Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Summary Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca²⁺-ATPase pump via its phosphorylation-dephosphorylation at Ser¹⁶. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.     

人胰岛素原C肽的合成及专一性抗体制备

利用固相多肽合成中的Boc途径合成了人胰岛素原C肽 ,经TSK柱层析一步纯化 ,产物达HPLC及毛细管电泳均一 ,蛋白质序列分析和质谱分析符合理论值 ,总回收率高达 41 %。为了提高寡肽抗体的专一性 ,我们将丙烯酰化的C肽经催化聚合形成了以聚丙酰为骨架 ,C肽为侧链的聚合体 ,分子量约为 2 5kD。免疫新西兰大白兔 1月 ,获得专一性抗体。用酶联免疫吸附法测得抗血清滴度达 2 .5× 1 0 4 ,与BSA无交叉反应。聚丙酰C肽抗原是制备C肽抗体的一种新的尝试 ,而且也可能成为新的多肽工程疫苗之一 ,为其他传染性疾病多肽疫苗的研究提供新的途径     

Synthesis of Chiral Heteronucleotide ONA Sequences by a Fmoc/Boc-Based Submonomeric Strategy

Peptide nucleic acid (PNA) is a DNA analog able to form hybridization complexes with complementary DNA or RNA strands. Many PNAs have been described in recent years, particularly chiral PNA analogs. Chiral heteronucleotide ONA (Orn backbone PNA) is an important tool in the antisensing field, but was not been fully explored yet. In the present work, we performed studies toward the synthesis of chiral heteronucleotide ONA sequences by utilizing a Fmoc/Boc-based submonomer approach on solid support. The desired oligomers with different nucleic content and length were obtained in very good yields and high purity. Specific binding to the complimentary ssDNA oligomers was demonstrated.     

小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响

为研究小鼠受精卵细胞早期发育过程中PKC对cdc2和cdc2 5C活性的影响 ,采用免疫印迹和电泳迁移率差异分析的方法 ,观察PKC的激活剂TPA及其抑制剂星形孢子素对小鼠受精卵一细胞期cdc2和cdc2 5C活性的影响 .10nmol L的TPA作用 10min后 ,小鼠受精卵一细胞期卵裂率明显大于对照组 (P <0 0 5 ) ,而星形孢子素作用后卵裂率显著下降 (P <0 0 1) .TPA处理后 ,受精卵中呈去磷酸化状态的活性cdc2明显增加 ,没有活性呈磷酸化状态的cdc2 5C明显减少 ;而星形孢子素处理的受精卵中没有活性的cdc2明显增加 ,有活性的cdc2 5C明显减少 .结果表明 ,TPA短时间作用可以促进小鼠一细胞期受精卵分裂 ,星形孢子素抑制受精卵的分裂 ;TPA可以促进cdc2的去磷酸化以及cdc2 5C的磷酸化 ,从而促进G2 M转换 ,星形孢子素则抑制cdc2和cdc2 5C的活性 ,阻止受精卵由G2 期进入M期     

Synthesis and Characterisation of a Multiphosphorylated Phosphophoryn Repeat Motif; H-[Asp-(Ser(<Emphasis Type="Italic">P</Emphasis>))<Subscript>2</Subscript>]<Subscript>3</Subscript>-Asp-OH

Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence H-[Asp-(Ser(P))₂]₃-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn using Fmoc-Ser(PO₃Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups. The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with good sensitivity (20 fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6 mol of calcium per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of a wide variety of multiphosphorylated peptides.     

Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Microwave-assisted Solid Phase Peptide Synthesis on High Loaded Resins

Kinetic of diffusion of reactants from the solution into the resin beads is a key factor of the efficiency of reaction in solid phase synthesis. We demonstrated in this paper that stirring is required to obtain homogeneous solution during the short coupling steps in microwave-assisted SPPS. Different types of resin, loading rate and coupling reactants were investigated to highlight this observation.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.     

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

Summary We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the - or -piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.     

Effect of histidine methylation on the recognition of OVA323-336 T-epitope: Synthesis of N-α-Fmoc-N-τ-methyl-L-histidine and its use for the synthesis of two histidine methylated analogues of OVA323-336

Summary N-α-Fmoc-N-τ-methyl-L-histidine was prepared in three steps fromN-α-Boc-L-histidine by treatment with methyliodine in DMF at−10°C, deprotection of theN-α position in pure TFA and subsequent reprotection by Fmoc-chloroformate in a 5% Na₂CO₃/dioxane mixture.N-α-Fmoc-N-τ-methyl-L-histidine was then used for the solid-phase synthesis of two analogues of the OVA_323–336 T-epitope, methylated on His₃₃₁ and on His_328/331, respectively. These peptides were tested for their ability to activate 3 DO-54.8 T-cell hybridoma when presented by fixed A-20.1.11 antigen presenting cells, and no significant difference was observed in IL-2 production.