De novo synthesis of modified saxitoxins for sodium ion channel study

Article abstract of DOI:10.1021/ja904179f

(Chemical Equation Presented) Access to novel forms of (+)-saxitoxin (STX), a potent and selective inhibitor of voltage-gated Na⁺ ion channels, has been made possible through de novo synthesis. Saxitoxin is believed to lodge in the outer mouth

Full text of DOI:10.1021/ja904179f

Published on Web 08/14/2009
De Novo Synthesis of Modified Saxitoxins for Sodium Ion Channel Study
Brian M. Andresen and J. Du Bois*
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
Received May 22, 2009; E-mail: jdubois@stanford.edu
Voltage-gated Na+ ion channels (NaV) serve an obligatory role
in the generation of bioelectricity and are essential for all of life’s
processes.1 Genes that encode for 10 unique channel isoforms
(NaV1.1-1.9, NaX) have been identified in mammalian cells.2
Differences in the biophysical properties of these protein subtypes,
their membrane concentrations and spatial distribution define the
signaling characteristics of a neuron.3 Aberrant NaV function and/
or expression is thought to be associated with numerous disease
states, including arrhythmia, epilepsy, neuropathic pain, and
congenital analgesia.4 Accordingly, chemical tools for exploring
protein structure, modulating the activity of specific NaV isoforms,
and tracking dynamic events associated with NaV regulation and
expression are sought to further understand the pathophysiologies
associated with channel function.5 Herein, we describe our initial
efforts to develop such agents, for which the shellfish poison (+)-
saxitoxin (STX) (1), a single-digit nanomolar inhibitor of certain
NaV subtypes, provides the molecular blueprint (Figure 1).6 Our
findings delineate a path for preparing novel carbamate-modified
forms of the toxin and establish that such structural changes do
not significantly influence substrate-receptor binding affinity.7
Specific contacts between the C13-carbamate unit, the C12-hydrated
ketone, the 1,2,3-guanidinium moiety, and the carboxylate residues
of the outer vestibule loop (E403, E758, D1532) are also high-
lighted.
Figure 2. (left) Current recordings at varying concentrations of 2 on
rNaV1.4 expressed in CHO cells. (right) Dose-response curves for (+)-
STX 1 (red) and 2 (black).
As a starting point for our investigations, we chose to examine
the contribution of the C13-carbamate as a hydrogen-bond donor
to the overall binding affinity of the toxin.8,9b Naturally occurring
decarbamoyl STX (dc-STX) displays a modest reduction (<10-fold)
in potency relative to STX.6 Previous efforts to alter this functional
group through semisynthetic modification of dc-STX have been
limited to a single succinate derivative.7a,c The availability of a de
novo synthesis of STX (see below) remedies this problem.
Accordingly, N,N-dimethyl-STX (2) was prepared and evaluated
for its ability to block Na+ current. Electrophysiology measurements
were performed in a whole-cell voltage-clamp format against the
R-subunit of the rat skeletal channel NaV1.4 (rNaV1.4) heterolo-
gously expressed in CHO cells.11 Figure 2 shows current recordings
following a 10 ms depolarizing pulse to 0 mV from a holding
potential of -100 mV. Increasing concentrations of 2 were perfused
into the external solution, resulting in decreased peak current. These
data were fit to a Langmuir isotherm to give an IC50 of 2.1 ( 0.1
nM for 2, a value nearly equal to the IC50 recorded for our synthetic
(+)-STX (2.9 ( 0.1 nM). This result seems to indicate that the
role of the C13-carbamate in the natural product is not as a
hydrogen-bond donor.9b Such a finding also provides the motivation
for exploring further the steric environment of the protein pore in
the vicinity of the carbamoyl residue.
A strategic modification to one of our previously published routes
to (+)-STX was devised in order to prepare alternate C13-carbamate
forms (Scheme 1).12 Tricyclic oxazolidinone 5 represents the
cornerstone of this new synthetic plan, our assumption being that
nucleophilic amines would open selectively this strained hetero-
cycle. This structure can be fashioned in just three steps from the
nine-membered-ring guanidine 3, a material that we now routinely
synthesize on >5 g scales. To access 5, sequential formation of the
C13-Troc carbonate and ring closure proved necessary; use of other
carbonylating agents (i.e., phosgene, carbonyldiimidazole) gave
almost exclusively the C4-C13 alkene. Catalytic ketohydroxylation
of 4 proceeded efficiently, albeit with modest selectivity, to generate
hemiaminal 5.13 Oxazolidinone ring opening with a 1° amine at
Figure 1. Model of (+)-STX bound in the NaV channel pore.9b
The fully functional voltage-gated Na+ channel consists of a large
heteromeric R-subunit (∼260 kDa) and one or two auxiliary
ꢀ-subunits (33-36 kDa).2 In the absence of protein crystallographic
data, small molecule pharmacological probes together with protein
mutagenesis and electrophysiology have provided much of the
structural insights that currently exist for this family of macromol-
ecules.8 These data together with X-ray structures of associated
K+ ion channels, Kcsa and MthK, have made possible the
construction of homology models of the NaV R-subunit.9 The outer
mouth of the channel includes the ion selectivity filter and is
considered to be the receptor site for STX and related guanidinium
poisons (Figure 1).2 Five carboxylate residues line this pore region
[D400, E755, E403, E758, and D1532 (NaV1.4 numbering)]; their
presence is critical for high-affinity STX binding, as shown by site-
directed mutagenesis studies.8,9b,10 Computational models by
Lipkind and Fozzard, Dudley, and Zhorov all posit that the 7,8,9-
guanidine of STX points toward the ring of four amino acids that
comprise the selectivity filter (also known as the DEKA loop).8,9
9
12524 J. AM. CHEM. SOC. 2009, 131, 12524–12525
10.1021/ja904179f CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
ambient temperature then furnished the corresponding carbamate
6. Two subsequent transformations, Lewis acid-mediated guanidine
ring closure and deprotection and C12 oxidation, completed the
assembly of the tailored saxitoxins.
Scheme 1. Synthetic Route to C13-Modified Saxitoxinsa
Figure 4. Final-step ligation strategy for STX modification.
natural product. New saxitoxins have been evaluated for their
efficacy in blocking NaV function using whole-cell voltage-clamp
techniques. These findings have revealed opportunities for re-
engineering the C13-carbamoyl unit of STX with any one of a
number of different structural groups. Studies of this type together
with the tools of molecular biology should allow us to map in
greater detail the three-dimensional arrangement of the channel pore.
We view these studies as a necessary step in a larger plan to utilize
designed chemical tools to interrogate dynamic processes associated
with NaV function.
Acknowledgment. B.M.A. is the recipient of graduate fellow-
ships from Amgen and the ACS Division of Organic Chemistry,
sponsored by Wyeth. We are grateful to Professors Richard Aldrich
(University of Texas) and Merritt Maduke (Stanford University)
for allowing use of their laboratory equipment and for many helpful
discussions. This work was supported by a grant from the NIH
and a gift from Pfizer.
a (a) Cl3CCH2C(O)Cl, C5H5N, 0 °C, 93%; (b) i-Pr2NEt, CH3CN, 86%;
(c) 10 mol % OsCl3, Oxone, 44%; (d) RNH2, THF, 77-99%; (e)
B(O2CCF3)3, 75–92%; (f) DCC, DMSO, C5H5NH+CF3CO2-, 25–63%. Mbs
) p-methoxybenzenesulfonyl.
IC50 values were determined for STX C13-derivatives 9-13
against rNaV1.4 (Figure 3). In spite of the steric, electronic, and
polar modifications to the C13-carbamate, all of these compounds
exhibited channel-blocking potency within 1-1.5 orders of mag-
nitude of the parent toxin. The most severe loss in affinity was
noted with acid 12, possibly the result of a charge interaction
between the carboxylate residue and one of the guanidinium
moieties.14 Other data, namely results from voltage-clamp record-
ings with 9 and 10, suggest that the carbamate appendage sits in a
narrow gorge between protein domains. Future experiments will
continue to test this hypothesis. Finally, we note that, to the best
of our knowledge, the benzophenone-labeled toxin 13 represents
the first such STX photoaffinity probe.15
The power of de novo synthesis to provide novel forms of STX
is further underscored with compounds such as 11 (Figure 4). The
presence of two guanidinium groups notwithstanding, 11 undergoes
selective coupling with an N-hydroxysuccinimide (NHS) benzoate
ester to give the desired amide 14. This final-step, “post-synthetic”
coupling reaction makes possible the attachment of structurally
complex payloads (e.g., fluorogenic groups, cofactors) to the STX
core, side-chain elements that might otherwise be incompatible with
the chemistries employed for guanidine deprotection and/or C12
oxidation (see Scheme 1).
Supporting Information Available: General experimental protocols
and characterization data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 3. Recorded IC50 values for novel STXs against rNaV1.4.
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