A Tandem Enzymatic sp2-C-Methylation Process: Coupling in Situ S-Adenosyl-l-Methionine Formation with Methyl Transfer

Article abstract of DOI:10.1002/cbic.201700115

A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-l-methionine (SAM), followed by alkylation of aromatic substrates by the C-methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled ¹³CH_3, ¹³CD₃ and CD₃ groups from their corresponding SAM analogues formed in situ.

Full text of DOI:10.1002/cbic.201700115

Accepted Article
Title: A Tandem Enzymatic sp2-C-Methylation Process: Coupling In
situ S-Adenosyl-L-Methionine Formation with Methyl Transfer
Authors: Joanna C Sadler, Luke D Humphreys, Glenn Burley, and
Radka Snajdrova
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To be cited as: ChemBioChem 10.1002/cbic.201700115
Link to VoR: http://dx.doi.org/10.1002/cbic.201700115
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10.1002/cbic.201700115
ChemBioChem
1 A Tandem Enzymatic sp2-C-Methylation Process: Coupling In Situ
2 S-Adenosyl-L-Methionine Formation with Methyl Transfer
3
Joanna. C. Sadler,[a],[b] Luke D. Humphreys,*[a],[c] Radka Snajdrova,[a] Glenn. A. Burley*[b]
Abstract:
A
one-pot, two-step biocatalytic platform for the
At present, the broader applicability of small-molecule MTs
is hampered by the inherent instability of SAM (942 min at pH 8)
as the corresponding methylating agent.[15,16] Additionally, the
synthesis of SAM by chemical methods results in the formation
of a diastereomeric mixture, which increases the cost of the
process and has the potential for undesirable C-MT inhibition by
the unwanted diastereomer.[17,18]
regiospecfic C-methylation and C-ethylation of aromatic substrates is
described. The tandem process utilizes SalL (Salinospora tropica)
for in situ synthesis of S-adenosyl-L-methionine, followed by
alkylation of aromatic substrates using the C-methyltransferase
NovO (Streptomyces spheroides). Application of this methodology is
demonstrated by regiospecific methyl, ethyl and isotopically-labelled
13CH3, 13CD3 and CD3 groups from their corresponding SAM
analogues formed in situ.
Regiospecific, late-stage methylation is a powerful strategy
for tuning the physical and biological properties of small
molecules and biomacromolecules.[1] At present, synthetic
methodologies that methylate C(sp2)-H bonds are predominantly
limited to transition metal-catalyzed strategies, which require
elevated temperatures and the need for additives such as
ligands and oxidants.[2–4] In addition, these methodologies are
typically regioselective rather than regiospecific, which further
restricts their scope.[5,6] In contrast, Nature routinely employs S-
adenosyl-L-methionine (SAM) dependent methyltransferases
(MTs) to methylate at O-, N-, S- and C- sites. Thus, MTs hold
considerable potential for the development of a biocatalytic
alkylation platform of small molecules.[7–9]
Of the various MTs available, C-MTs are particularly
attractive as they enable C-C bond formation under mild
conditions relative to traditional Friedel-Crafts reactions.[10,11]
One C-MT exemplar is NovO, which methylates the C-8 position
of 1a in the biosynthesis of the antibiotic novobiocin to form 2a
(Scheme 1a).[12] We and others have shown that regiospecific
methylation of the 8-position of 1b is catalyzed by a novel His-
Arg motif, which facilitates the deprotonation of the phenolic
proton in the 7-position, followed by methylation at position 8 by
SAM to form 2b.[13,14] NovO is also effective in catalysing Friedel-
Crafts alkylations using non-natural SAM analogues to alkylate
1b-d regiospecifically.[8,11] Furthermore, NovO accepts non-
natural substrates such as 3 to exclusively form 4 (Scheme 1b),
which is currently only accessible by this process.[11]
Scheme 1. Biocatalytic C-methylation of (a) coumarins (1a-d) and (b) 2,7-
dihydroxynaphthalene (3) catalysed by NovO. Both studies require the
formation of SAM prior to C-methylation. (c) Development of a one-pot, two-
enzyme process involving in situ SAM formation (SalL) and C-methylation
(NovO). SAM: S-adenosylmethionine. SAH: S-adenosylhomocysteine.
A powerful strategy that overcomes the need to prepare
and isolate SAM and SAM analogues would produce the
cofactor in situ, followed by alkyl transfer.[19–23] The enzyme SalL
(Salinospora tropica) is known to form SAM from 5’-deoxy-5-
chloroadenosine (ClDA, 5) and methionine (Met, 6).[24–28] As
ClDA is readily prepared from adenosine in a one-pot process,
the use of SalL offers an inexpensive and atom-efficient
alternative to the use of methionine adenosyl transferases,
which utilizes expensive ATP as the corresponding adenosyl
donor and have been used previously for in situ SAM
formation.[21,29–32]
At present, there have not been any reports on the utility of
the SalL-catalysed SAM production in tandem with C-MTs in a
one-pot procedure. Herein, we describe such a process for the
methylation of small molecule aromatic scaffolds using SalL and
the C-MT NovO (Scheme 1c). Additionally, we show that this
[a]
[b]
J. C. Sadler, L. D. Humphreys, R. Snajrova
GlaxoSmithKline Medicines Research Centre
Gunnels Wood Road, Stevenage, SG1 2NY (UK)
J. C. Sadler, Dr G.A. Burley*
WestCHEM, Department of Pure and Applied Chemistry
University of Strathclyde
295 Cathedral Street, Glasgow G1 1XL (UK)
Email: glenn.burley@strath.ac.uk
[c]
Dr L. D. Humphreys*
Current address: Gilead Alberta ULC
1021 Hayter Road NW, Edmonton, AB T6S 1A1, Canada.
Email: luke.humphreys@gilead.com
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700115
ChemBioChem
tandem strategy can be used to transfer ethyl groups and (Figure 1a, Entry E). To determine whether this was due to
isotopically-labeled methyl groups.[33–35]
residual SAM or Met present in the reaction mixture, the reaction
Initial investigations focused on determining the was also run in the absence of ClDA (Figure 1a, Entry F). In
compatibility of SalL and NovO to operate in a one-pot process. this case, no conversion to 2b was observed, which was
The model substrate 1b was used with an excess of ClDA (5, 2 indicative of background methylation being caused by residual
eq.) and Met (6, 50 eq.) using an E. coli cell-free extract from the Met present in the cell lysate and cell free extract (Table S3).
overexpression of NovO (Figure 1a; Entry A). Based on the
previously reported kinetic parameters for SAM formation
To address this issue, purified enzymes were used (Figure
1b and Table S2). Additionally, methylthioadenosine
nucleosidase (MTAN) or SAH hydrolase (SAHH) was added to
remove SAH from the reaction mixture, which is a known
inhibitor of many SAM dependent MTs.[18,37,38] Since ClDA
OH
O
(a)
(b)
OH
O
(i)
H
N
H
N
Ph
Ph
(i)-(ii)
inhibits both MTAN[39] and SAHH[40]
, SAM was pre-formed in
O
O
HO
O
HO
O
Me
situ before the addition of NovO and MTAN or SAHH. Initially,
the reaction was carried out using 2 eq. ClDA and 10 eq. Met
with either MTAN or SAHH (Figure 1b, Entries A and B). Whilst
only 68% conversion was achieved with SAHH, nearly
quantitative methylation of 1b was observed using MTAN as an
additive. Further optimisation enabled the reduction of the
number of equivalents of ClDA from 2.0 to 1.5 without loss of
conversion to 2b when 1 eq. DTT was added.[41] Carrying out the
reaction in the absence of MTAN decreased the conversion by
~40%, confirming the role of MTAN decreasing by-product
inhibition caused by SAH. Indeed, an IC50 value of 9.8 µM for
ClDA has been reported for MTAN from E. coli, which was used
in our study.[42] Finally, only 4% methylation was observed when
the reaction was carried out in the absence of Met, which may
be due to residual SAM bound in NovO (Figure 1b and Table
S2).
1b
2b
(a)
(b)
With optimised conditions for a one-pot, tandem SAM
formation C-methylation process in hand, we next explored the
scalability of the methodology. The tandem process was carried
out with Met, three isotopically labelled Met analogues and
ethionine; using 20 mg of 1b, 1c or 3 in each case. To the best
of our knowledge, this is the first time that a tandem process
which involved the in situ formation of SAM has been used on
preparative scale. For the transfer of an unlabelled methyl group,
crude E. coli cell lysate (NovO) and E. coli cell free extract (SalL)
was used, whilst purified enzymes were employed for the
transfer of isotopically labelled and ethyl groups. Moderate
(65%) to excellent (88-100%) levels of conversion were obtained
for transfer of CH3, 13CH3, CD3 and 13CD3, with 1c showing
quantitative conversion and isolated yields 76-91% in all cases
(Table 1). High levels of conversion (88-97%) were also
obtained for 1b, although isolated yields were lower due to poor
solubility of the corresponding products during work-up.
Alkylation of 3 was also successful on this scale to form 4 in high
conversions (69-87%) using purified SalL and NovO, whilst 65%
of 4 was formed using the cell lysate.
Figure 1. (a) Reaction optimization using substrate 1b. E. coli cell lysates
were used and % conversion determined by HPLC (area/area%) after 24 h.
Reagents and conditions: (i) ClDA, Met, BSA, DTT, NovO cell lysate in 50 mM
phosphate buffer, pH 6.5 resuspended at 10 mL/ g pellet, SalL cell free extract
in 50 mM phosphate buffer, pH 6.8 resuspended at 5 mL/ g pellet. A: 2 eq.
ClDA, 50 eq. Met; B: 1 eq. ClDA, 50 eq. Met; C: 2 eq. ClDA, 2 eq. Met; D: 2
eq. ClDA, 1 eq. Met; E: 2 eq. ClDA, no Met added; F: No ClDA or Met added.
(b) Reaction optimization using purified enzymes. Reagents and conditions: (i)
Met, SalL, BSA, 50 mM potassium phosphate buffer pH 6.5, ClDA, 37 °C, 8
hours. (ii) NovO, 37 °C, 16 hours. A: 2 eq. ClDA, 10 eq. Met, 0.5 M MTAN
added with NovO.; B: 2 eq. ClDA, 10 eq. Met; 2 uM SAHH added with NovO;
C: 1.5 eq. ClDA, 1.5 eq. Met, 0.5 uM MTAN added with NovO; D: 1.1 eq. ClDA,
1.1 eq. Met, 0.5 uM MTAN added with NovO; E: 1.5 eq. ClDA, 2 eq. Met, 1
mM DTT and 0.5 uM MTAN added with NovO; F: 1.5 eq. ClDA, no Met added;
0.5 uM MTAN added with NovO. Optimised conditions highlighted in blue.
In summary, we have demonstrated a scalable biocatalytic
platform for a Friedel-Crafts alkylation using SalL for in situ
cofactor SAM analogue synthesis from inexpensive starting
materials. Furthermore, to the best of our knowledge this is the
first example of using SalL for in situ cofactor production in
tandem with a MT for the site specific C-methylation/alkylation of
a small molecule. We envisage that our enzymatic platform
could form the basis of a valuable biocatalytic tool for late-stage,
regiospecific labelling of small molecules.[33,43–45]
compared to ClDA formation, low levels of chloride present in
the system were not anticipated to affect conversion levels.[36]
No other sources of chloride were added to the system.
This resulted in quantitative conversion of 1b into the
methylated product 2b in 24 hours. We then optimised the
reaction conditions, firstly by minimising the number of
equivalents of Met and ClDA relative to substrate 1b (Figure 1a
and Table S1). When the number of equivalents of ClDA was
reduced from 2 to 1.5 eq., a drop off in the formation of 2b was
observed (Table S1). Quantitative conversion was optimal with 2
equivalents of Met (Figure 1a, Entry C). Decreasing the amount
of Met further to 1 eq. reduced the conversion of 1b to 2b to
77% (Figure 1a, Entry D). However, when the reaction was run
without the addition of Met, 30% methylation of 1b was observed
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10.1002/cbic.201700115
ChemBioChem
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(i) SalL (0.5 mol%), BSA (1 mg/mL), DTT (1 eq.), 50 mM potassium phosphate
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(CH3, 13CH3, CD3 and 13CD3) or 32 hours (Et). (ii) NovO (4 mol%), MTAN (0.05
mol%), 37 °C, 16 hours. Reactions carried out using 20 mg of substrate.
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ChemBioChem
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Joanna. C. Sadler, Luke D. Humphreys,
Radka Snajdrova, Glenn. A. Burley*
Tandem SAM: A one-pot, two-enzyme
C-methylation process is described.
Linking SAM production using SalL (S.
Tropica) with the C-methyltransferase
NovO (S. Spheroides) enables the
synthesis a suite of methylated and
ethylated cuomarin products.
[a]
[b]
J. C. Sadler, L. D. Humphreys, R. Snajrova
GlaxoSmithKline Medicines Research Centre
Gunnels Wood Road, Stevenage, SG1 2NY (UK)
J. C. Sadler, Dr G.A. Burley*
WestCHEM, Department of Pure and Applied Chemistry
University of Strathclyde
295 Cathedral Street, Glasgow G1 1XL (UK)
Email: glenn.burley@strath.ac.uk
[c]
Dr L. D. Humphreys*
Current address: Gilead Alberta ULC
1021 Hayter Road NW, Edmonton, AB T6S 1A1, Canada.
Email: luke.humphreys@gilead.com
Supporting information for this article is given via a link at the
the document.
This article is protected by copyright. All rights reserved.