Chemoenzymatic Cascades toward Methylated Tetrahydroprotoberberine and Protoberberine Alkaloids

Article abstract of DOI:10.1021/acs.orglett.1c02110

Tetrahydroprotoberberine and protoberberine alkaloids are a group of biologically active natural products with complex molecular scaffolds. Isolation from plants is challenging and stereoselective synthetic routes, particularly of methylated compounds are

Full text of DOI:10.1021/acs.orglett.1c02110

pubs.acs.org/OrgLett
Letter
Chemoenzymatic Cascades toward Methylated
Tetrahydroprotoberberine and Protoberberine Alkaloids
Rebecca Roddan, Fabiana Subrizi, Joseph Broomfield, John M. Ward, Nicholas H. Keep,
and Helen C. Hailes*
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ABSTRACT: Tetrahydroprotoberberine and protoberberine alkaloids are a group of biologically active natural products with
complex molecular scaffolds. Isolation from plants is challenging and stereoselective synthetic routes, particularly of methylated
compounds are limited, reducing the potential use of these compounds. In this work, we describe chemoenzymatic cascades toward
various 13-methyl-tetrahydroprotoberberbine scaffolds using a stereoselective Pictet-Spenglerase, regioselective catechol O-
methyltransferases and selective chemical Pictet-Spengler reactions. All reactions could be performed sequentially, without the
workup or purification of any synthetic intermediates. Moreover, the naturally occurring alkaloids have the (+)-configuration and
importantly here, a strategy to the (−)-isomers was developed. A methyl group at C-8 was also introduced with some stereocontrol,
influenced by the stereochemistry at C-13. Furthermore, a single step reaction was found to convert tetrahydroprotoberberine
alkaloids into the analogous protoberberine scaffold, avoiding the use of harsh oxidizing conditions or a selective oxidase. This work
provides facile, selective routes toward novel analogues of bioactive alkaloids.
atural products and related analogues are a major source
Nof therapeutics, making up 42% of FDA-approved drugs
from 1981 to 2019.1 Many have a diverse range of molecular
scaffolds, often with multiple, defined stereocenters. However,
accessing natural products is challenging and different
approaches are used with varying successes. For example,
isolation from plant sources is hindered by low production and
issues with separation from other structurally similar
metabolites.2 Total synthesis is commercially viable in some
cases, although can be unattainable or not cost-effective for
more complex products.3 In vivo fermentation processes
involve significant bioengineering efforts and achieving high
enantiopurities can be challenging.4,5 High regio- and stereo-
selective control under benign conditions, with minimal side-
reactions can be achieved however by mimicking the
biosynthesis in vitro, using recombinantly expressed enzymes.6
Nevertheless, this approach can be limited by challenges such
as enzyme stability issues. Cascade processes, using a
combination of traditional synthetic methods and biocatalytic
enzymes, can alternatively lead to the generation of complex
products, in fewer steps with easier purification processes than
solely in vivo or organic synthetic routes.7−9
isolated alkaloids of this type as they possess a methyl group at
C-13.11,12 Many 13-Me-PB and 13-Me-THPB alkaloids have
been shown to have promising bioactivities,13 including as
inhibitors of reverse transcriptase activity14 and enterovirus
71.15 Others have been shown to be dopamine D-1 receptor
agonists and to have antihepatitis B activities.16,17
Synthetic routes toward racemic 13-Me-PB and 13-Me-
THPB alkaloids have been reported,18−20 and between them
only a few are stereoselective. The asymmetric synthesis of
natural 13-Me-THPBs has been described for example by
Zhou et al. (Scheme 1a).21,22 After the four-step synthesis of
an enantiopure PINAP ligand, the (13R,13aS)-13-Me-THPBs
were synthesized in three-steps in yields of 47−65% and high
selectivities (91−96% ee). Previous work in our group has
shown that the Pictet-Spenglerase23 norcoclaurine synthase
(NCS) can accept α-methyl-substituted aldehydes as sub-
strates.24 Notably, an active site variant of Thalictrum flavum
NCS (Tf NCS) M97V gave (1S,1′R)-tetrahydroisoquinolines
Received: June 25, 2021
Alkaloids are important nitrogen-containing natural prod-
ucts, many of which are biologically active.10 Protoberberine
(PB) and tetrahydroprotoberberine (THPB) alkaloids isolated
from plants of the Corydalis genus are unique among other
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aldehydes. Notably, it resulted in the generation of two
defined chiral centers in the resulting THIQs, with the major
product assigned as (1S,1′R).24 Aldehyde 2 was prepared in
three steps25 and after the final step taken through without
purification due to its oxidative sensitivity. TfNCS-M97V gave
the THIQ products with 2 in quantitative yield by HPLC
analysis, with the major diastereomer generated assigned as
(1S,1′R)-3a (d.r. = 96:4).24 The minor diastereomer observed
was (1S,1′S)-3a, as no racemic NCS background reaction was
observed and NCS has been shown to generate the S-
stereochemistry at C-1.26,27 Compound 3a was isolated by
preparative HPLC (with the reaction performed on a 0.10
mmol scale, with 20 mg 1a) for characterization purposes but
otherwise was taken through directly for the cascade process to
telescope the synthetic approach. While these initial experi-
ments used 2 equiv of 2, similar results and slightly higher
stereoselectivities were observed when using just 1 equiv
(>99% conversion, 18% isolated, d.r. = 97:3) as 2 can racemize
in situ.28 The challenges of THIQ product isolation have
previously been reported29 which can lower isolated yields,
highlighting the advantages of directly taking material through
to the next step using cascaded reaction sequences which is
described below. This reaction was also amenable to scale up
with conversions and stereoselectivities retained on a 50 mL,
10 mM scale.
The para-hydroxyl group of dopamine 1a is nonessential for
a productive NCS reaction,30 so the 7-OMe THIQ 3b was also
generated using a Tf NCS-M97V reaction between 2 and the 7-
OMe dopamine analogue, 1b,26 on a 0.10 mmol scale (with 20
mg of 1b). This reduced the oxidative sensitivity of the THIQ
scaffold. The product, (1S,1′R)-3b (Scheme 2), was generated
in high yield (>99% HPLC conversion, 81% isolated) and in a
reasonable d.r. (92:8).
To generate the analogous C6-OMe-THIQ (3c), use of
regioselective O-methyltransferases (O-MTs) were explored.
Previous work has shown that two promising O-MTs,
RnCOMT (isolated from Rattus norvegicus) and MxSafC
(isolated from Myxococcus xanthus), are capable of regiose-
lectively methylating the C6-OH or C7-OH of various 1-
benzylic and 1-aryl-THIQs.26,31−34 Both are S-adenosyl-L-
methionine (SAM) dependent enzymes, and although SAM is
expensive and has issues of instability, recent developments in
SAM supply systems have meant that such biocatalytic
methylations are viable on preparative scales.35,36 Here, we
used a previously described in situ SAM generation system
which utilizes ATP, L-methionine, and a methionine adenosyl
transferase from Escherichia coli (EcMAT E.C.2.5.1.6). After
the methylation reaction, S-adenosyl-L-homocysteine (SAH) is
generated which can inhibit the O-MT, so another enzyme
methylthioadenosine/SAH nucleosidase (EcMTAN
E.C.3.2.2.9) is used to breakdown SAH.35,37 The use of
other SAM generation systems was not attempted, but other in
situ SAM supply methods could be applied.38,39
Scheme 1. Routes towards 13-Me-THPB alkaloids (a)
Previous route using a chiral catalyst;21 (b) This work using
a chemoenzymatic approach
(THIQs) in high yields (up to 96%) and diastereomeric ratios
(d.r. = 98:2) in a single-step. Herein, the application of this
methodology in combination with other biocatalytic or
chemical steps is described to generate a range of 13-Me-
THPBs isolated from Corydalis plants. Importantly, the
products generated have opposing stereochemistry to those
isolated from plants, thus providing routes to useful natural
product analogues (Scheme 1b).
To generate the desired THIQ scaffold (Scheme 2), NCS-
mediated reactions between dopamine 1a and the aldehyde
Scheme 2. Stereoselective Synthesis of the THIQ Scaffold
Using Norcoclaurine Synthase
a
d.r. values correspond to the ratio of the two diastereomers formed,
1
(1S,1′R):(1S,1′S) and were determined by H NMR spectroscopy
b
and HPLC (method 2). Conversions were determined by HPLC
analysis, based upon calibration curves of the purified products (see
c
SI). Isolated yields after preparative HPLC purification.
The THIQ substrate 3a formed (using Tf NCS-M97V) was
directly lyophilized, and since the O-MT enzymes are known
to be highly selective toward the catechol moiety and no 1a
remained after the NCS reaction, there was no need for a
purification step. Using previously reported conditions,26,31
RnCOMT was used as clarified cell lysate and MxSafC was
used as a purified enzyme. Both O-MTs interestingly exhibited
high regioselectivity toward the 6-OH, generating the product
3c (Scheme 3) with complete conversions by HPLC analysis
and no observable methylation on the 7-OH.
224,25 were investigated. Variants of Tf NCS, M97V, L76V, and
M97F, which previously gave improved selectivities compared
with the wild type for the acceptance of α-methyl phenyl-
acetaldehyde, were explored (Supporting Information (SI)
Figure S6): when this racemic aldehyde had been used in NCS
catalyzed reactions, the R-enantiomer was accepted preferen-
tially over the S-isomer.24 This had been determined by
performing reactions with single enantiomer α-methyl
B
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a b c d
, , ,
Scheme 3. Regioselective Methylations, Chemical Pictet−Spengler Reactions and Generation of the 13-Me-PB Scaffold
a
b
Conversions were determined by HPLC analysis against product standards. Conversions were determined by HPLC analysis, based upon starting
c
d
material depletion (see calibration curves SI, Figures S7−S9). Isolated yields after preparative HPLC purification. Epimeric ratio determined
based upon previous studies in which the stereochemistries at C-13 and C-13a were determined, and NOESY H NMR analysis of the isolated
1
product (see SI). Isolated yields were lower than HPLC yields/conversions due to issues of oxidative sensitivity of the compounds generated and
minor impurities having similar retention times to the desired products by preparative HPLC. To obtain the compounds in high purity for
characterization purposes, isolated yields are lower than those typically reported.
Reactions were performed on a 0.10 mmol scale (20 mg 1a).
Since both enzymes exhibited similar reactivities, yet
RnCOMT could be used as clarified cell lysate, RnCOMT
was used in subsequent reactions. Regioselectivities were
established using Nuclear Overhauser Effect Spectroscopy
(NOESY). This result is consistent with the reported
regioselectivity of these enzymes toward THIQs with a phenyl
or cyclohexane ring at C-1.26,31 However, with substrates such
as dopamine, RnCOMT methylates the meta-OH and MxSafC
the para-OH.36 Such variations in the regioselectivity high-
lights that bulkier groups attached to C-1 can hinder
methylation at the C7-OH position in THIQs for steric
reasons. Reactions were performed on a preparative scale (20
mL, 5 mM) using RnCOMT to give (1S,1′R)-3c in
quantitative conversion by HPLC against product standards
and a 12% isolated yield.
Biosynthetic routes to give the THPB scaffold involve the
berberine bridge enzyme (BBE).40 The enantioselective
production of various (S)-THPBs from racemic, N-methylated,
1-benzylic THIQs using recombinant BBE has been achieved
in high conversions (98%) and selectivities (>97% ee).41
Although a highly productive route, there is the requirement to
generate the starting material and in situ deracemization of the
(R)-THIQ is needed, involving a selective monoamine oxidase
and borane reduction. Here, to generate the tetracyclic THPB
scaffold, chemical Pictet−Spengler (PS) reactions were
explored (Scheme 3a).42 Previous work has demonstrated
that a phosphate-mediated PS reaction with formaldehyde was
capable of converting 1-benzylic-THIQs into THPBs in high
yields and good regioselectivities (7:1 10,11-OMe:9,10-OMe
THPB).43 Both reported reaction conditions were attempted
here with 3c, but no conversion was observed, presumably
because the aromatic dimethoxy groups reduce the reactivity of
the substrates compared to phenolic groups. Instead, formic
acid catalyzed PS reactions were explored as described by Qian
et al.44 A telescoped synthesis was then performed, with crude,
lyophilized 3a or 3c used. Complete conversion, by monitoring
the consumption of starting material by HPLC to the products
(13S,13aR)-4a and (13S,13aR)-4b, was observed with 24%
and 23% isolated yields, over two or three steps, respectively.
Reactions were performed on a 5 mL, 10 mM scale. Complete
regioselectivity was observed on the D-ring (see Scheme 1),
with solely 10,11-dimethoxy-substituted products generated.
Nuclear Overhauser Spectroscopy (NOESY) analysis of the
products formed (4a and 4b) also provided further
confirmation of the stereochemistries at C-13 and C-13a, i.e.,
a syn-relationship between the two protons at these positions.
8-Me-THPB and 8-Me-PB alkaloids have been isolated from
C. ochotensis, and one 8-Me-PB has been shown to act as an
antileukemic agent.45 Stereoselective routes to 8-Me THPBs
are limited,46 and routes to analogues are useful for drug
discovery purposes. A chemical PS reaction in formic acid with
acetaldehyde was therefore explored, with the aim of
generating functionalized 8-THPBs. Crude, lyophilized 3c
was used again in a telescoped synthesis (0.050 mmol scale).
Product 4c was formed in a 56% conversion yield (starting
material consumption by HPLC) and 21% isolated yield over
two steps (Scheme 3). Two epimers at C-8 of the product
were observed in a 3:1 ratio by NMR spectroscopy. The
stereochemistries at C-13 and C-13a were retained, and the
major epimer was assigned by NOESY analysis as
(8S,13R,13aS)-4c. Modeling of both epimers was used to
rationalize these results. In both cases, the C-ring is held in a
half-chair conformation and, for the minor isomer, the two
methyl groups are cis to each other in pseudoaxial orientations
giving an unfavorable steric interaction (SI Figure S10).
Therefore, the stereochemistry at C-13 can lead to stereo-
control at C-8 in the cyclization to give 4c.
The regioselectivity of D-ring formation has been reported
to be solvent-mediated,47,48 with the other regioisomer of
product preferentially formed in apolar, aprotic solvents such
as toluene or dichloroethane. As 3c has poor solubilities in
such solvents, DMF was used with reactions performed at 120
°C on a 0.10 mmol scale (20 mg 1a). Complete consumption
C
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of 3c was observed after 18 h, and interestingly 5 was formed
via a cyclization and oxidation in 5% isolated yield (over three
steps from 1a and 2 with no purification of the intermediates).
The oxidation is presumably mediated by trace oxidants in the
crude material taken through and may prove useful in
generating the PB scaffold from the analogous THPB, as the
oxidation of the C-ring must occur after the PS reaction. It
would seem that the presence of activating methoxy groups on
the D-ring and the use of high temperatures also help to drive
the reaction. The unpurified THPB 4b (formed from 3c) was
also heated in DMF at 120 °C for 18 h with 86% of starting
material converted by HPLC, and this gave 5 as the only
product in 21% isolated yield. This is a useful route to PBs
from the analogous THPB, avoiding the addition of oxidase
enzymes49 or oxidants (I2/EtOH, reflux).50 Since the stereo-
chemistry is lost during this oxidation, the racemic THIQ
starting material could also be formed by a phosphate-
mediated PS reaction which has been shown to be
regioselective with α-methyl substituted aldehydes.24 The PB
scaffold is synthetically useful, as the iminium ion is susceptible
to nucleophilic attack, leading to C-8 functionalization.51
In summary, a range of novel (−)-13-Me-THPB alkaloids
have been generated with opposing stereochemistries at C-13
and C-13a to the naturally occurring 13-Me-THPB alkaloids
from Corydalis plants. Single regio- and diastereomer 13-Me-
THPBs were formed through two subsequent PS reactions:
one enzymatic reaction to generate a THIQ scaffold with two
defined stereocenters followed by a chemical PS reaction to
generate the tetracyclic scaffold. Regioselective methylation of
a single hydroxyl group of the THIQ was also achieved using
O-MTs. Heating the 13-Me-THPBs in DMF provided a facile
route to the analogous 13-Me-PB. All enzymatic reactions were
high yielding, and there was no requirement for the isolation of
material at each reaction step. High stereoselectivity was also
obtained without the need for chiral ligands or precursors, and
toxic reagents.
Joseph Broomfield − Department of Chemistry, Christopher
Ingold Building, University College London, London WC1H
0AJ, U.K.
John M. Ward − Department of Biochemical Engineering,
Bernard Katz Building, University College London, London
WC1E 6BT, U.K.
Nicholas H. Keep − Department of Biological Sciences,
Institute of Structural and Molecular Biology, Birkbeck
College, London WC1E 7HX, U.K.; orcid.org/0000-
0002-5042-1837
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c02110
Author Contributions
The manuscript was written through contributions of all
authors. R.R. and J.B. performed chemical syntheses. R.R.
performed synthesis and characterization, enzyme expression
and purification, and the chemoenzymatic cascades. F.S.
provided input on enzymatic reactions. The project was
supervised by H.C.H, N.H.K., and J.M.W. The manuscript was
written by R.R. and H.C.H. All authors have given approval to
the final manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Birkbeck College, for a
Birkbeck Anniversary PhD scholarship to R.R. Also, the
Biotechnology and Biological Sciences Research Council
(BBRSC) (BB/R021643/1) and 17-ERACoBioTech are
thanked for financial support of this work to F.S. as part of
the project BioDiMet. We also thank Jennifer N. Andexer
(Institute of Pharmaceutical Sciences, University of Freiburg)
and Michael Richter (Fraunhofer Institute for Interfacial
Engineering and Biotechnology, Straubing) for MTs
(RnCOMT, MxSafC), EcMAT and EcMTAN plasmids.
Furthermore, we gratefully thank the UCL Mass Spectrometry
and NMR Facilities in the Department of Chemistry and
EPSRC (EP/P020410/1) for 700 MHz NMR equipment
support.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c02110.
1
Experimental procedures and H/13C NMR spectra for
the compounds synthesized (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
Helen C. Hailes − Department of Chemistry, Christopher
Ingold Building, University College London, London WC1H
0AJ, U.K.; orcid.org/0000-0001-5574-4742;
Email: h.c.hailes@ucl.ac.uk
Authors
Rebecca Roddan − Department of Biological Sciences,
Institute of Structural and Molecular Biology, Birkbeck
College, London WC1E 7HX, U.K.; Department of
Chemistry, Christopher Ingold Building, University College
London, London WC1H 0AJ, U.K.
Fabiana Subrizi − Department of Chemistry, Christopher
Ingold Building, University College London, London WC1H
0AJ, U.K.
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