Stanford University

Total Synthesis

Our lab has described multi-step chemical syntheses of some of the world’s most infamous neurotoxins. Our synthesis of tetrodotoxin demonstrates the power of methods for C–H functionalization to alter strategic planning in small molecule assembly. Tetrodotoxin, which acts as a potent inhibitor of ion flux through voltage-gated Na+ channels (NaV), has inspired subsequent efforts in our lab to prepare other known modifiers of NaV function, including saxitoxin, gonyautoxins, zetekitoxin AB, batrachotoxin, and aconitine.

Each of these natural products poses its own unique set of challenges in total synthesis, and each has provided a framework for shaping our research in methods development. For example, interest in the guanidinium toxins has motivated us to develop oxidative tools for preparing 5-membered ring cyclic guanidine structures. These reactions rely on our expertise in the area of C–H bond functionalization. Batrachotoxin and related steroidal derivatives have led us to consider novel reaction processes for carbocyclic ring formation. Finally, a desire to synthesize aconitine, with its heavily oxygenated frame, has compelled us to seek selective methods for C–H hydroxylation in addition to new, convergent means for poly-carbocyclic ring construction. The uniqueness of these natural products as pharmacological agents for studying NaV structure/function is at the core of our interest in such molecules. The inability to access sufficient quantities of the guanidinium toxins and batrachotoxin and the difficulties associated with semi-synthetic manipulation of such compounds necessitates effective, modular synthetic protocols for their preparation.

We have described selective processes and new catalyst systems that enable site-specific modification of aliphatic C–H bonds. Such methods have the potential to alter the practice of assembling small molecules, which we have tried to demonstrate in syntheses of the puffer fish toxin, tetrodotoxin, manzacidins, and agelastatin.

Saxitoxin

Accessing the tricyclic core of bisguanidinium toxins of the Saxitoxin family has become paramount in the lab’s studies of sodium channels both to provide appropriate amounts of material as well as to enable further modulation.  Key steps include a diastereoselective Pictet–Spengler reaction and an intramolecular amination of an N-guanidyl pyrrole by a sulfonyl guanidine.

Zetekitoxin AB

We have undertaken efforts to synthesize Zetekitoxin AB, one of the more complex members of the Saxitoxin family due to its remarkable potency towards sodium channels. Initial studies have looked at the key C–C bond formation at C11 of the five-membered pyrrolidine, resulting in the synthesis of other Saxitoxin natural product, 11-Saxitoxinethanoic Acid.

Batrachotoxin (BTX)

Batrachotoxin (BTX), homobatrachotoxin, and batrachotoxinin A comprise a small family of complex steroidal alkaloids originally isolated in sparing amounts from the skin of Colombian poison dart frogs of the genus Phyllobates. BTX acts as a selective full agonist of voltage-gated sodium channels (NaVs) causing the channel to open more readily at hyperpolarized membrane potentials and blocking fast inactivation, and is among the most potent non-peptidic toxins known (LD50 in mice = 2 μg kg−1). BTX binding to the inner pore of the channel (Site II) elicits a multitude of functional responses, including hyperpolarization of threshold activation, inhibition of both fast and slow inactivation, and reduction in ion selectivity. While there exist other small molecule modulators of NaVs, arguably none show activity that is as multifaceted as BTX. Severely limited quantities of BTX, however, frustrate any efforts to evaluate structure–function relationships and to utilize BTX or select analogues to interrogate mechanisms of ion selectivity and channel gating. The uniqueness of BTX as a NaV agonist has motivated our efforts to understand the molecular details of its binding interactions with the channel and its structure–function properties.

As part of this program, we have prepared simplified BTX-like structures comprising the C, D, and E rings. Electrophysiology recordings with wild-type and mutant NaV isoforms demonstrate that these analogues block NaV current, a stark contrast to the behavior of BTX itself. Protein mutagenesis data suggest that our BTX analogues lodge in the inner pore lining of the channel, sharing a common receptor site with the parent compound. Surprisingly, both enantiomers of these simplified structures display nearly identical potency as NaV inhibitors. With our desire to use BTX and modified forms thereof for examining channel dynamics and ion gating mechanisms, these surprising results have motivated our efforts to obtain the natural product through de novo synthesis. Our now completed first-generation synthesis of BTX has enabled access to both the natural and unnatural antipodes for the first time. Electrophysiology studies on these toxins have shown striking divergent behavior between the natural and unnatural antipodes despite early evidence that they share an overlapping binding site.