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.

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.

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.

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.

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.

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.

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.

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.

Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132.

The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors.

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.

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.

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.

Ionisations within a subtilisin-glyoxal inhibitor complex.

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K(i)=2.3+/-0.2 microM at pH 7.0 and 25 degrees C. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 107.3 ppm by (13)C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the (13)C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK(a)<3. This is the first observation of oxyanion formation in a reversible subtilisin-inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK(a) of approximately 6.5 in the free enzyme and on a pK(a)<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol(-1). We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.

On the mechanisms of ageing suppression by dietary restriction-is persistent glycolysis the problem?

The mechanism(s) by which dietary restriction (DR) suppresses ageing and onset of age-related pathologies are discussed in relation to frequency of glycolysis, and the reactivity of glycolytic intermediates. Most glycolytic intermediates are potentially toxic and readily modify (i.e. glycate) proteins and other macromolecules non-enzymically. Attention is drawn to the reactivity of methyglyoxal (MG) which is formed predominantly from the glycolytic intermediates dihydroxyacetone- and glyceraldehyde-3-phosphates. MG rapidly glycates proteins, damages mitochondria and induces a pro-oxidant state, similar to that observed in aged cells. It is suggested that because DR animals' energy metabolism is less glycolytic than in those fed ad libitum, intracellular MG levels are lowered by DR The decreased glycolysis during DR may delay senescence by lowering intracellular MG concentration compared to ad libitum-fed animals. Because of the reactivity MG and glycolytic intermediates, occasional glycolysis could be hormetic where glyoxalase, carnosine synthetase and ornithine decarboxylase are upregulated to control cellular MG concentration. It is suggested that in ad libitum-fed animals persistent glycolysis permanently raises MG levels which progressively overwhelm protective processes, particularly in non-mitotic tissues, to create the senescent state earlier than in DR animals. The possible impact of diet and intracellular glycating agents on age-related mitochondrial dysfunction is also discussed.

Selection for high immunogenicity in drug-resistant sublines of murine lymphomas demonstrated by plaque assay.

The immunogenicity of lymphoma L1210 and three L1210 sublines, resistant to methylglyoxal bis(guanylhydrazone), 4,4'-diacetyldiphenylurea bis(guanylhydrazone), or guanazole (L1210/GZL), respectively, was evaluated. Syngeneic DBA/2J mice were given a single i.p. injection of serially diluted suspension of irradiated cells from L1210 or L1210 sublines. Five days later spleen cells from the immunized mice were tested for the presence of plaque-forming cells using the immunizing lymphoma cell lines as target. Sera collected from the animals were examined for cytolytic antibody activity by lysis in gel using the same target cells. For comparison, the H-2 immunogenicity of L1210 and its sublines was investigated in H-2-incompatible allogeneic mice. The following results were obtained. (a) All the sublines showed increased immunogenicity and susceptibility to lysis as compared to L1210 cells. The number of plaque-forming cells/spleen ranged from 100 for L1210 to 4450 for L1210/GZL, the most immunogenic subline, and the antibody titer ranged from 1/8 for L1210 to 1/128 for L1210/GZL. (b) All the sublines carried common tumor-associated antigens that apparently made primary contributions to the increased immunogenicity. (c) The common tumor-associated antigens were also expressed on L1210 cells, although in a lesser defree, as evidenced by the definite, albeit low, capacity of L1210 cells to absorb DBA/2J anti-L1210/GZL antibodies. (d) Spleen and thymus cells of DBA/2J mice as well as unrelated murine ascites tumor cells did not cause significant absorption of these antibodies. (e) Only a partial inverse relationship could be demonstrated between tumor-associated antigens but the lowest for H-2. The above results would seem compatible with the hypothesis that the increased immunogenicity of drug-resistant L1210 sublines is attributable to the selection of preexisting highly immunogenic cells during immunosuppression by treatments selecting for drug resistance.

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.

Studies on modification of tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone and its subunits.

Based on the regeneration of the hormonal activity following recombination, the alpha and beta subunits of human follicle-stimulating hormone have been designated as 'functional' or 'nonfunctional'. Chemical modifications of the tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone, luteinizing hormone, and the 'functional' human follicle-stimulating hormone alpha and beta subunits have indicated that the tryptophan in human follicle-stimulating hormone-beta and human luteinizing hormone-beta is essential for the biological activity. The iodination of human follicle-stimulating hormone-alpha did not interfere with the hormonal activity. The modification of arginine abolishes the biological activity of the hormones. The accessibility of tyrosine and methionine in human follicle-stimulating hormone-alpha, of arginine in both native hormones and subunits, and the non-availability of the tryptophan residues to 2-hydroxy 5-nitrobenzyl bromide suggest that the alpha subunit lies on the surface of the native molecule.

Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.

We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of E1 tetramer (alpha 2 beta 2) from modification. Circular dichroism spectral analysis indicated absence of any gross structural changes in the enzyme as a result of modification. Comparison of the peptide maps, monitored at 345 nm of unprotected and pyruvate plus TPP protected E1s after V8 digestion revealed that a peptide in the protected enzyme was labeled by pyreneglyoxal to a lesser extent than its counterpart in the unprotected enzyme. Sequence analysis of the peptide demonstrated that it corresponded precisely to amino-acid residues 235 to 246 in the human E1 beta sequence, with arginine residues at positions 239 and 242. Since Arg-239 is conserved in the beta-subunit of all presently known sequences of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex, it is strongly suggested that Arg-239 in the human E1 beta sequence is at or near the active site of bovine E1.