Chemical investigations into the biosynthesis of the gymnastatin and dankastatin alkaloids

Article abstract of DOI:10.1039/d1sc02613e

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strainGymnascella dankaliensishas provided an especially interesting collection of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore chemical and potentially biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding conditions that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can also be formed from aranorosin, a non-halogenated natural product also produced byGymnascellasp. fungi, using simple chloride salts thus offering an alternative hypothesis for the origins of these compounds in nature. Finally, growth inhibitory activity of multiple members against human triple negative breast cancer cells is reported.

Full text of DOI:10.1039/d1sc02613e

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Chemical investigations into the biosynthesis of the
gymnastatin and dankastatin alkaloids†
Cite this: Chem. Sci., 2021, 12, 8884
abc
Bingqi Tong,ab Bridget P. Belcher,ab Daniel K. Nomura
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
ab
*
and Thomas J. Maimone
Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein
function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an
especially interesting collection of halogenated cytotoxic agents derived from tyrosine which feature an
array of reactive functional groups. Herein we explore chemical and potentially biosynthetic relationships
between architecturally complex gymnastatin and dankastatin members, finding conditions that favor
formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural
products can also be formed from aranorosin, a non-halogenated natural product also produced by
Gymnascella sp. fungi, using simple chloride salts thus offering an alternative hypothesis for the origins
of these compounds in nature. Finally, growth inhibitory activity of multiple members against human
triple negative breast cancer cells is reported.
Received 12th May 2021
Accepted 31st May 2021
DOI: 10.1039/d1sc02613e
rsc.li/chemical-science
precursor. Gymnastatin and dankastatin alkaloids possess
a veritable treasure trove of distinctive electrophilic functional
Introduction
groups, including chloroenone, a-chloroketone, epoxyketone,
lactol, and a,b,g,d-unsaturated amide moieties; indeed, some
members contain as many as ve potential electrophilic sites.5
While detailed target identication studies are lacking, many of
these tyrosine-derived alkaloids are reported to possess signif-
icant anti-cancer activity.4
With over 20 members isolated possessing varying degrees of
oxygenation, halogenation, and cyclization, it is not unreason-
able to suspect that gymnastatin A (3) plays a central role in the
Natural products have long been a rich source of medicinal
agents and sources of inspiration for the design of numerous
clinical candidates and FDA-approved drugs.1 Among these,
members that interact with protein targets through covalent
bond formation have the potential to open up new areas of
druggable space, provide sustained target engagement, and
confer unique selectivity as a result of architectural comple-
mentarity to many fully synthetic small molecules.2 Moreover,
natural products featuring more than one covalent warhead
offer the prospect of engaging several nucleophilic protein
residues and potentially multiple protein partners.3
Against this backdrop, we became interested in the fasci-
nating array of chlorinated gymnastatin and dankastatin alka-
loids rst disclosed in 1997 from the fungal strain Gymnascella
dankaliensis isolated from the sponge Halichondria japonica
(Fig. 1).4 Presumably produced through the merger of tyrosine
and a 14-carbon polyketide fragment (see 1) to rst generate
gymnastatin N (2), electrophilic halogenation and various
oxidative cyclization reactions create a small library of archi-
tecturally complex natural products from a common and simple
aDepartment of Chemistry, University of California–Berkeley, Berkeley, CA 94720, USA.
E-mail: maimone@berkeley.edu
bNovartis-Berkeley Center for Proteomics and Chemistry Technologies, University of
California–Berkeley, Berkeley, CA 94720, USA
cDepartments of Nutritional Science and Toxicology, Cell and Molecular Biology, The
Innovative Genomics Institute, University of California–Berkeley, Berkeley, CA 94720,
USA
† Electronic supplementary information (ESI) available: Experimental procedures,
compound characterization data. See DOI: 10.1039/d1sc02613e
Fig. 1 Tyrosine-derived alkaloids from Gymnascella sp. fungi.
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Fig. 2 Selected chlorinated gymnastatin and dankastatin members and related natural product aranorosin.
biosynthesis of other tyrosine-derived alkaloids (see 4–14), growth inhibitory activity of six members spanning all three
possibly through chemistry which can be replicated without scaffold types (spirocyclic dienone, bicyclo[3.1.1]nonane, and
enzymes (Fig. 2). Indeed, biosynthetic logic has guided oxo-decalin) against human triple negative breast cancer cells.
synthetic routes to various gymnastatin members and related
alkaloids.6,7 Despite this, detailed chemical insight regarding
the formation, stereochemistry, and interconversion of various
Results and discussion
members is lacking. Given our interest in bicyclo[3.3.1]nonane-
containing natural products and covalently binding natural
products, especially those containing similar lipophilic amide
side chains,3 we sought to develop a unied synthetic platform
to these alkaloids as a gateway into studying their biological
targets.8,9 Herein we report simple synthetic solutions to
multiple chlorinated gymnastatin and dankastatin metabolites,
uncovering very subtle factors which favor the formation of
a given skeletal type. We also provide an unappreciated link
between this natural product family and the well-known fungal
natural product aranorosin (15) which has also been isolated
from a terrestrial variant of G. dankaliensis. Finally, we report
Bicyclo[3.3.1]nonane-containing gymnastatins and the oxo-
decalin-containing dankastatins are proposed to arise from 3
via aldol (see 16) and oxa-Michael (see 17) pathways respectively
(Fig. 3A).4 The presence of a C-9 methyl ether in gymnastatins F
(9) and Q (10) relative to a secondary hydroxyl group in gym-
nastatins G (11) raises questions regarding the identity of the
“OR” group that can trigger this process (i.e. H2O vs. MeOH), in
addition to stereochemical concerns arising from inter- vs.
intramolecular delivery of the oxygen nucleophile. Additionally,
dankastatins exists as two different sets of oxo-cis-decalin dia-
stereomers (compare 12 vs. 13/14); how (or if) nature controls
the formation of a given isomer is an intriguing question.
Fig. 3 Understanding ring formation in the biosynthesis of gymnastatin and dankastatin alkaloids. (A) Chemical and stereochemical possibilities.
(B) Stereochemical problems encountered when employing water as an oxygen nucleophile (C) optimization studies.
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We initially targeted gymnastatin G (11) owing to its potent two equivalents of base (entries 6 and 7).12 Of note, in entry 5,
reported activity against the P388 lymphocytic leukemia cell wherein the reaction was kept colder, we observed formation of
line, and reactive epoxyketone functionality.4a Inspired by the small amounts of the dankastatin scaffold (see 23) in addition
work of Nishiyama on ether-containing gymnastatins F/Q, we to 21/22. Finally, maintaining a ꢀ78 ꢁC reaction temperature
had hoped that simply substituting methanol with water would (entries 8–10) led to substantial quantities of 23 showing that
forge the bicyclo[3.3.1]nonane core of 11 in a biomimetic under carefully controlled conditions either scaffold can be
cascade (Fig. 3B).6d Known compound 18, derived from (L)- generated from 18.
tyrosine,6 was treated with aqueous KOH in MeCN yielding two
With conditions identied for construction of the key bicyclo
bicyclo[3.3.1]nonane-containing products, 19 and 20 in a 1.2 : 1 [3.3.1]nonane core with the correct C-9 stereocenter, we rein-
ratio. Surprisingly, however, the C-9 stereocenter was incorrectly vestigated the synthesis of gymnastatin G (11) (Scheme 1).
set during this process.10 Isomer 20 could be converted to 19 by While the epoxide found in 11 could be envisioned to arise from
treatment with catalytic amounts of base, suggesting that dia- the chloroenone motif of gymnastatin F/Q, we had been unable
stereomers at C-1 are formed in a reversible aldol reaction step, to realize this process using previously prepared isomer 19/20.11
and that, the oxa-Michael addition step, albeit producing an Given these observations, we proceeded to investigate a mono-
undesired outcome, was stereoselective. Presumably this chlorinated tyrosine derivative as a means to synthesize 11.
outcome arises from fast intramolecular oxa-Michael addition,
Carefully controlled mono-chlorination of Boc-tyrosine
wherein a hydrated aldehyde intermediate (see 17, R ¼ H) serves methyl ester (24) was achieved using sulfuryl chloride under
to deliver the oxygen nucleophile internally forming the cis-6,6- a stream of argon, by which the produced HCl could be removed
fused (dankastatin-type) bicyclic lactol. Subsequent lactol ring- thus preventing undesired removal of the Boc group under
opening then generates an aldehyde which participates in the acidic conditions. The resulting ester (25) was then reduced
aldol process. This observed reactivity questions the strategy with DIBAL providing aldehyde 26, which was subsequently
nature employs in setting the correct C-9 stereocenter if water is dearomatized with PIFA in the presence of TEMPO to provide
used as a nucleophile. From a synthetic standpoint, we were spirolactol 27 as a mixture of four diastereomers, two of which
also not successful in advancing 19/20 into gymnastatin G (11) are inconsequential. Using conditions discovered previously
(vide infra).11
(vide supra), treatment of 27 with allyl alcohol and KHMDS led
Given these results, we examined alternative alcohol-based to the bicyclo[3.3.1]nonane-containing product 28 in 35% yield
nucleophiles in order to prevent the proposed reaction as a mixture of diastereomers. Of note, the C-1 hydroxyl group,
pathway that leads to undesired C-9 stereochemistry; the formed in the aldol step, was a-disposed in the major product.
resulting alkyl ethers formed could in principle be deprotected Moreover, no isomeric bicyclo[3.3.1]nonane-containing prod-
and ultimately processed to 11 which we desired for biological ucts possessing an a-chloroenone motif were formed. The
testing (Fig. 3C). Dienone 18 was reacted with various quantities carefully-chosen allyl protecting group was removed under
of either allyl or benzyl alcohol using a variety of bases and reductive palladium-catalysis (Pd(PPh3)4, Bu3SnH) providing
subsequently quenched at various temperatures. Employing triol 29. Diastereoselective epoxidation of 29 with H2O2 and
sub stoichiometric quantities of Li-, Mg-, and Na-based bases Triton B afforded epoxyketone 30; notably the basic conditions
was ineffective at low temperatures (entries 1–3), but potassium employed also partially epimerized the C-1 stereocenter
bases employed in excess afforded substantial amounts of the (presumably via a retro-aldol/aldol reaction) to now favor b-
desired bicyclo [3.3.1]nonane 21 and isomeric counterpart 22 stereochemistry as found in 11. The mixture of epoxides were
(entries 4–7). The gymnastatin-type scaffold was favored under then exposed to TFA, removing the Boc group, and the free
these conditions, and optimal ratios of 21 were obtained using amine (31) coupled to known acid 32 (HATU, DIPEA) thus
Scheme 1 Total synthesis of gymnastatin G.
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Fig. 4 Studies towards the dankastatins. (A) Challenges in forming dankastatin A. (B) Total synthesis of dankastatin C.
delivering gymnastatin G (11) and its C1-epimer (33) in 60% chloroenone 37. In order to synthesize this structure, subtle
combined yield.13 Epimer 33 could also be converted into 11 in adjustments were made to the conditions for the intramolecular
50% yield by treatment with KHMDS.
oxa-Michael addition. Sodium methoxide was utilized as base
With access to the most complex gymnastatin member with MeOH as solvent and the reaction mixture maintained at
secured for biological testing, we turned our attention toward ꢀ20 ꢁC for a prolonged period—long enough for the double
the dankastatin family given that our initial screening of cycli- MeOH adduct (38) to be the major product but without signif-
zation conditions turned up conditions to favor this scaffold. icant bicyclo[3.3.1]nonane formation. If KHMDS was used as
Chlorinated dankastatin members (see 12–14), however, are base, bicyclo[3.3.1]nonane-containing products predominated.
produced with two different isomeric cis-decalin frameworks. Through this process, 38 was formed as a single diastereomer in
Notably, in dankastatin A (12) the tertiary alcohol and neigh- 42% yield along with 20% of 37. Finally, Boc deprotection of 38
boring proton (see C-4 and C-9) are on opposite faces as with TFA, followed by coupling with side chain 32 (HATU,
compared to dankastatins B (13) and C (14). In analogy to work DIPEA) forged dankastatin C (14).
in Fig. 3C, treating 18 with KHMDS/MeOH generated
Unlike dankastatin C, and in fact the majority of other
compound 37 and not the dankastatin A-type cis-fused skeleton tyrosine-derived alkaloids from Gymnascella, dankastatin B (14)
35 (Fig. 4A). We presume that in the cyclization of 18, an axial features an alcohol, rather than aldehyde, oxidation state at C-1.
conguration of the amide side chain (see 34 vs. 36), disfavors To access this natural product, Boc-tyrosine methyl ester (24)
formation of 35.14 Again, this raises the question as to how the was dichlorinated (SOCl2, HOAc) and reduced with DIBAL to
dankastatin A-type skeleton is prepared in nature. Fortunately, yield 40 (Fig. 5). With 40 in hand, we sought to nd suitable
isomer 37 does however, bear resemblance to dankastatins B oxidative dearomatization conditions that were compatible with
and C, thus offering a potential pathway to these targets the free hydroxy group. Aer some exploration, success was
(Fig. 4B).
Dankastatin C, a more recently isolated member of the the presence of cesium carbonate (see inset). The yields of this
dankastatin family,4c possesses
structure suggestive of process were initially quite low (entries 1–3), but in the presence
a hydration event on a biosynthetic intermediate akin to of PPh3 the dankastatin core (see 42) could be formed directly,
realized using singlet oxygen-based conditions (O2, TPP, hn) in
a
Fig. 5 Total synthesis of dankastatin B.
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Fig. 6 Aranorosin as a possible biosynthetic precursor to chlorinated alkaloids from Gymnascella sp.
albeit in low yield (entry 4). Interestingly, in addition to 42, we aranorosin.18 Notably, the two diastereomeric natural products
observed a minor isomer (iso-42) which corresponds to the (presumably generated via E1cB reactions of 45 and 46) were
dankastatin A scaffold (dr ꢂ 1.6 : 1). Through reductant and generated in nearly a 1 : 1 ratio—an apparent result of nonse-
temperature optimization (entries 5–11), we found that high lective epoxide opening; this observation echoes back to the two
yields of dienone 41 (70%) could be obtained using an electron- diastereomeric skeletons of dankastatins found in nature. Also
decient phosphine (P(3,5-(CF3)2C6H3)3) at low temperature. detected was a small amount of unsaturated aldehyde 47,
Isolated 41 could then be converted to 42 under basic condi- a structure reminiscent of prior C–C cleavage products.12 While
tions (KHMDS) in 78% yield. While this sequence requires two we are unaware of 47 being a real natural product, it is inter-
steps, the yield (78%) and diastereoselectivity (dr ¼ 8 : 1) were esting to consider whether Gymnascella dankaliensis might
substantially higher than the one-pot transformation. Depro- employ oxidized tyrosines as precursors to electrophilic small
tection of 42 (TFA) and coupling with acid 32 yield dankastatin molecules containing dehydroalanine-like motifs. In any event,
B (13).
The successful application of proposed biomimetic strate- and dankastatin alkaloid biosynthesis is appealing.
gies in the synthesis of dankastatin and gymnastatin alkaloids With access to these natural products, which contain all of
investigations into the enzymology surrounding gymnastatin
sheds some light on how nature might make these natural the relevant structural types found in this family, we evaluated
products and the challenges it faces and/or solves in doing so. their cytotoxicity against aggressive human triple negative
Yet problems we encountered in our pursuit of gymnastatin G breast cancer cells (231MFP cell line) (Fig. 7).19 As noted, many
and dankastatin A led us to consider alternative hypotheses for chlorinated tyrosine-derived alkaloids have shown strong anti-
the origins of these chlorinated alkaloids derived from tyrosine. cancer activity, although many of these studies have been
Specically, we were drawn to the bis-epoxyketone-containing
natural product aranorosin (15), which is not halogenated,
but bears clear structural, and likely biosynthetic, similarities to
3–14.4d Notably, the a-chloroenone in gymnastatin A and the
epoxyketone in aranorosin are of the same oxidation level and
we wondered if nature might use nucleophilic, chloride-
mediated chemistry and not electrophilic chloronium-induced
reactions in the construction of this alkaloid family.15,16
Commercially available aranorosin reacted with LiCl (1.5
equiv.) at room temperature in THF, forming a variety of chlo-
rinated products under very mild conditions (Fig. 6).17 Notably,
gymnastatin G (11) and 1-epi-gymnastatin G (33) were isolated
from the reaction mixture in 23% combined yield, presumably
through an aldol reaction of intermediate 46. In addition, two
more natural products, namely aranochlor A (44) and arano-
chlor B (43), which are oxidized variants of gymnastatins D (6)
and E (7) respectively, were also formed in the reaction (in 12%)
Fig. 7 Anti-triple negative breast cancer (231 MFP) activity of select
and can be viewed as links between gymnastatin A and
electrophilic alkaloids from Gymnascella sp.
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conducted in murine tumor cell lines.4 Dankastatin B exhibited analysis wherein support from NIH Shared Instrument Grant
the highest potency (EC50 ¼ 0.6 mM) followed closely by ara- (S10-RR027172) is also acknowledged.
norosin (EC50 ¼ 1.6 mM), gymnastatin A (EC50 ¼ 2.1 mM), and
nally dankastatin C (EC50 ¼ 5 mM). Interestingly, bicyclo[3.3.1]
Notes and references
nonane-containing gymnastatins Q and G, which notably also
contain only a single electrophilic site in the oxidized tyrosine
core, were far less active in this cellular context. Whether these
natural products have the same (or related) biological target
proles remains to be determined – work to address these
questions is currently underway and will be reported in due
course.
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Conclusion
In summary, we have completed the rst total syntheses of
gymnastatin G and dankastatins B and C, and in the process,
explored stereochemical and structural questions surrounding
the origins of chlorinated, tyrosine-derived alkaloids. During
our studies, we discovered that very small and subtle changes to
abiotic reaction conditions could be leveraged to promote the
formation of either oxo-decalin or bicyclo[3.1.1]nonane motifs;
how nature modulates these product ratios remains an open
and interesting question. Additionally, an alternative biosyn-
thetic hypothesis for the origins of chlorinated gymnastatin
alkaloids from the well-known fungal metabolite aranorosin
was also presented; notably this pathway can circumvent certain
stereochemical problems associated with the abiotic Michael/
aldol cascade approach. Finally, as a result of these investiga-
tions, dankastatin B has emerged as a potent, and easily
synthesized, small molecule hit against triple negative breast
cancer.
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Author contributions
¨
A. H. Aly, M. Abdel-Aziz, W. E. G. Muller, W. Lin,
T. J. M. and B. T. initiated the project and B. T. conducted all of
the synthetic experiments. B. P. B. and D. K. N. performed
biological assays. T. J. M. wrote the paper with the assistance of
B. T. All authors provided feedback and contributed to editing
the manuscript.
G. Daletos and P. Proksch, Cytotoxic acyl amides from the
soil fungus Gymnascella dankaliensis, Bioorg. Med. Chem.,
2015, 23, 712; (e) H. Wang, H. Dai, C. Heering, C. Janiak,
¨
W. Lin, R. S. Orfali, W. E. G. Muller, Z. Liu and P. Proksch,
Targeted solid phase fermentation of the soil dwelling
fungus Gymnascella dankaliensis yields new brominated
tyrosine-derived alkaloids, RSC Adv., 2016, 6, 81685; (f)
D. Xu, M. Luo, F. Liu, D. Wang, X. Pang, T. Zhao, L. Xu,
X. Wu, M. Xia and X. Yang, Cytochalasan and Tyrosine-
Derived Alkaloids from the Marine Sediment-Derived
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Conflicts of interest
This study was funded in part by the Novartis Institutes for
BioMedical Research and the Novartis-Berkeley Center for Pro-
teomics and Chemistry Technologies. Daniel K. Nomura is a co-
founder, shareholder, and adviser for Frontier Medicines.
5 Notably, many brominated variants of these alkaloids also
exist, see: ref. 4d and e.
6 For syntheses of various gymnastatins, see:(a) M. K. Gurjar
Acknowledgements
Financial support is acknowledged from the NIH NIGMS
(R01GM136945) and the Novartis Berkeley Center for Proteo-
mics and Chemistry Technologies (NB-CPACT). T. J. M.
acknowledges unrestricted nancial support from Novartis,
Bristol-Myers Squibb, Amgen, and Eli Lilly. We are grateful to Dr
Hasan Celik and Dr Jeffrey Pelton for NMR spectroscopic
assistance and NIH grants GM68933 and S10OD024998. Dr
Nicholas Settineri is acknowledged for X-ray crystallographic
and P. Bhaket, Total Synthesis of a Novel Cytotoxic
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incorrect
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structure assignment of the antitumor antibiotic
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our hands, this reaction did not occur using 19/20.
Additionally, chemoselective hydrodechlorination of the
vinyl chloride in the presence of the tertiary chloride was
not possible.
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these reactions was the cleavage reaction shown below. A
related process in proteins involving tyrosine has been
observed during thyroid hormone synthesis, see:
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13 While gymnastatin G was reported as quite labile, we found
our synthetic material was stable enough for full
characterization. Nevertheless, the synthetic gymnastatin G
was acylated with Ac2O, and the NMR resonances of the
resulting diacetate matched those reported by the Numata
group.
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R. Ho, H. S. Lehtola, M. To, X. Hu, T. R. Huffman, Y. Petri,
C. R. Altobelli, S. G. Demeulenaere, J. A. Olzmann,
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14 The same result was obtained using gymnastatin
A
indicating this outcome is not unique to the Boc
protecting group.
15 While there is no evidence that such a transformation is
related to the actual biosynthesis of these alkaloids, we
note that chloroperoxidases can also carry out P450-like
transformations. Given that an oxidase likely constructs
aranorosin's bis epoxide motif through double epoxidation
of a dienone (see ref. 4d), it is not inconceivable to
consider that outer sphere attack by chloride on an
initially oxidized product could be relevant to the
biosynthesis of these metabolites.
16 Studies by the Wipf lab on the reactivity of aranorosin with
sulfur nucleophiles has shown that bicyclo[3.3.1]nonane-
containing products can be formed, see: (a) P. Wipf,
P. Jeger and Y. Kim, Thiophilic ring-opening and
rearrangement reactions of epoxyketone natural products,
Bioorg. Med. Chem. Lett., 1998, 8, 351; (b) J. T. Hammill,
´
J. Contreras-Garcıa, A. M. Virshup, D. N. Beratan, W. Yang
and P. Wipf, Synthesis and chemical diversity analysis of
bicyclo[3.3.1]non-3-en-2-ones, Tetrahedron, 2010, 66, 5852.
17 Notably, these conditions are unusually mild for chloride-
induced opening/elimination of an epoxyketone, which
D. K. Nomura,
A Nimbolide-Based Kinase Degrader
Preferentially Degrades Oncogenic BCR-ABL, ACS Chem.
Biol., 2020, 15, 1788; (d) B. Tong, M. Luo, Y. Xie,
8890 | Chem. Sci., 2021, 12, 8884–8891
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typically require acid activation or elevated temperatures.
Production, Isolation, Structure Elucidation and Biological
Presumably the lithium cation is able to fulll this role
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given the highly activated aranorosin bis-epoxide system. 19 N. Jessani, M. Humphrey, W. H. Mcdonald, S. Niessen,
Importantly, when tetrabutyl ammonium chloride was
used, no conversion occurred under the same conditions.
18 T. Mukhopadhyay, R. G. Bhat, K. Roy, E. K. S. Vijayakumar
and B. N. Ganguli, Aranochlor A and Aranochlor B, Two
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New
Metabolites
from
Pseudoarachniotus
roseus:
© 2021 The Author(s). Published by the Royal Society of Chemistry
Chem. Sci., 2021, 12, 8884–8891 | 8891