Chemoenzymatic Total Synthesis of Deoxy-, epi-, and Podophyllotoxin and a Biocatalytic Kinetic Resolution of Dibenzylbutyrolactones

Article abstract of DOI:10.1002/anie.201900926

Podophyllotoxin is probably the most prominent representative of lignan natural products. Deoxy-, epi-, and podophyllotoxin, which are all precursors to frequently used chemotherapeutic agents, were prepared by a stereodivergent biotransformation and a biocatalytic kinetic resolution of the corresponding dibenzylbutyrolactones with the same 2-oxoglutarate-dependent dioxygenase. The reaction can be conducted on 2 g scale, and the enzyme allows tailoring of the initial, “natural” structure and thus transforms various non-natural derivatives. Depending on the substitution pattern, the enzyme performs an oxidative C?C bond formation by C?H activation or hydroxylation at the benzylic position prone to ring closure.

Full text of DOI:10.1002/anie.201900926

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Chemoenzymatic Total Synthesis of deoxy-, epi- and
Podophyllotoxin and a Biocatalytic Kinetic Resolution of
Dibenzylbutyrolactones
Authors: Mattia Lazzarotto, Lucas Hammerer, Michael Hetmann,
Annika Jasmin Eveliina Borg, Luca Schmermund, Lorenz
Steiner, Peter Hartmann, Ferdinand Belaj, Wolfgang Kroutil,
Karl Gruber, and Michael Fuchs
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900926
Angew. Chem. 10.1002/ange.201900926
Link to VoR: http://dx.doi.org/10.1002/anie.201900926
http://dx.doi.org/10.1002/ange.201900926

10.1002/anie.201900926
Angewandte Chemie International Edition
COMMUNICATION
Chemoenzymatic Total Synthesis of deoxy-, epi- and
Podophyllotoxin and a Biocatalytic Kinetic Resolution of
Dibenzylbutyrolactones
Mattia Lazzarotto,[a] Lucas Hammerer,[b] Michael Hetmann,[c] Annika Borg,[a] Luca Schmermund,[a]
Lorenz Steiner,[a] Peter Hartmann,[a] Ferdinand Belaj,[d] Wolfgang Kroutil,*[a] Karl Gruber[c] and
Michael Fuchs*[a]
This study is dedicated to Prof. Kurt Faber on the occasion of his retirement.
Abstract: Podophyllotoxin (1) is probably the most prominent
representative of lignan natural products. Deoxy-, epi- and
C-H bond via an iron-heme center at the expense of oxygen.[2]
While significant efforts have been dedicated to CYPs,[3] 2-
oxoglutarate dependent dioxygenases (2-ODDs) have gained
rather moderate attention thus far.[4] Predominantly, the
hydroxylation of aminoacids has been studied in a biocatalytic
context (e.g. hydroxylation of proline[5] and leucine/isoleucine[6]).
Nevertheless, recent reports demonstrate the high biocatalytic
potenital of these proteins (even on gram scale).[7] Besides these
studies, the portfolio of reactivity is far beyond “simple”
hydroxylation reactions: Many natural products’ biosyntheses rely
on 2-ODDs and reactions such as selective oxidation of sugar
moieties,[8] endo-peroxide formation,[9] ring expansion[9] and
contraction,[10] selective halogenations[11] and multi-step
oxidations[12] have been attributed to 2-ODDs. Recently, the
reactivity has been extended to the ring closure of the C-ring in
the podophyllotoxin (1) biosynthesis by Podophyllum
hexandrum[13] and Sinopodophyllum hexandrum.[14] The 2-ODD
enzyme – further referred to as 2-ODD-PH – catalyzes the
cyclization of yatein (2a) and yields deoxypodophyllotoxin (12a)
at the expense of 2-oxoglutaric acid and oxygen (see Figure 1).
podophyllotoxin
–
all precursors to frequently used
chemotherapeutic agents – were prepared via a stereodivergent
biotransformation and a biocatalytic kinetic resolution of the
corresponding dibenzylbutyrolactones by the same 2-
oxoglutarate dependent dioxygenase. The reaction can be
upscaled to two grams and the enzyme allows tailoring of the
initial “natural” structure transforming various non-natural
derivatives. Depending on the substitution, the enzyme performs
an oxidative C-C bond formation via C-H activation or
hydroxylation at the benzylic position prone to the ring closure.
The selective activation of inert C-H bonds represents one of the
most remarkable reactions in modern organic chemistry.[1] At the
same time it can be considered as one of the biggest challenges
as well. Organometallic chemistry has provided an impressive
assembly of examples requiring in most cases adjacent directing
groups to achieve selectivity.[1] In contrast to these endeavours,
nature provides selectivity for C-H activation by the enzyme’s
active site, which aligns the substrate(s) properly towards the
active center. The most prominent representatives are
cytochrome P450 monooxygenases (CYPs), which activate the
[a]
Msc. M. Lazzarotto, Msc. L. Hammerer, Msc. A. Borg, Msc. L.
Schmermund, L. Steiner, P. Hartmann, Prof. W. Kroutil, Dr. M.
Fuchs
Institute of Chemistry, Organic and Bioorganic Chemistry
University of Graz
Heinrichstrasse 28/II, 8010 Graz, Austria
E-mail: michael.fuchs@uni-graz.at
wolfgang.kroutil@uni-graz.at
[b]
[c]
Msc. L. Hammerer
Austrian Centre of Industrial Biotechnology, c/o University of Graz,
Graz, Austria
Msc. M. Hetmann, Prof. K. Gruber
Institute of Molecular Biosciences, University of Graz
University of Graz
Figure 1. Reaction of 2-ODD-PH in nature and structures of pharmaceutical
compounds.
Humboldtstraße 50/III, 8010 Graz, Austria
Prof. F. Belaj
[d]
Institute of Chemistry, Inorganic Chemistry
University of Graz
Schubertstraße 1/III, 8010 Graz, Austria
Podophyllotoxin (1) is the precursor of etoposide (4) and
teniposide (5), two chemotherapeutic agents used in several
clinical therapies. Both compounds inhibit the topoisomerase II, a
key enzyme in cell mitosis, by forming a ternary complex with the
DNA and isomerase enzyme. This prevents religation of the DNA
strands and ends up in cell apoptosis. As cancer cells proliferate
more rapidly, their cell death occurs preferentially.[15]
CCDC 1889727 contains the supplementary crystallographic data
for product 12h. These data are provided free of charge by The
Cambridge Crystallographic Data Centre.
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/anie.201900926
Angewandte Chemie International Edition
COMMUNICATION
within both substrate enantiomers. However, the new chiral
center formed during C-C bond formation possesses the same
configuration for both substrate enantiomers leading to an
enantiodivergent reaction (two diastereomers are formed).
Consequentially, the enzyme overrides the substrate controlled
stereopreference for the ring formation.[18a] The diastereomeric
mixture complicated the purification and pure products were
finally obtained via preparative HPLC only.
These results encouraged us to challenge the biocatalyst by a
substrate congener with an additional chiral center like the
hydroxyl group present in podophyllotoxin’s northern benzylic
position (see Figure 1). In order to obtain the “natural” relative
configuration of the OH group as present in podophyllotoxin (1),
we performed a Mitsunobu inversion on intermediate rac-2d, and
cleaved off the ester moiety in a two-step protocol yielding
substrate rac-2b (see Scheme 1b).
Etoposide (5) therefore became a member of the WHO’s list of
essential medicines.[16] Since other closely related compounds
exhibit interesting bioactivities too (e.g. cytotoxic, insecticidal,
antifungal,
antiviral,
anti-inflammatory,
neurotoxic,
immunosuppressive, antirheumatic, antispasmogenic and
hypolipidemic properties),[17] the structural motif has been the
subject of several synthetic research endeavours. Nevertheless,
the asymmetric construction of the yatein precursor (2a) and the
stereoselective ring closure of ring C still represent major
bottlenecks in the synthesis. Especially the construction of the
southern stereocenter with the appropriate configuration has
been remarkably difficult via the Friedel-Crafts approach, both
chemically[18] and via biotechnologic methods.[19] Some of these
challenges have been recently resolved, but require expensive
reagents or catalysts.[20] Therefore, a chemoenzymatic, scalable
route to the target compound podophyllotoxin (1) and the
biocatalyst’s substrate scope demonstrate an extremely tempting
scientific target.
Table 1. Biotransformations with 2-ODD-PH.
Aiming for an efficient synthesis by integrating biocatalysis[21] en
route towards podophyllotoxin, the racemic yatein precursor (rac-
2a) was prepared following a slightly modified literature protocol
(see Scheme 1a).[22] A simple switch to less polar solvents for the
allylation step gave better diastereomeric ratios in our hands
compared to literature and the target compound was obtained in
good yields (82% overall yield).
HPLC area
[%][a]
Yield [%][b]
(ee)
Substrate
ee of
2a-c
[%][c]
Entry
R1 =
R2 =
R3 =
R4 =
11
12
11
12
#
a-c
a-c
a-c
a-c
rac
-2a
H
H
1
2[f]
3
–CH2–
–CH2–
27[d]
-
29
5
19[e]
-
10
46
26
rac
-2b
H
OH
-
-
15
4
rac
-2c
Me
Me
OH
H
12
7
Reaction conditions: Cell-free extract (CFE, 44 vol-%), 20 mM substrate, 2-
oxoglutarate (1.75 eq.), sodium ascorbate (3 eq.), 23% DMSO as cosolvent;
[a] determined via peak area integration of the HPLC-UV chromatogram (at
215 nm); [b] Isolated yields of chromatographically pure and fully
characterized products are reported; [c] ee was determined via HPLC-UV
on a chiral stationary phase (for details see supporting information); [d] plus
37% of diastereoisomer 3; [e] 20% of compound 3 were isolated from the
same batch; []D20 values for 11a and 3 are in full consistency with literature
values;[24] [f] experiment was conducted on 50 mg substrate scale. Bold and
dashed lines refer to relative stereochemistry, bold and dashed wedges
refer to absolute stereochemistry.
Scheme 1. (a) Preparation of rac-yatein (rac-2a); (b) Preparation of substrate
rac-2b: installation of the “natural” stereoconfiguration. Bold and dashed lines
refer to relative stereochemistry.
When transforming substrate rac-2b the biocatalyst turned out to
be enantioselective leading to a kinetic resolution (see Table 1,
entry 2). Only one enantiomer was transformed, but instead of the
expected ring-closed aryltetralin structure, product 12b was
obtained with the benzylic position – formerly prone to cyclization
– hydroxylated. Probably substrate 2b may not be positioned
properly in the enzyme preventing the C-C bond formation and
favouring hydroxylation instead. Interestingly, substrate 2c (Table
1, entry 3), which bears two methoxy groups on the western
aromatic ring system, led to a mixture of products obtained from
ring-closure (11c) and hydroxylation (12c). These findings are in
consistency with a recent mechanistic study, which observes
related hydroxylated products.[23] In agreement with this report, a
cationic mechanism involving a Friedel Crafts alkylation scenario
is the most likely mechanistic pathway of the reaction.
Nevertheless, a radical pathway cannot be ruled out completely
(for a detailed outline of the catalytic mechanism please consult
For getting access to a sufficient amount of the enzyme 2-ODD-
PH, the gene was expressed in E. coli BL21(DE3) using a
pET21(a)+ vector system. Since first ca. 80% of the catalyst
remained insoluble and inactive, the expression was improved by
late induction (at OD600 = 1.2 - 1.4), yielding about fifty percent of
the enzyme in the soluble fraction at a high expression level (33
mg of soluble enzyme per 1.0 g of cells).
When performing the reaction with 2-ODD-PH (see supporting
information for further details) substrate rac-2a was recovered
with an ee of 10% (26% recovered material; see Table 1, entry 1),
while two enantiopure products were observed. We were able to
identify these two products as the diastereoisomers
deoxypodophyllotoxin (12a, 19% isolated yield) and
isodeoxypodophyllotoxin (3, 20% isolated yield). Thus, the
biocatalyst is non-stereoselective concerning the two chiral
centers present in the substrate 2a and forms the new C-C bond
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10.1002/anie.201900926
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COMMUNICATION
the ESI provided) and structural information of the enzyme is
required to provide further arguments for either way.
(enzyme:substrate 1:1.2) a phenyl substituent in the southern
position was shown to be accepted by the enzyme recently.[23]
As substrate rac-2b was transformed via a kinetic resolution, the
hydroxyl function at the northern benzylic position seems to play
a fundamental role in recognition. rac-2d possesses the opposite
relative configuration for this hydroxy group and therefore we
tested this compound too (see Table 2, top scheme). The results
clearly show, that with this stereoconfiguration the aromatic
moiety seems to be aligned in the proper way and the ring
formation was observed exclusively in a kinetic resolution of
substrate rac-2d. Only the enantiomer possessing the same
absolute configuration for the northern hydroxyl group as in
etoposide (4) was transformed. Consequently, product 11d bears
the same stereoconfiguration as the APIs etoposide (4) and
teniposide (5, see Figure 1) which may facilitate the follow up
steps towards these compounds.
Performing preparative reactions, the measured conversion was
confirmed by the isolated yield. We were able to obtain 770 mg
(39% isolated yield) of the target compound 11d from 235 mL
reaction mixture within 18 h. This corresponds to a space time
yield of 200 mg L-1 h-1 and an overall yield of 32% over the four
steps (note that a late stage kinetic resolution was performed,
which is limited to 50% yield by theory). The discrepancy in
conversion between the small scale and the upscale experiments
(see Table 2) results most likely from different surface to volume
ratios leading to a lower oxygen input as well as from deactivation
of the enzyme over time. Additionally, kinetics imply that the
reaction is actually performed within two to four hours only (see
Tables SI13a and SI13b in the supporting information; the upscale
experiment was performed for 16 h). This sums up to a space time
yield of 1.6 – 0.8 g L-1 h-1 for the analytical scale and we assume
that similar numbers can be obtained in upscale experiments.
Scheme 2. Preparation of podophyllotoxin (1) from its epi-congener 10b.
Despite the suitable configuration of the northern hydroxyl
function (vide supra), almost all substrates tested yield the
hydroxylated product type 12e-m (see Figure 2). Only substrate
2l, which has the methoxy substituent in para-position towards the
ring closing site, provided the aryltetraline product 11l as the
major product. It is worth to mention, that it could be shown that
the hydroxylated product 12l does not spontaneously cyclize
under these reaction conditions. A schematic overview about the
substrate alterations and their consequences can be found in
Figure 2 (for list of substrates, which were not accepted, please
consult the ESI provided):
Table 2. Biocatalytic transformation of substrate 2d by 2-ODD-PH.
11d [%]
Yield[b]
(+)-2d [%]
Yield[b] ee[c]
Conv.
[%][a]
Entry
Scale
ee[d]
99
[e]
[e]
1
2
3
1 mg
100 mg
2.0 g
50
43
41
-
-
99
72
66
38
39
95
50
45
95(>99[f])
Figure 2. Substrate Scope of the 2-ODD-PH enzyme; schematic overview of
the variable positions (top); two product structures (middle); substrate scope -
alterations to substrate 2d are indicated with black bold lines and letters
(bottom).
Reaction conditions: CFE (44 vol-%), 20 mM substrate, 2-oxoglutarate (1.75
eq.), sodium ascorbate (3 eq.), 23% DMSO as cosolvent, 18 h; absolute
configurations were determined via comparison to literature values;[24] [a] the
conversion was determined via calibrated HPLC-UV spectra. [b] Isolated
yields of chromatographically pure and fully characterized products are
reported; [c] ee was determined via HPLC-UV on a chiral stationary phase
(for details see supporting information); [d] ee was determined from
conversion and ee of the remaining substrate;[25] [e] no isolated yield was
determined; [f] ee determined via HPLC-UV analysis of the follow-up product
1 (see Scheme 2).
The lactone moiety and the southern aromatic fragment are
required for high enzyme activity. The northern hydroxyl function
can be removed, but may interfere with the stereorecognition and
the product outcome (vide supra). Lager substituents – namely
acetoxy moieties – gave no conversion (see ESI). The most
flexible part of the structural motif is the western aromatic moiety,
which accepts several substitution patterns in meta- and para-
position. Substitution in ortho-position ceases the catalytic activity
below the detection limit. In meta- and para-position electron
withdrawing as well as electron donating substituents are
accepted with similar yields of the obtained products. Even bulkier
substrates (e.g. naphthyl, substrate 2f) are accepted, whereas
heteroaromatic residues (in detail a furyl substituent) led to no
catalytic activity (see ESI).
Consequently, the actual natural product podophyllotoxin (1) was
prepared from 11d according to a literature protocol within two
additional steps (17 % overall yield over five steps, see Scheme
2).
Next, we focused on the substrate scope of the 2-ODD-PH
enzyme: The results are depicted in Figure 2. Whereas the
southern aromatic ring requires the trimethoxy motif for high
enzyme activity, the substituents on the western aromatic system
can be altered. Nevertheless, at high enzyme concentrations
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10.1002/anie.201900926
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COMMUNICATION
The crystal structure, which we obtained from product 12h
(isolated from the biotransformation of compound 2h), delivers
information of the accepted enantiomer and the stereoselectivity
of the hydroxylation (see Figure 2).
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Summarizing, the chemoenzymatic, target oriented synthesis of
podophyllotoxin (1) and its epi-congener 11d was successfully
achieved employing a steroselective biocatalytic C-C bond
formation as the key step. This enzymatic transformation lacks
analogies in conventional organic chemistry as it overrides the
stereopreference of the ring closure and provides a short way to
the precursor of etoposide (4) and teniposide (5). The latter are
key compounds in chemotherapies. The substrate screening
delivered
predominantly
products
12e-m,
and
thus
dibenzylbutyrolactones – another subclass of lignan natural
products – in enantiopure form. The biocatalytic C-C bond
formation was incorporated into a target oriented synthesis of epi-
podophyllotoxin (11d) and the kinetic resolution was performed
on a two gram substrate scale. The study opens a new avenue
into the synthesis of key therapeutic agents and homologues
using a biocatalytic C-C bond formation and represents one of the
rare examples of the target oriented application of this class of
enzymes.
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Acknowledgements
M. Fuchs and M. Lazzarotto are grateful for an FWF grant (P
31276-B21). L. Hammerer was financed by the Austrian FFG,
BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the
Austrian FFG-COMET-Funding Program. Philipp Neu is highly
acknowledged for the HRMS measurements and Bernd Werner
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Keywords: 2-Oxoglutarate Dependent Dioxygenase •
Podophyllotoxin • Lignans • Biocatalysis • Total Synthesis
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10.1002/anie.201900926
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
Taming the iron: Podophyllotoxin
and congeners were prepared via a
chemoenzymatic total synthesis
Mattia Lazzarotto, Lucas Hammerer,
Michael Hetmann, Annika Borg, Luca
Schmermund, Lorenz Steiner, Peter
Hartmann, Ferdinand Belaj, Wolfgang
Kroutil, Karl Gruber and Michael
Fuchs*
employing
the
2-oxoglutarate
dependent
Podophyllum
iron
enzyme
from
Via
hexandrum.
substrate engineering the reaction
outcome could be changed from an
Page No. – Page No.
enantiodivergent
to
a
kinetic
resolution process on a two gram
scale. Modifications of the substrate
allowed the kinetic resolution of
dibenzylbutyrolactones.
Chemoenzymatic Total Synthesis of
deoxy-, epi- and Podophyllotoxin
and a Biocatalytic Kinetic
Resolution of
Dibenzylbutyrolactones
This article is protected by copyright. All rights reserved.