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Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11, 12, 15(S)-THETA. Of the eight isomers, only 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid comigrated with the biological 11, 12, 15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer ([10.sup.-7] - [10.sup.-4] M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 [+ or -] 5%). These relaxations were blocked by apamin ([10.sup.-7] M), an inhibitor of small-conductance [Ca.sup.2+]-activated [K.sup.+] channels. In comparison, 11 (S), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA ([10.sup.-4] M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that [10.sup.-4] M 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current in isolated aortic smooth muscle cells by 119 [+ or -] 36% at +60 mV, whereas [10.sup.-4] M 11(R), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current by only 20 [+ or -] 2%. The 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA-stimulated increase in [K.sup.+] current was blocked by pretreatment with apamin. These studies suggest that 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating [K.sup.+] channels. The specific structural and stereochemical requirements for [K.sup.+] channel activation suggest that a specific binding site or receptor of 11, 12, 15-THETA is involved in these actions. endothelium-derived hyperpolarizing factor; smooth muscle cells; potassium channels; arachidonic acid

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid (AA) metabolite synthesized by cytochrome P-450 [omega]-oxidases, is reported to produce vasoconstriction in the cerebral circulation. However, we find that like 14,15-epoxyeicosatrienoic acid (14,15-EET), 20-HETE produces dilation of mouse basilar artery preconstricted with U-46619 in vitro. Indomethacin inhibited the vasodilation produced by 20-HETE but not by 14,15-EET, suggesting a cyclooxygenase (COX)-dependent mechanism. Metabolic studies indicated several mechanisms that may play a role in this process. Mouse brain endothelial cells (MBEC) converted 20-HETE to 20-OH-[PGE.sub.2], which was as potent as [PGE.sub.2] in dilating the basilar artery. 20-HETE also stimulated AA release and PGE2 and 6-keto-[PGF.sub.1[alpha]] production in MBEC. Furthermore, the basilar artery converted 20-HETE to 20-COOH-AA, which also produced COX-dependent dilation of the basilar artery. 20-COOH-AA increased AA release and PGEE and 6-keto-[PGF.sub.1[alpha]], production by the MBEC, but to a lesser extent than 20-HETE. Whereas the conversion of 20-HETE to 20-OH-[PGE.sub.2] and production of endogenous prostaglandins probably are primarily responsible for vasodilation, the production of 20-COOH-AA also may contribute to this process. 20-hydroxy-prostaglandin [E.sub.2]; 20-carboxy-arachidonic acid; prostaglandins; cerebral vascular tone

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine [A.sub.2A] receptors ([A.sub.2A]R) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of [G.sub.8[alpha], adenylyl cyclase, PKA, and [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channel activity, to the vasoactive responses to [A.sub.2A]R activation by a selective [A.sub.2A] agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 [micro]m) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 [micro]M), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 [micro]M) and found that both were inhibited by ~70% (P < 0.05), whereas the response to SNP (10 [micro]M) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 [micro]M), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 [micro]M), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 [micro]M) with PGMV resulted in an increase in cAMP levels (P < 0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of [K.sub.Ca] channel activity. Thus in rat PGMV activation of [A.sub.2A]R is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in [G.sub.s[alpha] protein activation. cytochrome P-450; rat arcuate artery

Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]. We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPAR[alpha] activator in a COS-7 cell expression system. Incubation with 10 [micro]M 14,15-DHET produced a 12-fold increase in PPAR[micro]-mediated luciferase activity, an increase similar to that produced by the PPAR[alpha] agonist Wy-14643 (20 [micro]M). Although 10 [micro]M 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPAR[alpha]. However, PPAR[alpha] was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[[sup.3]H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[[sup.3]H]EET. Binding studies indicated that 14,15-[[sup.3]H]DHET binds to the ligand binding domain of PPAR[alpha] with a [K.sub.d] of 1.4 [micro]M. Furthermore, 14,15-DHET increased the expression of camitine palmitoyltransferase lA, a PPAR[alpha]-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPAR[alpha]. cytochrome P-450; soluble epoxide hydrolase; 14,15-epoxyeicosatriconic acid analogs

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (E[D.sub.50] = [10.sup.-6] M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a [DELTA]8 double bond had full activity and potency. However, the analogs with only a [DELTA]5 or [DELTA]11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a [DELTA]8 double bond, and a 14(S),15(R)-(cis)-epoxy group. endothelium-derived hyperpolarizing factor; cytochrome P-450; arachidonic acid; epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs) are synthesized in the endothelial cells of vascular tissues. They are released from the endothelial cells and produce relaxation of the smooth muscle cells by hyperpolarization. The present findings demonstrate that EETs also regulate aromatase activity in vascular smooth muscle cells. Exposure of cultured rat aortic smooth muscle cells to either 1 [micro]M 14,15-EET or 1 [micro]M 11,12-EET inhibits dibutyryl cAMP-induced aromatase activity by 80-100%. 11,12-Dihydroxyeicosatrienoic acid, the hydration product of 11,12-EET, has no effect on dibutyryl cAMP-induced vascular smooth muscle aromatase activity. In contrast to 14,15-EET, the N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SA) was neither metabolized nor incorporated into cell lipids, but it retained the ability to inhibit cAMP-induced aromatase activity. Furthermore, the 14,15-EET-SA inhibition of cAMP-induced aromatase activity persisted when the sulfanilamide derivative of 14,15-EET was covalently tethered to silica beads (average diameter, 0.5 [micro]m), which restricted 14,15-EET-SA from entering the cell. These data are consistent with the presence of a receptor for EETs in the plasma membrane and support the hypothesis that the inhibition of aromatase by EETs is initiated by the interaction of EET with the putative plasma membrane receptor. epoxyeicosatrienoic acid; receptor; adenosine 3',5'-cylic monophosphate; estrogen

We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PI[P.sub.2]) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PI[P.sub.2] antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PI[P.sub.2] and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at both positions 4 and 5 of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu. phosphatidylinositol bisphosphate; pleckstrin homology domain; inwardly rectifying potassium channels; giant patch-clamp recording; kidney

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular [K.sub.+] to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward [K.sub.+] current and increased [K.sub.Ca] channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[[sup.14]C]EET methyl ester (Me) was converted to 14,15-[[sup.14]C]DHET-Me, 14,15-[[sup.14]C]DHET, and 14,15-[[sup.14]C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of [K.sub.Ca] channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity. endothelium-derived hyperpolarizing factor; epoxyeicosatrienoic acids; potassium channels; epoxide hydrolase

The effects of hormonal stimulation on the release of P-450 20-hydroxyeicosatetraenoic acid (HETE) in isolated perfused rabbit kidneys were analyzed by gas chromatography-mass spectrometry method. High levels of P-450 HETEs were detected from the isolated perfused kidneys after angiotensin-II-induced stimulation. However, the basal levels of P-450 HETE were not altered by equiprocessor concentrations of arginine vasopressin. Furthermore, bradykinin did not alter the release of P450 HETE from the isolated kidneys.

Enantiopure 2,3:5,6-di-O-cyclohexylidene-D-myo-inositol is used to prepare 1-O-({phosphonooxy}hydroxyphosphinyl)-2,3,4,5,6-pentakis-O-phosphono-D-myo- inositol and its antipode. In addition to the chiral precursor obtained from inositol, an efficient protocol is used for introducing protected pyrophosphates. The pyrophosphate unit is introduced in the presence of flanking phosphate triesters by the scission of a mixed phosphate methyl ester.

A synthesis scheme for the preparation of a fully functionalized taxol ring moiety is described. The method involves the biomimetic cation-olefin cyclization of chiral geraniol epoxide using SnCl4 in toluene. Fractional crytallization resulted in the endo form which was converted to an aldehyde by stereospecific epoxidation, secondary alcohol protection, and PCC oxidation. The enal compound was formed by NaOMe-mediated ring opening, and addition of lithium dithiane yielded the taxol ring A moiety as the sole product.

The synthesis of L-alpha-phosphatidyl-D-myo-inositol 3,4,5-trisphosphate (3,4,5-PIP3) from the differentially functionalized D-myo-inositol intermediate is described. The intermediate was derived from the unique cyclitol precursor dehydroshikimic acid. Key steps in the synthesis were the stereoselective hydride reduction of the shikimate ketone, exclusive osmylation from the alpha-face, controlled enolization and dioxane epoxidation with in situ rearrangement affording ketones. Two ether analogues of 3,4,5-PIP3 were also synthesized and characterized.

Triply convergent strategy is used for the sequential union of propargylic arsonium salts with pyranosides and furanose to enable the total asymmetric synthesis of 3-hydroxyleukotriene B4(3-OH-LTB4). Palladium-mediated coupling is the vital step in the process. 3(S)-isomer is the major component in the enzyme produced product. Chromatography and mass spectra are employed for stereochemical assignment.

CO catalysis by Pd and Cu(I) salts provides alpha-hetero-substituted ketones in moderate to good yields from Stille-type cross-coupling between alpha-alkoxy(amino)stannanes and acyl chlorides. The reaction applies to various tin compounds, particularly those having alpha-electron-withdrawing groups such as benzoyloxy or acetyloxy. The best yields result from aromatic acid chlorides. Retention of configuration accompanies coupling of chiral alpha-alkoxystannanes which are obtained by BINAL-H asymmetric reduction of acylstannanes.

A large number of dehydrative alkylations or annulations are influenced by bis-sulfones. Various processes of cyclization which form small- and medium-sized rings is seen as a facile process. Smooth progress of intermolecular alkylative dehydrations is observed. Functionalized carbocycles is availed in high quantities when extra reagents occur during the process of adding dehydrations to diols. Several normal functionalities reveal the compatible nature of the formation of carbon-carbon bonds in this manner, due to mild interaction conditions. Substrate appropriateness is attributed to basic secondary alcohols while lack of reactivity is observed in stericallly congested cases.

A complex of [[Ir(OMe)(cod)].sub.2] and 4,4-di-tert-butyl-2,2-bipyridine (dtbpy) has catalyzed the Z-selective, dehydrative silylation of terminal alkenes, but not 1,2-disubstituted alkenes, with triethylsilane or benzyldimethylsilane in THF. Yields and Z-stereoselectivity are improved by 2-norbornene, whereas [alpha],[beta]-unsaturated esters are unreactive and the reaction has proceeded through a Heck-type mechanism.

Scalemic [alpha]-cyanohydrin triflates have undergone Pd-catalyzed cross-coupling with aryl, heteroaryl, and vinyl boronic acids, where the coupling has proceeded with complete inversion of configuration at the stereogenic carbon. The resultant nitrile is converted into alternative functional groups of value in organic synthesis and has achieved higher level of molecular complexity than the products of traditional Suzuki reactions.

An unprecedented organocatalytic, enantioselective oxy-Michael addition to achiral [gamma]/[delta]-hydroxy-[alpha],[beta]-enones and its use in the preparation of the structural motif is described. The oxy-Michael adduct formed from (E)-4-hydroxy-1-phenyl-2-buten-1-one and phenylboronic acid under catalysis by quinine-based catalyst has acted as a template for the stereoselective addition of allenylboronic acid to the carbonyl.

Stereospecific cross-coupling of [alpha]-(thiocarbamoyl)organostannes with alkenyl, aryl, and heteroaryl iodides was studied. This reaction involves racemic and scalemic pyrroldinylthiocarbamoyl-protected [alpha]-hydroxystannanes cross-coupling with alkenyl/aryl/heteroiodides using copper(I) thiophene-2-carboxylate (CuTC) in THF at or below room temperature.

Fe(0) is investigated as a cost-effective, environmentally friendly alternative to Cr(II) for the olefination of carbonyls by activated polyhalides. A surprising resultant of stereoselectivity for aldehydes versus ketones is observed using both metals and the resultant alpha-halo-alpha,beta-unsaturated or alpha,beta-unsaturated carboxylic acids, esters, and nitriles are common structural elements in numerous compounds of interest as well as key intermediates in the preparation of other functionality.

A study demonstrates that the gated electron transfer (ET) reaction could be converted to a slower true ET reaction by removal of monovalent cations. The protonated methylammonium substrate could perform the role in regulating the rate and mechanism of ET from methylamine dehydrogenase.

The synthesis of base-free (C5R5)(sub 2)Zr((O(super t)Bu)(super +)) cations is reported. The observation of nonchelated alkene and alkyne adducts of the cations, and studies of the thermodynamic and dynamic properties of the novel species are done.

A highly enantioselective skeletal rearrangement of symmetrically alpha,alpha-distributed alpha-amino aldehydes is developed, using a chiral organoluminum Lewis acid. The method provides a new entry to active alpha-hydroxy ketones and 1,2-amino alcohols.

A highly stereoselective synthesis of (Z)-alpha-haloacrylates through CrCl2-mediated olefinations of aldehydes with commercial trihaloacetates is validated. The isolation of the dihalohydrin intermediate is also described.

Evidence for the internal proton return phenomenon with vinylidene carbenoids is provided.