Development of a Novel Chemoenzymatic Process for (S)-1-(Pyridin-4-yl)-1,3-propanediol

Article abstract of DOI:10.1021/acs.oprd.0c00403

We first developed a novel and efficient chemoenzymatic process to prepare (S)-1-(pyridin-4-yl)-1,3-propanediol, a vital HepDirect prodrug intermediate, from inexpensive and commercially available isonicotinic acid. Through this process, we provide a creative way to obtain the key chiral intermediate, β-hydroxyester, with ketoreductase (KRED) EA. After optimization of the process, we performed the reaction on a 100 g scale with a substrate concentration of up to 150 g/L, a yield of 93%, and an ee value of up to 99.9%. Additionally, we used a simple and effective NaBH4/MgCl2 reduction system to obtain (S)-1-(pyridin-4-yl)-1,3-propanediol with >99.9% ee and an 80% yield. This novel chemoenzymatic process has the potential to be a cost-effective and environmentally friendly process suitable for industrial use.

Full text of DOI:10.1021/acs.oprd.0c00403

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Development of a Novel Chemoenzymatic Process for (S)‑1-(Pyridin-
4-yl)-1,3-propanediol
Hong-Yi Wang, Jia-Wei Tang, Peng Peng, Hai-Jun Yan, Fu-Li Zhang,* and Shao-Xin Chen*
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ABSTRACT: We first developed a novel and efficient chemoenzymatic process to prepare (S)-1-(pyridin-4-yl)-1,3-propanediol, a
vital HepDirect prodrug intermediate, from inexpensive and commercially available isonicotinic acid. Through this process, we
provide a creative way to obtain the key chiral intermediate, β-hydroxyester, with ketoreductase (KRED) EA. After optimization of
the process, we performed the reaction on a 100 g scale with a substrate concentration of up to 150 g/L, a yield of 93%, and an ee
value of up to 99.9%. Additionally, we used a simple and effective NaBH4/MgCl2 reduction system to obtain (S)-1-(pyridin-4-yl)-
1,3-propanediol with >99.9% ee and an 80% yield. This novel chemoenzymatic process has the potential to be a cost-effective and
environmentally friendly process suitable for industrial use.
KEYWORDS: (S)-1-(pyridin-4-yl)-1,3-propanediol, ketoreductase (KRED), chemoenzymatic process, HepDirect prodrugs,
β-carbonyl ester, NaBH4/MgCl2
in the presence of cofactor NAD(P)H.12 With the increasing
1. INTRODUCTION
use of KREDs, an increasing number of chiral intermediates
HepDirect prodrugs have attracted the attention of many
are being synthesized by KREDs instead of chemical
researchers since the development of HepDirect technology by
catalysts.13,14 For example, Wei et al. reported an enzymatic
Erion et al.1,2 These prodrugs are 1,3-diol cyclic phosphates
process to synthesize (S)-2-chloro-1-(2,4-dichlorophenyl)
that are designed to undergo an oxidative cleavage reaction in
ethanol, the chiral intermediate of luliconazole, by a novel
the liver, catalyzed by cytochrome P450 (CYP).3−5 These
KRED mutant, LK08, from Lactobacillus kefiri.15 In 2004,
prodrugs selectively release the monophosphate of a nucleo-
Berendes Frank reported the bioreduction of ketone 4 to 5
side (NMP) in CYP3A4-expressing cells (e.g., hepatocytes),
using Saccharomyces cerevisiae, with a substrate concentration
while remaining intact in the plasma and extrahepatic tissues.6,7
of 20 mmol/L (3.9 g/L) and an of ee 81% (the methyl ester
derivative had an ee of 96%).16 Herein, we aimed to develop a
novel chemoenzymatic process for (S)-1-(pyridin-4-yl)-1,3-
As a vital prodrug intermediate, (S)-1-(pyridin-4-yl)-1,3-
propanediol (1) has found several applications in active
compounds as potential pharmaceuticals, such as MB-07133, a
propanediol to overcome the shortcomings of existing routes.
phase III liver cancer prodrug5 (Chart 1a). Additionally, other
We first developed the bioreduction process using KRED EA
to catalyze the transformation of substrate 4 to 5 with high
efficiency (150 g/L substrate concentration, >99.9% ee, and
preclinical compounds exist that use this intermediate (Chart
1b−d).8−10
The existing synthesis methods of (S)-1-(pyridin-4-yl)-1,3-
93% yield, Scheme 1c). Additionally, the starting material 2 is
propanediol have drawbacks such as low enantiomeric purity,
inexpensive and commercially available, and all reactions in our
low yield, and inconvenient operation. In 2006, Boyer first
process are mild with a simple purification procedure. After
reported the synthesis of (S)-1-(pyridin-4-yl)-1,3-propanediol
using isonicotinaldehyde as the starting material via the aldol
reaction, chiral separation, and LiAlH4 reduction to obtain the
target product, with an ee of 96% and an overall yield of 26%5
(Scheme 1a). In this way, the use of LDA, EDCI, LiAlH4, and
column chromatography for purification at every step limited
its applications in industries. In 2007, Erion selected 4-
acetylpyridine as a starting material, and through ester
condensation, asymmetric catalysis, and NaBH4 reduction,
the target compound was obtained but with a total yield of
only 16%11 (Scheme 1b). In this route, the expensive catalyst
RuCl(p-cymene)[(S,S)-Ts-DPEN] (s/c 500:1) and the low ee
value (87%) restricted its applications.
optimization of the reaction parameters, the new route showed
an overall yield of 55% with a product ee of >99.9% and purity
of up to 99.5%. This route provides great potency in the
industrial manufacturing of 1.
2. RESULTS AND DISCUSSION
2.1. Optimization of Esterification (2 to 3). Initially, we
carried out the esterification of isonicotinic acid with EtOH
Received: September 7, 2020
Ketoreductases (KREDs) catalyze reduction−oxidation
(redox) reactions between ketones and alcohols. The
biotransformation of ketones to chiral alcohols is carried out
https://dx.doi.org/10.1021/acs.oprd.0c00403
Org. Process Res. Dev. XXXX, XXX, XXX−XXX
© XXXX American Chemical Society
A

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Chart 1. Application of (S)-1-(pyridin-4-yl)-1,3-propanediol 1 in different drugs: (a) liver cancer drugs, Chiva Pharmaceuticals
& Ligand; (b) FXR/TGR5 agonists, Xi’An Biocare Pharma; (c) antihepatitis C virus drugs, Ligand; and (d) antihepatitis C
virus drugs, Merck & Co. and Ligand
Scheme 1. Synthesis of (S)-1-(Pyridin-4-yl)-1,3-propanediol
using 1.0 equiv of H2SO4.17 However, only 71% conversion
was obtained after 15 h (Table 1, entry 1). Increasing the
equivalents of H2SO4, we found that the reaction was
significantly accelerated, and the conversion increased (entries
2−4). When the H2SO4 was increased to 1.5 equiv (entry 3),
the reaction equilibrium was reached within 4 h, with 88%
conversion, and the extension of time had no effect on the
conversion. Although H2SO4 was increased to 2.0 equiv, we
observed that the conversion was not significantly improved
(entry 4). Moreover, the reaction required an equimolar
amount of Na2CO3 to quench H2SO4 during the reprocessing
operation. Considering the factors above, 1.5 equiv of H2SO4
was chosen for the reaction. The reaction was terminated at 4
h with an 88% isolated yield and a 99.7% purity (HPLC).
2.2. Optimization of Ester Condensation (3 to 4).
Initially, we performed the ester condensation reaction using
EtONa (1.5 equiv) and EtOAc under reflux conditions. EtONa
was prepared using NaH with EtOH in situ18 (Table 2, entry
B
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a
the reaction mixture to obtain the product; however, the
impurities and remaining raw materials were extracted
together, resulting in the purity of the crude product being
95%. Further crystallization (by EtOH/H2O 3:7) was
necessary to obtain a product purity of >99.0%, but this led
to yield loss and additional processing steps. Later, we were
surprised to find that when the reaction was quenched with
water, the unreacted ethyl isonicotinate was completely
hydrolyzed, but the product remained stable. After the
distillation of THF, the aqueous phase was gradually acidified
to a pH of 7.5 with 37% HCl, and the product precipitated out
with a purity of 99.6%. The simple precipitation method
avoided extraction and recrystallization and enabled one to
directly obtain products with a purity of 99.6% for the next
reaction. When we performed the condensation with the
optimized conditions on the 100 g scale, we found that the
hydrolysis impurity level was reduced to 5%, and the product
content was increased to 93% in HPLC. Finally, we obtained
the product with an 86% isolated yield and a 99.6% purity
(HPLC).
Table 1. Effect of H2SO4 Equivalents on the Reaction
b
HPLC (%)
entry
H2SO4 (equiv)
time (h)
2
3
1
2
3
4
1.0
1.2
1.5
2.0
15
12
4
28.7
14.8
12.0
10.1
71.3
85.2
88.0
89.9
4
a
Reaction conditions: all experiments were carried out with 20 equiv
of EtOH (10 V). Composition of the product was measured by
b
HPLC.
Table 2. Effects of Base and Solvents on Ester
a
Condensation
2.3. Optimization of Bioreduction Parameters (4 to
5). 2.3.1. Screening of KREDs for the Bioreduction of Ketone
4. We found no report of a ketoreductase that can be used to
catalyze substrate 4 to alcohol 5. To identify new
ketoreductases for the synthesis of alcohol 5, 16 KREDs
preserved in our laboratory were tested. Table 3 shows that
seven KREDs were found to be active toward substrate 4.
KREDs 736, EA and 1500 showed stereoselectivities of ee
>98% (Table 3, entries 3, 7, and 16), while KRED EA showed
the highest stereoselectivity of ee 99.6% (entry 7). Although
1306 showed high activity toward ketone 4, its ee value was
only 16.5% (entry 15). Therefore, further study of KRED EA
was carried out.
d
HPLC
entry
base (equiv)
solvent time (h)
3
2
4
b
1
2
3
4
5
6
EtONa (1.5)
EtONa (3.0)
t-BuONa (2.0)
t-BuOK (2.0)
t-BuOK (1.8)
t-BuOK (1.5)
none
none
THF
THF
THF
THF
17
17
2
2
2
23.3
8.2
5.3
1.3
1.9
5.7
4.6
7.2
12.9
8.7
8.9
9.1
71.1
84.5
79.3
89.6
89.2
85.0
b
c
c
c
c
2
a
Reaction conditions: all experiments were carried out on a 6 g scale.
Under reflux conditions with 2 equiv of EtOAc. With 1.2 equiv of
EtOAc and 2.0 M t-BuOK/t-BuONa solution in THF. The product
b
c
d
2.3.2. Optimization of the Substrate Concentration.
Optimization of the reaction parameters not only improves
the conversion efficiency but also minimizes production costs
to meet industrial requirements. The substrate concentration is
the first and most important factor that must be considered in
industrial production. The substrate loading study was carried
out with a concentration range of 25−200 g/L to investigate
the catalytic ability. The results showed that the bioreduction
can proceed completely with a substrate concentration lower
than 100 g/L. However, the conversion was found to decrease
sharply when the substrate concentration was increased to 150
g/L. In our previous research, we found that the pH and
temperature are essential for improving the enzyme activity
and the cosubstrate equivalent is a key factor for the
biocatalytic process.15,19 Thus, the process optimization for
the reaction conditions, including the pH, temperature,
cosubstrate loading, and catalyst loading, was subsequently
performed (Figure 1).
2.3.3. Optimization of the pH and Temperature. The pH
and temperature greatly influence the enzymatic activity. A pH
tolerance study of the reaction system including KRED EA and
GDH was carried out in the pH range of 4−9 (Figure 2a). The
results showed that when the pH is below 5, the catalytic
activity of the enzymes somewhat decreases, while above pH 7,
the enzyme activity decreases drastically. The optimum pH is
6−7. As shown in Figure 2b, the optimum temperature for the
reaction system was determined to be 35−40 °C. When the
temperature was above 40 °C, the conversion was found to
sharply decrease. Thus, the optimized temperature was chosen
to be 35 °C.
content was measured by HPLC.
1). However, we found that the product content was only 71%
in HPLC. Even with an increased amount of EtONa to 3.0
equiv, the product content was only 84% in 17 h (entry 2).
Moreover, we found that the purity of commercial EtONa was
only 96−98%, which contains 2−4% NaOH. This resulted in
the complete hydrolysis of 3. Therefore, EtONa must be
prepared using NaH or sodium with EtOH in situ, which limits
its application. To overcome these drawbacks, we aimed to
explore a commercially available base containing a lower
amount of hydroxide. We tested t-BuONa and t-BuOK; t-
BuOK showed better conversion and a higher product content
in HPLC (entries 3 and 4). Experiments for various
equivalents of t-BuOK showed that 1.8 equiv of t-BuOK was
a suitable choice for product content and reagent consumption
(entries 4−6). The hydrolysis of ethyl isonicotinate in the
reaction occurred caused by KOH, which was probably
generated by the weighing of t-BuOK in air. Water in THF
and EtOAc also accounted for the hydrolysis. Attempts to use
2-MeTHF (lower water content) as the solvent instead of THF
resulted in an extremely sticky reaction mixture; the reason for
this may be the poor solubility of t-BuOK in 2-MeTHF (<1
mol/L) and the good solubility in THF (2 mol/L). Therefore,
we decided to use THF as the reaction solvent. The optimum
conditions are shown in entry 5.
Additionally, we developed a stable and easy-to-operate
workup process to obtain a >99.5% pure product. Initially, we
quenched the reaction with 2 N HCl to neutral and extracted
C
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glucose equivalent amount was chosen as 1.5 equiv. However,
the conversion was merely 65−70% under the existing
conditions, and thus, we increased the KRED EA wet cell
dosage from 50 to 150 g/L. As shown in Figure 2d, the
reaction can proceed completely with 100 g/L wet cells, and
thus, 100 g/L wet cells was chosen.
Table 3. Screening of KREDs from Our Enzyme Library
2.3.5. Scale-Up of Bioreduction. With the optimized
parameters for the bioreduction process (150 g/L substrate
concentration, 100 g/L KRED EA wet cells, 1.5 equiv of
glucose, 20 g/L GDH wet cells, 0.5 g/L NADP+, 35 °C, and
pH 7.0), the reaction was tested on a 10 g scale. In the
reaction, the pH decreased gradually due to the generation of
gluconic acid during the cofactor regeneration cycle. When we
adjusted the pH using 10% Na2CO3, the addition of a base
solution led to enzyme inactivation. We resolved this problem
by the slow addition of 5% Na2CO3 to the reaction mixture.
After we verified the process again on the 30 g scale, we
performed the reaction on a 100 g scale. The reaction profile
obtained is shown in Figure 3. No negative impact on the
reaction was observed while operating on the 100 g scale. The
product was obtained with a 99.9% ee, 99.5% purity, and 93%
yield. The space−time yield of the bioreduction system
reached 222 mmol/L/h, indicating that when required, this
reaction can be scaled to larger quantities without the need for
further optimization. A comparison of two asymmetric
reduction systems is shown in Table 4. Our enzymatic process
has the potential to be a cost-effective and environmentally
friendly process suitable for industrial use.
2.4. Optimization of Ester Reduction (5 to 1). In
Erion’s work, the methyl ester was reduced by NaBH4 in
BuOH, but the reaction required heating to 93 °C for several
hours.11 We tried to change the reduction system to a lower
temperature and found that the combination of NaBH4/EtOH
could reduce the ester at a lower temperature (Table 5, entry
1). It has been reported that NaBH4 could be applied to ester
reduction with additives.20 We found that after adding MgCl2,
NaBH4 could reduce ester to alcohol quickly and completely
(entry 2). CaCl2 showed a slightly worse result (entry 3).
However, the addition of ZnCl2 led to a significant increase in
side reactions (entry 4). Hence, MgCl2 was chosen as the best
additive. We found that the conversion decreased and the
reaction rate slowed down when attempting to reduce the
equivalents of NaBH4 and MgCl2 (entries 5−7). Additionally,
we found that changing the temperature had no obvious
influence on the reaction (entry 8). Therefore, we chose the
reaction parameters to be 1.2 equiv of NaBH4 and 5% of
MgCl2, 10 V EtOH, at 20−40 °C for 3 h.
e
f
entry
enzyme
conversion (%)
ee (%)
configuration
a
1
2
3
4
5
6
7
8
223
<5
<5
N.D.
N.D.
98.5
95.1
97.8
92.4
99.6
N.D.
N.D.
N.D.
N.D.
N.D.
94.6
N.D.
16.5
98.1
N.D.
N.D.
S
S
S
a
500
a
736
>99
>99
>99
73
>99
<5
<5
<5
<5
<5
a
740
b
774
b
CK03
R
S
b
EA
787
c
N.D.
N.D.
N.D.
N.D.
N.D.
S
N.D.
S
S
c
9
1348
c
10
11
12
13
14
15
16
LSADH
c
LK08
d
701
d
902
>99
<5
>99
>99
d
1184
d
1306
d
1500
a
Reaction conditions: 10 g/L ketone 2, 20 g/L wet cells, 0.5 g/L
NAD+, 2 g/L GDH wet cells, 2 equiv of glucose in 2 mL of PBS buffer
b
(100 mM, pH 7.0) for 24 h at 25 °C and 220 rpm. 10 g/L ketone 2,
20 g/L wet cells, 0.5 g/L NADP+, 2 g/L GDH wet cells, 2 equiv of
glucose in 2 mL of PBS buffer (100 mM, pH 7.0) for 24 h at 25 °C
c
and 220 rpm. 10 g/L ketone 2, 20 g/L wet cells, 0.5 g/L NAD+, 3
equiv of IPA in 2 mL of PBS buffer (100 mM, pH 7.0) for 24 h at 25
d
°C and 220 rpm. 10 g/L ketone 2, 20 g/L wet cells, 0.5 g/L NADP+,
3 equiv of IPA in 2 mL of PBS buffer (100 mM, pH 7.0) for 24 h at
25 °C and 220 rpm. Measured by HPLC. Measured by a chiral IC
e
f
column with chiral HPLC.
During the workup process, there were two major problems:
one was the quenching method and the other was the
extraction efficiency. In Erion’s work,11 the reaction was
quenched with 10% K2CO3 (aq), and the boron complex
remained in the product (the purity of the product was
unreported). Additionally, the workup process described was
quite complicated. Moreover, the excellent water solubility of
diol makes it extremely difficult to extract it from the aqueous
phase. Aiming to solve the above problems, through
experimental runs and a literature search, we developed an
improved workup process. The reaction was quenched with
30% H2SO4 and stirred for 1−2 h to completely decompose
the boron complex into the product. Then, the aqueous phase
was adjusted to pH 8−9 with 20% NaOH; thus, the Na2SO4
was generated in situ to saturate the aqueous phase, which was
proven to be a better salting-out reagent than NaCl.21 Then,
Figure 1. Effect of the substrate concentration on the bioreduction of
ketone 2. Reaction conditions: various concentrations of substrate
ketone 4 (including 25, 50, 100, 150, and 200 g/L), 50 g/L EA wet
cells, 10 g/L GDH wet cells, 2.0 equiv glucose and PBS buffer (100
mM, pH 7.0) in a 2 mL reaction system for 18 h at 25 °C and 220
rpm.
2.3.4. Optimization of the Cosubstrate Equivalents and
Biocatalyst Loading. Cofactor regeneration is an essential step
for the bioreduction reaction. Glucose has been well used as a
substrate oxidized to gluconic acid by KREDs for the in situ
regeneration of NADPH from NADP+.12 The glucose
equivalents were set ranging from 1.1 to 3.0. As shown in
Figure 2c, no significant influence on conversion was observed.
Considering the conversion and glucose consumption, the
D
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Figure 2. (a) Effect of the pH on the bioreduction system. Reaction conditions: the reactions were performed with 20 g/L ketone 2, 20 g/L wet
cells, 4 g/L GDH wet cells, 2.0 equiv of glucose in a 2 mL reaction system for 30 min at 25 °C and 220 rpm in different pH buffer solutions,
including 100 mM sodium citrate (pH 4 and 5), 100 mM potassium phosphate (pH 6, 7, and 8), and 100 mM Tris−HCl buffers (pH 9). (b) Effect
of the temperature on the bioreduction system. Reaction conditions: the temperature study was performed with 20 g/L ketone 2, 20 g/L wet cells,
4 g/L GDH wet cells, 2.0 equiv of glucose, and PBS buffer (100 mM, pH 7.0) in a 10 mL reaction system for 30 min at 220 rpm. (c) Effects of the
glucose equivalents on conversion by KRED EA wet cells. Reaction conditions: various equivalents of cosubstrate glucose (including 1.1, 1.5, 2.0,
and 2.5 equiv), 150 g/L substrate ketone 2, 50 g/L KRED EA wet cells, 10 g/L GDH wet cells, and PBS buffer (100 mM, pH 7.0) in a 2 mL
reaction system for 6 h at 35 °C and 220 rpm. (d) Effects of the biocatalyst loading on conversion by wet cells. Reaction conditions: various KRED
EA wet cells in the range of 50−150 g/L, 150 g/L substrate ketone 2, 20 g/L GDH wet cells, and PBS buffer (100 mM, pH 7.0) in a 10 mL
reaction system at 35 °C and 220 rpm.
Table 4. Comparison of Two Asymmetric Reduction
a
Systems
RuCl(p-cymene)[(S,S)-Ts-
c
entry
solvent
KRED EA
water
DPEN]
DMF
catalyst loading
catalyst cost
cofactor cost
time
yield
purity
ee
0.66 kg (wet cells)
¥ 33
¥ 66 (3.3 g)
3.5 h
93%
99.5%
7.23 g (s/c 500:1)
¥ 1040
0
16 h
not isolated (>100%)
unreported
87%
b
Figure 3. Time curve for the bioreduction of ketone 4 to alcohol 5.
Reaction conditions: 100 g of ketone 4 (150 g/L), 67 g of KRED EA
wet cells (100 g/L), 14 g of GDH wet cells (20 g/L), 140 g of glucose
(1.5 equiv), 0.33 g of NADP+ (0.5 g/L), and 670 mL of PBS buffer
(100 mM, pH 7.0) were added to a 2 L reaction vessel, and the
reaction was carried out at 35 °C and 350 rpm.
99.9%
a
b
The data were calculated by catalyzing 1 kg of substrate. Referred to
NADP+. Erion Mark D’s work.11
c
with ethyl acetate to afford a white solid in an 80% isolated
yield with a purity of up to 99.5% and an ee of >99.9%.
EtOH was evaporated before extraction. Moreover, we also
compared the extraction efficiencies of 2-MeTHF, THF, and n-
BuOH, which revealed extraction efficiencies of 1:2:3 (HPLC).
The TLC indicated that the n-BuOH could extract the product
completely after two times, while the other solvents could not
extract the product completely after four rounds of extraction.
The organic phase was dried and evaporated under high
vacuum. In the end, ethyl acetate was added to the sticky
residue and evaporated again to remove the remaining n-
BuOH to obtain a solid. The resulting solid was recrystallized
3. CONCLUSIONS
In summary, we have designed and developed a new
chemoenzymatic process to prepare a key HepDirect drug
intermediate, (S)-1-(pyridin-4-yl)-1,3-propanediol. First, we
proposed and optimized a process for efficiently constructing a
β-carbonyl ester, a ketoreductase substrate, from inexpensive
and commercially available isonicotinic acid. The yield of the
two steps was 75%, and the purity of the product was up to
99.6%. Second, we developed an enzyme reduction process to
E
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a
Table 5. Optimization of Ester Reduction (5 to 1)
b
HPLC
c
d
entry
NaBH4 (equiv)
additive (equiv)
temperature (°C)
time (h)
1
5
impurities
1
2
3
4
5
6
7
8
1.5
1.5
1.5
1.5
1.2
1.0
1.2
1.2
none
40
40
40
40
40
40
40
20
3
1
1
1
1
3
3
3
49.1
99.6
97.8
10.5
99.3
84.6
99.4
99.2
46.5
0.2
1.4
70.3
0.4
14.6
0.2
4.4
0.2
0.9
19.2
0.3
0.8
MgCl2 (0.2)
CaCl2 (0.2)
ZnCl2 (0.2)
MgCl2 (0.2)
MgCl2 (0.2)
MgCl2 (0.05)
MgCl2 (0.05)
0.4
0.4
0.4
a
b
Reaction conditions: experiments were carried out in EtOH, and NaBH4 was added in two batches within 0.5 h. The product content was
measured by HPLC under 260 nm. Contains 1 and a boron complex of 1. Structures of impurities not identified.
c
d
a
obtain a chiral alcohol intermediate with a 93% yield, 99.5%
purity, and 99.9% ee. Finally, we found a simple and mild
reduction system to obtain (S)-1-(pyridin-4-yl)-1,3-propane-
diol with an 80% yield, 99.5% purity, and 99.9% ee. The overall
yield of this new route is up to 55%, and this novel
chemoenzymatic process is both economic and environ-
mentally friendly for industry applications.
Table 6. KREDs Used in This Study
references/genbank
number
entry enzyme
strain
1
2
3
4
5
6
7
223
500
736
740
774
CK03
EA
purchased
purchased
purchased
purchased
purchased
KC342003
Wada et al.22
purchased
purchased
AB213459
Wei et al.15
purchased
purchased
purchased
purchased
purchased
4. EXPERIMENTAL SECTION
Chryseobacterium sp. CA49
Exiguobacterium sp. F42
4.1. Materials and Methods. Isonicotinic acid 2 and
glucose were purchased from Aladdin (China). The other
chemicals were obtained commercially in analytical grade.
Except for the reported enzyme, the other enzymes were
purchased from the Huzhou Yihui Biological Technology Co.,
8
9
787
1348
10
11
12
13
14
15
16
LSADH Leifsonia sp. strain S749
LK08
701
Lactobacillus kefir
1
Ltd. (Zhejiang, China). The H NMR and 13C NMR spectra
were recorded using a Bruker AVANCE III 400 MHz
spectrometer with a DMSO-d6 solvent. The 1H NMR chemical
shift data were reported for δ (ppm) relative to tetramethylsi-
lane as the internal standard, and the 13C NMR chemical shift
data were reported for δ (ppm) relative to DMSO-d6. The
reaction was monitored by HPLC (Waters H-Class), and the
ee was determined by chiral HPLC (Dionex UltiMate 3000).
Optical rotations were measured by a WZZ-2B automatic
polarimeter.
4.2. Cloning and Expression. The codon-optimized
genes of CK03, EA, LSADH, LK08, and GDH (GenBank
accession number: AFQ56330.1) were cloned into a pET-
28a(+) vector using the ClonExpress Entry One Step cloning
kit. The resulting plasmids were transformed into E. coli
BL21(DE3) cells. The resulting recombinant E. coli was
cultured at 37 °C in LB medium (10 g/L tryptone, 5 g/L yeast
extract, and 10 g/L NaCl) containing 50 μg/mL kanamycin.
When the optical density of the culture measured at a
wavelength of 600 nm reached 0.6−0.8, 0.1 mM isopropyl β-D-
1-thiogalactopyranoside (IPTG) was added to the fermenta-
tion medium. The cultivation was continued overnight at 25
°C. Then, the cells were harvested by centrifugation (4000g, 4
°C, and 30 min) and stored at −20 °C for further use (Table
6).
902
1184
1306
1500
a
The purchased enzyme was provided by the Huzhou Yihui Biological
Technology Co., Ltd.23
g/L yeast extract, 12 g/L soybean peptone, 3 g/L NaCl, 5 g/L
glycerol, 2 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 50 μg/mL
kanamycin, and pH 7.0). When the optical density of the
culture measured at a wavelength of 600 nm reached 3.0, the
cultures were cooled to 25 °C, and 168 mg of IPTG was added
to induce the expression for 20 h. Then, the recombinant E.
coli/pET-28a(+)-EA cells were harvested by centrifugation
(4000g, 30 min, 4 °C) and stored at −20 °C. The E. coli/pET-
28a(+)-GDH cells were prepared by the same method.
4.4. Synthesis of Ethyl Isonicotinate 3. First, 123 g of
isonicotinic acid (1.0 mol) was placed into a 2 L four-necked
flask, followed by the addition of 0.92 kg (20 mol) of absolute
ethanol and mechanical stirring at 200 rpm. Then, 147 g (1.5
mol) of concentrated H2SO4 (98%) was added to the mixture
dropwise. Heating was turned on after the addition of H2SO4,
and the solution was refluxed at 80 °C. The reaction was
monitored by HPLC and terminated after 4 h when the ethyl
isonicotinate content was 88−89%. Then, the heating source
was removed and the inner temperature was below 30 °C.
Next, 500 mL of water was added, and 159 g (1.5 mol) of
sodium carbonate was added in batches. After ethanol was
4.3. Culture of E. coli/pET-28a(+)-EA Cells in a 10 L
Bioreactor. E. coli/pET-28a(+)-EA cells were precultured
overnight in 150 mL of LB medium containing 50 μg/mL
kanamycin at 37 °C. Then, the seed culture was transferred to
a 10 L bioreactor containing 7 L of fermentation medium (24
F
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Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
pubs.acs.org/OPRD
Article
evaporated under reduced pressure, the mixture was extracted
with ethyl acetate (2 × 300 mL). The organic phase was
washed with brine (200 mL), dried with anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. Finally, 128
g of crude product was obtained as a colorless liquid with a
yield of 88% and an HPLC purity of 99.7%. HPLC (method
1): the product ester 3 and starting material 2 in the reaction
solution were analyzed using a Waters BEH C18 column (2.7
× 50 mm2, 1.7 μm), a flow rate of 0.4 mL/min, 35 °C, mpA =
10 mM KH2PO4 pH 4.4, mpB = ACN. Gradient: 0−2.4 min,
5−65% B; 2.4−3.25 min, 65% B; and 3.3−4.5 min, 5% B. UV
phase: 25% IPA in hexane (V/V) isocratic. UV wavelength of
254 nm. RT: 11.0 min (S), 13.0 (R). [α]D25 = −30.3° (c 1.00,
1
MeOH). H NMR (400 MHz, DMSO-d6) δ 8.55−8.49 (m,
2H), 7.42−7.36 (m, 2H), 5.80 (d, J = 5.0 Hz, 1H), 5.00 (dt, J
= 9.1, 4.6 Hz, 1H), 2.76−2.54 (m, 2H), 1.17 (t, J = 7.1 Hz,
3H). 13C NMR (101 MHz, DMSO-d6) δ 170.28, 153.23,
149.40, 120.96, 68.18, 59.93, 43.50, 14.02.
4.7. Synthesis of (S)-1-(Pyridin-4-yl)-1,3-propanediol
1. First, 60.0 g (307.3 mmol) of (S)-3-hydroxy-3-(pyridin-4-
yl)propanoate 5 was added to a 1 L four-necked flask. Then,
600 mL of absolute ethanol was added, followed by mechanical
stirring. Next, 1.46 g (15.4 mmol) of anhydrous MgCl2 was
added slowly, with the inner temperature controlled to 20 °C.
Then, 13.95 g (123 mmol) of NaBH4 was added in batches
within 0.5 h. The reaction was monitored by HPLC until the
raw material was <0.5%. Next, 150 mL of 30% H2SO4 was
added to quench the reaction, the mixture was stirred for 1 h,
and the boron complex decomposed to the product
completely. Next, 20% NaOH was added to adjust the pH
to 8.5. The resulting salts were then filtered, and the ethanol
was removed under reduced pressure. The aqueous phase was
extracted with n-BuOH (240 mL × 2). The combined organic
phase was dried and evaporated under a high vacuum to obtain
a sticky colorless oil. EtOAc (240 mL) was added and
evaporated again, and then, EtOAc (180 mL) was added and
heated to reflux for 0.5 h to obtain a clear solution, followed by
gradual cooling to 0−5 °C for 1 h to precipitate the crystals.
Subsequently, filtration was carried out, and the obtained white
powder was washed with EtOAc (45 mL). The solid was then
dried in an air-drying oven to a constant weight to obtain 38.1
g (307.3 mmol) of a white powder with a yield of 80%. Melting
point: 98.1−99.3 °C. ee >99.9%. HPLC (method 2): RT, 0.5
min (1), 1.7 min (5). Chiral HPLC for 1: DAICEL IC (4.6 ×
250 mm2, 5 μm), mobile phase: 15% EtOH in hexane (V/V)
isocratic. UV at a wavelength of 254 nm. RT: 9.8 min (S), 11.7
1
at a wavelength of 220 nm. RT, 2.2 min (3), 0.3 min (2). H
NMR (400 MHz, DMSO-d6) δ 8.86−8.69 (m, 2H), 7.87−7.74
(m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). 13
C
NMR (101 MHz, DMSO-d6) δ 164.59, 150.72, 136.99, 122.45,
61.56, 13.96.
4.5. Synthesis of Ethyl 3-oxo-3-(pyridin-4-yl)-
propanoate 4. First, 650 mL of THF and 141 g of t-BuOK
(1.26 mol) were added to a 1 L four-necked glass, followed by
mechanical stirring at 300 rpm; the temperature was controlled
at 0 °C. Then, a mixture of 105.8 g (700 mmol) of ethyl
isonicotinate 3 and 74.0 g (840 mmol) of ethyl acetate was
added dropwise to the t-BuOK/THF solution within 30 min
with the temperature maintained below 10 °C. Once this
mixture was added, the reaction temperature was increased to
room temperature for 2 h; HPLC showed that the reactant was
less than 1%. Then, 300 mL of water was added to quench the
reaction, followed by concentration to remove THF. The
aqueous phase was acidified by 37% HCl to a pH of 7.5, and
the ketone precipitated out as the pH became lower. Then, the
mixture was cooled to 0−5 °C for 2 h to precipitate crystals
under stirring conditions. The crystals were filtered and
washed with cool water, followed by drying in an air-drying
oven to a constant weight to obtain 116.3 g of a white granular
solid with a yield of 86%. Melting point: 50.7−51.3 °C. HPLC
purity 99.6%. HPLC (method 1): RT, 2.0 min (4), 2.2 min
5
(R). [α]D = −51.5° (c 1.00, MeOH). 1H NMR (400 MHz,
DMSO-d6) δ 8.51−8.43 (m, 2H), 7.35−7.28 (m, 2H), 5.45 (d,
J = 4.9 Hz, 1H), 4.68 (dt, J = 7.6, 5.2 Hz, 1H), 4.57 (t, J = 5.2
Hz, 1H), 3.48 (ddt, J = 39.6, 10.7, 5.3 Hz, 2H), 1.78−1.57 (m,
2H). 13C NMR (101 MHz, DMSO-d6) δ 155.17, 149.28,
120.90, 68.17, 57.51, 41.83.
25
1
(3). H NMR (400 MHz, DMSO-d6) δ 8.89−8.75 (m, 2H),
7.85−7.80 (m, 2H), 4.26 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H),
1.16 (t, J = 7.1 Hz, 3H). Ketone/enol = 2.6:1. 13C NMR (101
MHz, DMSO-d6) δ 193.81, 167.22, 150.88, 141.70, 121.34,
45.61, 13.93.
4.6. Synthesis of Ethyl (S)-3-Hydroxy-3-(pyridin-4-
yl)propanoate 5. First, 670 mL of PBS buffer (100 mM, pH
7.0) was added to a 2 L four-necked flask, followed by
mechanical stirring at 350 rpm. Then, 140 g (776 mmol) of
glucose, 14 g of GDH wet cells, 67 g of EA wet cells, 100 g of
ketone 4 (518 mmol), and 0.33 g of NADP+ were added,
respectively. The reaction vessel was placed in a 35 °C water
bath. During the reaction, the pH value was closely monitored
using a pH meter. A 5% Na2CO3 solution was added dropwise
(slowly) to keep the pH level between 5.7 and 7.0. After 3.5 h
of reaction, HPLC analysis showed that the product content
was 99.6%, at which point the reaction was terminated. The
pH was adjusted to 7.5; the mixture was extracted with 2-
MeTHF (600 mL × 2); and the organic layer was washed with
saturated NaCl (400 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Finally, 94.0 g of
product was obtained as a light-yellow oil with a yield of
93%, an HPLC purity of 99.5%, and an ee value of 99.9%.
HPLC (method 2): the different parameters compared to
method 1 are as follows: mpA = 10 mM KH2PO4 pH 6.0, UV
wavelength of 260 nm. RT, 1.7 min (5), 2.2 min (4). Chiral
HPLC for 5: DAICEL IC (4.6 × 250 mm2, 5 μm), mobile
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.0c00403.
Chiral HPLC chromatograms of compounds 1 and 5
and NMR data of compounds 1, 3, 4, and 5 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Fu-Li Zhang − Shanghai Institute of Pharmaceutical Industry,
China State Institute of Pharmaceutical Industry, Shanghai
201203, China; orcid.org/0000-0002-4175-4795;
Email: zhangfuli1@sinopharm.com
Shao-Xin Chen − Shanghai Institute of Pharmaceutical
Industry, China State Institute of Pharmaceutical Industry,
Shanghai 201203, China; orcid.org/0000-0002-1604-
816X; Email: sxzlb@263.net
G
https://dx.doi.org/10.1021/acs.oprd.0c00403
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Organic Process Research & Development
pubs.acs.org/OPRD
Article
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Hong-Yi Wang − Shanghai Institute of Pharmaceutical
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Shanghai 201203, China; orcid.org/0000-0002-1663-
9808
Jia-Wei Tang − Department of Biological Medicines &
Shanghai Engineering Research Center of
Immunotherapeutics, Fudan University School of Pharmacy,
Shanghai 201203, China
Peng Peng − Collaborative Innovation Center of Yangtze
River Delta Region Green Pharmaceuticals, Zhejiang
University of Technology, Hangzhou 310014, China
Hai-Jun Yan − College of Chemistry and Chemical
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The authors declare no competing financial interest.
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