Exploiting the Potential of Meroterpenoid Cyclases to Expand the Chemical Space of Fungal Meroterpenoids

Article abstract of DOI:10.1002/anie.202011171

Fungal meroterpenoids are a diverse group of hybrid natural products with impressive structural complexity and high potential as drug candidates. In this work, we evaluate the promiscuity of the early structure diversity-generating step in fungal meroterpenoid biosynthetic pathways: the multibond-forming polyene cyclizations catalyzed by the yet poorly understood family of fungal meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were accessed chemoenzymatically using synthetic substrates. Their complex structures were determined by 2D NMR studies as well as crystalline-sponge-based X-ray diffraction analyses. The results obtained revealed a high degree of enzyme promiscuity and experimental results which together with quantum chemical calculations provided a deeper insight into the catalytic activity of this new family of non-canonical, terpene cyclases. The knowledge obtained paves the way to design and engineer artificial pathways towards second generation meroterpenoids with valuable bioactivities based on combinatorial biosynthetic strategies.

Full text of DOI:10.1002/anie.202011171

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Exploiting the Potential of Meroterpenoid Cyclases to Expand the
Chemical Space of Fungal Meroterpenoids
Authors: Ikuro Abe, Takaaki Mitsuhashi, Lena Barra, Zachary Powers,
Volga Kojasoy, Andrea Cheng, Feng Yang, Yoshimasa
Taniguchi, Takashi Kikuchi, Makoto Fujita, Dean J. Tantillo,
and John A. Porco
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202011171
Link to VoR: https://doi.org/10.1002/anie.202011171

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Exploiting the Potential of Meroterpenoid Cyclases to Expand the
Chemical Space of Fungal Meroterpenoids
Takaaki Mitsuhashi,+[a,b] Lena Barra,+[a] Zachary Powers,+[c] Volga Kojasoy,+[d] Andrea Cheng,[c] Feng
Yang,[c] Yoshimasa Taniguchi,[e] Takashi Kikuchi,[f] Makoto Fujita,[b,g] Dean J. Tantillo,*[d] John A. Porco,
Jr.,*[c] and Ikuro Abe*[a,h]
[a]
Dr. Takaaki Mitsuhashi,[+] Dr. Lena Barra,[+] Prof. Dr. Ikuro Abe
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
E-mail: abei@mol.f.u-tokyo.ac.jp
[b]
[c]
Dr. Takaaki Mitsuhashi,[+] Prof. Dr. Makoto Fujita
Division of Advanced Molecular Science, Institute for Molecular Science
National Institutes of Natural Sciences
5-1 Higashiyama, Myodaiji, Okazaki, 444-8787, Japan
Zachary Powers,[+] Andrea Cheng, Feng Yang, Prof. Dr. John A. Porco, Jr.
Department of Chemistry and Center for Molecular Discovery (BU-CMD)
Boston University
Boston, Massachusetts, 02215, United States
E-mail: porco@bu.edu
[d]
[e]
Volga Kojasoy,[+] Prof. Dr. Dean J. Tantillo
Department of Chemistry
University of California Davis
1 Shields Avenue, Davis, California 95616, United States
E-mail: djtantillo@ucdavis.edu
Dr. Yoshimasa Taniguchi
Central Laboratories for Key Technologies
Kirin Holdings Co. Ltd.
1-13-5, Fukuura Kana-zawa-ku, Yokohama-shi, Kanagawa, 236-0004 Japan
Dr. Takashi Kikuchi
[f]
Rigaku Corporation
3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan
Prof. Dr. Makoto Fujita
[g]
Department of Applied Chemistry, Graduate School of Engineering
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656
Prof. Dr. Ikuro Abe
Collaborative Research Institute for Innovative Microbiology
The University of Tokyo
Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
These authors contributed equally to this work
[h]
[+]
Supporting information for this article is given via a link at the end of the document.
Abstract: Fungal meroterpenoids are a diverse group of hybrid
natural products with impressive structural complexity and high
potential as drug candidates. In this work, we evaluate the promiscuity
of the early structure diversity-generating step in fungal
meroterpenoid biosynthetic pathways: the multibond-forming polyene
cyclizations catalyzed by the yet poorly understood family of fungal
meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were
accessed chemoenzymatically using synthetic substrates. Their
complex structures were determined by 2D NMR studies as well as
crystalline-sponge-based X-ray diffraction analyses. The results
Introduction
Fungal meroterpenoids have earned significant interest from the
scientific community as well as from the pharmaceutical and
chemical industry due to their remarkable structural architectures
and potent bioactivities.[1] Pyripyropene A (1), isolated from
Aspergillus fumigatus, is the strongest known inhibitor of acyl-
CoA:cholesterol acyltransferase and is being developed for the
treatment of atherosclerosis.[2] Additionally, 1 exhibits insecticidal
properties and
a
commercial insecticide based on the
pyripyropene core structure has been recently marketed.[3]
Derivatives of mycophenolic acid (2), isolated from Penicillium sp.,
are clinically used immunosuppressant drugs and inhibit inosine
obtained revealed
a high degree of enzyme promiscuity and
experimental results, together with quantum chemical calculations
provided a deeper insight into the catalytic activity of this new family
of non-canonical terpene cyclases. The knowledge obtained paves
the way to design and engineer artificial pathways towards second
generation meroterpenoids with valuable bioactivities based on
combinatorial biosynthetic strategies.
5‘-monophosphate dehydrogenase.[4] Andrastin
Penicillium albocoremium is an inhibitor of protein farnesyl
transferase and
potent anti-cancer agent,[5] whereas
tropolactone D (4) from Aspergillus sp. is a cytotoxic agent against
human colon carcinoma (Figure 1A).[6] The genetic basis for
fungal meroterpenoid biosynthesis has only been elucidated in
A (3) from
a
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
recent years, with the first biosynthetic gene cluster reported for
pyripyropene A in 2010.[7]
meroterpenoid cyclases is still lacking.[7-9] Phylogenetic analysis
of characterized meroterpenoid cyclases shows a close relation
to the group of Pyr4-like synthases involved in fungal indole
diterpene biosynthesis (LtmB, AtmB, and PaxB) and a distant
relation to the bacterial enzymes XiaE and DmtA1 (Figure 2).[10]
A
protein sequence alignment for selected enzymes is shown in
Figure S6A.
Figure 1. A) Selected examples of fungal meroterpenoids. B) Proposed
catalytic mechanism of meroterpenoid cyclases exemplified by Pyr4-mediated
reaction of epoxyfarnesyl-HPPO (5) to deacetyl pyripyropene E (6). The
polyketide portion is shown in green, terpenoid in blue.
Since then, the discovery of several related gene clusters
revealed a common modular assembly logic for all meroterpenoid
pathways, composed of i) building block generation (polyketide
synthase, oligoprenyl synthase), ii) assembly of building blocks
(prenyltransferase), iii) early structural diversification by the action
of a novel family of terpene cyclases in tandem with an epoxidase
and iv) late stage diversification by tailoring enzymes such as
cytochrome P450 monooxygenases and a-ketoglutarate-
dependent dioxygenases.[8] Their strong biological activities as
well as the conserved modular logic of their biosynthetic pathways
make meroterpenoids attractive targets for artificial pathway
engineering to generate novel structures with new or improved
activities. Herein, we set out to evaluate the potential of
meroterpenoid cyclases to generate novel scaffolds by employing
natural and unnatural synthetic substrate analogues. The results
obtained provide valuable information on matching pathway
combinations regarding the interchangeability of employed
meroterpenoid cyclases. At the same time, chemoenzymatic
access to eight new scaffolds, thus far unprecedented from
natural sources or chemical, biomimetic polyene cyclizations,
could be achieved.
Figure 2. Phylogenetic analysis of reported meroterpenoid cyclases and their
respective native substrates.
The substrates of known meroterpenoid cyclases are composed
of a linear epoxyoligoprenyl chain, in most cases derived from
farnesyl diphosphate (FPP) and a distinct non-terpenoid portion
usually generated by a designated polyketide synthase (Figure 2).
These biosynthetic intermediates are difficult to obtain from the
natural producer, since typically low concentrations are observed
and the frequently employed heterologous expression host
Aspergillus oryzae contains endogenous hydrolases producing
high amounts of a shunt diol product.[7] To overcome this limitation,
we recently developed a modular synthesis of the widespread 3,5-
dimethylorsellinic acid (DMOA)-containing substrate family. The
methodology
involves
base-mediated,
regioselective
dearomatization of DMOA with farnesyl electrophiles.[11] We
expanded the synthetic scope by employing enantiopure (10R)-
and (10S)-epoxyfarnesyl building blocks in this reaction to obtain
the naturally occurring substrate (10R)- and (10S)-(2E,6E)-5’-
DMOA methylester (7a, 7b), as well as (10R)- and (10S)-(2E,6E)-
3’-DMOA methylester (8a, 8b). Additionally, we also accessed the
(2Z,6E)-epoxyfarnesyl congeners (10R)- and (10S)-(2Z,6E)-5’-
DMOA methylester (9a, 9b) and (10R)- and (10S)-(2Z,6E)-3’-
DMOA methylester (10a, 10b) using the same strategy (Figure S1,
Figure 3). As dearomative alkylation of DMOA leads to formation
of two inseparable diastereoisomers with respect to 3’ and 5’ ring
substitution, the substrates obtained were used as diastereomeric
mixtures, exhibiting fixed stereochemistry for the epoxide moiety
(88% – 98% ee). With substrates in hand, we targeted nine
reported meroterpenoid cyclases: Pyr4[7], CdmG[12], AndB[13], AdrI’
Results and Discussion
Targeted Enzymes and Synthesis of Substrates
Non-canonical terpene cyclases involved in meroterpenoid
biosynthesis are integral membrane proteins of compact size (ca.
25 kDa).[7,8] Mutagenesis studies on the model cyclase Pyr4
involved in the pyripyropene biogenesis revealed two highly
conserved acidic amino acid residues (Glu63 and Asp218) crucial
for enzyme function and proposed to initiate polyene cyclization
by protonation of the priorly introduced terminal epoxide function
in the prenyl chain, triggering the subsequent polyene cyclization
(Figure 1B). The mechanism resembles that of type-II terpene
synthases of the 2,3-oxidosqualene-lanosterol cyclase type;
however, protein structural data to evaluate the mechanism of
[14], NvfL[15], PrhH[16], Trt1[17], AscF,[18] and MacJ[19]
.
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Pyr4, a cyclase which naturally utilizes the (10S)-configured
epoxide 5 (Figure 1B), was found to accept (10S)-(2E,6E)-5’-
DMOA (7a), (10S)-(2E,6E)-3’-DMOA (8a) as well as (10S)-
(2Z,6E)-3’-DMOA (10a) based on the detection of newly formed
peaks in the HPLC chromatogram (Table 1, Figure S2- S5). To
elucidate the structures of the putative new enzyme products, we
carried out preparative scale transformations in which case
reaction of Pyr4 with substrate 7a led to the isolation of
compounds 11 and 12 (Scheme 1A).
Figure 3. Synthetic approach and structures of obtained and tested DMOA
substrates (7a/7b – 10a/10b).
As we did not gain access to the MacJ producer strain, we cloned
the homologous protein MacJ’ from Penicillium chrysogenum MT-
12 (96% identity). The intron-free genes were expressed in the
heterologous host Saccharomyces cerevisiae INVSc1 and cell
free extracts were prepared and utilized for in vitro reactions with
synthetic substrates 7a/7b – 10a/10b. Monitoring of the reactions
by HPLC revealed a surprisingly high degree of promiscuity as
several new products were detected (Figure S2-S5, Table 1).
Table 1. Overview on productive enzyme substrate combinations.
(2E,6E)-5’-
(2E,6E)-3’-
(2Z,6E)-5’-
(2Z,6E)-3’-
DMOA
DMOA
DMOA
DMOA
Scheme 1. Structures of isolated meroterpenoids obtained from Pyr4- and
MacJ’-mediated reactions with A) (10S)-7a; B) (10S)-8a; C) (10S,3’R)-10a.
Pyr4
(10S)-7a
(10S)-7a
(10S)-7a
(10S)-7a
(10S)-8a
(10S)-8a
(10S)-8a
(10S)-8a
-
-
(10S)-10a
MacJ'
CdmG
AndB
Trt1
-
(10S)-10a
(10S)-9a
(10S)-9a
(10R)-9b
(10S)-10a
Both products exhibit a chair-chair conformation for the A/B ring
system, as is also found for the natural cyclization to form
pyripyropene E (6). However, the terminating cation-quenching
step differs for both products, leading to 11 after C-O bond
formation and 12 after C-C bond formation. These products were
also recently identified from chemical cyclization of rac-7 by using
EtAlCl2/Et2AlCl as Lewis acid promoter.[11] The [3.3.1] bridged
structure in 12 is also found in asperterpenes A and B, recently
isolated and potent BACE1 inhibitors from Aspergillus terreus.[20]
As can be delineated from the configuration of position 5’, Pyr4 is
able to accept both stereoisomers, (10S,5’S)-7a and (10S,5’R)-
7a, to form 11 and 12, respectively. The findings suggest that Pyr4,
which usually accepts the bulkier substrate 5, exhibits some
degree of promiscuity towards changes in the polyketide portion.
This is further demonstrated by the successful conversion of
(10S,3’R)-8a to 13, as well as (10S,3’S)-8a to 14. Substrate 8a
carries the epoxyfarnesyl chain connected to the 3’-position of the
DMOA-building block instead of the 5’-position as found in 7a.
-
-
(10S)-7a
(10R)-7b[a]
AdrI’
(10S)-7a
(10R)-7b[a]
-
(10R)-9b
-
PrhH
NvfL
AscF
(10R)-7b[a]
-
-
-
-
-
-
-
-
-
-
(10S)-10a
[a] Native enzyme substrate combination.
Substrate Scope of Pyr4 and MacJ’
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Products 13 and 14 are also accessed from chair-chair substrate
conformations, as observed for the natural reaction of Pyr4
towards the chair-chair product 6. These findings indicate that the
enzyme active site cavity provides a template for precise
arrangement of substrate conformation and guidance of a
stereochemically distinct polyene cyclization (Scheme 1B). Their
structures were determined by 1D and 2D NMR analyses, and
further confirmed by the recently developed crystalline sponge
(CS) method which enables 'crystallization-free' X-ray
crystallography. The method makes use of crystalline porous
metal complexes that can absorb and orient a compound of
interest. The neat alignment of the compound in the pores of the
complex makes them observable by conventional X-ray structure
analysis without the need for prior crystallization.[21]
(2E,6E)-configured substrate (10S)-8a led to formation of
compound 18, the 8,9-epimer of 13, derived from the (10S,3’S)-
8a isomer. The structure of 18 was determined by 2D NMR
analysis and was further supported by X-ray-CS-diffraction data.
The stereochemical outcome of the cyclization indeed
demonstrated a conserved chair-boat conformational control of
the substrate by the enzyme (Scheme 2B).
Reaction of CdmG with (10S)-9a represents the only case where
two products were found derived from one substrate stereoisomer,
in this case (10S,5’S)-9a, leading to the isolation of 19 and 20.
Whereas 19 is produced from a C-O bond forming event, product
20 is derived from C-C bond formation.
We were also interested in the flexibility of Pyr4 towards changes
in the farnesyl chain and therefore subjected substrate analogues
9a/9b and 10a/10b to Pyr4. Indeed, Pyr4 was able to convert
(10S,3’R)-10a to the new meroterpenoid 15 bearing a cis-fused
B/C ring system, presumably derived from a chair-chair substrate
conformation (Scheme 1C). The natural substrate of Pyr4
contains an (S)-configured epoxide functionality. Interestingly, all
productive enzyme substrate combinations were restricted to the
(10S)-series of substrates, as none of the analogous (10R)-epoxy
substrates (10R)-7b, (10R)-8b, (10R)-9b or (10R)-10b were
accepted. This finding indicates a strict recognition of the epoxide
within the substrate binding site of the enzyme. In addition, we
found that MacJ’ can also produce products 13 and 14 from (10S)-
8a and 15 from (10S,3’R)-10a (Scheme 1B/C). MacJ, naturally
involved in the biosynthesis of the drimane meroterpenoid
macrophorin A, is one of two known Pyr4-like cyclases which do
not require substrate activation by epoxidation. Instead, MacJ is
able to directly protonate the terminal double bond to initiate
cyclization (Figure 2, Figure S6B). It is therefore interesting to
note that MacJ’ also exhibits a clear preference for the (10S)-
stereoisomers of epoxide substrates evaluated (Table 1, Figure
S2-5).
Substrate Scope of CdmG and AscF
A meroterpenoid cyclase which is phylogenetically closely related
to Pyr4 is CdmG (Figure 2), utilized in the biosynthetic pathway
towards chrodrimanins from Penicillium verruculosum.[12]
Chrodrimanins exhibit strong inhibitory activities against protein
tyrosine phosphatase 1B (PTP1B) and are potential drug
candidates for the treatment of type 2 diabetes and obesity.[22]
Scheme 2. A) Natural Reaction of CdmG and isolated meroterpenoids obtained
from CdmG-mediated reactions with B) (10S,3’S)-8a; C) (10S,5’S)-9a; D) (10S)-
10a.
CdmG
naturally
catalyzes
the
formation
of
3-
hydroxypentacecilide A (17) from (S)-configured epoxide 16. In
contrast to Pyr4, CdmG controls the substrate conformation in a
chair-boat manner, leading to an inverted stereochemical
outcome for the trans-configured B/C ring system (Scheme 2A).
With this apparent change in conformational control, we were
interested in determining the substrate promiscuity of CdmG and
the putative differences in product formation.
In both cases, the same trans-cis configuration is observed for the
A/B/C-ring system (Scheme 2C). Compounds 19 and 20 have
also recently been identified from chemical cyclization where it
was further shown that 20 can be rearranged to 19 by formic acid
treatment.[11] To confirm the enzymatic origin of 19, 20 was
incubated under enzyme reaction conditions (KPP pH 7.4, 16 h,
30°C) but was found to not interconvert to 19. Reaction of CdmG
with (2Z,6E)-configured substrate 10a produced the new
meroterpenoids 21 and 15, derived from a chair-boat substrate
conformation (major) and a chair-chair substrate conformation
(minor), respectively. As observed for the promiscuity of Pyr4,
When CdmG was incubated with synthetic substrates, a high
degree of promiscuity was also observed as 7a, 8a, 9a, and 10a
were accepted by CdmG and led to the production of new
products (Figure S2-S5, Table 1). Whereas products from
substrate (10S)-7a were found to be too unstable for structural
characterization, products from substrates 8a, 9a, and 10a were
successfully isolated and structurally characterized. Reaction with
CdmG also had
a
strict preference for the epoxide
stereoconfiguration, as none of the (10R)-epoxides were
accepted by CdmG.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
AscF from the ascochlorin pathway (Figure S6B) was found to
also produce 21 and small amounts of 15 from (10S)-10a as the
only accepted substrate (Table 1, Figure S2-S5) and thus
represents a meroterpenoid cyclase with very low tolerance
towards alternative substrates.
Substrate Scope of AndB
The meroterpenoid cyclase AndB from the anditomin pathway[13]
utilizes DMOA-derived substrate 22 with an (S)-configured
epoxide to produce preandiloid
A
(23) with chair-boat
conformation (Scheme 3). In contrast to the results discussed for
CdmG, which also controls the conformation in a chair-boat
manner, AndB was found to exhibit a differing selectivity based
on the 3’- and 5’-stereocenters and therefore was found to
produce different products (Table 1, Figure S2-S5). Specifically,
AndB was found to accept (10S,3’R)-8a to produce the novel
meroterpenoid 24 (Scheme 3B). Similar to CdmG and Pyr4, the
native conformational control of the prenyl chain was conserved,
as a chair-boat substrate arrangement was found leading to 24.
AndB also accepted the (2Z,6E)-configured substrate (10S,5’R)-
9a leading to the isolation of meroterpenoid 25. The structure
elucidation for 25 was challenging as 2D NMR analysis did not
clearly reveal the connectivity between the terpenoid and non-
terpenoid portions. Additionally, the relative configuration
between the A/B and C/D ring systems were difficult to determine
due to ambiguous NOESY correlations. However, we were able
to fully establish the structure of 25 using crystalline sponge-X-ray
analysis which revealed the presence of an unprecedented 6-5-
ring system connected to a 5-6 ring system via a single C-C bond
(Scheme 3C).
Scheme 3. A) Natural reaction of AndB and structures of isolated
meroterpenoids obtained from AndB-mediated reactions with B) (10S,3’R)-8a;
C) (10S,5’R)-9a.
DFT Calculations for the Formation of 25, 20, CC, and 19
The latter finding was surprising, as all reactions in this study lead
to the formation of 6-6-ring systems for the A/B rings and also no
natural cyclization towards 6-5-ring systems has been reported
thus far. To gain further insight into the cyclization mechanism for
the formation of 25 from (10S,5R)-9a by AndB,
Figure 4. DFT calculations for the cyclization mechanism of (10S,5’R)-9a towards 25 by AndB. Two conformers of A were located, one connected to 9a-TS and
another connected to A-TS, which differ in energy by 0-3 kcal/mol, depending on the level of theory; see SI for details. Computed (B3LYP-D3(BJ)/6-
31G(d,p)//B3LYP/6-31G(d,p) in black (top), CPCM(H2O)-B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) in parentheses (bottom)) relative free energies (kcal/mol,
italics) for minima and transition state structures (TSSs) involved in formation of compound 25. Bond distances are in Angstroms (Å).
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 5. DFT calculations for the cyclization mechanism for the formation of 20. Computed (B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (on top), CPCM(H2O)-
B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (in parentheses) and MPW1PW91/6-31G(d,p)//B3LYP/6-31G(d,p) [in brackets]) relative free energies (kcal/mol,
italics) for minima and transition state structures (TSSs) and IRC traces for the respective TSSs. Bond distances are in Angstroms (Å).
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 6. DFT calculations for the cyclization mechanism for the formation of CC. Computed (B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (on top), CPCM(H2O)-
B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (in parentheses) and MPW1PW91/6-31G(d,p)//B3LYP/6-31G(d,p) [in brackets]) relative free energies (kcal/mol,
italics) for minima and transition state structures (TSSs) and IRC traces for the respective TSSs. Bond distances are in Angstroms (Å).
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
we conducted computational studies using density functional
theory (DFT) at the B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-
31G(d,p) and CPCM(H2O)-B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-
31G(d,p) levels[23] (see SI for details). The results (Figure 4)
revealed that the first intermediate in the reaction cascade
(modelled here in the absence of enzyme) is the monocyclic
tertiary cation A, generated by an endergonic process via
transition state 9a-TS. A is then converted, after a conformational
change, to 25-H+ by an exergonic concerted process consisting
of formation of the 5-membered B-ring in tandem with nucleophilic
attack of the adjacent oxygen functionality via transition state A-
TS. Inclusion of implicit solvent (CPCM(H2O), in parentheses;
single point calculations on previously optimized geometries in
gas phase) led to lower barriers (Figure 4).[24],[25] With either a
nonpolar (gas phase) or polar (water) surroundings, the barrier for
initial cyclization is high for a biological reaction,[26] however, in
the absence of enzyme, reactant (10S,5R)-9a-H+ relaxes to a
non-productive conformation with an internal hydrogen-bond
between the alcohol and epoxide; consequently, conformational
preorganization by the enzyme should lower the barrier and this
could be assisted by specific oriented noncovalent interactions
with the transition state structure. An alternative mechanism for
formation of the second ring could involve Markovnikov addition
to form a 6-6 intermediate, followed by ring-contraction in concert
with tetrahydrofuran ring formation. We find, however, that
Markovnikov addition leads directly to a “6-6-6-6” product (C) that
is not experimentally observed. We were able to optimize tertiary
carbocation B as a minimum and this species can then undergo
ring contraction to yield 25-H+, but accessing B would require
escape from the deep energy well associated with C and a large
conformational change. The enzyme would, however, have to
distinguish between A-TS and A’-TS, again by conformational
biasing and/or well-placed noncovalent interactions with the
transition state structure.
chair substrate conformation forming intermediary cation 26,
followed by Wagner-Meerwein rearrangement and a terminating
deprotonation of Ha. AdrI shares the intermediary cation 26 and
rearrangement, but differs in the terminating deprotonation side
(Hb) producing andrastin E (28). AusL and PrhH both catalyze the
formation of protoaustinoid
A (29) from 26 after direct
deprotonation of Hc (Scheme 4A). NvfL from the novofumigatonin
pathway utilizes a highly similar substrate as Trt1, AdrI, AusL, and
PrhH, carrying a free carboxylic acid instead of the methyl ester
in the DMOA-unit. The free acid is crucial for enzyme function and
the protein catalyzes formation of a spiro-center (Figure S6B).
Figure 7. DFT calculations for the interconversion of 20-H+ to 19-H+. Computed
(B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (on top), CPCM(H2O)-B3LYP-
D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p) (in parentheses) and MPW1PW91/6-
31G(d,p)//B3LYP/6-31G(d,p) [in brackets]) relative free energies (kcal/mol,
italics) for minima and transition state structures (TSSs). Bond distances are in
Angstroms (Å).
Formation mechanisms for 20 (Figure 5) and CC (Figure 6) from
protonated stereoisomers of 9a were also subjected to
computational analysis. For both reactions, we find highly
asynchronous but concerted pathways in which no discrete
carbocationic intermediates are formed. Similar concerted
polycyclizations have been reported for related systems.[27] Both
reactions also are predicted to be essentially barrierless once
productive reactant conformations are attained, suggesting that
preorganization controls which product is formed by a given
enzyme. Conversion of 20-H+ to 19-H+ is predicted to be an
endergonic process (Figure 7; neutral 19 is predicted to be
several kcal/mol lower in energy than neutral 20, however[11]), but
an appropriately positioned base in a restricted enzyme active site
could selectively deprotonate 19-H+. The barrier for the 20-
H+ to 19-H+ interconversion is also less certain than others
described above, since this reaction involves asynchronous bond-
breaking, C–C bond rotation, and bond-making that leads to a
“loose” transition state for which entropy and the effects of
externally-imposed conformational constraints are difficult to
estimate.
Scheme 4. A) Natural reaction of Trt1, AdrI/AdrI’ and AusL/PrhH. B) Isolated
meroterpenoids obtained from Trt1-mediated reactions with (10S,5’R)-7a.
Consistent with the obvious tight recognition of the polyketide
portion necessary to achieve these sophisticated and distinct
reactions, a comparable low promiscuity for this group of integral
membrane-bound enzymes was observed. Whereas PrhH and
NvfL did not accept any of the tested unnatural substrates, both
Trt1 and AdrI’ accepted (2Z,6E)-9b, with the natural 5’-
substitution pattern in the polyketide portion (Table 1, Figure S2-
S5).
Substrate Scope of Trt1, AdrI’, PrhH, and NvfL
Another phylogenetic clade is formed by Trt1, AdrI, AdrI’, AusL,
and PrhH (Figure 2). These enzymes share the DMOA substrate
(10R,5’R)-7b, but differ with regard to their product specificity.
Trt1 catalyzes the formation of preterretonin (27) via a chair-chair-
8
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
However, for these cases products were found to be too unstable
for structure determination. To our surprise, we found that Trt1 as
well as AdrI’ were able to convert (10S)-7a, the native substrate
with inverted stereochemistry with respect to the epoxide
functionality. We succeeded in the isolation of the Trt1-mediated
product from (10S,5’R)-7a and the structure was determined to
be 3-epi-preterretonin (30). The formation of this product can be
envisioned to occur via a boat-chair substrate conformation
(Scheme 4B).[28]
suitable heterologous production host. Whereas productive
enzyme combinations with naturally occurring substrates such as
7a and 7b provide a straightforward access by reconstituting
known pathways towards these substrates and interchanging the
introduced meroterpenoid cyclase, access to (2Z,6E)-configured
prenyl substrates require further engineering efforts, for example,
by employing a known bacterial (2Z,6E)-selective FPP synthase
from Mycobacterium[29] and optimization of downstream enzymes
towards substrates like 9a and 9b. Furthermore, the new
meroterpenoids obtained can be evaluated as substrates for
downstream tailoring enzymes such as a-ketoglutarate-
dependent dioxygenases. Since several protein crystal structures
have been reported in recent years, protein engineering by either
rational strategies or directed evolution represents an exciting
opportunity to create novel “unnatural” meroterpenoids with
valuable biological properties.[30]
Enzyme Kinetics of Trt1
To shed additional light on the substrate promiscuity of
meroterpenoid cyclases, we were interested in the comparison of
the KM values of the natural substrate to unnatural substrate
analogous. Synthetic (10R,5’R)-7b is the natural substrate of Trt1,
which was also found to accept unnatural (10S,5’R)-7a, and
(10R)-9b. Since Trt1 and other meroterpenoid cyclases cannot be
purified, the integral membrane bound enzyme was used as a
crude enzyme preparation. To ensure comparable reaction
conditions, Trt1 was freshly prepared and used for all kinetic
assays on the same day. The results revealed an apparent KM
value of 34 µM for the natural substrate 7b, whereas a 3-fold (95
µM) and 5-fold (144 µM) higher value was found for substrates 7a
and 9b, respectively. The Vmax values were determined as 22
µM/min, 7 µM/min and 0.5 µM/min, for 7b, 7a and 9b, respectively.
These findings indicate that the unnatural substrates have a lower
affinity for the enzyme, but the values are of the same order of
magnitude and thus further demonstrate the promiscuity
observed for this enzyme class.
Acknowledgements
We thank the National Institutes of Health (R35 GM-118173, J. A.
P., Jr.), a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan
(JSPS KAKENHI Grant Number JP16H06443 and JP20H00490
to I.A.; JP19H05461 to M. F.), and the MEXT Nanotechnology
Platform Program (Molecule and Material Synthesis) promoted at
IMS for research funding. We also thank the Deutsche
Forschungsgemeinschaft (DFG BA 6870/1-1) for a postdoctoral
research fellowship to L.B. Computational work was supported by
the National Science Foundation (CHE-1856416 and
supercomputing resources from the XSEDE program via CHE-
030089).
Conclusion
In summary, we have demonstrated chemoenzymatic access to
twelve complex, unnatural DMOA-derived meroterpenoids of
which eight represent novel compounds by exploiting the
surprisingly high promiscuity of fungal meroterpenoid cyclases.
Keywords: meroterpenoids • meroterpenoid cyclase •
chemoenzymatic synthesis • combinatorial biosynthesis •
crystalline-sponge x-ray diffraction
[1]
a) J. W. Cornforth, Chem. Br. 1968, 4, 102–106; b) R. Geris, T. J.
Simpson, Nat. Prod. Rep. 2009, 26, 1063–1094; c) L. Kaysser, P.
Bernhardt, S. J. Nam, S. Loesgen, J. G. Ruby, P. Skewes-Cox, P. R.
Jensen, W. Fenical, B. S. Moore, J. Am. Chem. Soc. 2012, 134, 11988–
11991; d) X. Peng, M. Qiu, Nat. Products Bioprospect. 2018, 8, 137–149.
a) S. Ōmura, H. Tomoda, Y. K. Kim, H. Nishida, J. Antibiot. 1993, 46,
1168–1169; b) H. Tomoda, Y. K. Kim, H. Nishida, R. Masuma, S. Ōmura,
J. Antibiot. 1994, 47, 148–153; c) A. Das, M. A. Davis, H. Tomoda, S.
Ômura, L. L. Rudel, J. Biol. Chem. 2008, 283, 10453–10460.
a) R. Horikoshi, K. Goto, M. Mitomi, K. Oyama, T. Sunazuka, S. Ōmura,
J. Antibiot. 2017, 70, 272–276; b) C. Dieleman, T. Knieriem, M. Krapp, P.
C. Kierkus, W. Xu, K. Benton, patent: WO 2012/035010 A1, 2012.
a) M. D. Sintchak, M. A. Fleming, O. Futer, S. A. Raybuck, S. P.
Chambers, P. R. Caron, M. A. Murcko, K. P. Wilson, Cell 1996, 85, 921–
930; b) P. W. Clutterbuck, A. E. Oxford, H. Raistrick, G. Smith, Biochem.
J. 1932, 26, 1441–1458; c) L. Canonica, W. Kroszczynski, B. M. Ranzi,
B. Rindone, C. Scolastico, J. Chem. Soc. D Chem. Commun. 1970, 6,
2639–2643; d) L. M. Shaw, I. Nowak, Ther. Drug Monit. 1995, 17, 685–
689.
Synthetic
3,5-dimethylorsellinic
acid
(DMOA)-containing
substrates were prepared using dearomative alkylation and
evaluated against nine meroterpenoid cyclases. The results
demonstrate tight recognition of the epoxide functionality by the
cyclase panel, but tolerance towards the polyketide portion. The
conserved cyclization mechanism initiated by epoxide protonation
and rigid control of the substrate conformation in the cyclase
active site cavity led to the formation of several variations of
naturally occurring meroterpenoid scaffolds, as well as generation
of a completely new scaffold, although the number of cyclization
steps that are concatenated into concerted processes appears to
be system-dependent. The challenging structure elucidation and
determination of relative and absolute configurations for the
obtained enzyme products was solved by combining 2D NMR
data analysis with the recently developed method of crystalline-
sponge-X-ray diffraction analysis. This method allowed us to
access the crystallographic data of non-crystalline enzyme
products such as 25 for unambiguous structure determination and
thus represents a powerful technique in combination with second
generation natural product discovery.
[2]
[3]
[4]
[5]
[6]
a) S. Ōmura, J. Inokoshi, R. Uchida, K. Shiomi, R. Masuma, T.
Kawakubo, H. Tanaka, Y. Iwai, S. Kosemura, S. Yamamura, J. Antibiot.
1996, 49, 414–417; b) R. Uchida, K. Shiomi, J. Inokoshi, T. Sunazuka,
H. Tanaka, Y. Iwai, H. Takayanagi, S. Ōmura, J. Antibiot. 1996, 49, 418–
424.
The knowledge obtained in this study can be used for the design
of artificial pathways by recombining biosynthetic genes in a
M. Cueto, J. B. MacMillan, P. R. Jensen, W. Fenical, Phytochemistry
2006, 67, 1826–1831.
9
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
[7]
T. Itoh, K. Tokunaga, Y. Matsuda, I. Fujii, I. Abe, Y. Ebizuka, T. Kushiro,
Nat. Chem. 2010, 2, 858–864.
S. Ehrlich, L. Goerigk, J. Comput. Chem. 2011, 32, 1456–1465; e) Y.
Takano, K. N. Houk, J. Chem. Theory Comput. 2005, 1, 70–77.
[8]
a) Y. Matsuda, I. Abe, Nat. Prod. Rep. 2016, 33, 26–53; b) L. Barra, I.
Abe, Y. Nat. Prod. Rep. 2020, 37, in press; c) Y. Matsuda, T. Awakawa,
T. Mori, I. Abe, Curr. Opin. Chem. Biol. 2016, 31, 1–7.
[24] We benchmarked our system using a variety of methods: CPCM(DCM)-
B3LYP-D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p), CPCM(MEOH)-B3LYP-
D3(BJ)/6-31G(d,p)//B3LYP/6-31G(d,p),
mPW1PW91/6-
[9]
J. D. Rudolf, C. Y. Chang, Nat. Prod. Rep. 2020, 37, 425–463.
31G(d,p)//B3LYP/6-31G(d,p) and CPCM(MEOH)- mPW1PW91/6-
31G(d,p)//B3LYP/6-31G(d,p). See SI for details. S. P. T. Matsuda, W. K.
Wilson, Q. Xiong, Org. Biomol. Chem. 2006, 4, 530–543.
[10] a) C. A. Young, M. K. Bryant, M. J. Christensen, B. A. Tapper, G. T.
Bryan, B. Scott, Mol. Genet. Genomics 2005, 274, 13–29; b) M. J.
Nicholson, A. Koulman, B. J. Monahan, B. L. Pritchard, G. A. Payne, B.
Scott, Appl. Environ. Microbiol. 2009, 75, 7469–7481; c) K. Tagami, C.
Liu, A. Minami, M. Noike, T. Isaka, S. Fueki, Y. Shichijo, H. Toshima, K.
Gomi, T. Dairi, H. Oikawa, J. Am. Chem. Soc. 2013, 135, 1260–1263; d)
H. Kato, Y. Tsunematsu, T. Yamamoto, T. Namiki, S. Kishimoto, H.
Noguchi, K. Watanabe, J. Antibiot. 2016, 69, 561–566; e) T. Yao, J. Liu,
Z. Liu, T. Li, H. Li, Q. Che, T. Zhu, D. Li, Q. Gu, W. Li, Nat. Commun.
2018, 9, 4091; f) K. Tsukada, S. Shinki, A. Kaneko, K. Murakami, K. Irie,
M. Murai, H. Miyoshi, S. Dan, K. Kawaji, H. Hayashi, E. N. Kodama, A.
Hori, E. Salim, T. Kuraishi, N. Hirata, Y. Kanda, T. Asai, Nat. Commun.
2020, 11, 1830; g) H. Li, Y. Sun, Q. Zhang, Y. Zhu, S. M. Li, A. Li, C.
Zhang, Org. Lett. 2015, 17, 306–309; h) J. Yaegashi, J. Romsdahl, Y. M.
Chiang, C. C. C. Wang, Chem. Sci. 2015, 6, 6537–6544.
[25] 9a is the conformer obtained from reverse 9a-TS intrinsic reaction
coordinate (IRC) analysis. 9a-TS forward IRC product undergoes a
conformational change to form A which is the reverse IRC product of A-
TS. See SI for details. A. C. Gonzalez, H. B. Schlegel, J. Phys. Chem.
1990, 94, 5523–5527; b) K. Fukui, Acc. Chem. Res. 1981, 14, 363–368;
c) S. Maeda, Y. Harabuchi, Y. Ono, T. Taketsugu, K. Morokuma, Int. J.
Quantum Chem. 2015, 115, 258–269.
[26] S. C. Wang, D. J. Tantillo, J. Org. Chem. 2008, 73, 1516–1523.
[27] a) D. J. Tantillo, J. Phys. Org. Chem. 2008, 21, 561–570; b) L. Smentek,
B. A. Hess, J. Am. Chem. Soc. 2010, 132, 17111–17117; c) C. McCulley,
M. J. Geier, B. M. Hudson, M. R. Gagne, D. J. Tantillo, J. Am. Chem.
Soc. 2017, 139, 11158–11164.
[28] I. Abe, M. Rohmer, J. Chem. Soc. Perkin Trans. 1 1994, 783–791.
[29] a) M. C. Schulbach, P. J. Brennan, D. C. Crick, J. Biol. Chem. 2000, 275,
22876–22881; b) M. C. Schulbach, S. Mahapatra, M. Macchia, S.
Barontini, C. Papi, F. Minutolo, S. Bertini, P. J. Brennan, D. C. Crick, J.
Biol. Chem. 2001, 276, 11624–11630.
[11] Z. Powers, A. Scharf, A. Cheng, F. Yang, M. Himmelbauer, T. Mitsuhashi,
L. Barra, Y. Taniguchi, T. Kikuchi, M. Fujita, I. Abe, J. A. Porco, Angew.
Chem. Int. Ed. 2019, 58, 16141–16146.
[12] T. Bai, Z. Quan, R. Zhai, T. Awakawa, Y. Matsuda, I. Abe, Org. Lett. 2018,
20, 7504–7508.
[30] a) Y. Nakashima, T. Mori, H. Nakamura, T. Awakawa, S. Hoshino, M.
Senda, T. Senda, I. Abe, Nat. Commun. 2018, 9, 104–114, b) H.
Nakamura, Y. Matsuda, I. Abe, Nat. Prod. Rep. 2018, 35, 633–645.
[13] a) Y. Matsuda, T. Wakimoto, T. Mori, T. Awakawa, I. Abe, J. Am. Chem.
Soc. 2014, 136, 15326–15336; b) Y. Nakashima, T. Mitsuhashi, Y.
Matsuda, M. Senda, H. Sato, M. Yamazaki, M. Uchiyama, T. Senda, I.
Abe, J. Am. Chem. Soc. 2018, 140, 9743–9750.
[14] a) Y. Matsuda, T. Awakawa, I. Abe, Tetrahedron 2013, 69, 8199–8204;
b) Y. Matsuda, Z. Quan, T. Mitsuhashi, C. Li, I. Abe, Org. Lett. 2016, 18,
296–299.
[15] Y. Matsuda, T. Bai, C. B. W. Phippen, C. S. Nødvig, I. Kjærbølling, T. C.
Vesth, M. R. Andersen, U. H. Mortensen, C. H. Gotfredsen, I. Abe, T. O.
Larsen, Nat. Commun. 2018, 9, 2587.
[16] a) Y. Matsuda, T. Iwabuchi, T. Fujimoto, T. Awakawa, Y. Nakashima, T.
Mori, H. Zhang, F. Hayashi, I. Abe, J. Am. Chem. Soc. 2016, 138, 12671–
12677; b) T. Zhang, J. Wan, Z. Zhan, J. Bai, B. Liu, Y. Hu, Acta Pharm.
Sin. B 2018, 8, 478–487.
[17] a) T. Itoh, K. Tokunaga, E. K. Radhakrishnan, I. Fujii, I. Abe, Y. Ebizuka,
T. Kushiro, ChemBioChem 2012, 13, 1132–1135; b) Y. Matsuda, T.
Awakawa, T. Itoh, T. Wakimoto, T. Kushiro, I. Fujii, Y. Ebizuka, I. Abe,
ChemBioChem 2012, 13, 1738–1741; c) C. J. Guo, B. P. Knox, Y. M.
Chiang, H. C. Lo, J. F. Sanchez, K. H. Lee, B. R. Oakley, K. S. Bruno, C.
C. C. Wang, Org. Lett. 2012, 14, 5684–5687; d) Y. Matsuda, T. Iwabuchi,
T. Wakimoto, T. Awakawa, I. Abe, J. Am. Chem. Soc. 2015, 137, 3393–
3401.
[18] a) Y. Araki, T. Awakawa, M. Matsuzaki, R. Cho, Y. Matsuda, S. Hoshino,
Y. Shinohara, M. Yamamoto, Y. Kido, D. K. Inaoka, K. Nagamune, K. Ito,
I. Abe, K. Kita, Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 8269–8274; b)
Z. Quan, T. Awakawa, D. Wang, Y. Hu, I. Abe, Org. Lett. 2019, 21, 2330–
2334.
[19] M. C. Tang, X. Cui, X. He, Z. Ding, T. Zhu, Y. Tang, D. Li, Org. Lett. 2017,
19, 5376–5379.
[20] C. Qi, J. Bao, J. Wang, H. Zhu, Y. Xue, X. Wang, H. Li, W. Sun, W. Gao,
Y. Lai, J. G. Chen, Y. Zhang, Chem. Sci. 2016, 7, 6563–6572.
[21] a) Y. Inokuma, S. Yoshioka, J. Ariyoshi, T. Arai, Y. Hitora, K. Takada, S.
Matsunaga, K. Rissanen, M. Fujita, Nature 2013, 495, 461–466, b) M.
Hoshino, A. Khutia, H. Xing, Y. Inokuma, M. Fujita, IUCrJ 2016, 3, 139–
151.
[22] H. Yamazaki, W. Nakayama, O. Takahashi, R. Kirikoshi, Y. Izumikawa,
K. Iwasaki, K. Toraiwa, K. Ukai, H. Rotinsulu, D. S. Wewengkang, D. A.
Sumilat, R. E. P. Mangindaan, M. Namikoshi, Bioorganic Med. Chem.
Lett. 2015, 25, 3087–3090.
[23] a) B. Miehlich, A. Savin, H. Stoll, H. Preuss, Chem. Phys. Lett. 1989, 157,
200–206; b) C. Lee, W. Yang, G. R. Parr, Phys. Rev. B 1988, 37, 785–
789; c) A. D. Becke, J. Chem. Phys. 1993, 98, 5648–5652; d) S. Grimme,
10
This article is protected by copyright. All rights reserved.

10.1002/anie.202011171
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Fungal meroterpenoid cyclases, a recently discovered family of membrane-integrated terpene cyclases, have been exploited as tools
for the synthesis of second generation meroterpenoid natural products. The results demonstrate a broad substrate tolerance leading
to several new meroterpenoid scaffolds. DFT calculations provided insights into the mechanism of the enzyme-mediated polyene
cyclizations.
11
This article is protected by copyright. All rights reserved.