Application Of LC-ESI-MS And NMR In The Reaction Of P₃m With Amino Acids And Synthesis Of Hydrogen Phosphonate Of D4T

Phosphorus is a basic element for the life.All of the life processes are related to phosphorus.For example,the phosphorylation of protein;the linking group of the nucleosides of DNA and RNA are phosphates;ATP as energy currency has three phosphorus atoms,which participates in energy metabolism,the process of oxidation and phosphorylation,the translation and expression of the genetic information,the expression of protein and the biosynthesis and decomposition for cell.Nobel laureate L.Todd said:"Where there's life,there's phosphorus." Because of its importance, phosphorus has been considered the adjusting center for life activities.Electrospray ionization mass spectrometry(ESI-MS) and high performance liquid chromatography(HPLC) coupled with ESI-MS as modern analytic methods are very powerful for the analysis of complicated mixtures and the investigation of the organic reaction mechanism.In this work,ESI-MS and LC-MS/MS combined with ³¹P NMR, ¹⁵N- and D- isotope techniques were used to investigate the mechanism of the reactions.First,the condensation reactions of single amino acid or each pair of amino acids such as glycine,L-alanine,β-alanine andγ-aminobutyric acid with sodium trimetaphosphate(P_3m) were reinvestigated by electrospray ion-trap mass spectrometry(ESI-MS) and high performance liquid chromatography.It was found that onlyα-amino acid could form peptide with inducement P_3m.Withoutα-amino acid,theβ-amino acid orγ-amino acid could not form peptide either by themselves or with their mixtures under the same conditions.The reaction of ¹⁵N labeled glycine with P₃m was traced by ³¹P NMR.A possible mechanism of hetero- anhydride was proposed.Second,the fragmentation pathway of protonated dipeptides was investigated by electrospray ionization tandem mass spectrometry.Protonated H-Gly-Gly-OH has a different dissociation pathway.On the contrary,other dipeptides,such as H-Ala-Ala-OH fragmented into form the[MH-CO]⁺ by the a₁-y₁ pathway and did not show such rearrangement reaction.Additional mechanistic information was obtained by exchanging the active hydrogens of protonated H-Gly-Gly-OH for deuterium.In order to illustrate the mechanism,the ¹⁵N labeled dipeptide ¹⁵N-Gly-¹⁵N-Gly was also investigated,and the structures of the fragment ions were confirmed by high-resolution electrospray ionization(ESI) tandem mass spectrometry.Third,the mechanism of the reaction of d4T with PCl₃/alcohol was investigated by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry(HPLC-ESI-MSⁿ) and ³¹P NMR.The reaction mixtures were separated by high performance liquid chromatography.And the sturctures of the mixture of the one-pot synthesis reaction,such as di-d4T hydrogen phosphonate and O-butyl H-phosphonate of d4T,were efficiently identified by on-line collision-induced dissociation(CID) tandem mass spectrometry.Furthermore,the reaction also was traced by ³¹P NMR.According to the reaction byproducts,a possible reaction mechanism was proposed.It was found that tert-butanol was not necessary for the synthesis of O-isopropyl H-phosphonate of d4T.Finally,a series of aryl and alkyl phosphoramidate derivatives of the anti-HIV drug 2',3'-dideoxy-2',3'-didehydrothymidine(d4T) were investigated by positive ion electrospray ionization mass spectrometry(ESI-MS).The fragmentation pathway of Na⁺ and K⁺ adducts were more complicated than proton adduct.In this work,the [M+Na]⁺ ion was investigated in detail by tandem mass spectrometric(MS/MS) techniques.With positive ion mode,many abundant characteristic fragment ions were found,and several rearrangement ions such as m/z 259,m/z 240 and m/z 160 were observed.A rearrangement reaction mechanism was proposed,which involved a five-membered ring of five valent phosphorus intermediate.The results are very useful for the structural elucidation of aryl and alkyl phosphoramidate derivatives of d4T in organic synthesis and drug metabolism.At the same time,it was indicated that ESI-MS is a powerful tool for the structure determination of phosphoramidate derivatives of d4T.