Asymmetric β-Methylation of l- and d-α-Amino Acids by a Self-Contained Enzyme Cascade

Article abstract of DOI:10.1002/anie.201916025

This report describes a modular enzyme-catalyzed cascade reaction that transforms l- or d-α-amino acids to β-methyl-α-amino acids. In this process an α-amino acid transaminase, an α-keto acid methyltransferase, and a halide methyltransferase cooperate in two orthogonal reaction cycles that mediate product formation and regeneration of the cofactor pyridoxal-5′-phosphate and the co-substrate S-adenosylmethionine. The only stoichiometric reagents consumed in this process are the unprotected l- or d-α-amino acid and methyl iodide.

Full text of DOI:10.1002/anie.201916025

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Asymmetric β-methylation of L- and D-α-amino acids by a self-
contained enzyme cascade
Authors: Cangsong Liao and Florian P. Seebeck
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916025
Angew. Chem. 10.1002/ange.201916025
Link to VoR: http://dx.doi.org/10.1002/anie.201916025
http://dx.doi.org/10.1002/ange.201916025

10.1002/anie.201916025
Angewandte Chemie International Edition
Asymmetric β-methylation of L- and D-α-amino acids by a self-contained
enzyme cascade
Cangsong Liao1 and Florian P. Seebeck1 *
1Department for Chemistry, University of Basel, Mattenstrasse 24a, BPR 1002, 4056, Basel,
Switzerland
*To whom correspondence should be addressed: florian.seebeck@unibas.ch
Abstract. This report describes a modular enzyme-catalyzed cascade reaction that transforms L- or D-
a-amino acids to b-methyl-a-amino acids. In this process an a-amino acid transaminase, an a-keto acid
methyltransferase and a halide methyltransferase cooperate in two orthogonal reaction cycles that
mediate product formation and regeneration of the cofactor pyridoxal-5'-phosphate and the co-substrate
S-adenosylmethionine. The only stoichiometric reagents consumed in this process are the unprotected
L- or D-a-amino acid and methyl iodide.
α-keto acid
SAM
α-amino acid
TA
TA
iodide
MT
HMT
PLP
methyl
iodide
SAH
β-methyl
α-keto acid
β-methyl
α-amino acid
1
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
Introduction. The development of new ways to make molecules is a central objective in chemical
research. In the comparison of competing synthetic strategies, criteria such as substrate scope, reaction
specificity, conversion efficiency and reagent economy are critical.[1] The number of preparative steps
that require isolation of intermediates is a central parameter. Hence, integration of multiple elementary
steps into cascade reactions has emerged as a promising approach to shorten synthetic routes.[2]
Biocatalysis is particularly well suited for the design of cascading reactions.[3] Most enzymes have
evolved to participate in multistep pathways and to process their substrates at low steady-state
concentrations without accumulation of unstable intermediates.[4] However, other aspects that are also
related to the cellular origin of enzymes make their application in chemical synthesis challenging. In
particular, enzymes that catalyze group-transfer and redox reactions depend on co-substrates or
cofactors such as nicotinamides (NAD(P)), nucleoside triphosphates (ATP, GTP), acyl-Coenzyme A,
S-adenosylmethionine (SAM), pyridoxal 5-phosphate (PLP) or riboflavins (FMN, FAD). In a living cell
regeneration of these reagents is coupled to central metabolism. In order to exploit these enzymes in
vitro it is therefore necessary to engineer artificial minimal metabolisms that afford co-substrate
regeneration instead.
Pioneering work on oxidoreductase biocatalysis has shown that cofactor regeneration can indeed be
implemented to drive difficult transformations without the support of a living cell.[5] [3a] These systems
generally depend on cyclic regeneration of NAD(P)H either by consuming sacrificial hydrogen donors
or acceptors, or by shuttling hydrides from the substrate to the final product in a process known as
hydrogen-borrowing.[5a, 5c, 6] Emerging technologies that enable regeneration of ATP, acyl-CoA or SAM
indicate that this approach may be extended to ligations and group-transfer reactions.[7] Capitalizing on
this idea we have developed an enzyme-based process that enables stereoselective production of L- or
D-b-methyl-a-amino acids (β-Me-a-aas) from unprotected L- or D-a-amino acids and methyl iodide.
R
R
O
TA
H2N
COOH
COOH
SAM
iodide
MT
HMT
PLP
PMP
methyl
iodide
SAH
R
R
O
TA
H2N
COOH
COOH
Figure 1. General scheme for stereoselective production of L- and D-b-methyl-a-amino acids by cascade biocatalysis: i) a-
amino acids are oxidized to a-keto acids by PLP-dependent L- or D-a-amino acid transaminases (L-TA or D-TA); ii) α-keto
acids are methylated by SAM-dependent methyltransferases (MT); iii) b-methyl-α-keto acids are reduced to the corresponding
L- or D-b-methyl-a-amino acids by the PMP-containing TA. S-adenosylhomocysteine (SAH) is remethylated to SAM by a
halide methyltransferase (HMT).
2
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
Results and discussion. The design of this system was motivated by the considerable potential of β-
Me-a-aas as building blocks in bioactive compounds, and by the inherent difficulty associated with their
chemical synthesis. L-β-Me-a-aas are common constituents of natural products, including the clinical
antibiotic daptomycin (Supplementary Figure 1).[8] The value of L-β-Me-a-aas as building block in
bioactive compounds is documented by the dramatic effect – the magic methyl effect – of strategically
placed methyl groups on bioactivity and stability.[9] Despite this potential, application of β-Me-a-aas in
research and development has been limited, not the least because their stereoselective synthesis remains
challenging.[10] A more direct biocatalytic approach to produce β-methyl-tryptophan derivatives has
been reported.[11] However, because this process depends on tryptophan synthases for stereoselective
carbon-carbon bond formation, the substrate scope is limited to indole-containing L-a-amino acids.
The methodology described in the following is more general. In a way, this system is a bionic replica of
the natural biosynthesis of L-β-Me Leu.[12] First, the substrate a-amino acid is oxidized to the
corresponding α-keto acid by a PLP-dependent L-amino acid transaminase (L-TA, Figure 1). The α-
keto acid is methylated by a SAM-dependent a-keto acid methyltransferase (MT) forming a new
stereocenter with R-configuration. Finally, the methylated a-keto acid reclaims its amino group from
the pyridoxamine (PMP)-containing L-TA to form a L-β-Me-a-aa. Because the reaction does not
contain additional amine acceptors, the steady state concentration of the two a-keto acids is limited by
the initial concentration of PLP. As a consequence, the accumulating product is the stable β-Me-a-aa
instead of the β-methylated a-keto acid, which is prone to racemization.[7b] The third enzyme in the
cascade is a halide methyltransferase. This enzyme regenerates SAM by stereoselective S-methylation
of S-adenosylhomocysteine (SAH) using methyl iodide as methyl donor.[7b]
To realize this concept we produced the branched chain L-amino acids transaminase (IlvE) from E.coli,
the a-keto acid methyltranferase (SgvM) from Streptomyces griseoviridis,[12] and the halide
methyltransferase (HMT) from Burkholderia xenovorans in the SAH nucleosidase-deficient strain E.
coli Δmtn (DE3). The purified enzymes (40 µM IlvE, 40 µM SgvM and 20 µM HMT) were combined
with 2 mM of L-norvaline (1), 4 mM methyl iodide, 40 µM SAH, 40 µM PLP in a 100 mM phosphate
buffer (pH 8.0) and incubated at 25 oC. This reaction converted 52 % after 24 hours and 75 % after 48
hours of L-norvaline to L-allo-isoleucine (11) as inferred by 1H NMR spectroscopy (Table 1, entry 1).
The diastereomer L-isoleucine (11a) was detectable as a minor product of 2 %.
IlvE and SgvM are both characterized by significant substrate promiscuity and may therefor support the
synthesis of other L-β-Me-a-aas.[13] Indeed, the same enzyme composition converted of 89 % L-leucine
(2) to (2S, 3R)-3-methyl-leucine (12) (entry 2) with a diastereomeric ratio of 98:2. a-amino acids with
longer and branched alkyl chains (entries 3 and 6) or with alkenyl side chains (entries 4, 5 and 7) were
3
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
Scheme 1
Table 1. Substrates and products of the methylation cascade.a
Entry
Substrate
L-norvaline (1)
L-leucine (2)
TA
IlvE
IlvE
IlvE
IlvE
IlvE
MT
Product
11
12
13
14
Conversion
52 %
3R:3S
1
2
3
4
5
SgvM
SgvM
SgvM
SgvM
SgvM
98:2
98:2
95:5
93:7
96:4
89 %
80 %
83 %
87%
L-norleucine (3)
L-allylglycine (4)
L-4,5 dehydroleucine (5)
L-2-amino-5-methyl
hexanoic acid (6)
L-2-amino-5-methyl
Hex-4-enoic acid (7)
L-methionine (8)
L-cyclopropylalanine (9)
L-tryptophan (10)
D-norvaline (21)
D-leucine (22)
D-norleucine (23)
D-allylglycine (24)
D-2-amino-5-methyl
hexanoic acid (25)
D-methionine (26)
D-cyclopropylalanine
(27)
15
6
7
IlvE
IlvE
SgvM
SgvM
16
17
82 %
70 %
98:2
99:1
8
9
IlvE
IlvE
SgvM
SgvM
MarI
SgvM
SgvM
SgvM
SgvM
18
19
20
28
29
30
31
47 %
96 %
>95 %
50 %
39 %
90 %
99:1
99:1
92:8
98:2
99:1
95:5
92:8
10
11
12
13
14
MarG
D-TA
D-TA
D-TA
D-TA
> 95 %
15
16
17
D-TA
D-TA
D-TA
SgvM
SgvM
SgvM
32
33
34
89 %
82 %
91:9
95:5
98:2
> 95 %
D-cyclopropylalanine
(27)
D-cyclopropylalanine
(27)
18b
19b
D-TA
D-TA
SgvM
SgvM
35
36
> 95 %
> 95 %
98:2
98:2
a All entries correspond to 2 ml reactions containing 4 µmol of substrate in 100 mM phosphate buffer, at pH 8.0 and at 25 °C.
All proteins were purified from E. coli Δmtn (DE3) over Ni(II)-NTA agarose. The identity of the product, and the conversion
efficiency and stereoselectivity of the reaction were inferred by 1H-NMR, 13C-NMR, 2D NMR and high-resolution electrospray
ionization mass spectrometry (HRESIMS, Supplementary figures 3 – 41).b Isotope labeled methyl iodide (13CH3I or CD3I )
was used to produce compounds 35 and 36.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
also methylated with similar efficiency and stereoselectivity. Notably, compound 17 is a naturally
occurring building block in cyclomarins, a class of anti-inflammatory cyclic peptides.[14] L-methionine
(8) was also accepted as a substrate (entry 8) and methylated to produce 18. The comparatively bulky
L-cyclopropylalanine (9) was converted to product with outstanding efficiency (96 % conversion) and
stereoselectivity (99:1, entry 9). To demonstrate that this cascade can be used for preparative synthesis,
we scaled this reaction to 200 ml from which we isolated 0.33 mmol of 19 (47 mg, 82% yield). One-
step purification by ion exchange chromatography was facilitated by the completeness of the reaction,
the high stereoselectivity and the simplicity of the reaction mixture. By comparison, a synthetic scheme
connecting 9 to 19 using traditional synthesis requires at least five steps, including protection,
installation of a directing group, stereoselective methylation of the protected a-amino acid and
deprotection.[10a]
Because of the modular architecture of this cascade reaction, its substrate scope could be expanded
simply by using TAs and a-keto acid methyltranferases with different substrate specificities. These
enzymes may be engineered variants of IlvE and SgvM,[15] or naturally evolved homologs. Enzymes
known for their involvement in the biosynthesis of other L-β-Me-a-aas such as L-β-Me-Glu, L-β-Me-
Trp, L-β-Me-Arg and L-β-Me-Phe provide an obvious starting point for diversification.[8b, 8c, 16] As an
illustration, we tested the ability of the Trp-specific transaminase MarG and the indole-3-pyruvate-
specific methyltransferase MarI to produce L-β-Me-Trp (20) in the context of our in vitro cascade.[8b]
Indeed, a cascade reaction containing these two enzymes in combination with HMT, SAH, PLP and
methyl iodide in phosphate buffer converted over 95 % of Trp to 20 (entry 10).
b-methylated D-a-amino acids (D-β-Me-a-aa) have not yet been observed as components of natural
products but may be used to optimize and diversify existing therapeutics. Therefore we surmised that
our technology could be used to produce some of these unexplored compounds. In the first step of the
cascade reaction described above the L-a-amino acid is deaminated to an achiral a-keto acid. The
stereoinformation, along with the amino group and the reduction equivalent is temporarily stored in the
transaminase, while the methyltranferase acts on an achiral substrate. Hence, replacing the L-TA with a
D-TA should allow the cascade to convert D-amino acids to D-β-Me-a-aas. To test this idea, we
produced a D-TA with a substrate scope that nearly mirrors that of IlvE.[17] The cascade containing D-
TA, SgvM, HMT, PLP, SAH and methyl iodide converted D-norvaline (21) and D-leucine (22) to
compounds 28 and 29 with moderate efficiency (entries 11 and 12) but with excellent stereoselectivity.
Residues with more bulky side chains were methylated with significantly higher efficiency (entry 13 -
17). Finally, as a demonstration that this methylation cascade could be used to produce isotope-labeled
13
2
β-Me-a-aa from comparatively cheap reagents we also produced C- and H-isotopologs of 34 using
13CH3I or CD3I as methylation agents (entries 18 and 19).
5
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
Conclusions. We have developed an enzyme-catalyzed one-pot process for stereoselective production
of L- or D-β-Me-a-aa. The high methyl-transfer potential of methyl iodide provides the free energy
required to drive this transformation to near completion. The completeness of the reaction, the high
stereoselectivity and the simplicity of the reaction mixture facilitates purification of the products. The
modularity of this system, compounded with the diversity of known D-TAs and L-TAs and the growing
abilities to modify the substrate specificity of enzymes by engineering bode well for the general
applicability of this method for the synthesis of β-Me-a-aa. The use of methyl iodide as a reagent is an
inherent weakness of this methodology. The toxicity and volatility of this reagent may complicate scale-
up efforts. However, it is important to note that the limited choices of methylation agents is a general
problem in organic chemistry. We are optimistic that future developments will eliminate this problem
by introducing more benign reagents to methylation biocatalysis.
Acknowledgements.
L.C. was a recipient of a SSSTC & CSC postdoctoral fellowship; F.P.S. is supported by the “Professur
für Molekulare Bionik”. This project was supported by a Proof of Concept grant from the European
Research Council (ERC-2018-PoC) and the “Innovationsraum Biokatalyse”.
Author contribution.
C.L. and F.P.S. contributed to this paper as follows: original conception and planning (F.P.S & C.L.);
empirical work (C.L.); data analysis and interpretation (C.L. & F.P.S.); writing of the manuscript (F.P.S
& C.L.).
6
This article is protected by copyright. All rights reserved.

10.1002/anie.201916025
Angewandte Chemie International Edition
References
[1]
[2]
[3]
aB. M. Trost, Science 1991, 254, 1471 - 1477; bP. A. Wender, B. L. Miller, Nature 2009, 460,
197 - 201.
aK. C. Nicolaou, D. J. Edmonds, P. G. Bulger, Angew. Chem. Int. Ed. Engl. 2006, 45, 7134 - 7186;
bC. Grondal, M. Jeanty, ., D. Enders, Nat. Chem. 2010, 2, 167 - 178.
aJ. H. Schrittwieser, S. Velikogne, M. Hall, W. Kroutil, Chem. Rev. 2018, 118, 270 - 348; bJ. M.
Sperl, V. Sieber, ACS Catal. 2018, 8, 2385 - 2396; cS. P. France, L. J. Hepworth, N. J. Turner, S.
L. Flitsch, ACS Catal. 2017, 7, 710 - 724.
[4]
[5]
C. T. Walsh, B. S. Moore, Angew. Chem. Int. Ed. Engl. 2019, 58, 6846 - 6879.
aF. G. Mutti, T. Knaus, N. S. Scrutton, M. Breuer, N. J. Turner, Science 2015, 349, 1525-1529;
bZ. C. Litman, Y. Wang, H. Zhao, J. F. Hartwig, Nature 2018, 560, 355 - 359; cM. Frese, N.
Sewald, Angew. Chem. Int. Ed. Engl. 2015, 54, 298 - 301; dR. Skirgaila, S. Grazulis, D. Bozic, R.
Huber, V. Siksnys, J. Mol. Biol. 1998, 279, 473-481.
[6]
[7]
aA. Bornadel, R. Hatti-Kaul, F. Hollmann, S. Kara, ChemCatChem 2015, 7, 2442 - 2445; bX.
Wang, T. Saba, H. H. P. Yiu, R. F. Howe, J. A. Anderson, J. Shi, Chem 2017, 2, 621 - 654.
aS. Mordhorst, J. Siegrist, M. Mueller, M. Richter, J. N. Andexer, Angew. Chem. Int. Ed. Engl.
2017, 56, 4037 - 4041; bC. Liao, F. P. Seebeck, Nat. Catal. 2019, 2, 696 - 701; cW. D. Fessner,
C. Walter, Angew. Chem. Int. Ed. Engl. 1992, 31, 614 - 616; dA. Kameda, T. Shiba, Y. Kawazoe,
Y. Satoh, Y. Ihara, M. M., K. Ishige, T. Noguchi, J. Biosci. Bioeng. 2001, 91, 557 - 563; eH. Zhao,
W. A. van der Donk, Curr. Opin. Biotechnol. 2003.
[8]
[9]
aS. D. Braun, J. Hofmann, A. Wensing, M. S. Ullrich, H. Weingart, B. Volksch, D. Spiteller, Appl.
Environ. Microbiol. 2010, 76, 2500-2508; bY. Zou, Q. Fang, H. Yin, Z. Liang, D. Kong, L. Bai, Z.
Deng, S. Lin, Angew. Chem. Int. Ed. Engl. 2013, 52, 12951 - 12955; cC. Mahlert, F. Kopp, J.
Thirlway, J. Micklefield, M. A. Marahiel, J. Am. Chem. Soc. 2007, 129, 12011 - 12008; dY. T.
Huang, S. Y. Lyu, P. H. Chuang, N. S. Hsu, Y. S. Li, H. C. Chan, C. J. Huang, Y. C. Liu, C. J. Wu, W.
B. Yang, T. L. Li, Chembiochem 2009, 10, 2480-2487; eU. Alcolombri, S. Ben-Dor, E. Feldmesser,
Y. Levin, D. S. Tawfik, A. Vardi, Science 2015, 348, 1466 - 1469; fR. H. Baltz, Curr Opin Chem Biol
2009, 13, 144-151.
aB. Y. Azizeh, M. D. Shenderovich, D. Trivedi, G. Li, N. S. Sturm, V. J. Hruby, J. Med. Chem. 1996,
39, 2449-2455; bC. Haskell-Luevano, K. Toth, L. Boteju, C. Job, A. M. d. L. Castrucci, M. E.
Hadley, V. J. Hruby, J. Med. Chem. 1997, 40, 2740-2749; cF.-D. T. Lung, J. P. Meyer, B.-S. Lou,
L. Xiang, G. Li, P. Davis, I. A. De Leon, H. I. Yamamura, F. Porreca, V. J. Hruby, J. Med. Chem.
1996, 39, 2456-2460; dG. Tóth, E. Ioja, C. Tömböly, S. Ballet, D. Tourwé, A. Péter, T. Martinek,
N. N. Chung, P. W. Schiller, S. Benyhe, A. Borsodi, J. Med. Chem. 2007, 50, 328-333; eE. J.
Barreiro, A. E. Kummerle, C. A. M. Fraga, Chem. Rev. 2011, 111, 5215 - 5246; fH. Schönherr, T.
Cernak, Angew. Chem. Int. Ed. 2013, 52, 12256 - 12267.
[10]
[11]
aS. Y. Zhang, Q. Li, G. He, W. A. Nack, G. Chen, J. Am. Chem. Soc. 2013, 135, 12135 - 12141;
bG. He, B. Wang, W. A. Nack, G. Chen, Acc. Chem. Res. 2016, 49, 635 - 645.
aC. E. Boville, R. A. Scheele, P. Koch, S. Brinkmann-Chen, A. R. Buller, F. H. Arnold, Angew.
Chem. Int. Ed. Engl. 2018, 57, 14764-14768; bM. Herger, P. van Roye, D. K. Romney, S.
Brinkmann-Chen, A. R. Buller, F. H. Arnold, J. Am. Chem. Soc. 2016, 138, 8388-8391.
Y. Xie, B. Wang, J. Liu, J. Zhou, J. Ma, H. Huang, J. Ju, Chembiochem 2012, 13, 2745-2757.
aC. Sommer-Kamann, A. Fries, S. Mordhorst, J. N. Andexer, M. Muller, Angew. Chem. Int. Ed.
Engl. 2017, 56, 4033-4036; bX. Yu, X. Wang, P. C. Engel, FEBS J. 2014, 281, 391-400.
A. W. Schultz, D.-C. Oh, J. R. Carney, R. T. Williamson, D. W. Udwary, P. R. Jensen, S. J. Gould,
W. Fenical, B. S. Moore, J. Am. Chem. Soc. 2008, 130, 4507-4516.
aI. Slabu, J. L. Galman, R. C. Lloyd, N. J. Turner, ACS Catal. 2017, 7, 8263 - 8284; bG. Guo, P.
Berglund, Green Chem. 2017, 19, 333 - 360.
aZ. Hu, T. Awakawa, Z. Ma, I. Abe, Nat. Commun. 2019, 10, 184; bS. D. Braun, B. Volksch, J.
Nuske, D. Spiteller, Chembiochem 2008, 9, 1913-1920.
[12]
[13]
[14]
[15]
[16]
[17]
J. Kobayashi, Y. Shimizu, Y. Mutaguchi, K. Doi, T. Ohshima, J. Mol. Catal. B-Ezym 2013, 94, 15-
22.
7
This article is protected by copyright. All rights reserved.