Enantioselective bioreduction of benzo-fused cyclic ketones with engineered: Candida glabrata ketoreductase 1-a promising synthetic route to ladostigil (TV3326)

Article abstract of DOI:10.1039/c7ob01803g

Biocatalysis has been recently emerging as a promising alternative to traditional chemical synthesis because of its "green" characteristics and comparable selectivities, which accord with the concept of sustainable development and demand for asymmetric synthesis. In this study, whole-cell biocatalysts containing glucose dehydrogenase (GDH) and Candida glabrata ketoreductase 1 (CgKR1) variants were constructed. These biocatalysts were applied to the reduction of benzo-fused cyclic ketones and showed good to high activities and enantioselectivities. Particularly, CgKR1 variants displayed high activities (90.6%-98.4% conversions) and enantioselectivities (>99.9% ee) towards 5a, a key intermediate of ladostigil (TV3326). Based on these results, a chemoenzymatic synthesis of (S)-5b was developed by using biocatalytic asymmetric reduction as a key step, giving the product with a total yield of 34.0% and 99.9% ee.

Full text of DOI:10.1039/c7ob01803g

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Organic & Biomolecular Chemistry
Enantioselective bioreduction of benzo-fused cyclic ketones with engineered
Candida glabrata ketoreductase 1 - A promising synthetic route to ladostigil
(TV3326)
Jingping Ou-yang,a Wenhe Zhang,b Fengyu Qin,b Weiguo Zuo,a Shaoyu Xu,a Yan Wang,b Bin Qin,c
Song You*b and Xian Jia*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Biocatalysis is recently emerging as a promising alternative to traditional chemical synthesis, because of its “green”
characteristics and comparable selectivities, which accord with the concept of sustainable development and demand for
asymmetric synthesis. In this study, whole-cell biocatalysts containing glucose dehydrogenase (GDH) and Candida glabrata
ketoreductases 1 (CgKR1) variants were constructed. These biocatalysts were applied to the reduction of benzo-fused
cyclic ketones and showed good to high activities and enantioselectivities. Particularly, CgKR1 variants displayed high
activities (90.6%-98.4% conversions) and enantioselectivities (＞99.9% ee) towards 5a, a key intermediate of ladostigil
(TV3326). Based on these results, a chemoenzymatic synthesis of (S)-5b was developed by using biocatalytic asymmetric
reduction as a key step, giving the product with a total yield 34.0% and 99.9% ee.
www.rsc.org/
this study (Figure 1). Among them, (S)-5b could be diversified into
corresponding (R)-amines, which could be used in the synthesis of
anti-Alzheimer’s drug ladostigil (TV3326).
Introduction
Enantiomerically pure alcohols are versatile building blocks in the
synthesis of pharmaceuticals and agricultural chemicals. Compared
with traditional metallo- and organocatalysis in chemical synthesis,
biocatalytic reduction of prochiral ketones using ketoreductases to
obtain chiral alcohols has been proved to be a practical and
environmentally friendly alternative1, because of several
advantages such as mild and environmentally benign reaction
conditions, high chemo-, regio- and stereo-selectivities. Therefore,
great efforts have been made to develop biocatalytic ketone
reduction processes in recent years.2
The indan structural motif can be found in many natural products,3
pharmaceuticals4 and functional materials.5 Recently we introduced
a chemoenzymatic route for synthesis of anti-Parkinson’s drug
rasagiline, in which the key intermediate (R)-2,3-dihydro-1-
indanamine was obtained through a dynamic kinetic resolution
(DKR) catalyzed by Candida antarctica lipase B (CALB) and a Pd
nanocatalyst.6 Indanones, which have the same indan structure,
have been emerged as substrates for reductatses.7 Given the fact
that the indanol motifs are synthetically useful,8 we became
interested in developing a novel process for the efficient
Figure 1. Asymmetric reduction of ketones 1a-5a.
For synthesis of ladostigil, several approaches have been reported.
In Chorev’s work, the costly (R)-1-aminoindan was used as starting
material. However, several steps in the synthetic route displayed
low yield, resulting in the waste of chiral materials (Figure 2-a).9
Later, Nudelman and coworkers reported a simple route, in which
the final product was obtained by resolution of racemic mixtures
(Figure 2-b).10 In the patent reported by Boulton et al, asymmetric
transfer hydrogenation (ATH) by Ru-Ts-DPEN was used to prepare
5b (Figure 2-c). A good yield and high enantioselectivity (up to 99%
ee) were obtained in ATH.11 Besides, (S)-5b could also be obtained
by using Ru–Cs-DPEN/surfactant system with 82% yield and 98% ee
in Luo et al.’s study.12 Ladostigil was then obtained via a
construction of these key motifs from readily available precursors.
Therefore, chiral indanols 1b-5b were selected as target products in
a.Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang
Pharmaceutical University), Ministry of Education, Shenyang 110016, China. E-
mail: jiaxian206@163.com. Tel: +86-24-13998835380
b.School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe, Shenyang 110016, China. E-mail:
yousong206@aliyun.com.
c. Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang
110016, China
nucleophilic substitution reaction. Because of the ever more
stringent regulatory restrictions on the level of metals allowed in
pharmaceutical products,12-13 the residual metal in the products
Electronic Supplementary Information (ESI) available: Experimental and spectral
(1H-NMR, MS and chiral HPLC) data. See DOI: 10.1039/x0xx00000x
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originating from the metal catalyst still presents a challenge.
Moreover, a series of novel dual inhibitors of AChE and MAO
derived from hydroxy aminoindan was reported, which possess
similar structure to ladostigil and could be taken as reference in our
synthetic work.14
Figure 2. Synthetic routes to ladostigil: a) Chorev’s synthetic route; b) Nudelman’s synthetic route; c) the method described in patent US
20060199974 A1.
In our previous studies, we have found that Candida glabrata
ketoreductase 1 (CgKR1) displays high stereoselectivity towards
substituted acetophenones.15 Moreover, the variants of CgKR1 also
displayed high activities and selectivities toward α halo ketones.16
In this study, eight CgKR1 mutants were employed for the reduction
of prochiral 1-indanone derivatives and several of them displayed
high stereoselectivities towards 6-hydroxy-2,3-dihydro-1H-indan-1-
one (2a) and N-ethyl-N-methyl-2-(3-oxo-2,3-dihydro-1H-indan-5-
yl)acetamide (5a). Further research on the chemoenzymatic
synthesis of key chiral intermediate (S)-5b of ladostigil was also
carried out, in order to explore the possibility of this method as an
alternative to chemical synthesis.
Figure 3. Docking of 1a into the binding pocket of WT CgKR1.
Results and discussion
Residues Phe92, Tyr208, Met144, and Phe145 were selected for
As reported in our previous study, CgKR1 wildtype displayed
mutation in this study.
moderate activity (65% conversion) towards 1-indanone (1a) and
gave (S)-1-indanol with high enantioselectivity (98% ee).15 To get a
To simplify the operation procedure and to maintain the enzymatic
better sense of substrate recognition of CgKR1, 1a was docked into
activity during the reduction, whole-cell biocatalysts were then
the modeled structure of CgKR1, which is reasonably designed
constructed by co-expression of CgKR1 variants and glucose
based on the crystal structure of NADPH-complex Gre2 (PDB ID:
4PVD),16 using AutoDock Vina program.17 As shown in Figure 3,
dehydrogenase (GDH, for NADPH regeneration) from Bacillus
subtilis. Asymmetric reduction of ketones (1a-5a) by these whole-
when 1a is in the appropriate position for catalysis, the phenyl
cell biocatalysts was shaken at 200 rpm using 10%
group of 1a is in the large binding pocket, which contains a bulky
dimethylsulfoxide (DMSO) as co-solvent at 50 mM substrate
residue Tyr208, while the cyclopentanone part is in the small
concentration, in which 7 equivalent glucose was added in order to
binding pocket, in which there is a bulky Phe92 hampering large
shift the reaction equilibrium toward the right (Figure 4.). As shown
groups from an effective binding. Further analysis of the docking
in Table 1, all variants catalyzed the asymmetric reduction of 1a
model suggests that the space in the large binding pocket might be
following the anti-Prolog’s rule, yielding (R)-1b with 12.6%-71.4%
reshaped by mutations of surrounding residues Met144 and
ee, which is opposite in the case of WT CgKR1, indicating that the
Phe145. Thus, four residues, Phe92, Tyr208, Met144, and Phe145,
benzyl group of 1a is now placed in the small binding pocket
were picked up and mutated to smaller residues to reshape and
created by Phe92Ala or Phe92Val substitution. This result indicated
enlarge the potential binding pocket for the five selected substrates
that a suitable substituent on the phenyl ring might lead to the
(Figure 3).
adoption of facing Re-site of other indanone derivatives to NADPH.
2 | Org. Biomol. Chem., 2017, 00, 1-3
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substrates. Meanwhile, the substitution at position 6 of 5a is big
enough, which forces Re-site of 5a to face NADPH (Figure 5-b).
Furthermore, the F92V variant displayed no reactivity towards 5a,
while the variants with mutations at Y208 showed good activities
and high enantioselectivities towards 5a. This result also proved
that mutations of Tyr208 is important for binding a large substrate.
Asymmetric reduction of 2a (6-hydroxyl substituted 1a) by these
eight variants displayed (S)-preference with 57.4%-96.4% ee. In case
of 6-methoxy-2,3-dihydro-1H-inden-1-one (3a), although high
activities were shown by seven variants (except F92V), low
stereoselectivities (8.8%-28.0% ee) were obtained. After the space
in the binding pocket was further increased by mutations at Met144
and Phe145, the ee value of (S)-3b was slightly enhanced from
13.9% (by F92V/Y208I variant) to 28.3% (by
Figure 4. Whole-cell biocatalysts containing GDH and CgKR1
variants.
To our delight, all variants except F92V displayed >99.9% ee
towards 5a, which is the key intermediate of ladostigil (TV3326),
yielding desired (S)-product with high conversion (90.6%-98.4%). To
throw some light on the excellent stereoselectivity in the
F92V/Y208I/M144V/F145V variant). The more Prelog
stereopreference of these variants towards 2a than 3a could be
attributed to the polarity of hydroxyl in 2a, since methoxyl has
bigger molecular volume than hydroxyl. Asymmetric reduction of
isatin (4a) catalyzed by all variants gave the product 4b with high
conversion (77.5%-99.7%), but no stereoselectivity. Although the
binding pocket has been further enlarged, variants at
asymmetric reduction, compound 1a was docked into the structure
of F92V variant and substrate 5a was docked into the structure of
F92V/Y208I variant. The structure of F92V and F92V/Y208I variants
were obtained in silico by mutations of WT enzyme in the target
position. The docking was performed using AutoDock Vina
program17 and related lowest-energy docked conformations are
shown in Figure 5. In the structure of F92V variant, the small
binding pocket is enlarged properly to accommodate the phenyl
ring of 1a, resulting in Si-site of 1a facing NADPH (Figure 5-a). Thus,
the (R)-1b was obtained as main product following anti-Prelog’s
rule. In the case of 5a docked into the structure of F92V/Y208I
variant, the space of both binding pockets are increased for large
F92/Y208/M144/F145 positions of CgKR1 didn’t show obvious
enhancements on the activity or enantioselectivity for the
reduction of 1a, 2a, 4a and 5a. These results suggested that
appropriate space is important for enzymes to conduct effective
catalysis.
Table 1. Asymmetric reduction of ketones 1a-5a by variants at F92, F92/Y208, and F92/Y208/M144/F145 positions of CgKR1.
S.
F92A
F92V
F92V/Y208I
F92V/Y208T
F92V/Y208I/M144L
/F145L
F92V/Y208I/M144
A/F145A
F92V/Y208I/M144
A/F145V
F92V/Y208I/M144V
/F145V
1a
2a
3a
98.4a, 48.3b(R)c
94.6, 94.8(S)
97.5, 8.8(S)
81.2, 71.4(R)
11.3, 57.4(S)
0, 0
99.0, 41.2(R)
94.6, 94.1(S)
96.6, 13.9(S)
99.2, 44.2(R)
94.1, 95.5(S)
96.6, 15.8(S)
98.9, 60.6(R)
96.7, 90.2(S)
97.3, 7.9(S)
98.5, 12.6(R)
92.3, 94.3(S)
98.6, 24.2(S)
98.6, 12.5(R)
94.9, 96.4(S)
97.6, 27.1(S)
99.4, 30.3(R)
93.4, 92.2(S)
98.5, 28.3(S)
4a
5a
95.3, 9.6d
77.5, 15.0
0, 0
94.7, 7.2
98.0, 6.3
99.7, 7.8
94.8, 7.8
95.7, 11.4
97.7, 7.5
98.4, >99.9(S)
98.1, >99.9(S)
96.9, >99.9(S)
98.2, >99.9(S)
90.6, >99.9(S)
93.0, >99.9(S)
96.6, >99.9(S)
a Conversion. bThe enantiomeric excess (ee) values of resultant alcohols (without purification), which were measured by chiral HPLC
analysis. cThe absolute configurations of resultant alcohols, which were identified by comparing the retention times of chiral HPLC with
authentic samples or literature data (Table S2. in the Supporting Information). dNo authentic samples of chiral HPLC analysis were found in
literature.
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Figure 5. a) Docking of 1a into the binding pocket of F92V variant. The small binding pocket is enlarged properly to accommodate the
phenyl ring. b) Docking of 5a into the binding pocket of F92V/Y208I variant. The big substitution in position 6 of 5a forces Re-site of 5a to
face NADPH.
Then we focused on the biocatalytic reduction process of 5a to
explore its potential of practical applications. First, we tried to
improve the substrate concentration. As shown in Table 1, F92A,
F92V/Y208I and F92V/Y208I/M144L/F145L variants, which
displayed good activity and selectivity at concentration of 50 mM
5a, were selected for reduction with higher substrate loading (Table
2). When the biocatalytic reduction was conducted at 100 mM
substrate concentration, the F92A, F92V/Y208I/M144L/F145L
variants gave (S)-5b with a conversion of 77.4% and 73.4%
respectively, whereas the F92V/Y208I variant gave the product with
88.4% conversion. In the subsequent test, the conversion fell to
55.5% when the substrate concentration increased to 150 mM.
Prolonging reaction time couldn’t improve the conversion, though
the stereoselectivity remained the same (＞99.9% ee). Then a
product inhibition test was carried out, and the result showed a
continuous decrease of conversion upon gradual increase of
additional (S)-5b (Figure S2.).
Second, the process was scaled up at gram levels to produce (S)-5b
with 81.4% yield and over 99.9% ee (detailed data are given in the
Supporting Information).
Figure 6. The chemoenzymatic synthetic route of the key
intermediate (S)-5b of ladostigil.
Based on above results, the enantiopure (S)-5b was synthesized in a
chemoenzymatic way started from p-methoxybenzaldehyde as
follows: reaction with Meldrum’s acid18 (HCO2H, Et3N) to give 3-(4-
methoxyphenyl)propanoic acid, which after being converted to the
acid chloride was cyclized to 3a.13 The latter was demethylated to
give 2a.19 Ketone 5a was prepared by reacting 2a with N-Ethyl-N-
methylcarbamic chloride20 in acetonitrile with potassium carbonate
as base.11 Finally, (S)-5b was prepared by bioreduction of 5a with an
overall yield of 34.0%, and ＞99.9% ee (Figure 6). The experimental
and spectral (1H-NMR, MS and chiral HPLC) data are included in the
Supporting Information.
Table 2. Asymmetric reduction of ketone 5a with high substrate
loading.
Variant
Concentration
(mM)
Time
(h)
Conversion
(%)
Yield
(%)
ee
(%)
100
150
200
100
150
200
100
150
200
9
77.4
43.6
39.1
88.4
55.5
47.9
73.4
49.3
46.6
55.4
42.1
36.6
72.3
51.3
49.6
52.4
45.2
44.4
＞99.9
＞99.9
＞99.9
＞99.9
＞99.9
＞99.9
＞99.9
＞99.9
＞99.9
11
13
9
F92A
11
13
9
Conclusions
F92V/Y208I
Biocatalysis has its intrinsic advantages over chemical
synthesis in a certain condition. In this study, through
mutations at residues Phe92, Tyr208, Met144, Phe145 of
CgKR1, which are important for the binding pocket,
engineered CgKR1s with high activity and stereoselectivity
towards benzo-fused cyclic ketones were obtained.
F92V/Y208I/
M144L/F145L
11
13
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Particularly, the whole-cell biocatalysts conducted by co-
expression of CgKR1 variants and GDH displayed excellent
stereoselectivity (>99.9% ee) towards N-ethyl-N-methyl-2-(3-
oxo-2,3-dihydro-1H-inden-5-yl)acetamide (5a), which is the
key intermediate of anti-Alzheimer’s drug ladostigil (TV3326).
Thus, a chemoenzymatic synthesis of enantiopure (S)-5b was
achieved via 5 steps with 34.0% overall yield starting from p-
methoxybenzaldehyde. Whole-cell biocatalysts suffered
substrate and product inhibitions at high substrate loading.
Due to this fact, further studies are needed to explore the
possibility of this chemoenzymatic method as an alternative to
chemical synthesis.
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Acknowledgements
This work was supported by the Science and Technology
Research Projects, Ministry of Education of the People's
Republic of China (213007A), Program for Liaoning Excellent
Talents in University (LR2014028), Young Elite Scientists
Sponsorship Program by CAST (2016QNRC001), National
Natural Science Foundation of China (81602993) and Project
from the Department of Education of Liaoning Province.
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5.
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01803G
ARTICLE
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T. Sun, Y. Zhang, Z. Cai and C. Zhu, CN 101580482B,
2009.
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