D. André d’Avignon

Structure elucidation of thioketone analogues of sildenafil detected as adulterants in herbal aphrodisiacs.

Two analogues of sildenafil were detected in herbal dietary supplements marketed as aphrodisiacs. Both compounds were identified as thioketone analogues of sildenafil in which the carbonyl group in the pyrimidine ring of sildenafil was substituted with a thiocarbonyl group. The first compound was identified as thiosildenafil, a compound that has recently been reported as an adulterant in health supplements. The structure of the second compound was established using LC-MS, UV spectroscopy, ESI-MS(n), NMR and a hydrolytic process. A detailed study of the hydrolysis products of sildenafil, thiosildenafil, and the second unknown compound proved that the second compound, named thiomethisosildenafil, had a structure analogous to sildenafil in which the N-methylpiperazine moiety had been replaced with 2,6-dimethylpiperazine and the oxygen atom of the carbonyl group in the heterocyclic ring had been replaced with a sulfur atom. Under the hydrolytic reaction conditions employed in this study, thioketones hydrolyze to ketones (e.g., thiosildenafil-->sildenafil), making this a valuable technique for the structure elucidation of thiosildenafil analogues. Ten herbal dietary supplements, each as a capsule dosage form, were found to contain 8-151 mg of thiomethisosildenafil per capsule, and one herbal dietary supplement was found to contain 35 mg of thiosildenafil per capsule.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.

Cardiomyocyte-specific deficiency of ketone body metabolism promotes accelerated pathological remodeling

Objective Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined. Methods Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed. Results While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals. Conclusions These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.

Temperature Dependence of the Folding and Unfolding Kinetics of the GCN4 Leucine Zipper via 13Cα-NMR

Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.

Site-specific thermodynamics and kinetics of a coiled-coil transition by spin inversion transfer NMR.

A 33-residue pseudo-wild-type GCN4 leucine zipper peptide is used to probe the equilibrium conformational population in proteins. 13Calpha-NMR shows that chain sites differ in structural content at a given temperature, and that two dimeric folded forms are evident at many sites. Spin inversion transfer experiments are reported bearing on the thermodynamics and kinetics of interconversion of the two dimeric folded forms (Fa <--> Fb) at the 13Calpha-labeled position L13. At each temperature, at conditions wherein the population of unfolded chains is quite small, inversion of the Fa spins via a tuned Gaussian pi-pulse is followed by a time interval (tau), interrogation, and recording of the free induction decay. Fifteen such inversions, with varying tau, provide the time course for recovery of equilibrium magnetization after inversion. Similar experiments follow inversion of the Fb spins. Re-equilibration is known to be modulated by four first-order rate constants: two (T1a(-1) and T1b(-1)) for spin-lattice relaxation intrinsic to the respective sites, and two (kab and kba) for the conformational change. All four follow from joint, Bayesian analysis of all the data at each temperature. The equilibrium constant at each temperature for this local transition, determined simply from the equilibrium relative magnetizations at Fa and Fb sites, agrees well with the kinetic ratio kab/kba. The standard Gibbs energies, enthalpy, and entropy follow. Activation parameters, both ways, are accessible from the rate constants and suggest a transition state with high Gibbs energy and enthalpy, but with entropy between those of Fa and Fb.

2,6-Dimethoxyphenylphosphirane Oxide and Sulfide and their Thermolysis to Phosphinidene Chalcogenides—Kinetic and Mechanistic Studies

Abstract 2,6-Dimethoxyphenylphosphirane is readily converted to its oxide and sulfide whose pyrolysis and (perhaps) photolysis lead to the generation of phosphinidene chalcogenides Ar–P Z (Z=O,S). Trapping experiments were carried out under conditions similar to the kinetic studies of the phosphirane chalcogenide pyrolyses that confirmed the formation of free Ar–P Z. The trapping experiments were in accord with carbene-like character for Ar–P Z, and the activation parameters suggest a non-least motion pathway for the addition of Ar–P Z to olefins. This represents quantitative evidence for the validity of the predictions of frontier-orbital theory for species that undergo reactions with small (or no) enthalpic barriers.

8-Nitroxanthine, a product of myeloperoxidase, peroxynitrite, and activated human neutrophils, enhances generation of superoxide by xanthine oxidase.

Reactive nitrogen and oxygen species are implicated in the damage of ischemic tissue that is reperfused. One important pathway may involve xanthine oxidase. Xanthine oxidase uses xanthine, a product of ATP degradation in ischemic tissue, to produce superoxide and hydrogen peroxide. Superoxide reacts rapidly with nitric oxide to form peroxynitrite, a powerful oxidant. Another potential source of reactive nitrogen species is the myeloperoxidase-hydrogen peroxide-nitrite system of activated phagocytes. We demonstrate that peroxynitrite and myeloperoxidase nitrate xanthine in vitro. Through 13C NMR spectroscopy, UV/visible spectroscopy, and mass spectrometry, the major product was identified as 8-nitroxanthine. Xanthine nitration by peroxynitrite was optimal at neutral pH and was markedly stimulated by physiological concentrations of bicarbonate. Xanthine nitration by myeloperoxidase required hydrogen peroxide and nitrite. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species. 8-Nitroxanthine was generated by a low, steady flux of peroxynitrite, and also by the myeloperoxidase-hydrogen peroxide-nitrite system of activated human neutrophils, suggesting that the reactions may be physiologically relevant. 8-Nitroxanthine may exert biological effects because it markedly increased the production of superoxide by the xanthine oxidase-xanthine system. Our observations suggest a mechanism for the enhanced formation of superoxide in reperfused tissue, which might increase the production of peroxynitrite and 8-nitroxanthine. Generation of 8-nitroxanthine by peroxynitrite and myeloperoxidase could represent a positive feedback mechanism that enhances further the production of both reactive oxygen and nitrogen species in ischemic tissue that is reperfused.

Enterobacteria secrete an inhibitor of Pseudomonas virulence during clinical bacteriuria

Escherichia coli and other Enterobacteriaceae are among the most common pathogens of the human urinary tract. Among the genetic gains of function associated with urinary E. coli isolates is the Yersinia high pathogenicity island (HPI), which directs the biosynthesis of yersiniabactin (Ybt), a virulence-associated metallophore. Using a metabolomics approach, we found that E. coli and other Enterobacteriaceae expressing the Yersinia HPI also secrete escherichelin, a second metallophore whose chemical structure matches a known synthetic inhibitor of the virulence-associated pyochelin siderophore system in Pseudomonas aeruginosa. We detected escherichelin during clinical E. coli urinary tract infection (UTI) and experimental human colonization with a commensal, potentially probiotic E. coli bacteriuria strain. Escherichelin production by colonizing enterobacteria may help human hosts resist opportunistic infections by Pseudomonas and other pyochelin-expressing bacteria. This siderophore-based mechanism of microbial antagonism may be one of many elements contributing to the protective effects of the human microbiome. Future UTI-preventive probiotic strains may benefit by retaining the escherichelin biosynthetic capacity of the Yersinia HPI while eliminating the Ybt biosynthetic capacity.

Methyl hypofluorite in the synthesis of 16-methoxyestradiol stereoisomers

The usual chemistry of methyl hypofluorite provides a previously unexplored route for functionalizing the 16-position of estradiol. Three isomers of 16-methoxyestradiol were prepared via two synthetic routes, each using methyl hypofluorite. The estrogen receptor binding affinity of these compounds was determined to evaluate their potential as positron emission tomographic (PET) imaging agents targeting estrogen receptor-positive breast cancer. Radiolabeled methyl hypofluorite ([11C]CH3OF) would allow the rapid preparation of novel carbon-11 PET imaging agents. The 17-trimethylsilyl enol ethers of 3-benzyloxy and 3-trifloxyestrone were prepared as substrates to react with methyl hypofluorite. Conditions for the reaction of methyl hypofluorite with simple substrates were optimized to provide reasonable reaction yields with the steroidal substrates. Following introduction of the methoxy substituent at the 16-position, reduction and deprotection conditions were manipulated to yield the various methoxyestradiol isomers. Two-dimensional NMR techniques (HMQC and HMQC-TOCSY) were instrumental in the characterization of the methoxyestradiol isomers. NOESY experiments confirmed the stereochemistry of the 16- and 17-positions. 16 alpha-Methoxyestradiol-17 beta and 16 beta-methoxyestradiol-17 beta each with the preferred beta orientation for the 17-alcohol, were determined to have relative binding affinities of 1.5% and 2.3%, respectively. The stereoisomer with the unfavored alpha orientation at the 17-position, 16 alpha-methoxyestradiol-17 alpha, exhibited only a 0.5% relative binding affinity for the estrogen receptor. The biological evaluation of these compounds was not pursued further because of their low binding affinities.

β-Cyclodextrin dimers as potential tumorpretargeting agents

A β-cyclodextrin dimer binds a di-tert-butylbenzyl–Cu–cyclen with high affinity, demonstrating potential as a receptor/ligand system for tumor pretargeting with monoclonal antibodies.