Deracemization of mexiletine biocatalyzed by ω-transaminases

Article abstract of DOI:10.1021/ol901834x

(S)- as well as (R)-mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral orally effective antiarrhythmic agent, was prepared by deracemizatlon starting from the commercially available racemic amine using ω-transaminases In up to >99% ee and conver

Full text of DOI:10.1021/ol901834x

ORGANIC
LETTERS
2009
Vol. 11, No. 21
4810-4812
Deracemization of Mexiletine
Biocatalyzed by ω-Transaminases
Dominik Koszelewski, Desiree Pressnitz, Dorina Clay, and Wolfgang Kroutil*
Department of Chemistry, Organic and Bioorganic Chemistry, Research Centre
Applied Biocatalysis, UniVersity of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
wolfgang.kroutil@uni-graz.at
Received August 7, 2009
ABSTRACT
(S)- as well as (R)-mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral orally effective antiarrhythmic agent, was prepared by
deracemization starting from the commercially available racemic amine using ω-transaminases in up to >99% ee and conversion with 97%
isolated yield by a one-pot two-step procedure. The absolute configuration could be easily switched to the other enantiomer, just by switching
the order of the applied transaminases. The cosubstrate pyruvate needed in the first oxidative step was recycled by using an amino acid
oxidase.
Mexiletine 1 is a therapeutically relevant chiral amine that
is clinically used as an antiarrhythmic, antimyotonic, and
analgesic oral drug in its racemic form.1-3 Activity studies
in vivo4 and in vitro5 of pharmacologically active mexiletine
indicate that its (R)-enantiomer binds preferentially to the
cardiac sodium channels. In addition, (R)-mexiletine is also
more active than (S)-mexiletine on native skeletal fibers.6
The use of mexiletine as a racemate in the treatment of
neuromuscular disorders is limited due to its possible side
effects.7 Therefore the development of methods to access
enantiomerically pure mexiletine is desirable.
Preparation of both enantiomers of mexiletine has previ-
ously been reported by several groups.8-14 Generally, the
methods involved resolution of racemic intermediates,
(1) (a) Koppe, H.; Zeile, K.; Kummer, W.; Stahle, H.; Danneberg, P.;U.S.
Patent 3,659,019, 1979. (b) Kalso, E.; Tramer, M. R.; McQuay, H. J.; Moore,
R. A. Eur. J. Pain 1998, 2, 3. (c) Cavalluzzi, M. M.; Catalano, A.; Bruno,
C.; Lovece, A.; Carocci, A.; Corbo, F.; Franchini, C.; Lentini, G.; Tortorella,
V. Tetrahedron: Asymmetry 2007, 18, 2409
.
(2) (a) De Luca, A.; Natuzzi, F.; Desaphy, J.-F.; Loni, G.; Lentini, G.;
Franchini, C.; Tortorella, V.; Conte Camerino, D. Mol. Pharmacol. 2000,
57, 268. (b) De Luca, A.; Natuzzi, F.; Duranti, A.; Lentini, G.; Franchini,
C.; Tortorella, V.; Conte Camerino, D. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 1997, 356, 777. (c) De Luca, A.; Pierno, S.; Natuzzi, F.;
Franchini, C.; Duranti, A.; Lentini, G.; Tortorella, V.; Jockush, H.; Conte
Camerino, D J. Pharmacol. Exp. Ther. 1997, 282, 93. (d) Fenster, P. E.;
(6) DeLuca, A.; Natuzzi, F.; Lentini, G.; Franchini, C.; Tortorella, V.;
Conte Camerino, D. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1995, 352,
653.
(7) Franchini, C.; Carocci, A.; Catalano, A. A.; Cavalluzzi, M. M.;
Corbo, F.; Lentini, G.; Scilimati, A.; Scilimati, A.; Tortorella, P.; Camerino,
D. C.; Luca, A. J. Med. Chem. 2003, 46, 5238.
Comes, K. A. Pharmacotherapy 1986, 6, 1
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(3) (a) Rudel, R.; Lehmann-Horn, F. Physiol. ReV. 1985, 65, 310. (b)
Cannon, S. C. Trends Neurosci. 1996, 19, 3. (c) Lehmann-Horn, F.; Rudel,
R. ReV. Physiol. Biochem. Pharmacol. 1996, 128, 195. (d) Jackson, C. E.;
(8) Turgeon, J.; Uprichard, A. C. G.; Belanger, P. M.; Harron, D. W. G.;
Grech-Belanger, O. J. Pharm. Pharmacol. 1991, 43, 630
(9) Franchini, C.; Cellucci, C.; Corbo, F.; Lentini, G.; Scilimati, A.;
Tortorella, V.; Stasi, F. Chirality 1994, 6, 590
(10) Phillips, G. T.; Shears, J. H. U.K. Patent GB 2246774 A1, 1992
(11) Gonzalez-Sabin, J.; Gotor, V.; Rebolledo, F. Tetrahedron: Asym-
metry 2000, 13, 1315
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Bason, R. J.; Ptacek, L. J. Muscle NerVe 1994, 17, 763
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10.1021/ol901834x CCC: $40.75
Published on Web 09/28/2009
 2009 American Chemical Society

enzymatic hydrolysis of an N-acyl derivative,10,11 or multi-
step stereospecific procedures.12 Flippin and co-workers13
reported a convenient procedure for the preparation of
stereoisomers of mexiletine, but the scope of products was
limited by the availability of chiral substrates and chromium
tricarbonyl complexes of aryl halides. Recently, optically
enriched (R)- and (S)-mexiletine was prepared by using
asymmetric reduction of oxime ethers and O-benzyl oximes
with borane-based catalysts.14
Scheme 1
.
Deracemization of rac-Mexiletine by a One-Pot
Two-Step Synthesis Procedure
Enzymes such as ω-transaminases have emerged as viable
biocatalysts for chiral amine preparation.15 ω-Transaminases
(ωTAs) can be used in two complementary ways, either (i)
in kinetic resolution of racemic amines with pyruvate as an
amine group acceptor or (ii) in the asymmetric synthesis from
ketones.16 We wish to report herein an deracemization
protocol for the synthesis of both enantiomers of mexiletine
employing ω-transaminases. Deracemization of amines via
dynamic kinetic resolution and cyclic deracemization are well
described.17 The deracemization protocol here is based on a
two-step one-pot process that consists of (i) a kinetic
resolution and (ii) a stereoselective amination employing
ω-transaminases (Scheme 1). Notably, the cosubstrate needed
in the first oxidation step (pyruvate) is recycled by an amino
acid oxidase18 (AAO). The advantage is that the formed
D-Ala or L-Ala in the kinetic resolution in the first step does
not interfere with the second step, where alanine of opposite
configuration is required. The deracemization approach
avoids the limitation of a kinetic resolution (50% of
conversion), thus leading to a theoretically quantitative yield
of optically pure amine starting with racemic amines by using
only a catalytic amount of amine acceptor pyruvate.
and from Vibrio fluVialis Vf-TA20) and four transaminases
described in the literature (from Bacillus megaterium BM-
ωTA,15e Alcaligenes denitrificans AD-ωTA,21 Chromobac-
terium Violaceum CV-ωTA,22 and a mutant termed CNB05-
01 originating from an Arthrobacter sp ArS-ωTA23) were
chosen as catalysts (Table 1). The four transaminases from
literature were used as a lyophilized whole-cell system
overexpressed in E. coli. The ω-transaminases catalyzed this
reaction efficiently giving the amine with up to >99% ee
with excellent enantioselectivity (E > 200). The important
point, however, was that transaminases ATA-113, Vf-ATA,
BM-ωTA, AD-ωTA, CV-ωTA, and ArS-ωTA showed (S)-
preference while ATA-117 displayed (R)-preference, thus
enantiocomplementary enzymes were available. This is
actually the precondition for the complete deracemization
sequence.
Testing first the kinetic resolution for racemic mexiletine,
three commercial ω-transaminases (ATA-113, ATA-117,19
In the kinetic resolution above a stoichiometric quantity
of pyruvate was applied, which could result in inhibition and
leads to accumulation of alanine. To avoid this, in situ
oxidation of alanine back to pyruvate by amino acid oxidase
(AAO) at the expense of molecular oxygen was considered.24
To find the most efficient system for the kinetic resolution
of rac-1 at 50 mM substrate concentration three enantio-
complementary amino acid oxidases (one D-AAO from
porcine kidney25 and two L-AAO from Crotalus adamanteus
and Crotalus atrox)26 were tested with the most interesting
transaminases employing only a catalytic amount of pyruvate
(2 mM; 4 mol %) (Table 2). For instance, rac-Mexiletine
was successfully resolved by ATA-117, giving the (S)-amine
with >99% ee at 53% conversion. When (S)-selective ω-TAs
(12) (a) Carocci, A.; Franchini, C.; Lentini, G.; Loiodice, F.; Tortorella,
V. Chirality 2000, 12, 103. (b) Carocci, A.; Catalano, A.; Corbo, F.; Duranti,
A.; Amoroso, R.; Franchini, C.; Lentini, G.; Tortorella, V. Tetrahedron:
Asymmetry 2000, 11, 3619.
(13) Loughhead, D. G.; Flippin, L. A.; Weikert, R. J. J. Org. Chem.
1999, 64, 3373.
(14) Huang, K.; Ortiz-Marciales, M.; Stepanenko, V.; De Jesus, M.;
Correa, W. J. Org. Chem. 2008, 73, 6928.
(15) (a) Hwang, B.-Y.; Cho, B.-K. H.; Koteshwar, Y. K.; Kim, B.-G. J.
Mol. Catal B: Enzym. 2005, 37, 47. (b) Kaulmann, U.; Smithies, K.; Smith,
M. E. B.; Hailes, H. C.; Ward, J. M. Enzyme Microbiol. Technol. 2007, 41,
628. (c) Yun, H.; Lim, S.; Cho, B.-K.; Kim, B.-G. Appl. EnViron. Microbiol.
2004, 70, 2529. (d) Ho¨hne, M.; Ku¨hl, S.; Robins, K.; Bornscheuer, U. T.
ChemBioChem. 2008, 9, 363. (e) Hanson, R. L.; Davis, B. L.; Chen, Y.;
Goldberg, S. L.; Parker, W. L.; Tully, T. P.; Montana, M. A.; Patel, R. N.
AdV. Synth. Catal. 2008, 350, 1367. (f) Koszelewski, D.; Clay, D.; Rozzell,
D.; Kroutil, W. Eur. J. Org. Chem. 2009, 2289.
(16) (a) Koszelewski, D.; Lavandera, I.; Clay, D.; Guebitz, G. M.;
Rozzell, D.; Kroutil, W. Angew. Chem., Int. Ed. 2008, 47, 9337. (b)
Koszelewski, D.; Lavandera, I.; Clay, D.; Rozzell, D.; Kroutil, W. AdV.
Synth. Catal. 2008, 350, 2761. (c) Truppo, M. D.; Rozzell, J. D.; Moore,
J. C.; Turner, N. J. Org. Biomol. Chem. 2009, 7, 395.
(20) Yun, H.; Cho, B.-K.; Kim, B.-G. Biotechnol. Bioeng. 2004, 87,
773.
(17) (a) Hoben, C. E.; Kanupp, L.; Ba¨ckvall, J.-E. Tetrahedron Lett.
2008, 49, 977. (b) Parvulescu, A. N.; De Vos, P. A. J. D. E. AdV. Synth.
Catal. 2008, 350, 113. (c) Parvulescu, A. N.; De Vos, P. A. J. D. E.
Chem.sEur. J. 2007, 13, 2034–2043. (d) Turner, N. J.; Carr, R. Biocatalysis
in the Pharmaceutical and Biotechnology Industries , Patel, R. N., Ed.;
CRC Press: Boca Raton, FL, 2007; p 743. (e) Thalen, L. K.; Zhao, D.;
Sortais, J.-B.; Paetzold, J.; Hoben, C.; Backvall, J.-E. Chem.sEur. J. 2009,
15, 3403.
(21) Yun, H.; Lim, S.; Cho, B.-K.; Kim, B.-G. Appl. EnViron. Microbiol.
2004, 70, 2529.
(22) Kaulmann, U.; Smithies, K.; Smith, M. E. B.; Hailes, H. C.; Ward,
J. M. Enzyme Microbiol. Technol. 2007, 41, 628.
(23) Pannuri, S.; Kamat, S. V.; Garcia, A. R. M. (Cambrex North
Brunswick Inc.) WO 2006/063336 A2, 2006 .
(24) Nakajima, N.; Esaki, N.; Soda, K. J. Chem. Soc., Chem. Commun.
1990, 947.
(18) Truppo, M. D.; Turner, N. J.; Rozzell, J. D. Chem. Commun. 2009,
2127.
(25) Pilone, M. S. Cell. Mol. Life Sci. 2000, 57, 1732.
(26) Servi, S.; Tessaroa, D.; Pedrocchi-Fantoni, G. Coord. Chem. ReV.
2008, 252, 715.
(19) ω-Transaminases 117 and 113 were purchased from Codexis Inc.
Org. Lett., Vol. 11, No. 21, 2009
4811

system and (ii) reduction to lactate, using lactate dehydro-
genase (LDH) (Scheme 1).16
Table 1. Kinetic Resolution of rac-1 Catalyzed by
ω-Transaminases Employing a Stoichiometric Amount of
Pyruvate
The reaction sequence was performed in the way that after
the kinetic resolution the second ω-transaminase with op-
posite stereopreference was added together with the corre-
sponding alanine enantiomer. To avoid interference from
using stereocomplementary ω-transaminases, a heat treatment
was performed prior to the second step. Thus after the kinetic
resolution, the sample was kept at 75 °C for 30 min, before
the enzyme required for the second step was added.
entry
ωTAa
convn (%)b
ee (%)c
Ed
1
2
3
4
5
6
7
117
113
Vf
BM
AD
CV
ArS
55
56
74
56
45
50
55
>99 (S)
>99 (R)
>99 (R)
>99 (R)
50 (R)
>99 (R)
>99 (R)
>200e
>200e
>200
>200
7
>200
>200
a Reaction conditions: rac- 1 (50 mM), sodium pyruvate (50 mM), and
ω-transaminase (6 mg commercial enzyme preparation or 20 mg of E. coli/
transaminase cells), phosphate buffer (100 mM, pH 7.0, 1 mM PLP), shaking
at 30 °C for 24 h. b Determined by GC. c Determined by GC on a chiral
phase. d Enantioselectivity was calculated from ee and conversion.
e Comparable results have been reported previously.15f
Table 3. Synthesis of Optically Pure Mexiletine via
Bioderacemization Protocol Employing ω-Transaminases
a
entry
ωTA1/ωTA2
convn (%)b
ee (%)c
1
2
3
4
5
6
117/CV
117/BM
117/113
117/Vf
CV/117
113/117
97
77
98
>99
>99
97
>99 (S)
95 (S)
>99 (S)
96 (S)
>99 (R)
>99 (R)
were used the opposite enantiomer of mexiletine was
obtained with up to >99% ee.
Having identified the suitable conditions for the kinetic
resolution of rac-mexiletine (1) we turned our attention to
couple the kinetic resolution with the stereoselective ami-
nation to achieve deracemization via a stepwise reaction
sequence without separation of intermediates.
a Order of adding ω-transaminase.; In the case of ATA-117 D-Ala was
used in the amination reaction and L-Ala for (S)-selective ω-TAs.
b Determined by GC. c Determined by GC analysis on a chiral phase.
Depending on the order of the ω-transaminases subjected,
the (R)- as well as the (S)-enantiomer was accessible with
up to >99% ee and excellent conversion >99% (Table 3).
Having optimized process conditions, a preparative trans-
formation of 100 mg of racemic mexiletine (1) at 28 mM
substrate concentration yielded (S)- or (R)-1 with complete
conversion after 48 h with >99% ee and 97% isolated yield.
In summary, a one-pot two-step deracemization sequence
leading to both enantiomers of optically pure mexiletine via
kinetic resolution combined with amino acid oxidase and
subsequent stereoselective amination catalyzed by enantio-
complementary ω-transaminases was described. For the
kinetic resolution only a catalytic amount of pyruvate was
required due to in situ recycling by the amino acid oxidase.
Table 2. Kinetic Resolution of rac-1 Catalyzed by a
ω-Transaminase/Amino Acid Oxidase System
entry
ωTAa
convn (%)b
ee (%)c
E
1
2
3
4
5
117
113
113d
Vf
53
55
40
48
51
>99 (S)
>99 (R)
66 (R)
97 (R)
>99 (R)
>200
>200
7.5
>200
>200
CV
a Reaction conditions: rac-1 (50 mM), sodium pyruvate (2 mM), L- or
D-AAO (15 mg, 4.5 U and 20 U, respectively), ω-TA (6 mg of crude enzyme
or 20 mg of E. coli/transaminase cells), phosphate buffer (100 mM, pH
7.0, 1 mM PLP), shaking at 30 °C for 24 h. b Determined by GC.
d
c Determined by GC analysis on a chiral phase. L-AAO from Crotalus
atrox (15 mg, 3 U).
Acknowledgment. Financial support by the FFG and the
Province of Styria is gratefully acknowledged. Codexis is
thanked for providing various enzymes.
Shifting the ketone-amine equilibrium to the amine side
is a challenge when employing ω-transaminases and
alanine.15d To shift the equilibrium the side product pyruvate
was removed by using two complementary systems: (i)
reduction by alanine dehydrogenase (AADH) and therefore
recycling of the cosubstrate alanine in a coupled reaction
Supporting Information Available: Experimental details
and achiral and chiral GC data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL901834X
4812
Org. Lett., Vol. 11, No. 21, 2009