Synthesis And ESI-MS Study On N-Dissoproyloxyphosphoryl Dipeptides

The peptides are very important to the life as basic materials and are involved with every process of lifecycle. Under many circumstances, the function of peptides is exclusive, especially phosphorylpeptides that is few and very necessary. They play very important role in the process of activity control of enzymes and synthesis of protein .In this article, we make use of triphenylphosphine and hexachloroethane as the active reagents to synthesize a series of dipeptides, and also investigate their racemization. Meanwhile, the fragmentation rule of these phosphoryl peptides in ESI-MS are analyzed.The reagents can be quantificationally translated into dipeptides with the existence of phosphous reagent in one-pot. We also found that the use of triethylamine as a base may lead a racemizaiton when using N-Cbz-amino acid as the reagent, but the despun becomes under control with another base N-Methylmorpholin. It is more interestinge that there is no despun discovered when we use phosphoryl amino acids to synthesize phosphoryl dipeptides. The process of racemizaiton is traced by 31P-NMR.. In a word, this new method of dipeptide synthesis is high-yield, easy-purification and high-optical purity.N-diisopropyloxyphosphoryl dipeptides were analyzed by ESI-MS together with high-resolution and tandem mass spectrometric techniques. The proposed mechanism gets supports by experiments on deuterium labeling. A novel rearrangement reaction with a carbonyl oxygen migration was observed in the ESI-MS/MS of N-Di-isopropyloxyphosphoryl dipeptides. A possible mechanism was proposed and supported by tandem mass spectrometry study, derivatization of different functional groups and deuterium labeling experiments. It revealed that metal ion could catalyze the rearrangement through a five-member-ring intermediate. And we find that the trend of carbonyl oxygen migration is different with different metal ion, as the order of Li~+> Na~+> K~+, but proton does not catalyze this rearrangement reaction. And we also found that for the cationized N-diisopropyloxyphosphoryl peptides were analyzed by ESI-MS/MS, a rearrangement reaction leads to hydroxy oxygen migration to carbonyl oxygens, and cationized N-phosphoamino acid was formed. The chain length of amino acids has no effect on this rearrangement. Two possible mechanisms were proposed. It was shown that the phosphoryl group has a strong affinity to the hydroxyl group in the gas phase.Strong affinity between phosphoryl group and oxygen atom in gas phase was proposed to result in this kind of rearrangement reaction, which might give us some basic information on the nature of phosphorylation. All these phenomenons studied here could be useful in the sequencing procedure in proteomics.