Total Synthesis of Aetokthonotoxin, the Cyanobacterial Neurotoxin Causing Vacuolar Myelinopathy

Article abstract of DOI:10.1002/chem.202101848

Aetokthonotoxin has recently been identified as the cyanobacterial neurotoxin causing Vacuolar Myelinopathy, a fatal neurologic disease, spreading through a trophic cascade and affecting birds of prey such as the bald eagle in the USA. Here, we describe the total synthesis of this specialized metabolite. The complex, highly brominated 1,2’-biindole could be synthesized via a Somei-type Michael reaction as key step. The optimised sequence yielded the natural product in five steps with an overall yield of 29 %.

Full text of DOI:10.1002/chem.202101848

Accepted Article
Accepted Article
Title: Total Synthesis of Aetokthonotoxin, the Cyanobacterial
Neurotoxin Causing Vacuolar Myelinopathy
Authors: Bernhard Westermann, Manuel Garcia Ricardo, Markus
Schwark, Dayma Llanes, and Timo H. J. Niedermeyer
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.202101848
Link to VoR: https://doi.org/10.1002/chem.202101848
01/2020

10.1002/chem.202101848
Chemistry - A European Journal
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Total Synthesis of Aetokthonotoxin, the Cyanobacterial
Neurotoxin Causing Vacuolar Myelinopathy
Manuel G. Ricardo,*[a] Markus Schwark,[b] Dayma Llanes,[a] Timo H. J. Niedermeyer, [b]
Bernhard Westermann*[a]
Dedicated to Prof. Ludger A. Wessjohann on the occasion of his 60th birthday
[a]
Dr. M. G. Ricardo,[+] M. Sc. D. Llanes, and Prof. Dr. B. Westermann
Leibniz Institute of Plant Biochemistry
Department of Bioorganic Chemistry,
Weinberg 3, 06120, Halle/Saale, Germany.
[*] E-mail: Bernhard.Westermann@ipb-halle.de
Dipl.-Pharm. M. Schwark, Prof. Dr. T. H. J. Niedermeyer
Martin-Luther-University Halle-Wittenberg
Institute of Pharmacy, Department of Pharmaceutical Biology
Hoher Weg 8, 06120 Halle, Germany
[b]
[+]
present address:
Max-Planck Institute of Colloids and Interfaces,
Department of Biomolecular Systems,
Am Mühlenberg 1, 14476 Potsdam, Germany.
[*] E-mail: Manuel.GarciaRicardo@mpikg.mpg.de
Supporting information for this article is given via a link at the end of the document.
Abstract: Aetokthonotoxin has recently been identified as the
cyanobacterial neurotoxin causing Vacuolar Myelinopathy, a fatal
neurologic disease, spreading through a trophic cascade and
affecting birds of prey such as the bald eagle in the USA. Here we
describe the total synthesis of this specialized metabolite; this
complex, highly brominated 1,2’-biindole could be synthesized via a
Somei-type Michael reaction as key step. The optimised sequence
yielded the natural product in five steps with an overall yield of 29%.
In Figure 1, the structure of Aetokthonotoxin (1) is depicted,
exhibiting several structural features rarely (if at all) found in
natural products: i) AETX is the only known naturally produced
1,2’-bi-1H-indole; ii) only one other natural product contains an
indole-3-carbonitrile.[2] iii) Most prominent, also from the isotope
pattern that is observed in mass spectrometry analyses, are the
five bromo substituents accounting for 61% of the molecular
weight.
Aetokthonotoxin (AETX) is a pentabrominated biindole alkaloid
produced by the cyanobacterium Aetokthonos hydrillicola, which
grows epiphytically on an invasive plant, Hydrilla verticillata, in
freshwater lakes.[1] The toxin traverses the food chain from
animals consuming the plant (and thus also the cyanobacterium)
like waterfowl or snails and then on to raptors such as eagles
(e.g. American bald eagle) and kites. While the mode-of-action
of AETX is still unknown, it has been shown to elicit Vacuolar
Myelinopathy in birds, which is characterized by a widespread
vacuolization of the myelinated axons in the white matter of the
brain and spinal cord of these species.[1] AETX has also been
found to be highly toxic to the nematode C. elegans (LC50 40
nM),[1] but susceptibility of mammals to this toxin has not been
studied, yet.
Scheme 1. Retrosynthetic analysis of AETX (1).
To enable detailed studies on the biological activity of this novel
cyanotoxin, and thus to assess its potential impact on human
health, a total synthetic route to 1 is a prerequisite. According to
our retrosynthetic analysis (Scheme 1), the most obvious
challenge is the coupling of two readily prepared indole subunits.
Methods for this uncommon 1,2’-biindole linkage have been
explored in a very limited extent, using metal-catalyzed ligation
or nucleophilic displacement protocols.[3–5] Due to side-reactions
Figure 1. Structure of Aetokthonotoxin (AETX, 1) with structural features
rarely found in natural products.
1
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10.1002/chem.202101848
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based on participation of the bromo-phenylic moieties in these
reactions, the use of such procedures seemed inappropriate for
our target. Therefore, we devised that a Somei-type Michael
coupling fusing advanced indole intermediates could offer a
straightforward alternative to access this 1,2’-biindole.[6–8] This
envisioned coupling would be the first example of nitrile-modified
Michael acceptors and extend the use of this method largely. In
addition, in previous literature discussions, it was believed that
this reaction could be a biomimetic approach to achieve this
hitherto unknown indole-linkage.[5]
Implementing the Somei coupling strategy, the two building
blocks would have to be indoles 2 and 3a-c (Scheme 1). The
key intermediate 2, which holds the Michael-acceptor reactivity
(3-cyano-N-methoxy substitution) demanded by the coupling
process, was planned to be obtained either by indole synthesis
starting from appropriate brominated precursors, or already
functionalized indoles to be brominated subsequently. For the
complementary Michael-donors, the brominated indoles 3a-c
were suggested, depending on the reactivity in the coupling
reaction. While coupling with 3c would lead directly to the fully
substituted natural product, coupling with 3a,b would require
further bromination steps.
deficient alkynes affording desired indole 2. Preceding the
cycloaddition, 2,4-dibromo aniline 7 was efficiently transformed
into the nitroso derivative 8.[11] The cyano acetylene 9 was
synthesized from commercially available propiol amide by
dehydration (see Supporting Information).[12] Conversely,
attempts to use cyano acetylene 9 for the synthesis of 2 led to
difficult to separate product mixtures after the cycloaddition
reaction due to the formation of polymerized byproducts (see
Supporting Information).
A second alternative, depicted in Scheme 2 (strategy B), was
envisioned to be based on the protocol developed by Somei et
al. for the concerted oxidative transformation of indoline
derivatives into N-methoxy indoles.[13–15] Here, 5,7-dibromo-
indoline 5 (available from indoline 6 in 83% yield) is transformed
to 4, in 72% yield following slightly modified protocols.[14] Very
gratifyingly, the procedure developed by Vorbrüggen et al. using
CSI led to conversion of 4 to the wanted nitrile 2 in an isolated
yield of 95%.[16] Overall, the synthesis of 2 could be achieved in
gram scale in only three steps starting from commercially
available educts.
In the best case, the synthesis of AETX (1) would have been
achieved by coupling
2 with tribrominated indole 3c via
intermediate 12 by an initial Michael-reaction (Scheme 3). To
achieve the 1,4-C-N coupling, carbonyl moieties have been
reported on the Micheal acceptor,[14] here for the first time a
cyano group is used. The biindole is restored by a loss of the
methoxy moiety and rearomatization. The synthesis of this
tribrominated indole, however, proved to be quite tedious, as
most of the common bromination reagents gave only mixtures of
multi-brominated products of two (3,5; 3b), tri (2,3,5; 3c) and
tetra (2,3,5,6)-substituted bromoindoles. Thus, using bromine as
brominating agent, 3c could be obtained in 45% overall yield
starting from 3a (Scheme 2).[17] The synthesis of indole 3b has
been evaluated following numerous established protocols,
revealing that the method with PyHBr3 was most efficient in our
hands (Scheme 2).[18-20]
For the crucial biindole formation, the coupling step establishing
the N1-C2’ linkage could not be achieved when using indole 3c;
steric or stereoelectronic effects may be accountable for this.
Different conditions were applied unsuccessfully, with the
undesired 2-methoxy-3-cyano-5,7-dibromoindole 14 being the
only compound observed, as reported in the literature when poor
nucleophiles are used (Scheme 3, Supporting Information).[6][8]
Scheme 2. Synthesis of building blocks 2, and 3b,c. Reagents and conditions:
a) (NH4)2SO4 (5 equiv), H2SO4 (5 equiv), 0°C, 4h, 61%; b) 9 (5 equiv), K2CO3
(10 equiv), (CH3)2SO4 (10 equiv), toluene, 60°C, 6h, no reaction; c) PyHBr3
(2.2 equiv), DCM, 48 h, 83%; d) i: H2O2 (30%, 10 equiv), Na2WO4 (cat.),
MeOH/H2O/THF 6:1:2, 0-5°C, 1 h; ii: K2CO3 (3 equiv), (CH3)2SO4, (4 equiv),
0-5°C, 1 h, 72% (two steps); e) CSI (1 equiv), MeCN, 0°C, 1 h, then DMF,
0°C, 1 h, 95%; f) NaH (1.2 equiv), ClCO2Me (1.2 equiv), DMF, 0 °C, 2 h,
94%; g) Br2 (8 equiv), CCl4, r. t., 12 h, 55%, h) NaH (1.5 equiv), MeOH, reflux;
2h, 88%; i) PyHBr3 (1,1 equiv), DCM, 12h, 77%.
For the synthesis of the Michael acceptor 2, we initially
considered a protocol by Penoni et al. to be particularly
convenient for the creation of N-methoxy-3-substituted
indoles.[9,10] As depicted in Scheme 2 (strategy A), this approach
consisted of the cycloaddition of nitroso benzenes with electron
2
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Scheme 4. Synthesis of AETX (1). Reaction conditions: a) 3a (1.2 equiv), NaH
(1.1 equiv), 2 (1 equiv), DMF, 0°C to r. t., 12h; b) SEM-Cl (2.5 equiv), Cs2CO3,
(2 equiv) , r. t., 12 h, 74% (two steps); d) PyHBr3 (3 equiv), DCM, 12 h, 85%
(17); e) DBDMH (3 equiv), DCE, reflux, 12 h, 29%; f) Br2 (5 equiv), DCE, r. t., 1
h, 68% (1)
.
To extend the bromination alternatives, building block 2 was
coupled with 3a, and subsequently, instead of acidic “work-up”,
intermediate 12 was quenched with SEM-chloride to yield 16 in
74% yield (Scheme 4). The SEM group was chosen as ideal
protecting group to avoid interference of the NH-proton in the
required bromination steps and to boost hydrophobicity and,
therefore, increase solubility and facilitate purification. According
with our previous experiences in brominating procedures, we
tried PyHBr3 in DCM first, observing a clean reaction to tetra-
brominated 17. At this stage, attempts to selectively brominate
at position C-2 also failed. In further attempts, we turned our
attention to DBDMH in DCE, as it was reported to achieve C-2
and C-3-dibrominaton of indoles.[17] To our delight, we were
able, for the first time, to isolate a pentabromo biindole 18,
followed by SEM deprotection using 50% TFA in DCM, afforded
the natural product 1. Unfortunately, under these conditions, the
yield was quite low. Optimization efforts yielded the final product
in only 29% despite full consumption of the starting material, as
side reactions were observed. However, this result was
Scheme 3. Somei-Michael coupling attempts. Reagents and conditions: a)
General conditions of Somei reaction: 3 (1.2 equiv), NaH (1.1 equiv), 2 (1
equiv), DMF, 0°C to r. t., 12h; b) NH4Cl, 13a (65%), 13b (53%), for 3c: 14
detected instead of biindole; c) All brominating attempts modify only C-3 in 13a
to form 13b, when NBS in THF are used, C-6’ is then brominated to form 15.
Consequently, indoles 3a and 3b were employed in the coupling
reactions. Considered as alternatives to the desired coupling,
further bromination of the subsequent dimer would be necessary.
The Somei couplings performed with mono-brominated 3a and
di-brominated 3b, using NaH as base, confirmed the expected
reactivity of the indole 2, affording the desired N1-C2’ biindoles
13a and 13b in 65% and 53% yield, respectively (Scheme 3).
With these results, the choice of using the Somei-Michael
reaction for the synthesis of AETX became a realistic strategy.
Having in hand advanced biindoles, lacking bromine atoms only
at C-3 (13b) or C-2 and C-3 (13a) with respect to the natural
product; brominations at these most reactive positions were
considered achievable due to methods already carried out in our
studies (e.g. 11).[17,19,21] However, the very poor solubility of
13a,b in most common organic solvents reduced the possibilities
for the final bromination substantially, as the polarity of the
solvent is reported to be crucial in the selectivity of these
reactions.[17,19] All bromination attempts, regardless of the agent
used, did not afford 1 starting from 13a,b using THF or DMF as
solvents. Position C-3 of 13a was always smoothly brominated
to form 13b, while the missing bromine substitution at C-2
neither occurred nor took place at other positions rather than C-
2. We speculated that the electronic influence of the highly
ionizable NH group (1H-NMR signal over 12 ppm) along with a
different puckering of the two indole units in such polar solvents
would lead to form considerably electron enriched biindole
species that guide the bromination to other, unwanted positions.
The presence of a bromine atom at C-6’ in a pentabromo
biindole obtained in an experiment conducted with 13a, using
NBS in THF, agreed to this hypothesis (Scheme 3).[22]
inspiring,
as
it
indicated
the
feasibility
of
the
coupling/bromination strategy. Finally, applying 5 equivalents of
Br2 in DCE,[19,23-24] a clean reaction could be obtained, leading to
the satisfactory bromination at C-2 and C-3 at the same time.
Most pleasingly, the reaction conditions also cleaved the SEM-
protecting group (triggered by HBr formed in situ) to afford 1
directly
and
in
a
single
one-pot
reaction.
The
bromination/deprotection sequence could be accomplished in
68% yield. All spectroscopic and analytical data of synthesized
AETX were in accordance with the isolated natural product.
In summary, we could achieve the first total synthesis of
Aetokthonotoxin in five steps with an overall yield of 29%. The
crucial biindole-linkage could be realized via a Somei-Michael
reaction, the protection/deprotection reactions were both carried
out on the fly as quenching reactions. The highly convergent and
flexible synthesis presented here may facilitate remarkable
opportunities for further biological studies upon derivatization of
this alkaloid.
3
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10.1002/chem.202101848
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Experimental Section
Experimental procedures, HR-MS and spectroscopic data are
available in the Supporting Information.
Acknowledgements
Part of this work has been funded by the Deutsche Forschungs-
gemeinschaft (DFG, German Research Foundation, NI 1152/3-
1; INST 271/388-1).
Keywords: indole • biindole-coupling • natural products • total
synthesis • bromination
[1]
S. Breinlinger, T. J. Phillips, B. Haram, J. Mareš, J. A. Martínez
Yerena, P. Hrouzek, R. Sobotka, W. M. Henderson, P. Schmieder,
S. Williams, J. D. Lauderdale, H. D. Wilde, W. Gerrin, A. Kust, J. W.
Wahington, C. Wagner, B. Geier, M. Liebeke, H. Enke, T. H. J.
Niedermeyer, S. B. Wilde, Science, 2021, 371, eaax9050.
X. Fu, F. J. Schmith, J. Nat. Prod. 1995, 58, 1950–1954.
X. Tian, L. Song, M. Rudolph, F. Rominger, T. Oeser, A. S. K.
Hashmi, Angew. Chem. 2019, 131, 3627–3631; Angew. Chemie -
Int. Ed. 2019, 58, 3589–3593.
[2]
[3]
[4]
[5]
M. F. Comber, C. J. Moody, Synthesis 1992, 731–733.
T. Newhouse, C. A. Lewis, K. J. Eastman, P. S. Baran, J. Am.
Chem. Soc. 2010, 132, 7119–7137.
[6]
[7]
F. Yamada, D. Shinmyo, M. Somei, Heterocycles 1994, 38, 273–
276.
M. Somei, A. Tanimoto, H. Orita, F. Yamada, T. Ohta, Heterocycles
2001, 54, 425–432.
[8]
[9]
[10]
F. Yamada, D. Shinmyo, M. Nakajou, M. Somei, Heterocycles 2012,
86, 435–453.
A. Penoni, G. Palmisano, G. Broggini, A. Kadowaki, K. M. Nicholas,
J. Org. Chem. 2006, 71, 823–825.
G. Ieronimo, A. Mondelli, F. Tibiletti, A. Maspero, G. Palmisano, S.
Galli, S. Tollari, N. Masciocchi, K. M. Nicholas, S. Tagliapietra, G.
Cravotto, A. Penoni, Tetrahedron 2013, 69, 10906–10920.
A. M. Churakov, O. Y. Smirnov, S. L. Ioffe, Y. A. Strelenko, V. A.
Tartakovsky, Russ. Chem. Bull. 1994, 43, 1532–1535.
R. J. Halter, R. L. Fimmen, R. J. McMahon, S. A. Peebles, R. L.
Kuczkowski, J. F. Stanton, J. Am. Chem. Soc. 2001, 123, 12353–
12363.
[11]
[12]
[13]
[14]
M. Somei, T. Kawasaki, Heterocycles 1989, 29, 1251–1254.
K. Aoki, Y. Nagahama, K. Sugaya, Y. Maeda, H. Sato, K.
Nakagawa(Goto), M. Somei, Heterocycles 2019, 98, 236–270.
M. Somei, K. Yamada, M. Hasegawa, M. Tabata, Y. Nagahama, H.
Morikawa, F. Yamada, Heterocycles 1996, 43, 1855–1858.
H. Vorbrüggen, K. Krolikiewicz, Tetrahedron 1994, 50, 6549–6558.
J. Yan, T. Ni, F. Yan, Tetrahedron Lett. 2015, 56, 1096–1098.
V. Bocchi, G. Palla, Synthesis 1982, 1096–1097.
[15]
[16]
[17]
[18]
[19]
O. R. Suárez-Castillo, L. Beiza-Granados, M. Meléndez-Rodríguez,
A. Álvarez-Hernández, M. S. Morales-Ríos, P. Joseph-Nathan, J.
Nat. Prod. 2006, 69, 1596–1600.
[20]
M. R. Brennan, K. L. Erickson, F. S. Szmalc, M. J. Tansey, J. M.
Thornton, Heterocycles 1986, 24, 2879–2885.
[21]
[22]
S. Tang, J. H. Li, Y. X. Xie, N. X. Wang, Synthesis 2007, 1535–1541.
M. Hussain, S. M. Tengho Toguem, R. Ahmad, D. Thanh Tùng, I.
Knepper, A. Villinger, P. Langer, Tetrahedron 2011, 67, 5304–5318.
I. Saikai, A. J. Borah, P. Phukan, Chem. Rev. 2016, 116, 6837–
7042.
[23]
[24]
K. Piers, C. Meimaroglou, R. V. Jardine, R. K. Brown, Can. J. Chem.
1963, 41, 2399–2401.
4
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Entry for the Table of Contents
CN Br
NATURAL
SYNTHETIC
Br
Br
N
N
H
Br
Br
AETX
Synthesizing the eagle murder’s weapon. With the identification of the alkaloid Aetokthonotoxin (AETX) as the neurotoxin causing
wildlife deaths in the USA, further studies to characterize the biological effects of this natural product are mandatory. These studies
will be enabled by a total synthesis of this natural product, which has been achieved in only five steps overall.
Twitter: Pers: @bernis_lab; Inst: @ipb_halle
5
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