Highly enantioselective bioreduction of 1-(3,4-difluorophenyl)-3-nitropropan-1-one: Key intermediate of ticagrelor

Article abstract of DOI:10.1039/c5ra25948g

A simple, highly effective and economical whole-cell mediated process was developed for the biocatalytic reduction of a ketone, intermediate in the synthesis of platelet inhibiting drug ticagrelor. Sixteen different microorganisms were screened for the bi

Full text of DOI:10.1039/c5ra25948g

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Highly enantioselective bioreduction of 1-(3,4-
difluorophenyl)-3-nitropropan-1-one: key
intermediate of ticagrelor†
Cite this: RSC Adv., 2016, 6, 35086
Manpreet Singh,a Hare Krishnen,a Uday Kumar Neelam,a Kavitha Charugondla,a
a
b
a
*
Goverdhan Gilla, Karen Holt-Tiffin and Rakeshwar Bandichhor
A simple, highly effective and economical whole-cell mediated process was developed for the biocatalytic
reduction of a ketone, intermediate in the synthesis of platelet inhibiting drug ticagrelor. Sixteen different
microorganisms were screened for the bioreduction of 1-(3,4-difluorophenyl)-3-nitropropan-1-one (1)
to (S)-1-(3,4-difluorophenyl)-3-nitropropan-1-ol (2). Growing as well as resting cells of Candida
parapsilosis 104659 exhibited high conversion (>99.0%) and enantioselectivity (98.0%) for the
S-enantiomer.
Received 5th December 2015
Accepted 30th March 2016
DOI: 10.1039/c5ra25948g
www.rsc.org/advances
cyclopropylamine-4 can be derived from (S)-1-(3,4-
diuorophenyl)-3-nitropropan-1-ol (2). The (S)-nitro alcohol (2)
1. Introduction
can be chemically prepared by utilizing chiral catalysts such as
CBS, however, the overall yields and enantioselectivity are oen
low and the catalyst is expensive (Scheme 1).19 Biological
synthesis of optically active compounds especially alcohols is
more stereospecic, environmentally friendly and efficient in
comparison to conventional chemical methods. Thus we
envisaged a route to the (S)-nitro alcohol (2) via enantioselective
bioreduction of the prochiral ketone (1).
In recent years, we have been investigating the microbial
reduction of different prochiral ketones used as intermediates
for the synthesis of active pharmaceutical ingredients.20 In the
present study, sixteen different microorganism strains obtained
Chiral alcohols are useful intermediates in the synthesis of
active pharmaceutical ingredients.1,2 Biocatalytic reduction of
prochiral ketones is nding foothold in industry and is
replacing traditional chemocatalysis.3 The use of wild type
whole cells,4,5 recombinant cells6–8 and isolated enzymes9 has
been reported on a variety of substrates. In comparison to the
isolated enzymes, whole cells offer the advantage of internal
co-factor regeneration and are usually more stable and elimi-
nate the cost of enzyme purication. However, the limiting
factor in the case of whole cell processes is the low volumetric
productivity. Limited solubility of the ketone starting material
in water can also contribute to low productivity of the system.
Medium engineering and modied substrate addition strate-
gies lead to enhanced substrate utilization and play an impor-
tant role in establishing whole cell processes at industrial
scale.10–12
Ticagrelor (Scheme 1, (5)) is a novel P2Y12 receptor antagonist
which functions by blocking adenosine diphosphate-mediated
platelet aggregation. There are several published reports on
the synthesis of this carbocyclic nucleoside and analogs,13–16
however insufficient research has been targeted on the key
chiral intermediate diurophenyl cyclopropylamine (4).17
Zhang et al. reported the synthesis of different derivatives of
ticagrelor
especially
the
prodrugs.18
The
chiral
aIntegrated Product Development, Innovation Plaza, Dr. Reddy's Laboratories Ltd.,
Bachupalli, Qutubullapur, R. R. Dist., 500 072 Andhra Pradesh, India. E-mail:
rakeshwarb@drreddys.com; Fax: +91 40 44346164; Tel: +91 40 44346430
bChirotech Technology Ltd., 410 Cambridge Science Park, Milton Road, Cambridge,
Cambridgeshire, UK, CB4 OPE
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra25948g
Scheme 1
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Table
1
Bioreduction of (1) to (2) using different strains of Table 2 Bioreduction of (1) to (2) using several species of Candidaa
microorganismsa
Microbial strains
Conv.b (%)
Rc
Sc
Microbial strains
Conv.b (%)
Rc
Sc
C. glabrata 104523
82.0 C. boidinii 103655
22.0
5.0
7.0
5.0
8.0
10.0
>98.0
76.0
>99.0
50.0
>98.0
68.0
32.0
—
—
Candida tropicalis 104656
Bacillus licheniformis 103023
Saccharomyces cerevisiae 103168
Rhodococcus sp. 103102
98.0
0.0
6.0
18.0
—
100.0
—
100.0
100.0
40.0
62.0
50.0
7.0
93.0
17.0
60.0
9.0
—
—
—
C. sonorensis 104061
C. magnoliae 104517
C. apis 104519
60.0
38.0
50.0
93.0
7.0
83.0
40.0
91.0
0.0
Pichia methanolica 103660
Rhodotorula sp. 103024
Candida rugosa 103332
73.0
41.0
96.0
73.0
20.0
9.0
27.0 C. maltosa 104532
80.0 C. parapsilosis 104659
91.0 C. etchellsii 104521
C. tropicalis 104656
a
Reaction conditions: 0.09 mol of (1) was dissolved in 100 ml of
methanol and added to 24 h old fermentation broth (50.0 ml)
consisting of potato dextrose broth (24.0 g lꢀ1). Samples were taken
aer 48 h and extracted with equal volume of ethyl acetate and dried
C. utilis 104952
C. rugosa 103332
a
Reaction conditions: 0.09 mol of (1) was dissolved in 100 ml of
methanol and added to 24 h old fermentation broth (50.0 ml)
consisting of potato dextrose broth (24.0 g lꢀ1). Samples were taken
aer 48 h and extracted with equal volume of ethyl acetate and dried
b
using nitrogen. Determined by HPLC (Zorbex Eclipse Plus C-18,
210 nm); mobile phase A: 0.02 M KH2PO4 buffer pH 3.0/mobile phase
c
B: ACN : H2O (90 : 10 v/v) – see ESI. Determined by chiral HPLC
b
[Chiralcel OD-H column, hexane/iPrOH/TFA (92 : 7.9 : 0.1)] – see ESI.
using nitrogen. Determined by HPLC (Zorbex Eclipse Plus C-18,
210 nm); mobile phase A: 0.02 M KH2PO4 buffer pH 3.0/mobile phase
c
B: ACN : H2O (90 : 10 v/v) – see ESI. Determined by chiral HPLC
[Chiralcel OD-H column, hexane/iPrOH/TFA (92 : 7.9 : 0.1)] – see ESI.
from Chirotech Technology Centre, Cambridge, UK were
screened for their potential activity in the asymmetric reduction
of 1-(3,4-diuorophenyl)-3-nitropropan-1-one (1). In the rst
stage of screening seven (Candida tropicalis, Bacillus lichen-
iformis, Saccharomyces cerevisiae, Rhodococcus sp., Pichia meth-
anolica, Rhodotorulla sp. and Candida rugosa) organisms of
bacterial and fungal origin were investigated for their perfor-
mance in the bioreduction reaction. The initial screening was
carried out by supplementing the substrate in 24 h old
fermentation media itself. Candida species demonstrated
superior activity (>96.0%) and selectivity ($82.0%) in compar-
ison to other selected strains (Table 1). Rhodotorula sp. showed
moderate conversion, whilst S. cerevisiae and P. methanolica
showed preference for other enantiomer.
bioreduction reaction. The best bioreduction was in the pres-
ence of THF with >99.0% conversion and >94.0% enantiose-
lectivity aer 48 h of incubation (Table 3). The reason behind
the increased enantioselectivity in the presence of THF might
be inhibition of other KREDs or a change in the conformation of
the enzyme active site.15 Evaluation of different concentrations
of THF in the bioreduction revealed the optimum activity was at
1.0% THF concentration; further increase in concentration
decreased the conversion rate.
2.3. Effect of substrate concentration
Substrate loading can have a substantial effect on the activity of
enzymes; hence different concentrations of (1) were tested. As
shown in Fig. 1, the highest conversion was achieved at only
0.5 g lꢀ1 of (1) concentration aer 48 h of incubation. Further
increase in (1) concentration decreased the conversion rate. We
2. Results and discussions
2.1. Screening
Since the Candida species displayed better enantioselectivity
and excellent conversion for the bioreduction of (1), eleven
different types of Candida sp. were then screened for the
biotransformation of (1) to (2). Table 2 shows the conversion
and enantioselectivity obtained from the diverse species of
Candida. All the species have different activity and selectivity for
(1). C. parapsilosis and C. rugosa had the highest conversion
(>98.0%) and enantioselectivity (>82.0%), while C. tropicalis
showed excellent conversion (>99.0%) but poor selectivity
(66.0%). Candida etchellsii showed good conversion but prefer-
ence for the R-enantiomer (86.0%).
Table 3 Effect of different co-solvents on the bioreduction of (1) using
C. parapsilosisa
Solvents
Conv.b (%)
eec (%)
Methanol
Ethanol
THF
IPA
MTBE
98.0
90.0
>99.0
98.0
76.0
86.0
85.0
94.0
92.0
80.0
Based on the screening results, it was concluded that the C.
parapsilosis and C. rugosa were the best organisms for the bio-
reduction of (1) to (2). Candida parapsilosis was chosen for
further studies because of its better enantioselectivity.
a
Reaction conditions: 0.09 mol of (1) was dissolved in 100 ml solvent
and added in to 24 h old fermentation broth (50.0 ml) consisting of
potato dextrose broth (24.0 g lꢀ1). Samples were taken aer 48 h and
extracted with equal volume of ethyl acetate and dried using nitrogen.
b
Determined by HPLC (Zorbex Eclipse Plus C-18, 210 nm); mobile
2.2. Effect of co-solvents
phase A: 0.02 M KH2PO4 buffer pH 3.0/mobile phase B: ACN : H2O
c
(90 : 10 v/v) – see ESI. Determined by chiral HPLC [Chiralcel OD-H
To determine the effect of co-solvents, different solvents
column, hexane/iPrOH/TFA (92 : 7.9 : 0.1)] – see ESI.
(methanol, ethanol, IPA, THF, and MTBE) were tested in the
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Fig. 1 The effect of substrate concentration on the conversion of (1)
to (2) using 24 h old fermentation broth of C. parapsilosis. Reaction
conditions: 0.3–0.7 g lꢀ1 of (1) was dissolved in 100 ml of THF and
added to 24 h old fermentation broth (50.0 ml) consisting of potato
dextrose broth (24.0 g lꢀ1). Samples were taken after 48 h and
extracted with equal volume of ethyl acetate and dried using nitrogen.
Determined by HPLC (Zorbex Eclipse Plus C-18, 210 nm); mobile
phase A: 0.02 M KH2PO4 buffer pH 3.0/mobile phase B: ACN : H2O
(90 : 10 v/v) – see ESI.†
Fig. 2 The effect of substrate concentration on the biotransformation
of (1) using resting cells of C. parapsilosis. Reaction conditions: cells
were harvested from 48 h old culture by centrifugation (6000 rpm ꢁ
10 min). 1.0 g cells were taken, washed with de-ionized water and then
re-dissolved in 5% glucose solution. Different concentrations (0.5–6.0
g lꢀ1) of (1) were dissolved in THF and then charged to the glucose
solution containing cells. The reactions were incubated at 30 ꢂC and
200 rpm in an orbital shaker for 48 h. Determined by HPLC (Zorbex
Eclipse Plus C-18, 210 nm); mobile phase A: 0.02 M KH2PO4 buffer pH
3.0/mobile phase B: ACN : H2O (90 : 10 v/v) – see ESI.† c Determined
by chiral HPLC [Chiralcel OD-H column, hexane/iPrOH/TFA
(92 : 7.9 : 0.1)] – see ESI.†
knew we would need to increase the substrate concentration to
allow a cost effective process to be developed.
in fermentation media containing molasses (8.0%) as the sole
carbon source. Aer the completion of reaction the product was
isolated at an overall yield of 85.0% with an ee > 98.0% and
>99% purity of (2).
2.4. Reduction using resting cells of C. parapsilosis
The next strategy to increase the productivity of the bio-
reduction was to carry out the reaction using media free resting
cells of Candida parapsilosis. Taking into account the effect of
glucose as a co-substrate in bioreduction reactions, the experi-
ments were conducted by charging resting cells of C. para-
psilosis in H2O containing glucose and (1) dissolved in THF. The
reactions were carried out at different concentrations of (1)
(0.5 to 6.0 g lꢀ1). As illustrated in Fig. 2, the resting cell reaction
yielded excellent conversion (>99.0%) and enantioselectivity
(98.0%) at a substrate concentration of 4.0 g lꢀ1. Further
increase in substrate concentration however was not tolerated
under these conditions.
3. Conclusions
In conclusion, the Candida sp. mediated reduction of 1-(3,4-
diuorophenyl)-3-nitropropan-1-one (1) is a convenient way to
prepare a key chiral alcohol intermediate to ticagrelor with high
enantioselectivity. C. parapsilosis has shown complete conver-
sion and excellent enantioselectivity (>98.0%) at 4.0 g lꢀ1
substrate concentration and the organism is robust enough to
grow on natural media. Our whole-cell catalyzed system has the
advantage of not requiring external cofactors or cofactor recy-
cling systems, thus reducing costs.
2.5. Molasses as sole carbon source
We then turned our attention to optimize the fermentation
media, since the cost of media components play a major role in
any industrial scale fermentation process. Due to enormous
utilization of sugar in India there are numerous sugar indus-
tries, therefore their byproduct, molasses is low cost in
comparison to synthetic substrates. Several experiments were
conducted by utilizing different concentrations of molasses as
a sole fermentation media for the growth of C. parapsilosis.
Pleasingly molasses as fermentation media yielded the same
degree of growth, wet cell-mass and activity as seen in synthetic
media made of potato infusion and dextrose.
4. Materials and methods
4.1. General method
All the microbial strains utilized in this study were obtained
from Chirotech Technology Centre, Cambridge, UK. 1-(3,4-
Diuorophenyl)-3-nitropropan-1-one, chirally pure standards
and
(R,S)-1-(3,4-diuorophenyl)-3-nitropropan-1-ol
were
synthesized and obtained from the chemistry department,
IPDO, Dr. Reddy's Labs, Hyderabad, India following reported
procedures.19 The chemicals used were of analytical grade and
procured from commercial sources. The synthetic medium
used in fermentation process was procured from Himedia
2.6. Preparative-scale bioreduction using C. parapsilosis
With the aim of scale-up, the bioreduction of (1) was carried out Laboratories Pvt. Ltd. Mumbai. The molasses used in the opti-
at 10.0 g scale using the resting cells of C. parapsilosis produced mization study was obtained from the sugar industries in and
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1
around Hyderabad, Telengana, India. H NMR and 13C spectra solvent was removed by rota-vaporization and the samples were
were recorded in CDCl3 solution on a Bruker AVANCE III HD analyzed for purity and chiral purity by HPLC (Section 4.3.). Also
NanoBay 400 MHz instrument. Infra-red spectra was recorded the samples were submitted for recording FT-IR and LC-MS
on Perkin Elmer Spectrum 100 instrument. HPLC analysis was data.
performed on Agilent 1100 series instrument equipped with
UV-detector. The conversion of (1) and (2) was determined using
reverse phase Zorbex Eclipse plus C-18 column with ow rate of
1.0 ml minꢀ1 using gradient ow consisting of 0.02 M potas-
sium phosphate buffer pH 3.0 and acetonitrile water (90 : 10) as
eluent. The chiral purity was determined by running the
samples against chiral standards using Chiralcel OD-H column
with ow rate of 0.5 ml minꢀ1 using hexane, IPA and TFA in the
ratio of 92 : 7.9 : 0.1 as eluent. All the samples were analyzed at
210 nm. LC mass spectrum was obtained on Agilent LC ABSciex
4000 with Q. Trap system.
4.5. 1-(3,4-Diuorophenyl)-3-nitropropan-1-one
1
Dark brown colored liquid, H NMR (400 MHz, CDCl3) d 3.62
(2H, J ¼ 5.92 Hz, t), 4.83–4.81 (2H, m), 7.17–7.15 (1H, m)
7.3–7.82 (2H, m); 13C NMR (100 MHz, CDCl3): 196.6 (1C), 155.5
(1C), 153.0 (1C), 132.8 (1C), 125.3 (1C), 118.0 (1C), 117.7 (1C),
69.0 (1C), 34.7 (1C).
4.6. 1-(3,4-Diuorophenyl)-3-nitropropan-1-ol
1
Dark brown colored liquid H NMR (400 MHz, CDCl3) d 2.31–
2.30 (2H, m), 4.50–4.49 (2H, m), 4.67–4.60 (2H, m), 4.83 (2H, J ¼
6.65 Hz, 2.65 Hz, dd), 7.07–7.19 (3H, m), EI-mass: m/z 216 [M ꢀ
4.2. Medium and cultivation
H]+l; IR (KBr): 877.2, 1283.3, 3435.0 cmꢀ1
.
ꢂ
The microbial cultures preserved as glycerol stocks in ꢀ80 C
were thawed and transferred to solid medium consisting of
potato dextrose agar (PDA) for further use. The seed culture was
prepared by inoculating single colony from agar medium to
liquid medium (PDB, pH 7.0) in a 100 ml Erlenmeyer ask.
Consequently, 5.0% of the seed culture was transferred into
Acknowledgements
The authors greatly appreciate K. Lavanya and Subbaiah Arla for
providing analytical support.
100 ml of the production medium (PDB, pH 7.0) and incubated
ꢂ
in an orbital shaker at 30 C and 200 rpm for 48 h. The media Notes and references
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and the cell pellet was washed twice with de-ionized water and
again separated by centrifugation.
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