Synthesis of Anhydroryanodol.

A stereoselective entry to ryanoids is described that culminates in the synthesis of anhydroryanodol and thus the formal total synthesis of ryanodol. The pathway described features an annulation reaction conceived to address the uniquely complex and highly oxygenated polycyclic skeleton common to members of this natural product class. It is demonstrated that metallacycle-mediated intramolecular coupling of an alkyne and a 1,3-diketone can proceed with a highly functionalized enyne and with outstanding levels of stereoselection. Furthermore, the first application of this technology in natural product synthesis is demonstrated here. More broadly, the advances described demonstrate the value that programs in natural product total synthesis have in advancing organic chemistry, here through the design and realization of an annulation reaction that accomplishes what previously established reactions do not.

Studies Targeting Ryanodol Result in an Annulation Reaction for the Synthesis of a Variety of Fused Carbocycles.

An annulation reaction is described to access a range of polycyclic and highly oxygenated carbocycles. First developed in an approach to the synthesis of ryanodol, metallacycle-mediated annulative diketone-alkyne coupling defines a framework for realization of new retrosynthetic relationships for complex molecule synthesis. In addition to demonstrating this reaction in the context of forging distinct carbocyclic systems, including those featuring a seven-membered ring, the choice of quenching reagent leads to unique reaction outcomes.

Ryanodine Receptors for Drugs and Insecticides: An Overview.

Ryanodine receptors (RyRs) are calcium channels located on the endo(sarco)plasmic reticulum of muscle cells and neurons. They regulate the release of stored intracellular calcium and play a critical role in muscle contraction. The N-terminal part of these receptors accounts for roughly 80% and contains the binding sites for diverse RyRs modulators. The C-terminal domain contains the transmembrane region. This review summarizes the current knowledge about the molecular biology of insect RyRs, chemicals targeting mammal or insect RyRs, and the reasons for mammal RyR-related diseases and diamides resistances. It may lay the foundation for effective management of mammal RyR-related diseases and diamides resistances.

Toward the Total Synthesis of Ryanodol via Oxidative Alkyne-1,3-Diketone Annulation: Construction of a Ryanoid Tetracycle.

A synthetic strategy conceived with the intent of establishing a novel approach to the de novo construction of ryanoids is described that is based on a recently developed metallacycle-mediated intramolecular oxidative alkyne-1,3-diketone coupling reaction. In short, a one-pot annulation/oxidation sequence is shown to be capable of establishing a densely oxygenated polycyclic intermediate that could be converted to a composition of matter that contains the ABCD tetracyclic ring system present in the ryanoid family of natural products.

A 15-step synthesis of (+)-ryanodol.

(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and cardiac muscle excitation-contraction coupling and synaptic transmission. Chemical derivatization of these diterpenoids has demonstrated that certain peripheral structural modifications can alter binding affinity and selectivity among ryanodine receptor isoforms. Here, we report a short chemical synthesis of (+)-ryanodol that proceeds in only 15 steps from the commercially available terpene (S)-pulegone. The efficiency of the synthesis derives from the use of a Pauson-Khand reaction to rapidly build the carbon framework and a SeO2-mediated oxidation to install three oxygen atoms in a single step. This work highlights how strategic C-O bond constructions can streamline the synthesis of polyhydroxylated terpenes by minimizing protecting group and redox adjustments.

Chemical Conversion of Ryanodol to Ryanodine.

Ryanodine (1) is a plant-derived natural product with powerful pharmacological and insecticidal action, and is a potent modulator of intracellular calcium release channels. Compound 1 possesses a 1H-pyrrole-2-carboxylate ester at the C3-position of heptahydroxylated terpenoid ryanodol (2). Whereas 2 was readily obtained from 1 by basic hydrolysis, 1 has never been synthesized from 2, due to the extreme difficulty in selectively introducing the bulky pyrrole moiety at the severely hindered C3-hydroxyl group of heptaol 2. Here we report chemical conversion of 2 to 1 for the first time. The derivatization was realized through the use of a new protective group strategy and the application of on-site construction of the pyrrole-2-carboxylate ester from the glycine ester and 1,3-bis(dimethylamino)allylium tetrafluoroborate.

Quantification of the effects of a ryanodine receptor channel mutation on interaction with a ryanoid.

Understanding the nature of the interaction of the plant alkaloid ryanodine with its receptor channel (RyR) is important to aid interpretation of physiological studies and provide structure-function information about RyR. We present here the first quantitative description of the relative single-channel kinetic effects of a single-point mutation in RyR2. We exploit the well-characterized ryanoid 8beta-amino-9alpha-hydroxyryanodine that displays reversible kinetics with RyR2. We explicitly show that the effect of the Q4863A mutation is to increase the apparent dissociation constant by increasing the apparent dissociation rate of the ryanoid. The voltage-dependence of the interaction displays no change. We infer that Q4863 is not involved with the voltage-drop but is able to influence ryanoid-bound structural changes. We discuss structural mechanisms by which this mutation could affect ryanoid interaction.

The interaction of an impermeant cation with the sheep cardiac RyR channel alters ryanoid association.

In previous studies, we have demonstrated that the interaction of ryanoids with the sarcoplasmic reticulum Ca(2+)-release channel [ryanodine receptor (RyR)] incorporated into planar lipid bilayers reduced the effectiveness of tetraethylammonium (TEA(+)) as a blocker of K(+) translocation (J Gen Physiol 117: 385-393, 2001). In the current study, we investigated both the effect of TEA(+) on [(3)H]ryanodine binding and the actions of this impermeant cation on the interaction of the reversible ryanoid 21-amino-9alpha-hydroxyryanodine with individual, voltage-clamped RyR channels. A dose-dependent inhibition of [(3)H]ryanodine binding was observed in the presence of TEA(+), suggesting that the cation and alkaloid compete for access to a common site of interaction. Single channel studies gave further insights into the mechanism of the competition between the two classes of ligands. TEA(+) decreases the association rate of 21-amino-9alpha-hydroxyryanodine with its receptor, whereas the dissociation rate of the ryanoid from the channel was unaffected. Our results demonstrate that TEA(+) inhibits both K(+) translocation through RyR, and ryanoid interaction at the high affinity ryanodine site on the channel. These actions involve binding of TEA(+) to different, but weakly interacting, sites in the RyR channel.

Localisation, function and composition of primary Ca(2+) spark discharge region in isolated smooth muscle cells from guinea-pig mesenteric arteries.

Smooth muscle cells (SMCs) contain numerous calcium release domains, grouped into regions discharging as a single unit. Laser scanning confocal microscopy, voltage clamp and immunocytochemistry of single SMCs from small mesenteric arteries of guinea-pig were used to study the localisation, function and macromolecular composition of such calcium discharge regions (CDRs). Use of the Ca(2+)-sensitive fluorescent dye fluo-3 or fluo-4 with BODIPY TR-X ryanodine (BTR), a fluorescent derivative of ryanodine, showed spontaneous Ca(2+) sparks originating from regions stained by BTR, located immediately under the plasma membrane, in the arch formed by the sarcoplasmic reticulum surrounding the nucleus. Membrane depolarisation or application of noradrenaline or alpha,beta-methylene ATP, a P2X purinoceptor agonist, elicited Ca(2+) sparks from the same, spontaneous Ca(2+) spark-discharging region. The most active (primary) CDR accounted for nearly 60% of spontaneous transient outward currents at -40 mV and these were of significantly higher amplitude than the ones discharged by secondary CDRs. Immunocytochemical staining for type 1 IP(3) receptors, BK(Ca) channels, P2X(1) purinoceptors or alpha(1) adrenoceptors revealed their juxtaposition with BTR staining at the location typical of the primary CDR. These data suggest the existence of a primary calcium discharge region in SMCs; its position can be predicted from the cell's structure, it acts as a key region for the regulation of membrane potential via Ca(2+) sparks and is a potential link between the external, neurohumoral and the cell's internal, calcium signalling system.

The Gln4863Ala mutation within a putative, pore-lining trans-membrane helix of the cardiac ryanodine receptor channel alters both the kinetics of ryanoid interaction and the subsequent fractional conductance.

The specific, high-affinity interaction of the plant toxin ryanodine with its molecular target the ryanodine receptor channel (RyR) has been instrumental in RyR research. Alanine scanning of putative pore regions of mouse RyR2 has highlighted the amino acid Gln4863, predicted to lie within trans-membrane helix TM10, as an important determinant of ryanodine binding. We have investigated the effects of several ryanodine derivatives, guanidinopropionylryanodine, 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine, 8beta-amino-9alpha-hydroxyryanodine, and 21-amino-9alpha-hydroxyryanodine, with the mouse Q4863A RyR2 mutant at the single-channel level. Our results demonstrate that the rate of dissociation of all ryanoids investigated is increased by the mutation. The modification of channel function after ryanoid binding is qualitatively similar for wild-type and mutant, but in several cases, single-channel conductances were increased with Q4863A. These novel findings have been interpreted within the framework of existing comparative molecular field analysis studies on ryanoids. We suggest that replacement of a glutamine by an alanine residue at position 4863 causes RyR2 to simultaneously alter interactions with both ends of the ryanoid molecule.

Voltage-sensitive equilibrium between two states within a ryanoid-modified conductance state of the ryanodine receptor channel.

We have investigated the influence of transmembrane holding potential on the kinetics of interaction of a cationic ryanoid, 8beta-amino-9alpha-hydroxyryanodine, with individual ryanodine receptor (RyR) channels and on the functional consequences of this interaction. In agreement with previous studies involving cationic, neutral, and anionic ryanoids, both rates of association and dissociation of the ligand are sensitive to transmembrane potential. A voltage-sensitive equilibrium between high- and low-affinity forms of the receptor underlies alterations in rates of association and dissociation of the ryanoid. The interaction of 8beta-amino-9alpha-hydroxyryanodine with RyR influences the rate of cation translocation through the channel. With this ryanoid bound, the channel fluctuates between two clearly resolved subconductance states (alpha and beta). We interpret this observation as indicating that with 8beta-amino-9alpha-hydroxyryanodine bound, the pore of the RyR channel exists in two essentially isoenergetic conformations with differing ion-handling properties. The equilibrium between the alpha- and beta-states of the RyR-8beta-amino-9alpha-hydroxyryanodine complex is sensitive to transmembrane potential. However, the mechanisms determining this equilibrium differ from those responsible for the voltage-sensitive equilibrium between high- and low-affinity forms of the receptor.

The interaction of ryanoids with individual ryanodine receptor channels.

Ryanodine binds with high affinity and specificity to a class of Ca(2+)-release channels known as ryanodine receptors (RyR). The interaction with RyR results in a dramatic alteration in function with open probability (Po) increasing markedly and rates of ion translocation modified. We have investigated the features of ryanodine that govern the interaction of the ligand with RyR and the mechanisms underlying the subsequent alterations in function by monitoring the effects of congeners and derivatives of ryanodine (ryanoids) on individual RyR2 channels. While the interaction of all tested ryanoids results in an increased Po, the amplitude of the modified conductance state depends upon the structure of the ryanoid. We propose that different rates of cation translocation observed in the various RyR-ryanoid complexes represent different conformations of the channel stabilized by specific conformers of the ligand. On the time scale of a single channel experiment ryanodine binds irreversibly to the channel. However, alterations in structure yield some ryanoids with dissociation rate constants orders of magnitude greater than ryanodine. The probability of occurrence of the RyR-ryanoid complex is sensitive to trans-membrane voltage, with the vast majority of the influence of potential arising from a voltage-driven alteration in the affinity of the ryanoid-binding site.

An anionic ryanoid, 10-O-succinoylryanodol, provides insights into the mechanisms governing the interaction of ryanoids and the subsequent altered function of ryanodine-receptor channels.

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.

Diketopyridylryanodine has three concentration-dependent effects on the cardiac calcium-release channel/ryanodine receptor.

By interacting with more than one site, ryanoids induce multiple effects on calcium-release channels. To date, the kinetics of interaction of only one of these sites has been characterized. Using C(4),C(12)-diketopyridylryanodine in both [(3)H]ryanodine binding and single channel experiments we characterized another site on the cardiac ryanodine receptor (RyR2) with which ryanoids interact. Competitive binding of this ryanoid to RyR2 implied a minimal two-site binding model. At the single channel level, C(4),C(12)-diketopyridylryanodine induced three distinct effects. At nanomolar concentrations, it increased channel open probability severalfold without inducing a subconductance. This effect was independent of membrane holding potential. As for other ryanoids, low micromolar concentrations of C(4),C(12)-diketopyridylryanodine readily induced a subconductance state. The major subconductance had a current amplitude of 52% of fully open, it was reversible, and its time to induction and duration were voltage- and concentration-dependent, affording Hill slopes of >2. At higher micromolar concentrations C(4),C(12)-diketopyridylryanodine induced long lasting, yet reversible shut states. Using a pharmacological strategy we have discerned an additional ryanoid-binding site on RyR2 that triggers an increase in channel activity. This site likely resides outside the strict confines of the transmembrane conducting pathway.

Excess noise in modified conductance states following the interaction of ryanoids with cardiac ryanodine receptor channels.

The interaction of ryanodine with the ryanodine receptor (RyR) produces profound changes in channel function. Open probability increases dramatically and conductance is reduced. In this report we describe differences in the properties of reduced conductance states produced by the interaction of ryanodine derivatives with RyR channels. Some reduced conductance states are considerably noisier than the normal open state of the RyR channel. Inspection and analysis of these events reveals that the excess noise arises from transitions between two conductance states. Following the interaction of certain ryanodine derivatives, RyR channels undergo transitions between two conformations with slightly different ion-handling properties.

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site.

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca(2+) signaling.

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Pseudoreceptor model for ryanodine derivatives at calcium release channels.

This paper describes the generation of a pseudoreceptor model for ryanodine receptor (RyR) modulating ryanoids in rabbit skeletal muscle. For this purpose, the molecular modelling software PrGen was applied to correlate experimentally determined and calculated free energies of binding for a set of 15 ryanodine derivatives. The final model indicates a narrow cleft with hydrogen bond donor and acceptor capacities (represented by an Asn) as most crucial for binding the pyrrole carboxylate substituent at C3 of ryanodine. In addition, hydrophobic residues flank the aromatic pyrrole ring (Tyr, Phe, and Ile). Two of those residues (Tyr and Ile) interact with the 2-isopropyl moiety, which seems to contribute to binding. Opposite to the pyrrole locus, a second hydrophobic region (represented by a Leu) restricts ryanodine derivatives in their longitudinal axis and leads to the discrimination of equatorial and axial positioned methyl groups and of polar substituents at C9. Finally, a charged glutamate residue generates strong hydrogen bonding and electrostatic interactions with the hydroxyl groups at C10 and C15. For this binding-site model--composed of six amino acid residues--a correlation for the training set ligands of R = 0.99 (Q2 = 0.975) and a root mean square (rms) deviation of 0.568 kcal/mol for the prediction of the binding energies of four test set ligands was obtained. Based on this pseudoreceptor model the putative topology of the real binding site of ryanoids will be discussed.

The interaction of a neutral ryanoid with the ryanodine receptor channel provides insights into the mechanisms by which ryanoid binding is modulated by voltage.

In an earlier investigation, we demonstrated that the likelihood of interaction of a positively charged ryanoid, 21-amino-9alpha-hydroxyryanodine, with the sarcoplasmic reticulum Ca(2+)-release channel (ryanodine receptor, RyR) is dependent on holding potential (Tanna, B., W. Welch, L. Ruest, J.L. Sutko, and A. J. Williams. 1998. J. Gen. Physiol. 112:55-69) and suggested that voltage dependence could result from either the translocation of the charged ligand to a site within the voltage drop across the channel or a voltage-driven alteration in receptor affinity. We now report experiments that allow us to assess the validity of these alternate mechanisms. Ryanodol is a neutral ryanoid that binds to RyR and induces modification of channel function. By determining the influence of transmembrane potential on the probability of channel modification by ryanodol and the rate constants of ryanodol association and dissociation, we demonstrate that the influence of voltage is qualitatively the same for both the neutral and positively charged ryanoids. These experiments establish that most, if not all, of the modification of ryanoid interaction with RyR by transmembrane holding potential results from a voltage-driven alteration in receptor affinity.

Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels.

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9alpha-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9alpha-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9alpha-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.