N,N-Dimethyl-histidine methyl ester

The objective of this study was to identify the Fatty Acid Methyl Ester (FAME) from Neurospora intermedia N-1 that isolated from Indonesian red peanut cake (oncom). FAME profiles have been used as biochemical characters to study many different groups of organisms, such as bacteria and yeasts. FAME from N. intermedia N-1 was obtained by some stages of extraction the orange spores and fractination using a chromatotron. The pure compound (1) was characterized by 500 mHz NMR (1H and 13C), FTIR and LC-MS. Summarized data's of 1H and 13C NMR spectra of compound 1 contained 19 Carbon, 34 Hydrogen and 2 Oxygen (C19H34O2). The position of the double bonds at carbon number 8 and 12 were indicated in the HMBC spectrum (2D-NMR). LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is visible by the presence of protonated molecular ion [M+H] at m/z 295. Methyl esters of long chain fatty acids was presented by a 3 band pattern of IR spectrum with bands near 1249, 1199 and 1172 cm(-1). We suggested that the structure of the pure compound 1 is methyl octadeca-8,12-dienoate. The presence methyl octadeca-8,12-dienoate in N. intermedia is the first report.

The biosynthesis of N-hydroxy pipecolic acid (NHP) has been intensively studied, though knowledge on its metabolic turnover is still scarce. To close this gap, we discovered three novel metabolites via metabolite fingerprinting in Arabidopsis thaliana leaves after Pseudomonas infection and UV-C treatment. Exact mass information and fragmentation by tandem mass spectrometry (MS/MS) suggest a methylated derivative of NHP (MeNHP), an NHP-OGlc-hexosyl conjugate (NHP-OGlc-Hex), and an additional NHP-OGlc-derivative. All three compounds were formed in wild-type leaves but were not present in the NHP-deficient mutant fmo1-1. The identification of these novel NHP-based molecules was possible by a dual-infiltration experiment using a mixture of authentic NHP and D9-NHP standards for leaf infiltration followed by UV-C treatment. Interestingly, the signal intensity of MeNHP and other NHP-derived metabolites increased in ugt76b1-1 mutant plants. For MeNHP, we unequivocally determined the site of methylation at the carboxylic acid moiety. MeNHP application by leaf infiltration leads to the detection of a MeNHP-OGlc as well as NHP, suggesting MeNHP hydrolysis to NHP. This is in line with the observation that MeNHP infiltration is able to rescue the fmo1-1 susceptible phenotype against Hyaloperonospora arabidopsidis Noco 2. Together, these data suggest MeNHP as an additional storage or transport form of NHP.

Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. Nε-Carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) are considered to be the advanced glycation end-products (AGEs) of L-lysine (Lys) with GO and MGO, respectively. Here, we investigated the reaction of free L-lysine (Lys) with GO and MGO in phosphate buffer (pH 7.4) at 37 °C and 80 °C in detail in the absence of any other chemicals which are widely used to reduce Schiff bases. The concentrations of Lys, GO and MGO used in the experiments were 0.5, 2.5, 5.0, 7.5 and 10 mM. The reaction time ranged between 0 and 240 min. Experiments were performed in triplicate. The concentrations of remaining Lys and of CML and CEL formed in the reaction mixtures were measured by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS). Our experiments showed that CML and CEL were formed at higher concentrations at 80 °C compared to 37 °C. CML was found to be the major reaction product. In mixtures of GO and MGO, MGO inhibited the formation of CML from Lys (5 mM) in a concentration-dependent manner. The highest CML concentration was about 300 µM corresponding to a reaction yield of 6% with respect to Lys. An addition of Lys to GO, MGO and their mixtures resulted in strong reversible decreases in the Lys concentration up to 50%. It is assumed that free Lys reacts rapidly with GO and MGO to form many not yet identified reaction products. Reaction mixtures of Lys and MGO were stronger colored than those of Lys and GO, notably at 80 °C, indicating higher reactivity of MGO towards Lys that leads to polymeric colored MGO species. We have a strong indication of the formation of Nε-(hydroxymethyl)-lysine (HML) as a novel reaction product of Lys methyl ester with MGO. A mechanism is proposed for the formation of HML from Lys and MGO. This mechanism may explain why Lys and GO do not react to form a related product. Preliminary analyses show that HML is formed at higher concentrations than CEL from Lys methyl ester and MGO. No Schiff bases or their hydroxylic precursors were identified as reaction products. In their reactions with Lys, GO and MGO are likely to act both as chemical oxidants on the terminal aldehyde group to a carboxylic group (i.e., R-CHO to R-COOH) and as chemical reductors on labile Schiff bases (R-CH=N-R to R-CH2-NH-R) presumably via disproportionation and hydride transfer. Our study shows that free non-proteinic Lys reacts with GO and MGO to form CML, CEL and HML in very low yield. Whether proteinic Lys also reacts with MGO to form HML residues in proteins remains to be investigated. The physiological occurrence and concentration of HML in biological fluids and tissues and its relation to CML and CEL are elusive and warrant further investigations in health and disease. Chemical synthesis and structural characterization of HML are expected to advance and accelerate the scientific research in this topic.