N-(1-Deoxy-d-xylulos-1-yl)-glutathione: A Maillard Reaction Intermediate Predominating in Aqueous Glutathione-Xylose Systems by Simultaneous Dehydration-Reaction.

The effect of simultaneous dehydration-reaction (SDR) on Amadori rearrangement product (ARP) N-(1-deoxy-d-xylulos-1-yl)-glutathione and its key degradation products, 3-deoxyxylosone (3-DX) and 1-deoxyxylosone (1-DX), were investigated in an aqueous glutathione-xylose (GSH-Xyl) system. The yield of ARP was increased to 67.98% by SDR compared with 8.44% by atmospheric thermal reaction at 80 °C. Reaction kinetics was applied to analyze the mechanism and characteristics of ARP formation and degradation under SDR. ARP formation and degradation rate was highly dependent on temperature, and the latter was more sensitive to temperature. By regulating the reaction conditions of temperature and pH, the ratio of ARP formation rate constant to its degradation rate constant could be controlled to achieve an efficient preparation of ARP from GSH-Xyl Maillard reaction through SDR.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

Near-Infrared Fluorescence Probe for Monitoring the Metabolic Products of Vitamin C in HepG2 Cells under Normoxia and Hypoxia.

Vitamin C (ascorbic acid; AA) is a well-known reducing agent and has been evaluated for its antitumor activity. However, the mechanism for its antitumor action remains unclear. Tracking the metabolism of AA may help to elucidate its antitumor mechanism. In this study, a near-infrared fluorescent probe (Arg-Cy) for monitoring the metabolic products of AA in living cells was developed based on the reaction of the guanidine group in Arg-Cy with the adjacent diketone involved in the metabolites of AA. Consequently, the probe can respond to L-xylosone, a metabolite of AA, with high selectivity and sensitivity and was successfully used to visualize the real-time changes of L-xylosone levels in living cells incubated under normoxic conditions. Considering that the tumor microenvironment suffers from hypoxia, the L-xylosone levels in the process of HepG2 cell death induced by pharmacological doses of AA were also monitored under hypoxic conditions. Surprisingly, no obvious fluorescence change appeared during this process. Furthermore, detection of the intracellular redox state using a reported H2O2 probe confirmed that AA can be metabolized to L-xylosone only under normoxic conditions due to the oxidative stress, but not under hypoxic conditions. Therefore, we hypothesize that the mechanism for cell death induced by AA under hypoxia is different from that under normoxia. Thus, the developed probe can provide a tool for monitoring the metabolism of AA and may help to clarify the mechanism for the antitumor activity of vitamin C in the tumor microenvironment.

Structure- and concentration-specific assessment of the physiological reactivity of α-dicarbonyl glucose degradation products in peritoneal dialysis fluids.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Antioxidant capacity of 1-deoxy-D-erythro-hexo-2,3-diulose and D-arabino-hexo-2-ulose.

The antioxidant capacity of two 1,2-dicarbonyl compounds, 1-deoxy-d-erythro-hexo-2,3-diulose (1-deoxyglucosone) and d-arabino-hexo-2-ulose (d-glucosone), was investigated. Both compounds are key intermediates of the Maillard reaction, and both possess a reductone-like structure. The reductive potential of the reductones was measured with the trolox equivalent antioxidant capacity (TEAC) assay and the Folin-Ciocalteu reagent (FCR) assay. Their antioxidant capacity set them apart from their precursors and other typical Maillard reaction products. Using electron paramagnetic resonance (EPR) spectroscopy, the special radical scavenging behavior of 1-deoxyglucosone and d-glucosone was measured. Both exhibited a slow, but constant, scavenging ability over the course of several hours, even days. It was postulated that this characteristic behavior is caused by the isomeric composition and the transformation to the particular antioxidant form. Reaction mixtures of 1-deoxyglucosone showed a correlation between the decrease of antioxidant properties and the decomposition of 1-deoxyglucosone.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line.

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,ß-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Two new tryptamine derivatives, leptoclinidamide and (-)-leptoclinidamine B, from an Indonesian ascidian Leptoclinides dubius.

Two new tryptamine-derived alkaloids, named as leptoclinidamide (1) and (-)-leptoclinidamine B (2), were isolated from an Indonesian ascidian Leptoclinides dubius together with C²-α-D-mannosylpyranosyl-L-tryptophan (3). The structure of 1 was assigned on the basis of spectroscopic data for 1 and its N-acetyl derivative (4). Compound 1 was an amide of tryptamine with two ß-alanine units. Although the planar structure of 2 is identical to that of the known compound (+)-leptoclinidamine B (5), compound 2 was determined to be the enantiomer of 5 based on amino acid analysis using HPLC methods. Compounds 1 to 4 were evaluated for cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma) cells, but none of the compounds showed activity.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.

Correlating changes that occur in chemical properties with the generation of antioxidant capacity in different sugar-amino acid Maillard reaction models.

UNLABELLED: We investigated the development of antioxidant activity relative to the change of pH, fluorescent intensity, ultraviolet (UV) absorbance (A294), browning (A420), and alpha-dicarbonyl compounds in sugar-amino acid Maillard reaction (MR) model systems comprising fructose, glucose, or ribose each with glycine (Fru-Gly, Glu-Gly, and Rib-Gly) or lysine (Fru-Lys, Glu-Lys, and Rib-Lys), respectively, which were heated at 121 °C for 5 to 90 min. For hexose models, the change in pH was shown to fit a second-order polynomial regression with A294 and A420. Antioxidant activity was significantly and positively correlated with UV absorbance (r = 0.905, P < 0.001) and browning products (r = 0.893, P < 0.001) rather than with fluorescent products or the alpha-dicarbonyl compounds. Type of sugar was most important in evoking a change in UV absorbance, browning, alpha-dicarbonyl compounds, and antioxidant activity of MR products (MRPs). In conclusion, the antioxidant activity of MRPs in six model systems was more closely associated with products derived at the intermediate-to-late stages of the reaction and influenced mostly by the type of sugar. PRACTICAL APPLICATION: We report on the different factors and their interactions that are important for understanding the functional attributes of food components that comprise the generation of Maillard browning products and the associated antioxidant activities generated during high-temperature food processing.

Identification of aminoreductones as active components in Maillard reaction mixtures inducing nuclear NF-kappaB translocation in macrophages.

Coffee, a highly processed food, and Maillard mixtures are able to activate nuclear factor kappaB translocation in macrophages via generation of hydrogen peroxide. In this study, a substructure library was prepared and used to identify Maillard products that are responsible for this effect. Three different Maillard reaction products with aminoreductone substructure (C(6)-aminoreductone, C(4)-aminoreductone, and aminohexose reductone) strongly induce nuclear factor kappaB translocation in macrophages. The effect was almost completely blocked by co-incubation with catalase, indicating that cellular activation was mediated by the ability of the test compounds to generate hydrogen peroxide. The cellular effect of a Maillard mixture, which was produced under conditions favoring aminoreductone formation, could be almost completely related to the presence of C(6)-aminoreductone.

Synthesis, chemical and biological studies on new Fe(3+)-glycosilated beta-diketo complexes for the treatment of iron deficiency.

A simple synthetic pathway to obtain glycosilated beta-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe(3+)-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH(3)) are characterized by means of spectroscopic (UV, (1)H and (13)C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t(1/2)>100h) and show a low cytotoxicity also in high concentrations (IC(50)>400 microM). They are able to bind Fe(3+) ion in acid condition (pH approximately 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (logP>-0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca(2+), and in particular 6-GlcOCH(3) is able to inhibit lipid peroxidation.

Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method.

The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.

Detection of Amadori compounds by capillary electrophoresis coupled to tandem mass spectrometry.

Capillary electrophoresis coupled to mass spectrometry (CE-MS) is reported for the first time as an alternative and powerful analytical method for the characterization and monitoring of N-substituted 1-amino-1-deoxyketoses (Amadori compounds). It allows rapid separation and identification of Amadori compounds, while benefiting from the well-known advantages of MS, such as specificity and sensitivity. Amadori compounds of several amino acids, such as glycine, valine, isoleucine, methionine, proline, and phenylalanine, as well as a cysteine-derived compound, were separated and/or discriminated using CE-MS/MS under standard conditions. The technique may also be useful to study the stability and degradation kinetics of other labile charged Maillard intermediates that play an important role in food and medical science.

Selective oxidation of histidine residues in proteins or peptides through the copper(II)-catalysed autoxidation of glucosone.

Glucosone has been identified as the main intermediate sugar moiety product of the copper(II)-catalysed autoxidation of the Amadori compound [Kawakishi, Tsunehiro & Uchida (1991) Carbohydr. Res. 211, 167-171]. Oxidative fragmentation of the model protein, especially selective degradation of the histidine residue in protein or peptides mediated by the copper(II)-catalysed autoxidation of glucosone, is discussed in this paper. The oxidative damage to protein could be retarded by catalase (EC 1.11.1.16) and EDTA, while superoxide dismutase (EC 1.15.1.1) and hydroxyradical scavengers showed little effect. Through the process of the oxidative degradation of N-benzoylhistidine and other histidine-containing peptides, the oxidation of the imidazole ring in histidine caused by the glucosone-copper(II) system was the same as that by the ascorbate-copper(II) system. These facts suggest that the copper-catalysed autoxidation of glucosone could generate some active-oxygen species causing oxidative damage to protein similar to that caused by the ascorbate-copper(II) system.