CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.     

2-(4-Chlorophenyl)sulfonylethoxycarbonyl (Cps) amino-protecting group for the synthesis of peptides in a solution. Synthesis of [Leu]enkephalin and its [D-ala2]analog

Solution syntheses of [Leu]enkephalin and its [D-Ala2]analogue were accomplished using a new 2-(4-chlorophenyl)sulfonylethoxycarbonyl (Cps) base-labile group for amino protection and a chromogenic acid-labile 4-(4-phenylazo)benzyloxybenzyl (Abz) group for carboxyl protection. The syntheses were performed by stepwise attachment of Cps-amino acids, which were introduced as pentachlorophenyl esters or as dicyclohexylammonium salts in the presence of tris(dimethylamino)chlorophosphonium perchlorate. To remove Cps-group, Abz-esters of Cps-peptides were treated with two molar equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene in dimethylformamide followed by neutralization of the base with an excess of 1-hydroxybenzotriazole; the deblocked amino components were then used without isolation. Fully deblocked pentapeptides were purified and characterized by HPLC, FAB mass spectra and amino acid composition.     

Two proteins homologous to the N- and C-terminal domains of the bacterial glycosyltransferase Murg are required for the second step of dolichyl-linked oligosaccharide synthesis in Saccharomyces cerevisiae

Two highly conserved eukaryotic gene products of unknown function showing homology to glycosyltransferases involved in the second steps of bacterial peptidoglycan (Murg) and capsular polysaccharide (Cps14f/Cps14g) biosynthesis have been identified in silico. The amino acid sequence of the eukaryotic protein that is homologous to the lipid acceptor- and membrane-associating N-terminal domain of Murg and the Cps14f beta4-galactosyltransferase enhancer protein is predicted to possess a cleavable signal peptide and transmembrane helices. The other eukaryotic protein is predicted to possess neither transmembrane regions nor a signal peptide but is homologous to the UDP-sugar binding C-terminal domain of Murg and the Cps14g beta4-galactosyltransferase. Both the eukaryotic proteins are encoded by essential genes in Saccharomyces cerevisiae, and down-regulation of either causes growth retardation, reduced N-glycosylation of carboxypeptidase Y, and accumulation of dolichyl-PP-GlcNAc. In vitro studies demonstrate that these proteins are required for transfer of [3H]GlcNAc from UDP-[3H]GlcNAc onto dolichyl-PP-GlcNAc. To conclude, two gene products showing homology to bacterial glycosyltransferases are required for the second step in dolichyl-PP-oligosaccharide biosynthesis.     

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.     

Ripening of Nectarine Fruit (Changes in the Cell Wall,Vacuole, and Membranes Detected Using Electrical Impedance Measurements)

Electrical impedance measurements were used to characterize changes in intracellular and extracellular resistance as well as changes in the condition of membranes during ripening of nectarines (Prunus persica [L.] Batsch cv Fantasia). These measurements were related to changes in fruit texture assessed by flesh firmness and apparent juice content. An electrical model indicated that, during ripening (d 1-5) of freshly harvested fruit, the resistance of the cell wall and vacuole declined by 60 and 26%, respectively, and the capacitance of the membranes decreased by 9%. Accurate modeling of the impedance data required an additional resistance component. This resistance, which declined by 63% during ripening, was thought to be associated with either the cytoplasmic or membrane resistance. Changes in tissue resistance measured using low frequencies of alternating current were closely related to flesh firmness. After storage at 0[deg]C for 8 weeks, the nectarines developed a woolly (dry) texture during ripening at 20[deg]C. The main difference between these chilling-injured nectarines and fruit ripened immediately after harvest was the resistance of the cell wall, which was higher in woolly tissue (4435 [omega] after 5 d at 20[deg]C) than in nonwoolly tissue (2911 [omega] after 5 d at 20[deg]C). The results are discussed in relation to physiological changes that occur during the ripening and development of chilling injury in nectarine fruit.     

Sterol biosynthesis by the sea urchin Echinus esculentus

1. The 4-demethyl sterols of Echinus esculentus consisted of cholesterol as the major component, with lower concentrations of nine other C(26), C(27), C(28) and C(29) Delta(5) sterols. 2. [2-(14)C]Mevalonic acid was readily incorporated by the urchin into squalene, lanosterol and desmosterol but only to a small extent into cholesterol. 3. [26-(14)C]Desmosterol did not appear to be reduced to give cholesterol, but conversion of 5alpha-[2-(3)H(2)]lanost-8-en-3beta-ol into cholesterol was observed. 4. No C-24 dealkylation of [4-(14)C]sitosterol or metabolism of [4-(14)C]cholesterol could be detected.     

Ultraviolet irradiation of nucleic acids: formation, purification, and solution conformational analyses of oligothymidylates containing cis-syn photodimers

Acetone-photosensitized UV irradiation of three thymine oligomers, d(TpT), d(TpTpT), and d(TpTpTpT), forms predominantly cis-syn cyclobutyl photodimers. C-18 reverse-phase high-performance liquid chromatography is used to purify the following positional isomers: d(TpT[p]T), d(T[p]TpT), d(TpTpT[p]T), d(TpT[p]TpT), d(T[p]TpTpT), and d(T[p]TpT[p]T), where T[p]T represents the cis-syn photodimer. Conformational properties of the cis-syn dimers and adjacent thymine nucleotides have been investigated in solution by using 1H, 13C, and 31P NMR spectroscopy. These studies show that (1) the photodimer conformation in longer oligothymidylates is similar to that in the dinucleoside monophosphate and (2) the cis-syn dimer induces alterations to a greater degree on the 5' side than on the 3' side of the photodimer. Specifically, the photodimer distorts the exocyclic bonds epsilon(C3'-O3') in Tp- and gamma(C5'-C4') in -pT[p]- on the 5' side and slightly alters the furanose equilibrium of the -pT nucleotide on the 3' side of the dimer.     

Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS

COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.     

Steroidal saponins of Yucca filamentosa: Yuccoside C and protoyuccoside C

Two new saponins, yuccoside C and protoyuccoside C, have been isolated from the methanolic extract of Yucca filamentosa root and their structures elucidated. Yuccoside C is 3-O-[α-d-galactopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl]-(25S)-5β-spirostan-3β-ol, whereas protoyuccoside C is 3-O-[α-d-galactopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl]-26-O-[β-d-glucopyranosy]-(25S)-5β-furostan-3β,22α,26-triol.     

CpsB is a modulator of capsule-associated tyrosine kinase activity in Streptococcus pneumoniae

Tyrosine phosphorylation is associated with polysaccharide synthesis in a number of Gram-positive and Gram-negative bacteria. In Streptococcus pneumoniae, CpsB, CpsC, and CpsD affect tyrosine phosphorylation and are critical for the production of a mature capsule in vitro. To characterize the interactions between these proteins and the phosphorylation event they modulate, cps2B, cps2C, and cps2D from the capsule type 2 S. pneumoniae D39 were cloned and expressed both individually and in combination in Escherichia coli. Cps2D purified from E. coli was not phosphorylated unless it was co-expressed with its cognate transmembrane domain, Cps2C. Purified phosphorylated Cps2D had tyrosine kinase activity and could phosphorylate both dephosphorylated Cps2D and an exogenous substrate (poly-Glu-Tyr) in the absence of ATP. Cps2B exhibited phosphatase activity against both purified phosphorylated Cps2D and p-nitrophenyl phosphate. An additional role for Cps2B as an inhibitor of Cps2D phosphorylation was demonstrated in both co-expression experiments in E. coli and in vitro experiments where it blocked the transphosphorylation of Cps2D even in the presence of the phosphatase inhibitor sodium orthovanadate. cps2C and cps2D deletion mutants in S. pneumoniae produced no detectable mature capsule during laboratory culture. Both were avirulent in systemic mouse infections and were unable to colonize the nasopharynx, suggesting that the failure to produce capsule was not dependent on the environment. Based on these results, we propose a model for capsule regulation where CpsB, CpsC, CpsD, and ATP form a stable complex that enhances capsule synthesis.     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

Validation of deuterium-labeled fatty acids for the measurement of dietary fat oxidation during physical activity

Measurement of 13C-labeled fatty acid oxidation is hindered by the need for acetate correction, measurement of the rate of CO2 production in a controlled environment, and frequent collection of breath samples. The use of deuterium-labeled fatty acids may overcome these limitations. Herein, d31-palmitate was validated against [1-13C]palmitate during exercise. Thirteen subjects with body mass index of 22.9 +/- 3 kg/m2 and body fat of 19.6 +/- 11% were subjected to 2 or 4 h of exercise at 25% maximum volume oxygen consumption (VO2max). The d31-palmitate and [1-13C] palmitate were given orally in a liquid meal at breakfast. The d3-acetate and [1-13C]acetate were given during another visit for acetate sequestration correction. Recovery of d31-palmitate in urine at 9 h after dose was compared with [1-13C] palmitate recovery in breath. Cumulative recovery of d31-palmitate was 10.6 +/- 3% and that of [1-13C]palmitate was 5.6 +/- 2%. The d3-acetate and [1-13C]acetate recoveries were 85 +/- 4% and 54 +/- 4%, respectively. When [1-13C]acetate recovery was used to correct 13C data, the average recovery differences were 0.4 +/- 3%. Uncorrected d31-palmitate and acetate-corrected [1-13C]palmitate were well correlated (y=0.96x + 0; P <0.0001) when used to measure fatty acid oxidation during exercise. Thus, d31-palmitate can be used in outpatient settings as it eliminates the need for acetate correction and frequent sampling.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM).     

Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D approximately P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D approximately P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis.     

Role of the [65-72] disulfide bond in oxidative folding of bovine pancreatic ribonuclease A

To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond. There is no bias (over the entropic influence) for the three native disulfide bonds, [26-84], [40-95], and [58-110]. Previous oxidative folding results for wt-RNase A indicated the predominance of the des [40-95] intermediate over des [65-72] after the rate-determining step in the regeneration process. Considering that there is no preferential distribution of disulfides in the 1S ensemble of [C65S, C72S], in contrast to the preferential population of the [65-72] disulfide bond in wt-RNase A, these results indicate a critical role for the [65-72] disulfide bond in the regeneration of wt-RNase A. Furthermore, analysis of the disulfide distribution of the 1S intermediates of [C65S, C72S] compared to that of wt-RNase A lends support for a physicochemical basis for the previously observed slow folding rate of this mutant, compared to its analogue (des [65-72]) of wt-RNase A.     

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.     

Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Radiosynthesis of [N-methyl-(11)C](S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide ([(11)C]BBAC or [(11)C]3) and [N-methyl-(11)C] (S)-N-([1,1'-biphenyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([(11)C]BBPC or [(11)C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate (S)-2-([1,1'-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [(11)C]CH(3)OTf in the presence of base in acetone afforded [(11)C]3 and [(11)C]4 in 30±5% yield (EOS) with >99 % radiochemical purities with a specific activity ranged from 2.5±0.5 Ci/μmol (EOB). The logP of [(11)C]3 and [(11)C]4 were determined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [(11)C]3 and [(11)C]4 cannot be used as PET tracers for imaging orexin2 receptors.