Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A

Abstract

Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.

Figure 1

Natural pinnatoxins A and G and synthetic analogue PnTX AK with the acyclic amino ketone that are the subject of the synthetic and biological studies described in this article.

Figure 2

Hydrogen-bonding stabilization in spiroketal intermediates.

Figure 3

Effect of PnTX A and PnTX AK on nAChRs. ACh-evoked current was recorded at a holding potential of −60 mV in Xenopus oocytes expressing (a) human α7, (b) Torpedo α1₂βγδ, and (c) human α4β2 nAChRs, before (black tracing) and after (red tracing) the action of PnTX A. The lines above current tracing indicate the time ACh was perfused. Note that PnTx A did not affect the rate of desensitization in the various nAChR subtypes. (d) Concentration-dependent inhibition of ACh-evoked currents by PnTX A or PnTX AK in Xenopus oocytes expressing the human α7 (solid circles, black curve) and α4β2 (solid triangles, red curve) neuronal nAChRs, or having incorporated into their membranes the Torpedo muscle type α1₂βγδ nAChR (solid diamonds, blue curve). Amplitudes of the ACh-current peak (mean ± SEM; 5 oocytes per concentration), recorded at a holding membrane potential of −60 mV, in the presence of either PnTX A or PnTX AK were normalized to control currents and fitted to the Hill equation (see Table 2 for IC₅₀ values). Note the low activity of PnTX AK (open symbols; same colors apply for the curves as with PnTX A).

Figure 4

Effect of PnTX A and PnTX AK on various nAChRs. Inhibition of specific [¹²⁵I]α-BTX or (±)-[³H]epibatidine binding by increasing concentrations of PnTX A or PnTX AK on (a) Torpedo and neuronal α7-5HT₃ or (b) heteropentameric α3β2 and α4β2 nAChRs. The results are expressed as the ratio of the specific radiotracer binding measured with (B) or without (B₀) competitive ligands, expressed as a percentage. Curve fitting was based on a nonlinear regression analysis using the Hill equation. Data are mean values ± SEM of at least three inhibition experiments.

Figure 5

Inhibition of interaction of [³H]NMS with five human mAChR subtypes by various ligands. The results for each receptor subtype are expressed as the ratio of the specific [³H]NMS binding measured with (B) or without (B₀) the competitive ligands, expressed as a percentage. B_T is [³H]NMS total binding, and NS is nonspecific binding in the presence of 50 μM atropine. Note that the effect of PnTX A and PnTX AK was evaluated at 1 μM concentration.

Figure 6

Representative protein–ligand interactions in PnTX A–nAChR complexes obtained by molecular modeling: (a) human α7 (green, α7–α7 interface), (b) human α4β2 (magenta, α4–β2 interface), and (c) Torpedo α1β1γδ (cyan, α1–δ interface). Only amino acids interacting through hydrogen bonds with the ligand and the residues from equivalent positions in the sequence alignment are shown (see Supporting Information, Table 2–1, for further information). PnTX A is colored in (a) yellow, (b) light blue, and (c) violet, respectively. For each complex, two different views, rotated by 90°, are presented. Nonpolar hydrogen atoms of the ligand are not shown for clarity.

Scheme 1

Overview of Synthesis Plan

Scheme 2

Key Ireland–Claisen Rearrangement

Scheme 3

Examples of Stereoselective Generation of Acyclic α,α-Disubstituted Enolates

Scheme 4

Synthesis of G-Ring Aldehyde 6

Scheme 5

Early Approach to BCD-Bisketal

Scheme 6

Assembly of BCD-Dispiroketal

Scheme 7

Completion of Synthesis of the BCD-Bisketal Fragment

Scheme 8

Completion of Synthesis of PnTX A

Scheme 9

Total Synthesis of PnTX G

Scheme 10

Synthesis of Amino Ketone PnTX AK