ReportsChemistry

Asymmetric synthesis of batrachotoxin: Enantiomeric toxins show functional divergence against NaV

See allHide authors and affiliations

Science  18 Nov 2016:
Vol. 354, Issue 6314, pp. 865-869
DOI: 10.1126/science.aag2981
  • Fig. 1 Background and synthetic plan.

    (A) The structures of lipophilic site 2 toxins (−)-batrachotoxin (BTX), aconitine, and veratridine. (B) A pore model of NaV with (−)-BTX (depicted as spheres) docked at site 2. The structure is based on Magnetococcus marinus NaV crystallographic data (Protein Data Bank accession code 4F4L) (9, 19). Domain I, orange; domain II, red; domain III, gray; domain IV, teal. (C) Retrosynthetic analysis of BTX and BTX C-20 ester analogs. LD50, half-maximal lethal dose; Me, methyl; tBu, tert-butyl; Et, ethyl; TBS, tert-butyldimethylsilyl.

  • Fig. 2 Enyne radical cyclization to furnish the steroidal core of BTX.

    Reagents, conditions, and product yields for steps a to p are as follows: (A) a, t-BuLi, THF, −90°C, then 4 (see Fig. 1) (65%); b, K2CO3, MeOH (94%); c, Et3B, air, n-Bu3SnH. (B) d, Me3SiC≡CSiEt2Cl, imidazole, CH2Cl2 (93%); e, O2, n-Bu3SnH, Et3B, Ph2O, 150°C (75%); f, n-Bu4NF, THF, 60°C (94%); g, 2-iodoxybenzoic acid, t-BuOH, 65°C, then OsO4 [7 mol %], NaIO4, pyridine, H2O (57%); h, MeNH2, CH2Cl2; NaB(O2CCF3)3H, CH2Cl2, −78°C, then ClCH2COCl, 2,6-lutidine, −78 to 0°C (52%); i, NaOEt, EtOH, 1:1 THF/C6H6 (92%); j, KN(SiMe3)2, PhNTf2, THF, −78 to 0°C (94%); k, CuCl2, O2, 1,4-dioxane, 73°C (85%); l, NaClO2, NaH2PO4, dimethyl sulfoxide/H2O; m, SOCl2, pyridine, CH2Cl2; n, NaN3, acetone/H2O; o, aqueous AcOH, 1,4-dioxane, 90°C (57% over four steps); p, p-TsOH, 4-Å molecular sieves, p-methoxyphenethyl alcohol (PMBCH2OH), C6H6 (89%). THF, tetrahydrofuran; Ph, phenyl; Tf, trifluoromethansulfonate; Ts, p-toluenesulfonate; Ac, acetate.

  • Fig. 3 Completion of the synthesis.

    Reagents, conditions, and product yields for the preparation of (A) (–)-BTX (steps q to t) and (B) BTX-B and its enantiomer (steps q to s, then u) are as follows: q, LiCl, CuCl, Pd(PPh3)4, tributyl(1-ethoxyvinyl)tin, THF, 60°C, then 1 M oxalic acid, 0°C (77%); r, AlH3, THF, −78 to 0°C (33%); s, p-TsOH, 3:2 acetone/H2O (83%); t, (ethyl carbonic)-2,4-dimethyl-1H-pyrrole-3-carboxylic anhydride, Et3N, C6H6, 45°C (79%); u, benzoic (ethyl carbonic) anhydride, Et3N, C6H6, 45°C (70%).

  • Fig. 4 Effects of synthetic BTX-B and ent-BTX-B on wild-type rat NaV1.4 function.

    (A) Representative trace for rat NaV1.4 (rNaV1.4) current before (black) and after (red) steady-state binding of 10 μM BTX-B. Current was evoked by a 150-ms test pulse from –120 to 0 mV after establishment of steady-state inhibition by repetitive depolarizing pulses to 0 mV. (B) Voltage dependence of activation for rNaV1.4 in the presence of 10 μM BTX-B (open circles) compared with control conditions (filled circles) for n ≥ 3 cells (mean ± SD). (C) Representative trace for rNaV1.4 current before (black) and after (red) steady-state binding of 5 μM ent-BTX-B. Current was evoked by a 24-ms test pulse from –120 to 0 mV after establishment of steady-state inhibition by repetitive depolarizing pulses to 0 mV. (D) Voltage dependence of activation for rNaV1.4 in the presence of 10 μM ent-BTX-B (open circles) compared with control conditions (filled circles) for n ≥ 3 cells (mean ± SD). (E) rNaV1.4 homology model highlighting residues that have previously been shown to abolish (–)-BTX activity. (F) Percent current inhibition of rNaV1.4 mutants by 5 μM ent-BTX-B (mean ± SD). WT, wild type; F, phenylalanine; K, lysine; L, leucine; N, asparagine.

Supplementary Materials

  • Asymmetric synthesis of batrachotoxin: Enantiomeric toxins show functional divergence against NaV

    Matthew M. Logan, Tatsuya Toma, Rhiannon Thomas-Tran, J. Du Bois

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S12
    • Tables S1 to S14
    • References

Navigate This Article