两种树脂对固相法合成丙型肝炎疫苗多肽的影响

本实验采用固相法合成丙型肝炎疫苗多肽,比较Wang树脂和二氯三苯甲基树脂（以下简称二氯树脂）作为载体时的连接率以及目标肽的产率和纯度。通过质谱鉴定分析所得目标多肽的分子量,用茚三酮法对两种树脂与氨基酸的连接率进行比较,用反相高效液相色谱（RP-HPLC）进行粗肽的分析和纯化。结果显示以二氯树脂作载体,第一个氨基酸的连接率,以及目标肽的纯度和产率都要明显高于Wang树脂,因此二氯树脂作载体合成丙型肝炎疫苗多肽更适合大规模工业化生产的要求。     

Fmoc固相合成JFT的工艺研究

目的:研究多肽JFT的合成工艺。方法:本实验采用固相合成法(spps),以Fmoc—氨基酸为原料,TBTU\HoBt\DIEA混合试剂缩合,用三氟乙酸\苯甲硫醚\巯基乙醇\苯酚\水脱保护,将多肽从MBHA树脂上切割下来。结果:粗肽的收率为62%,经RP-HPLC纯化,即可获得纯度在98%以上的目标肽。经MALDI—MS质谱分析其分子量与理论值一致。结论:此工艺操作简单,便于推广,适合大规模生产。     

杂环肽FIK的Fmoc固相合成法研究

为研究二硫键成环的杂环肽FIK的合成工艺, 以Fmoc氨基酸为原料, 采用固相合成法, 经TBTU/HOBT/DIEA复合缩合剂催化合成直链肽, 再经I2氧化肽链上两个半胱氨酸的巯基生成分子内二硫键而得到目标环肽, 将其用切割试剂切割脱离树脂得到粗产品, MALDI-MS和RP-HPLC进行鉴定, 分析和纯化。产率可以达到18%, 纯化后纯度达97%以上, 经MALDI-MS和Ellman试剂检测确定为目标肽。该合成法高效, 简便, 快速, 目标肽收到较理想产率, 适合大批量生产。     

杂环肽FIK的Fmoc固相合成法研究

为研究二硫键成环的杂环肽FIK的合成工艺, 以Fmoc氨基酸为原料, 采用固相合成法, 经TBTU/HOBT/DIEA复合缩合剂催化合成直链肽, 再经I2氧化肽链上两个半胱氨酸的巯基生成分子内二硫键而得到目标环肽, 将其用切割试剂切割脱离树脂得到粗产品, MALDI-MS和RP-HPLC进行鉴定, 分析和纯化。产率可以达到18%, 纯化后纯度达97%以上, 经MALDI-MS和Ellman试剂检测确定为目标肽。该合成法高效, 简便, 快速, 目标肽收到较理想产率, 适合大批量生产。     

两种树脂固相合成机体保护肽的比较

目的:比较Wang树脂和二氯三苯甲基树脂(以下简称二氯树脂)在固相合成机体保护肽中的表现,为工业化生产提供理论依据。方法:通用的Fmoc固相合成法制备机体保护肽,通过比色法对两种树脂与氨基酸的偶联率进行比较,用质谱(MALDI-TOFMS)对粗肽进行鉴定分析,用反相高效液相色谱(RP-HPLC)进行粗肽的分析和纯化。结果:显示以二氯树脂作载体,第一个氨基酸的连接率,以及机体保护肽的纯度和产率都要明显高于Wang树脂。结论:二氯树脂作载体合成机体保护肽更适合大规模工业化生产的要求。     

生物活性肽SSDI固相合成工艺的研究

目的:研究生物活性肤SSDI的固相合成工艺,并为大规模合成目标肽提供理论依据.方法:采用固相合成法,原料氨基酸以Fmoe形式保护,用Wang树脂为载体,经1-氧3-双二甲胺羰基苯骈三氮唑四氟化硼盐(TBTU)\1-羟基-苯并-三氮唑(HOBt)\二-异丙基乙胺(DIEA)混合试剂缩合,20%哌啶的DMF溶液脱保护,用三氟乙酸\茴香硫醚\巯基乙醇\苯酚\水混合作为切割试剂将多肽从Wang树脂上切割下来.结果:多肽粗品的得率高达70%,经RP-HPLC纯化,可获得纯度在98%以上的目标肽,经MALDI-MS质谱鉴定其分子量与理论值一致.结论:此合成方法操作简单,产品得率高,适合大规模合成目标肽.     

固相法合成多肽AN-M的工艺研究

目的:探讨生物活性肽AN-M的固相合成工艺,并为工业化合成目的肽提供理论依据。方法采用固相法,以Fmoc-保护基保护的α-氨基酸和Wang树脂为原料,经1—氧—3—双二甲胺羧基苯骈三氮唑四氟化硼盐(TBTU)、1—羟基苯并三氮唑(HoBt)、二异丙基乙胺(DIEA)缩合,20%哌啶的DMF溶液脱保护,用切割试剂将AN-M粗品从Wang树脂上切割下来。结果经反相高效液相色谱分析纯化,可得目的肽的得率大于67．00%,最终成品的纯度在98．78%以上,经质谱鉴定其分子量与理论值一致。结论该合成方法步骤简便、便于操作、产品得率高,可用于大规模合成目的肽。     

用混合氨基酸接肽的方法合成OX型肽亚库

肽库合成是药物研究组合化学策略的重要技术之一。建立合成OX型肽亚库的固相合成方法 ,接肽反应采用等摩尔的Fmoc氨基酸混合物和DIC HOBt缩合方法 ,以高浓度的缩合剂和不断缩减溶剂的策略促进偶联反应进行完全。产物的氨基酸组成分析结果显示 ,所使用的常见氨基酸都能以相近的摩尔比例接肽至X位置。推测经多次接肽后最终形成的肽亚库中 ,含低活力氨基酸较多的肽其浓度虽然会较低一些 ,但影响不会太大 ,且本合成方法成本相对较低 ,故可为抗原表位分析、多肽药物筛选及构效关系分析提供一种有用的工具。     

解淀粉芽孢杆菌Q-426群体感应系统ComX信息素前体肽的合成研究

采用Fmoc固相合成法合成了解淀粉芽孢杆菌Q-426群体感应系统Com X信息素的前体五肽。利用反向半制备色谱(RP-HPLC)和液质联用仪(LC-MS)对合成的五肽进行了分离纯化及纯度分析。建立了检测游离氨基酸的新方法,并详细考察了合成条件对树脂上肽链连接效率的影响。结果表明,新型NNA试剂(茚三酮+正丁醇+乙酸溶液)可替代传统的Kaiser试剂用于Fmoc固相合成中游离氨基的检测;在35℃下,使用DCM和DMF对树脂交替溶胀1h后,树脂上肽链的连接效率最高。合成的Com X信息素前体五肽粗品产率为98%,纯度为58.60%;经半制备型高效液相纯化后,纯度达99%。抑菌实验表明,Com X信息素前体五肽具有明显的生物活性。     

降钙素基因相关肽的合成

降钙素基因相关肽是一个包含有37个氨基酸残基的具有较强降血压生理功能的活性多肽。我们采用常规的固相合成方法,以简单的装置最后经无水氟化氢处理,将肽从树脂上切下,同时脱除所有侧链保护基。粗产物在30％乙酸溶液中用碘作为氧化剂使二个半胱氨酸氧化,形成二硫键。合成的α-人降钙素基因相关肽经反向高效液相层析分离,获得在高效液相层析为单一峰的产物。经酸水解氨基酸分析证明与理论值相符并具有全部生理活性。     

Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry

Peptide and peptidomimetic molecule arrays are emerging powerful tools for parallel screening of binding in proteomics and pharmaceutical discovery research. Up to now the common method of preparing peptide arrays was based on spotting on glass using a library of presynthesized peptides. However, due to the large number of monomers (amino acids) it is not possible to have combinatorial libraries which include all combinations of natural and synthetic amino acids. We describe a very flexible on-chip oligopeptide synthesis method which uses the well developed t-Boc based solid state synthesis chemistry. A very high degree of flexibility is achieved by using light photo generated acids and maskless projection lithography for spatially directed deprotection. Use of microfluidic chips enables moderately high densities, short reaction times and off-the-shelf chemicals. Examples are given from synthesis of metal ion binding peptides and epitope binding assays.     

Individually addressable parallel peptide synthesis on microchips

Miniaturized, spatially addressable microchips of peptides and peptidomimetics are powerful tools for high-throughput biomedical and pharmaceutical research and the advancement of proteomics. Here we report an efficient and flexible method for the parallel synthesis of peptides on individually addressable microchips, using digital photolithography and photogenerated acid in the deprotection step. We demonstrate that we are able to synthesize thousands of peptides in a 1 cm(2) area on a microchip using 20 natural amino acids as well as synthetic amino acid analogs, with high stepwise yields and short reaction-cycle times. Epitope screening experiments using a p53 antibody (PAb240) produced clearly defined binding patterns. The peptidomimetic sequences on the microchip show specific antibody binding and provide insights into the molecular details responsible for specificity of epitope binding. Our approach requires just a conventional synthesizer and a computer-controllable optical module, thereby allowing potential development of peptide microchips for various pharmaceutical and proteomic applications in routine research laboratories.     

Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation.

A completely general method for the O-phosphorylation of peptides of any given composition using solid-phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis(benzyloxy)(diisopropylamino)phosphine and tert.-butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O-phosphopeptides of up to 15 residues in length is described.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

The amino acid sequence of plastocyanin from Vicia faba L. (broad bean)

The amino acid sequence of plastocyanin from broad bean was determined. It consists of a single polypeptide chain of 99 residues. The sequence was determined by using a Beckman 890C sequencer and by dansyl-phenyl isothiocyanate analysis of peptides obtained by the enzymic cleavage of purified cyanogen bromide fragments. Some parts of the sequence depend on the results of Edman degradation of peptides for which amino acid analyses were not obtained. The evidence for one overlap is not strong.     

Isolation and characterization of an organic solvent soluble polypeptide component from photoreceptor complexes of Rhodospirillum rubrum.

An organic solvent soluble polypeptide has been isolated from photoreceptor complexes and chromatophores of Rhodospirillum rubrum. After extraction of the protein from lyophilized samples with 1:1 chloroform-methanol, it was purified by column chromatography. Its isoelectric point determined by isoelectric focusing was 7.10. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified polypeptide ran as a single band of an apparent molecular weight of 12 000. However, according to amino acid analysis, the minimal molecular weight based on one histidine residue per polypeptide is 19 000. The polypeptide contains no cysteine and no tyrosine. Amino acid analysis indicated that three methionines were present per histidine residue and cyanogen bromide cleavage gave four smaller peptides which were isolated by two-dimensional electrophoresis and chromatography. Spectroscopic analysis indicated the presence of three tryptophan residues per histidine and N-bromosuccinamide cleavage also gave four smaller peptides which could be isolated by two-dimensional electrophoresis and chromatography. The C-terminal amino acid was shown to be glycine by two methods, while the N-terminal amino acid appears to be blocked. The organic solvent soluble polypeptide accounts for approximately 50% of the chromatophore protein and seems to bind the antenna bacteriochlorophyll and carotenoid molecules. Using this procedure, organic solvent soluble polypeptides were isolated from several photosynthetic bacteria and were found to have substantially different amino acid contents.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.     

Preparation of multifunctional and multireactive polypeptides via methionine alkylation

We report the development of a new "click"-type reaction for polypeptide modification based on the chemoselective alkylation of thioether groups in methionine residues. The controlled synthesis of methionine polymers and their alkylation by a broad range of functional reagents to yield stable sulfonium derivatives are described. These "methionine click" functionalizations are compatible with deprotection of other functional groups, use an inexpensive, natural amino acid that is readily polymerized and requires no protecting groups, and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.