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

Article abstract of DOI:10.1021/ja201254c

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.

Full text of DOI:10.1021/ja201254c

ARTICLE
pubs.acs.org/JACS
Total Synthesis of Pinnatoxins A and G and Revision of the Mode of
Action of Pinnatoxin A
Romulo Araoz,† Denis Servent,‡ Jordi Molgꢀo,† Bogdan I. Iorga,§ Carole Fruchart-Gaillard,‡ Evelyne Benoit,†
Zhenhua Gu,^ Craig Stivala,^ and Armen Zakarian*,^
†Institut de Neurobiologie Alfred Fessard, FRC2118, Laboratoire de Neurobiologie et Dꢀeveloppement, UPR-3294, and §Institut de
Chimie des Substances Naturelles, UPR 2301, Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette cedex, France
‡Service d’Ingꢀenierie Molꢀeculaire des Protꢀeines, Laboratoire de Toxinologie Molꢀeculaire et Biotechnologies, Commissariat ꢁa l'Energie
Atomique, F-91191 Gif-sur-Yvette, France
^Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
S Supporting Information
b
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 IrelandꢀClaisen rearrangement of
a complex R,R-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 R7 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.
’ INTRODUCTION
are available, although none of the spiroimine toxins are
presently regulated.
Pinnatoxins are naturally occurring cyclic imines that com-
prise a newly emerging group of marine toxins with worldwide
distribution.1ꢀ3 Typically, marine toxins are classified and
regulated on the basis of their toxicological profiles and
mechanism of action; they can also be grouped on the basis
of their chemical structures.4 Many of the toxins have become
indispensable reagents for fundamental research in biology.
Regulatory control of marine algal toxins requires availability of
pure, high-quality standards for toxicological and other relevant
studies, as well as detailed information on the mechanism of
biological activity. Previous studies with natural spirolides and
gymnodimine—other members of the spiroimine group—
revealed unique toxicological effects for this class of toxins in
mice, where few symptoms are observed until a lethal dose is
reached.5,6 It is expected that a similar profile would be
pronounced in humans, although no fatal cases involving the
consumption of shellfish contaminated with cyclic imine toxins
have been conclusively established thus far. Certified standards
for pure spirolides and gymnodimine, but not for pinnatoxins,
Current data indicate that cyclic imine toxins are substantially
less toxic by oral administration than by injection, with one
probable exception being pinnatoxins.7,8 Oral toxicity data are
especially relevant for risk assessment of shellfish destined for
human consumption. For pinnatoxins, extremely limited infor-
mation is available due to their low and unreliable availability
from natural sources, undermined by the fact that their produ-
cing organism has not been identified until recently in 2010.7
Notwithstanding their highly intricate molecular structure, a
chemical synthesis of pinnatoxins can provide an alternative
source of high-quality material for biological and toxicological
studies while also enabling fundamental studies aimed at a deeper
understanding of their mode of action. It has been initially
suggested that the mode of action of pinnatoxins involves
calcium-channel activation.9,10 However, there is a growing body
Received: February 9, 2011
Published: June 06, 2011
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of evidence that a different mechanism of activity may be
operational.11
substantial quantities of pinnatoxin A (PnTX A). With reliable
access to synthetic PnTX A on scale, extensive electrophysio-
logical, computational, and competition binding studies have
been carried out that provide compelling evidence for its mode
of action as an inhibitor of various subtypes of nicotinic
acetylcholine receptors (nAChRs), redefining the originally
proposed mechanism of bioactivity. In addition, we have found
that PnTX A interacts selectively with the human neuronal
R7 subtype of nicotinic receptors. We experimentally con-
firmed that the spiroimine fragment is the pharmacophore
required for biological activity by examination of amino keto
acid 2 (PnTX AK), which contains an open form of the imino
ring A. The total synthesis of newly discovered pinnatoxin G,
the putative biosynthetic progenitor for all pinnatoxins, is also
reported. It has been proposed that the more toxic PnTX A is
generated through metabolic oxidation of PnTX G after inges-
tion by shellfish.8
The intricate molecular architecture of pinnatoxins creates
abundant opportunities for innovation at the level of strategy,
tactics, and invention of synthetic methods (Figure 1). Sig-
nificant effort has been devoted to developing a chemical
synthesis of pinnatoxins.12ꢀ35 In 2008, we disclosed our
original approach that resulted in completion of the total
synthesis of (+)-pinnatoxin A.32 In order for the methods and
strategies developed for the synthesis of this complex structure
to have a broader impact on biological and toxicological
investigations, they must meet certain standards of efficiency
and scalability. The previous synthetic endeavors that suc-
ceeded in reaching the natural product reported the production
of only ∼1 mg quantities of pinnatoxins. In this article, we
report the total synthesis of pinnatoxin A that has a capacity to
satisfy the requirement of efficiency and scalability, furnishing
’ RESULTS AND DISCUSSION
Synthesis of (+)-Pinnatoxins A and G. Synthesis Design.
The structure of pinnatoxin A is composed of the characteristic
AG-spiroimine and BCD-dispiroketal domains connected by the
C7ꢀC11 acyclic carbon chain and the bridged EF-ketal, suggest-
ing a convergent synthesis plan as outlined in Scheme 1. The
general strategy was developed during the first-generation synth-
esis and is based on (1) the diastereoselective IrelandꢀClaisen
rearrangement for the construction of the challenging quaternary
stereogenic center at C5; (2) fragment union by a direct
alkyllithium addition to aldehyde to form the C6ꢀC7 bond;
(3) the assembly of the 27-membered carbocycle by ring-closing
metathesis introducing the C25ꢀC26 double bond; and (4) EF-
ketalization and installation of the imine ring (Scheme 1).32
Several problematic transformations identified in the first-gen-
eration synthesis have been revised in order to enable the
reproducible production of PnTX A on sufficient scale. The
most important of these include modification of the substrate for
ring-closing metathesis and the entire late-stage reaction se-
quence starting from compound 5 and culminating in the target
molecule. The advanced 27-membered macrocyclic enone 5 was
assembled by a convergent approach from two major building
blocks, a G-ring aldehyde (6) and a BCD-bisketal fragment (7).
Synthesis of G-Ring Fragment. Synthesis of the G-ring
aldehyde 6 incorporates another point of convergence beginning
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.
Scheme 1. Overview of Synthesis Plan
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Scheme 2. Key IrelandꢀClaisen Rearrangement
Scheme 3. Examples of Stereoselective Generation of Acyclic
r,r-Disubstituted Enolates
cost associated with its large-scale production from inexpensive
benzylamine. Each base can be recovered in high yield by a
simple extraction with aqueous acid.
The efficient transfer of chirality in the rearrangement of ester
10 is enabled by a highly stereoselective enolization of the acyclic
R,R-disubstituted ester affording a stereodefined tetrasubstituted
enolate (A). The unusual mode of enolization, based on the
chirality match between the ester and the reagent, was developed
originally to address the problem of stereoselective installation of
the C5 quaternary stereogenic center in the spiroimine toxins.38
To date, this is the only general method for stereoselective
generation of acyclic R,R-disubstituted ester enolates, although
several methods for the synthesis of corresponding amide
enolates have been developed recently (Scheme 3). Gleason
and co-workers39,40 pioneered a reductive enolization of thiaze-
pinones such as i (Scheme 3) where the sense of stereoselectivity
is determined by the ground-state conformation of the substrate.
In 2008, Myers and co-workers41 described the stereoselective
generation of R,R-disubstituted enolates of amides ii (Scheme 3)
derived from pseudoephedrine. The geometry of the enolate is,
in part, an outcome of the relative configuration of the pseudoe-
phedrine fragment and the R-position of the amide carbonyl
with a coupling of nearly equimolar amounts of carboxylic acid
831 and allylic alcohol 9, affording ester 10 in 84% yield
(Scheme 2). We have found that the Yamaguchi protocol is
superior to that employed originally [1.3 equiv of 8, 1-ethyl-
3-[3-(dimethylamino)propyl]carbodiimide (EDC), CH2Cl2,
94% yield based on 9, 71% yield based on 8] with equivalent
amounts of 8 and 9.36
A key operation in the synthesis is the installation of the
quaternary stereogenic center at C5 (PnTX numbering), which
was accomplished in the next step by a diastereoselective
IrelandꢀClaisen rearrangement using a chiral lithium amide
base. N-Trifluoroethyl-substituted lithium amide [(S)-11-CF3]
and benzyl-substituted lithium amide [(S)-11-Ph] gave compar-
able results with essentially complete stereocontrol in the
formation of the Z-enolate (A).37 After reduction of the inter-
mediate carboxylic acid with LiAlH4, the isolated yields of alcohol
12 were in the range of 81ꢀ85%. On scales in excess of 5 g of
ester 10, lithium amide (S)-11-Ph was preferred due to a lower
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Scheme 4. Synthesis of G-Ring Aldehyde 6
Scheme 5. Early Approach to BCD-Bisketal
compounds prior to the proton transfer event in enolization
reactions.43ꢀ46
With alcohol 12 in hand, diol 13 was secured in four additional
steps that include benzoylation of the primary hydroxy group,
oxidative removal of the p-methoxybenzyl protecting group,
ozonolysis in the presence of N-methylmorpholine N-oxide
according to the method of Dussault and co-workers,47 and
reduction with sodium borohydride (Scheme 4). The last
reduction of the hydroxy aldehyde (not shown) to diol 13 was
necessary because its direct oxidation to desired dialdehyde 14
was not successful with a variety of oxidants. A simultaneous
oxidation of the two primary hydroxy groups in 13 to dialdehyde
14 was achieved under Swern conditions. Subsequent aldol
cyclocondensation completed the formation of the G-ring. In
this reaction, dibenzylammonium trifluoroacetate induced a
cleaner transformation than piperidinium acetate, affording an
89% isolated yield of the desired product over two steps. The
cyclocondensation was characterized by a rapid cyclization to
aldol adduct 15, which was followed by a slow dehydration to
R,β-unsaturated aldehyde 16.
The subsequent seven steps were necessary to prepare this
fragment for coupling with BCD-precursor 7. After reduction of
the aldehyde, the allylic hydroxyl was protected as a methoxy-
methyl ether, the primary triisopropylsilyl (TIPS) ether was
cleaved, and the resultant primary hydroxy group was oxidized to
aldehyde, which was methylenated with the Wittig reagent to
afford 17 in an 87% yield over five steps. Reductive removal of
the benzoate followed by oxidation completed the synthesis of
aldehyde 6 fully functionalized for fragment coupling in 16%
overall yield, 26 linear steps from (S)-citronellic acid.
Synthesis of BCD-Bisketal Fragment. Synthesis of the BCD-
bisketal fragment started with an efficient enzymatic resolution
of t-butyl 3-hydroxy-4-pentenoate (Scheme 5).17 Alcohol 19
was advanced to borane 20 in two steps, while acetate 21 was
converted to Weinreb amide 23 via acetonide 22 in six steps
following our original approach. During scaleup, we have found
that the chain extension using 3-propyn-1-ylmagnesium bro-
mide (24) was not reliable due to difficulties in efficient
generation of the Grignard reagent. Various attempts to im-
prove reproducibility of this transformation have not been
productive, and as a recourse we opted for a direct installation
of a more functionalized substituent incorporating a vinylsilane
group. In the procedure developed in our laboratory, the con-
figuration of the enolate is defined by the relative configuration of
the ester and the chiral lithium amide. For example, enolization of
3-phenylpropyl (S)-2-methylbutyrate with (S)-11-Ph gives the
E-enolate with 98% selectivity, whereas its enolization with the
enantiomeric (R)-11-Ph produces the Z-enolate with 96%
selectivity (Scheme 3).38
According to our hypothesis, the chiral base controls the
conformational preference in the transition complex between
the substrate and the reagent preceding the proton-transfer
event. This conformational preference translates into the geo-
metrical selectivity in enolate formation upon proton transfer.
In Scheme 3, this concept is depicted by use of chairlike
structures proposed by Ireland et al.,42 although alternative
conformationally defined transition structures are feasible, if
not more plausible. Nonetheless, as long as the proton transfer
occurs after precomplexation between the lithium amide and
the ester, proceeding within a highly organized chiral system,
the same logic will be applicable. There is strong evidence for
precomplexation between lithium amide bases and carbonyl
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Scheme 6. Assembly of BCD-Dispiroketal
Table 1. Effect of Selective Silylation on Relative Stability of
C19 Epimers
entry
R1
R2
33-Si:34-Si
1
2
3
4
H
H
4.0:1
1.7:1
13:1
8.3:1
H
i-Pr3Si
H
Et3Si
Et3Si
i-Pr3Si
that served as a precursor to an iodoalkene required for further
elaboration.
Figure 2. Hydrogen-bonding stabilization in spiroketal intermediates.
Iodide 27, prepared from alcohol 26, was subjected to lithiumꢀ
iodine exchange that generated alkyllithium reagent 28, which
was coupled with amide 23 (Scheme 6). After two additional
steps (NaBH4, TBSCl), silane 29 was prepared in a scalable
and reproducible manner. Stereoselective iododesilylation48 and
SuzukiꢀMiyaura coupling of the resultant iodoalkene with bor-
ane 20 in the presence of Pd(dppf)Cl2 and Ph3As afforded 30
in excellent yield.49 Sharpless asymmetric dihydroxylation of the
trisubstituted alkene,50 desilylation, and double oxidation afforded
diketone 31 in preparation for the BCD spirotricylic ketal forma-
tion. Removal of the acetal protecting groups ([tetrahydropyranyl
(THP) and acetonide] upon exposure to acidic methanol at
room temperature was accompanied by rapid ketalization of
intermediate tetraol 32. Upon extended reaction times, the
desired diastereomer 33 is produced as a major product; however,
the C19 epimer is formed initially and thus is favored under kinetic
control.
A change of solvent from methanol to cyclohexane was
performed to maximize diastereoselectivity in favor of 33 versus
its C19 epimer. We initially ascribed the influence of the solvent
on selectivity to the well-known enhancement of the anomeric
effect in media of low polarity.17 Upon superficial analysis, isomer
33 fully stabilized by anomeric effect should be increasingly more
favored over its epimer 34 lacking anomeric stabilization at C19
as the medium polarity decreases, and indeed, this was consistent
with our observations. Insightful contributions from the groups
of Murai,14,33,34 Hirama,27,30 and Kishi20 also suggest that the
effect of intramolecular hydrogen-bonding stabilization should
be considered. The C19 epimer 34 can be stabilized by a
hydrogen bond between the tertiary hydroxy group at C15 and
the D-ring oxygen, while the desired isomer is stabilized by long-
range hydrogen bonding between the terminal ester and hydro-
xyl groups. The unusual long-range hydrogen bonding is sup-
ported by crystallographic studies reported by Hirama and co-
workers27 on enantiomeric bisketal 35 closely related to 33. In
order to further ascertain the effect of hydrogen bonding on the
selectivity of spiroketalization, we carried out equilibration
experiments with selectively silylated substrates. In all cases,
the desired configuration at C19 was favored, but the level of
preference varied substantially. As shown in Table 1, equilibra-
tion of the diol (entry 1) in the presence of camphorsulfonic acid
in toluene at room temperature for 48 h resulted in a 4.0:1
mixture of diastereomers. The long-range H-bonding was dis-
rupted by silylation of the primary hydroxy group (entry 2),
which resulted in decrease in ratio to 1.7:1. Silylation of the
tertiary hydroxyl group (Et3Si) increased the ratio significantly to
13:1 (entry 3). When both hydroxyl groups were silylated, an
8.3:1 ratio of the isomers was recorded at equilibrium (entry 4).
These results indicate that when only the anomeric effect is
operational in the fully silylated substrate (entry 3), the thermo-
dynamic ratio of 8.3:1 is observed in toluene, and hydrogen
bonding from both tertiary and primary hydroxyl groups as
depicted in Figure 2 has a significant effect on the position of the
equilibrium.
In anticipation of the fragment coupling, the ester group in
the fully silylated BCD-bisketal was reduced to aldehyde 36.
Addition of 3-(p-methoxybenzyloxy)-1-propyllithium followed
by oxidation and methylenation afforded 37 in 76% yield over
the previous seven steps (Scheme 7). We have found that the
two-step Peterson olefination51 procedure was substantially
better suited for large-scale operations rather than the originally
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Scheme 7. Completion of Synthesis of the BCD-Bisketal
Fragment
the EF-ketal and introduction of a nitrogen substituent at the C1
position. Originally, EF-ketal formation was accomplished first.
followed by debenzylation and azidation atC1. Wediscovered that
the overall efficiency and scalability is substantially improved if the
C1-azido group is introduced first. Two critical steps were
facilitated by this reversal. First, open-chain EF-ketal precursor
43 is better suited for oxidative debenzylation because of its
apparently higher resistance to oxidative decomposition in the
presence of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). Sec-
ond, azide 44 proved to be a superior substrate for multiple
deacetalization and EF-ketal formation under aqueous acidic
conditions compared to its C1-benzyloxy analogue, delivering
azido triol 4 in 71% yield in a reproducible fashion. The prepara-
tion of a total of 0.37 g (0.50 mmol) of 4 was enabled by this
approach.
Synthetic (+)-PnTX A was obtained in six additional steps that
began with oxidation of the allylic hydroxy group to carboxylic
acid, followed by formation of the methyl ester. Although a free
carboxyl group is present in the final compound, introduction of
the methyl ester significantly improved the efficiency of material
purification after reduction of the azide to primary amine, which
was achieved by hydrogenation in the presence of Pd-CaCO3.
The imine formation was readily accomplished under modified
Kishi conditions (triethylammonium mesitoate, PhMe, 85 ꢀC,
60 h). Final hydrolysis of the methyl ester with lithium hydroxide
in aqueous tetrahydrofuran (THF) delivered the natural product
(Scheme 8). For the purposes of biological studies, 22 mg of the
synthetic toxin has been prepared from 55 mg of 4.
Synthesis of newly discovered pinnatoxin G (PnTX G) was
completed by diverting some of the stock of azido triol 4 for this
purpose (Scheme 9). The vinyl group was introduced in two
steps by selective oxidation of the allylic hydroxy group to
aldehyde [2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), PhI-
(OAc)2] and Wittig olefination (76% overall yield). Staudinger
reduction, imine ring closure, and purification by reverse-phase
HPLC afforded pure PnTX G as its trifluoroacetate. NMR data of
the synthetic material were consistent with those reported for
natural PnTX G.8,55
In order to determine whether the imine ring is required for
biological activity of PnTX A, an amino ketone analogue (PnTX
AK) was prepared by modifying the final steps of the total
synthesis (Scheme 10). Azido acid 46 was obtained in two steps
from azido triol 4 and subjected to hydrogenation in the presence
of the Lindlar catalyst. The requisite amino acid was isolated after
purification by reverse-phase HPLC in 35% yield. During azide
reduction studies, we have found that both hydrogenolysis and
Staudinger reduction conditions led to the formation of variable
amounts of dimeric urea 47, presumably formed due to the
presence of CO2 dissolved in the reaction medium.
PnTX A Blocks Human r7 and r4β2, and Torpedo r12βγδ
nAChRs in Xenopus Oocytes. With reliable access to synthetic
PnTXA established, wecarried out pharmacological investigations
guided by the flaccid paralysis effect observed after injection of
PnTX A in mice, suggesting its potential interaction with nAChRs.
The hypothesis is supported by recent reports that other toxins in
the cyclic imine family, gymnodimine A and 13-desmethylspir-
olide C, block nAChRs.11,56 Initial experiments were performed in
oocytes expressing either the human R7 or R4β2 nAChRs. A good
expression of both nAChR subtypes was obtained 2ꢀ4 days after
injection of the corresponding cDNA, as judged by the peak
amplitude of currents elicited by ACh perfusion. Thus, in oocytes
expressing the R7 nAChR, the perfusion of 350 μM ACh
employed one-step Takai olefination.52 The Wittig protocol
was accompanied by ∼30% epimerization at the C12 position.
The synthesis of fully elaborated BCD-fragment 7 was com-
pleted in two additional steps, affording 7 in 13% overall yield
over 26 steps from acrolein (Scheme 7).
Fragment Coupling and Completion of Total Synthesis.
Unification of the two major fragments on a multigram scale
was accomplished by direct addition of the complex organo-
lithium reagent generated from 7 by lithiumꢀiodine exchange to
aldehyde 6, with a 1:1 stoichiometry maintained for the two
intermediates (78% yield). Unreacted aldehyde 6 was recovered
in 16% yield. From 38, the substrate for ring-closing metathesis
was obtained in three steps involving selective desilylation,
oxidation to ketoaldehyde, and chemoselective addition of
vinylmagnesium bromide, giving a 1:1 mixture of diastereomeric
alcohols 39 in 76% overall yield.
Preserving the silylation of the tertiary hydroxy group at
C15 was essential for high regioselectivity in the ring-closing
metathesis of 39, a crucial transformation leading to the 27-
membered carbocyclic framework of pinnatoxins. With catalyst
40 (20 mol %),53 the ring closure was accomplished with >12:1
selectivity favoring 27-membered carbocycle 41 (75% isolated
yield). The 17-membered macrocyclic byproduct 42 (6% iso-
lated yield) was formed by ring-closing metathesis that engaged
the methylene group at C10. The relative amount of the
regioisomeric product was substantially larger with a free
C15-hydroxy group. When the tertiary alcohol was desilylated,
the ring-closing metathesis occurred with 3:1 regioselectivity
under a variety of reaction conditions. The reasons for the
change in selectivity are unclear but can possibly be understood
as a result of subtle conformational differences between 39 and
its desilylated analogue.
The final stereogenic center was established by oxidation of 41
to enone (85%) and conjugate addition of lithium methylcya-
nocuprate in the presence of boron trifluoride etherate (86%),
which took place in an anti fashion relative to the C28 alkoxy
substituent with high stereoselectivity.54
The objective of the next set of transformations was assembly of
the macrocyclic framework of pinnatoxins through construction of
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Scheme 8. Completion of Synthesis of PnTX A
Scheme 9. Total Synthesis of PnTX G
Scheme 10. Synthesis of Amino Ketone PnTX AK
interval) for PnTX A and 5.06 nM (3.84ꢀ6.67, 95% confidence
interval) for PnTX G]. The blocking action of PnTX A on R7
nAChR was not reversible after a 10ꢀ15 min washout of the
molecule from the medium. Furthermore, when applied on its
own, PnTX A (0.1ꢀ50 nM) did not evoke any current, indicating
that it has no direct agonist effect on the R7 nAChR subtype
(n= 6). PnTX A also blocked the human R4β2 nAChR expressed in
Xenopus oocytes, as shown in Figure 3C. However, higher PnTX
A concentrations were needed to block the ACh-evoked currents
(Table 2), indicating that the toxin was less potent in this neuronal
receptor subtype, compared to the R7 nAChR (Figure 3D).
Microtransplantation of Torpedo nAChR into Xenopus oocytes
has proven to be an elegant method to study its function and the
(corresponding to the EC50 determined experimentally) elicited
peak currents that varied between oocytes and ranged from 1 to
3 μA at ꢀ60 mV holding membrane potential (n = 54 oocytes
tested, from five donors). As shown in a typical recording
(Figure 3A), the ACh-evoked currents started to desensitize
within about 300 ms, but PnTX A did not appear to affect the
rate of desensitization. In contrast, the ACh-evoked currents were
markedly reduced in amplitude with pico- to nanomolar PnTX A
concentrations, as shown in the concentrationꢀinhibition curves
(Figure 3D). PnTx G also blocked ACh-evoked currents in oocytes
expressing the human R7 nAChR but was significantly less potent
than PnTX A when their concentrationꢀinhibition curves were
compared [IC50 = 0.107 nM (0.086ꢀ0.132, 95% confidence
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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 R7, (b) Torpedo R12βγδ, and (c) human R4β2
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 R7
(solid circles, black curve) and R4β2 (solid triangles, red curve) neuronal
nAChRs, or having incorporated into their membranes the Torpedo muscle
type R12βγδ 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).
Table 2. Inhibition Constants for PnTX A on ACh-Evoked
Nicotinic Currents in Xenopus Oocytesa
Figure 4. Effect of PnTX A and PnTX AK on various nAChRs.
Inhibition of specific [125I]R-BTX or (()-[3H]epibatidine binding by
increasing concentrations of PnTX A or PnTX AK on (a) Torpedo and
neuronal R7-5HT3 or (b) heteropentameric R3β2 and R4β2 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.
nAChR
PnTX A IC50b (nM)
R7 (human)
0.107 (0.086ꢀ0.132)
30.4 (19.4ꢀ47.5)
5.53 (4.5ꢀ6.8)
R4β2 (human)
R12βγδ (Torpedo)
a Oocytes expressed human neuronal R7 or R4β2 nAChR subtypes or
were microtransplanted with muscle-type R12βγδ nAChR. b Mean
values from concentrationꢀresponse curves were recorded on 46ꢀ50
oocytes for each experimental condition; 95% confidence intervals are
shown in parentheses. For further details, see Figure 3.
wereperformedwithPnTXAKaminoketoacid(2), an analogue of
PnTX A that contains an open form of the imine ring A. As shown
in Figure 3D, PnTX AK had no action on the different nAChR
subtypes expressed or incorporated into the membrane of Xenopus
oocytes in the range of concentrations in which PnTX A blocked
nAChRs. These results indicate that the spiroimine component is
an important structural factor for blocking nAChRs by PnTX A.
Binding Competition Experiments between PnTX A and
Radiolabeled Ligands. To get a deeper insight into the interac-
tion between PnTX A and nAChR subtypes, competition binding
studies were performed in HEK-293 cells expressing the chimeric
chicken neuronal R7-5HT3 and the human R4β2 and R3β2
nAChR subtypes and in Torpedo membranes expressing the
muscle-type (R12βγδ) nAChR, with both [125I]R-bungarotoxin
(R-BTX) and (()-[3H]epibatidine as radiotracers.
effect of pharmacological agents.57 Therefore, subsequent ex-
periments were performed in oocytes with the Torpedo R12βγδ
muscle-type nAChR incorporated into their membranes. PnTX
A blocked the ACh-evoked nicotinic currents in muscle-type
nAChR in a concentration-dependent manner (Figure 3D).
Table 2 contains the IC50 data for PnTX A in both muscle-
and neuronal-type nAChRs studied. An important finding is that
the inhibitory potency of PnTX A is dependent on the nAChR
receptor subtype. The order of potency established by the
functional evaluation of PnTX A on nAChRs is R7 (human) >
R12βγδ (Torpedo) > R4β2 (human), with IC50 values spanning
3 orders of magnitude from 0.1 to 30.4 nM (Table 2).
To determine whether the spiroimine fragment in PnTX A is
required for the blocking action in nAChR subtypes, experiments
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Table 3. Affinity Constants for PnTX A and Its Amino Keto
Analogue (PnTX AK) on Muscle and Neuronal nAChR
Subtypesa
Ki ( SEMb (nM)
R12βγδ
R7ꢀ5HT3
(chick)
R4β2
R3β2
ligand
(Torpedo)
(human)
(human)
PnTX A
2.8 ( 0.03
0.35 ( 0.04
15.6 ( 5.2
9.4 ( 1.9
PnTX AK
>1000
>10 000
>2000
>2000
a Determined by equilibrium competition binding experiments. b Mean
values ( SEM from three distinct experiments performed in duplicate.
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. BT 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 proteinꢀligand interactions in PnTX Aꢀ
nAChR complexes obtained by molecular modeling: (a) human R7
(green, R7ꢀR7 interface), (b) human R4β2 (magenta, R4ꢀβ2 inter-
face), and (c) Torpedo R1β1γδ (cyan, R1ꢀδ 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 90ꢀ, are presented.
Nonpolar hydrogen atoms of the ligand are not shown for clarity.
Competition binding curves obtained with PnTX A on
muscle-type and homopentameric R7 neuronal receptors are
shown in Figure 4A. These data tally well with competition for a
single class of binding sites, and the apparent affinity constants
(Ki) deduced from these experiments are reported in Table 3.
PnTX A interacts with Torpedo and R7-5HT3 receptors with
high affinities respectively equal to 2.8 and 0.35 nM. Further-
more, PnTX A binds to the R3β2 and R4β2 heteropentameric
neuronal receptors with affinities in the nanomolar range
(Figure 4B, Table 3), and its order of potency on the various
nAChRs subtypes is R7-5HT3 > Torpedo > R3β2 = R4β2,
confirming the results obtained with electrophysiological ex-
periments. Interestingly, PnTX A displaces approximately half
the [3H]epibatidine in the R4β2 receptor, revealing a partial
antagonist property on this receptor subtype. Studies on the
binding potency of PnTX AK on the muscle and neuronal
nAChRs subtypes showed that this toxin produces no signifi-
cant displacement of the radioactive tracer, even at high
concentrations (Figure 4A,B), suggesting a decrease of at least
3 orders of magnitude in affinity as compared to PnTX A. Thus,
disruption of the imine ring in PnTX A is responsible for the
drastic loss of affinity of this compound for the various nAChR
subtypes.
potential inhibitory activity of 1 μM PnTX A or PnTX AK was
evaluated on CHO cells stably expressing the distinct human
muscarinic acetylcholine receptor (mAChR) subtypes. As shown
in Figure 5, both PnTX A and PnTx AK had no significant effect
on 3H-NMS binding to M1, M2, M3, and M4 mAChR subtypes,
whereas PnTX A (1 μM) induced 35% radiotracer displacement
in M5 mAChR (Figure 5), indicating a low micromolar affinity
for this receptor subtype. Here again, this property was lost for
the PnTX AK analogue that contains an open form of the imine
ring A (2).
Computational Modeling of the PnTX A-nAChR interac-
tions. Structural modeling of the PnTX A-nAChRs complexes
revealed that the toxin is embedded within loops C and F of the
receptor, involving mainly aromatic side chains of the principal
and complementary faces (see Supporting Information for more
details on molecular modeling protocols). The main proteinꢀ
ligand interactions are shown in Figure 6, and a detailed analysis
of residues surrounding PnTX A in the binding site is provided in
Supporting Information, Table 2-1. The ligand is positioned in a
similar manner in all three complexes, with differences in binding
affinity arising mainly from the residue variability in key positions
of the binding site. PnTX A establishes an important number
of hydrogen bonds (seven) with R7 nAChR, which are well
distributed in the binding site and involve most of the ligand’s
While the ability of cyclic imines to affect drastically the
nicotinic function has now been clearly demonstrated, their
action on other cholinergic neurotransmission pathways re-
mained elusive due to the lack of purified PnTX A. With the
ready access to pure PnTX A established in this study, the
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functional groups. Conversely, only five hydrogen bonds are
observed in the R4β2 complex, covering approximately 50%
of the binding site at a close distance. The R12β1γδ complex
shows intermediate behavior, with the five hydrogen bonds more
equally distributed in the binding site. The latter complex also
benefits from important hydrophobic contacts with the comple-
mentary δ subunit; these interactions are substantially reduced in
the R4β2 complex. Most of the residues present in the binding
site are conserved among the three nAChRs subtypes considered
(residues 93, 143, 147, 149, 188, 190, 191, and 195 from the
principal side and residues 55, 79, and 118 from the comple-
mentary side, highlighted in Supporting Information, Table 2-1),
while in a few positions with amino acid variability the neighbor-
ing residues are able to compensate for the missing interaction
(e.g., in R12β1γδ, the carboxylate group of PnTX A is not able to
establish an ionic interaction with Val186 as it does with the
equivalent residue Arg186 in R7 and R4β2 nAChR subtypes;
instead, it interacts with Lys143). However, in some cases,
differences in amino acid sequence are responsible for changes
in the proteinꢀligand interaction pattern. One example is
Gln116 from the R7 subtype, which interacts with an oxygen
atom from the bis-spiroketal core, while the equivalent residues
Phe116 from R4β2 and Leu116 from the R12β1γδ subtype are
unable to establish similar interactions. Similarly, Gln57 from the
R7 subtype and Asp57 from the R12β1γδ subtype form hydro-
gen bonds with a hydroxy group at the C28 position of PnTX A
near the solvent-exposed site, whereas the side chain of the
equivalent Thr57 residue from the R4β2 subtype is too short to
form analogous interactions. Overall, the proteinꢀligand inter-
actions identified in these three complexes are in very good
agreement with the experimentally determined biological prop-
erties, with PnTX A showing notably stronger interactions with
the R7 nAChR subtype compared to the R4β2 subtype, while the
R12β1γδ subtype displayed intermediate binding potency with
significant hydrophobic contribution from the complementary
side.
Pinnatoxin A: Selective Inhibition of nAChRs. The produc-
tion of several milligrams of PnTX A enabled us to perform the
first broad receptor binding screening and comprehensive func-
tional and pharmacological characterization of this natural cyclic
imine toxin with nicotinic acetylcholine receptors.58 Indeed, until
now, the mode of action of pinnatoxins was ascribed to action on
calcium channels.9 However, in our studies, even at 10 μM
PnTX A, no significant binding activity was detected for calcium
channels.58 The symptoms observed upon intraperitoneal injec-
tion of a lethal dose of PnTX A in mice give a toxicological profile
characterized by transient mouse hyperactivity, followed by a
decrease in respiratory rate with prominent abdominal breathing
leading to death due to respiratory paralysis. Such a toxicological
profile is also seen with agents affecting nicotinic receptors in the
central nervous system and in the skeletal neuromuscular junc-
tion. Furthermore, there is growing evidence that other structu-
rally related cyclic imine toxins affect both muscle- and neuronal-
type nAChRs.11,56 The electrophysiological and binding studies
described herein demonstrate the ability of PnTX A to interact
very efficiently with various nAChR subtypes. The selectivity
profile and affinity values determined by the two approaches
were similar: R7 (0.1ꢀ0.3 nM) > Torpedo (3ꢀ5 nM) > R3β2 =
R4β2 (10ꢀ30 nM), demonstrating the ability of this toxin to
recognize muscle as well as neuronal subtypes that potentially is
at the origin of the toxicity of this compound. Furthermore, the
apparent irreversibility of the PnTX AꢀnAChR interaction may
reinforce its toxicological effect.
To determine whether the spiroimine fragment in PnTX A is
required for its potent blockage of nAChR subtypes, experiments
were performed with the PnTX AK analogue, which contains an
acyclic form of the imine ring A. Both electrophysiological and
binding assays showed the inactivity of PnTX AK with different
nAChRs, highlighting the crucial role of the cyclic imine for the
biological activity of PnTX A. This inactivity can be explained by
the existence of conformers strongly stabilized by an intramole-
cular ionic interaction between the ammonium and carboxylate
groups of PnTX AK in solution (see Supporting Information,
Figure 2-2). These conformations, which are unable to bind
directly to the nAChR subunit interface, would require an
energetically unfavorable disruption of the intramolecular ion
pair to produce an “open” form of the ligand, which could
potentially interact with the binding site. These observations
reinforce the functional role of the cyclic imine and the im-
portance of its structural integrity for the interaction with
nAChRs. The cyclic imine moiety represents the key feature in
this family of toxins and its role in PnTX A binding to nAChRs,
according to the modeling, is dual. First, in conjunction with the
C28 hydroxy group of the bridged EF-ketal, it anchors the ligand
to the binding site through hydrogen bonds in a conformation
ideally positioned to optimize the interactions with neighboring
residues. Moreover, the hydrogen bond established by this group
with the backbone carbonyl of Trp147 is observed not only in all
three docking complexes obtained in this work but also in the two
crystal structures of the complexes between ACh binding protein
(AChBP) and gymnodimine A or 13-desmethyl spirolide C.11
Second, binding of the closed imino ring A in PnTX A appears to
be morefavorable, bothstericallyand energetically, than that ofthe
corresponding open amino ketone form in PnTX AK. The
presence of a protonated nitrogen could serve as a common
functional signature in the cyclic imine phycotoxins, interacting
with nicotinic receptors as has been shown for PnTX A (this work),
gymnodimine A, and 13-desmethyl spirolide C11,56 and that has
yet to be demonstrated for pteriatoxins and prorocentrolides.
Finally, aside from their clear effect on nAChRs, interaction of
cyclic imine toxins such as 13-desmethyl spirolide C with other
targets of the cholinergic neurotransmission pathway, especially
the muscarinic receptors (mAChRs), has also been reported
recently.59 Therefore, interaction of PnTX A and PnTX AK with
the five human mAChR subtypes has been further evaluated at
1 μM concentration. Although no interaction was observed with
M1, M2, M3, and M4 mAChR subtypes, a weak effect of PnTX A
was measured for the M5 subtype, suggesting a low micromolar
affinity of this toxin for the M5 receptor subtype. This weak
interaction with the M5 receptor is at least 3 orders of magnitude
lower compared to the interaction of PnTX A with various
nAChRs and seems unlikely to be correlated with its toxicological
profile.
’ CONCLUSION
The comprehensive study described here encompasses the
scalable synthesis and thorough biological evaluation of PnTX A
to conclusively establish its mechanism of toxicity as primarily
due to its potent and effectively irreversible inhibition of nicotinic
acetylcholine receptors. With respect to total synthesis, reliable
access to PnTX A and PnTX G on scale was enabled by the
development of new enolization technique based on a chiral
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match between the ester substrate and the base. This allowed
for a reliable control of stereoselectivity in the installation of the
spiroimine fragment of the marine toxins. Late-stage opera-
tions including ring-closing metathesis, EF-ketal formation, and
closure of the imine ring were developed and optimized, resulting
in a relatively high overall yield of the natural products. Thus,
PnTX A was accessed in a 1.4% overall yield, which is a several-
fold improvement compared to previously described syntheses.
By use of the chemistry developed in this synthesis effort, an
analogue with an open imine ring, amino ketone PnTX AK, was
prepared to determine the significance of the AG-spiroimine
subunit as a pharmacophore. Subsequent comprehensive elec-
trophysiological and competition binding studies confirmed the
hypothesis that the spiroimine component of pinnatoxins is an
important structural factor for blocking nAChRs. Furthermore,
computational modeling studies provided further insight into the
functional determinants and binding regions for both PnTX A
and nAChR subtypes that are important for strong molecular
interactions. Finally, chemical synthesis of the pinnatoxins and
knowledge of their mode of action on nicotinic acetylcholine
receptors pave the way for the production of certified standards
to be used for mass spectrometry determination of toxins in
marine matrices and for the development of tests to detect these
toxins in contaminated shellfish.
(mAChRs) were kindly provided by Professor P. O. Couraud
(Institut Cochin de Gꢀenꢀetique Molꢀeculaire, Paris, France).
Expression of nAChRs in Human Embryonic Kidney (HEK-
293) Cells. A chimeric cDNA of the neuronal type R7-5HT3
nAChR was transfected into HEK-293 cells by calcium phos-
phate precipitation, as previously described.56,61 Briefly, the chick
cDNA (15 μg of R7-5HT3) was transfected by calcium pre-
cipitation with careful control of the pH (6.95). The cells were
placed at 37 ꢀC under 5% CO2, and 2 days after transfection, cells
were harvested in phosphate-buffered saline (PBS) with 5 mM
ethylenediaminetetraacetic acid (EDTA) and resuspended in
3 mL/plate of this buffer for binding experiments. For human
R3β2 or R4β2 nAChR subtypes, 24 h after the calcium phos-
phate transfection (7 μg of R3 or R4, and 7 μg of β2) the cells
were placed for 2 days at 30 ꢀC, 5% CO2 before being collected
for binding assays, which used a cell density adjusted to speci-
fically bind e10% of radioligand.
CHO Cells Expressing Human M1ꢀM5 mAChRs and Mem-
brane Preparation. CHO cells stably expressing M1ꢀM5
mAChRs were grown in plastic Petri dishes (Falcon) and
incubated at 37 ꢀC in an atmosphere of 5% CO2 and 95%
humidified air in Ham F12 medium precomplemented with
L-glutamine and bicarbonate and supplemented with 10% fetal
calf serum and 1% penicillin/streptomycin. At 100% confluence,
the medium was removed and cells were harvested in Versen
buffer (PBS + 5 mM EDTA). They were washed with ice-cold
phosphate buffer and centrifuged at 1700g for 10 min (4 ꢀC).
The pellet was suspended in ice-cold buffer (1 mM EDTA,
25 mM sodium phosphate, and 5 mM MgCl2, pH 7.4) and
homogenized with a PotterꢀElvehjem homogenizer (Fisher
Scientific Labosi, Elancourt, France). The homogenate was
centrifuged at 1700g for 15 min (4 ꢀC). The sediment was
resuspended in buffer, homogenized, and centrifuged at 1700 g
for 15 min (4 ꢀC). The combined supernatants were centrifuged
at 35000g for 30 min (4 ꢀC) and the pellet was suspended in
the same buffer (0.1 mL/dish). Protein concentrations were
determined according to the Lowry method with bovine serum
albumin (BSA) as standard. The membrane preparations were
aliquotted and stored at ꢀ80 ꢀC.
’ EXPERIMENTAL SECTION
Pinnatoxins A, G, and AK and Radioligands. PnTX A, PnTX
G, and the ketamine analogue (PnTX AK) were prepared by total
synthesis as described herein. Detailed procedures and spectral
data are presented in Supporting Information. Samples of
synthetic PnTX A, PnTX G and PnTX AK were dissolved in
ethanol and diluted in physiological solutions. The total ethanol
concentration in the test solution did not exceed 0.15%. All
physical data for synthetic PnTX A, PnTX AK, and PnTX G,
including 1H and 13C NMR spectroscopy, high-resolution
mass spectrometry (HRMS), HPLC, and optical rotation data
showed both compounds to be of high purity and identical to
natural samples. [125I]R-Bungarotoxin ([125I]R-BTX) (210ꢀ250
Ci mmolꢀ1), (()-[3H]epibatidine (55 Ci mmolꢀ1), and N-
Binding Assays. The affinity of PnTX A and AK for the
chimeric R7-5HT3 nAChR was determined as previously
described.61,62 Briefly, cells expressing the R7-5HT3 receptors
were preincubated with different PnTX A concentrations and
filtered through GF/C filters 6 min after the addition of 1 nM
3
3
methyl-[3H]scopolamine methyl chloride ([3H]NMS) (78 Ci
3
mmolꢀ1) were purchased from Perkin-Elmer (Courtaboeuf,
France). All other chemicals were obtained from SigmaꢀAldrich
(Saint Quentin Fallavier, France) or other usual sources.
Animals and Biological Materials. Adult female Xenopus
laevis frogs were obtained from the Centre de Ressources
Biologiques Xꢀenope, CNRS (Universitꢀe de Rennes 1, France).
Live Torpedo marmorata fishes were purchased from the Service
Modꢁeles Biologiques of the Station Biologique de Roscoff
(France). Live animals were maintained in the Animal Campus
facility of Gif-sur-Yvette. Experiments were performed in accor-
dance with European Community guidelines for laboratory
animal handling and with the official edict presented by the
French Ministry of Agriculture and the recommendations of the
Helsinski Declaration. The cDNAs coding for chick chimeric R7-
5HT3, human R4β2,and human R3β2 were kindly provided by
Dr. P. J. Corringer (Pasteur Institute, Paris, France) and by
Professor O. Steinlein (Institute of Human Genetics, Bonn,
Germany). The human R7 cDNA60 was a generous gift from
Professor I. Bermudez(OxfordBrookes University, Oxford, U.K.).
Chinese hamster ovary (CHO) cells stably expressing the
M1ꢀM5 cloned human muscarinic acetylcholine receptors
[
125I] R-BTX. The filters were washed with cold PBS and
counted on a γ counter (LKB-Multigamma 1261, Uppsala,
Sweden). The protection constant (Kp) calculated by fitting
the competition data by the empirical Hill equation was shown
to correspond to the dissociation constants.63 Competition
experiments with Torpedo nAChRs were performed at equilib-
rium by incubating (for at least 4 h) 0.05 μg of Torpedo
membrane with different concentrations of PnTX A and
[
125I]R-BTX (0.25ꢀ0.35 nM). The mixture was filtered, rinsed,
and counted as previously described for the chimeric R7-5HT3
nAChR. Equilibrium binding experiments on R4β2 and R3β2
subtypes used [3H]epibatidine as radioactive tracer. Cells ex-
pressing these receptor subtypes were incubated with 0.5ꢀ1 nM
[3H]epibatidine and various concentrations of PnTX for 4 h.
After filtration, GF/C filters were dried and 6 mL of scintillation
solution (Ultima Gold F, PerkinElmer, France) was added
before the filters were counted on a Liquid Scintillation Analy-
zers (Tri-Carb 2300 TR, Perkin-Elmer, France).
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In equilibrium competition experiments, IC50 values were
determined by fitting the competition data by the empirical
Hill equation and converted to Ki constants via the equation
Ki = IC50/(1 + L*/Kd),64 with Kd for R-BTX on muscle-type
receptor of 50 pM and Kd for epibatidine on human R3β2 and
R4β2 equal to 35 and 20 pM, respectively. All experiments were
done at least three times in duplicate.
0.5ꢀ1.5 MΩ tip resistance when filled with 3 M KCl. Data were
acquired with a pCLAMP-9/Digidata-1322A system (Molecular
Devices, Union City, CA). The recording chamber (capacity
300 μL) was superfused (8ꢀ12 mL/min; 20 ꢀC) with a modified
Ringer’s solution containing (in millimolar units) NaCl 100, KCl
2.8, BaCl2 0.3, and HEPES 5 (pH 7.4), where BaCl2 substitution
for CaCl2 prevents secondary activation of a Ca2+-dependent
Clꢀ current.66 Oocytes were initially incubated for 3 min with the
respective PnTX A or its amino keto analogue and then ACh was
applied, for 3 s in oocytes expressing the human R7 nAChR or for
15 s for human R4β2 as well as for the Torpedo (R12βγδ)
nAChR incorporated to the oocyte membrane, by use of a
computer-controlled solution exchange system (VC-6; Warner
Instruments). Between successive ACh applications, 4-min per-
fusion intervals with modified Ringer’s were maintained to
ensure receptor recovery from desensitization.
ConcentrationꢀInhibition Analysis of ACh Currents.
Doseꢀresponse curves for agonist activation were analyzed
by using the equation I = Imax[L]n/(EC50 + [L])nH, where I is
the measured agonist-evoked current, [L] is the agonist
concentration, EC50 is the agonist concentration that evoked
half the maximal current (Imax), and nH is the Hill coefficient.
I values were normalized to the Imax value recorded from the
same oocyte, to yield fractional (percent) response data. The
IC50 was determined from doseꢀresponse curves by fitting
to the equation F = 1/[1 + ([X]/IC50)nH], where F is the
fractional response obtained in the presence of the inhibitor at
concentration [X] and IC50 is the inhibitor concentration that
reduced the ACh-evoked amplitude by half.
The effect of PnTX on the equilibrium binding of [3H]NMS
on the five mAChRs subtypes (M1ꢀM5) was determined in
inhibition experiments on 96-well plates. With [3H]NMS as
tracer, membrane protein concentrations was adjusted so that
no more than 10% of added radioligand was specifically bound
(around 1500ꢀ2500 cpm), and the plate was incubated in
PBSꢀBSA at 25 ꢀC for 120 min with micromolar concentra-
tions of ligands and [3H]NMS (0.5 nM), in a final assay volume
of 100 μL. Nonspecific binding was determined in the presence
of 50 μM atropine. The reaction was stopped by filtration of
the 96-well plate simultaneously through a GF/C plate pre-
soaked in 0.5% poly(ethylenimine), by use of a FilterMate
harvester (Perkin-Elmer, France). The filters were washed
twice with ice-cold buffer (PBS) and dried, and the bound
radioactivity was counted, after the addition of 25 μL of
MicroScint/well, by scintillation spectrometry on a TopCount
β counter (PerkinElmer, France). Each experiment was done at
least two times in duplicate.
Expression of Human r7 nAChR in Xenopus Oocytes.
Oocytes were surgically removed from mature female Xenopus
laevis frogs under anesthesia with ethyl-3-aminobenzoate metha-
nesulfonate salt (SigmaꢀAldrich, Saint Quentin Fallavier, France)
solution (1.5 g/L), were treated for 10 min with 1 mg mLꢀ1
3
collagenase type I (SigmaꢀAldrich) in calcium-free medium
containing (in millimolar units) NaCl 88, KCl 2.5, MgCl2 1, and
N-(2-hydroxyethyl)piperidine-N0-2-ethanesulfonic acis (HEPES)
5 (pH 7.6), and were manually defolliculated. Following an
extensive washing with this solution, oocytes were transferred to
Barth’s solution containing (in millimolar units) NaCl 88, KCl 1,
MgSO4 0.33, CaCl2 0.41, MgSO4 0.82, Ca(NO3)2 0.33, NaHCO3
2.4, and HEPES 10 (pH 7.2) supplemented with streptomycin
’ ASSOCIATED CONTENT
S
Supporting Information. (1) Full experimental proce-
b
dures for the synthesis of pinnatoxins A and G; (2) text, two
figures, and one table with details of computational molecular
modeling; (3) 1H and 13C NMR and HPLC spectra; and (4) one
table and one figure showing binding assay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
sulfate and 0.01 mg mLꢀ1 penicillin G. Oocytes were maintained
3
at 19 ꢀC for up to 4 days in this solution. Stage VꢀVI oocytes were
selected and microinjected with 50 nL of human R7 cDNA
’ AUTHOR INFORMATION
(1 μg μLꢀ1) or R4β2, as reported previously, by use of a
3
Corresponding Author
zakarian@chem.ucsb.edu
Nanoliter 2000 Micro 4 Controller (World Precision Instruments,
Inc., U.K.) mounted on a microscope. Recordings were performed
3ꢀ4 days after injection.
Torpedo Membrane Preparation and nAChR Microtrans-
plantation to Oocytes. Torpedo marmorata specimens kept in
conventional artificial seawater were anesthetized with tricaine
(SigmaꢀAldrich), at a concentration of 0.03% in seawater,
before surgical excision of electric organs. The R12βγδ
nAChR-rich membranes were prepared at 4 ꢀC from freshly
dissected and sliced Torpedo electric organs by procedures
previously described,65 resuspended in 5 mM glycine, and stored
aliquotted at ꢀ80 ꢀC until use. Microtransplantation of Torpedo
nAChR used microinjection into the oocyte cytoplasm of a
membrane suspension (50 nL at 3.5 mg/mL protein) from a
Nanoliter2000 Micro4 controller mounted on a microscope.
Voltage-Clamp Recording on Oocytes. ACh-evoked cur-
rents were recorded with a standard two-microelectrode voltage-
clamp amplifier (OC-725B, Warner Instrument Corp., Hamden,
CT) at a holding potential of ꢀ60 mV. Voltage and current
microelectrodes were pulled from borosilicate glass to reach
’ ACKNOWLEDGMENT
Receptor binding data for over 40 receptors, transporters, and
ion channels were generously provided by the National Institute
of Mental Health’s Psychoactive Drug Screening Program,
Contract HHSN-271-2008-025C (NIMH PDSP). The NIMH
PDSP is directed by Bryan L. Roth. M.D., Ph.D., at the University
of North Carolina at Chapel Hill and Project Officer Jamie
Driscol at NIMH, Bethesda, MD. We thank Dr. Jon Evans (UNC
Chapel Hill) for assistance. This research was supported by the
U.S. National Institutes of Health (NIGMS, GM077379 to A.Z.
with subcontract KK1036 to J.M.) and by the CNRS and the
CEA. We are grateful to P. Villeneuve and G. Esnault (CNRS)
and E. Bindundu (CEA) for technical assistance. C.S. is sup-
ported by Tobacco-Related Disease Research Program Disserta-
tion Award and the UCSB Dean’s Fellowship. Eli Lilly and
Amgen provided additional unrestricted support (A.Z.).
10510
dx.doi.org/10.1021/ja201254c |J. Am. Chem. Soc. 2011, 133, 10499–10511

Journal of the American Chemical Society
ARTICLE
’ REFERENCES
(37) Gu, Z.; Herrmann, A. T.; Stivala, C. E.; Zakarian, A. Synlett
2010, 1717–1722.
(38) Qin, Y.-c.; Stivala, C. E.; Zakarian, A. Angew. Chem., Int. Ed.
2007, 46, 7466–7469.
(39) Manthorpe, J. M.; Gleason, J. L. J. Am. Chem. Soc. 2001,
123, 2091–2092.
(1) Cembella, A.; Krock, B. In Seafood and Freshwater Toxins, 2nd
ed.; Botana, L. M., Ed.; CRC Press: Boca Raton, FL, 2008; pp 561ꢀ580.
(2) Gueret, S. M.; Brimble, M. A. Nat. Prod. Rep. 2010, 27,
1350–1366.
(3) Munday, R. In Seafood and Freshwater Toxins, 2nd ed.; Botana,
L. M., Ed.; CRC Press: Boca Raton, FL, 2008; pp 581ꢀ594.
(4) Aune, T. In Seafood and Freshwater Toxins, 2nd ed.; Botana,
L. M., Ed.; CRC Press: Boca Raton, FL, 2008; pp 3ꢀ20.
(5) Hu, T.; Burton, I. W.; Cembella, A. D.; Curtis, J. M.; Quilliam,
M. A.; Walter, J. A.; Wright, J. L. C. J. Nat. Prod. 2001, 64, 308–312.
(6) Munday, R.; Towers, N. R.; Mackenzie, L.; Beuzenberg, V.;
Holland, P. T.; Miles, C. O. Toxicon 2004, 44, 173–178.
(7) Rhodes, L.; Smith, K.; Selwood, A. I.; McNabb, P.; van Ginkel,
R.; Holland, P. T.; Munday, R. Harmful Algae 2010, 9, 384–389.
(8) Selwood, A. I.; Miles, C. O.; Wilkins, A. L.; van, G. R.; Munday,
R.; Rise, F.; McNabb, P. J. Agric. Food Chem. 2010, 58, 6532–6542.
(9) Zheng, S.; Huang, F.; Chen, S.; Tan, X.; Zuo, J.; Peng, J.; Xie, R.
Zhongguo Haiyang Yaowu 1990, 9, 33–35.
(10) Uemura, D.; Chou, T.; Haino, T.; Nagatsu, A.; Fukuzawa, S.;
Zheng, S.-z.; Chen, H.-s. J. Am. Chem. Soc. 1995, 117, 1155–1156.
(11) Bourne, Y.; Radic, Z.; Araoz, R.; Talley, T. T.; Benoit, E.;
Servent, D.; Taylor, P.; Molgꢀo, J.; Marchot, P. Proc. Natl. Acad. Sci. U.S.A.
2010, 107, 6076–6081.
(12) Hao, J.; Matsuura, F.; Kishi, Y.; Kita, M.; Uemura, D.; Asai, N.;
Iwashita, T. J. Am. Chem. Soc. 2006, 128, 7742–7743.
(13) Ishihara, J.; Horie, M.; Shimada, Y.; Tojo, S.; Murai, A. Synlett
2002, 403–406.
(14) Ishihara, J.; Sugimoto, T.; Murai, A. Synlett 1998, 603–606.
(15) Ishihara, J.; Tojo, S.; Kamikawa, A.; Murai, A. Chem. Commun.
2001, 1392–1393.
(16) Ishiwata, A.; Sakamoto, S.; Noda, T.; Hirama, M. Synlett
1999, 692–694.
(17) Lu, C.-D.; Zakarian, A. Org. Lett. 2007, 9, 3161–3163.
(18) Matsuura, F.; Hao, J.; Reents, R.; Kishi, Y. Org. Lett. 2006,
8, 3327–3330.
(19) Matsuura, F.; Peters, R.; Anada, M.; Harried, S. S.; Hao, J.;
Kishi, Y. J. Am. Chem. Soc. 2006, 128, 7463–7465.
(20) McCauley, J. A.; Nagasawa, K.; Lander, P. A.; Mischke, S. G.;
Semones, M. A.; Kishi, Y. J. Am. Chem. Soc. 1998, 120, 7647–7648.
(21) Nakamura, S.; Inagaki, J.; Kudo, M.; Sugimoto, T.; Obara, K.;
Nakajima, M.; Hashimoto, S. Tetrahedron 2002, 58, 10353–10374.
(22) Nakamura, S.; Inagaki, J.; Sugimoto, T.; Kudo, M.; Nakajima,
M.; Hashimoto, S. Org. Lett. 2001, 3, 4075–4078.
(40) Tiong, E. A.; Gleason, J. L. Org. Lett. 2009, 11, 1725–1728.
(41) Kummer, D. A.; Chain, W. J.; Morales, M. R.; Quiroga, O.;
Myers, A. G. J. Am. Chem. Soc. 2008, 130, 13231–13233.
(42) Ireland, R. E.; Mueller, R. H.; Willard, A. K. J. Am. Chem. Soc.
1976, 98, 2868–2877.
(43) Ramirez, A.; Sun, X.; Collum, D. B. J. Am. Chem. Soc. 2006,
128, 10326–10336.
(44) Sun, X.; Collum, D. B. J. Am. Chem. Soc. 2000, 122, 2452–2458.
(45) Sun, X.; Collum, D. B. J. Am. Chem. Soc. 2000, 122, 2459–2463.
(46) Sun, X.; Kenkre, S. L.; Remenar, J. F.; Gilchrist, J. H.; Collum,
D. B. J. Am. Chem. Soc. 1997, 119, 4765–4766.
(47) Schwartz, C.; Raible, J.; Mott, K.; Dussault, P. H. Org. Lett.
2006, 8, 3199–3201.
(48) Ilardi, E. A.; Stivala, C. E.; Zakarian, A. Org. Lett. 2008,
10, 1727–1730.
(49) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457–2483.
(50) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev.
1994, 94, 2483–2547.
(51) Peterson, D. J. J. Org. Chem. 1968, 33, 780–784.
(52) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986,
108, 7408–7410.
(53) Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am.
Chem. Soc. 2000, 122, 8168–8179.
(54) Yamamoto, Y.; Chounan, Y.; Nishii, S.; Ibuka, T.; Kitahara, H.
J. Am. Chem. Soc. 1992, 114, 7652–7660.
(55) Kuduk, S. D.; DiPardo, R. M.; Chang, R. K.; Ng, C.; Bock, M. G.
Tetrahedron Lett. 2004, 45, 6641.
(56) Kharrat, R.; Servent, D.; Girard, E.; Ouanounou, G.; Amar, M.;
Marrouchi, R.; Benoit, E.; Molgꢀo, J. J. Neurochem. 2008, 107, 952–963.
(57) Eusebi, F.; Palma, E.; Amici, M.; Miledi, R. Progress in Neuro-
biology 2009, 88, 32–40.
(58) Receptor binding data for over 40 receptors, transporters, and
ion channels were generously provided by the National Institute of
Mental Health’s Psychoactive Drug Screening Program (see Acknowl-
edgment). See Supporting Information 4 for additional details.
(59) Wandscheer, C. B.; Vilarino, N.; Espina, B.; Louzao, M. C.;
Botana, L. M. Chem. Res. Toxicol. 2010, 23, 1753–1761.
(60) Maskell, P. D.; Speder, P.; Newberry, N. R.; Bermudez, I. Br. J.
Pharmacol. 2003, 140, 1313–1319.
(61) Servent, D.; Winckler-Dietrich, V.; Hu, H.-Y.; Kessler, P.; Drevet,
P.; Bertrand, D.; Mꢀenez, A. J. Biol. Chem. 1997, 272, 24279–24286.
(62) Eisele, J.-L.; Bertrand, S.; Galzi, J.-L.; Devillers-Thiery, A.;
Changeux, J.-P.; Bertrand, D. Nature 1993, 366, 479–483.
(63) Weber, M.; Changeux, J.-P. Mol. Pharmacol. 1974, 10, 1–14.
(64) Cheng, Y..; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099–3108.
(65) Krieger, F.; Mourot, A.; Araoz, R.; Kotzyba-Hibert, F.; Molgꢀo,
J.; Bamberg, E.; Goeldner, M. ChemBioChem 2008, 9, 1146–1153.
(66) Sands, S. B.; Costa, A. C.; Patrick, J. W. Biophys. J. 1993,
65, 2614–2621.
(23) Nakamura, S.; Inagaki, J.; Sugimoto, T.; Ura, Y.; Hashimoto, S.
Tetrahedron 2002, 58, 10375–10386.
(24) Nakamura, S.; Kikuchi, F.; Hashimoto, S. Tetrahedron: Asym-
metry 2008, 19, 1059–1067.
(25) Nakamura, S.; Kikuchi, F.; Hashimoto, S. Angew. Chem., Int. Ed.
2008, 47, 7091–7094.
(26) Nitta, A.;Ishiwata, A.;Noda, T.;Hirama, M. Synlett 1999, 695–696.
(27) Noda, T.; Ishiwata, A.; Uemura, S.; Sakamoto, S.; Hirama, M.
Synlett 1998, 298–300.
(28) Pelc, M. J.; Zakarian, A. Org. Lett. 2005, 7, 1629–1631.
(29) Pelc, M. J.; Zakarian, A. Tetrahedron Lett. 2006, 47, 7519–7523.
(30) Sakamoto, S.; Sakazaki, H.; Hagiwara, K.; Kamada, K.; Ishii, K.;
Noda, T.; Inoue, M.; Hirama, M. Angew. Chem., Int. Ed. 2004,
43, 6505–6510.
(31) Stivala, C. E.; Zakarian, A. Tetrahedron Lett. 2007, 48, 6845–6848.
(32) Stivala, C. E.; Zakarian, A. J. Am. Chem. Soc. 2008, 130, 3774–3776.
(33) Sugimoto, T.; Ishihara, J.; Murai, A. Tetrahedron Lett. 1997, 38,
7379–7382.
(34) Sugimoto, T.; Ishihara, J.; Murai, A. Synlett 1999, 541–544.
(35) Suthers, B. D.; Jacobs, M. F.; Kitching, W. Tetrahedron Lett.
1998, 39, 2621–2624.
(36) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.
Bull. Chem. Soc. Jpn. 1979, 52, 1989–1993.
10511
dx.doi.org/10.1021/ja201254c |J. Am. Chem. Soc. 2011, 133, 10499–10511