The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between a-COOH and b-COOH in phosphoryl aspartic acid was studied by the...The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between a-COOH and b-COOH in phosphoryl aspartic acid was studied by theoretical study (Hartree-Fock and Density Functional methods) in this paper. The intermediates II containing five-membered ring were more stable than III with six-membered ring. While for intermediates III, the isomers with six-membered ring in apical-equatorial spanning arrangement were more stable than those with di-equatorial spanning arrangement. At B3LYP/6-31G** level, it was shown that transition states IV and V involving a-COOH or b-COOH group had energy barriers of DE = 58.67 kJmol-1 and 103.94 kJmol-1, respectively. These results were in agreement with the experimental data. So the a-COOH group was involved in form of the intramolecular penta-coordinate phosphoric-carboxylic mixed anhydride intermediates, but not b-COOH group.展开更多
A series of N-phosphoryl branched peptides were synthesized by coupling of various N-phosphoryl amino acids to L-Lysine methyl ester, and their structures were confirmed by 31P NMR, 1H NMR, MS and elemental analysis....A series of N-phosphoryl branched peptides were synthesized by coupling of various N-phosphoryl amino acids to L-Lysine methyl ester, and their structures were confirmed by 31P NMR, 1H NMR, MS and elemental analysis. The results of cell biological tests indicated that compound 1d and 1e obviously inhibited the growth of both K562 and A2780 cells.展开更多
The β-carboxylic group plays an important role in the peptide formation,esterification and the ester exchange at the phosphoryl group of N-phosphorylated aspartic acid.
The reactions of four different N-(O,O'-diisopropyl) phosphoamino acids (DIPP-aa), such as N-phosphoryl-L-α-alanine (DIPP- L-α-Ala), N-phosphoryl-D-α-alanine (DIPP-D-α-A1a), N-phosphoryl-β-alanine (DIPP...The reactions of four different N-(O,O'-diisopropyl) phosphoamino acids (DIPP-aa), such as N-phosphoryl-L-α-alanine (DIPP- L-α-Ala), N-phosphoryl-D-α-alanine (DIPP-D-α-A1a), N-phosphoryl-β-alanine (DIPP-β-A1a) and N-phosphoryl-γ-amino butyric acid (DIPP-γ-Aba), and four nucleosides, adenosine (A), guanosine (G), cytidine (C) and uridine (U), were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and HPLC/ESI-MS. DIPP-L-α-A1a and DIPP-D-α-A1a produced the same phosphorylated nucleosides, dinucleotides and phosphoroligopeptide. However, DIPP-β-A1a and DIPP-γ-Aba gave no relevant products.展开更多
In the presence of histidine, N-(O,O-diisopropyl)phosphoryl serine could catalyze the cleavage of RNA in aqueous solution at neutral pH. The results were detected by sub-marine agarose gel electrophoresis and capillar...In the presence of histidine, N-(O,O-diisopropyl)phosphoryl serine could catalyze the cleavage of RNA in aqueous solution at neutral pH. The results were detected by sub-marine agarose gel electrophoresis and capillary zone electrophoresis. It was found that at high concentration histidine could cleave the RNA slightly. While the participation of DIPP-Ser could significantly accelerate the cleavage reaction. N-phosphodipeptide---N-(O,O-diisopropyl) Ser-His are proposed to be involved in the mechanism.展开更多
The oligouridylates formation with N-(O,O-diisopropyl)-phosphoryl alanine in the presence of poly((-(N7-adeninyl)ethyl methacrylate) was studied. An increased yield by the presence of the polymeric nucleic acid analo...The oligouridylates formation with N-(O,O-diisopropyl)-phosphoryl alanine in the presence of poly((-(N7-adeninyl)ethyl methacrylate) was studied. An increased yield by the presence of the polymeric nucleic acid analog was confirmed by anion exchange column HPLC, C18 reverse phase HPLC, and turbo ionspray MS.展开更多
The peptide formation of N-phosphoryl aminoacids with amino acids proceeds in aqueous solution withoutany coupling reagents. After being separated in sephadex gelcolumn, the phosphoryl dipeptides were analyzed by thee...The peptide formation of N-phosphoryl aminoacids with amino acids proceeds in aqueous solution withoutany coupling reagents. After being separated in sephadex gelcolumn, the phosphoryl dipeptides were analyzed by theelectrospray ionization tandem mass spectrometry (ESIMS/MS). The result demonstrates that phosphoryl dipeptideswere datected in all the reaction systems. It is found tkat theformation of N-phosphoryl dipeptides is oriented: theN-terminal amino acid residues of the N-phosphoryl dipep-tides are from N-phosphoryl amino acids, and the peptideelongation happened at the C-terminal. Only adipeptide, noβ-dipeptide, is formed in the N-phosphoryl dipeptides,showing that α-carboxylic group is activated selectively byN-pbosphorylation. Theoretical calculation shows that thepeptide formation of N-phosphoryl amino acids might hap-pen through a pentu-coordinate carboxylic-phosphoric in-termediate in solution. These results might give some clues tothe stlidy on the origin of proteins and protein展开更多
The hydrolysis reactions of N-(O,O'diisopropyl)phosphoryl-L-α-alanine (DIPP-L-α-Ala), N-(O,O'diisopropyl)- phosphoryl-D-α-alanine (DIPP-D-α-Ala), N-(O,O'-diisopropyl)phosphoryl-β-alanine (DIPP-β-Al...The hydrolysis reactions of N-(O,O'diisopropyl)phosphoryl-L-α-alanine (DIPP-L-α-Ala), N-(O,O'diisopropyl)- phosphoryl-D-α-alanine (DIPP-D-α-Ala), N-(O,O'-diisopropyl)phosphoryl-β-alanine (DIPP-β-Ala) and N-(O,O'-diisopropyl)phosphoryl-γ-amino butyric acid (DIPP-γ-Aba), were studied by HPLC and their hydrolysis reaction kinetic equations were obtained. Under acid conditions, the reaction rate of DIPP-L-α-Ala was close to that of DIPP-D-α-Ala and the same rule was true between DIPP-β-Ala and DIPP-γ-Aba. Meantime, the reaction rate of DIPP-L/D-α-Ala was as 10 times as that of DIPP-β-Ala or DIPP-γ-Aba. Under basic conditions, the hydrolysis reactions of DIPP-β-Ala and DIPP-γ-Aba almost did not take place and the reaction rate of DIPP-L/D-α-Ala was about 1/10 of that under acid conditions. Moreover, theoretical calculation further illuminated the differences of the hydrolysis rate from the view of energy. The results would provide some helpful clues to why nature chose a-amino acids but not other kinds of analogs as protein backbones.展开更多
Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Be...Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Be- sides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M+H]+, [M+Na]+ and [M-H]- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.展开更多
The interactions of oxidative radicals (Br2?-, HO?etc.) with N-phosphoryl dipeptide derivatives (NDM-TrpOMe and NDT-MetOMe) have been investigated by using pulse radiolysis at different pH values. It has been found th...The interactions of oxidative radicals (Br2?-, HO?etc.) with N-phosphoryl dipeptide derivatives (NDM-TrpOMe and NDT-MetOMe) have been investigated by using pulse radiolysis at different pH values. It has been found that Br2?- and HO? radicals oxidize the Met-site and Trp-site in the dipeptide derivatives via formation of the three-electron-bonded intermediate and indolyl radical simultaneously. Then the intramolecular electron transfer along the peptide backbone occurs. The rate constants of electron transfer, k, have been determined and the reaction mechanism has been deduced.展开更多
Arginine phosphorylation(p Arg)is recently discovered as a ubiquitous protein N-phosphorylation in bacteria.However,its prevalence and roles in mammalian cells remain largely unknown due to the lack of established wor...Arginine phosphorylation(p Arg)is recently discovered as a ubiquitous protein N-phosphorylation in bacteria.However,its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of phosphoramidate(P–N)bond.Emerging evidences suggest that N-phosphorylation may extensively exist in eukaryotes and play crucial roles.We report a phosphoproteomic workflow,which allows for the first time revealing the widespread occurrence of p Arg in human cells by mass spectrometry.By virtue of this approach,we identified 152 high-confidence p Arg sites derived from 118 proteins.Remarkably,the discovered p Arg phosphorylation motif and gene ontology hint a possible cellular function of arginine phosphorylation which may regulate the favorability of propeptide convertase substrate.The obtained p Arg dataset paves a way for a better understanding of the biological functions of eukaryotic p Arg in the future.展开更多
Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond(phos...Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond(phosphorylation of histidine, arginine and lysine) has impaired the progress of N-phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling(ReDD) strategy for the isolation and identification of phosphorylated lysine(pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.展开更多
The amino acids’ side chains act as the relay device to modulate the chemical reactivity of the N-phosphoryl amino acids. The N-dialkyl phosphoryl cysteine is stable, but the N-dialkyl phosphoryl serine or threoine w...The amino acids’ side chains act as the relay device to modulate the chemical reactivity of the N-phosphoryl amino acids. The N-dialkyl phosphoryl cysteine is stable, but the N-dialkyl phosphoryl serine or threoine was converted into many kinds of products at 40℃. The N-dialkyl phosphoryl gltamic acid is a stable compound, while the N-dislkyl phosphoryl aspartic acid was transferred into the peptides, esters and the phosphoryl ester-exchanged products under mild conditions. The N-dialkyl phosphoryl histidine has the similar reactivity through the co-participation of the side chain, carboxyl and phosphoryl groups. A hexacoordinate phosphorus was proposed to account for this differentiation and promotion effect.展开更多
LC-ESI-MS method was used to analyze the formed di and tri-peptide in the reaction system of N-(O, O-diisopropyl) phosphoryl aspartic acid and adenosine. Cluster ions of the peptides were given in the ESI-MS. The stru...LC-ESI-MS method was used to analyze the formed di and tri-peptide in the reaction system of N-(O, O-diisopropyl) phosphoryl aspartic acid and adenosine. Cluster ions of the peptides were given in the ESI-MS. The structures of these small peptides were confirmed by LC-MS-MS analysis. Compared with the traditional HPLC-UV detection, this method showed good sensitivity and selectivity for peptide in the presence of compounds with strong UV absorption, such as nucleoside and nucleotide.展开更多
基金the National Natural Science Foundation of China (No. 29802006) the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of MOE P.R.C. and Tsinghua University.
文摘The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between a-COOH and b-COOH in phosphoryl aspartic acid was studied by theoretical study (Hartree-Fock and Density Functional methods) in this paper. The intermediates II containing five-membered ring were more stable than III with six-membered ring. While for intermediates III, the isomers with six-membered ring in apical-equatorial spanning arrangement were more stable than those with di-equatorial spanning arrangement. At B3LYP/6-31G** level, it was shown that transition states IV and V involving a-COOH or b-COOH group had energy barriers of DE = 58.67 kJmol-1 and 103.94 kJmol-1, respectively. These results were in agreement with the experimental data. So the a-COOH group was involved in form of the intramolecular penta-coordinate phosphoric-carboxylic mixed anhydride intermediates, but not b-COOH group.
基金This work is supported by Natural Science Foundation of Beijing City (7002006). Elemental analyses were performed by Institute of Chemistry Chinese Academy of Science.
文摘A series of N-phosphoryl branched peptides were synthesized by coupling of various N-phosphoryl amino acids to L-Lysine methyl ester, and their structures were confirmed by 31P NMR, 1H NMR, MS and elemental analysis. The results of cell biological tests indicated that compound 1d and 1e obviously inhibited the growth of both K562 and A2780 cells.
文摘The β-carboxylic group plays an important role in the peptide formation,esterification and the ester exchange at the phosphoryl group of N-phosphorylated aspartic acid.
基金The authors would like to thank the financial supports from the National Natural Science Foundation of China(No.20132020)the Ministry of Science and Technology.the Chinese Ministry of Education and Tsinghua University.
基金the National Natural Science Foundation of China(Nos.20572061,20672104)the Chinese Ministry of Education and Zhengzhou University for financial support.
文摘The reactions of four different N-(O,O'-diisopropyl) phosphoamino acids (DIPP-aa), such as N-phosphoryl-L-α-alanine (DIPP- L-α-Ala), N-phosphoryl-D-α-alanine (DIPP-D-α-A1a), N-phosphoryl-β-alanine (DIPP-β-A1a) and N-phosphoryl-γ-amino butyric acid (DIPP-γ-Aba), and four nucleosides, adenosine (A), guanosine (G), cytidine (C) and uridine (U), were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and HPLC/ESI-MS. DIPP-L-α-A1a and DIPP-D-α-A1a produced the same phosphorylated nucleosides, dinucleotides and phosphoroligopeptide. However, DIPP-β-A1a and DIPP-γ-Aba gave no relevant products.
文摘In the presence of histidine, N-(O,O-diisopropyl)phosphoryl serine could catalyze the cleavage of RNA in aqueous solution at neutral pH. The results were detected by sub-marine agarose gel electrophoresis and capillary zone electrophoresis. It was found that at high concentration histidine could cleave the RNA slightly. While the participation of DIPP-Ser could significantly accelerate the cleavage reaction. N-phosphodipeptide---N-(O,O-diisopropyl) Ser-His are proposed to be involved in the mechanism.
基金This work was support by the National Natural Science Foundation, the National Science and Technology Committee, and the Educati
文摘The oligouridylates formation with N-(O,O-diisopropyl)-phosphoryl alanine in the presence of poly((-(N7-adeninyl)ethyl methacrylate) was studied. An increased yield by the presence of the polymeric nucleic acid analog was confirmed by anion exchange column HPLC, C18 reverse phase HPLC, and turbo ionspray MS.
基金the National Natural Science Foundation of China (Grant Nos. 20072023 and 20175026)the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institute of Ministry of Education of China
文摘The peptide formation of N-phosphoryl aminoacids with amino acids proceeds in aqueous solution withoutany coupling reagents. After being separated in sephadex gelcolumn, the phosphoryl dipeptides were analyzed by theelectrospray ionization tandem mass spectrometry (ESIMS/MS). The result demonstrates that phosphoryl dipeptideswere datected in all the reaction systems. It is found tkat theformation of N-phosphoryl dipeptides is oriented: theN-terminal amino acid residues of the N-phosphoryl dipep-tides are from N-phosphoryl amino acids, and the peptideelongation happened at the C-terminal. Only adipeptide, noβ-dipeptide, is formed in the N-phosphoryl dipeptides,showing that α-carboxylic group is activated selectively byN-pbosphorylation. Theoretical calculation shows that thepeptide formation of N-phosphoryl amino acids might hap-pen through a pentu-coordinate carboxylic-phosphoric in-termediate in solution. These results might give some clues tothe stlidy on the origin of proteins and protein
基金Project supported by the National Natural Science Foundation of China (Nos. 20572061, 20672104).
文摘The hydrolysis reactions of N-(O,O'diisopropyl)phosphoryl-L-α-alanine (DIPP-L-α-Ala), N-(O,O'diisopropyl)- phosphoryl-D-α-alanine (DIPP-D-α-Ala), N-(O,O'-diisopropyl)phosphoryl-β-alanine (DIPP-β-Ala) and N-(O,O'-diisopropyl)phosphoryl-γ-amino butyric acid (DIPP-γ-Aba), were studied by HPLC and their hydrolysis reaction kinetic equations were obtained. Under acid conditions, the reaction rate of DIPP-L-α-Ala was close to that of DIPP-D-α-Ala and the same rule was true between DIPP-β-Ala and DIPP-γ-Aba. Meantime, the reaction rate of DIPP-L/D-α-Ala was as 10 times as that of DIPP-β-Ala or DIPP-γ-Aba. Under basic conditions, the hydrolysis reactions of DIPP-β-Ala and DIPP-γ-Aba almost did not take place and the reaction rate of DIPP-L/D-α-Ala was about 1/10 of that under acid conditions. Moreover, theoretical calculation further illuminated the differences of the hydrolysis rate from the view of energy. The results would provide some helpful clues to why nature chose a-amino acids but not other kinds of analogs as protein backbones.
基金Project supported by the National Natural Science Foundation of China (No. 20175026).
文摘Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Be- sides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M+H]+, [M+Na]+ and [M-H]- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.
基金Project supported by the National Natural Science Foundation of China (No. 39800036)the Youth Science Foundation of the University of Science and Technology of China.
文摘The interactions of oxidative radicals (Br2?-, HO?etc.) with N-phosphoryl dipeptide derivatives (NDM-TrpOMe and NDT-MetOMe) have been investigated by using pulse radiolysis at different pH values. It has been found that Br2?- and HO? radicals oxidize the Met-site and Trp-site in the dipeptide derivatives via formation of the three-electron-bonded intermediate and indolyl radical simultaneously. Then the intramolecular electron transfer along the peptide backbone occurs. The rate constants of electron transfer, k, have been determined and the reaction mechanism has been deduced.
基金supported by National Natural Science Fundation of China(21977085,21502159 to C.Fu,91856126,21778042 to YF Zhao).
文摘Arginine phosphorylation(p Arg)is recently discovered as a ubiquitous protein N-phosphorylation in bacteria.However,its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of phosphoramidate(P–N)bond.Emerging evidences suggest that N-phosphorylation may extensively exist in eukaryotes and play crucial roles.We report a phosphoproteomic workflow,which allows for the first time revealing the widespread occurrence of p Arg in human cells by mass spectrometry.By virtue of this approach,we identified 152 high-confidence p Arg sites derived from 118 proteins.Remarkably,the discovered p Arg phosphorylation motif and gene ontology hint a possible cellular function of arginine phosphorylation which may regulate the favorability of propeptide convertase substrate.The obtained p Arg dataset paves a way for a better understanding of the biological functions of eukaryotic p Arg in the future.
基金supported by the National Key Research and Development Program of China (2017YFA0505003, 2016YFA0501401)the National Natural Science Foundation of China (21505133, 21725506, 91543201)+1 种基金the CAS Key Project in Frontier Science (QYZDY-SSW-SLH017)Innovation Program from DICP, Chinese Academy of Sciences (DICP TMSR201601)
文摘Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond(phosphorylation of histidine, arginine and lysine) has impaired the progress of N-phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling(ReDD) strategy for the isolation and identification of phosphorylated lysine(pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.
基金This work was supported by the National Natural Science Foundation of China, the National Science and Technology Committee of China and Tsinghua University, and the Chinese National Educational Ministry Special Grant for YFZ (1990, 1992).
文摘The amino acids’ side chains act as the relay device to modulate the chemical reactivity of the N-phosphoryl amino acids. The N-dialkyl phosphoryl cysteine is stable, but the N-dialkyl phosphoryl serine or threoine was converted into many kinds of products at 40℃. The N-dialkyl phosphoryl gltamic acid is a stable compound, while the N-dislkyl phosphoryl aspartic acid was transferred into the peptides, esters and the phosphoryl ester-exchanged products under mild conditions. The N-dialkyl phosphoryl histidine has the similar reactivity through the co-participation of the side chain, carboxyl and phosphoryl groups. A hexacoordinate phosphorus was proposed to account for this differentiation and promotion effect.
基金Project supported by the National Natural Science Foundation of China (No. 39870415 and 29672022), National ScienceTechnology Committee of China, Education Ministry of China and Tsinghua University
文摘LC-ESI-MS method was used to analyze the formed di and tri-peptide in the reaction system of N-(O, O-diisopropyl) phosphoryl aspartic acid and adenosine. Cluster ions of the peptides were given in the ESI-MS. The structures of these small peptides were confirmed by LC-MS-MS analysis. Compared with the traditional HPLC-UV detection, this method showed good sensitivity and selectivity for peptide in the presence of compounds with strong UV absorption, such as nucleoside and nucleotide.