Radical-free hyperpolarized MRI using endogenously occurring pyruvate analogues and UV-induced nonpersistent radicals.

It was recently demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with UV light, enabling radical-free dissolution dynamic nuclear polarization. Although pyruvate is endogenous, the presence of pyruvate may interfere with metabolic processes or the detection of pyruvate as a metabolic product, making it potentially unsuitable as a polarizing agent. Therefore, the aim of the current study was to characterize solutions containing endogenously occurring alternatives to pyruvate as UV-induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha-ketovalerate (αkV) and alpha-ketobutyrate (αkB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing αkV and αkB were studied with UV-visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with electron spin resonance and compared with pyruvate. The addition of 13 C-labeled substrates to the sample matrix altered the radical yield of the precursors. Using αkB increased the 13 C-labeled glucose liquid-state polarization to 16.3% ± 1.3% compared with 13.3% ± 1.5% obtained with pyruvate, and 8.9% ± 2.1% with αkV. For [1-13 C]butyric acid, polarization levels of 12.1% ± 1.1% for αkV, 12.9% ± 1.7% for αkB, 1.5% ± 0.2% for OX063 and 18.7% ± 0.7% for Finland trityl, were achieved. Hyperpolarized [1-13 C]butyrate metabolism in the heart revealed label incorporation into [1-13 C]acetylcarnitine, [1-13 C]acetoacetate, [1-13 C]butyrylcarnitine, [5-13 C]glutamate and [5-13 C]citrate. This study demonstrates the potential of αkV and αkB as endogenous polarizing agents for in vivo radical-free hyperpolarized MRI. UV-induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality-control tests in clinical applications.

3-Bromopyruvate as a potent anticancer therapy in honor and memory of the late Professor André Goffeau.

3-Bromopyruvate (3BP) is a small, highly reactive molecule formed by bromination of pyruvate. In the year 2000, the antitumor properties of 3BP were discovered. Studies using animal models proved its high efficacy for anticancer therapy with no apparent side effects. This was also found to be the case in a limited number of cancer patients treated with 3BP. Due to the "Warburg effect," most tumor cells exhibit metabolic changes, for example, increased glucose consumption and lactic acid production resulting from mitochondrial-bound overexpressed hexokinase 2. Such alterations promote cell migration, immortality via inhibition of apoptosis, and less dependence on the availability of oxygen. Significantly, these attributes also make cancer cells more sensitive to agents, such as 3BP that inhibits energy production pathways without harming normal cells. This selectivity of 3BP is mainly due to overexpressed monocarboxylate transporters in cancer cells. Furthermore, 3BP is not a substrate for any pumps belonging to the ATP-binding cassette superfamily, which confers resistance to a variety of drugs. Also, 3BP has the capacity to induce multiple forms of cell death, by, for example, ATP depletion resulting from inactivation of both glycolytic and mitochondrial energy production pathways. In addition to its anticancer property, 3BP also exhibits antimicrobial activity. Various species of microorganisms are characterized by different susceptibility to 3BP inhibition. Among tested strains, the most sensitive was found to be the pathogenic yeast-like fungus Cryptococcus neoformans. Significantly, studies carried out in our laboratories have shown that 3BP exhibits a remarkable capacity to eradicate cancer cells, fungi, and algae.

Amine-Catalyzed Decarboxylative Aldol Reaction of ß-Ketocarboxylic Acids with Trifluoropyruvates.

Decarboxylative aldol reaction of aliphatic carboxylic acids is a useful method for C-C bond formation because carboxylic acids are an easily available class of compounds. In this study, we found that the decarboxylative aldol reaction of tertiary ß-ketocarboxylic acids and trifluoropyruvates proceeded smoothly to yield the corresponding aldol products in high yields and with high diastereoselectivity in the presence of a tertiary amine catalyst. In this reaction, we efficiently constructed a quaternary carbon center and an adjacent trifluoromethylated carbon center. This protocol was also extended to an enantioselective reaction with a chiral amine catalyst, and the desired product was obtained with up to 73% enantioselectivity.

In ovo feeding of creatine pyruvate modulates growth performance, energy reserves and mRNA expression levels of gluconeogenesis and glycogenesis enzymes in liver of embryos and neonatal broilers.

The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the growth performance, energy reserves and mRNA expression levels of gluconeogenesis and glycogenesis enzymes in liver of late-term embryos and neonatal broilers were investigated. After candling on 16 day of incubation, a total of 960 eggs were randomly assigned to three treatments: (i) non-injected control, (ii) saline group injected with 0.6 ml of 0.75% physiological saline and (iii) Creatine pyruvate group injected with 0.6 ml of physiological saline containing 12 mg CrPyr/egg. After hatching, 120 male chicks with average body weight (BW) were randomly allocated into each treatment group for a 7-day feeding trial. The results showed that broilers subjected to CrPyr treatment had higher BW than those of the control and saline groups on 1, 3 and 7 day post-hatch, as well as the yolk sac weight on 19 day of incubation (19 E), the day of hatch and 3 day post-hatch (p < .05). Compared with the control and saline groups, IOF of CrPyr increased the plasma creatine concentration on the day of hatch, and the plasma pyruvate concentration on the day of hatch and 3 day post-hatch (p < .05). Moreover, IOF of CrPyr increased the liver pyruvate and glucose concentrations on 19 E and the day of hatch, and the liver glycogen concentration during the experiment (p < .05). Broilers in the CrPyr group showed increased mRNA expression levels of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glycogen synthase 2 (GYS2) on 19 E and the day of hatch (p < .05). These results indicated that IOF of CrPyr increased energy reserves in liver of embryos and neonatal broilers possibly through upregulating the mRNA expression levels of PC, PEPCK and GYS2, which could benefit the increase of BW in broilers on 7 day post-hatch.

Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Previous work has demonstrated that the enzyme cycles between a catalytically inactive open state and a catalytically active closed state. The transition of the enzyme between these states requires the transition of several active site loops to shift from mobile, disordered structural elements to stable ordered states. The mechanism by which these disorder-order transitions are coupled to the ligation state of the active site however is not fully understood. To further investigate the mechanisms by which the mobility of the active site loops is coupled to enzymatic function and the transitioning of the enzyme between the two conformational states, we have conducted structural and functional studies of point mutants of E89. E89 is a proposed key member of the interaction network of mobile elements as it resides in the R-loop region of the enzyme active site. These new data demonstrate the importance of the R-loop in coordinating interactions between substrates at the OAA/PEP binding site and the mobile R- and Ω-loop domains. In turn, the studies more generally demonstrate the mechanisms by which the intrinsic ligand binding energy can be utilized in catalysis to drive unfavorable conformational changes, changes that are subsequently required for both optimal catalytic activity and fidelity.

Functional and structural characterization of an unusual cofactor-independent oxygenase.

The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2(18)O, (18)O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.

Chiral bis(imidazolinyl)phenyl NCN pincer rhodium(III) catalysts for enantioselective allylation of aldehydes and carbonyl-ene reaction of trifluoropyruvates.

Chiral NCN pincer rhodium(III) complexes with bis(imidazolinyl)phenyl ligands were found to be effective catalysts for the allylation of a variety of electronically and structurally diverse aldehydes with allyltributyltin, giving the corresponding optically active homoallylic alcohols in high yields with enantioselectivities of up to 97% ee. These complexes were also applied in the carbonyl-ene reaction of ethyl or methyl trifluoropyruvate with various 2-arylpropenes. With the aid of silver trifluoromethanesulfonate, the pincer rhodium(III) catalysts could catalyze the reaction to provide the corresponding chiral α-hydroxy-α-trifluoromethyl esters in good yields with high stereoselectivities (up to 95% ee).

Diverse reactivity in microwave-promoted catalyst-free coupling of substituted anilines with ethyl trifluoropyruvate and biological evaluation.

Diverse reactivity by coupling of substituted anilines with ethyl trifluoropyruvate was developed under microwave irradiation without catalysts to generate 3-trifluoromethyl-3-hydroxy oxindoles, aromatic hydroxy trifluoromethyl esters, and 1,2-dicarbonyl compounds in a fast and efficient manner. The plausible mechanism for obtaining different products was proposed. Furthermore, the anti-HIV activity of aromatic hydroxy trifluoromethyl esters was first reported. The best inhibitory activity against wild-type HIV-1 IIIB was exemplified by trifluoromethyloxindole 3q with an IC50 = 5.8 µM, which also displayed potential activity against Y181C mutant virus with an IC50 = 7.5 µM. More significantly, the activities of oxindoles 3q and 3r to inhibit K103N/Y181C double mutant HIV-1 reverse transcriptase (RT) are probably similar to that of the second-generation nonnucleoside inhibitor HBY 097 by docking calculation.

Asymmetric synthesis of the carbon-14-labeled selective glucocorticoid receptor modulator using cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate.

We describe in this study the asymmetric synthesis of radioisotope (RI)-labeled selective glucocorticoid receptor modulator. This synthesis is based on optimization of the cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate and Negishi coupling of zinc cyanide to the 6-bromoindole moiety. [¹4C] Labeled (-)-{4-[(1-{2-[6-cyano-1-(cyclohexylmethyl)-1H-indol-3-yl]-3,3,3-trifluoro-2-hydroxypropyl}piperidin-4-yl)oxy]-3-methoxyphenyl}acetic acid (-)-1 was synthesized successfully with high enantioselectivity (>99% ee) and sufficient radiochemical purity.

Copper-catalyzed [4+2] oxidative annulation of α,ß-unsaturated ketoxime acetates with ethyl trifluoropyruvate.

A novel route for tandem C-N/C-O formation via copper-catalyzed [4+2] oxidative annulation of α,ß-unsaturated ketoxime acetates with ethyl trifluoropyruvate to synthesize valuable trifluoromethyl-containing 2H-1,3-oxazines in moderate to good yields is developed. This procedure represents the first [4+2] oxidative annulation of oxime derivatives with activated CO bonds and provides an alternative route towards functionalized 2H-1,3-oxazines.

The antibiotic dehydrophos is converted to a toxic pyruvate analog by peptide bond cleavage in Salmonella enterica.

The metabolic processing of dehydrophos, a broad-spectrum peptide antibiotic containing an unusual vinyl-phosphonate moiety, was examined by using a panel of Salmonella enterica mutants deficient in peptide uptake and catabolism. Dehydrophos bioactivity is lost in opp tpp double mutants, demonstrating a requirement for uptake via nonspecific oligopeptide permeases. Dehydrophos bioactivity is also abolished in a quadruple Salmonella mutant lacking the genes encoding peptidases A, B, D, and N, showing that hydrolysis of the peptide bond is required for activity. (31)P nuclear magnetic resonance spectroscopy was used to assess the fate of dehydrophos following in vitro digestion of the antibiotic with purified PepA. The results suggest that the initial product of peptidase processing is 1-aminovinyl-phosphonate O-methyl ester. This phosphonate analogue of dehydroalanine undergoes rearrangement to the more stable imine, followed by spontaneous hydrolysis to yield O-methyl-acetylphosphonate, a structural analogue of pyruvate. This compound is a known inhibitor of pyruvate dehydrogenase and pyruvate oxidase and is probably the active species responsible for dehydrophos bioactivity.

Straightforward synthesis of enantiopure (R)- and (S)-trifluoroalaninol.

Two efficient routes are reported for the synthesis of both enantiomers of trifluoroalaninol in enantiopure form. The first pathway involves a Strecker-type reaction performed from a chiral trifluoromethyloxazolidine (Fox). The second route, which is more direct, involves, as a key step, the reduction of chiral oxazolidines or imines derived from ethyl trifluoropyruvate.

Joint experimental and DFT study of the gas-phase unimolecular elimination kinetic of methyl trifluoropyruvate.

The elimination kinetics of methyl trifluoropyruvate in the gas phase was determined in a static system, where the reaction vessel was always deactivated with allyl bromide, and in the presence of at least a 3-fold excess of the free-radical chain inhibitor toluene. The working temperature range was 388.5-430.1 degrees C, and the pressure range was 38.6-65.8 Torr. The reaction was found to be homogeneous and unimolecular and to obey a first-order rate law. The products of the reaction are methyl trifluoroacetate and CO gas. The Arrhenius equation of this elimination was found to be as follows: log k(1) (s(-1)) = (12.48 +/- 0.32) - (204.2 +/- 4.2) kJ mol(-1)(2.303RT)(-1) (r = 0.9994). The theoretical calculation of the kinetic and thermodynamic parameters and the mechanism of this reaction were carried out at the B3LYP/6-31G(d,p), B3LYP/6-31++G(d,p), MPW1PW91/6-31G(d,p), MPW1PW91/6-31++G(d,p), PBEPBE/6-31G(d,p), and PBEPBE/6-31G++(d,p) levels of theory. The theoretical study showed that the preferred reaction channel is a 1,2-migration of OCH(3) involving a three-membered cyclic transition state in the rate-determining step.

Asymmetric direct aldol reactions of pyruvic derivatives.

Simple chiral primary-tertiary diamine-Brønsted acid conjugates such as 1e can effectively catalyze the direct aldol reactions of pyruvic derivatives with excellent syn diastereoselectivities and enantioselectivities, thus functionally mimicking the pyruvate-dependent type I aldolases.

Affinity labeling of a catalytic site, cysteine(247), in rat mercaptopyruvate sulfurtransferase by chloropyruvate as an analog of a substrate.

A bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (EC 2.8.1.2), is inactivated by 3-chloropyruvate, an analog of 3-mercaptopyruvate serving as a sulfur-donor and -acceptor substrate. To elucidate a reaction mechanism of the enzyme, the inactivation kinetic studies using 3-chloropyruvate were carried out. However, 3-chloropyruvate cannot be mixed with 3-mercaptopyruvate, 2-mercaptoethanol and thiosulfate because these substrates decompose 3-chloropyruvate. Thus, 3-mercaptopyruvate sulfurtransferase was incubated with 3-chloropyruvate, and then the remaining activity was measured separately in the assay system containing 3-mercaptopyruvate and 2-mercaptoethanol. The inactivation kinetics was analyzed by Kitz and Wilson method (J. Biol. Chem. 237 (1962) 3245-3248). The inactivation of mercaptopyruvate sulfurtransferase by 3-chloropyruvate proceeded in one-on-one manner and exhibited pseudo first-order kinetics with k(inact) = 0.068 +/- 0.003 min(-1) and K(I) = 4.0 +/- 0.2 mM (n = 3, mean +/- S.D.). Further, SH titration using DTNB revealed that MST was inactivated by 3-chloropyruvate in a 1:1 stoichiometry. Site-directed mutagenesis for binding sites of 3-mercaptopyruvate (Arg(187)-->Gly or Arg(196)-->Gly) (J. Biol. Chem. 271 (1996) 27395-27401) did not critically affect the inactivation. These findings suggest that 3-chloropyruvate behaves as an affinity label and directly tags the catalytic site, Cys(247). An ESI-LC/Q-TOF mass spectrometric study suggests that a pyruvate adduct is formed at Cys(247), which mimics a reaction intermediate.

Spectroscopic studies of diketoacids-metal interactions. A probing tool for the pharmacophoric intermetallic distance in the HIV-1 integrase active site.

The interactions with divalent cations of 4-phenyl-4-oxo-2-hydroxybuten-2-oic acid (benzoylpyruvic acid (BPA)), the pharmacophore of HIV-1 integrase inhibitors, were investigated using spectroscopic tools. In the absence of the enzyme, a 2:2 metal-ligand complex was characterized with an intermetallic distance of 4-6 A. Molecular modeling allowed us to propose a compatible structure for the metal-ligand complex. BPA does not inhibit the reactions catalyzed by HIV-1 IN, emphasizing the importance of the aromatic ring substitution in the antiviral activity.

Rational design, synthesis, and structure-activity relationships of novel factor Xa inhibitors: (2-substituted-4-amidinophenyl)pyruvic and -propionic acids.

An inhibitor of factor Xa (fXa), the m-substituted benzamidine AXC1578 (1a), was structurally modified with the aim of increasing its potency. In particular, pyruvic acid and propionic acid substituents were incorporated into the P1 benzamidine moiety to introduce a favorable interaction with the oxy-anion hole in the catalytic triad region of fXa. This strategy was based on computational docking studies using the extracted active site of fXa. The validity of the computational model was supported by the acquisition of X-ray crystal structures of the 1a-trypsin and 3b-trypsin complexes (the homology around the active sites of fXa and trypsin is high). The above modifications significantly increased the inhibitory activity toward fXa, whereas the high selectivity for fXa versus thrombin was maintained or enhanced. Compounds 3b, 3c, 3e, and 4b are considered to be potential lead compounds for the development of orally active anticoagulant drugs because they demonstrated potent activity when administered orally to cynomolgus monkeys.

Lead removal onto cross-linked low molecular weight chitosan pyruvic acid derivatives.

Adsorption capacity of cross-linked low molecular weight chitosan pyruvic acid derivatives CSnPA-GLA (n=8, 11) were examined by employing 2(3) factorial design method. Three (3) factors and two (2) levels of adsorbent dose (A) (0.05 and 0.1 g), adsorbent type (B) (CS8PA-GLA and CS11PA-GLA) and concentration of lead solution (C) (1 and 3 mg/L) were considered. From the statistical analysis, all the main parameters (A, B and C) and some interactions of the main parameters (AC and ABC) had influence on the adsorption process at 5% significance level. The adsorption process was greatly influenced by the adsorbent type (B). The adsorption equilibrium results correlated well with the Freundlich isotherm model. The adsorption kinetic data also correlated well with the pseudo second order. The thermodynamic studies also revealed that the nature of lead adsorption was spontaneous and endothermic. The findings suggest that CS8PA-GLA is better than CS11PA-GLA for lead sorption.

ZnO nanoparticles-catalyzed cyclocondensation reaction of arylmethylidenepyruvic acids with 6-aminouracils.

A new, efficient, and solvent-free cyclocondensation reaction of arylmethylidenepyruvic acids with 6- aminouracils is presented that uses a catalytic amount of ZnO nanoparticles (ZnO Nps) as a recyclable catalyst at 70 °C. This protocol has the advantages of high yields (91-98%), easy work-up, very short reaction time (2 h), and using environmentally friendly procedure.

Differential resuscitative effect of pyruvate and its analogues on VBNC (viable but non-culturable) Salmonella.

An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost culturability by the addition of 3 mM H2O2 to cultures grown in Luria-Bertani (LB) medium; however, some H2O2-treated bacteria regained their culturability in M9 minimal medium, if sodium pyruvate was present at at least 0.3 mM. In addition, most pyruvate analogues, such as bromopyruvate or phenylpyruvate, did not show restoration activity similar to that of pyruvate, except in the case of α-ketobutyrate. Further analysis of the mechanism underlying the resuscitation by pyruvate revealed that although many of the bacteria showed respiratory activity on CTC (5-cyano-2,3-di-(p-tolyl) tetrazolium chloride) reduction with or without pyruvate, the biosynthesis of DNA and protein synthesis were quite different in the presence or absence of pyruvate, i.e., pyruvate endowed the cells with the ability to incorporate much more radio-label into precursors during the resuscitation process. These results suggest that pyruvate is one of the key molecules working in the resuscitation process by taking bacteria from the non-culturable state to the growing and colony-forming state by triggering the synthesis of macromolecules such as DNA and protein.