Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 50-hydroxy- Cite this: Chem. Sci., 2020, 11, 11927 0 † All publication charges for this article 6 -deoxy-amanitin have been paid for by the Royal Society of Chemistry Alla Pryyma, Kaveh Matinkhoo, Antonio A. W. L. Wong and David M. Perrin * Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 50-hydroxy- Received 29th July 2020 60-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of a-amanitin, Accepted 2nd October 2020 that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we DOI: 10.1039/d0sc04150e meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and rsc.li/chemical-science diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide. drug-tolerant cell subpopulations.7 Examples include ADCs for Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Introduction targeting human epidermal growth factor receptor 2 and pros- 8 9 a-Amanitin, the deadliest of the amatoxins produced by the tate specic membrane antigen. With but a few exceptions, death-cap mushroom Amanita phalloides, is a potent, orally nearly all bioconjugates to a cytotoxic amanitin have emerged 1 a available inhibitor of RNA polymerase II (pol II) (Ki 10 nM), from naturally-sourced -amanitin. To date, conjugation that has been validated as a payload for targeted cancer handles used for a-amanitin-based bioconjugates include the d- 2a therapy.2 First described in 1907 3 and isolated in 1941,4 a- hydroxyl of (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle), the 10 0 amanitin is a compact bicyclic octapeptide that has been asparagine side chain, and the 6 -hydroxyl of the tryptathio- 11 indispensable for probing RNA pol II-catalysed transcription in nine staple. Of these, increasingly, the latter has been favored This article is licensed under a eukaryotes. While the toxin makes numerous contacts with RNA as a site for bioconjugation to the intact toxin. 12 pol II, the lack of synthetic access has limited the ability to As biosynthetic fermentation is currently low-yielding, generate discrete analogs to probe these contacts, most notably synthesis will likely serve as the main means for accessing large the role of the 60-hydroxyl group on the indole. Of the few amounts of toxin needed to address structure–activity rela- Open Access Article. Published on 15 October 2020. Downloaded 10/1/2021 2:37:59 AM. naturally occurring amatoxins wherein the 60-OH is replaced by tionships and provide new handles for bioconjugation. This H, amaninamide and amanin are only 25–50% as toxic as the heightened interest in a-amanitin has inspired three total 13 natural product (Fig. 1) indicating moderate importance of the syntheses that address synthetic challenges embedded in OH group.5 three key components: (i) an enantioselective synthesis of 0 Its putative contribution to affinity is understood from two DHIle, (ii) creation of an oxidatively challenged 6 -OH- apparent interactions seen in a recent co-crystal structure of a- tryptathionine-sulfoxide that demands formal oxidation at amanitin-RNA pol II: (i) the 60-hydroxyl group forms an H-bond ff with Ser-782 accounting for the di erence in Ki's between mammalian and yeast RNA pol II; (ii) the hydroxyl group increases the electron density of the indole which forms a cation–p interaction with Arg-749.6 Besides contributing to toxicity, the 60-OH provides a preferred handle for chemical functionalization for graing to antibodies. Recently, a-amanitin has been showcased as a highly effec- tive payload in antibody drug conjugates (ADCs).2a Unlike other ADC payloads, a-amanitin targets both dividing and quiescent cells and shows potential for preventing cancer relapse from Fig. 1 Chemical structure of a/b-amanitin and structure–activity Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, relationships of natural and synthetic amatoxins with indole modifi- B.C., V6T 1Z1, Canada. E-mail: [email protected] cations; a 2–4 fold-increase in Ki as a result of indole modification is 0 † Electronic supplementary information (ESI) available. See DOI: relative to the 6 -OH containing counterparts; commonly used 10.1039/d0sc04150e conjugation sites are highlighted. This journal is © The Royal Society of Chemistry 2020 Chem. Sci.,2020,11, 11927–11935 | 11927 View Article Online Chemical Science Edge Article positions 2 and 6 of the indole, and (iii) stereoselective oxida- tion of the thioether to give (R)-sulfoxide, which is approxi- mately 8-fold more toxic than the (S)-sulfoxide.13a In 2018, Perrin et al. opted for a 6-boronated-L-tryptophan as a masked phenol equivalent that underwent oxidative uo- rocyclization followed by Savige–Fontana reaction to form the tryptathionine staple14 with the pendent boronate.13a Oxidative deborylation, peptide elongation, and macrocyclization gave the S-deoxy-amanitin that was readily sulfoxidized with moderate diastereoselectivity (ca. 2 : 1) favoring the (R)- sulfoxide. In 2020, Sussmuth¨ et al. described a convergent “5+1+2” strategy whereby the tryptathionine staple was synthesized rst by reacting protected cysteine-sulfenyl chloride with orthogo- nally protected 6-OBn-L-tryptophan. The tryptathionine was then elaborated to a pentapeptide followed by addition of DHIle and Pro–Asn and macrolactamization.13b A third report advanced a scalable synthesis of 6-OAc-Trp, and utilized Savige– Fontana reaction14 to introduce the 60-OH-tryptathionine sta- 0 0 ple.13c While all three reports provide access to 60-OH- Fig. 2 Docking pose of 5 -OH-6 -deoxy-amanitin 2 showing favor- 0 0 – tryptathionine, they noted the severe challenges of selective able interactions with RNA-pol II; 5 -OH-6 -deoxy-amanitin 2 orange, a-amanitin 1 – magenta, green dashed line – distance oxidative thiolation of the electron-rich indole. 0 between Ser-782 and indole alcohol. Creative Commons Attribution-NonCommercial 3.0 Unported Licence. In contrast to the immediate interest in accessing the 6 - hydroxyl group, interest in generating the (R)-sulfoxide remains academic. Nevertheless, because Nature exclusively produces also engage in an H-bonding interaction with Ser-782. A dock- the (R)-sulfoxide, logic suggests that the (R)-sulfoxide may 0 0 ing study validated this hypothesis: the 5 -OH-6 -deoxy-amanitin provide privileged pharmacological/toxicological properties. As (2) was docked into the RNA pol II using Autodock Vina such, diastereoselective access of the (R)-sulfoxide continues to 15 molecular docking program. pose a signicant synthetic challenge that was met with but 0 0 The most energetically favorable pose of 5 -OH-6 -deoxy- moderate selectivity in the rst total synthesis and remained amanitin was compared to the crystal structure pose of a- unprogressed in the two subsequent ones. amanitin (Fig. 2). Further assessment using PyMOL Molecular This article is licensed under a Despite these synthetic efforts, there remains a demand for Graphics System conrmed that the aforementioned key inter- biologically active and more synthetically accessible a-amanitin actions between the enzyme and the indole ring are still analogs that: (i) capture the putative p–cation interaction for achievable and the modied indole does not alter the overall enhanced affinity and (ii) provide an indole-situated conjuga- 16 Open Access Article. Published on 15 October 2020. Downloaded 10/1/2021 2:37:59 AM. conrmation of the t. Importantly, the docking pose sug- tion handle for ADC development. Towards these ends, herein 0 gested that the 5 -hydroxyl group should still form the antici- we report the synthesis of a constitutional isomer of near-native pated H-bond with Ser-782 (Fig. 2, green line). Based on these toxicity that introduces a 50-OH-tryptathionine, which is readily observations, we reasoned that the incorporation of 5-OH- achieved from cheap, commercially available 5-OH-L-trypto- tryptophan should maintain the toxicity of the new amanitin phan (5-OH-Trp). analog. In addition, we identify new methodologies to create the (R)- 0 Encouraged by this result, we undertook a synthesis of 5 - sulfoxide with high diastereoselectivity (>15 : 1) along with the 0 OH-6 -deoxy-amanitin (2) that would rely on two key compo- (S)-sulfoxide diastereomer and sulfone for comparison. Antici- nents: (i) introduction of the fully protected DHIle-NHS ester as pating success with chemoselective conjugation akin to afore- previously reported, and (ii) synthesis and characterization of mentioned conjugates to the 60-OH-indole, this work now the 3a-uoro-pyrroloindoline produced from 5-OH-tryptophan expands the toolbox of available synthetic amanitin analogs for that could be subjected to late-stage Savige–Fontana reaction consideration as payloads for use in translational applications. 0 to give the 5 -OH-tryptathionine (Scheme 1). In addition to this synthetic feat, we addressed mid- and late-stage sulfoxidation Results with high diastereoselectivity to afford the desired (R)-sulfoxide. Following the approach used in the rst total synthesis of a- In approaching the synthetic challenge of creating a hydroxy- 13a amanitin and supported by more recent work on the oxidative tryptathionine staple, we appreciated the high natural abun- uorocyclization of tryptophan to give uoropyrroloindoline dance of 5-OH-tryptophan in a number of biosynthetic path- 17 (FPI) for Savige–Fontana tryptathionylation, we started with ways along with its commercial availability as a single commercially available 5-OH-tryptophan. Following Fmoc- enantiomer. Hence, we hypothesized that 5-OH-L-tryptophan protection, oxidative uorocyclization with N-uoro-2,4,6- could be introduced in lieu of the synthetically-demanding 6- 0 trimethylpyridinium tri ate (FP-T300) gave a diastereomeric OH-L-tryptophan. We also reasoned that the 5 -hydroxyl could 11928 | Chem.