A Convergent Total Synthesis of the Death Cap Toxin α-Amanitin

Abstract

The toxic bicyclic octapeptide α-amanitin is mostly found in different species of the mushroom genus Amanita, with the death cap (Amanita phalloides) as one of the most prominent members. Due to its high selective inhibition of RNA polymerase II, which is directly linked to its high toxicity, particularly to hepatocytes, α-amanitin received an increased attention as a toxin-component of antibody-drug conjugates (ADC) in cancer research. Furthermore, the isolation of α-amanitin from mushrooms as the sole source severely restricts compound supply as well as further investigations, as structure-activity relationship (SAR) studies. Based on a straightforward access to the non-proteinogenic amino acid dihydroxyisoleucine, we herein present a robust total synthesis of α-amanitin providing options for production at larger scale as well as future structural diversifications.

Keywords: amatoxins; asymmetric synthesis; total synthesis; tryptathionine; α-amanitin.

Figure 1

Structure of α‐Amanitin (1). The numbers in blue, turquoise and red refer to the number of amino acids of the building blocks (depicted blue, turquoise and red) employed in this total synthesis strategy [5+1+2].

Scheme 1

Retrosynthetic analysis of α‐amanitin (1). Final assembly of α‐amanitin from three building blocks and enantioselective synthesis of the hydroxylated amino acid building blocks.

Scheme 2

Synthesis of N‐Teoc‐6‐benzyloxy‐l‐tryptophan (8) by dynamic kinetic resolution: a) l‐serine, Ac₂O, AcOH, 75 °C, 2 h, 82 %; b) 40 % NaOH, MeOH/H₂O, c) chiral ligand 16, Ni(NO₃)₂ 6H₂O, MeOH, reflux, 16 h, 84 % over two steps, d) 6 m HCl, MeOH, 70 °C, 2 h, e) TeocOSu, Et₃N, DMF, 60 °C, 4 d, quant.

Scheme 3

Synthesis of the C‐ and N‐terminally deprotected monocyclic tryptathionine peptide 5: a) Boc₂O, NaHCO₃, dioxane/H₂O, r.t., 16 h, 99 %; b) SO₂Cl₂, CHCl₃, r.t., 1 h; c) 8, NaHCO₃, CHCl₃, 0 °C to r.t., quant., d) H‐Gly‐Ile‐Gly‐OH, CO(OSu)₂, collidine, acetonitrile/H₂O, r.t., 2 h; e) PTSA, THF, r.t., 2 h, 70 % over 2 steps; f) T3P, DIPEA, DMF, r.t., 16 h, 70 %, g) TFA/H₂O (7:3), r.t., 2 h, quant.

Scheme 4

Synthesis of side chain‐protected Fmoc‐(3R,4R)‐4,5‐l‐dihydroxyisoleucine (6) a) (+)‐DIPT, Ti(OiPr)₄, TBHP, DCM, −20 °C, 4 h; b) TsCl, Et₃N, DMAP, DCM, −10 °C, 30 h, 67 %; c) NaI, Zn(Cu), THF, 70 °C, 2 h; d) Boc₂O, NaH, THF, 0 °C to r.t., 16 h, 75 %; e) ethyl trifluoroacetate, NEt₃, MeOH, r.t., 16 h, quant.; f) LHMDS, ZnCl₂, PPh₃, [(p‐cymene)RuCl₂]₂, 22, THF, −72 °C to r.t., 16 h, 88 %; g) NaBH₄, MeOH, r.t., 1 h; h) FmocOSu, Et₃N, dioxane, r.t., 4 h, 82 %; i) K₂OsO₄⋅H₂O, NMO, CHCl₃/H₂O, r.t., 6 h, 40 %; j) TBSCl, pyridine/DMF(1:9), r.t., 24 h, 95 %; k) TMSOTf, 2,6‐lutidine, 0 °C to r.t., 2 h, 90 %.

Scheme 5

Fragment couplings and cyclizations to the target molecule α‐Amanitin (1): a) MSA, COMU, DIPEA, DMA, 0 °C to r.t. 3 h; b) (a and b: onepot), H‐Asn‐Hyp‐OFm⋅HCl (4), HATU, DIPEA, DMF, 0 °C to r.t., 2 h; c) Et₂NH, DMF, 1 h, r.t.; d) 1 m TBAF, THF, r.t., 2 h, 77 % over four steps; e) HATU, DIPEA, DMF, r.t., 16 h, 68 %, f) BF₃⋅OEt₂, EtSH, r.t., 2 h; g) mCPBA, iPrOH/EtOH (2:1), r.t., 30 min, 35 % over two steps.

Figure 2

CD spectra of natural and synthetic α‐amanitin (1).