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R A Pascal

Publications and source records attributed to R A Pascal.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of an extremely crowded naphthalene via a stable norbornadienone.

Computational studies at the HF/3-21G and B3LYP/6-31G(d) levels suggest that 5,6,8-tri(tert-butyl)-1,2,3,4-tetraphenylnaphthalene (2) is perhaps the most crowded naphthalene derivative that will show normal stability; more highly congested naphthalenes will prefer to exist as the corresponding Dewar isomers. Initial attempts to prepare 2 by reacting 3,4,5,6-tetraphenylbenzyne with 2,3,5-tri(tert-butyl)cyclopentadienone at 83 degrees C gave instead a stable norbornadienone, 1,2,4-tri(tert-butyl)-5,6,7,8-tetraphenyl-9-oxo-1,4-dihydro-1,4-methanonaphthalene (3), which was characterized by X-ray crystallography. The experimental and calculated (HF/3-21G) activation energies (E(a)) for the decarbonylation of 3 were quite high: 39 and 46 kcal/mol, respectively, a manifestation of the substantially increased strain in the transition state as the tert-butyl groups are forced together. The naphthalene 2 was obtained in good yield by heating 3 in refluxing toluene, and its X-ray structure showed exceptional distortions from a normal naphthalene geometry. However, 2 is not completely stable, and it decomposes upon prolonged heating in xylenes.

Journal Article↗
PubMed2001

Determining absolute configuration in flexible molecules: a case study.

Assigning absolute configuration of molecules continues to be a major problem. Determining absolute configuration in conformationally flexible systems is challenging, even for experts. Here, we present a case study in which we use a combination of molecular modeling, solution NMR, and X-ray crystallography to illustrate why it is difficult to use solution methods alone for configuration assignment. For the case examined, a comparison of calculated and experimental optical rotatory dispersion (ORD) data provides the most straightforward way to assign the absolute configuration.

Benzoates↗
PubMed1994

Synthesis of new C-19-functionalized cholesterols.

A series of new derivatives of cholesterol bearing polar functional groups on carbon-19 were synthesized, including 19-oximino-, 19-amino-, 19-(methylamino)-, 19-mercapto-, and 19-(methylthio) cholesterol, and 3 beta-hydroxycholest-5-en-19-oic acid, as well as an unusual cyclosterol, 5,19-cyclocholest-6-en-3 beta-ol.

Cholesterol↗
PubMed1991

Structures of 3 beta-tetrahydropyranyloxy-5 alpha-cholesta-20(21),24-diene and 3 beta-tetrahydropyranyloxy-21-nor-5 alpha-ergost-24-en-20-one.

3 beta-Tetrahydropyranyloxy-5 alpha-cholesta-20(21),24-diene: C32H52O2, Mr = 468.77, orthorhombic, P2(1)2(1)2(1), a = 6.710 (4), b = 11.361 (4), c = 37.812 (11) A, V = 2882 (2) A3, Z = 4, Dx = 1.08 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 4.60 cm-1, F(000) = 1040, T = 224 K, final R = 0.088 for 1729 unique observed reflections. 3 beta-Tetrahydropyranyloxy-21-nor-5 alpha-ergost- 24-en-20-one: C32H52O3, Mr = 484.77, triclinic, P1, a = 6.640 (2), b = 9.589 (2), c = 12.202 (3) A, alpha = 111.33 (2), beta = 101.22, gamma = 90.27 (2) degrees, V = 707.4 (3) A3, Z = 1, Dx = 1.14 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.8 cm-1, F(000) = 268, T = 225 K, final R = 0.058 for 2208 unique observed reflections. The configuration at C(17) of these synthetic sterol derivatives, which had been uncertain, is unambiguously established to be 'normal' (possessing a 17 alpha-H).

Cholestadienes↗
PubMed1990

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata.

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.

Animals↗
PubMed1989

Effects of thiastearic acids on growth and on dihydrosterculic acid and other phospholipid fatty acyl groups of Leishmania promastigotes.

Thiastearic acid positional isomers (8, 9, 10, 11) were examined for their ability to inhibit population growth and the biosynthesis of a phosphatidylethanolamine cyclopropane fatty acyl group, cis-9,10-methyleneoctadecanoic acid (dihydrosterculic acid), by promastigotes of Leishmania species. Thiastearic acids are candidate chemotherapeutic agents, since cyclopropane fatty acids are not formed by vertebrate cells. 8- and 10-thiastearic acids strongly inhibited the growth of strains containing the most dihydrosterculic acid (Leishmania tropica and Leishmania donovani; 25-35% phosphatidylethanolamine fatty acyl groups) and less strongly inhibited strains containing no dihydrosterculic acid (Leishmania major). The 11-thiastearic acid was less effective and 9-thiastearic acid ineffective. Strains containing 1-15% dihydrosterculic acid (L. donovani, Leishmania braziliensis, Leishmania aethiopica and Leishmania mexicana mexicana) were with few exceptions not inhibited by any of the isomers. All the thiastearic acid isomers caused a dose-dependent loss of dihydrosterculic acid. This was accompanied by a loss of phosphatidylethanolamine in the case of dihydrosterculic acid-rich leishmanial strains exposed to the 8- and 10-isomers. The 8- and 10-thiastearic acids also caused a loss of C18 unsaturated fatty acyl groups and increases in palmitic and stearic acids in the phosphatidylethanolamine and phosphatidylcholine of the dihydrosterculic acid-rich and dihydrosterculic acid-free leishmanial strains. 11-Thiastearic acid was much less effective and 9-thiastearic acid ineffective. These changes were not evident in those strins which contained 1-15% dihydrosterculic acid and whose growth was not inhibited by the thiastearic acid isomers. It is concluded that thiastearic acid isomers may inhibit both dihydrosterculic acid biosynthesis and fatty acid desaturation, with the 9-isomer having the highest specificity for dihydrosterculic acid biosynthesis. Population growth of promastigotes of Leishmania species in culture is not dependent upon dihydrosterculic acid biosynthesis but is dependent upon fatty acid desaturation.

Animals↗
PubMed1988

Effects of sulfur-containing analogues of stearic acid on growth and fatty acid biosynthesis in the protozoan Crithidia fasciculata.

A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations. The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration. The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived.

Animals↗
PubMed1988

Synthesis of 24-heteroatom-substituted cholestanols.

Short syntheses of 24-thia-5 alpha,20 xi-cholestan-3 beta-ol, 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol, and 24-nor-5 alpha,20 xi-cholan-3 beta-ol from 3 beta-hydroxy-5 alpha-pregnan-20-one are described. The products and synthetic intermediates have been fully characterized by the results of proton NMR, infrared, and high and low resolution mass spectral studies.

Cholestanols↗
PubMed1986

10-Thiastearic acid inhibits both dihydrosterculic acid biosynthesis and growth of the protozoan Crithidia fasciculata.

10-Thiastearic acid is a specific inhibitor of the biosynthesis of dihydrosterculic acid (9,10-methyleneoctadecanoic acid) in the trypanosomatid protozoan Crithidia fasciculata. A 50% inhibition of the biosynthesis of dihydrosterculate is observed in the presence of 4 microM 10-thiastearate in the protozoan growth medium, but little effect is seen on the distribution of the other fatty acids. In addition, the growth of the protozoa is slowed by the presence of 10-thiastearate, with 50% growth inhibition produced at about 10 microM. A possible mechanism of this inhibition and the implication of this result with regard to the design of antiprotozoal agents are discussed.

Chromatography, Gas↗
PubMed1986

Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases.

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.

Catechol 1,2-Dioxygenase↗
PubMed1986

Synthesis of heteroatom-substituted analogues of stearic acid.

We report herein the syntheses of several analogues of stearic acid in which oxygen or sulfur atoms or sulfoxide groups have replaced the methylene groups at either position 9 or position 10 of the polymethylene chains. These compounds have been fully characterized by the results of proton and carbon 13 nuclear magnetic resonance, and low and high resolution mass spectral studies.

Chromatography, Gas↗
PubMed1985

Resonance Raman spectroscopy of pyridoxal Schiff bases.

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.

Hydrogen-Ion Concentration↗
PubMed1985

Alternate substrates and inhibitors of bacterial 4-hydroxyphenylpyruvate dioxygenase.

A variety of analogues of (4-hydroxyphenyl)pyruvic acid were synthesized, and the reactions of these compounds with the 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 were examined. Several of the ring-substituted substrate analogues are reversible inhibitors of the enzyme, the most potent being the competitive inhibitor (2,6-difluoro-4-hydroxyphenyl) pyruvate (Ki = 1.3 microM). Two substrate analogues (2-fluoro-4-hydroxyphenyl)pyruvate and [(4-hydroxyphenyl)thio]pyruvate proved to be alternate substrates for the enzyme. The former compound is converted to (3-fluoro-2,5-dihydroxyphenyl)acetate in an essentially normal catalytic sequence including oxidative decarboxylation, ring hydroxylation, and side-chain migration. The latter compound, however, undergoes oxidative decarboxylation and sulfoxidation to give [(4-hydroxyphenyl)sulfinyl]acetate; ring oxidation is not observed. The implications of these results with regard to the catalytic mechanism of 4-hydroxyphenylpyruvate dioxygenase are discussed.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1984

Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata.

Deuterium-labeled dihydroorotates bearing one, two, or three deuteriums at the pair of C4 and C5 positions have been synthesized in high isotopic and chiral purity and characterized by NMR and mass spectroscopy. These substrates have been used with the FMN-containing biosynthetic dihydroorotate oxidase from Crithidia fasciculata [Pascal, R., Trang, N., Cerami, A., & Walsh, C. (1983) Biochemistry 22, 171] to probe stereochemistry and mechanism. At pH 6.0 the (4RS)-[5,5-2H2]dihydroorotate shows a Vmax isotope effect (DV) of 2.83; since the (4S,5R)-[5-2H]dihydroorotate shows a DV of no more than 1.1, a secondary effect, the overall stereochemistry of desaturation is anti as previously reported for the degradative orotate reductase from Clostridium oroticum. The (4RS)-[4-2H]dihydroorotate shows a DV of 2.97, indicating removal of the C4-H is also partially rate limiting at pH 6.0. When trideuterio (4RS)-[4,5,5-2H3]dihydroorotate was tested, a DV of 8.0, a value close to the product of the separate isotope effects at the 4- and 5S-positions, was observed. At this pH then, both C-H cleavage steps are partly rate limiting in catalysis. Under anaerobic conditions without an electron acceptor the enzyme catalyzes the preferential exchange of the 5S hydrogen with solvent protons. The aggregate isotope effects on Vmax (DV) and on Vmax/Km [D(V/K)] are analyzed and suggest a stepwise rather than a concerted mechanism for this biosynthetic desaturation in pyrimidine biosynthesis.

Animals↗
PubMed1983

Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei.

Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Animals↗
PubMed1981

Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp.

The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species.

Butyrates↗
PubMed1981

Inhibitors of sterol synthesis. Differential effects of 14 alpha-hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol and 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol on sterol synthesis in cell-free homogenates of rat liver.

14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol and 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol have been shown to be potent inhibitors of the synthesis of digitonin-precipitable sterols in mouse L-cells and in primary cultures of fetal mouse liver cells and to cause a reduction in the levels of activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the same cells (Schroepfer, G. J., Jr., Parish, E. J., Pascal, R. A., Jr., and Kandutsch, A. A. (1980) J. Lipid Res. 21, 571-584). In the present study, we have found that both sterols have a second, but distinct, site of action, distal to the formation of mevalonic acid. 14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol has been found to be a potent inhibitor of the synthesis of digitonin-precipitable sterols from labeled mevalonate in cell-free preparations of rat liver. This inhibition was accompanied by a striking accumulation of labeled lanosterol and 24,25-dihydrolanosterol. The latter sterols were fully characterized by the results of chromatographic and co-crystallization experiments. In contrast, 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol had only a slight effect on the synthesis of digitonin-precipitable sterols from labeled mevalonate in cell-free rat liver preparations. The delta 6-3 beta, 15 alpha, 32-triol had no apparent effect on the metabolism of lanosterol and 24,25-dihydrolanosterol but caused a substantial accumulation of labeled 5 alpha-cholest-8-en-3 beta-ol which was fully characterized by the results of chromatographic and co-crystallization experiments. These findings are compatible with a specific inhibition of the metabolism of lanosterol and 24,25-dihydrolanosterol by the delta 7-3 beta, 15 alpha, 32-triol and a specific inhibition of the delta 8 leads to delta 7 isomerase by the delta 6-3 beta, 15 alpha, 32-triol. [2,4]3H]14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, prepared by chemical synthesis, was not convertible to cholesterol upon incubation, under aerobic conditions, with a cell-free homogenate preparation of rat liver. The labeled delta 7-3 beta, 15 alpha, 32-triol was, however, metabolized to several polar compounds.

Animals↗