Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton

Article abstract of DOI:10.1002/anie.202007280

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Full text of DOI:10.1002/anie.202007280

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced
Cyclization of an Epoxyalcohol Affording a Characteristic
Lolitremane Skeleton
Authors: Yulu Jiang, Taro Ozaki, Mei Harada, Tadachika Miyasaka,
Hajime Sato, Kazunori Miyamoto, Junichiro Kanazawa,
Chengwei Liu, Jun-ichi Maruyama, Masaatsu Adachi, Atsuo
Nakazaki, Toshio Nishikawa, Masanobu Uchiyama, Atsushi
Minami, and Hideaki Oikawa
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.202007280
Link to VoR: https://doi.org/10.1002/anie.202007280

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced
Cyclization of an Epoxyalcohol Affording a Characteristic
Lolitremane Skeleton
Yulu Jiang,[a] Taro Ozaki,[a] Mei Harada,[b,c] Tadachika Miyasaka,[d] Hajime Sato,[b,c] Kazunori
Miyamoto,[b] Junichiro Kanazawa,[b] Chengwei Liu,[a] Jun-ichi Maruyama,[e] Masaatsu Adachi,[f] Atsuo
Nakazaki,[d] Toshio Nishikawa,[d] Masanobu Uchiyama,[b,c] Atsushi Minami,*[a] and Hideaki Oikawa*[a]
[a]
Y. Jiang, Dr. T. Ozaki, Dr. C. Liu, Dr. A. Minami, Prof. Dr. H. Oikawa
Department of Chemistry, Faculty of Science
Hokkaido University
Sapporo 060-0810, Japan
E-mail: aminami@sci.hokudai.ac.jp, hoik@sci.hokudai.ac.jp
Dr. C. Liu Present address: College of Life Sciences, Northeast Forestry University, Harbin 150040, China.
M. Harada, Dr. H. Sato, Dr. K. Miyamoto, Dr. J. Kanazawa, Prof. Dr. M. Uchiyama
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
M. Harada, Dr. H. Sato, Prof. Dr. M. Uchiyama
Cluster for Pioneering Research (CPR), Advanced Elements Chemistry Laboratory
RIKEN
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
T. Miyasaka, Dr. A. Nakazaki, Prof. Dr. T. Nishikawa
Graduate School of Bioagricultural Sciences
Nagoya University
Chikusa, Nagoya 464-8601, Japan
Prof. Dr. J. Maruyama
Department of Biotechnology, Graduate School of Agricultural and Life Sciences
The University of Tokyo
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Dr. M. Adachi
[b]
[c]
[d]
[e]
[f]
Graduate School of Pharmaceutical Sciences
Tohoku University
6-3. Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
Supporting information for this article is given via a link at the end of the document.
Abstract: Lolitrems are tremorgenic indole diterpenes that exhibit a
unique 5/6 bicyclic system of the indole moiety. Although genetic
analysis has indicated that prenyltransferase LtmE and cytochrome
P450 LtmJ are involved in the construction of this unique structure,
the detailed mechanism remains to be elucidated. Herein, we
reconstitute the biosynthetic pathway for lolitrems employing a
recently established genome-editing technique for the expression
host Aspergillus oryzae. Heterologous expression and bioconversion
of the various intermediates revealed that LtmJ catalyzes multistep
oxidation to furnish the lolitrem core. We also isolated the key reaction
intermediate with an epoxyalcohol moiety. This observation allowed
us to establish the mechanism of radical-induced cyclization, which
was firmly supported by density functional theory calculations and a
model experiment with a synthetic analog.
Introduction
Indole diterpenes (IDTs) are representative fungal secondary
metabolites with remarkable structural diversity and a wide range
of biological activities, such as inhibition of calcium-activated
potassium channels, tremorgenic activity, progesterone receptor
Figure 1. Representative fungal indole diterpenes.
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 2. Biosynthetic pathway for terpendoles and lolitrems. A) The biosynthetic pathway for 3. B) The biosynthetic pathway for 8 and 7 from 2 and 6. PT:
prenyltransferase; FMO: flavin-dependent monooxygenase; TC: terpene cyclase.
agonistic activity, and anti-MRSA activity. [1,2] To understand the
mechanism of their structural diversification, extensive studies
have been conducted on their biosynthesis. Among more than a
hundred natural indole terpenes, paspaline (1) -derived IDTs are
the largest group, comprising 70% of all isolated IDTs (Figure 1).
The core structure of IDTs is diversified mainly by cytochromes
P450, such as PaxPQ, involved in the biosynthesis of paxilline (2),
or their homologs.[3.4] In the late stage of biosynthesis, prenylation
of the indole ring and further modification also contribute to
structural diversification. Previously, we elucidated the
biosynthetic pathway of penitrem A and shearinines/janthitrems,
in which prenyltransferases and flavoproteins are involved in the
construction of a bicyclic system on indole rings (Figure 1). [5,6]
However, little is known about the biosynthesis of lolitrems, which
have a unique 5/6 bicyclic system (Figure 1). Lolitrems are
tremorgenic mycotoxins produced by the fungal endophyte
Epichloë festucae var. lolii. A major congener, lolitrem B (3), is
classes of compounds, including more than 30 congeners. Most
lolitrems are structurally characterized by the presence of trans-
fused 5/6 bicyclic systems (A/B rings), while minor congeners can
also have cis-fused systems (Figure 1)[2]. The biosynthetic gene
cluster of lolitrems, which is composed of ten genes and splits into
three loci,[9,10] contains four genes, ltmGCMB (Figure S1), which
are responsible for the biosynthesis of 1. [11] In the biosynthesis of
terpendole C (4), which has an identical structure to the IDT core
of 3, a key intermediate 1 undergoes a series of modifications
catalyzed by TerPQFK to yield 4 via terpendole E (5), J, and I (6)
as intermediates (Figure 2A),[12] suggesting that the orthologous
enzymes LtmPQFK are responsible for the same conversions in
lolitrem biosynthesis.[11] Prenyltransferase LtmE and cytochrome
P450 LtmJ are specific to the biosynthesis of lolitrems and are
likely involved in the construction of their A/B rings. [11] However,
the exact mechanism is still unknown. In this study, we elucidated
the mechanism of P450-catalyzed oxidative cyclization employing
known as a causative agent of ryegrass staggers.[7,8] Among IDTs, heterologous expression, density functional theory (DFT)
lolitrems/terpendoles are known as one of the most diverse calculation, and a model experiment with a synthetic analog.
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
that 20 also had the same partial structure. Comparing the
chemical shifts of 1H and 13C NMR spectra with those of synthetic
analogs, we determined the relative stereochemistry to be syn
(Tables S2–S4).[16] Although the NMR spectra of 18, which was
Results and Discussion
Based on the biosynthetic studies of 2,[3] we initially introduced
four genes, paxG, paxC, paxM, and paxB, into Aspergillus oryzae
NSPlD1 employing CRISPR/Cas9-based genome editing to
produce the putative intermediate 1 (Figure S2, Table S1).[13] This
screening-free method readily enabled us to obtain the AO-
paxGCMB which produced 1 (133 mg kg−1), as we reported
previously[3] (Figure 3A-ii). Given the expected transformant,
cytochrome P450 genes terP and/or terQ were introduced into
AO-paxGCMB. The obtained transformants, AO-paxGCMB/terQ
and AO-paxGCMB/terPQ, produced 5 (109 mg kg−1) and 6 (148
mg kg−1), respectively (Figure 3A-iii and iv). Two additional genes,
terF and terK, were introduced. The obtained transformant, AO-
paxGCMB/terPQFK, which produced 4 (52 mg kg−1), confirmed
that TerF and TerK exerted the expected activities (Figure 3A-v).
Finally, we introduced ltmE and ltmJ. The resulting AO-
previously identified by the bioconversion of
2 by AO-
spdE/ltmJ,[17] were nearly identical to those of sespelline (S4), an
unnatural IDT produced by the bioconversion of 2 by AO-
spdE/spdJ,[17] the difference in their retention times suggested a
diastereomeric relationship (Figure S6).
paxGCMB/terPQ/ltmEJ
and
AO-paxGCMB/terPQFK/ltmEJ
produced lolitriol (7)[14] (6 mg kg−1) and lolitrem B (3) (0.3 mg kg−1),
respectively (Figure 3A-vi and vii). These results showed that
LtmE and LtmJ are responsible for the construction of the A/B
rings of lolitrems. The low production of 4 and 3 can be explained
by the substrate promiscuity of prenyltransferase TerF (Figure
3A-v and vii). Competition between several enzymes is also likely
(e.g., 6 is a substrate for both TerF and LtmE).
Considering that more than 10 lolitremanes/lolitrem congeners
have been isolated in nature, we hypothesized that LtmEJ can
accept a diverse array of IDTs. To investigate their substrate
scope, we constructed the transformant AO-ltmEJ. Indole
terpenes 6, 4, and 2 were converted to naturally occurring
lolitrems 7, 3, and lolilline (8),[15] respectively (Figure 3B). Among
the compounds tested, paspalinine (9) was converted to the
putative lolitremane 10, while 13-desoxypaxilline (11) was not
converted (Figure 4A, 4B, and S3), suggesting that the 13-
hydroxy group was important for substrate recognition. This
hypothesis was supported by the observation that terpendole D
(12) and emindole SB (13) were not converted in the same
condition (Figure 4C and 4D). Interestingly, it was possible to
apply the same empirical rule to indole sesquiterpenes (ISTs):
lecanindole B (14), which has a 9-hydroxy group, was also
converted to the putative product 15, while prelecanindole (16)
was not (Figure 4E, 4F, and S3). These results indicated that
LtmE and LtmJ can be applied to the modification of IDTs/ISTs
with 13/9-hydroxy groups (Figure S4). Conversion of 5 was
extremely low, but MS spectra and chromatograph profiles
indicated that lolicine A (17) was formed (Figure 4G, S3).[14]
Insufficient conversion of 5 suggested the other structural
features may also be important for their recognition by the
enzymes. In the case of 6, we also detected a trace amount of
isomeric metabolites (Figure 3B), suggesting that the products
with cis-fused A/B rings were formed in the same condition.
LC-MS analysis of the bioconversion of 2, 6, and 4 revealed
that products 8, 7, and 3 were always accompanied by the
compounds 18, 19, and 20, respectively, whose deduced
molecular weights were two mass units larger than those of the
corresponding lolitrems (Figures 3B and S3). Similar compounds
were also observed in the cases of 5, 9, and 14 (Figure 4A, E, G,
S3). We determined the planar structures of 18 and 19 to be
epoxyalcohols (Scheme 1A, Figure S5, Table S2), suggesting
Figure 3. HPLC analyses of IDTs produced by the transformants. A) Traces of
the metabolites extracted from the transformants. The peaks marked with
asterisks indicate putative monoprenylated derivatives of 4. B) Bioconversion of
6, 4, and 2 by AO-WT or AO-ltmEJ. All chromatograms were extracted at 265
nm. The black circle indicates a putative 35-epimer of 7 based on its MS
spectrum.
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 4. HPLC profiles of feeding experiments to AO-ltmEJ with 9 (A), 11 (B), 12 (C), 13 (D), 14 (E), 16 (F), and 5 (G). All chromatograms were extracted at 265
nm. White circles indicate putative intermediates with epoxyalcohol moiety.
Subsequently, we conducted bioconversion of 18 and 19 by
AO-ltmJ and obtained 8 and 7, respectively (Figures S6 and S7).
In addition, 20,21-diprenylterpendole I (21), obtained by the
bioconversion of 6 by AO-ltmE, was also converted to 7 by AO-
ltmJ (Figure S8, Table S2). These results unequivocally indicated
that 18 and 19 are the intermediates of 3-step oxidation catalyzed
by LtmJ (Figure 2B). Interestingly, S4 was not converted to 8 or
other related compounds (Figure S6), suggesting that the
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
stereochemistry of epoxyalcohol is important for recognition by
LtmJ.
monooxygenase LtmJ is solely responsible for the formation of a
bicyclic system via epoxyalcohol. Therefore, we propose a
mechanism of oxidative cyclization, shown in Scheme 1A. Based
on previous studies that reported radical-induced epoxide ring
opening,[20] we hypothesized that H30 would be abstracted by
LtmJ to generate substrate radical RT, which would be rapidly
converted to give alkoxy radical IM1. [21] Radical-induced epoxide
opening was used for the isomerization of a,b-epoxyketones to
carbocycles (Scheme 1B).[22] A subsequent 1,5-H shift would give
the isomeric alkoxy radical IM2,[23] which would undergo
intramolecular addition to the double bond in a 6-exo manner. The
resulting tertiary radical PD would undergo one electron oxidation
to give a tertiary cation, [24–28] which would be trapped by the tert-
hydroxy group, furnishing the A/B rings (Scheme 1A, path a).
Alternatively, the tertiary radical would couple the alkoxy radical
which would be generated by abstraction of second hydrogen
from the hydroxy group (Scheme 1A, path b).
To examine the feasibility of this hypothesis, we next performed
DFT calculations. The results of several examinations indicated
that the most thermodynamically and kinetically favorable
pathway for radical-induced 6-membered ring (B-ring) formation
is the multistep reaction cascade shown in Figure 5, which is
essentially the same framework as that described above: 1) all
activation barriers are low enough for the reactions to proceed
smoothly at ambient temperature (no step with a barrier greater
than 20 kcal mol–1), 2) the first radical-induced epoxide ring-
opening reaction is the rate-determining step with the highest
energy barrier (12.1 kcal mol–1) in this cyclization cascade, and 3)
the overall exothermicity is very large (nearly 30 kcal mol–1). The
initial epoxide ring-opening reaction of RT via TS1 is an
endothermic process due to the instability of oxy radicals,
affording an allylic alkoxy radical (IM1), which smoothly
undergoes a 1,5-H shift via TS2 with very low activation energy
(2.0 kcal mol–1) to yield the more stable radical intermediate (IM2)
with a large exothermicity. Judging by the elongated C–C bond
distance (1.44 Å) and the shortened C–O bond distance (1.24 Å)
of the enol moiety, we consider IM2 to be an a-ketoalkyl radical,
in which the secondary carbon radical is stabilized by the
resonance with an a-carbonyl π bond rather than an enoyl
(vinyloxy) radical structure (shown in parentheses in Figure 5).
The next step branches into two possible routes to afford cis- and
trans-1-tetralone derivatives (PD-c and PD-t). Our computational
study suggested that the intramolecular trans-annulation of IM2 is
kinetically more favorable than the cis-pathway by 8.5 kcal/mol–1,
in line with the experimental findings that a selective formation of
trans-fused A/B rings with/without only a trace amount of cis-fused
congeners. Essentially the same results were obtained when we
used a model substrate 22 with a benzene core (Figure S9).
Scheme 1. Proposed mechanism of A) LtmJ-catalyzed reaction and B) the
related radical cyclization.
Epoxide opening–triggered cyclizations with epoxide hydrolases
are often found in natural product biosynthesis.[18,19] However, the
successful conversion of epoxyalcohol into a bicyclic ketone in
bioconversion with AO-ltmJ strongly suggests that P450
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 5. Computed reaction pathway from RT to PD. Potential energies relative to RT are shown in parentheses (kcal mol−1; Gibbs free energies calculated at the
UM06-2X/6-31G(d) level).
reaction, while product 23c was formed by hydrogen abstraction
of the enolate radical intermediate from Bu3SnH. The conditions
of low temperature and short reaction time despite the low
conversion were due to suppression of the retro aldol reaction.[29]
The formation of 23a and 23b thus strongly supports the proposed
enzyme reaction mechanism.
Conclusion
In this study, we achieved total biosynthesis of 3 and elucidated
the mechanism of the construction of characteristic A/B rings.
Bioconversion of various indole terpenes suggested that both
Scheme 2. A model experiment of conversion of 22 to 23a. Reagents and
conditions: Bu3SnH (4 equiv), Et3B (1 equiv), THF, 40°C, 1 h. cis-23a and trans-
23a were obtained as inseparable mixtures with 22 and 23c, respectively. The
yields were calculated from the ratios of integration values of 1H NMR spectra
of these mixtures and described based on the recovered starting material (brsm)
(49% of 22 was recovered).
LtmE and LtmJ show relaxed substrate specificities, which
account for the diversity of the reported lolitrems. As a synthetic
route for 5, a key intermediate in the biosynthesis, has already
been established, [30] other structurally related terpendoles can be
synthesized in either natural or unnatural forms. Bioconversion of
those synthetic analogs with AO-ltmEJ would be effective for the
biosynthesis of lolitrem derivatives.[31]
To validate the proposed cyclization mechanism of the 6-
membered ring from epoxyalcohols 18 and 19, we designed a
model experiment using a simplified prenyl benzene (22)
possessing epoxyketone at the ortho position, as it has been
reported that reaction of epoxyketone with a tin radical induces
ring opening of the epoxide via a ketyl radical, a proposed
intermediate in the enzymatic reaction (Scheme 1B). After several
experiments, we found that epoxyketone 22 was heated with
Bu3SnH in the presence of Et3B at 40°C for 1 h to give the
expected product 23a along with 23b and 23c (Scheme 2). A
careful separation and NMR analysis enabled us to determine the
yield of the products as shown in Scheme 2. The relative
stereochemistry of trans-23a was determined by extensive NMR
analysis of the alcohol obtained by stereoselective reduction of
trans-23a[29]. Product 23b was formed from 23a by a retro aldol
In fungal metabolite biosynthesis, we often observe the
conversion of secondary alcohol to ketone via a carbinyl radical,
such as RT. An intriguing point of LtmJ-catalyzed oxidative
cyclization is that RT did not end up forming ketone. Instead, rapid
epoxide opening preceded O-rebound to give enol radical IM1.
This branch point completely changed the LtmJ-catalyzed
reaction pathway to one distinct from conventional P450-
catalyzed reactions. Our proposed pathway is firmly supported by
DFT calculations and chemical demonstration of the last oxidative
cyclization. Our findings add a radical-induced mechanism to the
repertoire of biosynthetic cascade cyclization, which is usually
initiated by acid-catalyzed epoxide opening.
Acknowledgements
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
[26] Y.-H. Chooi, Y. J. Hong, R. A. Cacho, D. J. Tantillo, Y. Tang, J. Am.
Chem. Soc. 2014, 136, 4426-4436.
This work was supported by JSPS KAKENHI (B) (No. 19H02891
(H.O.), No. 19H02896 (T.N.)), JSPS KAKENHI (S) (No.
17H06173 (M.U.)), JSPS Grant-in-Aid for Scientific Research on
Innovative Areas (No. 16H06446 (A.M.), No. 19H04635 (T.O.),
No. 17H05430 (M.U.), No. 17H06406 (T.N.), No. 19H04644
(J.M.)). Allotment of computational resource (Project G19012)
from HOKUSAI BigWaterfall (RIKEN) is gratefully acknowledged.
We are grateful to Dr. H. Osada and Dr. T. Motoyama (RIKEN) for
the helpful discussion about terpendole biosynthesis and Prof. D.
Takemoto (Nagoya University) for the genomic DNA sample of a
lolitrem producer. We also thank S. Yamaguchi (Nagoya
University) for the preparation of compound 22.
[27] H.-C. Lin, Y. Tsunematasu, S. Dhingra, W. Xu, M. Fukutomi, Y.-H. Chooi,
D. E. Cane, A. M. Calvo, J, K. Watanabe, Y. Tang, J. Am. Chem. Soc.
2013, 135, 16805-16808.
[28] A. Tazawa, Y. Ye, T. Ozaki, C. Liu, Y. Ogasawara, T. Dairi, Y. Higuchi,
N. Kato, K. Gomi, A. Minami, H. Oikawa, Org. Lett. 2018, 20, 6178-6182.
[29] cis-23a was isolated as the corresponding alcohol by reduction of the
mixture with NaBH4 in MeOH. trans-23a was isolated from the mixture by
exclusive reduction of 23c with NaBH(OAc)3 in THF. The details are
given in ESI.
[30] T. Teranishi, T. Murokawa, M. Enomoto, S. Kuwahara, Biosci. Biotechnol.
Biochem. 2015, 79, 11-15.
[31] R. Tanifuji, A. Minami, H. Oguri, H. Oikawa, Nat. Prod. Rep. 2020, DOI
10.1039/C9NP00073A.
Keywords: biosynthesis • radical reactions • natural products •
cytochrome P450 • lolitrem
[1]
[2]
A. Minami, C. Liu, H. Oikawa, Heterocycles 2016, 92, 397-421.
C. Liu, A. Minami, T. Ozaki, H. Oikawa in Comprehensive Natural
Products III: Chemistry and Biology, (Eds.: T. P. Begley, H.-W. Liu),
Elsevier Science, 2020, vol. 2.
[3]
K. Tagami, C. Liu, A. Minami, M. Noike, T. Isaka, S. Fueki, Y. Shichijo,
H. Toshima, K. Gomi, T. Dairi, et al., J. Am. Chem. Soc. 2013, 135, 1260-
1263.
[4]
[5]
K. Tagami, A. Minami, R. Fujii, C. Liu, M. Tanaka, K. Gomi, T. Dairi, H.
Oikawa, ChemBioChem 2015, 16, 2076-2080.
C. Liu, K. Tagami, A. Minami, T. Matsumoto, J. C. Frisvad, H. Suzuki, J.
Ishikawa, K. Gomi, H. Oikawa, Angew. Chem. Int. Ed. 2015, 54, 5748-
5752; Angew. Chem. 2015, 127, 5840-5844.
[6]
[7]
[8]
[9]
C. Liu, A. Minami, T. Dairi, K. Gomi, B. Scott, H. Oikawa, Org. Lett. 2016,
18, 5026-5029.
R. T. Gallagher, A. D. Hawkes, P. S. Steyn, R. Vleggaar, J. Chem. Soc.
Chem. Commun. 1984, 614-616.
R. T. Gallagher, E. P. White, P. H. Mortimer, N. Z. Vet. J. 1981, 29, 189-
190.
C. A. Young, M. K. Bryant, M. J. Christensen, B. A. Tapper, G. T. Bryan,
B. Scott, Mol. Genet. Genomics 2005, 274, 13-29.
[10] C. A. Young, S. Felitti, K. Shields, G. Spangenberg, R. D. Johnson, G. T.
Bryan, S. Saikia, B. Scott, Fungal Genet. Biol. 2006, 43, 679-693.
[11] S. Saikia, D. Takemoto, B. A. Tapper, G. A. Lane, K. Fraser, B. Scott,
FEBS Lett. 2012, 586, 2563-2569.
[12] T. Motoyama, T. Hayashi, H. Hirota, M. Ueki, H. Osada, Chem. Biol.
2012, 19, 1611-1619.
[13] C. Liu, A. Minami, T. Ozaki, J. Wu, H. Kawagishi, J. I. Maruyama, H.
Oikawa, J. Am. Chem. Soc. 2019, 141, 15519-15523.
[14] S. C. Munday-Finch, A. L. Wilkins, C. O. Miles, J. Agric. Food Chem.
1998, 46, 590-598.
[15] S. C. Munday-Finch, A. L. Wilkins, C. O. Miles, H. Tomoda, S. Omura, J.
Agric. Food Chem. 1997, 45, 199-204.
[16] M. Adachi, K. Higuchi, N. Thasana, H. Yamada, T. Nishikawa, Org. Lett.
2012, 14, 114-117.
[17] K. Kudo, C. Liu, T. Matsumoto, A. Minami, T. Ozaki, H. Toshima, K. Gomi,
H. Oikawa, ChemBioChem 2018, 19, 1492-1497.
[18] A. Minami, H. Oguri, K. Watanabe, H. Oikawa, Curr. Opin. Chem. Biol.
2013, 17, 555-561.
[19] J. J. Fernández, M. L. Souto, M. Norte, Nat. Prod. Rep. 2000, 17, 235-
246.
[20] P. Dowd, W. Zhang, Chem. Rev. 1993, 93, 2091-2115.
[21] D. H. R. Barton, R. S. H. Motherwell, W. B. Motherwell, J. Chem. Soc.
Perkin Trans. 1 1981, 2363-2367.
[22] V. H. Rawal, V. Krishnamurthy, A. Fabre, Tetrahedron Lett. 1993, 34,
2899-2902.
[23] S. Kim, J. S. Koh, J. Chem. Soc. Chem. Commun. 1992, 1377-1378.
[24] D. D. M. Wayner, D. J. McPhee, D. Griller, J. Am. Chem. Soc. 1988, 110,
132-137.
[25] D. Zhu, M.-J. Seo, H. Ikeda, D. E. Cane, J. Am. Chem. Soc. 2011, 133,
2128-2131.
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202007280
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Lolitrem B is a tremorgenic indole diterpene produced by a fungal endophyte. During its biosynthesis, LtmJ, a P450 monooxygenase,
catalyzes a 3-step oxidation of a diprenylated intermediate to construct the 5/6 bicyclic system. Given the reaction intermediate with
an epoxyalcohol, we establish the mechanism of radical-induced cyclization, supported by density functional theory calculations and
a model experiment.
8
This article is protected by copyright. All rights reserved.