Asymmetric Biocatalytic Amination of Ketones at the Expense of NH3 and Molecular Hydrogen

Article abstract of DOI:10.1021/acs.orglett.5b01154

A biocatalytic system is presented for the stereoselective amination of ketones at the expense of NH₃ and molecular hydrogen. By using a NAD⁺-reducing hydrogenase, an alanine dehydrogenase, and a suitable ω-transaminase, the R- as well as the S-enantiomer of various amines could be prepared with up to >99% ee and 98% conversion. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.orglett.5b01154

Letter
pubs.acs.org/OrgLett
Asymmetric Biocatalytic Amination of Ketones at the Expense of NH3
and Molecular Hydrogen
Anja K. Holzer,†,§ Katharina Hiebler,†,§ Francesco G. Mutti,†,⊥ Robert C. Simon,† Lars Lauterbach,‡
Oliver Lenz,‡ and Wolfgang Kroutil*,†
†Department of Chemistry, University of Graz, NAWI Graz, Heinrichstraße 28, 8010 Graz, Austria
‡Department of Chemistry, Technische Universitat Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
̈
S
* Supporting Information
ABSTRACT: A biocatalytic system is presented for the stereoselective amination of
ketones at the expense of NH3 and molecular hydrogen. By using a NAD+-reducing
hydrogenase, an alanine dehydrogenase, and a suitable ω-transaminase, the R- as
well as the S-enantiomer of various amines could be prepared with up to >99% ee
and 98% conversion.
Scheme 2. Biocatalytic System for the Asymmetric S-
Amination of Ketones Consuming NH3/H2 (*Switch in Cahn-
Ingold-Prelog Priority)
he spectrum of catalytic methods to prepare optically pure
1
Tα-chiral primary amines encompasses organo-,
metal-
organo-,2 and biocatalysts.3,4 Various biocatalytic methods using
ω-transaminases5,6 have been developed requiring different
amine sources7 such as 2-propylamine, amino acids (e.g.,
alanine), or some special donors.6a,e A biocatalytic system just
using ammonia as the amine source required the use of reducing
agents such as formate or glucose.8 Here an alternative option is
shown using molecular hydrogen as reducing agent (Scheme 1),
thereby improving the atom economy of the asymmetric
reductive amination in comparison to the NH3/glucose or
formate system.
Scheme 1. Asymmetric Bioamination of Ketones at the
Expense of NH3 and Molecular Hydrogen
To design a biocatalytic system for the asymmetric amination
of ketones at the expense of ammonia and hydrogen, alanine was
used as a direct amine donor for the ω-transaminase, leading to
the formation of pyruvate as the side product (Scheme 2). To
remove pyruvate from the equilibrium, it was recycled back to
alanine; this step is catalyzed by an alanine dehydrogenase that
consumes ammonia and reduced nicotinamide adenine
dinucleotide (NADH). The NADH is finally recycled by the
hydrogenase at the expense of molecular hydrogen. The O2-
tolerant NAD+-reducing hydrogenase from Ralstonia eutropha
H16 was employed as an efficient catalyst for the H2-driven
reduction of NAD+ to NADH.9
In contrast to commonly used cofactor regeneration systems,
H2-based NADH recycling by hydrogenase is 100% atom-
efficient and relies on a cheap, carbon-free reducing agent.9b In
contrast to most other hydrogenases, the R. eutropha enzyme is
O2-tolerant; that is, it sustains full catalytic activity even in the
presence 20% O2,9c which makes its utilization in cofactor
regeneration particularly convenient.
The L-alanine dehydrogenase (L-AlaDH) from Bacillus
subtilis10 was used for the recycling of L-alanine from pyruvate.
When various ω-transaminases in the system were tested with
the hydrogenase and the L-AlaDH for the amination of various
ketones 1a−g, the following S-selective transaminases turned out
to be the most suitable: For instance, the ω-transaminase from
Pseudomonas fluorescens (PF-ω-TA)10b,11 transformed ketone 1a
to the corresponding (S)-amine 2a with 86% conversion at 10
mM substrate concentration within 8 h (Table 1). The ω-
Received: April 20, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b01154
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Asymmetric S-Amination of Ketones 1a−g at the
Table 2. Biocatalytic R-Amination at the Expense of NH3 and
H2
a
a
Expense of NH3 and H2
b
c
d
b
c
d
substrate
ω-TA
conversion (%)
ee (%)
substrate
ω-TA
conversion (%)
ee (%)
1a
1b
1c
1d
1e
1f
Pf
86
96
90
92
98
76
77
92 (S)
93 (S)
>99 (S)
1a
1b
1c
1d
1e
1f
ArR
At
70
69
87
51
94
32
49
>99 (R)
>99 (R)
>99 (R)
Bf
Cv
Bm
Cv
Cv
Vf
ArR
ArR
ArR
ArR
ArR
e
e
>99 (R)
92 (S)
>99 (S)
>99 (R)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
1g
1g
a
a
Reaction conditions: substrate (10 mM), lyophilized Escherichia coli
Reaction conditions: Same as described in Table 1, but using D-
b
cells containing the overexpressed ω-TA (20 mg), K-phosphate buffer
(100 mM, pH 7.5), PLP (1 mM), NAD+ (1 mM), FMN (0.5 mM), L-
alanine (250 mM), AlaDH (12 U), NH4Cl (150 mM), hydrogenase
(1.81 U), 8 h, 30 °C, 120 rpm, 2 bar p(H2). ω-TA from Pf,
Pseudomonas fluorescens; Bm, Bacillus megaterium; Cv, Chromobacte-
alanine instead of L-alanine. ω-TA from ArR, (R)-transaminase from
Arthrobacter sp.; At, Aspergillus terreus. Conversions were measured by
GC on an achiral phase. Enantiomeric excess was determined by GC
c
d
b
on a chiral phase after derivatization to the corresponding acetamide.
e
Switch in Cahn-Ingold-Prelog priority.
c
rium violaceum; Vf, Vibrio fluvialis. Conversions were measured by GC
on an achiral phase. Enantiomeric excess was determined by GC on a
d
hydrogen (2 bar). The corresponding amine was isolated with
62% yield.
chiral phase after derivatization to the corresponding acetamide.
e
Switch in Cahn-Ingold-Prelog priority.
In conclusion, it was successfully shown that the asymmetric
amination of ketones can be performed with NH3 as the nitrogen
source and H2 as the reducing agent by employing a biocatalytic
system comprising a hydrogenase, an alanine dehydrogenase,
and a ω-transaminase. Conversion up to 98% was reached, giving
the corresponding amines with up to >99% ee.
transaminase from Chromobacterium violaceum (CV-ω-TA)12
transformed 1c and 1e with over 90% conversion and reached an
ee of >99% for 1c. Also, the transaminase from Bacillus
megaterium13 successfully converted 1d to the optically pure
chiral amine 2d with 92% conversion. Amine (S)-2g was
obtained with >99% ee by employing the ω-transaminase from
Vibrio fluvialis.14
The experiments were performed at 2 bar p(H2) as the
optimum because higher (3 bar H2) as well as lower pressure (1
bar H2) led to decreased conversion within the reaction time.
To apply the NH3/H2 system for the preparation of (R)-
amines, R-selective transaminases were investigated (Scheme 3).
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and characterization data. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.orglett.5b01154.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wolfgang.kroutil@uni-graz.at.
Present Address
Scheme 3. Biocatalytic System for the R-Amination of
Ketones at the Expense of NH3/H2 (*Switch in Cahn-Ingold-
Prelog Priority)
⊥F.G.M.: Manchester Institute of Biotechnology, Faculty of Life
Sciences, University of Manchester, Manchester M1 7DN, UK.
Author Contributions
§A.K.H. and K.H. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financed by the European Commission within
FP7 (Project: AmBioCas, Grant Agreement No. 245144,
THEME KBBE-2009-3-3-02), and the DFG EXC-314 “Unifying
Concepts in Catalysis” (to L.L. and O.L.). John Whittall
(University of Manchester) is thanked for all his efforts within
AmBioCas and other projects. COST Action CM1303 “Systems
Biocatalysis” and support by NAWI Graz are acknowledged.
Since these enzymes accept D-alanine as a direct amine donor,
ideally, a D-alanine dehydrogenase would be required. Since no
suitable enzyme was at hand, the formed pyruvate was instead
removed to L-alanine by L-AlaDH. Alanine racemase(s) present
in the E. coli15 preparation of the ω-TAs allows the racemization
of alanine to a certain extent.16
Using the NH3/H2 biocatalytic system for the amination of
ketones 1a−g, all amines 2a−g were obtained in optically pure
form (>99% ee) using the R-selective ω-transaminase from either
Arthrobacter sp.17 or Aspergillus terreus18 (Table 2). Conversion
up to 94% was reached in the best case (entry 5).
REFERENCES
■
(1) Selected reviews and examples: (a) Muller, C. E.; Schreiner, P. R.
Angew. Chem., Int. Ed. 2011, 50, 6012−6042. (b) Weiner, B.; Szyman
̈
ski,
W.; Janssen, D. B.; Minnaard, A. J.; Feringa, B. L. Chem. Soc. Rev. 2010,
39, 1656−1691. (c) Bertelsen, S.; Jørgensen, K. A. Chem. Soc. Rev. 2009,
38, 2178−2189. (d) Huang, K.; Ortiz-Marciales, M.; Stepanenko, V.; De
Jesus, M.; Correa, W. J. Org. Chem. 2008, 73, 6928−6931.
́
A preparative transformation of ketone 1a (50 mg) with the ω-
TA from P. fluorescens reached 97% conversion within 8 h under
(2) Selected examples: (a) Guan, Z. H.; Huang, K.; Yu, S.; Zhang, X.
Org. Lett. 2009, 11, 481−483. (b) Steinhuebel, D.; Sun, Y.; Matsumura,
B
DOI: 10.1021/acs.orglett.5b01154
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
K.; Sayo, N.; Saito, T. J. Am. Chem. Soc. 2009, 131, 11316−11317.
(c) Milczek, E.; Boudet, N.; Blakey, S. Angew. Chem., Int. Ed. 2008, 47,
6825−6828. (d) Ouellet, S. G.; Walji, A. M.; Macmillan, D. W. C. Acc.
Chem. Res. 2007, 40, 1327−1339.
(3) For reviews on biocatalytic methods, see: (a) Ghislieri, D.; Turner,
N. J. Top. Catal. 2014, 57, 284−300. (b) Kohls, H.; Steffen-Munsberg,
F.; Hohne, M. Curr. Opin. Chem. Biol. 2014, 19, 180−192. (c) Huisman,
̈
G. W.; Collier, S. J. Curr. Opin. Chem. Biol. 2013, 17, 284−292.
(d) Turner, N. J.; Truppo, M. Chiral Amine Synthesis: Methods. In
Developments and Applications; Nugent, T. C., Ed.; Wiley-VCH:
Weinheim, Germany, 2010; pp 431−459. (e) Hohne, M.;
̈
Bornscheuer, U. T. ChemCatChem 2009, 1, 42−51.
(4) For a review on chemical methods, see: (a) Nugent, T. C.; El-
Shazly, M. Adv. Synth. Catal. 2010, 352, 753−819.
(5) Selected recent reviews: (a) Simon, R. C.; Richter, N.; Busto, E.;
Kroutil, W. ACS Catal. 2013, 4, 129−143. (b) Kroutil, W.; Fischereder,
E.-M.; Fuchs, C. S.; Lechner, H.; Mutti, F. G.; Pressnitz, D.; Rajagopalan,
A.; Sattler, J. H.; Simon, R. C.; Siirola, E. Org. Process Res. Dev. 2013, 17,
751−759. (c) Mathew, S.; Yun, H. ACS Catal. 2012, 2, 993−1001.
(d) Malik, M. S.; Park, E.-S.; Shin, J.-S. Appl. Microbiol. Biotechnol. 2012,
94, 1163−1171. (e) Rudat, J.; Brucher, B. R.; Syldatk, C. AMB Express
2012, 2:11, 1−10.
(6) Selected recent examples: (a) Green, A. P.; Turner, N. J.; O’Reilly,
E. Angew. Chem., Int. Ed. 2014, 53, 10714−10717. (b) Limanto, J.;
Ashley, E. R.; Yin, J.; Beutner, G. L.; Grau, B. T.; Kassim, A. M.; Kim, M.
M.; Klapars, A.; Liu, Z.; Strotman, H. R.; Truppo, M. D. Org. Lett. 2014,
16, 2716−2719. (c) Andrade, L. H.; Kroutil, W.; Jamison, T. F. Org. Lett.
2014, 16, 6092−6095. (d) O’Reilly, E.; Iglesias, C.; Ghislieri, D.;
Hopwood, J.; Galman, J. L.; Lloyd, R. C.; Turner, N. J. Angew. Chem., Int.
Ed. 2014, 53, 2447−2450. (e) Richter, N.; Simon, R. C.; Kroutil, W.;
Ward, J. M.; Hailes, H. C. Chem. Commun. 2014, 50, 6098−6100.
(f) Shin, G.; Mathew, S.; Shon, M.; Kim, B.-G; Yun, H. Chem. Commun.
2013, 49, 8629−8631. (g) Wang, B.; Land, H.; Berglund, P. Chem.
Commun. 2013, 49, 161−163. (h) Tufvesson, P.; Lima-Ramos, J.;
Jensen, J. S.; Al-Haque, N.; Neto, W.; Woodley, J. M. Biotechnol. Bioeng.
2011, 108, 1479−1493.
(7) Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends
Biotechnol. 2010, 28, 324−332.
(8) (a) Koszelewski, D.; Lavandera, I.; Clay, D.; Guebitz, G. M.;
Rozzell, D.; Kroutil, W. Angew. Chem., Int. Ed. 2008, 47, 9337−9340.
(b) Truppo, M. D.; Rozzell, J. D.; Moore, J. C.; Turner, N. J. Org. Biomol.
Chem. 2009, 7, 395−398.
(9) (a) Schneider, K.; Schlegel, H. G. Biochim. Biophys. Acta 1976, 452,
66−80. (b) Lauterbach, L.; Lenz, O.; Vincent, K. A. FEBS J. 2013, 280,
3058−3068. (c) Lauterbach, L.; Lenz, L. J. Am. Chem. Soc. 2013, 135,
17897−17905.
(10) (a) Siranosian, K. J.; Ireton, K.; Grossman, A. D. J. Bacteriol. 1993,
175, 6789−6796. (b) Mutti, F. G.; Fuchs, C. S.; Pressnitz, D.; Turrini, N.
G.; Sattler, J. H.; Lerchner, A.; Skerra, A.; Kroutil, W. Eur. J. Org. Chem.
2012, 1003−1007.
(11) Kawano, S.; Ito, N.; Yasohara, Y. Kaneka Corp., Patent No. WO
2007/139055, 2007.
(12) Kaulmann, U.; Smithies, K.; Smith, M. E. B.; Hailes, H. C.; Ward,
J. M. Enzyme Microb. Technol. 2007, 41, 628−637.
(13) 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,
1367−1375.
(14) Shin, J.-S.; Yun, H.; Jang, J.-W.; Park, I.; Kim, B.-G. Appl. Microbiol.
Biotechnol. 2003, 61, 463−471.
(15) (a) Wang, E.; Walsh, C. Biochemistry 1978, 17, 1313−1321.
(b) Strych, U.; Benedik, M. J. J. Bacteriol. 2002, 184, 4321−4325.
(16) Richter, N.; Farnberger, J. E.; Pressnitz, D.; Lechner, H.; Zepeck,
F.; Kroutil, W. Green Chem. 2015, DOI: 10.1039/C4GC02363C.
(17) (a) Iwasaki, A.; Yamada, Y.; Ikenaka, Y.; Hasegawa, J. Biotechnol.
Lett. 2003, 25, 1843−1846. (b) Iwasaki, A.; Yamada, Y.; Kizaki, N.;
Ikenaka, Y.; Hasegawa, J. Appl. Microbiol. Biotechnol. 2006, 69, 499−505.
(18) Hohne, M.; Schatzle, S.; Jochens, H.; Robins, K.; Bornscheuer, U.
̈
̈
T. Nat. Chem. Biol. 2010, 6, 807−813.
C
DOI: 10.1021/acs.orglett.5b01154
Org. Lett. XXXX, XXX, XXX−XXX