalyzed by CtPDC1C223E
a
Standard reaction conditions. Reactions were performed in 1 mL system: Substrate 1 (4.5 mM), glyoxylic acid (15 mM), E. coli (CtPDC1C223E) 30
g/L, Mg2+ 5 mM, ThDP 1 mM, 0.5% DMSO, MOPS buffer (2.5 M, pH 6.5), reaction temperature 30 °C, and 24 h. Yield determined via GC−MS
with 5a as a standard.
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Figure 9. Biosynthesis of (S)-PED and (R)-PED using the respective engineered E. coli whole-cell catalysts.
mining and the enzyme activity, the NADH-dependent SCR
from Gluconobacter oxydans, the RCR from Bacillus subtilis
168,8 and BmGDH from Bacillus megatherium20 were used to
construct engineered E. coli strains harboring pET-Deut-
GoSCR-BmGDH or pET-Deut-BsRCR-BmGDH plasmids (E.
coli WL03 and E. coli WL04, respectively) (Figure S5 and
Table 4).
with an ee value of 99.9%. The results showed that 26.3% of
the 2-HAP intermediate accumulated during the reaction,
which could not be further transformed. This indicated that
the catalytic activity of GoSCR toward 2-HAP was the rate-
limiting step of the cascade reaction.
The 1-pot 2-stage cascade reaction for (R)-PED synthesis
combined CtPDC1C223E and BsRCR as catalysts. For the
subsequent reduction reaction, 60.4% substrate conversion of
60.4% and 99.9% ee value for 24 h was observed, with the
overall conversion of the cascade reaction at 56.0% (Figure
10b). Additionally, 44.0% of the 2-HAP intermediate
accumulated during the reaction. The results suggested that
the reduction reaction remained the rate-limiting step;
therefore, our future work will focus on improving the speed
of this cascade reaction.
Table 4. Specific Enzyme Activity and ee Values of Carbonyl
Reductases
enzyme
specific enzyme activity (U/mg protein)
ee (%)
BsRCR
PpRCR
GoSCR
0.39
1.28
0.53
>99.9
33.5
>99.9
The enzymatic−chemical cascade route for preparative scale
reactions with E. coli expressing CtPDC1C223E as a whole-cell
biocatalyst (30 g/L, wet cells) was tested in a 100 mL reaction
volume. Under optimal conversion conditions (2.5 M MOPS
buffer [pH 6.5], 10 mM MgSO4, 10 mM ThDP, 5% DMSO;
benzaldehyde and glyoxylic acid were added in 20 mM and 60
mM batches every 2 h, respectively), this route converted the
substrates benzaldehyde (100 mM, 10.6 g/L) and glyoxylic
acid (300 mM, 22.2 g/L) to 12.3 g/L of 2-HAP after 24 h at
30 °C and 200 rpm, with a 90.0% conversion and 81% (996
mg) isolated yield, respectively. Additionally, preparative scale
reactions were conducted on (S)-PED and (R)-PED.
Subsequently, (S)-PED was obtained with a 71.2% (9.8 g/L)
overall conversion, 99.9% ee value, and 63% (617 mg) isolated
yield, and (R)-PED was obtained with a 55.4% (7.7 g/L)
overall conversion, 99.9% ee value, and 60% (462 mg) isolated
yield.
Evaluation of the transformation modes of the cascade
reaction revealed two modes: a 1-pot 1-stage mode
(CtPDC1C223E and E. coli WL03 participate in the reaction
together) and a 1-pot 2-stage mode (CtPDC1C223E and E. coli
WL03 participate in the reaction in order). The yield and
enantiomeric excess (ee) values for the 1-pot 1-stage mode
were 57.0% and 99.9%, respectively, whereas those for the 1-
pot 2-stage mode were 73.7% and 99.9%, respectively.
Additionally, the byproduct benzyl alcohol was identified in
both modes, with the former mode producing 0.7 mM and the
latter 0.1 mM. Therefore, the 1-pot 2-stage mode was chosen
for the cascade reaction (Table 5).
Table 5. Comparison of the Two Transformation Modes
mode
yield (%)
ee (%)
byproduct (mM)
1-pot 1-stage
1-pot 2-stage
57.0
73.7
99.9
99.9
0.7
0.1
DISCUSSION
■
In this study, the design and evaluation of a cascade reaction
combining enzymatic and spontaneous chemical reactions to
produce 2-HAP were described. On the basis of retrosynthetic
analysis, a spontaneous proton-transfer reaction was designed
to generate a synthetic route for 2-HAP from 4a through
computational simulation. Furthermore, the unnatural sub-
strate 2 was introduced into the C−C ligation reaction
catalyzed by CtPDC1 in order to construct the route for 4a
synthesis. Using 2-HAP as the starting substrate, routes to
The 1-pot 2-stage cascade reaction for (S)-PED synthesis
combines CtPDC1C223E and the (S)-selective GoSCR as
catalysts. At the first stage, a nonisolated yield (92.7%, from
4.5 mM substrate) of 2-HAP was obtained in 3.5 h. In the
second stage, the reaction was initiated by adding E. coli WL03,
0.2 mM NAD+, and 6 mM D-glucose, resulting in substrate
conversion of 79.5% and an ee value of 99.9% (24 h) (Figure
10a). The overall conversion of the cascade reaction was 73.7%
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Figure 10. 1-pot 2-stage cascade reaction from 1a and 2 to (S)-and (R)-PED. Time courses of (a) (S)-PED and (b) (R)-PED synthesis using the 1-
pot 2-stage mode. Reaction conditions: step 1:2.5 M MOPS (pH 6.5), 5 mM Mg2+, 1 mM ThDP, 0.5% DMSO, 4.5 mM 1a, 15 mM 2, 30 g/L E.
coli (CtPDC1C223E), 30 °C, 200 rpm, and 3.5 h; Step 2: pH 7, 0.2 mM NAD+, 6 mM D-glucose, 30g/L E. coli WL03 or E. coli WL04, 2.5 M MOPS,
30 °C, 200 rpm, and 24 h.
produce (S)- and (R)-1-phenyl-1,2-ethanediol were also
generated. The cascade reaction presented here represents a
low-cost method for limiting byproduct production and the
substrate toxicity associated with 2-HAP synthesis.
addresses the potential mass-transfer problem between the
two catalysts.
In general, the natural substrates of PDCs are pyruvate and
1a;16 however, previous studies focused on expanding the
catalytic availability of PDCs to include 1a derivatives as
substrates [i.e., PDC-mediated catalysis of the reaction of
pyruvate with 2,5-dimethoxybenzaldehyd34 and a halogenated
1a35 to produce 1-hydroxy-1-(2,5-dimethoxyphenyl)propan-2-
one and the corresponding halogenated phenylacetylcarbinol].
In fact, pyruvate as the smallest keto acid substrate has multiple
structural analogs. In the present study, 2 was used as a
substrate for CtPDC1 to promote its reaction with 1a, resulting
in the product 4a. To the best of our knowledge, this is the first
report using 2 as a smaller unnatural substrate to participate in
a C−C carboligation reaction to produce aromatic α-hydroxy
ketones. The expanding number of pyruvate-like substrates has
led to increased numbers of aromatic α-hydroxy ketones
synthesized by PDCs. Furthermore, biotransformations with
CtPDC1C223E have a broad substrate scope, where
CtPDC1C223E accepts various forms of ortho-, para-, or meta-
substituted benzaldehyde as the substrate and catalyzes the
synthesis of the corresponding 2-HAP derivatives. For the
enzyme catalysis, expanding the substrate scope of a specific
enzyme represents an efficient strategy for acquiring new
products. For example, by expanding the substrate tunnel of
CgTD, the substrate scope of CgTD could potentially show
affinity for the bulky substrate phenylserine.36 Additionally, a
previous study produced a series of noncanonical amino acids
by engineering tryptophan synthase to enlarge its substrate
scope to include nitroalkane nucleophiles.37
Here, spontaneous chemical reactions were used as a
synthetic strategy in a cascade reaction to produce chemicals
in order to (1) maximize reaction compatibility, (2) minimize
the need for the reaction to be driven by an outside energy
source, and (3) remove barriers to mass transfer. One example
is a spontaneous ring-closing reaction using cascading
asymmetric reductive amination to synthesize biaryl-bridged
NH lactams under Ru-catalyzed conditions.21 A second
example is in the synthetic pathway of macromolecular natural
products quinolone alkaloids catalyzed by dioxygenase AsqJ, a
spontaneous nonenzymatic rearrangement and elimination
reaction is required to achieve the synthesis of the final
product.22 Recently, chemical reactions have increasingly
combined with biocatalysis to synthesize a series of high-
value chemicals, including terpenes and their analogues
(arabinomannan, glycoconjugates, and glycopeptide antibiotic
analogues).23−25 However, chemical−enzymatic cascade re-
actions are often limited by poor compatibility in areas,
including temperature, solvent, pH, and reaction condi-
tions,26−29 that make it difficult to achieve one-pot, continuous
catalysis under the same condition. In the present study,
efficient and environmentally friendly synthesis of 2-HAP was
achieved through introducing a spontaneous chemical reaction
after CtPDC1-catalyzed C−C ligation reaction, where the
presence of H+ and Mg2+ in solution promoted the
spontaneous reaction. Furthermore, compared with chemical
reactions requiring higher temperatures, high pressure, and
specific light conditions,13,26,30 this spontaneous chemical
reaction does not require external energy to drive the
transformation, which involves conversion of 4a into 2-HAP
via proton transfer, thereby promoting the concepts of “atom
economy” and “green chemistry”.31,32 Furthermore, compared
with other enzymatic−chemical cascade reactions involving
two or more catalysts,23,24,26−28,33 this spontaneous chemical
reaction does not require an additional catalyst, which
The goal of this process was to insert a new reaction into a
cascade reaction to promote synthesis of new products. A
previous study described dihydroxylation of benzene catalyzed
by P450-BM3 for synthesis of the natural product arbutin via a
cascading glucosyltransferase reaction.38 In the present study,
stereospecific SCR and RCR were used to produce the
optically pure compounds (S)- and (R)-1-phenyl-1,2-ethane-
diol, respectively. Additionally, under optimized conditions,
the preparative scale transformations for the synthesis of (S)-
and (R)-PED were achieved. This pathway has three
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ACS Catal. 2021, 11, 2808−2818

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pubs.acs.org/acscatalysis
Research Article
characteristics: (1) it realizes the transformation process of
“achiral compounds−prochiral compounds−chiral com-
pounds” to synthesize high-value chemicals through chiral-
group resetting;39 (2) compared with the starting substrates 4-
phenyl-1,3-dioxolan-2-one, styrene, and 2-HAP40−42 for syn-
thesis of (S)- or (R)-1-phenyl-1,2-ethanediol, it uses the achiral
substrates 1a and 2, which are readily available, inexpensive,
and represent sustainable alternatives for producing optically
pure 1-phenyl-1,2-ethanediol; and (3) this one-pot enzymatic−
chemical cascade reaction can incorporate other enzymes, such
as transaminases, to synthesize high-value amino alcohols from
1a and 2.43,44
In summary, an enzymatic−chemical cascade synthetic
pathway was designed combining CtPDC1-catalyzed unnatural
reaction and a spontaneous proton transfer for synthesizing 2-
HAP. This method represents a platform for synthesizing
aromatic α-hydroxy ketones from low-cost starting materials
and offers an attractive strategy and reference for the synthesis
of other chemicals.
Cong Gao − State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi 214122, P. R. China
Jia Liu − State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi 214122, P. R. China
Xiulai Chen − State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi 214122, P. R. China
Jinghua Chen − School of Pharmaceutical Sciences, Jiangnan
University, Wuxi 214122, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.0c04961
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was financially supported by the National Natural
Science Foundation of China (22038005, 22008089, and
32021005), the National Key R& D Program of China
(2018YFA0901400), the national first-class discipline program
of Light Industry Technology and Engineering (LITE2018-
08).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c04961.
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AUTHOR INFORMATION
■
Corresponding Author
Liming Liu − State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi 214122, P. R. China;
Phone: +86-510-85197875; Email: mingll@
jiangnan.edu.cn
Authors
Lei Wang − State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi 214122, P. R.
China; orcid.org/0000-0002-5243-2185
Wei Song − State Key Laboratory of Food Science and
Technology and School of Pharmaceutical Sciences, Jiangnan
University, Wuxi 214122, P. R. China; orcid.org/0000-
0002-2278-2205
Binju Wang − State Key Laboratory of Physical Chemistry of
Solid Surfaces and Fujian Provincial Key Laboratory of
Theoretical and Computational Chemistry, Xiamen
University, Xiamen 360015, P. R. China; orcid.org/0000-
0002-3353-9411
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