Characterization of electrospray ionization mass spectrometry for N-diisopropyloxyphosphoryl dipeptide methyl esters.

A systematic study of the fragmentation pattern of N-diisopropyloxyphosphoryl (DIPP) dipeptide methyl esters in an electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was presented. A combination of accurate mass measurement and tandem mass spectrometry had been used to characterize the major fragment ions observed in the ESI mass spectrum. It was found that the alkali metal ions acted as a fixed charge site and expelled the DIPP group after transferring a proton to the amide nitrogen. For all the N-phosphoryl dipeptide methyl esters, under the activation of a metal ion, the rearrangement product ion at m/z 163 was observed and confirmed to be the sodium adduct of phosphoric acid mono-isopropyl esters (PAIE), via a specific five-membered penta-co-ordinated phosphorus intermediate. However, no rearrangement ion was observed when a beta-amino acid was at the N-terminal. This could be used to develop a novel method for differentiating isomeric compounds when either alpha- or beta-amino acid are at the N-terminus of peptides. From the [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters (DIPP Xaa1 Xaa2 OMe), the peaks corresponding to the [M+Na Xaa1 C3H6]+ were observed and explained. The [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters with Phe located in the C-terminal, such as DIPPValPheOMe, DIPPLeuPheOMe, DIPPIlePheOMe, DIPPAlaPheOMe and DIPPPhePheOMe, had characteristic fragmentation. Two unusual gas-phase intramolecular rearrangement mechanisms were first proposed for this fragmentation. These rearrangements were not observed in dipeptide methyl ester analogs which did not contain the DIPP at the N-terminal, suggesting that this moiety was critical for the rearrangement.

[Apoptosis of K562 cell induced by N-phosphoryl dipeptide methyl ester].

BACKGROUND & OBJECTIVE: Phosphorus plays a crucial role in metabolism. Some N-phosphoryl amino acids and N-phosphopeptides have important biological activities and medicinal value. The current study was designed to investigate the apoptosis of K562 cells induced by N-phosphoryl dipeptide methyl ester. METHOD: Cell growth inhibition of K562 cells induced by 15 kinds of N-phosphoryl dipeptide methyl esters was analyzed by using MTT assay. The nuclei were stained by Hoechst 33,258 and the morphologic changes were observed. DNA agarose gel electrophoresis, double-staining, and flow cytomery were used to detect early apoptosis of K562 cells. RESULT: (DIPP-L-Leu)2-L-Lys-OCH3 was the compound screened with MTT method that had better growth inhibiting activity with the IC50 of 22.66 mumol/L. Chromatin condensation and nuclear fragmentation were seen under fluorescence microscope in the cells treated with Hoechst 33,258. DNA agarose gel electrophoresis showed nuclear fragmentation (DNA ladder). Early apoptotic cells were also detected by flow cytometry. CONCLUSION: These results suggest that(DIPP-L-Leu)2-L-Lys-OCH3 could induce the apoptosis of K562 cells.