Communication pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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.3 Unlike most cytotoxins that act primarily on rapidly dividing cells, α-amanitin kills both rapidly growing and quiescent cells by inhibiting Pol II, causing its rapid degradation12 leading to apoptosis.13 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 common chemotherapeutics.14 Impressively, antibody−drug conjugates (ADCs) of α-amanitin cure mice of pancreatic cancer xenografts and are advancing toward human trials.15 As fermentation yields are low,16 a synthetic source of the toxin is needed to prepare ADCs for use in clinical oncology.15 Moreover, the lack of a synthetic route to α-amanitin presents a critical barrier to the design of new analogues for systematic SAR analysis.17 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 enantioselective 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. The first challenge is the most formidable as L-Trp must be selectively oxidized at positions 6 and 3 to afford a 6-hydroxytryptathionine 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 peptide macrocycle in the case of phalloidin derivatives.18−20 A tryptathionine cross-link may also be introduced in peptides containing Cys/Trp by treatment with Hg/HOAc.21 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 phalloidin22 and amanitin23,24 involves condensation of a cysteine-thiol with a pendant 3a-hydroxy-indoline (i.e., 1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indole-2-carboxamide),25 that is generated from Nα-protected L-Trp by an oxidant, e.g., a peracid,26,27 or dimethyldioxirane (DMDO), which may be generated in situ.28,29 Whereas this strategy would be expected 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 inconvenient reaction of L-Trp with H2O2 in HSbF6.30
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)4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation. α-Amanitin, one of the deadliest toxins known to humankind (LD50 = 50−100 μg/kg), is featured in modern biochemistry textbooks,1 underscoring a rich scientific history.2,3 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.4,5 Isolated over 60 years ago,6 α-amanitin is a potent, orally available, highly selective allosteric inhibitor of RNA polymerase II (Pol II).7 Its bicyclic octapeptide structure contains two oxidized amino acids that are key to its toxicity: trans-4-hydroxy-proline (Hyp) and notably (2S,3R,4R)-4,5dihydroxy-isoleucine (DHIle), along with a cross-link comprising 6-hydroxy-tryptathionine-(R)-sulfoxide that is unique among natural products. β-Amanitin, in which Asp replaces the Asn, afforded a crystal structure (Figure 1)8 that matched
Figure 1. (A) α-Amanitin: in red, DHIle; in blue, 6-hydroxytryptathionine; in green, (R)-sulfoxide. (B) Crystal structure of βamanitin.8
the 1H NMR solution structure of α-amanitin.9 XRD studies on α-amanitin-Pol II co-crystals revealed multiple interactions, including key H-bonds to the hydroxyl groups of Hyp and DHIle10,11 thus confirming certain structure−activity relationships (SARs) gleaned formerly from a limited number of © XXXX American Chemical Society
Received: November 30, 2017
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DOI: 10.1021/jacs.7b12698 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
together with the expected Nα-Boc-2-oxo-L-Trp-OMe and a small amount of the 3a-hydroxy-indoline (
