Leslie S. Jimenez

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1–1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro andin vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1–2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590–609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1–2s in both Western blots and immunoprecipitation assays and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590–609.

Probing a putative dantrolene-binding site on the cardiac ryanodine receptor

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Isolation and Structural Determination of Steroidal Glycosides from the Bulbs of Easter Lily (Lilium longiflorum Thunb.)

The bulbs of the Easter lily ( Lilium longiflorum Thunb.) are used as a food and medicine in several Asian cultures, and they are cultivated as an ornamental plant throughout the world. A new steroidal glycoalkaloid and two new furostanol saponins, along with two known steroidal glycosides, were isolated from the bulbs of L. longiflorum. The new steroidal glycoalkaloid was identified as (22R,25R)-spirosol-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-[6-O-acetyl-beta-d-glucopyranosyl-(1-->4)]-beta-d-glucopyranoside. The new furostanol saponins were identified as (25R)-26-O-(beta-d-glucopyranosyl)-furost-5-en-3beta,22alpha,26-triol 3-O-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranosyl-(1-->3)-beta-d-glucopyranoside and (25R)-26-O-(beta-d-glucopyranosyl)-furost-5-en-3beta,22alpha,26-triol 3-O-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-xylopyranosyl-(1-->3)-beta-d-glucopyranoside. The previously known steroidal glycosides, (22R,25R)-spirosol-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-beta-d-glucopyranosyl-(1-->4)-beta-d-glucopyranoside and (25R)-26-O-(beta-d-glucopyranosyl)-furost-5-en-3beta,22alpha,26-triol 3-O-alpha-l-rhamnopyranosyl-(1-->2)-beta-d-glucopyranosyl-(1-->4)-beta-d-glucopyranoside were identified in L. longiflorum for the first time. These new compounds from L. longiflorum and the isolation methodologies employed can be used for studies on the biological role of steroidal glycosides in plant development and plant-pathogen interactions, as well as for studies in food and human health, for which little is known.

A total synthesis of (±)-Mitomycin K. Oxidation of the mitosene C9-9a double bond by (hexamethylphosphoramido)oxodiperoxomolybdenum (VI) (MoO5 · HMPA)

Abstract A synthesis of (±)-Mitomycin K has been achieved in thirteen steps from commerically available 2,5-dimethylanisole in a 1.36% overall yield. One of the key steps is the oxidation of the C9-9a double bond of 1 by (hexamethylphosphoramido)oxodiperoxomolybdenum (VI) to give 5a and b . This allows for the facile introduction of the C-9a methoxy group into the mitomycin skeleton.

A camphor-derived vinylsulfonium salt as a reagent for a cycloannulation

Abstract A chiral, non-racemic vinylsulfonium salt 6 was prepared from camphorquinone in seven steps and reacted with indole-2-carboxaldehyde to give a tricyclic azido alcohol 2 in a 35% yield and 43% e.e. and a 40% recovery of the chiral sulfide.

REACTIVITY AND REARRANGEMENTS OF DIALKYL- AND DIARYLVINYLSULFONIUM SALTS WITH INDOLE-2- AND PYRROLE-2-CARBOXALDEHYDES

Abstract Various dialkyl- and diarylvinylsulfonium salts react with indole-2-carboxaldehydes in the presence of sodium hydride and sodium azide to form tricyclic azido alcohols analogous to 2 . With pyrrole-2-carboxaldehyde, [2,3] sigmatropic and other rearrangements occur except in the case of diphenylvinylsulfonium trifluoromethanesulfonate where the annulation reaction does take place to give a low yield of 29 .

Synthesis of 1H-2,3-dihydropyrrolizine derivatives as precursors of bifunctional alkylating agents

Two 1H-2,3-dihydropyrrolizine derivatives bearing a nitro group at the 6 position have been synthesized and an improved method for nitrating pyrroles using potassium nitrate in trifluoroacetic acid was developed. An efficient, two-step synthesis of the butterfly pheromone, Danaidone, was also developed with an overall 33% yield.

Synthesis of the FR900482 Tetracyclic Ring System by Oxidation of A 2,3-Dihydro-1H-Pyrrolo[1, 2-a]Indole Derivative

Abstract The tetracyclic FR900482 ring skeleton has been synthesized from the 2,3-dihydro-1H-pyrrolo[1,2-a]indole derivative 5. Treatment of 5 with osmium tetraoxide, hydrogen sulfide, magnesium monoperoxyphthalic acid (MMPP), and triphenylphosphine resulted in the formation of 12.

Annulation reactions of chiral vinylsulfonium salts with 2-indolecarboxaldehyde

Abstract The preparation of the chiral uinylsulfonium salts 4, 5, and 6 is described. Their use in an annulation with 2-indolecarboxaldehyde and sodium azide proceeds to form the azido alcohol 2 with low asymmetic induction. These results represent an initial investigation of the asymmetric induction obtained from enantiomerically pure vinylsulfonium salts.