Synthesis and Biological Activities of N-Phosphoryl Branched Peptides

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.

Theoretical Study on the Activity of a-COOH and b-COOH of N-Phosphoryl Aspartic Acids

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.

A Facile Synthesis of N-Phosphoryl Amino Acids Containing Hydroxy Group

three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent and tetrabutyl ammonium bromide as the phase transfer catalyst in basic aqueous media.

Synthesis of N-Phosphoryl Branched Peptides

H-phosphonates were conveniently prepared by direct transesterification of diphenyl phosphite (DPP) with the corresponding alcohols, without further purification they were reacted with branched peptide methyl ester (L-Leu2-L-LysOMe) through Atherton-Todd method, a series of different substituted alkyloxy (N-phosphoryl-L-Leu)2-L-LvsOMe were synthesized, and their structures were confirmed by P NMR, ESI-MS, H NMR,13 C NMR, IR and elemental analysis. 31 1 The approach possesses the advantages of easy operation, high yield and inexpensive phosphorylating reagent.

Interaction between N-phosphoryl-α-,β-and γ-amino acids and nucleosides

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.

Analysis of Hydrolysis Reaction of N-Phosphoryl Phenylalanine by HPLC-ESI-MS/MS

Hydrolysis procedure of N-phosphoryl phenylalanine (DIPP-Phe) was studied by HPLC-ESI-MS/MS. The results showed that (HO)(i-PrO)P(O)Phe was the main intermediate and the hydrolysis of DIPP-Phe also occurred through a penta-coordinate transition state.

INTRAMOLECULAR β-CARBOXYLIC ACTIVATING OF N-PHOSPHORYL ASPARTIC ACID

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.

Construction and Analysis of N-phosphoryl Peptide Libraries

N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

RNA Cleavage by N-Phosphoryl Serine in Histidine Buffer

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.

Nphos:Database and Predictor of Protein N-phosphorylation

Protein N-phosphorylation is widely present in nature and participates in various biological processes.However,current knowledge on N-phosphorylation is extremely limited compared to that on O-phosphorylation.In this study,we collected 11,710 experimentally verified N-phosphosites of 7344 proteins from 39 species and subsequently constructed the database Nphos to share up-to-date information on protein N-phosphorylation.Upon these substantial data,we characterized the sequential and structural features of protein N-phosphorylation.Moreover,after comparing hundreds of learning models,we chose and optimized gradient boosting decision tree(GBDT)models to predict three types of human N-phosphorylation,achieving mean area under the receiver operating characteristic curve(AUC)values of 90.56%,91.24%,and 92.01%for pHis,pLys,and pArg,respectively.Meanwhile,we discovered 488,825 distinct N-phosphosites in the human proteome.The models were also deployed in Nphos for interactive N-phosphosite prediction.In summary,this work provides new insights and points for both flexible and focused investigations of N-phosphorylation.It will also facilitate a deeper and more systematic understanding of protein N-phosphorylation modification by providing a data and technical foundation.Nphos is freely available at http://www.bio-add.org/Nphos/and http://ppodd.org.cn/Nphos/.

Orientation of the peptide formation of N-phosphoryl amino acids in solution

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

N-phosphoryl amino acid models for P–N bonds in prebiotic chemical evolution

Post-translational modification of proteins by N-phosphorylation of the basic amino acid residues plays important roles in biological processes. The high-energy P–N bond might have contributed to the evolution of prebiotic chemistry. N-phosphoryl amino acids(PAAs) can serve as interesting small molecular models for the study of P–N bonds in prebiotic chemical evolution. PAAs are capable of simultaneously producing several important biomolecules such as polypeptides and oligonucleotides under mild reaction conditions. In this review, we describe the chemistry of PAAs, discusse their likely prebiotic origins and their reactivity and how they relate to biological P–N bond species. We also depict a possible prebiotic scenario mediated by PAAs in which PAAs may have acted as one of the essential forces driving prebiotic biomolecules to the first protocell.

Hydrolysis Reaction of N-Phosphoryl-α-, β- and γ-amino Acids Studied by HPLC

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.

Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

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.

Interactions of oxidative radicals with N-phosphoryl dipeptide derivatives

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.

蛋白质N-磷酸化修饰富集方法进展

蛋白质磷酸化作为一种最普遍和最重要的翻译后修饰调控着几乎所有的生命过程。随着高效富集方法和生物质谱技术的快速发展,低丰度的蛋白质O-磷酸化修饰获得了规模化鉴定,从而使其生物学功能得到较为透彻的研究。而发生在组氨酸、赖氨酸和精氨酸侧链氨基的N-磷酸化修饰,由于P-N键化学稳定性差,导致其在酸和热条件下不稳定。而目前依赖酸性条件的O-磷酸化富集方法难以适用N-磷酸化富集,导致蛋白质N-磷酸化生物功能研究严重滞后。因此,迫切需要发展针对蛋白质N-磷酸化的高效富集方法。本文首先介绍了蛋白质N-磷酸化的结构特征和已报道的生物学功能,重点综述并分析了近20年来蛋白质N-磷酸化修饰富集方法,并对每一种富集方法的优缺点进行了评述,最后对潜在的富集方法进行了展望。

复方芩柏汤调控ERK/JNK信号通路治疗溃疡性结肠炎的效应及机制研究

目的 研究复方芩柏汤对细胞外信号调节激酶(extracellular regulated protein kinases, ERK)/c-Jun氨基末端激酶(cJun N-terminal kinase, JNK)信号通路的调控作用,以及对溃疡性结肠炎(ulcerative colitis, UC)小鼠血清中炎症因子的影响。方法 将60只SPF级健康雄性BALB/C小鼠利用3%葡聚糖硫酸(dextran sulfate sodium, DSS)建立UC模型,造模成功后随机分为模型组(以生理盐水灌肠)、美沙拉嗪组(以0.196 g/kg美沙拉嗪药液灌肠)、复方芩柏汤组(以1.092 g/kg复方芩柏颗粒剂药液灌肠),每组20只,每天保留灌肠2次,连续3周。另取20只正常饲养小鼠作为空白组。在治疗前和治疗后第7、14、21天,观察小鼠体质量、大便性状、便血情况,并计算疾病活动指数(disease activity index, DAI),且于给药结束后进行麻醉取血并取结肠组织。采用HE染色观察各组小鼠结肠组织病理变化情况;ELISA法检测各组小鼠血清中炎症因子白细胞介素(interleukin)-22、IL-6、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)含量变化情况;采用Western blot法检测各组小鼠结肠组织中的p90核糖体蛋白S6激酶(p90 ribosomal protein S6 kinase, p90RSK)、JNK、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase, p-JNK)、细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2, ERK 1/2)、磷酸化细胞外调节蛋白激酶1/2(phospho extracellular regulated protein kinases, p-ERK 1/2)蛋白的表达。结果 在治疗的第7、14、21天,美沙拉嗪组、复方芩柏汤组小鼠体质量均明显高于模型组(P<0.05,P<0.01),DAI评分明显低于模型组(P<0.05,P<0.01)。光镜下,与模型组相比,美沙拉嗪组及复方芩柏汤组小鼠结肠组织病理改变呈不同程度的恢复,炎症浸润减轻。给药结束后,与空白组比较,模型组p90RSK、p-JNK、p-ERK 1/2蛋白以及IL-6、TNF-α蛋白表达明显升高(P<0.01);与模型组比较,美沙拉嗪组和复方芩柏汤组p90RSK、p-JNK、p-ERK 1/2蛋白以及IL-6、TNF-α蛋白表达明显降低(P<0.05,P<0.01),抗炎因子IL-22、IL-10含量明显升高(P<0.01)。结论 复方芩柏汤可能通过抑制ERK/JNK信号通路,促进抗炎因子IL-22、IL-10的表达,抑制促炎因子IL-6、TNF-α的表达,减少肠道炎症反应,促进肠上皮细胞增殖,改善肠黏膜屏障,促进UC小鼠肠道黏膜组织损伤修复。

电针对脑缺血大鼠前扣带皮质HMGB1和p-JNK蛋白表达的影响

目的:观察电针对脑缺血大鼠前扣带皮质高迁移率族蛋白1(high mobility group protein 1,HMGB1)和磷酸化的c-Jun氨基酸末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)的表达影响,探讨电针对脑缺血大鼠前扣带皮质的保护作用及机制。方法:将24只雄性SD大鼠随机分为假手术组、模型组、电针组和假电针组,6只/组。采用右侧大脑中动脉栓塞法制备脑缺血大鼠模型,电针组选取“百会”穴、左侧“足三里”穴进行电针刺激,1次/d,30 min/次,持续14 d;假电针组仅浅刺入两穴位皮下,接电针仪但不通电。采用Longa评分评估各组大鼠神经功能损伤情况;Nissl染色观察右侧前扣带皮质神经元的形态与分布情况;免疫组化检测右侧前扣带皮质HMGB1和p-JNK蛋白的表达情况。结果:与假手术组相比,模型组和假电针组大鼠神经功能缺损评分升高(P<0.01),右侧前扣带皮质区Nissl阳性神经元数量减少(P<0.01),HMGB1和p-JNK蛋白表达增加(P<0.01);与模型组相比,电针组大鼠在脑缺血第7天、14天时神经功能缺损评分降低(P<0.05),Nissl阳性神经元数量增加(P<0.01),HMGB1和p-JNK蛋白表达降低(P<0.01)。结论:电针可能通过抑制脑缺血后HMGB1和p-JNK的过表达,减轻前扣带皮质的损伤。

益气聪明汤在光学离焦性近视大鼠巩膜组织MMP-2、p-JNK表达中的作用

目的探究中药方剂益气聪明汤在光学离焦性近视(LIM)大鼠巩膜组织基质金属蛋白酶2(MMP-2)、磷酸化氨基末端蛋白激酶(p-JNK)表达中的作用。方法选取SPF级健康无眼疾21 d龄SD大鼠60只,所有大鼠均选择右眼为实验眼。雌雄不拘,随机分为空白(BG)组、模型(MG)组、中药低剂量(LG)组、中药中剂量(MD)组、中药高剂量(HG)组以及西药(WG)组,每组各10只。将-5 D近视离焦透镜打磨至直径约1.7 cm的圆形,在两侧打一小孔,分别缝于MG、LG、MD、HG、WG组大鼠右眼上下眼睑皮肤处建立LIM模型。在造模4周后开始灌胃,WG组大鼠用0.1 g·L^(-1)阿托品滴眼液滴眼,BG组大鼠不做任何干预,LG、MD、HG组给予益气聪明汤灌胃,MG、BG组给予生理盐水灌胃。记录每组大鼠造模前、造模4周及8周后的眼轴长度。造模8周后处死大鼠,苏木素-伊红(HE)染色观察各组大鼠巩膜组织形态学变化,Western blot检测各组大鼠巩膜组织中MMP-2、p-JNK蛋白表达,免疫组织化学检测各组大鼠巩膜组织中MMP-2阳性表达,透射电镜观察各组大鼠巩膜胶原纤维直径大小。结果造模4周和8周后,与BG组比较,MG组大鼠眼轴长度均明显增加(均为P<0.001);与MG组比较,WG、LG、MD、HG组大鼠眼轴长度均减小(均为P<0.05)。造模8周后,BG大鼠的巩膜层间胶原纤维排列规则,走行正常;与BG组比较,MG组大鼠巩膜胶原纤维疏松有明显空隙,排列紊乱;与MG组比较,MD、HG组大鼠巩膜胶原纤维排列较规则,纤维间空隙变小,LG、WG组大鼠巩膜各层间胶原纤维相对清晰紧实。造模8周后,与BG组比较,MG组大鼠巩膜中MMP-2、p-JNK蛋白相对表达水平均明显升高(均为P<0.001);与MG组比较,WG、LG、MD、HG组大鼠巩膜中MMP-2、p-JNK蛋白相对表达水平均降低(均为P<0.05)。造模8周后,与BG组比较,MG组大鼠巩膜中MMP-2蛋白阳性表达升高(P<0.001);与MG组比较,WG、LG、MD、HG组大鼠巩膜中MMP-2蛋白阳性表达均降低(均为P<0.05)。透射电镜下观察可见,BG组大鼠巩膜胶原纤维排列整齐致密,大小均匀;与BG组比较,MG组大鼠巩膜胶原纤维溶解;与MG组比较,WG组及LG组可见胶原纤维溶解减少,密度相对增加;与MG组比较,MD和HG组可见大鼠巩膜中胶原纤维直径较均匀,空隙减少,无纤维溶解。结论益气聪明汤可通过调节LIM大鼠巩膜中MMP-2、p-JNK的表达,减缓近视的发生发展。