Proton nuclear magnetic resonance study of the conformation and configuration of the cyclized pyridine nucleotide adducts.

We have closely examined by high-frequenty 1H nuclear magnetic resonance spectroscopy the structure of the adducts which form when various carbonyl compounds react with pyridine nucleotides at elevated pH. These studies show that the adducts of N-(2,6-dichlorobenzyl)nicotinamide-acetone, n-(2,6-dichlorobenzyl)nicotinamide-pyruvate, NMN-pyruvate, NAD-pyruvate, NAD-acetaldehyde, and NAD-oxaloacetate form with identical structural features as well as configuration. The following structural features are observed: (1) the adducts are pyridine N-4-substituted compounds; (2) a second six-membered ring forms by addition of the nicotinamide amido to the carbonyl group of the compound forming the addition complex; (3) cyclization occurs stereospecifically, indicating that the stereochemistry is predetermined by the initial attack at the N-4 position; (4) two diastereomeric forms are observed for each nucleotide adduct. Finally, the determination of configuration at all symmetric carbon atoms in these adducts will be discussed.     

Asymmetric synthesis of functionalized carbocycles and heterocycles

Anions of 1-halo-4-hexenyl phosphonamides derived from chiral, enantiopure C2 symmetrical 1,2-diamino cyclohexane react at the gamma-position in conjugate addition reactions with alpha, beta-unsaturated carbonyl compounds such as cyclopentenone, 4-(H)-furanone, pyrroline-2-one, and cinnamates to give functionalized adducts. Addition to imines is also possible. The adducts can be transformed into enantiopure or enriched carbocyclic and heterocyclic compounds bearing useable functionality.     

Alpha-aminophosphonates as novel organocatalysts for asymmetric Michael addition of carbonyl compounds to nitroolefins

A cyclic alpha-aminophosphonate was found to be a novel organocatalyst for Michael type addition reactions of carbonyl compounds to nitroolefins to afford the corresponding adducts in high enantio- and diastereoselectivities.     

A rapid method to evaluate potential vasodilatory activity of organic nitrates

The kinetics of interaction between organic nitrates (3,3-bis(nitroxymethyl)oxetane) and cysteine were evaluated by the rate of nitrite ion formation at various concentrations of reagents and pH. The activities of natural reducing agents, including cysteine, glutathione, and NADH, in generating the nitrite ion from organic nitrates (3,3-bis(nitroxymethyl)oxetane) were compared. Cysteine was shown to be the most potent reducing agent. Studying the effectiveness of nitrates (trinitroglycerol, 3,3-bis(nitroxymethyl)oxetane, and nicorandil) at a concentration of 3 mM showed that the rate of nitrite ion accumulation in the reaction with 10 mM cysteine is 1.66, 0.37, and 0.02 μM/min, respectively. The reaction of organic nitrate with cysteine (Cys) is used as a test system for analyzing the effectiveness of nitrates in nitrite ion formation, which correlates with vasodilatory activity of these compounds (dilation of blood vessels).     

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalyzed by a human placental NADPH-linked carbonyl reductase.

Polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathionyl adducts of quinones undergo oxidation-reduction (redox) cycling in the presence of NADPH and the NADPH-linked human placental carbonyl reductase. K-region and non-K-region o-quinones and their glutathione adducts are the best substrates of this enzyme; they are reduced to hydroquinones. Under aerobic conditions, the hydroquinones are autoxidized with the formation of potentially hazardous semiquinones and the superoxide anion. Because of these reactions it is unlikely that polycyclic aromatic hydrocarbon quinones or their glutathione adducts are inert products of detoxication in tissues that contain the carbonyl reductase or another enzyme with similar substrate specificity. If superoxide dismutase is added to reaction mixtures containing the carbonyl reductase and quinones, it inhibits redox cycling. Presumably this results from destruction of the superoxide anion which acts as a chain propagator in these reactions.     

2-Isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195) treatment inhibits the development of intimal thickening after balloon injury of rat carotid artery: role of glycoxidation and lipoxidation reactions in vascular tissue damage

We have pursued the hypothesis that the carbonyl modification of proteins by glycoxidation and lipoxidation reactions plays a role in atherogenesis. Human atherosclerotic tissues with fatty streaks and uremic arteriosclerotic tissues were examined, with specific antibodies, to detect protein adducts formed with carbonyl compounds by glycoxidation or lipoxidation reactions, i.e. advanced glycation end products (AGEs) or glycoxidation products, such as carboxymethyllysine (CML) and pentosidine, and lipoxidation products, such as malondialdehyde (MDA)-lysine and 4-hydroxy-nonenal (HNE)-protein adduct. All the four adducts were identified in the proliferative intima and in macrophage-rich fatty streaks. If the carbonyl modification is not a mere result but is a contributor to atherogenesis, inhibition of glycoxidation and lipoxidation reactions might prevent vascular tissue damage. We tested this hypothesis in rats following balloon injury of their carotid arteries, a model exhibiting a remarkable intimal thickening, which are stained positive for all the four adducts. Oral administration of 2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanili de (OPB-9195), an inhibitor of both glycoxidation and lipoxidation reactions, in rats following balloon injury effectively prevented the intimal thickening. These data suggest a role for the carbonyl modification of proteins by glycoxidation and lipoxidation reactions in most, if not all, types of vascular tissue damage ('carbonyl stress'), and the usefulness of inhibitors of carbonyl reactions for the treatment of vascular tissue damage.     

Synthesis and some reactions of 1,3:4,6-di-O-benzylidene-d-threo-2,5-hexodiulose

Oxidation of 1,3:4,6-di-O-benzylidene-d-mannitol in ethyl acetate with ruthenium tetraoxide gave the corresponding diketone as its hydrate, which was dehydrated to the free diketone. These compounds were treated with a variety of reagents containing hydride, oxygen, nitrogen, and carbon nucleophiles. Only in reactions with methylmagnesium iodide and with diazomethane was the symmetrical bis-adduct obtained. In all other cases, the reactivity of the two carbonyl groups was different. Hydride addition occurred with opposite stereoselectivity at each carbonyl group. With oxygen and nitrogen nucleophiles, cyclic adducts were obtained, arising from bridging across the two carbonyl groups by one molecule of nucleophilic reagent. The configurations of the ring fusions were deduced by p.m.r spectroscopy, and the cis,cis-structure was preferred in compounds containing one five- and two six-membered rings. In the reactions with other carbon nucleophiles, complex mixtures were obtained due, at least in the case of the reaction with phenylmagnesium bromide, to elimination reactions.     

Carnosine and related dipeptides as quenchers of reactive carbonyl species: from structural studies to therapeutic perspectives

Carnosine (beta-alanyl-L-histidine) and related peptides such as homocarnosine (gamma-amino-butyryl-histidine), balenine beta-alanyl-L-3-methylhistidine) and anserine beta-alanyl-L-1-methylhistidine) are histidine-containing dipeptides (HD) particularly abundant in excitable tissues such as nervous system and skeletal muscle. Although their biochemical role is still unknown, several evidences indicate that these endogenous compounds act as quenchers of reactive and cytotoxic carbonyl species. In this presentation we will review the structural evidences and ex vivo data supporting this hypothesis. We first elucidated the reaction mechanism of carnosine as quencher of alpha, beta-unsaturated aldehydes such as 4-hydroxy-trans-2,3-nonenal (HNE) and acrolein (ACR) and then demonstrated the efficacy of carnosine and related peptides as detoxifying agents of HNE in spontaneously oxidized rat skeletal muscle, by detecting the corresponding HNE-Michael adducts in the crude biological matrix by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Finally, we set-up and validated a sensitive, selective and specific LC-ESI-MS/MS method for the determination of HD and of the corresponding HNE-Michael adducts to monitor their profile in physiological (aging) and pathological conditions (diabetes, atherosclerosis) characterized by a carbonyl-mediated degenerative overload. The results obtained, beside to give a contribution to the understanding of the biochemical role of histidine-dipeptides, provide a strong rational to the design of novel derivatives, active as exogenous agents able to detoxify carbonyl compounds.     

The antihypertensive hydralazine is an efficient scavenger of acrolein

Recent work indicates the highly toxic alpha,beta-unsaturated aldehyde acrolein is formed during the peroxidation of polyunsaturated lipids, raising the possibility that it functions as a 'toxicological second messenger' during oxidative cell injury. Acrolein reacts rapidly with proteins, forming adducts that retain carbonyl groups. Damage by this route may thus contribute to the burden of carbonylated proteins in tissues. This work evaluated several amine compounds with known aldehyde-scavenging properties for their ability to attenuate protein carbonylation by acrolein. The compounds tested were: (i) the glycoxidation inhibitors, aminoguanidine and carnosine; (ii) the antihypertensive, hydralazine; and (iii) the classic carbonyl reagent, methoxyamine. Each compound attenuated carbonylation of a model protein, bovine serum albumin, during reactions with acrolein at neutral pH and 37 degrees C. However, the most efficient agent was hydralazine, which strongly suppressed carbonylation under these conditions. Study of the rate of reaction between acrolein and the various amines in a protein-free buffered system buttressed these findings, since hydralazine reacted with acrolein at rates 2-3 times faster than its reaction with the other scavengers. Hydralazine also protected isolated mouse hepatocytes against cell killing by allyl alcohol, which undergoes in situ alcohol dehydrogenase-catalysed conversion to acrolein.     

Stimulation of photosynthesis by carbonyl compounds and chelators

A number of carbonyl compounds including bicarbonate, ethylene carbonate, dimethylcarbonate, propylene carbonate, bis-pentamethylene urea, and glycidol, and several chelators were tested for their effect on photosynthetic reactions in isolated spinach chloroplasts. It was found that carbonyl compounds inhibited the DCMU-insensitive silicomolybdate reduction by photosystem II but stimulated the O₂ evolution associated with ferricyanide reduction in presence of DBMIB and the H₂O→methylviologen reaction. Many chelators behaved in the same manner except 1,10-phenanthroline which shows the opposite effect. The carbonyl compounds did not uncouple because they stimulated the proton gradients associated with noncyclic photophosphorylation, whereas some chelators, such as bathocuproine or bathophenanthroline inhibited the proton gradients 100%. Electron transport in presence of ADP and inorganic phosphate showed a stimulation of rates beyond that obtained in presence of an uncoupler. The data are discussed in terms of inhibition of cyclic electron flow around PS II which leads to increased electron transport rates toward PS I.     

Carbonyl scavenger and antiatherogenic effects of hydrazine derivatives

Reactive carbonyl compounds (RCC) generated by polyunsaturated fatty acid oxidation alter progressively cellular and tissular proteins by forming adducts on free amino groups and thiol residues (carbonyl stress). Carbonyl scavengers may neutralize RCC, but their protective effect in atherosclerosis has not been extensively studied. We report the carbonyl scavenger and antiatherogenic properties of hydrazine derivatives, namely hydralazine, an antihypertensive drug, isoniazid, an antituberculosis agent, and two antidepressants, phenelzine and iproniazid. These drugs were poorly efficient in preventing the oxidation of LDL mediated by smooth muscle cells (SMCs), but inhibited the toxicity of UV-oxidized LDL (oxLDL) and of 4-hydroxynonenal (4-HNE). Hydrazine derivatives prevented the formation of foam cells resulting from LDL oxidation in human macrophagic U937 cells, and blocked the carbonyl stress in SMCs, by inhibiting the decrease in free amino group content, the increase in carbonylated proteins, and the formation of 4-HNE adducts on PDGFR. Experimental studies carried out on apoE-/- mice supplemented with drugs (30 mg/L in drinking water) showed a significant carbonyl stress inhibition correlated with a net reduction of atherosclerotic lesion development. In conclusion, these data indicate that hydrazine derivatives exhibit carbonyl scavenger and antiatherogenic properties, which opens novel therapeutical approaches for atherosclerosis and its cardiovascular complications.     

Direct asymmetric Michael addition of thioether‐based aryl sulfanyl‐propan‐2‐one to nitroalkenes catalyzed by a chiral amine‐thiourea bifunctional organocatalyst

Although the organocatalytic direct asymmetric Michael reactions of carbonyl compounds to nitroalkenes have been investigated intensely, the Michael reaction of the thioether‐based donors remains a rather undeveloped field. This work concerns the asymmetric Michael addition of aryl sulfanyl‐propan‐2‐one to nitroalkenes with benzoic acid as an additive, and chiral amine‐thiourea as a bifunctional organocatalyst. The reactions provided the highly functionalized chiral adducts with excellent enantioselectivities (up to 96% ee) and good yields. Moreover, the further transformed products exhibited excellent diastereoselectivity. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

重组基因工程菌在不对称还原羰基化合物中的应用

手性醇是药物合成的重要手性砌块,利用生物催化剂不对称还原羰基化合物是手性醇制备的重要方法。介绍了生物催化还原羰基化合物的反应原理及特点,综述了重组基因工程菌的构建及其在不对称还原羰基化合物中的应用情况,展望了今后研究发展的方向。     

Carbohydrate binding specificity of monoclonal antibodies raised against lactose-protein Maillard adducts.

Three hybridoma antibodies (L101, L104, and L117) specific for lactose-protein amino carbonyl products (Maillard adducts) were obtained by immunizing mice with the lactose-ovalbumin Maillard adduct and by screening with the lactose-bovine serum albumin (BSA) adduct. They reacted with the Maillard adducts of lactose with several different proteins, but not with the adducts of several other reducing sugars. L101 reacted well with the lactose-BSA adducts formed by 2- to 16-day incubation, whereas L104 and L117 reacted with the advanced stage reaction products but not with the adducts of 2-day incubation. The competitive inhibition of the antibody binding by several mono- and disaccharides showed that lactulose (4-O-beta-D-galactopylanosyl-D-fructose) was the best inhibitor for all three antibodies, and that L104 and L117 were inhibited by methyl-beta-D-galactoside more effectively than L101. These results suggested that different components produced during the progress of the Maillard reaction could be antigenic determinants, and that the carbohydrate moiety including the terminal galactosyl residue played an important role in the antibody binding to the lactose-protein Maillard adducts.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

Urinary aldehydes as indicators of lipid peroxidation in vivo

The excretion of malondialdehyde (MDA), lipophilic aldehydes and related carbonyl compounds in rat and human urine was investigated. MDA was found to be excreted mainly in the form of two adducts with lysine, indicating that its predominant reaction in vivo is with the lysine residues of proteins. Adducts with the phospholipid bases serine and ethanolamine and the nucleic acid bases guanine and deoxyguanosine also were found. Except for the adduct with deoxyguanosine (dG-MDA), the excretion of these compounds increased with peroxidative stress imposed in the form of vitamin E deficiency or the administration of iron or carbon tetrachloride. Marked differences in the concentration of dG-MDA in different tissues were correlated with their content of fatty acids having three or more double bonds, the putative source of MDA. Fourteen nonpolar and eleven polar lipophilic aldehydes and other carbonyl compounds were identified as their 2,4-diphenylhydrazine derivatives in rat urine. The excretion of five nonpolar and nine polar compounds was increased under conditions of peroxidative stress. The profile of lipophilic aldehydes obtained for human urine resembled that for rat urine. Except for a reported 4-hydroxynon-2-enal conjugate with mercapturic acid, the conjugated forms of the lipophilic aldehydes excreted in urine remain unidentified. Aldehyde excretion is influenced by numerous factors that affect the formation of lipid peroxides in vivo such as energy status, physical activity and environmental temperature, as well as by wide variations in the intake of peroxides in the diet. Consequently, urinalysis for aldehydic products of lipid peroxidation is an unreliable indicator of the general state of peroxidative stress in vivo.     

Radical scavenging and electron-transfer reactions in Polyporus Versicolor laccase: A pulse radiolysis study

The interaction of the radicals OH^?, t-BuO^?, e_aq^?, CO₂^XXX and O₂^XXX with the copper oxidase. laccase. from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeplide part of the protein amongst R-S-S-R. histidine and aromatic residues. Interaction with the carbonyl group of some of the peptide bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, in a subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu²⁺ with rates varying between 10³ and 10⁴ s^?1. Further reduction of the type-1 Cu²⁺ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO^XXX₂ and O^XXX₂, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO^XXX₂ or O^XXX₂. The oxidant radicals OH^?. Br^XXX₂ and (SCN)^XXX₂, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.