The reactive pyruvate metabolite dimethylglyoxal mediates neurological consequences of diabetes.

Complications of diabetes are often attributed to glucose and reactive dicarbonyl metabolites derived from glycolysis or gluconeogenesis, such as methylglyoxal. However, in the CNS, neurons and endothelial cells use lactate as energy source in addition to glucose, which does not lead to the formation of methylglyoxal and has previously been considered a safer route of energy consumption than glycolysis. Nevertheless, neurons and endothelial cells are hotspots for the cellular pathology underlying neurological complications in diabetes, suggesting a cause that is distinct from other diabetes complications and independent of methylglyoxal. Here, we show that in clinical and experimental diabetes plasma concentrations of dimethylglyoxal are increased. In a mouse model of diabetes, ilvb acetolactate-synthase-like (ILVBL, HACL2) is the enzyme involved in formation of increased amounts of dimethylglyoxal from lactate-derived pyruvate. Dimethylglyoxal reacts with lysine residues, forms Nε-3-hydroxy-2-butanonelysine (HBL) as an adduct, induces oxidative stress more strongly than other dicarbonyls, causes blood-brain barrier disruption, and can mimic mild cognitive impairment in experimental diabetes. These data suggest dimethylglyoxal formation as a pathway leading to neurological complications in diabetes that is distinct from other complications. Importantly, dimethylglyoxal formation can be reduced using genetic, pharmacological and dietary interventions, offering new strategies for preventing CNS dysfunction in diabetes.

Copper(I)-catalyzed tandem synthesis of 2-acylquinolines from 2-ethynylanilines and glyoxals.

An efficient one-step synthesis of 2-acylquinolines using a copper-catalyzed tandem reaction of 2-ethynylanilines with glyoxals in the presence of piperidine has been developed. This new protocol successfully avoids multi-step operation and the use of highly toxic cyanides required in traditional methods, and provides a practical tool for synthetic and pharmaceutical chemists. Various 2-acylquinolines are obtained with perfect regioselectivity in moderate to good yields (up to 86%). The potential synthetic utility of this method is exemplified by a large-scale experiment and synthetic transformation of the products.

Complexes of Formaldehyde and α-Dicarbonyls with Hydroxylamine: FTIR Matrix Isolation and Theoretical Study.

The interactions of formaldehyde (FA), glyoxal (Gly) and methylglyoxal (MGly) with hydroxylamine (HA) isolated in solid argon and nitrogen were studied using FTIR spectroscopy and ab initio methods. The spectra analysis indicates the formation of two types of hydrogen-bonded complexes between carbonyl and hydroxylamine in the studied matrices. The cyclic planar complexes are stabilized by O-H⋯O(C), and C-H⋯N interactions and the nonplanar complexes are stabilized by O-H⋯O(C) bond. Formaldehyde was found to form with hydroxylamine, the cyclic planar complex and methylglyoxal, the nonplanar one in both argon and nitrogen matrices. In turn, glyoxal forms with hydroxylamine the most stable nonplanar complex in solid argon, whereas in solid nitrogen, both types of the complex are formed.

A chemiluminescence resonance energy transfer strategy and its application for detection of platinum ions and cisplatin.

A novel chemiluminescence resonance energy transfer (CRET) system was developed and combined with a structure-switching aptamer for the highly sensitive detection of platinum. Platinum was chosen as a model analyte to demonstrate the generality of the new CRET system. This aptameric platform consisted of a streptavidin labeled aptamer against platinum and a streptavidin-coated magnetic bead for the selective separation of platinum-bound aptamer. The platinum-aptamer probe contained several guanine (G) bases bound to the 3,4,5-trimethoxyphenyl-glyoxal (TMPG) donor group at the 5' end, a fluorescent acceptor (6-carboxy-2',4,7,7'-tetrachlorofluorescein, TET) at the 3' end, and a streptavidin aptamer sequence in which several base pairs were replaced by the G-G mismatch to induce the platinum-oligonucleotide coordination. The chemiluminescence (CL) generated by TMPG/G bases is transferred to the acceptor (TET). In the presence of platinum, the platinum-aptamer probe was folded such that the G bases at the 5' end and TET at the 3' were in close proximity. The complex was separated using streptavidin-coated magnetic beads by the addition of TMPG to form the TMPG/G bases complex. The ultraweak CL from the TMPG/G bases was strongly enhanced by TET. This novel CRET-based method can be easily performed with high limit of detection (50 ng·mL-1) and selectivity over other metal ions. This technique provides a novel method for simple, fast, and convenient point-of-care diagnostics for monitoring proteins and metal ions. Graphical abstract Schematic presentation of chemiluminescence resonance energy transfer (CRET) detection of platinum(II) by Pt-base pair coordination to the aptamer. TMPG: 3,4,5-trimethoxyphenyl-glyoxal, fluorophore TET: 6-carboxy-2',4,7,7'-tetrachlorofluorescein.

Toxicity of dihydroxyacetone is exerted through the formation of methylglyoxal in Saccharomyces cerevisiae: effects on actin polarity and nuclear division.

Dihydroxyacetone (DHA) is the smallest ketotriose, and it is utilized by many organisms as an energy source. However, at higher concentrations, DHA becomes toxic towards several organisms including the budding yeast Saccharomyces cerevisiae In the present study, we show that DHA toxicity is due to its spontaneous conversion to methylglyoxal (MG) within yeast cells. A mutant defective in MG-metabolizing enzymes (glo1Δgre2Δgre3Δ) exhibited higher susceptibility to DHA. Intracellular MG levels increased following the treatment of glo1Δgre2Δgre3Δ cells with DHA. We previously reported that MG depolarized the actin cytoskeleton and changed vacuolar morphology. We herein demonstrated the depolarization of actin and morphological changes in vacuoles following a treatment with DHA. Furthermore, we found that both MG and DHA caused the morphological change in nucleus, and inhibited the nuclear division. Our results suggest that the conversion of DHA to MG is a dominant contributor to its cytotoxicity.

Effect of glycation derived from α-dicarbonyl compounds on the in vitro digestibility of ß-casein and ß-lactoglobulin: A model study with glyoxal, methylglyoxal and butanedione.

α-Dicarbonyl compounds, which are widely found in common consumed food, are one of the precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) or butanedione (BU) on the in vitro digestibility of ß-casein (ß-CN) and ß-lactoglobulin (ß-Lg) was investigated. Glycation from α-dicarbonyl compounds reduced the in vitro digestibility of studied proteins in both gastric and intestinal stage. In addition, glycation substantially altered the peptides released through gastric and gastrointestinal digestion, as detected by liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from BU considerably reduced the sensitivity of glycated ß-Lg towards digestive proteases, albeit to a lesser degree in glycated ß-CN due to its intrinsic unordered structure. By contrast, non-crosslinked AGEs that formed adjacent to enzymatic cleavage sites did not block the enzymatic reaction in several cases, as evidenced by the corresponding digested peptides modified with glycation structures. These findings expand our understanding of the nutritional influence of α-dicarbonyl compounds and health impact of relevant dietary AGEs.

Access to 3-Acyl-(2H)-indazoles via Rh(III)-Catalyzed C-H Addition and Cyclization of Azobenzenes with α-Keto Aldehydes.

The rhodium(III)-catalyzed direct C-H functionalization of azobenzenes with ethyl glyoxalate and aryl glyoxals is described. This protocol provides the facile and efficient formation of various C3-acylated-(2H)-indazoles in moderate to high yields.

Modification of ß-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

BACKGROUND: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human ß-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO). METHODS: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells. RESULTS: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO. CONCLUSIONS: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

Convenient access to polyfunctional pyrazoles via a highly efficient and regioselective multicomponent reaction.

A multicomponent reaction has been developed for the synthesis of polyfunctional pyrazole derivatives from readily available arylglyoxal monohydrates, tosylhydrazine, and aldehydes or ketones. This synthetic method has significant advantages in broad substrate scope, excellent regioselectivity, and simple operation.

Structure-activity relationships of substituted 1-pyridyl-2-phenyl-1,2-ethanediones: potent, selective carboxylesterase inhibitors.

Inhibition of intestinal carboxylesterases may allow modification of the pharmacokinetics/pharmacodynamic profile of existing drugs by altering half-life or toxicity. Since previously identified diarylethane-1,2-dione inhibitors are decidedly hydrophobic, a modified dione scaffold was designed and elaborated into a >300 member library, which was subsequently screened to establish the SAR for esterase inhibition. This allowed the identification of single digit nanomolar hiCE inhibitors that showed improvement in selectivity and measured solubility.

Rutin metabolites: novel inhibitors of nonoxidative advanced glycation end products.

Glycation is a nonenzymatic condensation reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, leading to the formation of advanced glycation end products (AGEs) including fluorescent (argpyrimidine) and nonfluorescent (N(epsilon)-carboxymethyllysine; CML) protein adducts and protein cross-links. AGEs are formed via protein glycation and correlate with processes resulting in aging and diabetes complications. Reactive carbonyl species such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism that potently induce the formation of AGEs by nonenzymatic protein glycation and may achieve plasma concentrations of 0.3-1.5 micromol/L. In this in vitro study histone H1 glycation by glyoxal, methylglyoxal, or ADP-ribose was used to model nonoxidative protein glycation, permitting us to distinguish specific AGE inhibition from general antioxidant action. Rutin derivatives were tested as AGE inhibitors because rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, is metabolized by gut microflora to a range of phenolic compounds that are devoid of significant antioxidant activity and achieve blood concentrations in the mumol/L range. Our data show that in a 1:1 stoichiometry with glyoxal or methylglyoxal, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3,4-dihydroxytoluene (DHT) are powerful inhibitors of CML and argpyrimidine histone H1 adduct formation, respectively. Furthermore, when DHPAA and DHT were tested as inhibitors of histone H1 glycation by the powerful glycating agent ADP-ribose, they inhibited glycation as effectively as aminoguanidine. These results suggest that dietary flavonoids may serve as effective AGE inhibitors and suggest mechanisms whereby fruit- and vegetable-rich diets contribute to the prevention of processes resulting in aging and diabetes complications.

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: detection of a new tetrahedral adduct.

The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be approximately 10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 ppm and 200.5 ppm are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp(3) and sp(2) hybridised respectively. The signal at 107.2 ppm from the carbon atom attached to the hemiketal oxyanion is formed in a slow exchange process that involves the dehydration of the glyoxal aldehyde carbon. Two additional signals are observed one at 108.2 ppm and the other at 90.9 ppm for the glyoxal keto and aldehyde carbons respectively at pHs 6-8 demonstrating that subtilisin forms an additional tetrahedral adduct with Z-Ala-Ala-Phe-glyoxal in which both the glyoxal keto and aldehyde carbons are sp(3) hybridised. For the first time we can quantify oxyanion stabilisation in subtilisin. We conclude that oxyanion stabilisation is more effective in subtilisin than in chymotrypsin. Using (1)H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pK(a) of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results is discussed.

One- and two-electron reduced 1,2-diketone ligands in [Zn(II)(L*)2(Et2O)], [Co(II)(L*)2(Et2O)], and Na2(Et2O)4[Co(II)(LRed)2].

The reaction of 1,2-diketone bis(2,6-diisopropylphenyl)glyoxal (L(Ox)) with ZnCl(2) or CoCl(2) (ratio 2:1) in dry diethyl ether with 2 equiv of sodium (per transition-metal ion) afforded the neutral complexes [Zn(II)(L(*))(2)(Et(2)O)] (1) and [Co(II)(L(*))(2)(Et(2)O)] (2), which were characterized by X-ray crystallography, magnetochemistry, IR, electron paramagnetic resonance, and UV-vis spectroscopy. When 4 equiv of sodium were added, complex Na(2)(Et(2)O)(4)[Co(II)(L(Red))(2)] (4) was isolated, which included some crystals of a minor (<2%) product Na(Et(2)O)(2)[Co(III)(L(Red))(2)] (3). (L(*))(-) represents the pi-radical monoanion of the 1,2-diketone, and (L(Red))(2-) is its enediolate(2-) analogue. The electronic structures of 1, 2, and 4 have been elucidated by spectroscopy, and results are corroborated by broken-symmetry density functional theory calculations using the B3LYP functional. 1 possesses an S = 0 ground state with an excited triplet state that is 130 cm(-1) higher in energy; 2 and 4 have an S = 1/2 ground state. These complexes corroborate the notion that acyclic 1,2-diketones are redox noninnocent ligands.

Synthesis of unsymmetrical benzil licoagrodione.

A synthesis of unsymmetrical 1,2-diarylethane-1,2-dione is reported involving the intramolecular cyclization of anionic benzylic ester of the aryl benzyl ether followed by oxidation employing dioxirane. With the use of microwave irradiation, licoagrodione was prepared from Claisen rearrangement of the corresponding allyl phenyl ether 1,2-diketone readily available from the Lindlar's reduction of the corresponding alkyne derivative. Subsequent removal of protecting groups then furnished the desired product.

alpha-Dicarbonyl compounds--key intermediates for the formation of carbohydrate-based melanoidins.

The Maillard reaction of carbohydrates and amino acids is the chemical basis for flavor and color formation in many processed foods. Dicarbonyl compounds, such as 1-, 3-deoxyosones and 1,4-dideoxyosones, as well as short-chain dicarbonyls, such as methylgyoxal or glyoxal, are key compounds of the Maillard browning reaction. The alpha-dicarbonyls are also starting materials for polymerization reactions which lead to formation of carbohydrate-based melanoidins. With regard to the dicarbonyl compound, different possible chemical structures of melanoidins will be discussed. The analysis by size-exclusion chromatography revealed that those colored compounds differ in their molecular size and are directly associated with reactions having specific alpha-dicarbonyl compounds.

Suppression of renal alpha-dicarbonyl compounds generated following ureteral obstruction by kidney-specific alpha-dicarbonyl/L-xylulose reductase.

Renal unilateral ureteral obstruction (UUO) causes acute generation of alpha-dicarbonyl stress substances, such as glyoxal, 3-deoxyglucosone, and methylglyoxal, in the kidneys. These alpha-dicarbonyl compounds are prone to form advanced glycation end products (AGEs) via the nonenzymatic Maillard reaction. Using transgenic (Tg) mice overexpressing a kidney-specific short-chain oxidoreductase, alpha-dicarbonyl/L-xylulose reductase (DCXR), we measured generation of alpha-dicarbonyls following UUO by means of electrospray ionization/liquid chromatography/mass spectrometry in their kidney extracts. The accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of the mice Tg for DCXR compared to their wild-type littermates, demonstrating 4.91 +/- 2.04 vs. 6.45 +/- 1.85 ng/mg protein (P = 0.044) for the obstructed kidneys, and 3.68 +/- 1.95 vs. 5.20 +/- 1.39 ng/mg protein (P = 0.026) for the contralateral kidneys. Despite the reduction in accumulated alpha-dicarbonyls, collagen III content in kidneys of the Tg mice and their wild-type littermates showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal alpha-dicarbonyl compounds under oxidative circumstances, but it is not sufficient to suppress acute renal fibrosis during 7 days UUO.

Planarity and constraint of the carbonyl groups in 1,2-diones are determinants for selective inhibition of human carboxylesterase 1.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P < 0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors.

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.

Unusual mixed valent FeIIIFeII complex (St = 3/2) stabilised by a reduced bulky 1,2-diketone.

The reduction of the bulky 1,2-diketone bis(2,6-diisopropylphenyl)glyoxal () and FeBr(2) with 1.5 equivalents of Na results in a Class 2 mixed valent H.S. Fe(II) L.S. Fe(III) complex (2) with two five-coordinate Fe centres which are antiferromagnetically exchange-coupled to give a total spin S(t) = 3/2 ground state and an S(t) = 5/2 excited state that are separated by about 25 cm(-1) (for Delta(J) approximately 5J).

Development of quantitative analysis of 8-nitroguanine concomitant with 8-hydroxydeoxyguanosine formation by liquid chromatography with mass spectrometry and glyoxal derivatization.

Under inflammatory conditions, both 8-nitroguanine (NO2Gua) and 8-hydroxydeoxyguanosine (8-OHdG) are found in tissues. Measurements of the two types of damaged bases on nucleotides are expected to provide information pointing to the possible correlation between inflammation and carcinogenesis. For the establishment of an in vivo model, in this study, a sensitive and precise method for the determination of NO2Gua, which uses liquid chromatography with mass spectrometry (LC-MS) and 6-methoxy-2-naphthyl glyoxal (MTNG) derivatization, was developed in vitro. The procedure for DNA digestion in this method is identical to that widely used for 8-OHdG measurement, which enables us to detect the two damaged bases in the same DNA sample. In order to validate our method, we measured NO2Gua levels in DNA sample using LC-MS. A mass spectrometer equipped with an electrospray atmospheric pressure ionization source and operated in the negative ion mode (ESI-) was set up with selective ion monitoring at m/z 391 and 394 for NO2Gua-MTNG and [13C, 15N2]-NO2Gua-MTNG as surrogate standard, respectively. The average recoveries from DNA samples spiked with 25, 50 and 250 nM NO2Gua were 99.4, 99.8 and 99.1% with correction using the added surrogate standard, respectively. The limit of quantification was 3.0 nM for NO2Gua. To ascertain the applicability of our method to DNA samples harboring the two damaged bases, we measured NO2Gua and 8-OHdG levels in calf thymus DNA treated with ONOO-. As a result, both NO2Gua and 8-OHdG levels were clearly increased with ONOO- dose dependency, the amount of NO2Gua at the high dose ONOO- being almost the same as those of 8-OHdG. LC-MS was able to determine NO2Gua in a small amount of DNA sample, and is therefore expected to be a very powerful tool for the evaluation of DNA damage induced by reactive nitrogen species.