Biocatalytic C3-Indole Methylation—A Useful Tool for the Natural-Product-Inspired Stereoselective Synthesis of Pyrroloindoles

Article abstract of DOI:10.1002/anie.202107619

Enantioselective synthesis of bioactive compounds bearing a pyrroloindole framework is often laborious. In contrast, there are several S-adenosyl methionine (SAM)-dependent methyl transferases known for stereo- and regioselective methylation at the C3 position of various indoles, directly leading to the formation of the desired pyrroloindole moiety. Herein, the SAM-dependent methyl transferase PsmD from Streptomyces griseofuscus, a key enzyme in the biosynthesis of physostigmine, is characterized in detail. The biochemical properties of PsmD and its substrate scope were demonstrated. Preparative scale enzymatic methylation including SAM regeneration was achieved for three selected substrates after a design-of-experiment optimization.

Full text of DOI:10.1002/anie.202107619

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Biocatalytic C3-Indole Methylation – A Useful Tool for Natural
Product Inspired Stereoselective Synthesis of Pyrroloindoles
Authors: Pascal Schneider, Birgit Henßen, Beatrix Paschold, Benjamin
P. Chapple, Marcel Schatton, Florian Seebeck, Thomas
Classen, and Joerg Pietruszka
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202107619
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RESEARCH ARTICLE
Biocatalytic C3-Indole Methylation – A Useful Tool for the Natural
Product Inspired Stereoselective Synthesis of Pyrroloindoles
Pascal Schneider,[a] Birgit Henßen,[b] Beatrix Paschold,[a] Benjamin P. Chapple,[a] Marcel Schatton,[a]
Florian Seebeck,[c] Thomas Classen,[b] and Jörg Pietruszka[a,b]*
Dedicated to the memory of Volker Jäger
[a]
P. Schneider, B. Paschold, B. P. Chapple, M. Schatton, Prof, Dr. J. Pietruszka
Institut für Bioorganische Chemie
Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich
Stetternicher Forst, Geb. 15.8, 52426 Jülich, Germany, and Bioeconomy Science Center (BioSC).
E-mail: j.pietruszka@fz-juelich.de
[b]
[c]
B. Henßen, Dr. T. Classen, Prof. Dr. J. Pietruszka
Institut für Bio- und Geowissenschaften: Biotechnologie (IBG-1)
Forschungszentrum Jülich GmbH
52428 Jülich, Germany
Prof. Dr. Florian Seebeck
Department of Chemistry
University of Basel
Mattenstrasse 24a, CH-4058 Basel (Switzerland)
Supporting information for this article is given via a link at the end of the document
Abstract: Enantioselective synthesis of bioactive compounds bearing
a pyrroloindole framework is often laborious. In contrast, there are
several S-adenosyl methionine (SAM) dependent methyl transferases
known for stereo- and regioselective methylation at the C3 position of
various indoles, directly leading to the formation of the desired
pyrroloindole moiety. Herein, the SAM dependent methyl transferase
PsmD from Streptomyces griseofuscus, a key enzyme in the
biosynthesis of physostigmine, is characterized in detail. The
biochemical properties of PsmD and its substrate scope was
demonstrated. Preparative scale enzymatic methylation including
SAM regeneration was achieved for three selected substrates after a
design-of-experiment optimization.
strategies towards the tricyclic scaffold 8 can be divided into two
groups, either following a biomimetic approach – electrophilic C3-
alkylation followed by cyclisation – or utilizing oxindoles 9 (Figure
1, B).[8,9] Biosynthesis is an alternative for the natural product itself,
as has been demonstrated for acetylcholinesterase inhibitor
physostigmine (1) that is produced by
a Streptomyces
griseofuscus strain from tryptophan (10) with titers of up to 790
mg/L.[10]
Introduction
In the past twenty years, biocatalysis was shown to be a valuable
tool within the toolbox of an organic chemist.[1] Besides
organocatalysts and metal catalysts, biocatalysts are state of the
art in medicinal chemistry:[2] They are not only employed in the
synthesis of dedicated (chiral) building blocks, but in particular in
late stage functionalization of active agents. After overcoming
initial limitations, e.g., with respect to cofactor recycling systems
or scaling, the proven versatility of enzymes even in industrial
processes increased the demand for new biocatalysts in recent
years.[3,4] Also with regard to natural product synthesis, enzymes
exhibit great potential for challenging synthetic tasks such as
stereoselective synthesis and late stage functionalization under
mild conditions.[5]
One particular class of natural products and derivatives
thereof, namely drugs based on pyrroloindolines 1-5,[6] involve
methyl transferases as key enzymes within their biosynthesis
(Figure 1, A).[7] The quaternary stereogenic carbon center is
formed by C-alkylation of indoles 6 or 7, a reaction that has
attracted considerable attention recently.[6c,6q] The major synthetic
Figure 1. A. Selected examples of (natural) drugs bearing the pyrroloindoline
framework (blue). B. Common retrosynthetic approaches towards
pyrroloindolines. C. The methyl transferase (MT) PsmD investigated in this work
represents the key enzyme in the biosynthesis of physostigmine (1). For a
proposed mechanism of the PsmD reaction see supporting information (Figure
S1). (SAM: S-Adenosyl methionine; SAH: S-Adenosyl homocysteine).
1
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10.1002/anie.202107619
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RESEARCH ARTICLE
The corresponding gene cluster encoding for the eight enzymes
was resolved in 2014, also suggesting the central role of PsmD
for the stereoselective methylation (Figure 1, C).[6c] While
fermentation proved to be versatile for physostigmine (1)
production, derivatives thereof of the corresponding biosynthetic
intermediates are less readily available and the stereoselective
access remains challenging. Thus, there is still a demand for
efficient, robust and mild methods to access the pyrroloindole
framework in an enantiomerically pure fashion and we anticipated
that the methyl transferase PsmD might serve as the required tool
to overcome these limitations.
range (6.0-8.5, 100 mM KPi buffer). In the following, the optimized
parameters were used to elucidate the kinetics of the PsmD
catalyzed methylation. For this, the commercially available
bioluminescence-based Mtase Glow Assay (Promega) was used
for the SAM dependent methyl transferase activity screening.[23]
.
Six classes of methyl transferases have been identified, the most
common representatives belonging to the family of the S-
adenosyl methionine (SAM) dependent methyl transferases
including N-, O-, S- as well as C- methyl transferases.[11-13] They
are involved in many different process including DNA and RNA
regulation,[14,15] posttranslational protein modification,[16] and
biosynthesis of several bioactive natural compounds.[7,17,18]
Despite their undeniable potential, their application in organic
synthesis was hampered by the lack of a scalable recycling
system of SAM, one of the most expensive cofactors available.[19]
Only recently, promising protocols have been established: Apart
from utilizing the complete SAM regeneration cycle,[20] mainly two
approaches are used to address this limitation. On the one hand
there is the so called “supply chain”, which uses methionine
adenosyl transferases (MAT) to form SAM from methionine and
adenosine triphosphate (ATP).[20] Even though this reaction
requires stoichiometric amounts of ATP and methionine, these
chemicals are comparably cheap and available in large quantities.
On the other hand, there is the direct recycling by using halide
methyl transferases (HMT), which consume methyl iodide to
convert S-adenosyl homocysteine (SAH) to SAM directly.[21]
In this work, we report the application of the C3-methyl
transferase PsmD with regard to potential application for total
synthesis of physostigmine derivatives on an enzymatic
preparative scale applying only catalytic amounts of the cofactor
by direct SAM recycling.[21]
Scheme 1. A. Substrate synthesis; DMAP - 4-dimethylaminopyridine. B.
Experimental setup used for the reaction characterisation (for details see
supporting information). C. Summary of the parameters of PsmD. [a] Calculation
based on the amino acid sequence. [b] Kinetic parameters were obtained by
application of the Mtase glo assay (Promega; for details see supporting
information).
Results and Discussion
Optimized conditions for PsmD reaction. To evaluate the
proposed reaction of the methyl transferase PsmD (Figure 1C),
the enzyme was expressed and purified as described by Liu et
al.[6] The natural substrate was synthesized starting from the
commercially available 5-benzyloxyindole-3-acetonitrile (11)
(Scheme 1A): reductive acylation provided amide 12 that could
be de-O-benzylated furnishing phenol 13a.[22] The consecutive
carbamoylation led to methyl carbamate 7a in high yield (90%;
65% yield over three steps).
Next, the proposed enzymatic transformation of indole 7a was
performed in vitro and product 8a formation was confirmed by
HPLC-MS (Scheme 1B). Based on the initial results, the optimal
reaction conditions for the biocatalytic methylation were
systematically tested. Thus, the temperature optimum was
determined by comparing relative conversion from compound 7a
to compound 8a after 4 hours by HPLC-MS. The tested
temperature ranged from 25°C to 40°C; the enzyme showed best
conversion at 35°C. The pH optimum was shown to be at 7.5 in
KPi buffer (100 mM) (see supporting information for details). Even
though only slight differences were observed in the tested pH
For determining the kinetic parameters, substrate 7a was tested
with concentrations ranging from 0.1 µM to 100 µM. The SAM
concentration was fixed to 60 µM and 1 µg of enzyme (or 0.1 µg
for low substrate concentrations from 0.1 µM to 1 µM) was used in
total for each well (20 µL reaction volume). The reaction was
stopped after 5, 7, and 10 minutes by addition of 0.5%
trifluoroacetic acid. After addition of the Mtase reagent and Mtase
detection solution the bioluminescent signal based on the formed
SAH concentration was measured. Since this is an indirect
method, it is assumed that SAH formation is exclusively based on
the consumption of SAM by the methyl transferase reaction. To
rule out autodemethylation of SAM, for each run a negative
control with 60 µM SAM and 1 µg enzyme was used. Reaction
rate was calculated and plotted against the tested concentration.
Applying a least-square-fit with the Michaelis-Menten-equation
the maximal velocity is 18.3 µmol·min-1·g-1 with a KM value of
11.3 µM (see supporting information for details; the validity of the
2
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10.1002/anie.202107619
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RESEARCH ARTICLE
method was independently confirmed by HPLC reaction control).
The biochemical data obtained for PsmD are summarized in
Scheme 1C.
Substrate scope. In order to test the scope of PsmD beyond the
natural substrate 7a, a set of 16 selected substrates 7a-p was
synthesized first (see supporting information for details) and then
evaluated in the enzymatic transformation towards compounds
8a-p. Enzyme activities were measured as triplicates as
described for the determination of kinetic parameters using the
Mtase glow assay. The substrate concentration was set to 20 µM.
To validate that conversion always resulted from the respective
reaction from substrate to product, all samples were also
monitored by HPLC-MS. First, the influence of the carbamate was
evaluated (Scheme 2). While the convenient formation of
compounds 8a-d could be confirmed, it was apparent that
increasing the steric bulk resulted in a decrease in activity,
ultimately leading to no (tert-butyl product 8e) or almost no
(phenyl carbamate 8f) product formation. Similarly, an analogous
trend was observed when varying the amide group (products
8a,g-k): However, while a decrease in activity from the acetyl
group (in compound 8a) to the pivalate 8j or benzoate 8k can be
observed, the remaining activity should still qualify for preparative
scale transformation and thus allowing to test the limits of the
enzymatic approach towards physostigmine derivatives.
Surprisingly, omitting the carbamate completely (products 8l-p,
see box in Scheme 2) was feasible and transformation was
detectable, albeit with low residual relative activity (for 8l-n). Since
the latter examples are still important with respect to further
application of the methyl transferase in organic syntheses, we
decided to include the target compound 8m in the set of
experiments for enzymatic preparative conversion as well. While
the carbamate group is crucial for high activity, the volume of the
substrates is another important parameter as can be seen from
the correlation of the PsmD-activity with the Connolly solvent
excluded volume (Figure 2).
Scheme 2. Substrate scope and schematic procedure for evaluation of enzyme
promiscuity. Relative PsmD activity is displayed in % (relative to the natural
reaction 7a to 8a). The results represent the mean of three experiments and
details are shown in the supporting information.* Conversion is displayed as
relative conversion based on the substrate to product ratio and was determined
by HPLC-MS as described in the supporting information. Reactions were
stopped after 16 hours and performed as described for pH and T screening.[a]
N-acetyl-L-tryptophan methyl ester (10a) (not shown).
Figure 2. Correlation between the volume of the tested compounds and relative
PsmD activity [%] (compounds 8a-n). The volume was calculated using
Chem3D Connolly solvent excluded volume (probing radius 1.4 Å).[24] The non-
carbamoylated compounds have been highlighted in yellow.
PsmD - stereoselectivity. Since the natural compound
physostigmine (1) is enantiomerically pure, it can be assumed that
3
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10.1002/anie.202107619
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RESEARCH ARTICLE
(Scheme 4A). The plasmid containing the gene encoding for the
His-tagged CtHMT and the SAH nucleosidase deficient strain
BL21 mtn (DE3), which was shown to be superior to the
commercially available strains for the cofactor recycling, were in
general used as described previously.[21] However, the reaction
conditions had to be adapted to the chosen substrate as well as
PsmD, and hence a systematic optimization was performed to find
the best parameters for the conversion keeping the enzyme load
as low as possible (Figure 3). First, a factorial design was
performed testing the following factors: SAH (20 µM & 50 µM),
methyl iodide (2 mM & 10 mM), EDTA (0 mM & 1 mM), temperature
(25°C & 35°C), pH (7.5 & 8.0). Three parameters turned out to
influence the reaction outcome dominantly and were optimized in
a second round using a response surface design of experiment.
The parameters were in particular: The methyl iodide
concentration, the PsmD amount, and the CtHMT concentration.
The conversion of compound 7a to 8a can be modelled by the
hypersurface shown in Figure 3. By this, we were not only able to
define ideal parameters for the conversion [35°C, pH 7.5, 1 mM
EDTA, 2 mM substrate, 13 mM MeI, 20 mM SAH, 6.7 µM PsmD
(16 U·g-1), 262 µL CtHMT lysate (0.09 U·mL-1)], but furthermore
were able to even reduce the demand for SAH to 20 µM.
the PsmD-mediated methylation is also enantioselective. In order
to validate this, three representatives 8a,j+m have been selected
for the synthesis of the respective racemic chromatographic
standard (Scheme 3A): For the key step, the dearomative
cyclisation of the tryptamine, an adapted protocol by Yi et al. was
used forming 8m as well as 15a+b in good yield (67-84%).[6g] It
should be noted that under these reaction conditions the direct
transformation of carbamates 7 led to decomposition of the
starting material. Next, deprotection and reprotection allowed the
selective phenol carbamoylation leading to intermediates 16a+b.
As the N-unsubstituted hexahydropyrroloindoles 8a+j are prone
to ring-opening of the heterocycle, the benzyl-deprotection has to
be performed under mild conditions (-40 - 23°C) to suppress this
side reaction. Although the non-optimized reaction sequence
mimics the biosynthesis in terms of the dearomative cyclisation, it
lacks both the elegance of a one-step procedure and the
efficiency of the enzyme-mediated reaction. However, with the
racemic standards 8a,j+m at hand, it was unambiguously proven
that the corresponding enzymatic in vitro conversions shown in
Scheme 2 were indeed highly enantioselective (>99% ee;
Scheme 3B+C as well as supporting information).
Figure 3. The diagram shows a 3D-cut of the hypersurface for the 4D-
optimization. The red maximum corresponds to 100% conversion. For detailed
information on the tested conditions for recycling optimization, see supporting
information. The hypersurface is described by the equation: conversion =
−1
−1
125.5% − 7.5898%µM−1 ∙ c MeI + 0.5684%µL ∙ V PsmD − 0.0184%µL
∙
Scheme 3. A Synthesis of the racemic standard compounds 8a,j+m. B+C
HPLC-traces for the ee-analytics of compounds 8a+j (in blue - racemic standard,
in orange - compounds obtained after enzymatic methylation with >99% ee; for
details see supporting information).
(
)
(
)
V 퐶푡HMT + 0.2074%µM−1µg−1c MeI ∙ V PsmD + 0.0058%µM−1µL−1
∙
(
)
(
)
(
)
c MeI ∙ V 퐶푡HMT − 0.00418%µL−1µL−1 ∙ V(PsmD) ∙ V(퐶푡HMT).
(
)
(
)
After successful screening on 2 mM scale, we decided to use the
best conditions for preparative scale enzymatic methylation (50
mg scale; see Scheme 4B). While the focus was on the natural
substrate 7a, we also decided to test the scope with two indoles
7j+m that proved to be poor substrates in the initial tests (see
Scheme 2). Pleasingly, the key-intermediate 8a within the
physostigmine (1) biosynthesis was isolated after quantitative
conversion of tryptamine 7a in 84% yield of isolated pure product.
We were surprised to find that also the non-natural products 8j+m
could be obtained, albeit as expected in poor yield of pure isolated
Preparative scale enzymatic methylation. While the PsmD-
catalyzed methylation proved to be mild, protecting group-free,
and highly selective providing a short-cut in the synthesis of
pyrroloindolines 8, its synthetic utility remains unclear until the
stoichiometric demand for SAM can be overcome. Here, we
based the recycling on the halide methyl transferase from
Chloracidobacterium
thermophilum
(CtHMT)
described
previously.[21] This enzyme transfers a methyl group from methyl
iodide onto SAH to form SAM needed for the PsmD-reaction
4
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10.1002/anie.202107619
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RESEARCH ARTICLE
product (28% and 13%, respectively): With the same amount of
PsmD (6.7 µM; 16 U·g-1) and PsmD showing the lowest residual
activity for compound 7m, only 4% conversion was observed after
16 hours (reaction was monitored by TLC, after 16 hours no
further conversion was observed). A tenfold excess of PsmD was
necessary to increase the conversion (19%), also making
enantiomerically pure product 8m accessible (8 mg; 13%).
the µM range were determined, showing on the one hand that
increasing the bulk at the amide residue leads to a sterical clash
within the target enzyme and on the other hand that the carbamoyl
moiety seems to be necessary for proper binding to the enzyme.
In summary, we were able to apply the methyl transferase PsmD
in the direct and scalable synthesis of enantiomerically pure and
bioactive physostigmine derivatives.
Scheme 5. Bioactivity assay used for in vitro activity profiling of the
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) determined with
the Ellman method.[26] General assay used in this study (for detailed description,
see supporting information) and summary of the results obtained in this study.
Values are displayed as IC50 [µM] and represent the mean of three experiments.
Conclusion
In the present study, PsmD, the key enzyme in the biosynthesis
of physostigmine (1) that is responsible for the enantioselective
dearomative C3-methylation of tryptamine 7a, is heterologously
expressed and biochemically characterized for the first time. It
was demonstrated that it shows remarkable substrate promiscuity
by converting twelve out of sixteen selected tryptamine
derivatives with SAM as cofactor. Compared to conventional
synthesis, the reaction conditions proved to be much milder, also
providing the products in excellent enantioselectivity (>99% as
determined by HPLC analysis). The objective could be reached
by successfully implementing cofactor recycling of SAM in an
enzymatic preparative scale transformation by using the halide
methyl transferase from Chloracidobacterium thermophilum
(CtHMT): Methyl iodide was the stoichiometric reagent in the
methylation of SAH. Optimal reaction conditions were obtained
upon a design of experiments investigation proving at the same
time that an excess of methyl iodide does have a negative
influence on the overall conversion. Thus, PsmD was
demonstrated to be a versatile tool in the enantioselective
synthesis of pyrroloindolines 8, compounds that were shown to
display remarkable bioactivity as inhibitors of the acetylcholine-
and butyrylcholine-esterase.
Scheme 4. A SAM cofactor recycling as applied in the enzymatic preparative
scale synthesis of methylated compounds 8. B Enzymatic preparative scale
methylation of substrates 7a,j+m. All reactions were carried out with 50 mg of
the respective substrate utilizing the optimized conditions: 35°C, pH 7.5, 1 mM
EDTA, 2 mM substrate, 13 mM MeI, 20 mM SAH 6.7 µM PsmD (0.8 U·µg-1), 262
µL CtHMT lysate (0.09 U·mL-1) in a closed Schott flask. Yield is given as yield
of isolated pure product and conversion is displayed as relative conversion
based on the substrate to product ratio determined by HPLC. * A 10-fold excess
of PsmD was used.
Bioactivity testing. The compounds 8a, 8j and 8m were tested
for their inhibition of the acetylcholine- and butyrylcholine-
esterase. Even though physostigmine (1) is a known and potent
inhibitor of these medicinally important enzymes, it has poor
pharmacokinetics and is thereof no longer considered for
Alzheimer’s disease treatment.[25] Nevertheless, because of the
rising importance and thus demand for Alzheimer targeting drugs
and the known potential of the hexahydropyrrolo indole scaffold,
we decided to test the three obtained compounds with the
Ellmanns method (Scheme 5).[26] We were able to determine the
respective IC50 values by applying a nonlinear dose response
curve (for detailed information and assay procedure see
supporting information). As a reference compound, commercially
available physostigmine (1) and its analogue rivastigmine were
used. Compound 8a proved its potential as an inhibitor for the
AChE as well as for the BChE: At least in the in vitro setting,
superior values relative to the reference compounds were
measured. Furthermore, for compound 8j and 8m IC50 values in
Acknowledgements
We gratefully acknowledge the state of North Rhine Westphalia
(NRW) and the European Regional Development Fund (EFRE)
for funding the project within the ‚CLIB-Kompetenzzentrum
5
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10.1002/anie.202107619
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RESEARCH ARTICLE
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10.1002/anie.202107619
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Utilizing the promiscuous C3-indole methyl transferase PsmD enabled the enantioselective synthesis of various pyrroloindolines. By
additionally applying the halo methyl transferase CtHMT not only the expensive SAM cofactor could be recycled, but also the
enzymatic preparative scale transformation was achieved.
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