Synthesis of the Death-Cap Mushroom Toxin α-Amanitin

Article abstract of DOI:10.1021/jacs.7b12698

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.

Full text of DOI:10.1021/jacs.7b12698

Communication
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin
Kaveh Matinkhoo, Alla Pryyma, Mihajlo Todorovic, Brian O. Patrick, and David M. Perrin*
Chemistry Department, The University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T-1Z1, Canada
*
S Supporting Information
derivatives of amanitin along with partially active synthetic
analogues.
Unlike most cytotoxins that act primarily on rapidly dividing
3
ABSTRACT: α-Amanitin is an extremely toxic bicyclic
octapeptide isolated from the death-cap mushroom,
Amanita phalloides. As a potent inhibitor of RNA
polymerase II, α-amanitin is toxic to eukaryotic cells.
Recent interest in α-amanitin arises from its promise as a
payload for antibody−drug conjugates. For over 60 years,
A. phalloides has been the only source of α-amanitin. Here
we report a synthesis of α-amanitin, which surmounts the
key challenges for installing the 6-hydroxy-tryptathionine
sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-
cells, α-amanitin kills both rapidly growing and quiescent cells
12
by inhibiting Pol II, causing its rapid degradation leading to
13
apoptosis. Recently, it was shown that α-amanitin, when
injected intraperitoneally at sublethal doses, prevents cancer
relapse in mice bearing tumor xenografts that are resistant to
14
common chemotherapeutics. Impressively, antibody−drug
conjugates (ADCs) of α-amanitin cure mice of pancreatic
1
5
cancer xenografts and are advancing toward human trials.
16
4,5-dihydroxy-isoleucine, and diastereoselective sulfoxida-
As fermentation yields are low, a synthetic source of the
toxin is needed to prepare ADCs for use in clinical oncology.
15
tion.
Moreover, the lack of a synthetic route to α-amanitin presents a
critical barrier to the design of new analogues for systematic
17
α-Amanitin, one of the deadliest toxins known to humankind
SAR analysis. That α-amanitin has resisted synthesis is
reflected in three implicit challenges: (i) a synthetic route to the
oxidatively delicate 6-hydroxy-tryptathionine, (ii) an enantio-
selective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and
(iii) a diastereoselective sulfoxidation to favor the (R)-sulfoxide.
Herein, we overcome these challenges in a synthesis of this
venerated toxin, for which the retrosynthesis is given in Figure
2.
(
LD50 = 50−100 μg/kg), is featured in modern biochemistry
1 2,3
textbooks, underscoring a rich scientific history. It is the
principal toxin of the amatoxin family of peptides produced by
Amanita phalloides, the notorious “death-cap” mushroom,
which has been used for murder and suicide dating to
Roman times. Isolated over 60 years ago, α-amanitin is a
potent, orally available, highly selective allosteric inhibitor of
4
,5
6
7
RNA polymerase II (Pol II). Its bicyclic octapeptide structure
The first challenge is the most formidable as L-Trp must be
selectively oxidized at positions 6 and 3 to afford a 6-hydroxy-
tryptathionine cross-link (Figure 2, bottom panel, left), which
must undergo sulfoxidation in a later step. Routes to a
tryptathionine cross-link have involved the reaction of
tryptophan with a cysteine−sulfenyl chloride or iodide, either
with orthogonally protected monomers or in the context of a
contains two oxidized amino acids that are key to its toxicity:
trans-4-hydroxy-proline (Hyp) and notably (2S,3R,4R)-4,5-
dihydroxy-isoleucine (DHIle), along with a cross-link compris-
ing 6-hydroxy-tryptathionine-(R)-sulfoxide that is unique
among natural products. β-Amanitin, in which Asp replaces
8
the Asn, afforded a crystal structure (Figure 1) that matched
18−20
peptide macrocycle in the case of phalloidin derivatives.
A
tryptathionine cross-link may also be introduced in peptides
2
1
containing Cys/Trp by treatment with Hg/HOAc. Yet none
of these methods has been applied to amanitins, nor toward
tryptathionylation with 6-hydroxy-L-tryptophan. Historically,
the preferred method for tryptathionine synthesis for
22
23,24
phalloidin and amanitin
involves condensation of a
cysteine-thiol with a pendant 3a-hydroxy-indoline (i.e.,
2
5
1
,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indole-2-carboxamide),
α
that is generated from N -protected L-Trp by an oxidant, e.g., a
26,27
Figure 1. (A) α-Amanitin: in red, DHIle; in blue, 6-hydroxy-
tryptathionine; in green, (R)-sulfoxide. (B) Crystal structure of β-
peracid,
generated in situ.
or dimethyldioxirane (DMDO), which may be
28,29
Whereas this strategy would be expected
8
amanitin.
to fail owing to the presence of the electron-releasing 6-OH
that would promote oxindole formation (Figure 2, right bottom
panel), testing this assertion would be challenging since the
only reported synthesis of 6-hydroxy-L-Trp requires the
1
9
the H NMR solution structure of α-amanitin. XRD studies on
α-amanitin-Pol II co-crystals revealed multiple interactions,
including key H-bonds to the hydroxyl groups of Hyp and
30
inconvenient reaction of L-Trp with H O in HSbF .
2
2
6
1
0,11
DHIle
thus confirming certain structure−activity relation-
Received: November 30, 2017
ships (SARs) gleaned formerly from a limited number of
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b12698
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
α
together with the expected N -Boc-2-oxo-L-Trp-OMe and a
small amount of the 3a-hydroxy-indoline (<10%), which
proved particularly fragile (see Supporting Information).
Instead, we envisioned retaining the boronate at position 6 to
serve as a latent hydroxyl group that could be revealed
following oxidation at position 3. Yet, DMDO degraded 1 to
α
N -Boc-2-oxo-L-Trp-OMe with the 6-boronyl-pinacolate intact,
along with several unidentified byproducts. Trifluoroborates
prepared from 1 suffered similar fates (data not shown).
As a solution, we turned to halogenating agents since these
react less readily with arylboronates and would selectively
oxidize position 3. Given the relative stability of 3a-fluoro-
33,34
35,36
indolines
compared to other 3a-halo-indolines,
we
opted for electrophilic fluorocyclization of 4 (Figure 1) to
create 3a-fluoro-6-MIDA-boronyl-1,2,3,3a,8,8a-hexa-hydropyr-
α
rolo-[2,3-b]indolyl-N -Boc-2-carboxylate (5). Although fluoro-
3
3,34
cyclization of tryptophan derivatives is known,
the
feasibility of converting a 3a-fluoro-indoline to a tryptathionine
37
cross-link is unprecedented. Following desilylation and de-
esterification, 1 was converted to MIDA-boronate ester 4,
which gratifyingly fluorocyclized cleanly in the presence of 1-
fluoro-2,4,6-trimethylpyridinium triflate to afford 5 as an
inseparable pair of diastereomers (syn-cis and anti-cis) in 85%
yield (Scheme 1). Of note, 1 also underwent fluorocyclization
in similar yields (data not shown), yet because subsequent
products were prone to pinacol loss and deborylation, we
3
8
favored the MIDA-boronate.
Figure 2. Retrosynthetic analysis: (top, middle) challenges in selective
oxidation of L-Trp and in (bottom left) oxidizing 6-hydroxy-L-Trp to
give a key 3a-hydroxy-indoline (bottom right).
A priori, the use of a diastereomeric mixture of 5 would not
28
be problematic. Hence 5 was coupled directly to the
hexapeptide on the solid-phase to give heptapeptide 6. TFA/
CH Cl treatment resulted in simultaneous deprotection, resin
cleavage, and to our delight, formation of two tryptathionine-
linked products: MIDA-boronate 7a and boronic acid 7b
Figure 1). Of note, the acidic buffer used to purify 7a and 7b
0.1% HCO H in H O:MeCN) resulted in real-time proto-
deborylation that necessitated pH-neutralization upon elution.
In lieu of a detailed mechanistic study, it stands to reason that
ipso protonation at position 6, followed by hydration of boronic
2
2
As an alternative, Baran’s elegant report on the Ir-catalyzed
α
ε
borylation of N -Boc-N TIPS-L-Trp-OMe to give the corre-
31
sponding 6-boronyl-pinacolate 1 (see Scheme 1) provided
(
(
α
access to N -Boc-6-hydroxy-L-Trp-OMe in 45% yield over two
2
2
steps via oxidation with sodium perborate (see Supporting
3
2
Information). Whereas this decomposed on standing,
immediate reaction with DMDO gave myriad byproducts
39
acid leads to deborylation as seen for anisylboronic acids.
a
Scheme 1
Threatened by deborylation, we employed a very brief aqueous
base treatment to convert remaining 7a to 7b, which was
immediately oxidized to give heptapeptide 8.
With 8 in hand, we turned to procuring enantiopure
2S,3R,4R)-4,5-dihydroxy-isoleucine (DHIle), a deceptively
(
simple molecule with challenges owing to the (i) anti
configuration at positions 2−4, (ii) high degree of functionality,
(
iii) significant steric congestion upon protection, (iv) difficulty
in separating diastereomers, and (v) propensity for lactoniza-
tion. In 1982, Bartlett reported a racemic synthesis based on
diastereoselective aza-Claisen rearrangement of Phth-glycine-
(
3
Z)-crotyl ester to give (2S,3S)- and (2R,3R)-2-N-phthalimidyl-
40
-methylpent-4-enoic acid.
Diastereoselective iodo-
lactonization (8:1), trifluoroacetate displacement, and saponi-
fication furnished a racemate of the desired diastereomer, but
41
not without ∼10% epimerization at Cα. Curiously, there has
been little traction on DHIle since 1982. A report on the
synthesis of S-deoxy amanitin, also lacking the 6-hydroxy-group,
employed an inseparable mixture of four DHIle diastereomers
that had been synthesized by a variation of Bartlett’s method;
these were elaborated to a diastereomeric mixture of four
bicyclic octapeptides that were ultimately resolved by HPLC,
the most active of which was assumed ipse dixit to possess the
a
Reagents and conditions: (a) TBAF (1 M in THF, 1.1 equiv), THF,
1
9−21 °C, 30 min, 74%; (b) LiOH (2 equiv), THF/H O 1:1, 4 °C, 4
2
h; (c) N-methyliminodiacetic acid (5 equiv), DMSO, 110 °C, 16h,
2
equiv), THF, 65 °C, 4 h, 85% for both syn-cis and anti-cis
5% over two steps; (d) 1-fluoro-2,4,6-trimethylpyridinium triflate (2
diastereomers; (e) TFA/DCM 1:1, 19−21 °C, 1 h, 70%; (f) KOH
(
0.5 M in H O), EtOH, acetone, 19−21 °C, 5 min; (g) mCPBA (1.1
2
42
equiv) in EtOH, 0−4 °C, 5 min, 58% over two steps.
stereochemistry of the natural product. Hence, we sought a
B
DOI: 10.1021/jacs.7b12698
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
a
practical route to (2S,3R,4R)-4,5-dihydroxy-isoleucine suitable
Scheme 3
for peptide coupling as shown in Scheme 2.
a
Scheme 2
a
a
Reagents and conditions: (a) ref 43; (b) 2,6-lutidine (3.5 equiv),
Reagents and conditions: (a) 20 (3.8 equiv), 8 (1 equiv) DIPEA (to
NH, DMF, 19−21 °C, 2 h;
TBDMSOTf (1.5 equiv), DCM, 4 °C, 1 h, 19−21 °C, 1 h, 65%; (c)
NMO (3 equiv), OsO (4% aqueous solution, 4 mol %), acetone/H O
2
1
equiv), KCN (1 equiv), MeOH/H O 1:1, 0 °C to 19−21 °C, 48h,
4
pH ∼8.5), DMF, 19−21 °C, 48 h; (b) Et
2
(c) TBAF (1 M in THF, excess) and HOAc to pH ∼5, DMF, 19−21
°C, 1 h; (d) HATU (7 equiv), (7 equiv), DIPEA (to pH 9), DMA,
19−21 °C, 2 h, ∼15% over four steps; (e) mCPBA (1.3 equiv),
iPrOH/EtOH 2:1, 78% for both diastereomers.
4
2
:1, 19−21 °C, 16 h, 48%; (d) NaIO (1.2 equiv), MeOH/H O 3:1,
4
2
9−21 °C, 30 min, 75%; (e) (S)-α-phenethylamine-HCl (1 M aq, 1
2
0% for correct diastereomer, 75% overall, dr = 1.14:1); (f) 6 N HCl,
reflux, 6 h, 46%; (g) H (1 atm), Pd−C 10%, MeOH (HCl to pH 4),
would be driven by these stereoelectronic considerations, we
chose mCPBA as a bulkier oxidant in the hope of favoring the
2
1
9−21 °C, 16 h, quant.; (h) LiOH (1.5 equiv), H O, 19−21 °C, 2 h,
2
quant.; (i) imidazole (10 equiv), TBDMSCl (8 equiv), DMA 19−21
(
R)-sulfoxide. Surprisingly, various solvents greatly influenced
°
C, 18 h; (j) sat’d aq. Na CO (pH ∼9), Fmoc-OSu (1.3 equiv), 1,4-
2
3
the diastereoselectivity; for example, DMF afforded the
unnatural (S)-sulfoxide in near exclusivity. In contrast, the
use of mCPBA in isopropanol-ethanol (8:3 v/v) favored the
dioxane, 19−21 °C, 5 h, 50−65% over two steps; (k) disuccinimidyl
carbonate (1.97 equiv), 2,4,6-collidine (2.8 equiv), EtOAc/MeCN 2:1,
4
°C to 19−21 °C, 5 h, 68%.
(
R)-sulfoxide in moderate excess (∼2.5:1, see Supporting
Information for HPLC traces). Access to the less-toxic (S)-
sulfoxide may be of use in ADC syntheses for which attenuated
toxins may be of interest. The stereochemical assignment of the
While Brown crotylation gave high enantioselectivity, a
mismatched Strecker reaction gave lower diastereoselectivity
and yield. Nonetheless, this route afforded good separation of
nitrile 14. NOESY correlations on 15 suggested the desired
configuration while XRD on lactone 16 confirmed the desired
stereochemistry (see Supporting Information, including CIF
file). Lactone 16 was converted to the desired monomer 20,
which was then coupled with 8 to give the octapeptide 21a. A
two-step deprotection gave 21c, which was cyclized to give the
1
sulfoxides was further buttressed by H NMR spectroscopic
analysis. Notable chemical shift differences were observed for
4
α
the β-CH of Cys, the HC of the tryptathionine, and the
2
indole-NH, all consistent with values reported for sulfoxides
48
derived from the natural product. The CD-spectrum (Figure
1
3
A) as well as the H NMR spectrum of the natural product
42
very toxic S-deoxy amanitin (22) (Scheme 3).
Finally, we turned our attention to sulfoxidation. It is known
that the thioether, (R)-sulfoxide, and sulfone are equally toxic
44
whereas the (S)-sulfoxide is ∼10-fold less toxic. Previously,
sulfoxides of α-amanitin were prepared by K MoCl -mediated
3
6
reductive deoxygenation of naturally sourced α-amanitin to give
2, which was reoxidized by H O or CH CO H to the (R)-
2
2
2
3
3
45
and the (S)-sulfoxide in a 1:2 ratio along with the sulfone. In
our hands, treatment of 22 with H O gave 23a and 23b in a
2
2
similar 1:2 ratio, along with minor amounts of several side
products. As 23a and 23b have very similar UV−vis spectra and
identical masses, their identity was addressed by HPLC; co-
injection of natural α-amanitin as a known standard
demonstrated that 23a is indeed the natural product whereas
Figure 3. (A) Circular dichroism spectra of 23a and naturally sourced
α-amanitin. (B) IC50 cytotoxicity assays on CHO cells (MTT assay).
coincided with that of the synthetic one (Supporting
Information). To assay cytotoxicity, 5−20 μg (i.e., 5−22
nmol) of 23a and 23b were accurately quantified by UV−vis
spectroscopy based on the known extinction coefficient for
2
3b is the (S)-sulfoxide diastereomer (Supporting Informa-
tion).
We then sought to control the diastereoselectivity of the
46
sulfoxidation. From the crystal structure, the pro-R lone pair on
sulfur appears to be considerably more solvent exposed
compared to the pro-S lone pair, which is oriented toward
the macrolactam interior (Figure 1). Assuming that selectivity
amanitin: the cytotoxicity profile of 23a matched that of the
natural product whereas that of 23b was 8-fold less toxic
(Figure 3B). With these data in hand, we concluded the first
total synthesis of α-amanitin.
C
DOI: 10.1021/jacs.7b12698
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Key to this synthesis is the retention of a 6-boronate ester on
tryptophan, which, as a latent hydroxyl group, curtails indole
electron density to enable selective fluorination at position 3.
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Alla Pryyma: 0000-0003-2124-2853
Mihajlo Todorovic: 0000-0002-2568-6331
Brian O. Patrick: 0000-0002-2921-8439
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The authors acknowledge funding from the Natural Sciences
and Engineering Research Council, the Canadian Institutes for
Health Research #220656, and the Canadian Cancer Society
Research Initiative Grant #703374, and thank Dr. Maria Ezhova
for help with NMR acquisition, Dr. Elena Polischuk and Ms.
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