A regio- and stereoselective ω-transaminase/monoamine oxidase cascade for the synthesis of chiral 2,5-disubstituted pyrrolidines

Article abstract of DOI:10.1002/anie.201309208

Biocatalytic approaches to the synthesis of optically pure chiral amines, starting from simple achiral building blocks, are highly desirable because such motifs are present in a wide variety of important natural products and pharmaceutical compounds. Herein, a novel one-pot ω-transaminase (TA)/monoamine oxidase (MAO-N) cascade process for the synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The reactions proceeded with excellent enantio- and diastereoselectivity (>94 % ee; >98 % de) and can be performed on a preparative scale. This methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the transaminase is not affected by the monoamine oxidase, and highlights the potential of this multienzyme cascade for the efficient synthesis of chiral building blocks. Mirror mirror on the wall: A ω-transaminase (ω-TA)/monoamine oxidase (MAO-N) cascade process for the asymmetric synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the TA reaction is not affected by the MAO-N catalyzed step. Copyright

Full text of DOI:10.1002/anie.201309208

Angewandte
Chemie
DOI: 10.1002/anie.201309208
Biocatalysis
A Regio- and Stereoselective w-Transaminase/Monoamine Oxidase
Cascade for the Synthesis of Chiral 2,5-Disubstituted Pyrrolidines**
Elaine OꢀReilly, Cesar Iglesias, Diego Ghislieri, Jennifer Hopwood, James L. Galman,
Richard C. Lloyd, and Nicholas J. Turner*
Abstract: Biocatalytic approaches to the synthesis of optically
pure chiral amines, starting from simple achiral building
blocks, are highly desirable because such motifs are present in
a wide variety of important natural products and pharmaceut-
ical compounds. Herein, a novel one-pot w-transaminase (TA)/
monoamine oxidase (MAO-N) cascade process for the syn-
thesis of chiral 2,5-disubstituted pyrrolidines is reported. The
reactions proceeded with excellent enantio- and diastereose-
lectivity (> 94% ee; > 98% de) and can be performed on
a preparative scale. This methodology exploits the comple-
mentary regio- and stereoselectivity displayed by both
enzymes, which ensures that the stereogenic center established
by the transaminase is not affected by the monoamine oxidase,
and highlights the potential of this multienzyme cascade for the
efficient synthesis of chiral building blocks.
reductive amination of prochiral ketones, thereby providing
the corresponding chiral amines.[3a–e] MAO-N catalyzes the
oxygen-dependent conversion of amines into imines and is
typically selective for the (S)-enantiomer.[3f–l] Variants of
MAO-N have been exploited for the deracemization of
primary, secondary, and tertiary amines with diverse struc-
tural motifs.[3a,f–j] The development of several chemoenzy-
matic routes[3c,4] to industrially important target molecules by
employing these two enzyme classes is testament to the
advances in protein engineering[1a,5] that have resulted in the
development of biocatalysts with the desired substrate scope,
selectivity, and stability.
2,5-Disubstituted pyrrolidines are important scaffolds in
pharmaceutical compounds[6] and natural products,[7] and
considerable efforts have been devoted to developing asym-
metric routes to both cis- and trans-disubstituted derivatives
that show moderate to good stereoselectivity.[8] The lack of
stereofacial bias induced by the preexisiting C2-stereocenter
means that obtaining the trans-diastereomers through reduc-
tion of the corresponding imine in high de is not straightfor-
ward. Our approach (Scheme 1) features a highly selective
T
he exquisite chemo-, regio- and stereoselectivity displayed
by enzymes has led to their widespread application as
catalysts for stereocontrolled organic synthesis.[1] These
properties, coupled with their ability to catalyze reactions
under similar conditions, has enabled the development of
elegant multienzyme cascade processes, in which the product
formed by the action of the first enzyme becomes the starting
material for the subsequent biotransformation.[2] Such
tandem processes alleviate the need for protecting-group
manipulations and intermediate purification steps, thus
providing cost-effective routes to target molecules.
Among the most synthetically useful biocatalysts for the
synthesis of chiral amines are the w-transaminase (TA) family
and variants of monoamine oxidase from Aspergillus niger
(MAO-N).[3] TAs are capable of mediating the selective
Scheme 1. A chemoenzymatic approach for the synthesis of 2,5-
disubstituted pyrrolidines by employing an w-transaminase (TA) and
a monoamine oxidase (MAO-N).
[*] Dr. E. O’Reilly, Dr. D. Ghislieri, Dr. J. Hopwood, Dr. J. L. Galman,
Prof. N. J. Turner
School of Chemistry, University of Manchester
Manchester Institute of Biotechnology
131 Princess Street, Manchester, M1 7DN (UK)
E-mail: nicholas.turner@manchester.ac.uk
TA-mediated reductive amination of an achiral 1,4-diketone
to generate an optically active pyrroline followed by dia-
stereoselective chemoenzymatic conversion into the corre-
sponding pyrrolidine by MAO-N/NH3·BH3.
Initially, we examined the w-TA-mediated selective
monoamination of commercially available 1,4-diketone 1a,
which bears a small methyl substituent and a large phenyl
substituent (Scheme 2). The first example of the asymmetric
bioamination of 1,5-diketones was recently reported with
excellent regio- and stereoselectivity achieved.[9] We found
the commercially available (S)-selective transaminase
ATA113 to be highly regioselective in mediating the reductive
C. Iglesias
Facultad de Quꢀmica, Universidad de la Repfflblica (Uruguay)
Dr. R. C. Lloyd
Chirotech Technology Ltd, 410 Cambridge Science Park
Milton Road, Cambridge, CB40PE (UK)
[**] The research leading to these results has received funding from the
European Union Seventh Framework Programme (FP7/2007-2013)
under grant agreements n8 266025 (BIONEXGEN) and n8 245144
(AMBIOCAS). Funding also provided by BBSRC (CASE award to JH)
and ANII MOV_CA_2012_1_7759 and CSIC-UdelaR-Uruguay.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201309208.
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
idines owing to poor diastereoselectivity in the reduction step.
We envisaged using MAO-N variants in combination with
NH3·BH3 for the asymmetric synthesis of 4a. Two MAO-N
variants (D5 and D9) were selected based on their known
activity and excellent selectivity towards structurally related
amine frameworks, including pyrrolidines and piperidines.[3h,k]
Our strategy relies upon MAO-N variants displaying com-
plete regio- and stereoselectivity to avoid stereorandomiza-
tion of the C2-stereocenter generated by the (S)-selective w-
TA. Imine 3a is in equilibrium with the open-chain amino
ketone (S)-2a and hence optimization of the MAO-N/
NH3·BH3 oxidation/reduction cycle was necessary in order
to prevent the formation of undesired amino alcohol as a side
product. Ketone reduction was minimized by lowering the
concentration of the MAO-N biocatalyst during the reaction
while maintaining a high concentration of NH3·BH3. Under
these conditions, rapid reduction of pyrroline 3a occurred,
thus ensuring that a minimal concentration of the amino
ketone was present during the biotransformation.
Treatment of (S)-3a with NH3·BH3 afforded 4a initially as
a mixture of diastereoisomers with a slight excess of the
(2S,5S)-isomer (de ꢀ 10%; Scheme 3a). Both MAO-N var-
iants mediated the oxidation of the (2S,5S)-4a diastereo-
isomer exclusively and displayed complete regioselectivity for
the more bulky phenyl side of the pyrrolidine. Following
successive rounds of selective oxidation with the MAO-N D5
variant and nonselective reduction with NH3·BH3, (2S,5R)-4a
was isolated in greater than 99% de. Despite a bias towards
the formation of the cis diastereoisomer upon reduction with
NH3·BH3, the combination with MAO-N yielded solely the
trans reduction product (2S,5R)-4a. The complementary
regioselectivity displayed by the w-TA and MAO-N variants
circumvents epimerization of the (S)-C2-center and provides
a method for accessing optically pure 2,5-pyrrolidines.
Having developed efficient individual biocatalytic routes
for the synthesis of optically pure pyrroline 3a and the target
chiral 2,5-pyrrolidine 4a, we next sought to combine the w-TA
and MAO-N biocatalysts in a one-pot cascade (Scheme 3c).
Diketone 1a was exposed to ATA 113 followed by MAO-N
and NH3·BH3, and the target (2S,5R)-4a was obtained in 82%
yield and > 99% de.
Scheme 2. Preparative-scale (25 mm 1a) reductive amination of di-
ketone 1a mediated by (S)-selective ATA113 or (R)-selective ATA117,
followed by spontaneous cyclization.
amination of 1a exclusively on the methyl ketone at
a substrate concentration of 25 mm with l-alanine as the
amine donor. The resulting 1,4-amino ketone (S)-2a subse-
quently underwent spontaneous cyclization to provide pyrro-
line (S)-3a in high yield (91%) and excellent ee (> 99%). The
lactate dehydrogenase (LDH)/glucose dehydrogenase
(GDH) system was used to drive the equilibrium towards
the product and recycle the NAD+ cofactor (see the
Supporting Information). The (R)-selective transaminase
ATA117 also catalyzed the regio- and stereoselective mono-
amination of 1a to afford pyrroline (R)-3a in 65% yield and
> 99% ee.
Having established an effective means of accessing
optically pure pyrrolines on a preparative scale, we subse-
quently explored a route for the diastereoselective synthesis
of 2,5-disubstituted pyrrolidine 4a starting from 3a
(Scheme 3). 2,6-Disubstituted piperidines have been pre-
pared through a chemoenzymatic route employing an w-
transaminase followed by diastereoselective hydrogenation
using Pd/C.[9] However, the same strategy is not applicable to
the diastereoselective synthesis of 2,5-disubstituted pyrrol-
To allow access to the (2R,5R)-4a diastereoisomer, the
(R)-3a enantiomer, derived from the use of ATA117, was
treated with the NH3·BH3/MAO-N combination (Scheme 3b).
Following nonselective reduction to give a mixture of
(2R,5S)-4a and (2R,5R)-4a, both MAO-N variants mediated
the selective oxidation of the (2R,5S)-isomer to provide
(2R,5R)-4a exclusively after successive rounds of oxidation/
reduction. The stereochemistry at C2 has a minimal effect on
the activity of the MAO-N enzyme and no effect on the
stereoselectivity; the target (2R,5R)-3a was isolated in > 99%
de.
The generality of the TA/MAO-N cascade process was
investigated by examining a series of diketones (1b–g) using
ATA113, as well as a novel transaminase (pf-ATA) from
Pseudogulbenkiania ferrooxidans[10] (Table 1). Pf-ATA shares
95% sequence identity with the transaminase from Chromo-
bacterium violaceum (cv-ATA; ATCC 12472).[11] ATA113
mediated the reductive amination of diketones 1b–g, the
Scheme 3. a,b) Analytical-scale synthesis of (2S,5R)- and (2R,5R)-4a
mediated by MAO-N D5. c) One-pot TA/MAO-N cascade for the
preparative-scale asymmetric synthesis of (2S,5R)-4a. Reduction of the
starting diketone by NH3·BH3 prevented the addition of all of the
reagents concurrently.
2
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Table 1: TA-mediated reductive amination of 1a–g.
Table 2: MAO/NH3·BH3-mediated asymmetric synthesis of (2S,5R)-
4a–g.
Entry
Substrate
w-TA
Conv. [%]
ee [%]
Entry
Substrate
MAO-N variant
de [%]
(2S,5R)
1
2
3
4
5
6
7
8
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
ATA113
P. ferrooxidans
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
60
>99 (S)
75 (S)
>99 (S)
>78 (S)
>99 (S)
68 (S)
>99 (S)
76 (S)
>99 (S)
78 (S)
1a
1b
1c
1d
1e
1 f
1
2
3
4
5
6
7
8
D5
D9
D5
D9
D5
D9
D5
D9
D5
D9
D5
D9
D5
D9
>99
96
88
3a
3b
3c
3d
3e
3 f
3g
98
>99
>99
>99
>99
68
>99
64
>99
56
9
10
11
12
13
14
9
96 (S)
76 (R)
94 (S)
46 (R)
10
11
12
13
14
>99
>99
75
1g
96
products of which spontaneously cyclized to yield pyrrolines
3b–g as the sole regioisomers with excellent conversion and
high ee values. Unsurprisingly, replacement of the small
methyl group by a larger ethyl substituent resulted in a slightly
reduced ee (entries 11 and 13). The biotransformations
performed with Pf-ATA proceeded with reduced selectivity,
with ee values lower than those achieved with ATA113.
Interestingly, replacing the methyl substituent by an ethyl
group resulted in a switch in stereoselectivity to give (R)-3 f,g
as the predominant enantiomers (entries 12 and 14, see the
Supporting Information for absolute configuration and ee
determination). We also compared the selectivity observed
with pf-ATA to that of the related cv-ATA against diketones
1a and 1d,e and noted comparable conversion and selectivity
(see the Supporting Information). The (R)-selective ATA117
also mediated the reductive amination of diketones 1a and
1d,e in > 99% conversion and ee (see the Supporting
Information).
The efficiency of the MAO-N/NH3·BH3 step with the
isolated pyrrolines 3b–g was next examined (Table 2). In
general, the D9 variant showed higher selectivity and employ-
ing either the D5 or D9 MAO-N variants allowed access to all
of the Me/Ar and Et/Ar substituted pyrrolidines in excellent
de. We have also extended the one-pot TA/MAO-N cascade
for the synthesis of (2S,5R)-4b, (2S,5R)-4d, and (2S,5R)-4e in
> 99% conversion and > 99% de when starting from the
corresponding diketones (Table 3), thus demonstrating the
generality of this one-pot approach.
Table 3: ATA113/MAO-N one-pot cascade for the synthesis of (2S,5R)-
4a, (2S,5R)-4b, (2S,5R)-4d, and (2S,5R)-4e.
Ketone
w-TA
MAO-N
Conv. [%]
de [%]
1a[a]
1b[b]
1d[b]
1e[b]
ATA113
ATA113
ATA113
ATA113
D5
D9
D9
D9
>99
>99
>99
>99
>99 (2S,5R)-4a
>99 (2S,5R)-4b
>99 (2S,5R)-4d
>99 (2S,5R)-4e
[a] 25 mm substrate; [b] 5 mm substrate.
corresponding pyrrolidine. The compatibility of the two
biocatalysts means that the reaction can be performed in
one pot without the need for costly intermediate purification
steps. The chemoenzymatic approach exploits four distinct
biocatalytic operations and takes advantage of the comple-
mentary regioselectivity displayed by the w-TA and MAO
variants to establish two stereogenic centers. All of the
biocatalysts described herein are commercially available[12]
and hence readily accessible for practical application.
Received: October 22, 2013
Revised: December 11, 2013
Published online: && &&, &&&&
Keywords: biocatalysis · cascade reactions ·
.
monoamine oxidase · pyrrolidines · transaminase
In summary, the combination of two complementary
biocatalysts has been demonstrated in a novel one-pot
chemoenzymatic cascade for the regio- and stereoselective
synthesis of a panel of 2,5-disubstituted pyrrolidines from the
corresponding 1,4-diketones. The transaminase w-TA is
highly selective for the sterically less demanding methyl
ketone while the monoamine oxidase MAO-N shows an
overwhelming preference for the more bulky portion of the
[1] a) M. T. Reetz, J. Am. Chem. Soc. 2013, 135, 12480 – 12496;
b) N. J. Turner, E. Oꢀ Reilly, Nat. Chem. Biol. 2013, 9, 285 – 288.
[2] a) J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti, W. Kroutil,
Curr. Opin. Chem. Biol. 2011, 15, 249 – 256; b) I. Oroz-Guinea,
E. Garcꢁa -Junceda, Curr. Opin. Chem. Biol. 2013, 17, 236 – 249;
c) E. Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal.
2011, 353, 2239 – 2262; d) J. H. Sattler, M. Fuchs, K. Tauber, F. G.
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
Mutti, K. Faber, J. Pfeffer, T. Haas, W. Kroutil, Angew. Chem.
2012, 124, 9290 – 9293; Angew. Chem. Int. Ed. 2012, 51, 9156 –
9159.
[5] a) U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz,
J. C. Moore, K. Robins, Nature 2012, 485, 185 – 194; b) N. J.
Turner, Nat. Chem. Biol. 2009, 5, 567 – 573.
[3] a) M. Hçhne, U. Bornscheuer, ChemCatChem 2009, 1, 42 – 51;
b) S. Mathew, H. Yun, ACS Catal. 2012, 2, 993 – 1001; c) C. K.
Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R.
Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N.
Devine, G. W. Huisman, G. J. Hughes, Science 2010, 329, 305 –
309; d) D. Koszelewski, K. Tauber, K. Faber, W. Kroutil, Trends
Biotechnol. 2010, 28, 324 – 332; e) L. Frodsham, M. Golden, S.
Hard, M. N. Kenworthy, D. J. Klauber, K. Leslie, C. Macleod,
R. E. Meadows, K. R. Mulholland, J. Reilly, C. Squire, S. Tomasi,
D. Watt, A. S. Wells, Org. Process Res. Dev. 2013, 17, 1123 – 1130;
f) M. Alexeeva, A. Enright, M. J. Dawson, M. Mahmoudian,
N. J. Turner, Angew. Chem. 2002, 114, 3309 – 3312; Angew.
Chem. Int. Ed. 2002, 41, 3177 – 3180; g) R. Carr, M. Alexeeva, A.
Enright, T. S. C. Eve, M. J. Dawson, N. J. Turner, Angew. Chem.
2003, 115, 4955 – 4958; Angew. Chem. Int. Ed. 2003, 42, 4807 –
4810; h) C. J. Dunsmore, R. Carr, T. Fleming, N. J. Turner, J. Am.
Chem. Soc. 2006, 128, 2224 – 2225; i) I. Rowles, K. J. Malone,
L. L. Etchells, S. C. Willies, N. J. Turner, ChemCatChem 2012, 4,
1259 – 1261; j) A. Znabet, M. M. Polak, E. Janssen, F. J. J.
de Kanter, N. J. Turner, R. V. A. Orru, E. Ruijter, Chem.
Commun. 2010, 46, 7918 – 7920; k) D. Ghislieri, A. P. Green,
M. Pontini, S. C. Willies, I. Rowles, A. Frank, G. Grogan, N. J.
Turner, J. Am. Chem. Soc. 2013, 135, 10863 – 10869; l) D.
Ghislieri, D. Houghton, A. P. Green, S. C. Willies, N. J. Turner,
ACS Catal. 2013, 3, 2869 – 2872.
[6] a) J. B. Brenneman, S. F. Martin, Org. Lett. 2004, 6, 1329 – 1331;
b) F. Xu, Org. Lett. 2013, 15, 1324 – 1345; c) S. Hanessian, M.
Bayrakdarian, X. Luo, J. Am. Chem. Soc. 2002, 124, 4716 – 4721.
[7] a) V. K. Aggarwal, C. J. Astle, M. Rogers-Evans, Org. Lett. 2004,
6, 1469 – 1471; b) S. Zhang, L. Xu, L. Miao, H. Shu, M. L.
Trudell, J. Org. Chem. 2007, 72, 3133 – 3136; c) A. Goti, S. Cicchi,
V. Mannucci, F. Cardona, F. Guarna, P. Marino, T. Tejero, Org.
Lett. 2003, 5, 4235 – 4238; d) B. M. Trost, D. B. Horne, M. J.
Woltering, Angew. Chem. 2003, 115, 6169 – 6172; Angew. Chem.
Int. Ed. 2003, 42, 5987 – 5990; e) E. A. Severino, C. R. D.
Correia, Org. Lett. 2000, 2, 3039 – 3042.
[8] a) F. A. Davis, M. Song, A. Augustine, J. Org. Chem. 2006, 71,
2779 – 2786; b) G. S. Lemen, J. P. Wolfe, Org. Lett. 2010, 12,
2322 – 2325; c) M. G. Moloney, T. Panchal, R. Pike, Org. Biomol.
Chem. 2006, 4, 3894 – 3897; d) C. Enkisch, C. Schneider, Eur. J.
Org. Chem. 2009, 5549 – 5564; e) K. R. Campos, A. Kalpars, J. H.
Waldman, P. G. Dormer, C.-y. Chen, J. Am. Chem. Soc. 2006,
128, 3538 – 3539; f) F. A. Davis, J. Zhang, H. Qui, Y. Wu, Org.
Lett. 2008, 10, 1433 – 1436.
[9] R. C. Simon, B. Grischek, F. Zepeck, A. Steinreiber, F. Belaj, W.
Kroutil, Angew. Chem. 2012, 124, 6817 – 6820; Angew. Chem. Int.
Ed. 2012, 51, 6713 – 6716.
[10] K. G. Byrne-Bailey, K. A. Weber, J. D. Coates, J. Bacteriol. 2012,
194, 2400 – 2401.
[11] U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes, J. M.
Ward, Enzyme Microb. Technol. 2007, 41, 628 – 637.
[12] a) MAO-N Screening Kits: http://www.discovery-bc.co.uk/
monoamineoxidase.php (2013); b) Transaminases: http://www.
codexis.com.
[4] a) T. Li, J. Liang, A. Ambrogelly, T. Brennan, G. Gloor, G.
Huisman, J. Lalonde, A. Lekhal, B. Mijts, S. Muley, L. Newman,
M. Tobin, G. Wong, A. Zaks, X. Zhang, J. Am. Chem. Soc. 2012,
134, 6467 – 6472; b) B. de Lange, D. J. Hyett, P. J. D. Maas, D.
Mink, F. B. J. van Assema, N. Sereinig, A. H. M. de Vries, J. G.
de Vries, ChemCatChem 2011, 3, 289 – 292.
4
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Biocatalysis
E. O’Reilly, C. Iglesias, D. Ghislieri,
J. Hopwood, J. L. Galman, R. C. Lloyd,
N. J. Turner*
&&& — &&&
A Regio- and Stereoselective w-
Transaminase/Monoamine Oxidase
Cascade for the Synthesis of Chiral 2,5-
Disubstituted Pyrrolidines
Mirror mirror on the wall: A w-trans-
aminase (w-TA)/monoamine oxidase
(MAO-N) cascade process for the asym-
metric synthesis of chiral 2,5-disubsti-
tuted pyrrolidines is reported. The meth-
odology exploits the complementary
regio- and stereoselectivity displayed by
both enzymes, which ensures that the
stereogenic center established by the TA
reaction is not affected by the MAO-N
catalyzed step.
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!