Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes

Article abstract of DOI:10.1002/anie.202014239

Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst-controlled pyrazole alkylation by a cyclic two-enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non-natural analogs of the common cosubstrate S-adenosyl-l-methionine. A second engineered enzyme transfers the alkyl group in highly selective C?N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme-library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole-alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.

Full text of DOI:10.1002/anie.202014239

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: Engineered enzymes enable selective N-alkylation of pyrazoles
with simple haloalkanes
Authors: Ludwig L. Bengel, Benjamin Aberle, Alexander-N. Egler-
Kemmerer, Samuel Kienzle, Bernhard Hauer, and Stephan
Christian Hammer
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202014239
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10.1002/anie.202014239
Angewandte Chemie International Edition
RESEARCH ARTICLE
Engineered enzymes enable selective N-alkylation of pyrazoles
with simple haloalkanes
Ludwig L. Bengel,[b] Benjamin Aberle,[b] Alexander-N. Egler-Kemmerer,[b] Samuel Kienzle,[b] Bernhard
Hauer[b] and Stephan C. Hammer*[b],[a]
[a]
Prof. Dr. S. C. Hammer
Faculty of Chemistry, Organic Chemistry and Biocatalysis
Bielefeld University
Universitätsstraße 25, 33615 Bielefeld (Germany)
E-mail: stephan.hammer@uni-bielefeld.de
[b]
L. L. Bengel, B. Aberle, A.-N. Egler-Kemmerer, S. Kienzle, Prof. Dr. B. Hauer
Department of Technical Biochemistry
Institute of Biochemistry and Technical Biochemistry, University of Stuttgart
Allmandring 31, 70569 Stuttgart (Germany)
Supporting information for this article is given via a link at the end of the document.
Abstract: Selective alkylation of pyrazoles could solve a challenge
in chemistry and streamline synthesis of important molecules. Here
we report catalyst-controlled pyrazole alkylation by a cyclic two
enzyme cascade. In this enzymatic system, a promiscuous enzyme
uses haloalkanes as precursors to generate non-natural analogs of
substrates including small molecules, peptides, proteins and
DNA. Enzymes achieve excellent selectivities in such
challenging reactions because their active sites offer a high level
of molecular recognition to accurately pre-organize substrates
prior to catalysis. A broad application of alkyl transferases in
synthesis is, however, hampered by the complexity of the used
alkyl donors.
the common cosubstrate S-adenosyl-L-methionine.
A second
engineered enzyme transfers the alkyl group in highly selective C–N
bond formations to the pyrazole substrate. The cosubstrate is
recycled and only used in catalytic amounts. Key is a computational
enzyme library design tool that converted
a
promiscuous
methyltransferase into a small enzyme family of pyrazole alkylating
enzymes in one round of mutagenesis and screening. With this
enzymatic system, pyrazole alkylation (methylation, ethylation,
propylation) was achieved with unprecedented regioselectivity
(>99%), regiodivergence and in a first example on preparative scale.
Introduction
The regioselective alkylation and functionalization of
molecules bearing multiple heteroatoms with similar properties is
a particular challenge in synthesis.[1] This is especially true for N-
heterocyclic compounds such as pyrazoles, triazoles or
pyridones where tautomerization leads to heteroatoms with
comparable reactivity. Alkylation of such N-heterocyclic
compounds is under substrate control and produces product
mixtures that are often laborious to separate (Fig. 1A and
Fig. S1).[2] Selective alkylation depends on protecting group
strategies[3] and a general catalyst-controlled alkylation does not
exist.[4,5] Given the significant number of C–N alkylations
conducted in medicinal chemistry[6,7] and based on the
importance of such N-alkylated heterocycles in biologically
active agents (Fig. S2),[8] it becomes clear why selective
alkylation methods are on top of many wish lists for organic
chemistry.[4,5,9] New methods that form such C–N bonds
selectively could shorten current synthetic routes and even
make new molecules accessible that were previously difficult to
prepare.
Figure 1. (A) Pyrazole structures can be described as two different tautomers.
Tautomerization results in N-atoms with very similar reactivity that are
challenging to discriminate in chemical reactions. (B) SAM-dependent
methyltransferases can transfer methyl groups with very high selectivity to
complex substrates. In this reactions S-adenosyl-L-methionine (SAM) serves
as cosubstrate and methyl donor. S-adenosyl-L-homocysteine (SAH) is
generated as byproduct and can be used by halide methyltransferases to
regenerate SAM using iodomethane as methyl source.[12] (C) The research
outlined here uses promiscuous halide methyltransferases to generate and
recycle non-natural SAM analogs (NSA) from simple haloalkanes. In
combination with engineered alkyl transferases highly selective pyrazole
alkylation is envisioned.
In nature, selective heteroatom alkylation is carried out by
various well-known enzymes with unprecedented precision and
activity.[10,11] Prenyl-, methyl- and glycosyltransferases assemble
C–heteroatom bonds with high selectivity on a large set of
1
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10.1002/anie.202014239
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RESEARCH ARTICLE
Figure 2. Engineering of NMT. (A) Promiscuous activity of homo sapiens nicotinamide N-methyltransferase (NMT) in the methylation of 3-methylpyrazole (1). (B)
Enzyme engineering of NMT was performed using FuncLib[13], a computational enzyme library design tool that calculates the sequence space of tolerated active
site mutations. An advantage to classical directed evolution approaches such as iterative saturation mutagenesis[14] is that FuncLib allows large and meaningful
steps in sequence space by introducing multiple active site mutations simultaneously. Fig. 2B is inspired by recently published, excellent reviews.[15,16] (C)
Structure of NMT (pdb 2iip): Active site amino acids that have been randomized using FuncLib are shown as colored sticks. Cocrystallized SAH is shown as black
sticks and the shape of the substrate binding pocket is illustrated as grey shadow.
Enzyme-catalyzed selective heteroatom alkylation largely
depends on alkyl donors with leaving groups that are
synthetically difficult to access, unstable and/or lead to low atom
economy in the overall reaction (Fig. S3). If we were able to
combine enzymatic alkylation with the utilization of simple and
readily available alkyl donors, solutions for many challenging
reactions could be envisioned.
time SAH is regenerated and is therefore only required in
catalytic concentrations to shuttle the methyl group between
both enzymes. Currently, there is very little evidence that this
system can be used beyond methylation to synthesize and
recycle NSA directly from simple haloalkanes (Fig. S4).
Here we report selective N-alkylation of pyrazoles by
engineered enzymes in a cyclic two enzyme cascade (Fig. 1C).
The method was implemented based on two major findings. First,
an identified promiscuous halide methyltransferase can
synthesize and recycle NSA using haloalkanes as sole
stoichiometric reagents. Second, a computational enzyme library
design tool[13] successfully transformed a second promiscuous
MT into a pyrazole-alkylating enzyme family in one round of
mutagenesis and screening. The enzyme system catalyzed
desired C–N bond formations with unprecedented selectivities
using simple haloalkanes as starting materials.
Recently, significant progress towards enzymatic alkylation
chemistry using “off the shelf” alkylation reagents has been
made,
in
particular
exploiting
SAM-dependent
methyltransferases (MTs). MTs use the cosubstrates S-
adenosyl-L-methionine (SAM) and a natural carboxy-analog[17] of
SAM in highly selective methylation and carboxymethylation
reactions. Multiple studies have further demonstrated that MTs
are functional with non-natural SAM analogs (NSA) to selectively
transfer a huge variety of alkyl groups.[11,18] However, the
synthesis of NSA is currently not particularly straight forward
(Fig. S4). Several elegant strategies for enzymatic synthesis of
NSA have been developed.[19–21] These approaches still depend
on the laboratory synthesis of methionine analogs as NSA
precursors that limits their application.[11,18,22] Liao and Seebeck
have recently shown that SAM can be enzymatically synthesized
and recycled (Fig. 1B).[12] In this cyclic enzyme cascade, a
halide methyltransferase charges S-adenosyl-L-homocysteine
(SAH) with iodomethane to generate SAM. A second MT uses
SAM to methylate a substrate with high selectivity. At the same
Results and Discussion
Enzyme
engineering
to
generate
pyrazole
methyltransferases
Because pyrazoles are extremely rare in nature[23]
,
pyrazole MTs are currently not known. As a result, we aimed to
access this enzyme function by engineering a promiscuous
2
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10.1002/anie.202014239
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RESEARCH ARTICLE
natural MT. In order to identify a suitable starting point for
enzyme engineering, we examined several MTs for promiscuity
towards pyrazole methylation using SAM as cosubstrate. In
particular, we studied the homo sapiens nicotinamide N-
tautomers differ only in the relative position of a small methyl
group (see Fig. 1A). High regioselectivities and in part
regiodivergence were also found for 3-substituted pyrazoles
bearing cyclopropyl (3) and (hetero)-aromatic groups (4 and 5)
as well as for pyrazoles with bulky substituents on the 4-position
(6) (see Fig. 3 and Fig. S15-S20 for details). Please note that
methyltransferase[24]
methyltransferase[25] and histamine N-methyltransferase[26,27]
,
phenylethanolamine
N-
due to their reported substrate flexibility. Out of these 3 enzymes, NMT wild-type (Fig. 3) as well as chemical methylations with
only the homo sapiens nicotinamide N-methyltransferase (NMT)
revealed promiscuous activity with 3-methylpyrazole (1) as
substrate (Fig. 2A). The respective products were formed with
14% as mixture of regioisomers (67:33 for 1a:1b). This
illustrates that wild-type NMT can hardly discriminate between
the two N-atoms of 1.
standard reagents (Fig. S1) generate product mixtures for all the
tested pyrazole substrates.[3,30] There are currently no catalysts
known that control pyrazole methylation or alkylation with such
precision.[4,5]
In addition to regiocontrol, we studied the activities of the
selected pyrazole MTs using stoichiometric amounts of SAM as
cosubstrate. Because MTs in general and the homo sapiens
nicotinamide N-methyltransferase in particular are known to
suffer from inhibition of the SAH by-product (Fig. 1B),[31,32] we
decided to validate activity on the bases of initial rate
We sought to improve the enzyme’s promiscuous activity
by using computational enzyme library design.[16] While
traditional directed evolution protocols optimize promiscuous
activity for one substrate towards one chosen product,[28] we
aimed to generate a whole panel of transferases with activity for
a broad range of substrates potentially accessing both product
isomers with high selectivity. To this end, we used a recently
developed algorithm by Sarel Fleishman and coworkers called
FuncLib.[13] This protein design method can efficiently calculate
the theoretically tolerated sequence space in enzyme active
sites.[28] Phylogenetic and atomistic calculations are combined to
analyze the stability of multiple active site mutations in silico. We
have chosen 12 active site amino acids of NMT to
computationally explore the stability of multiple mutations in the
active site. These 12 amino acids build the substrate binding
pocket but do not directly interact with the SAM cosubstrate
(Fig. 2C and Fig. S5). The FuncLib algorithm was applied as
described in the supporting information.
determination
using
uniform
substrate
and
enzyme
concentrations (Fig. 3). Activity comparison by initial rate
determination is most likely not biased by potential SAH
inhibition. In general, many designs showed activity
enhancements by a factor >10 compared to the NMT wild-type
(Fig. 3 and Fig. S21-S26). In some cases, very high increase in
activity up to a factor of 72 and 118 was found. This is
remarkable because the variants performed with excellent
selectivities at the same time. Such activity enhancements are
outstanding and usually not observed with conventional
mutagenesis strategies.[33] Iterative saturation mutagenesis or
random mutagenesis often show activity enhancements by a
factor of 2-4 per round of evolution.[28,34–37]
Six different enzyme variants (v17, v22, v28, v36, v40,
v49) with increased activity and selectivity for the six pyrazoles
substrates were studied in detail (Fig. 3). In these six designs, 7
out of the 12 target active site amino acids were mutated
(Fig. S6). Three positions (D167, S201, S213) were altered in all
of the six variants and two positions (A198, N249) were mutated
in ³50%. Interestingly, these five amino acids are spatially all
located next to each other (see top of the active site in Fig. 2C)
and might generate the binding pocket for the different
substituents on the pyrazole ring. These mutable amino acids
are part of the loops connecting the 4-5, 5-6 and 6-7 b-strands of
the methyltransferase Rossmann-fold.[38] The introduced
beneficial mutations at these five hot-spot positions are very
diverse (D167C/H, A198L/T, S201A/C/I/Q, S213A/C/H/M and
N249C/A) and general beneficial mutations for pyrazole
methylation cannot be identified. As recently reported, the
multiple mutations introduced by FuncLib often show non-
additive behavior.[13] Such epistasis explains large steps in
activity and selectivity, but makes it difficult to generalize the
mode of action. We believe that the single set of mutations in
each top variant enables selective binding of the pyrazole
substrate in a reactive conformation. This is in agreement with
literature highlighting that methyltransferases bind their
substrates in a near-attack conformation to achieve efficient
catalysis.[39]
In short, the phylogenetic analysis and mutational stability
calculations at these 12 amino acid positions reduced the active
site sequence space to 473.294 possible variants. This pool was
then further analyzed in silico by calculating the stability of the
designs bearing 3-5 active site mutations. The most stable
designs were clustered based on their sequence diversity
(cluster criterion: amino acid difference ³ 3). We decided to test
the top 50 sequences, all bearing 3-5 active site mutations and
differ in at least 3 residues (see Fig. S6). In this way, large and
potentially meaningful steps in the active site sequence space
can be experimentally investigated without depending on
ultrahigh-throughput screening methods[29] (Fig. 2B).
The enzyme panel (v1 – v50) was bought as synthetic
DNA, produced in E. coli and tested for methyl transfer activity
using a set of six structurally different pyrazoles as substrates
(Fig. 3). Screening of the library was performed using cell lysate
in deep-well plate format with SAM as cosubstrate (Fig. S7). The
performance of the variants was analyzed using gas
chromatography. Even though the designs introduced 3-5 active
site mutations simultaneously, >90% of the designs were active
and converted at least one pyrazole substrate (Fig. S8-S13).
Depending on the substrate, 10-30% of the variants showed
increased activity and/or selectivity compared to the NMT wild-
type enzyme. Next, we purified a selected set of the best
enzymes (Fig. S14) to characterize the selectivities and activities
in detail. The selected pyrazole MTs perform C–N bond
formation with very high catalytic control, in some cases with
regioselectivities of >99% (Fig. 3 and Fig. S15-S20). Catalyst
Identification of promiscuous halide methyltransferases
control could even be demonstrated for
dimethylpyrazole (2) as substrates for which the pyrazole
1
and 3,4-
After establishing the selective methylation of pyrazoles, we
aimed to expand the system towards selective alkylation. The
3
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RESEARCH ARTICLE
Figure 3. (A) Selected variants were characterized by analyzing the initial rates using 2 mM pyrazole, 2 mM SAM and 50 µM enzyme. All enzymatic reactions
were performed in triplicates. Please note that all chemical methylations using reagents as well as reactions with NMT wild-type generate product mixtures (see
Fig. S1 and S15-S20). a) Activity increase is calculated by dividing the initial rate of the variant by the initial rate of NMT wild-type. (B) As an example, GC
chromatograms for the conversion of 6 with selected variants (red) are shown and compared to wildtype (black) as well as product standards (top). These
examples demonstrate the efficiency of FuncLib to convert a promiscuous starting point (NMT wild-type) into highly selective variants with significant increase in
activity in just one round of mutagenesis and screening. GC retention time 6a: 6.4 minutes. GC retention time 6b: 6.9 minutes (see Fig. S20).
remarkable SAM cofactor recycling system by Liao and
Seebeck[12] (Fig. 1B) has the potential to enzymatically
synthesize and recycle non-natural SAM analogs (NSA) from
readily available haloalkanes as alkyl donors.[40] While this
system is currently limited to methylation,[12,41,42] we envisioned
to expand its application towards general alkyl transfer. Key in
this endeavor is to find promiscuous halide methyl transferases
(HMT) that accept different haloalkanes apart from iodomethane
as substrates. This would enable synthesis and recycling of
NSAs directly from SAH (see Fig. 1B). As a step in this direction,
we report here a fungal HMT from Aspergillus clavatus that
accepts haloethane, -propane and -butane as substrates for
NSA synthesis. In the following, we will refer to this enzyme as
NSA-synthase. The NSA-synthase was identified by studying
the substrate scope of six literature-known HMTs, including
enzymes from bacterial, fungal und plant origin (Fig. S28). The
enzymes were bought as synthetic DNA and produced in the
SAH-nucleosidase deficient E. coli. strain JW0155.[12] Substrate
4
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RESEARCH ARTICLE
scope screening was performed with cell lysates using SAH and
several haloalkanes as substrates (haloalkanes include
NSA-synthase, we used iodomethane as alkyl donor and studied
the productivity of the system using different SAH concentrations.
Surprisingly, our first experiments revealed that residual SAH
bound to the purified enzymes (v36 and NSA-synthase) was
sufficient for efficient methylation of 3. Further addition of SAH
(0.05 equiv. with respect to the substrate) did not change the
productivity of the system (Fig. S32). Next, we examined the
formation of the methylated product as
a
function of
iodomethane excess. Highest product formation of 3b (62%,
Fig. 4) was achieved using 10 equivalents of iodomethane.
However, equimolar concentrations of iodomethane with respect
to the substrate 3 reduced the product formation only slightly to
47% (Fig. S33). In all cases 3 was methylated with very high
regioselectivity (>99%) to generate 1-methyl-5-cyclopropyl
pyrazole (3b). To demonstrate that enzymatic pyrazole
alkylation can be performed on a preparative scale (1.0 mmol),
the methylated pyrazole 3b was synthesized using iodomethane
as alkyl donor (Fig. S34). The product was generated with 37%
isolated yield and with very high regioselectivity (97%). 55% of
pure starting material was recovered after the reaction.
Finally, we aimed to prove general enzymatic alkyl transfer
using iodoethane, bromoethane and 1-iodopropane as starting
material. Even though v36 was originally selected for methyl
transfer, 3 was ethylated and propylated using the enzyme
system (Fig. 4). The enzymatic alkylation of 3 generated 1-ethyl-
5-cyclopropylpyrazole (7b) and 1-propyl-5-cyclopropylpyrazole
(8b) with very high regioselectivites of >98% and >97%
(Fig. S35 and S36). The moderate to low yields achieved in
these initial enzyme cascade reactions might originate from
several causes and require detailed analysis. In a simple
scenario, v36 is efficient in methylation but inefficient in
ethylation and propylation of 3. It is believed that SAM-
dependent methyltransferases function by binding the substrate
and cosubstrate in a reactive conformation, pre-aligning the
orbitals for efficient and selective catalysis.[39] A variant chosen
for methylation may therefore be unproductive in binding
pyrazole 3 in the same reactive conformation in the presence of
Figure 4. Enzymatic selective alkylation of pyrazoles using haloalkanes. v36
and NSA-synthase (50 µM each) was used as catalyst. The reactions were
performed with 2 mM pyrazole (3) as substrate, 10 equiv. of haloalkane and
30°C (iodo-, bromoethane and iodopropane) as well as 37°C (iodomethane).
The reaction time was 30 h using iodomethane, 40 h in the iodomethane
preparative scale reaction, 72 h in using haloethanes and 50 h with 1-
iodopropane. *This reaction was performed on preparative scale (1.0 mmol),
please see SI for details. Regioselectivity is described as regioisomeric ratio
(r.r.).
iodomethane, iodoethane, bromoethane, 1-iodopropane, 1-
iodobutane and 1-bromobutane, see Fig. S28). While most of
the tested HMTs were very restrictive accepting only
iodomethane as substrate, the identified NSA-synthase
converted haloethanes, -propanes and -butanes. Notably, next
to iodoalkanes the corresponding bromoalkanes are also
substrates of the NSA-synthase. Control experiments without
enzyme, SAH or alkyl donor confirmed that the NSA formation is
enzyme-catalyzed (Fig. S29). As a general trend, we observed
that the activity of enzymatic NSA synthesis decreased with
increasing alkyl chain length of the haloalkanes (Fig. S30). While
the ethyl analog was synthesized with >80% product formation,
the propyl analog was generated with 12% and the butyl analog
with 3.6%. After confirming the formation of the NSA by mass
spectrometry (Fig. S31), we aimed to combine the NSA-
synthase with the engineered pyrazole MTs to study enzymatic
pyrazole alkylation.
a
larger ethyl or propyl group. Alternatively, the
methyltransferase could suffer from product inhibition. Finally,
the alkylation of SAH can produce NSA in two epimeric forms as
such sulfonium species have a stereochemically active lone pair.
It is currently not clear which epimer is generated by the NSA
synthase, and it is also unknown whether the engineered
methyltransferases such as v36 accept both epimers as
cosubstrate. These questions will be addressed in future studies.
Conclusion
Our studies highlight a potentially generalizable approach
to obtain catalyst-control in challenging alkylation reactions. In
particular, we show that selective alkylation of pyrazoles can be
achieved using simple haloalkanes, engineered enzymes and an
expanded cosubstrate pool. An important step was the
identification of a promiscuous halide methyltransferase (named
NSA-synthase) that enzymatically generates and recycles non-
natural analogs of SAM. An additional central part was the
engineering of a second promiscuous transferase that is active
on pyrazoles as substrates. We believe that this approach
together with important data published elsewhere[12,42] will open
up interesting new avenues for enzyme engineering to enable
Enzymatic pyrazole alkylation using simple haloalkanes
Since NSA formation was most efficient for ethyl and
propyl analogs (Fig. S30), we focussed our proof-of-concept
studies on the enzymatic ethylation and propylation using
enzyme v36 and 3-cyclopropylpyrazol (3) as substrate (Fig. 3).
This enzyme-substrate combination was selected based on the
high activity and selectivity in pyrazole methylation (Fig. S27).
To set up the cyclic enzyme cascade consisting of v36 and the
5
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10.1002/anie.202014239
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RESEARCH ARTICLE
Opin. Chem. Biol. 2017, 37, 97–106.
selective C-heteroatom bond formation in various complex
molecules. Many of these reactions are highly desired and have
so far eluded catalytic-selective synthesis.[4,5,9]
[12]
[13]
C. Liao, F. P. Seebeck, Nat. Catal. 2019, 2, 696–701.
O. Khersonsky, R. Lipsh, Z. Avizemer, Y. Ashani, M. Goldsmith,
H. Leader, O. Dym, S. Rogotner, D. L. Trudeau, J. Prilusky, et
al., Mol. Cell 2018, 72, 178–186.
The research outlined here also supports that
computational enzyme library design tools such as FuncLib[13]
are very effective. Over 90% of the designs are active, 10-30%
of the variants were more active and/or selective than the
wildtype enzyme and we identified highly selective variants with
significant activity improvements at the same time. The strength
of FuncLib is to successfully introduce multiple active site
mutations simultaneously. This enables large steps in sequence
space accompanied with drastic increase in activity and
selectivity. FuncLib transformed a promiscuous starting point
(NMT wild-type) into a small pyrazole alkylating enzyme family in
just one round of mutagenesis and screening. This artificial
enzyme family accepts structurally diverse pyrazoles as
substrates, is highly regioselective and in many cases
regiodivergent (Fig. 3). It will be interesting to see whether
computational enzyme library design tools can be combined with
machine learning[15] or performed iteratively to further speed up
directed enzyme evolution.[43]
[14]
[15]
M. T. Reetz, J. D. Carballeira, Nat. Protoc. 2007, 2, 891–903.
K. K. Yang, Z. Wu, F. H. Arnold, Nat. Methods 2019, DOI
10.1038/s41592-019-0496-6.
[16]
[17]
J. Weinstein, O. Khersonsky, S. J. Fleishman, Curr. Opin.
Struct. Biol. 2020, 63, 58–64.
A. J. Herbert, S. A. Shepherd, V. A. Cronin, M. R. Bennett, R.
Sung, J. Micklefield, Angew. Chem. Int. Ed. 2020, 59, 14950–
14956.
[18]
[19]
[20]
[21]
T. D. Huber, B. R. Johnson, J. Zhang, J. S. Thorson, Curr. Opin.
Biotechnol. 2016, 42, 189–197.
M. Thomsen, S. B. Vogensen, J. Buchardt, M. D. Burkart, R. P.
Clausen, Org. Biomol. Chem. 2013, 11, 7606–7610.
S. Mordhorst, J. Siegrist, M. Müller, M. Richter, J. N. Andexer,
Angew. Chem. Int. Ed. 2017, 56, 4037–4041.
F. Michailidou, N. Klöcker, N. V. Cornelissen, S. K. Rohit, A.
Peters, A. Ovcharenko, D. Kümmel, A. Rentmeister, Angew.
Chem. Int. Ed. 2020, DOI 10.1002/anie.202012623.
[22]
[23]
[24]
[25]
I. J. W. McKean, P. A. Hoskisson, G. A. Burley, ChemBioChem
2020, DOI 10.1002/cbic.202000187.
G. Zhao, S. Yao, K. W. Rothchild, T. Liu, Y. Liu, J. Lian, H. Y.
He, K. S. Ryan, Y. L. Du, ChemBioChem 2020, 21, 644–649.
Y. Peng, D. Sartini, V. Pozzi, D. Wilk, M. Emanuelli, V. C. Yee,
Biochemistry 2011, 50, 7800–7808.
Acknowledgements
N. Drinkwater, C. L. Gee, M. Puri, K. R. Criscione, M. J.
McLeish, G. L. Grunewald, J. L. Martin, Biochem. J. 2009, 422,
463–471.
We thank Dr. Wendy Escobedo, Lorenzo Favilli, Sebastian
Gergel, Lea Rapp, Sven Richter, Andreas Schneider and Julian
Wissner for fruitful discussions. This work was supported by the
Bundesministerium für Bildung und Forschung (031B0359B) and
Deutsche Forschungsgemeinschaft via the Emmy Noether
program (fellowship HA 7668/2-1 to S.C.H.).
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
J. R. Horton, K. Sawada, M. Nishibori, X. Zhang, X. Cheng,
Structure 2001, 9, 837–849.
C. Tongsook, J. Niederhauser, E. Kronegger, G. Straganz, P.
Macheroux, Biochim. Biophys. Acta 2017, 1863, 188–199.
S. C. Hammer, A. M. Knight, F. H. Arnold, Curr. Opin. Green
Sustain. Chem. 2017, 7, 23–30.
H. A. Bunzel, X. Garrabou, M. Pott, D. Hilvert, Curr. Opin.
Struct. Biol. 2018, 48, 149–156.
Conflict of interest
S. Ouk, S. Thiébaud, E. Borredon, B. Chabaud, Synth.
Commun. 2005, 35, 3021–3026.
The authors declare no conflict of interest.
D. K. Liscombe, A. R. Usera, S. E. O’Connor, Proc. Natl. Acad.
Sci. USA 2010, 107, 18793–18798.
Keywords: enzyme engineering • alkylation • enzyme cofactors
H. S. Loring, P. R. Thompson, Biochemistry 2018, 57, 5524–
5532.
• pyrazoles • biocatalysis
E. M. J. Gillam, J. N. Copp, D. F. Ackerley, Directed Evolution
Library Creation, Springer New York Heidelberg Dordrecht
London, 2014.
[1]
[2]
[3]
[4]
J. W. Bode, J. Mahatthananchai, A. M. Dumas, Angew. Chem.
Int. Ed. 2012, 51, 10954–10990.
L. C. Behr, R. Fusco, C. H. Jarboe, in Chem. Heterocycl.
Compd. (Ed.: A. Weissberger), John Wiley & Sons, 1967.
[34]
[35]
[36]
[37]
[38]
S. C. Hammer, G. Kubik, E. Watkins, S. Huang, H. Minges, F.
H. Arnold, Science 2017, 358, 215–218.
R. Goikhman, T. L. Jacques, D. Sames, J. Am. Chem. Soc.
2009, 131, 3042–3048.
C. K. Prier, R. K. Zhang, A. R. Buller, S. Brinkmann-Chen, F. H.
Arnold, Nat. Chem. 2017, 9, 629–634.
D. C. Blakemore, L. Castro, I. Churcher, D. C. Rees, A. W.
Thomas, D. M. Wilson, A. Wood, Nat. Chem. 2018, 10, 383–
394.
R. K. Zhang, K. Chen, X. Huang, L. Wohlschlager, H. Renata, F.
H. Arnold, Nature 2019, 565, 67–72.
Z. Sun, Y. Wikmark, J.-E. Bäckvall, M. T. Reetz, Chem. Eur. J.
2016, 22, 5046–5054.
[5]
[6]
K. Krämer, “A Wish List for Organic Chemistry,” can be found
under https://www.chemistryworld.com/news/the-five-reactions-
on-every-organic-chemists-wish-list/3010150.article, 2019.
B. P. S. Chouhan, S. Maimaiti, M. Gade, P. Laurino,
Biochemistry 2019, 58, 166–170.
S. D. Roughley, A. M. Jordan, J. Med. Chem. 2011, 54, 3451–
[39]
[40]
[41]
T. C. Bruice, Acc. Chem. Res. 2002, 35, 139–148.
J. Micklefield, Nat. Catal. 2019, 2, 644–645.
3479.
[7]
[8]
D. G. Brown, J. Boström, J. Med. Chem. 2016, 59, 4443–4458.
M. A. Beliaeva, R. Burn, D. Lim, F. P. Seebeck, Angew. Chem.
Int. Ed. 2020, DOI 10.1002/anie.202011096.
E. Vitaku, D. T. Smith, J. T. Njardarson, J. Med. Chem. 2014,
57, 10257–10274.
[42]
[43]
C. Liao, F. P. Seebeck, Angew. Chem. Int. Ed. 2020, 59, 7184–
7187.
[9]
D. J. C. Constable, P. J. Dunn, J. D. Hayler, G. R. Humphrey, J.
L. Leazer, Jr., R. J. Linderman, K. Lorenz, J. Manley, B. A.
Pearlman, A. Wells, et al., Green Chem. 2007, 9, 411–420.
M. D. Truppo, ACS Med. Chem. Lett. 2017, 8, 476–480.
[10]
[11]
L. A. Wessjohann, J. Keim, B. Weigel, M. Dippe, Curr. Opin.
Chem. Biol. 2013, 17, 229–235.
M. R. Bennett, S. A. Shepherd, V. A. Cronin, J. Micklefield, Curr.
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This article is protected by copyright. All rights reserved.

10.1002/anie.202014239
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Enzyme-controlled N-alkylation: Biological alkylation is highly selective, yet, it depends on complex leaving groups. Now,
promiscuous and engineered enzymes achieve selective enzymatic alkylation using simple haloalkanes.
Institute and/or researcher Twitter usernames: @Stephan_Hammer
7
This article is protected by copyright. All rights reserved.