Development of an analytical method for the unambiguous structure elucidation of cyclic peptides with special appliance for hepatotoxic desmethylated microcystins

The periodical occurrence of harmful algal blooms (HABs) in freshwater lakes requires the determination of potential cyanobacterial toxins, especially microcystins (MCs). On demand of an adequate risk assessment, the high diversity of these hepatotoxic cyclic heptapeptides implicates the need of an unambiguous detection of their specific structural variants. Therefore, LC-MS and LC-MS/MS methods are the approaches of choice for determination of MCs. In contrast, even tandem mass spectromic fragmentation patterns are not even sufficient in any kind of structural determination requirements, whereas NMR methods require very high amounts of MCs. In this study, we present a novel method for chromatographic separation of desmethylated microcystins (dm-MCs). Based on the isolation of the specific structural variants using semi-preparative HPLC, a method was developed for the structure elucidation of cyclic peptides with special appliance for the determination of dm-MCs via analysis of the specific amino acid composition after peptide hydrolysis followed by stereospecific detection of the amino acids and resulting keto acids. On the basis of this method it is demonstrated that dm-MC-RR with the structure [Dha⁷]MC-RR represented the major compound in the microcystin pattern of Microcystis aeruginosa bloom events in 2005 and 2006 in Lake Senftenberg, Germany.     

N—酰基去氢丙氨酸衍生物对体外培养细胞的抗辐射损伤效应

N-酰基去氢丙氨酸衍生物,最近被推荐为新型抗辐射剂。本研究用体外培养细胞在乏氧条件下检验了其防护效应。结果表明:在低浓度下化合物无防护作用;在高浓度时则显示一定的防护效应。作者认为如果进一步改善其化学结构,使之增加水溶性而又不增加毒性,则可能有更强的抗辐射的效应。     

Dehydroalanine and lysinoalanine in thermolyzed casein do not promote colon cancer in the rat.

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.     

Glutathione conjugation of busulfan produces a hydroxyl radical-trapping dehydroalanine metabolite

1. The Phase 2 drug metabolism of busulfan yields a glutathione conjugate that undergoes a β-elimination reaction. The elimination product is an electrophilic metabolite that is a dehydroalanine-containing tripeptide, γ-glutamyldehydroalanylglycine (EdAG). In the process, glutathione lacks thiol-related redox properties and gains a radical scavenging dehydroalanine group.

2. EdAG scavenged hydroxyl radical generated in the Fenton reaction in a concentration-dependent manner was monitored by electron paramagnetic resonance (EPR) spectroscopy. The apparent rate of hydroxyl radical scavenging was in the same range as published values for known antioxidants, including N-acyl dehydroalanines.

3. A captodatively stabilized carbon-centered radical intermediate was spin trapped in the reaction of EdAG with hydroxyl radical. The proposed structure of a stable product in the Fenton reaction with EdAG was consistent with that of a γ-glutamylserylglycyl dimer.

4. Observation of the hydroxyl trapping properties of EdAG suggests that the busulfan metabolite EdAG may contribute to or mitigate redox-related cytotoxicity associated with the therapeutic use of busulfan, and reaffirms indicators that support a role in free radical biology for dehydroalanine-containing peptides and proteins.