Asymmetric Synthesis of Florfenicol by Dynamic Reductive Kinetic Resolution with Ketoreductases

Article abstract of DOI:10.1002/ejoc.201800658

A chemoenzymatic synthesis of the veterinary antibiotic florfenicol is described. The key step involves the dynamic reductive kinetic resolution (DYRKR) of a keto ester by using a ketoreductase-02 (KRED-02) to afford the two contiguous stereocenters of the (2S,3R)-cis-1,2-amino alcohol intermediate in >99 % ee and a diastereomeric ratio (dr) of 99 %. This green biocatalysis is environmental friendly with high enantioselectivity and product yields. Two methods for the nucleophilic fluorination step involved the use of aziridines and cyclic sulfates to safely prepare fluoroamines with high regioselectivity. Additional studies have indicated that KRED-02 can also be used to afford chiral alcohol (S)-21 in good yields with high enantioselectivity. This study shows that the integration of biocatalysis into organic synthesis can be useful and provide industrial opportunities for applications of florfenicol.

Full text of DOI:10.1002/ejoc.201800658

A Journal of
Accepted Article
Title: Asymmetric Synthesis of Florfenicol via Dynamic Reductive
Kinetic Resolution with Ketoreductases
Authors: Jie Zou, Guowei Ni, Jiawei Tang, Jun Yu, Luobin Jiang,
Dianwen Ju, Fuli Zhang, and Shaoxin Chen
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800658
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800658
Supported by

10.1002/ejoc.201800658
European Journal of Organic Chemistry
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Asymmetric Synthesis of Florfenicol via Dynamic Reductive
Kinetic Resolution with Ketoreductases
Jie Zou+,[a] Guowei Ni+,[a,b] Jiawei Tang,[a] Jun Yu,[a] Luobin Jiang,[a] Dianwen Ju,[b] Fuli Zhang*[a], and
Shaoxin Chen*[a]
Dedication ((optional))
Abstract: A novel chemo-enzymatic synthesis of the veterinary
antibiotic florfenicol is described. The key step was the dynamic
reductive kinetic resolution (DYRKR) of a keto ester with a
ketoreductase
(KRED-02)
to
afford
two
contiguous
stereocenters of (2S,3R)-cis-1,2-amino alcohol in >99% ee and
99% dr in one step. This biocatalysis is green and environmental
friendly with high enantioselectivity and productivity.
Subsequently two new methods of nucleophilic fluorination via
aziridines and cyclic sulfates were developed to prepare the
fluoro amine in high regioselectivity, greatly reducing the safety
risk. Additional studies have indicated that KRED-02 can also be
used to afford chiral alcohol S-21 with a good yield and
stereoselectivity. By integrating biocatalysis into organic
synthesis, it has an opportunity to be useful in industrial
applications for florfenicol.
Figure 1. Structures of florfenicol (1) and thiamphenicol (2)
Recently, Chen developed two asymmetric methods of
florfenicol based on sharpless epoxidation5c,f and catalytic
aziridination5d. Lin performed a new chemo-enzymatic route with
hydroxynitrile lyase to establish the first chiral carbon and
generated the second by DIBAL reduction and hydrocyanation.5e
However lengthy steps and expensive cost of catalysts limited
their industrial application. Later on, Chen disclosed a new route
of synthesizing florfenicol, involving DKR process with
asymmetric hydrogenation catalysted by Ru and ligands, which
afforded (2S, 3S)-1,2-amino alcohol with low stereoselectivity in
92% de and 78%ee，therefore its hydroxyl still needed to be
inversed for the florfenicol.6 Asymmetric hydrogenation
combined with dynamic kinetic resolution (DKR) is particularly
attractive for constructing two adjacent stereocenters in one
step.7 Ketoreductases have been widely applied as an efficient
biocatalyst for pharmaceutical industry becausese of its high
stereoselectivity and bioreductive activity.8 However few
literaures reported DKR with ketoreductases （ KREDs ） in
organic synthesis.9 Herein we report a novel chemo-enzymatic
approach via DYRKR with ketoreductases and two new methods
of introducing fluorine are designed for the synthesis of
florfenicol.
Introduction
Florfenicol (1) was discovered as a 3’-fluoro derivative of
thiamphenicol (2), which had been widely used as a veterinary
antibiotic for its significant superiority in antibacterial spectrum
and activity (Figure 1).1 Previous investigations, which focused
on the deoxyfluorination, had identified the Ishakawa reagent,
which severely corroded the equipment and was relatively high
in cost, as the chief successful reagent for completing this
transformation.2 Construction of the two stereocenters was
based upon the aldol reactions involving classical chemical
resolution, which caused waste water pollution.3 Since the large
productivity (4000 ton per year) and environmentally inefficient
industrial process, there has been considerable attention on
using different strategies for the fluorination4 and two adjacent
stereocenters of cis-1,2-amino alcohol5.
[a]
J. Zou, G. Ni, J. Tang, J. Yu, L. Jiang, F. Zhang, S. Chen
Shanghai Institute of Pharmaceutical Industry, China State Institute
of Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai
201203, China.
E-mail: zhangfuli1@sinopharm.com; sxzlb@263.net
G. Ni, D. Ju
School of Pharmacy, Fudan University, 826 Zhang Heng Road,
Shanghai 201203, China.
[b]
[+]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Retrosynthetic Analysis of Florfenicol (1)
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10.1002/ejoc.201800658
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We envisioned that florfenicol (1) could be accomplished
acid catalyzed by glucose dehydrogenase (GDH) or using i-
through nucleophilic fluorination of the corresponding aziridine (8) propanol as co-substrate to acetone, delivered the hydride
and cyclic sulfate (14, 15) intermediates with the two
stereocenters established already. Further, the aziridines and
cyclic sulfates might be synthesized from (2S, 3R)-5a. The two
adjacent stereocenters could be elaborated via asymmetric
bioreduction of the corresponding amino ketone (4) through
enzymatic DYRKR (Scheme 1).
source to selectively reduce (S)-4. The enzymatic DKR
reduction with the selected KRED-02 was carried out by treating
in an aqueous phosphate buffer in the presence of glucose at
25℃. NaOH (1M) was added to neutralize the gluconic acid
formed during the reaction and maintain the reaction pH at 7.5 to
accomplish a fast epimerization of 6, thereby achieving 98%
conversion at 24 h and 4-(methylsulfonyl)benzoic acid was the
main byproduct as the unstability of the compound 4 in the basic
condition.
Results and Discussion
For the above synthetic strategy, an approach was developed to
prepare the desired α-amino β-keto ester 4 for our investigation
on ketoreductases (Scheme 2). Treatment of glycine methyl
ester with benzoyl chloride 3 at 0 ℃, in the presence of Na2CO3,
gave the corresponding benzamide. The subsequent protection
of the benzamide with (Boc)2O under DMAP catalysis in
acetonitrile at room temperature afforded the N-Boc amide. The
N-Boc amide was then subjected to
a
base-promoted
intramolecular N→C benzoyl migration reaction under t-BuOK in
THF at 0 ℃. Thus β-keto ester (4) was prepared in 71% yield via
three steps (Scheme 1).10
Table 1. Stereoselectivity of ketoreductase-catalyzed of keto ester 4.
Scheme 2. Synthesis of β-keto ester 4
KRED
Conversion[e]
(%)
Diastereomeric ratio (%)[f]
5a
43.8
28.2
65.6
0
5b
6.7
7.8
0
5c
49.5
64.0
34.4
0
5d
0
Before our screening of KREDs, adequate chiral HPLC
analyses were developed for (rac)-5 to achieve a reliable
method to measure the enantiomeric excesses of the final
product from the ketoreductase-catalyzed reaction. Accordingly,
four stereoisomers (2S, 3R)-5a, (2R, 3S)-5b, (2S, 3S)-5c and
(2R, 3R)-5d were prepared to comfirm the corresponding peak
in the chiral HPLC.11
1
2
3
4
5
6
774[a]
0902[b]
740[c]
05[a]
95
85
80
95
90
98
0
0
86.9
0
13.1
0
Next, we chose to evaluate the putative DYRKR of 4 to
prepare (2S, 3R)-5a. It is accepted that DKR, which combines
asymmetric bioreduction and in situ racemization of the
unreacted enantiomer, is a promising process to overcome the
limitations in chemical resolution of 50% yield.12 Fortunately 4
was found to be easily racemized with the ranging from pH 6-8
which is suitable for bioreduction. Using keto esters 4 as starting
materials, KREDs were screened to carry out reductions that
were stereoselective.13 The selected data for the conversion and
stereoselectivity of ketoreductases are presented in Table 1,
where two out of the four stereoisomers were formed in optically
pure form using different enzymes. KRED-02 was identified to
perform the desired DKR bioreduction to afford cis-(2S, 3R)-5a
in 91% yield with >99% ee and 99% dr, as judged by chiral
HPLC. Trans-(2R, 3R)-5c could also be prepared with KRED-10
in high selectively >99% ee and 96.8% dr, which exhibited the
diversity of reductases. The attainment of the catalytic cycle for
the enzymatic DKR reduction, by consuming glucose to gluconic
10[d]
1.6
98.4
0.5
02[a]
99.5
0
0
[a] 0.05g ketone 4, 0.2g wet cells, 0.2g glucose, 1mg NADP+ and PBS buffer
(100 mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [b]
0.05g ketone 4, 0.2g wet cells, 1mg NADP+, 2mL IPA and PBS buffer (100
mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [c] 0.05g
ketone 4, 0.2g wet cells, 0.2g glucose, 1mg NAD+ and PBS buffer (100 mM ,
pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [d] 0.05g
ketone 4, 0.2g wet cells, 1mg NAD+, 2mL IPA and PBS buffer (100 mM , pH
7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [e] Measured by
HPLC. [f] Measured by chiral HPLC using a chiral IB-3 column.
The poor solubility of 4 in buffer solutuin restricted the scale-
up of the bioreduction by using KRED-02. We therefore tried to
append organic co-solvents into the aqueous medium, DMSO
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10.1002/ejoc.201800658
European Journal of Organic Chemistry
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and toluene were chose to represent miscible and immiscible
solvents with water. 50% DMSO or toluene relative to the buffer
solution was well tolerated by KRED-02, which was also desired
for improving the solubility of 4 and the concentration of the
compound 6 was elevated to 50g/L from 5 g/L which showed the
prospectivity for industrial application (Table 2).
Table 2. DYRKR of keto ester 4 in phosphate buffer/ organic co-solvent (v/v).
Co-solvent(%)
-
Conversion(%)[c]
ee (%)[d]
>99
dr (%)[d]
99
Scheme 3. Synthesis of Florfenicol 1 via N-Boc aziridines
1[a]
2[a]
3[a]
4[a]
5[b]
20%
54%
95%
98%
98%
Octadecane(50%)
Toluene(50%)
DMSO(50%)
DMSO(50%)
>99
99
Our first approach was to obtain the corresponding aziridine
and react with nucleophilic fluorinating reagents for fluorination
(Scheme 3).16 The reduction with NaBH4 led to aminodiol 6,
followed by the selective masking of the primary hydroxy group
with mesyl chloride to afford 7 without purification for the next
step. The step of cyclization to the aziridine ring was carried out
by potassium carbonate and aziridine 8 was obtained in 61%
>99
99
>99
99
>99
99
[a] 0.2g ketone 4, 0.2g KERD-02, 0.5g glucose, 2mg NADP+ and PBS buffer
(100 mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [b]
0.5g ketone 4, 0.5g KERD-02, 0.8g glucose, 5mg NADP+ and PBS buffer (100
mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [c]
Measured by HPLC. [d] Measured by chiral HPLC using a chiral IB-3 column.
yield over two steps. By treating
8
with triethylamine
trihydrofluoride in 1,2-dichloroethane (DCE) under 70℃, 8 was
regioselectively converted into the desired 9 in 77% yield. The
fluorination of β-hydroxyl aziridine 8 took place at the less
hindered side leaving the hydroxyl group untouched; 19F NMR
confirmed the regioselectivity of fluorination. Other common HF-
based reagents, DMPU-HF (65%HF in DMPU) and Olah’s
reagent (70% HF in pyridine), gave no reaction at all due to
higher acidity. After deprotection of 9 with EtOAc-HCl at ambient
temperature, the fluoro amine 10 HCl salt was directly isolated
from EtOAc in 90% yield. Finally, florfenicol (1) was obtained in
95% yield by acylation of 10 with methyl dichloroacetate.
The high stereoselectivity of (2S, 3R)-5a, which is generally
not readily prepared through chemical reduction, was prepared
through a powerful DKR bioreduction of racemic substrate 4 in
one step. The application of biotechnology into classical organic
synthesis is a practical and efficient tool to deal with synthetic
problems.10
With (2S, 3R)-5a in hand, we attempted to develop new
methods of introducing fluorine atom. Many of these strategies
rely on the hydroxyl group replaced by fluorine with
(diethylamino)sulfur trifluoride (DAST) or Ishakawa reagent.2 In
contrast, aziridine ring-opening by fluoride offers a direct route to
fluoamines. The studies of Doyle demonstrated that the
hydrofluorination of aziridines to provide fluoroamines using
amine-HF reagents was presented with regioselectivity.14 Xu
and Hammond reported conveniently achieving regioselectivity
in the fluorination of the aziridine substrate under acidic
conditions.15 Additionally, studies on the ring-opening of
aziridines are highly sought after.
Scheme 4. Synthesis of the Intermediate 13
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10.1002/ejoc.201800658
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Taking advantage of the efficient ring-opening reaction, the
other synthesis of florfenicol was developed as outlined in
Scheme 4. The N-Boc group of (2S, 3R)-5a was cleaved by
using HCl-EtOAc, and the resulting amine was then acetylized
by dichloroacetic chloride in the presence of saturated sodium
bicarbonate solution to afford 11. Reduction of ester 11
successfully gave the aminodiol 2 in good yield. The subsequent
protection of the primary hydroxy group afforded O-tosyl
derivative 12. In constract , the intramolecular cyclization of the
O-tosyl derivative 12 with potassium carbonate to prepare
aziridine was attempted. After being stirred for 2h at 60℃, the
starting material disappeared and a new product was observed
by HPLC. To our surprise, the cyclic compound 13, which is a
vital intermediate for the synthesis of florfenicol (1), was the
main product and the same result was obtained when the
tosylation product was treated with KOH or other strong alkali in
acetonitrile.
Hydrolysis of a sulfate ester was reported by Kim and Sharpless
using catalytic concentrated sulfuric acid and 0.5 to 1.0 equiv of
water in THF.23 In our case, hydrolysis of the sulfate could be
accomplished while deprotecting by treating the crude reaction
mixture with EtOAc-HCl to gave the fluoro amine 10 in 74% yield.
Finally, florfenicol (1) was obtained in 95% yield by acylation of
10 with methyl dichloroacetate.
Scheme 6. Synthesis of Florfenicol 1 via N-COCHCl2 cyclic sulfates 15
The foregoing method utilizing aziridines to obtain florfenicol 1,
could avoid waste water pollution caused by the present process.
In addition, triethylamine hydrofluoride as a fluorinated ring-
opening reagent, might improve safety compared to anhydrous
HF and reduce corrosion of equipment.17
Although these routes were an improvement over fluorinations,
there are still multiple chromatographic separations required. We
searched for alternative fluorination methods through six-
membered cyclic sulfates.18 Gao and Sharpless reported
efficiently opening cyclic sulfates with nucleophiles including
fluoride.19 Furthermore, Marc observed that a cyclic sulfate was
opened by the nucleophile at the primary position while no
secondary substitution product could be detected.20 Opening
cyclic sulfates from diols resulted in a high selectivity for the
primary position. These results led us to try this approach on
1,3- diols.
We envisaged that cyclic sulfates would be good substrates
for substitution. The straightforward purification and facile
separation encouraged us to look for a shorter route to
synthesize florfenicol 1 (Scheme 6). Treatment of 2 with thionyl
chloride gave a mixture of sulfites that was oxidized without
further purification with RuCl3/NaIO4 to the corresponding cyclic
sulfate 15, which was obtained in 72% yield. Treatment of the
key precursor 15 with Et3N-3HF as the nuclephile, followed by
acidic hydrolysis, gave florfenicol 1.
Scheme 5. Synthesis of Florfenicol 1 via N-Boc cyclic sulfates 14
According to our strategy shown in Scheme 5, the cyclic
sulfate 14 was obtained in 71% yield by reaction of diol 6 with
SOCl2/TEA followed by in situ oxidation with NaIO4/RuCl3.
Attempts to perform direct sulfation of 6 using SO2Cl2/TEA also
afford 14 but in lower yields.21 Regioselective ring-opened
reactions of the key precursor 14 using nucleophilic fluorinating
reagents were studied. Et3N-3HF in DCE worked best. We found
that the phenyl group at the C4 position of 14 would sterically
hinder approaching of the fluoride nucleophile to C4, directly
attacking to the C6 position (cyclic sulfate numbering).22
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Scheme 7. Synthetic routes towards florfenicol 1
Once the process was validated, direct application of our
ketoreductases to an interesting intermediate 22 (Table 4),13
which is an useful intermediate for the synthesis of florfenicol (1)
and thiamphenicol (2), was tested. Compared with the previous
chemical synthetic method through sharpless asymmetric
epoxidation25, the bioreduction with KRED-02 worked well in
buffered aqueous solution, the yields are excellent and
enantiomeric ratios almost in favor of allylic alcohol (S)-21
(Scheme 8). This finding further demonstrated that recuction
with ketoreductases showed the potential for industrial
application.
The synthetic routes towards florfenicol 1 (Scheme 7) were
evaluated via using decision analysis as a tool based on a
weighing of the desired characteristics for the synthesis and an
evaluation of each route against these characteristics. 4c, 24
Table 3. Evaluation of routes to florfenicol 1.
Criteria
number of steps
health and safety
environment
yield
Weight[a]
Route 1[b]
Route 2[b]
Route 3[b]
2
5
5
3
2
4
2
2
4
3
4
3
Table 4. Bioreduction of ketone 22.
KRED
774[a]
0902[b]
740[c]
05[a]
Conversion(%)[f]
ee (%)[g]
25.1
88.6
30.9
22.2
37.3
74.6
>99
Configuration
2
3
3
1
2
95
92
92
90
95
90
95
98
75
50
98
98
S
S
S
S
S
S
R
R
R
S
S
S
3
4
2
flexibility
4
4
3
3
purity
3
3
2
4
total[c]
53
70
58
5
10[d]
[a] Scores for each weighting were given from 1 = least important to 5 = most
important. [b] Scores for each category were given from 1 = meets the criteria
least successfully to 5 = meets the criteria most successfully. [c] The total
score was given by the sum of the products of the weighting and the scoring
for each category.
6
1306[b]
1500[b]
1184[b]
787[d]
1348[d]
02[a]
7
8
>99
9
>99
The evaluation shows clearly route 2 performs the best in this
analysis (Table 3), primarily due to the nucleophilic fluorination
via cyclic sulfates. The development of a new fluorination
strategy, using Et3N-3HF as a fluorinating reagent, improved the
safety for the synthesis of florfenicol 1. The application of
enzymatic DYRKR significantly reduced the pollution of the
process by chemical resolution and achieved an excellent yield.
In developing a process we have therefore made the process
operationally green and safer.
10
11
12
94.5
95.3
95.3
02[e]
[a] 0.05g ketone 22, 0.2g wet cells, 0.2g glucose, 1mg NADP+ and PBS buffer
(100 mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [b]
0.05g ketone 22, 0.2g wet cells, 1mg NADP+, 2mL IPA and PBS buffer (100
mM , pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [c] 0.05g
ketone 22, 0.2g wet cells, 0.2g glucose, 1mg NAD+ and PBS buffer (100 mM ,
pH 7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [d] 0.05g
ketone 22, 0.2g wet cells, 1mg NAD+, 2mL IPA and PBS buffer (100 mM , pH
7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [e] 0.5g ketone
22, 0.5g wet cells, 0.8g glucose, 5mg NADP+ and PBS buffer (100 mM , pH
7.0) in 10mL reaction system for 24h at 25 °C and 200 rpm. [f] Measured by
HPLC. [g] Measured by chiral HPLC using a chiral IC-3 column.
Conclusions
In summary, novel chemo-enzymatic routes has been developed
for the synthesis of florfenicol, which features the application of
enzymatic DYRKR to establish the two stereo centers of cis-1,2-
amino alcohol (2S, 3R)-5a with >99% ee and 99% dr in one step.
For the introduction of key fluorine atom, two new methods were
developed through aziridines and cyclic sulfates with
nucleophilic fluoride reagent Et3N-3HF in a highly regioselective
Scheme 8. Application of KRED-02 to intermediate 22
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To a solution of phosphate buffer (pH 7.0, 10mL) was added dextrose
(0.8g) followed by KRED-02 wet cells (0.5g), 5mg NADP+, ketone 4
(0.5g), DMSO (5mL). The resulting solution was pH adjusted to a
minimum of 7.5 with 1M NaOH prior to use. The reaction was carried out
at 30 ℃ and shaken at 200 rpm shaker for 24 h. The reaction mixture
was then extracted with DCM (10 mL×3), and the DCM layers were
combined. The organic layers were dried over Na2SO4 and concentrated
under reduced pressure, obtaining (2S, 3R)-5a (0.46 g, 91 %) as a white
solid. Mp: 143.7-144.5 ℃ . 1H NMR (600MHz, CDCl3): δ=7.85(d,
J=8.16Hz, 2H), 7.57(d, J=8.28Hz, 2H), 5.37(s, 1H), 5.34(d, J=9.06Hz,
1H), 4.55(d, J=8.52Hz, 1H), 3.80(s, 3H), 3.27(s, 1H), 3.00(s, 3H), 1.29(s,
9H); 13C NMR (150MHz, CDCl3): δ=170.75, 155.53, 146.39, 139.74,
127.28, 127.16, 80.33, 73.19, 59.09, 52.83, 44.49, 28.08; HR-MS calcd
for C16H27N2O7S [M+NH4]+ m/z 391.1533, found m/z 391.1532. The
chiral HPLC was performed using a CHIRALPAK IB-3 (4.6×250 mm,
manner. Additionally we found that KRED-02 can be used to
afford (S)-21 with a good yield and stereoselectivity. The
integration of asymmetric dynamic bioreduction for the synthesis
of other analogues and further studies involved with the
evolution of KRED-02 are in progress.
Experimental Section
Materials and methods
Materials, reagents and solvents were obtained commercially and used
without additional purification. All the KRED genes (the codon-optimized
open reading frames encoding proteins) were synthesized by Bioligo
(Shanghai). ClonExpress® Entry One Step Cloning Kit used for
molecular cloning was from Vazyme Biotech (Nanjin, China) and other
enzymes from Takara (Dalian, China). BCA Protein Assay Kit was
purchased from cwbiotech (Beijin, China). All the KREDs were used in
the form of wet cells. Column chromatography was performed on silica
gel (GENERAL-REAGENT®, 200-300 mesh). After chromatography the
fractions were pooled, evaporated to dryness and dried in vacuo. Melting
points were determined on a WRS-3 digital melting-point apparatus and
are uncorrected. NMR spectras were recorded on Bruker Advance 600
3µm, DAICEL, Shanghai) column with
a mobile phase of 95%
0.2%(DEA+TFA) Hex-5% EtOH. The product was detected at 220 nm,
and the retention times of diastereoisomers were 34.4 min, 36.1 min,
39.5 min ((2S, 3R)-5a), and 43.2 min, respectively.
tert-Butyl
((1R,2R)-1,3-dihydroxy-1-(4-methylsulfonyl)phenyl)
propan-2-yl carbamate (6)
NaBH4 (4.0 g, 105.7 mmol) was added to a solution of (2S, 3R)-5a (13.5
g, 34.8 mmol) in MeOH (60 mL) at 0℃. The mixture was stirred for 5h
and H2O (15 mL) was added and the mixture was stirred further for 1h.
The mixture was then filtered and the filtrate was concentrated. The
residue was diluted with CH2Cl2 (80 mL), washed with brine (30 mL),
dried (Na2SO4), filtered, and concentrated under vacuum. The residue
was purified by flash column chromatography [silica gel petroleum ether-
MHz instrument. Chemical shifts are reported in ppm, relative to TMS
1
(1H; internal standard, δH = 0.00 ppm) or solvent peaks (CDCl3: H, δH
=
7.26 ppm and 13C, δC = 77.0 ppm; DMSO-d6: H, δH = 2.49 ppm and 13C,
δC = 39.5 ppm; MeOD: 1H, δH = 3.31 ppm and 13C, δC = 49.0 ppm ). 19F
NMR spectra were recorded on a Agilent spectrometer (400 MHz) using
CCl3F (19F, δ = 0.0 ppm) as an externalstandard. High resolution mass
1
1
EtOAc (2:1)] to give a white solid (11.3 g, 94 %). Mp: 144.3-145.9 ℃. H
spectra (HRMS) were recorded on
a Bruker MicroTOF mass
NMR (600MHz, DMSO-d6): δ=7.85(d, J=8.16Hz, 2H), 7.55(d, J=8.16Hz,
2H), 6.11(d, J=9.3Hz, 1H), 5.51(d, J=5.52Hz, 1H), 4.92-4.91(m, 1H),
4.75(t, J=5.46Hz, 1H), 3.68-3.64(m, 1H), 3.54-3.50(m, 1H), 3.32-3.28(m,
1H), 3.14(s, 3H) , 1.23(s, 9H); 13C NMR (150MHz, DMSO-d6): δ=155.62,
150.27, 139.44, 127.52, 126.82, 78.12, 70.42, 61.14, 58.02, 44.11,
28.51; HR-MS calcd for C15H23NNaO6S [M+Na]+ m/z 368.1138, found
m/z 368.1138.
spectrometer. HPLC analyses were carried out on a Dionex UItiMate
3000 HPLC instrument.
Methyl-2-((tert-butoxycarbonyl)amino)-3-(4-(methylsulfonyl)phenyl)-
3- oxopropanoate (4)
To a suspension of Na2CO3 (7.6 g, 71.7 mmol) in water (80 mL) and
EtOAc (120 mL) at 0 ℃ was added glycine methyl ester hydrochloride
(8.3 g, 66.1 mmol) in portions over 30 min. The mixture was stirred for
additional 30min, 3 (12.0 g, 54.9 mmol) was then added in portions over
30min at 0 ℃. The reaction mixture was warmed to 25 ℃ and formed a
homogenous biphasic solution. The separated organic phase was
evaporated in vacuo and dissloved in MeCN (150 mL). DMAP (0.04 g,
0.3 mmol) and a solution of (Boc)2O (18.0 g, 82.5 mmol) in MeCN was
added at ambient temperature dropwise over 1 h. The reaction mixture
(1R,2R)-2-((tert-Butoxycarbonyl)amino)-1-hydroxy-1-(4-
(methylsulfonyl)phenyl)propyl methanesulfonate (7)
Compound 6 (7.0 g, 20.3 mmol) was dissolved in dry dichloromethane
(70 mL), triethylamine (7.2 g, 71.2 mmol) and methanesulfonyl chloride
(7.0 g, 61.1 mmol) were added at 0℃, and the reaction mixture was
stirred for 1 h. The reaction mixture was quenched with ice-cold water
and extracted with DCM (3 × 20 mL) , the solvent was removed under
vacuum. The crude compound was used in the next step without further
purification.
was stirred at ambient temperature for
3 h and the solvent was
evaporated under reduced pressure. THF (200 mL) was added. Then, a
solution of t-BuOK (13.5 g, 120.3 mol) in THF was added at 0 ℃
dropwise over 1 h. After aging at 0 ℃ for 1h, 10 % citric acid was added
to adjust to pH = 7 at 0 ℃. The organic phase was washed with brine (50
mL × 3) and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (petroleum ether / ethyl acetate 1:1, V/V) to
give 4 (14.5 g, 71 %) as a white solid. Mp: 135.7-136.6 ℃. 1H NMR
(600MHz, CDCl3): δ=8.28(d, J=8.22Hz, 2H), 8.09(d, J=8.34Hz, 2H),
5.96(d, J=7.92Hz, 1H), 5.87(d, J=7.62Hz, 1H), 3.75(s, 3H), 3.10(s, 3H),
3.27(s, 1H), 1.45(s, 9H); 13C NMR (150MHz, CDCl3): δ=191.53, 166.90,
154.92, 145.04, 138.44, 130.23, 127.89, 81.15, 59.37, 53.46, 44.27,
28.21; HR-MS calcd for C16H21NNaO7S [M+Na]+ m/z 394.0931, found
m/z 394.0938.
(S)-[4-(methylsulfonyl)phenyl][(R)-1-(tert-
Butoxycarbonyl)amino)aziridine] methanol (8)
To a solution of 7 (obtained in the previous step) in acetonitrile (80 mL)
was added K2CO3 (8.0 g, 57.9 mmol), the reaction mixture was stirred at
50℃ for 5h. The solvent was evaporated in vacuo and the residue was
diluted with CH2Cl2 (80 mL), washed with brine (30 mL), dried with
Na2SO4, filtered, and concentrated under vacuum. the residue was
purified by flash chromatography ( petroleum ether / ethyl acetate 1:1,
V/V) to afford 8 (4.0 g, 61%, over two steps) as a colorless oil. 1H NMR
(600MHz, CDCl3): δ=7.94(d, J=8.4Hz, 2H), 7.68(d, J=8.22Hz, 2H), 4.52(t,
J=5.25Hz, 1H), 3.26(d, J=5.04Hz, 1H), 3.04(s, 3H), 2.71-2.69(m, 1H),
2.39(d, J=5.94Hz, 1H), 2.29(d, J=3.66Hz, 1H), 1.44(s, 9H); 13C NMR
(2S,3R)-Methyl-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-
(methylsulfonyl)phenyl)propanoate ((2S, 3R)-5a)
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800658
European Journal of Organic Chemistry
FULL PAPER
(150MHz, CDCl3): δ=161.66, 146.93, 140.11, 127.66, 127.28, 82.36,
73.00, 44.53, 42.35, 29.29, 27.85; HR-MS calcd for C15H21NNaO5S
[M+Na]+ m/z 350.1033, found m/z 350.1032.
allowed to warm to room temperature and stirred for 2h. The resulting
slurry was filtered, and the cake was washed with EtOAc. The cake was
suspended in dichloromethane (40 mL). Saturated sodium bicarbonate
solution (20 mL) and a solution of dichloroacetic chloride (2.0 g, 13.6
mmol) was added and the mixture was stirred for 1h at 0 ℃. The reaction
mixture was quenched with water and extracted with DCM (3 × 20 mL) ,
the DCM layer was removed under vacuum. to leave a residue. The
crude product was recrystallized with petroleum ether / ethyl acetate (1:1,
V/V) to give white solid (4.9 g, 95 %). Mp: 164.3-166.2 ℃. 1H NMR
(600MHz, DMSO-d6): δ=8.93(d, J=9.12Hz, 1H), 7.85(d, J=8.34Hz, 2H),
7.66(d, J=8.4Hz, 2H), 6.56(s, 1H), 6.37(d, J=4.38Hz, 1H), 5.35(t,
J=3.63Hz, 1H), 4.72(dd, J=9.12Hz, 2.76Hz, 1H), 3.72(s, 3H), 3.16(s, 3H);
13C NMR (150MHz, DMSO-d6): δ=169.93, 164.24, 147.42, 140.19,
127.64, 126.98, 71.79, 66.27, 58.66, 52.93, 44.00; HR-MS calcd for
C13H15Cl2NNaO6S [M+Na]+ m/z 405.9889, found m/z 405.9889.
tert-butyl((1R,2S)-3-fluoro-1-hydroxy-1-(4-
(methylsulfonyl)phenyl)propan-2-yl) carbamate(9)
The aziridine 8 (3.0 g, 9.2 mmol) was dissolved in DCE (30 mL).
Triethylamine trihydrofluoride (12.0 g, 74.4 mmol) was added and the
reaction mixture was stirred at 70℃ for 10h. The reaction was quenched
with saturated aqueous NaHCO3 and extracted with CH2Cl2 (20 mL × 3).
The combined organic extracts were dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash chromatography
( petroleum ether / ethyl acetate 1:1, V/V) to afford 9 (3.1 g, 77 %) as a
white solid. Mp: 175.2-175.7 ℃. 1H NMR (600MHz, DMSO-d6): δ=7.86(d,
J=8.88Hz, 2H), 7.58(d, J=8.22Hz, 2H), 6.63(d, J=9.24Hz, 1H), 5.74(d,
J=5.16Hz, 1H), 4.87(t, J=4.02Hz, 1H), 4.61-4.50(m, 1H), 4.35-4.24(m,
1H), 4.01-3.94(m, 1H), 3.15(s, 3H), 1.23(s, 9H); 13C NMR (150MHz,
DMSO-d6): δ=155.62, 148.98, 139.77, 127.70, 126.87, 82.95(d,
J=167.85Hz), 78.38, 70.61(d, J=6.03Hz), 55.95(d, J=19.67Hz), 44.08,
28.48; HR-MS calcd for C15H22FNNaO5S [M+Na]+ m/z 370.1095, found
m/z 370.1096.
2,2-dichloro-N-((1R,2R)-1,3-dihydroxy-1-(4-
(methylsulfonyl)phenyl)propan-2-yl)acetamide (2)
NaBH4 (1 g, 26.4 mmol) was added to a solution of 11 (3.5 g, 8.8 mmol)
in MeOH (20 mL) at 0℃. The mixture was stirred for 5h and H2O (5 mL)
was added and the mixture was stirred for a further 1h. The mixture was
then filtered and the filtrate was concentrated. The residue was diluted
with CH2Cl2 (40 mL), washed with brine (20 mL), dried (Na2SO4), filtered,
and concentrated under vacuum. The residue was purified by flash
column chromatography [silica gel petroleum ether-EtOAc (1:1)] to give a
(1R,2S)-2-amino-3-fluoro-1-[4-(methylsulfonyl)phenyl]-1-
propanol(10)
1
white solid (2.8 g, 91 %). Mp: 164.4-164.9 ℃. H NMR (600MHz, DMSO-
To a solution of 9 (3.0 g, 8.6 mmol) in EtOAc (10 mL) was added 2N HCl
in EtOAc (10mL) at 0℃. The reaction mixture was allowed to warm to
room temperature and stirred for 2h. The resulting slurry was filtered, and
the cake was washed with EtOAc. The wet cake was dried at 45℃ in
vacuum to give HCl salt 10 (2.1 g, 90 %). Mp: 155.9-157.3 ℃. 1H NMR
(600MHz, DMSO-d6): δ=8.54(s, 3H), 7.95(d, J=8.46Hz, 2H), 7.68(d,
J=8.34Hz, 2H), 6.67(d, J=4.5Hz, 1H), 4.91-4.89(m, 1H), 4.68-4.58(m, 1H),
4.35-4.25(m, 1H), 3.64-3.57(m, 1H), 3.22(s, 3H); 13C NMR (150MHz,
DMSO-d6): δ=146.64, 141.00, 128.42, 127.65, 81.31(d, J=167.96Hz),
69.61(d, J=5.43Hz), 56.15(d, J=18.15Hz), 43.97; HR-MS calcd for
C10H15FNO3S [M+H]+ m/z 248.0751, found m/z 248.0752.
d6): δ=8.30(d, J=9.06Hz, 1H), 7.84(d, J=8.4Hz, 2H), 7.58(d, J=8.28Hz,
2H), 6.49(s, 1H), 5.97(d, J=4.38Hz, 1H), 5.03-5.01(m, 1H), 4.98-4.96(m,
1H), 3.94-3.90(m, 1H), 3.60-3.56(m, 1H), 3.36-3.32(m, 1H), 3.16(s, 3H);
13C NMR (150MHz, DMSO-d6): δ=163.89, 149.65, 139.72, 127.49,
126.93, 69.50, 66.96, 60.65, 57.35, 44.06; HR-MS calcd for
C12H16Cl2NO5S [M+H]+ m/z 356.0121, found m/z 356.0121.
(2R,3R)-2-(2,2-dichloroacetamido)-3-hydroxy-3-(4-
(methylsulfonyl)phenyl) propyl 4-methylbenzenesulfonate (12)
Compound 2 (3 g, 8.4 mmol) was dissolved in dry dichloromethane (30
mL), pyridine (10 mL) and 4-methylbenzenesulfonyl chloride (1.9 g, 10.0
mmol) were added at 0℃, and the reaction mixture was stirred for 8 h.
The reaction mixture was quenched with ice-cold water and extracted
with DCM (3 × 20 mL) , the solvent was removed under vacuum. The
residue was purified by flash column chromatography [silica gel
petroleum ether-EtOAc (1:1)] to give a white solid (3.8 g, 88 %); 1H NMR
(600MHz, CDCl3): δ=7.81-7.77(m, 4H), 7.50(d, J=8.28Hz, 2H), 7.38(d,
J=8.04Hz, 2H), 6.95(d, J=8.46Hz, 1H), 5.74(s, 1H), 5.19(s, 1H), 4.32-
4.25(m, 2H), 4.06-4.04(m, 1H), 3.60(d, J=4.02Hz, 1H), 3.01(s, 3H),
2.45(s, 3H); 13C NMR (150MHz, CDCl3): δ=164.31, 146.39, 145.68,
139.84, 132.05, 130.20, 128.01, 127.46, 126.94, 69.95, 67.25, 65.94,
54.09, 44.50, 21.73; HR-MS calcd for C19H21Cl2NNaO7S2 [M+Na]+ m/z
532.0029, found m/z 532.0029.
2,2-dichloro-N-((1R,2S)-3-fluoro-1-hydroxy-1-(4-
(methylsulfonyl)phenyl)propan-2-yl)acetamide(1)
A solution of 10 (5.0 g, 17.6 mmol) in dry MeOH (25 mL), Et3N (6.1 g,
60.3 mmol) and methyl dichloroacetate (7.2 g, 50.4 mmol) was stirred at
50℃ for 10h. The solvent was evaporated in vacuo and the residue was
purified by flash chromatography ( petroleum ether / ethyl acetate 1:1,
V/V) to afford 1 (7.1 g, 95 %) as a white solid. Mp: 155.7-156.2 ℃. 1H
NMR (600MHz, DMSO-d6): δ=8.62(d, J=9.06Hz, 1H), 7.86(d, J=8.4Hz,
2H), 7.62(d, J=8.28Hz, 2H), 6.46(s, 1H), 6.16(d, J=4.38Hz, 1H), 4.99(t,
J=3.6Hz, 1H), 4.71-4.60(m, 1H), 4.44-4.33(m, 1H), 4.31-4.25(m, 1H),
3.16(s, 3H); 13C NMR (150MHz, DMSO-d6): δ=164.19, 148.39, 140.02,
127.62, 126.97, 82.83(d, J=168.96Hz), 69.8 (d, J=6.0Hz), 66.72, 55.09(d,
J=19.71Hz), 44.04; HR-MS calcd for C12H14Cl2FNNaO4S [M+Na]+ m/z
379.9897, found m/z 379.9900. The chiral HPLC was performed using a
CHIRALPAK IC-3 (4.6×250 mm, 3µm, DAICEL, Shanghai) column with
a mobile phase of 70% Hex-30% IPA. The product was detected at
224nm, and the retention times of diastereoisomers were 45.6 min,
48.2min (1), 51.1min and 57.4 min, respectively.
(R)-((R)-2-(dichloromethyl)-4,5-dihydrooxazol-4-yl)(4-
(methylsulfonyl)phenyl) methanol (13)
To a solution of 12 (4 g, 7.8 mmol) in acetonitrile (40 mL) was added
K2CO3 (4.3 g, 31.1 mmol), the reaction mixture was stirred at 50℃ for 2h.
The reaction mixture was quenched with water and extracted with DCM
(20 × 3 mL), the solvent was removed under vacuum, the residue was
purified by flash chromatography ( petroleum ether / ethyl acetate 1:1,
V/V) to afford 13 (2.3 g, 85 %) as a white solid. Mp: 129.7-130.4 ℃. 1H
NMR (600MHz, CDCl3): δ=7.93(d, J=8.4Hz, 2H), 7.61(d, J=8.16Hz, 2H),
6.22(s, 1H), 4.82-4.80(m, 1H), 4.59-4.55(m, 1H), 4.43(t, J=9.39Hz, 1H),
(2S,3R)-methyl2-(2,2-dichloroacetamido)-3-hydroxy-3-(4-
(methylsulfonyl) phenyl)propanoate (11)
To a solution of (2S, 3R)-5a (5 g, 13.4 mmol) in EtOAc (20 mL) was
added 2N HCl in EtOAc (20 mL) at 0℃. The reaction mixture was
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800658
European Journal of Organic Chemistry
FULL PAPER
4.36(t, J=8.4Hz, 1H), 3.04(s, 3H), 2.92(d, J=3.78Hz, 1H); 13C NMR
(150MHz, CDCl3): δ=164.13, 145.73, 140.38, 127.81, 127.68, 74.71,
71.83, 70.84, 60.95, 44.49; HR-MS calcd for C12H14Cl2NO4S [M+H]+ m/z
338.0015, found m/z 338.0015.
J=10.26Hz, 1H), 3.03(s, 3H), 2.41(s, 1H); 13C NMR (150MHz, CDCl3):
δ=148.76, 139.45, 139.32, 127.57, 127.12, 116.57, 74.63, 44.55; HR-MS
calcd for C10H12NaO3S [M+Na]+ m/z 235.0399, found m/z 235.0403. The
chiral HPLC was performed using a CHIRALPAK IC-3 (4.6×250 mm,
3µm, DAICEL, Shanghai) column with a mobile phase of 90% Hex-10%
EtOH. The product was detected at 220 nm, and the retention times of
diastereoisomers were 47.2 min, and 49.7 min (S-21), respectively.
tert-butyl((4R,5R)-4-(4-(methylsulfonyl)phenyl)-2,2-dioxido-1,3,2-
dioxathian-5-yl)carbamate (14)
To a stirred solution of diol 6 (5.0 g, 14.48 mmol) in anhydrous
dichloromethane (50 mL) and triethylamine (40 mL) at 0℃ was slowly
added thionyl chloride (13.8 g, 116.00 mmol). After 30min, the reaction
was diluted with dichloromethane (40 mL) and washed with dilute
hydrochloric acid (25 mL × 2) and brine (50 mL). The organic layer was
concentrated under reduced pressure. the crude product was dissolved
in a mixture of water (10 mL), MeCN (10 mL), and CH2Cl2 (15 mL). NaIO4
(4.0 g, 18.70 mmol) and RuCl3 hydrate (3 mg, 0.01 mmol) were added
and the solution was vigorously stirred for 7h at ambient temperature.
EtOAc (50 mL) was added to the cooled mixture. The organic layer was
removed, dried (Na2SO4), and concentrated. The crude product was
chromatographed to give the corresponding compound 14 (4.2g, 71 %)
as a white solid. Mp: 232.8-234.8 ℃ . 1H NMR (600MHz, CDCl3):
δ=7.99(d, J=8.4Hz, 2H), 7.57(d, J=8.22Hz, 2H), 6.19(s, 1H), 5.31(d,
J=9.9Hz, 1H), 5.15(dd, J=11.79Hz, 1.77Hz, 1H), 4.66(dd, J=11.76Hz,
Acknowledgements
We thank Rongsheng Tao (Yi-hui Biological Technology Co. Ltd)
for supply of ketoreductases. We are grateful to Shanghai
Institute of Pharmaceutical Industry for financial support.
Keywords: Florfenicol • Dynamic Reductive Kinetic Resolution •
Ketoreductases • Fluorination
[1]
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75, 4330-4333.
1.2Hz, 1H), 4.38(dd, J=9.93Hz, 1.53Hz, 1H), 3.03(s, 3H), 1.23(s, 9H); 13
C
a) D. P. Schumacher, J. E. Clark, B. F. Murphy, P. A. Fischer, J. Org.
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NMR (150MHz, CDCl3): δ=154.49, 141.34, 138.82, 127.75, 126.73,
86.33, 81.13, 47.06, 44.39, 27.96; HR-MS calcd for C15H21NNaO8S2
[M+H]+ m/z 430.0601, found m/z 430.0606.
[3]
[4]
2,2-dichloro-N-((4R,5R)-4-(4-(methylsulfonyl)phenyl)-2,2-dioxido-
1,3,2- dioxathian-5-yl)acetamide (15)
To
a stirred solution of diol 2 (5.0 g, 14.0 mmol) in anhydrous
[5]
a) J. E. Clark, P. A. Fischer, D. P. Schumacher, Synthesis, 1991, 10,
891-894; b) B. Kaptein, T. J. G. M. Dooren, W. H. J. Boesten, T. Sonke,
A. L. L. Duchateau, Q. B. Broxterman, J. Kamphuis, Org. Process Res.
Dev., 1998, 2, 10-17; c) W. Y. Lu, P. R. Chen, G. Q. Lin, Tetrahedron,
2008, 64, 7822-7827; d) Z. H. Wang, F. Li, L. Zhao, Q. Q. He, F. E.
Chen, C. Zheng, Tetrahedron, 2011, 67, 9199-9203; e) F. Li, Z. H.
Wang, L. Zhao, F. J. Xiong, Q. Q. He, F. E Chen, Tetrahedron:
Asymmetry, 2011, 22, 1337-1341; f) Z.-H. Wang, C. Zheng, F. Li, L.
Zhao, F.-E. Chen, Q.-Q. He, Synthesis, 2012, 44, 699-704.
X. Wang, L. Xu, L. Yan, H. Wang, S. Han, Y.Wu, F. Chen, Tetrahedron,
2016, 72, 1787-1793.
dichloromethane (50 mL) and triethylamine (40 mL) at 0℃ was slowly
added thionyl chloride (13.4 g, 112.6 mmol). After 30min, the reaction
was diluted with dichloromethane (40 mL) and washed with dilute
hydrochloric acid (2 ×25 mL) and brine (50 mL). The organic layer was
concentrated under reduced pressure. the crude product was dissolved
in a mixture of water (10 mL), MeCN (10 mL) and CH2Cl2 (15 mL). NaIO4
(4.0 g, 18.70 mmol) and RuCl3 hydrate (3 mg, 0.01 mmol) were added
and the solution was vigorously stirred for 7h at ambient temperature.
EtOAc (50 mL) was added to the cooled mixture. The organic layer was
removed, dried (Na2SO4), and concentrated. The crude product was
chromatographed to give compound 15 (4.3g, 72 %) as a white solid. Mp:
187.0-189.0 ℃. 1H NMR (600MHz, DMSO-d6): δ=9.48(d, J=9.6Hz, 1H),
7.96(d, J=8.52Hz, 2H), 7.61(d, J=8.28Hz, 2H), 6.54(d, J=1.92Hz, 1H),
6.36(s, 1H), 5.17(dd, J=12.09Hz, 2.01Hz, 1H), 4.85(dd, J=12.09Hz,
1.41Hz, 1H), 4.71(dd, J=9.63Hz, 1.77Hz, 1H), 3.17(s, 3H); 13C NMR
(150MHz, DMSO-d6): δ=163.98, 141.52, 139.08, 127.50, 127.19, 86.95,
77.98, 66.11, 46.04, 43.89; HR-MS calcd for C12H13Cl2NNaO7S2 [M+Na]+
m/z 439.9403, found m/z 439.9402.
[6]
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a) H. Pellissier, Tetrahedron, 2003, 59, 8291-8327; b) E. Vedejs, M.
Jure, Angew. Chem., 2005, 117, 4040-4069; Angew. Chem. int. ed.,
2005, 44, 3972-4001.
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a) S. K. Ma, J. Gruber, C. Davis, L. Newman, D. Gray, A. Wang, J.
Grate, G. W. Huisman, R. A. Sheldon, Green Chem., 2010, 12, 81-86;
b) J. Liang, J. Lalonde, B. Borup, V. Mitchell, E. Mundorff, N. Trinh, D.
A. Kochrekar, R. N. Cherat, G. G. Pai, Org. Process Res. Dev., 2010,
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2012, 13, 13542-13553; d) H. Li, J. Moncecchi, M. D. Truppo, Org.
Process Res. Dev., 2015, 19, 695-700; e) G.-P. Xu, H.-B. Wang, Z.-L.
Wu, Process Biochem., 2016, 51, 881-885.
(S)-1-(4-(methylsulfonyl)phenyl)prop-2-en-1-ol (S-21)
[9]
N. Urano, S. Fukui, S. Kumashiro, T. Ishige, S. Kita, K. Sakamoto, M.
Kataoka, S. Shimizu, J. Biosci. Bioeng., 2011, 111, 266-271.
To a solution of phosphate buffer (pH 7.0, 10mL) was added dextrose
(0.8g) followed by KRED-02 wet cells (0.5g), 5mg NADP+, ketone 22
(0.5g). The resulting solution was pH adjusted to a minimum of 7.5 with
2M NaOH prior to use. The reaction was carried out at 30 ℃ and shaken
at 200 rpm shaker for 24 h. The reaction mixture was then extracted with
DCM (10 mL×3), and the DCM layers were combined. The organic layers
were dried over Na2SO4 and concentrated under reduced pressure,
obtaining 22 (0.48 g, 95 %) as a white solid. Mp: 54.8-55.8 ℃. 1H NMR
(600MHz, CDCl3): δ=7.89(d, J=8.34Hz, 2H), 7.57(d, J=8.28Hz, 2H), 6.01-
5.93(m, 1H), 5.38(d, J=17.1Hz, 1H), 5.29(d, J=5.7Hz, 1H), 5.25(d,
[10] F. Xu, J. Y. L. Chung, J. C. Moore, Z. Liu, N. Yoshikawa, R. S. Hoerrner,
J. Lee, M. Royzen, E. Cleator, A. G. Gibson, R. Dunn, K. M. Maloney,
M. Alam, A. Goodyear, J. Lynch, N. Yasuda, P. N. Devine, Org. Lett.,
2013, 15(6), 1342–1345.
[11] To prepare four stereoenantimers (2S, 3R)-5a, (2R, 3S)-5b, (2S, 3S)-5c
and (2R, 3R)-5d, see the Supporting Information.
[12] a) R. Noyori, T. Ikeda, T. Ohkuma, M. Widhalm, M. Kitamura, H.
Takaya, S. Akutagawa, N. Sayo, T. Saito, T. Taketomi, H.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800658
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10.1002/ejoc.201800658
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Jie Zou, Guowei Ni, Jiawei Tang, Jun
Yu, Luobin Jiang, Dianwen Ju, Fuli
Zhang*, and Shaoxin Chen*
Page No. – Page No.
Asymmetric Synthesis of Florfenicol
via Dynamic Reductive Kinetic
Resolution with Ketoreductases
Chemo-enzymatic synthesis: The new synthesis of florfenicol features the
application of enzymatic DYRKR to establish the two stereo centers of cis-1,2-
amino alcohol with >99% ee and 99% dr in one step. Two new methods via
aziridines and cyclic sulfates with Et3N-3HF in high regioselectivuty to introduce
fluorine atom were developed.
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