Construction of a meroterpenoid-like compound collection by precursor-assisted biosynthesis

Article abstract of DOI:10.1039/d0ob01235a

Natural products (NPs) and their derivatives play a pivotal role in drug discovery due to their complexity and diversity. The strategies to rapidly generate NP-like compounds offer unique opportunities to access bioactive compounds. Here we present a new approach, precursor-assisted biosynthesis (PAB), for the creation of NP-like compounds by combination of artificial supplementation of common precursors and divergent post-modifications of precursor-deficient fungi. This method was applied to construct a meroterpenoid-like compound collection containing 43 compounds with diverse molecular scaffolds. Extensive bioactive screening of the collection revealed novel STING (stimulator of interferon genes) inhibitors, cytotoxic and antifungal compounds. This result indicates that PAB is an effective methodology for producing compound collections for the purpose of drug discovery.

Full text of DOI:10.1039/d0ob01235a

Organic &
Biomolecular Chemistry
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Construction of a meroterpenoid-like compound
collection by precursor-assisted biosynthesis†
Cite this: DOI: 10.1039/d0ob01235a
a
a
a
a
a
b
Panlong Ren,‡ Xinyu Miao,‡ Ting Tang,‡ Yueting Wu, Jing Wang, Ying Zeng,
a
a
a
Yun Li,
Kun Gao* and Yan-Long Yang
*
Natural products (NPs) and their derivatives play a pivotal role in drug discovery due to their complexity
and diversity. The strategies to rapidly generate NP-like compounds offer unique opportunities to access
bioactive compounds. Here we present a new approach, precursor-assisted biosynthesis (PAB), for the
creation of NP-like compounds by combination of artificial supplementation of common precursors and
divergent post-modifications of precursor-deficient fungi. This method was applied to construct a mero-
terpenoid-like compound collection containing 43 compounds with diverse molecular scaffolds.
Extensive bioactive screening of the collection revealed novel STING (stimulator of interferon genes)
inhibitors, cytotoxic and antifungal compounds. This result indicates that PAB is an effective methodology
for producing compound collections for the purpose of drug discovery.
Received 15th June 2020,
Accepted 14th July 2020
DOI: 10.1039/d0ob01235a
rsc.li/obc
Biosynthetic machineries usually make use of divergent
pathways to produce a variety of structurally complex and
Introduction
Natural products (NPs) and their derivatives remain a rich diverse NPs from only a handful of fundamental building
source of inspiration in the discovery and development of blocks. Large numbers of NP-like compounds have been gener-
1
potential drugs and biological probes. The scaffolds of NPs ated by various combinations of chemical and biosynthetic
8
,9
represent the biologically relevant and privileged fractions of approaches.
Unfortunately, most of them possess similar
chemical space because they have been naturally selected in scaffolds with original natural products. Since skeletal diver-
evolution to specifically interact with biological macro- sity is a major issue associated with the development of a
2
molecules. It has been suggested that NP-like compound col- high-value compound library for screening of promising drug
3
lections are enriched in biochemical and biological activity.
leads and biological probes, a general strategy for the creation
Thus, the construction of NPs inspired compound collections of NP-like compounds with diverse molecular scaffolds needs
and their evaluation is a highly promising strategy for the to be formulated.
identification of unique bioactive compounds. Based on this
The common biosynthetic precursors can be divergently
reasoning, a number of chemical synthesis strategies have modified to produce distinct skeletons in different organ-
been developed to rapidly generate NP-like compounds includ- isms and therefore show significant potential to increase the
4
ing diversity-oriented synthesis (DOS), biology-oriented syn- skeletal diversity of NPs. During the evolution of secondary
5
6
thesis (BIOS), diversity-enhanced extracts and the complex- metabolism, the emergence of enzymes responsible for the
7
ity-to-diversity (CtD) strategy. However, the synthetic accessi- biosynthesis of common precursors and post modifications
bility and efficiency of these strategies may face challenges sometimes happened separately.
1
0
Besides, non-specific
associated with their structural complexity.
enzymes could modify natural products and their biosyn-
1
1
thetic precursors to increase the chemical diversity. Thus,
although some organisms possess post modification
enzymes, the deficiency of the ability to synthesize the
common precursors precludes the production of NPs.
Artificial supplementation of the precursors to these organ-
isms may lead to diverse NP-like compounds. We therefore
focused on the development of a precursor-assisted biosyn-
thesis (PAB) that combines artificial supplementation of
common precursors and divergent post-modifications of pre-
cursor-deficient fungi to produce a large number of NP-like
compounds.
a
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou 730000, China.
E-mail: npchem@lzu.edu.cn, ylyang@lzu.edu.cn
b
State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
†
Electronic supplementary information (ESI) available: Experimental details,
supplementary figures, spectral data and CIF files. CCDC 1997727–1997731. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
d0ob01235a
‡
These authors contributed equally to this work.
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Meroterpenoids are natural products of mixed biosynthetic
Organic & Biomolecular Chemistry
Results and discussion
1
2
origin that are partially derived from terpenoids. Particularly,
meroterpenoids derived from the shikimate and terpenoid Prenylhydroquinone (p1a) is a common precursor of meroter-
pathway produced by plants, mushrooms and marine invert- penoids with diverse scaffolds. To test whether this common
ebrates display high scaffold diversity and significant precursor could be used for the generation of meroterpenoid-
1
3–16
bioactivity.
Therefore, constructing a diverse compound like compounds by PAB, p1a was synthesized and fed to the
collection based on shikimate derived meroterpenoids would culture of five precursor-deficient basidiomycete fungi
1
7
increase the likelihood of discovering new drug candidates.
Agrocybe pediades, Marasmius graminum, Stereum histurum,
Intriguingly, a large number of members with diverse scaffolds Psathyrella candolleana and Trametes hirsute on the seventh day
of this family were biosynthesized from only several simple after inoculation, respectively. After an additional two days of
common precursors, prenylhydroquinone (p1a), geranylhydro- culture, the culture broth was harvested and extracted with
quinone (p1b) and farnesylhydroquinone (p1c) by divergent ethyl acetate, and the extracts were analyzed by HPLC. Notably,
pathways (Fig. 1). For example, a biogenetic hypothesis for some new peaks were observed in the HPLC profiles of the
representative members is outlined in Fig. 1, where the preny- crude extracts of p1a supplemented culture of all five strains
lation and oxidative decarboxylation of 4-hydroxybenzoate compared with controls (Fig. S1–S5†). To elucidate the struc-
(4-HBA) lead to the formation of common precursors p1a–c. tures of the transformed products, p1a was supplemented in
Divergent modification including oxidation, cyclization and the scaled-up culture of these fungi. Systematic purification of
dehydration of p1a–c directly generates a vast chemical diver- their ethyl acetate extracts by a combination of column chrom-
sity and produces panepoxydone, shikonin, clavilactone A, atography over silica gel and Sephadex LH-20 and semi-pre-
1
3–16
conicol, longithorone B, pleurotin and rossinone B.
parative reversed-phase HPLC afforded eight (1–8), two (9–10),
Biosynthesis of structurally distinct meroterpenoids from two (7–8), two (7–8) and three (1, 11–12) meroterpenoid-like
common precursors showed the huge potential of post-modifi- compounds from these fungi, respectively (Fig. 2). Their struc-
cation to create chemical diversity.
tures were elucidated on the basis of extensive spectroscopic
In this paper, we reported the construction of a meroterpe- analysis (ESI-MS, HR-ESI-MS, and 1D and 2D NMR) (Tables
noid-like compound collection containing 43 compounds with S1–S3†) and by comparison of their NMR data with those
twelve distinct scaffolds by feeding chemical synthesized reported in the literature. The absolute configuration of 1 was
common precursors, p1a–c, and their analogues, p2–5, of mer- deduced by comparing its specific rotation, −43.5 (c 0.23,
oterpenoids to five precursor-deficient basidiomycete fungi MeOH), with that of (S)-4-hydroxy-3-methyl-2-cyclohexen-1-one,
1
8
19
which do not produce meroterpenoids under normal lab con- −35.2 (c 1.0, CHCl3) and speciosin K, −14.5 (c 0.13, CHCl ).
3
1
ditions. Among these compounds, STING (stimulator of inter- The relative configurations of 2–3 were deduced from H NMR
feron genes) inhibitors, cytotoxic and antifungal compounds coupling constants (Table S1†) and their absolute configur-
were obtained through extensive bioactive screening.
ations were assigned by octant rule analysis of the Cotton
effects observed in the CD spectra (Fig. S23 and S24†). The
relative configuration of 10 was determined by single-crystal
X-ray diffraction analysis (Fig. S16 and Table S15†). The
1
β-xylose unit in 11–12 was elucidated based on the H NMR
2
0
coupling constants (Table S3†). Compounds 1–3 and 10–12
are new compounds, while 4–6, 7, 8 and 9 were isolated pre-
2
1
22
viously from Hexagonia crinigera, Acremonium sp., Aplidium
2
3
24
californicum, and M. graminum, respectively. Compounds
–6 are assumed to be formed by successive reduction of pre-
1
nylquinone (A) as shown in Fig. 3. Oxidation of p1a followed
by reduction of the C5–C6 double bond and the C1 ketone gen-
erates 1 and its enantiomer 1a. Further reduction of 1a at the
C2–C3 double bond afforded 2–3 and subsequent reduction of
2
7
–3 at the C4 ketone led to the formation of 4–6. Compounds
–8 seem to be formed by epoxidation of p1a followed by cycli-
2
5
zation and dehydration. Compound 9 is assumed to be pro-
duced by 6π electrocyclization followed by dehydration of inter-
mediate D, while compound 10 seems to be formed by radical
addition of intermediate C. The formation of 11–12 may be
catalyzed by a beta-xylosidase.
2
6
2
7
Meromonoterpenoids derived from geranylhydroquinone
(
p1b) display greater chemical diversity than p1a derived mero-
terpenoids, owing to the geranyl offering more modification
Fig. 1 Proposed divergent biosynthetic pathways of shikimate derived
meroterpenoids.
sites. To obtain meromonoterpenoid-like compounds, we syn-
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Fig. 2 Meroterpenoid-like compounds isolated from p1a–c supplemented culture of precursor-deficient fungi.
thesized p1b and fed it to the culture of A. pediades, (13–19, 22 and 28), three (24–26), nine (13–17, 21–23 and 25)
M. graminum, S. histurum, P. candolleana and T. hirsute as and two (20–21) compounds were isolated from those of
described for p1a. HPLC analysis of the extracts indicated M. graminum, S. histurum, P. candolleana and T. hirsute,
obvious new peaks emerged from the crude extracts of p1b respectively (Fig. 2). The structures including the relative
supplemented culture of all five strains compared with con- stereochemistry of these compounds were established based
trols (Fig. S6–S10†). The crude extracts derived from the p1b on NMR spectroscopy and single-crystal X-ray diffraction ana-
supplemented culture of five strains were separated by lysis (Fig. S17–S20, Tables S4–S8 and S16–S19†). The specific
repeated column chromatography and semi-preparative rotation, +4.2 (c 0.95, CHCl ) and the absence of CD Cotton
3
reversed-phase HPLC. Eleven meroterpenoid-like compounds effects indicated that 13 is likely a racemate which was further
(
13–17, 20–21, 23–25 and 27) were isolated from the extracts of confirmed by chiral HPLC analysis and single-crystal X-ray
p1b supplemented culture of A. pediades (Fig. 2), whereas nine data (Fig. S17 and S21†). Considering the same biosynthesis
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Fig. 3 Plausible biosynthetic pathways for the meroterpenoid-like compounds obtained by PAB.
pathway as well as their specific rotation and CD spectra, com- Diels–Alder reactions (VQDA) of intermediate G. Several mero-
pounds 14–22 were also deduced to be racemate. Compounds terpenoids containing this rare 6,6,5-ring system, pycnanthu-
1
3–19 have a rare linearly fused 6,6,5-tricyclic carbon skeleton, quinones A–C and rossinone B, have been reported and the
which seems to be formed by intramolecular vinyl quinone biomimetic synthesis of (−)-pycnanthuquinone C was accom-
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2
8
plished by the Trauner group in 2010. The fact that 13–22 are
To obtain merosesquiterpenoid-like compounds, p1c (Z, E
racemate accords with proposed nonenzymatic biosynthetic mixture) was synthesized and fed to the culture of A. pediades,
pathways as shown in Fig. 3. Hydroxylation of p1b at C-7 fol- M. graminum, S. histurum, P. candolleana and T. hirsute as
lowed by rearrangement of hydroxyl and oxidation generates described for p1a. Unfortunately, new peaks were observed
the vinyl quinone G. The intramolecular VQDA reaction and only in the crude extracts of p1c supplemented culture of
subsequent addition of water at C-7 afforded 14 and 15. S. histurum (Fig. S11–S15†). After a 10 L scale culture, (E)-dic-
Methylation of C-7 hydroxyl could yield 16–17. On the other tyochromenol (29) and (Z)-dictyochromenol (30) were isolated
hand, aerobic oxidation of 14–15 afforded 18–19. Compounds as transformed products of p1c (Fig. 2). Dictyochromenol was
2
0–22 are assumed to be produced by cleavage of the C-8/C-9 reported previously as an antifeedant with potent activity com-
carbon bond of 14–15 depicted in Fig. 3. Compound 23 pos- parable to that of zonarol and zonarone from Dictyopteris undu-
3
1
sesses a rare benzo[b]cyclopenta[e]oxepine ring system, which lata. Compounds 29–30 seem to be formed by oxidative cycli-
2
5
was assumed to be formed by 8,12 cyclization of J. The optical zation of p1c.
rotation for 23 was −2.7 (c 0.10, MeOH), as well as the CD spec- The production of compounds 1–30 from common precur-
trum showed no Cotton effect indicating that 23 was also a sors p1a–c showed the huge potential of precursor-deficient
racemic mixture, which was further confirmed by chiral HPLC basidiomycete fungi to create the chemical diversity of mero-
analysis (Fig. S22†). Epiconicol (24), conidione (25), conitriol terpenoids by divergent modification. To further expand the
(
26) and cordiachromene A (27) were isolated previously from chemical diversity of meroterpenoid-like compounds, ana-
Ascidian Aplidium conicum and 24–26 were likely formed by logues, p2–5, of p1b were synthesized as described in support-
2
9
7
,12 cyclization of F. Compound 28 was reported as a syn- ing materials and fed to the culture of A. pediades. As a result,
thetic compound and seems to be formed by 6π electrocycliza- four (31–34), three (35–37), two (38–39) and four (40–43) mero-
3
0
tion followed by dehydration of intermediate L.
terpenoid-like compounds were isolated as the transformed
Fig. 4 Meroterpenoid-like compounds isolated from p2–5 supplemented culture of A. pediades.
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products of p2, p3, p4 and p5, respectively (Fig. 4). The struc-
tures including the relative configurations of these compounds
Acknowledgements
were established based on NMR spectroscopy (Tables S9–S14 This work was financially supported by the National Natural
and Fig. S122–S197†). Compounds 31, 38 and 40 are assumed Science Foundation of China (No. 21807054); the State Key
to be formed by carbocation-initiated cyclization. Compounds Laboratory of Phytochemistry and Plant Resources in West
3
2, 39 and 41–42 seem to be formed by intramolecular de- China (No. P2017-KF05) and the Fundamental Research Funds
hydration. The formation of compounds 35–37 is similar to for the Central Universities (No. lzujbky-2019-ct03).
that of 4–6.
To verify the usefulness of the collection of meroterpenoid-
like compounds for drug discovery and to discover new bio-
active compounds, the collection was extensively screened for
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Conflicts of interest
The authors declare no competing financial interest.
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