脱苏氨醇奥曲肽的微波辅助合成

利用自制100 ml微波反应管通过微波辅助固相肽合成技术制得奥曲肽原料药中关键有关物质脱苏氨醇奥曲肽(1)。采用Fmoc策略以2-Cl-Trt-树脂为固相载体,经上载、缩合、脱保护、裂解等操作合成还原型脱苏氨醇奥曲肽,再用空气氧化法并加入活性炭快速氧化得到1,总收率62.9%,单批次产量达3.5 g,中间体及终产物经高分辨质谱及氨基酸分析确证,并通过圆二色谱法研究了环合前后多肽构象的变化。该方法比传统固相合成方法快捷,为研究奥曲肽的关键有关物质提供了可行的合成方法。     

抗肝癌药物酪丝亮肽的工艺研究

［摘要］目的 探索一类抗肝癌新药酪丝亮肽的固相合成路线,并与申报的液相合成路线进行优缺点比较. 方法 采用Fmoc/Wang树脂固相合成方法,逐步接肽,然后用裂解液（TFA：Tis:H2O,95.0：2.5：2.5）从树脂切割得到粗品,最后制备液相纯化脱盐,冻干得到产品. 结果 终产品经质谱、磁共振检测确证,纯度为99.5%,产率为82.0%. 结论 Fmoc固相合成方法可以得到与液相合成一致的产品,相比液相路线,该合成方法便捷易操作,生产周期短,产率高,但是成本相对较高.     

奥曲肽固相合成工艺研究

目的优化奥曲肽固相合成工艺。方法本研究采用Fmoc保护基固相合成策略合成奥曲肽直链肽[D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol)],并在此基础上进行裂解、成环、纯化、制备奥曲肽。本研究着重对裂解条件进行优化,包括裂解液成分、比例、反应温度和时间等。结果与结论以TFA∶TIS∶H_2O∶DTT=90∶2.5∶2.5∶8为裂解体系,在10℃下反应2～3 h得到纯度92%(HPLC)的直链肽粗品。本文优化的裂解条件提高了奥曲肽直链肽纯度,同时避免了乙二硫醇的使用,使得生产过程对环境更加友好,更适合工业化生产。     

47肽的微波辅助固相合成工艺研究

目的合成一长链肽47肽。方法采用Fmoc/t-Bu正交保护多肽固相合成策略,使用9-芴甲氧羰基(Fmoc)保护氨基酸的α-氨基,用Liberty微波多肽合成仪,每个耦合循环用20%哌啶脱除Fmoc保护基团,连续添加氨基酸,并用茚三酮法检测反应终点,有效检测反应的进度,最后用82.5%的三氟乙酸将多肽从树脂上切割下来,得到47肽粗品。经过反相高效液相制备系统纯化得到多肽纯品,用反相高效液相分析仪和傅里叶高分辨质谱仪验证该产物。结果高分辨质谱仪验证了该产物,纯品质量分数达98%。结论微波可促进多肽特别是长链肽的合成。     

西曲瑞克的固相合成

目的 优化西曲瑞克的固相合成条件及纯化方法。方法 采用Fmoc固相合成法,以Rink Amide-AM Resin为固相载体,以DIC/HOBt或DIC/HOAt为缩合剂,采用制备型反相高效液相色谱法进行纯化。 结果 合成西曲瑞克粗肽,粗品纯度为94.8%。粗肽经制备型反相高效液相色谱纯化,所得精肽的纯度高达99%,总收率为62%。结论 该合成方法简单易行,产品纯度及收率都很高,适合用于西曲瑞克的工业化生产。     

芋螺毒素B179的化学合成及其氧化折叠的研究

目的合成新型芋螺毒素B179,并对其氧化折叠进行研究。方法采用Fmoc固相合成法合成芋螺毒素多肽B179,研究了不同的氧化缓冲液、谷胱甘肽浓度、还原型与氧化型谷胱甘肽的比例、以及温度对氧化折叠的影响。结果采用Fmoc固相合成法可以成功合成线性肽,其氧化折叠产物中主要为含一对二硫键的产物,但还出现了其他副产物,可能是含有分子间二硫键的折叠产物;在高浓度还原型谷胱甘肽环境下,折叠副产物更易形成;随着氧化反应温度升高,折叠反应速率加快。结论固相合成法可以有效合成芋螺毒素B179,且不同因素对其二硫键的形成有着较大的影响,本研究结果可为新型芋螺毒素的合成及其氧化折叠提供借鉴。     

固相合成血管紧张素Ⅱ及其反相高效液相色谱纯化与分析

目的用固相方法合成血管紧张素Ⅱ,建立其纯化和分析条件。方法以Fmoc-Phe-Wang树脂为固相载体,采取Fmoc/tBu正交保护固相合成策略,粗品的纯化采用制备型RP-HPLC。结果经过分析型RP-HPLC分析最后纯品的纯度>98%,并用电喷雾接口质谱分析证实了分子质量的正确性。结论用Fmoc固相法合成血管紧张素Ⅱ快速简便、操作方便而且回收率很高,最后通过RP-HPLC纯化,可以得到纯度很高的血管紧张素Ⅱ。     

作为乙肝疫苗的新型结构肽的设计与合成

在乙肝病毒表面抗原结构研究的基础上,自行设计了三个新型结构的肽疫苗,采用Boc化学和Fmoc化学相匹配的策略,应用固相肽逐步合成法合成,产物经氨基酸分析鉴定。     

人胸腺肽α1的固相合成及体外活性研究

目的对胸腺肽α1的固相合成工艺进行研究,并对合成产物进行活性评价.方法采用对碱敏感的Nα-芴甲氧羰基(Fmoc)作为α-氨基的保护基,以Fmoc-Asn(Trt)-Wang Resin为起点,逐个延伸固相合成法合成胸腺肽α1.与Nα-Fmoc-基团配套的保护策略还有:叔丁氧羰基(Boc)保护Lys的侧链氨基,叔丁酯基(OtBu)保护Asp、Glu的侧链羧基,叔丁氧基(tBu)保护Ser、Thr的侧链羟基,三苯甲基(Trt)保护Asn的侧链酰胺基.采用DCC-HOBt缩合剂法进行接肽反应,茚三酮定性显色法和水杨醛定量自由氨基检测法控制反应进程,肽段最后用TFA-DCM(V∶V=1∶1)定量地从树脂上切除.结果胸腺肽α1总产率为33.2%.纯化后的产物,经SDS-PAGE和RP-HPLC鉴定,纯度为98.8%以上,活性与日达仙对照品相当.结论 Nα-芴甲氧羰基保护策略的固相合成方法操作简便、条件温和、副反应少,产品纯度高、收率高.     

固相片段法合成利拉鲁肽

目的 研究利拉鲁肽的固相合成。方法 通过汇聚式合成策略，利用固相片段缩合法制备利拉鲁肽。以芴甲氧羰基(Fmoc)保护的氨基酸为原料，分别合成4个全保护多肽片段，依照肽序依次偶联得到全保护利拉鲁肽主链[GLP-1(7-37)],经缩合反应在第20位赖氨酸引入侧链N^α-棕榈酰基-L-谷氨酰基，最后将其从树脂上切割并脱保护得到利拉鲁肽。结果与结论利拉鲁肽粗肽收率为25.65%,纯度为52.35%。采用制备型反相高效液相色谱技术进行分离纯化后，利拉鲁肽纯度为98.42%。该合成工艺反应条件温和、操作简单、合成周期短，是实验室规模下较好的利拉鲁肽制备方法。     

Application of the allyloxycarbonyl protecting group for the indole of Trp in solid-phase peptide synthesis*

The synthesis and stability of allyloxycarbonyl (Aloe) indole-protected Trp derivatives and their application in solid-phase peptide synthesis are reported. The study shows that the Aloe protection on the indole moiety is suitable for orthogonal protection in the Fmoc/tBu strategy if the Fmoc group is cleaved with DBU. Several tryptophan-containing peptides have been synthesized including dynorphin A-(1-13), which has been intensively studied with respect to side reactions during the final TFA cleavage procedure. The results demonstrate the protective function of the Aloe group on the Trp during final deprotection. Furthermore, it could be demonstrated that Trp(Aloe)-containing peptides can be isolated and that the Aloe group can then be removed in a second step. The synthesis of phosphorylated delta sleep inducing peptide (P-DSIP) using the global phosphorylation approach provides another example in which Trp indole protection by Aloe prevents the formation of oxidative side products.     

Synthesis of hydrazinopeptides using solid-phase N-electrophilic amination: extension to the Fmoc/tert-butyl strategy and chemistry of the N–N bond in strong acid media

Abstract: The synthesis of hydrazinopeptides using solid-phase N-electrophilic amination was extended to the Fmoc/tert-butyl strategy. Both Boc/benzyl and Fmoc/tert-butyl strategies led to the isolation of by-products arising from the partial instability of the N–N bond during the final cleavage and deprotection step. Two paths of decomposition have been shown: the cleavage of the N–N bond leading to the regeneration of the amine and a Hofmann-type elimination yielding original dianisyl adducts. Our data suggest that the Fmoc/tert-butyl strategy is better suited for the synthesis of hydrazinopeptides.     

Cyclic disulfide analogues of the complement component C3a Synthesis and conformational investigations

The flexible C-terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C-terminus, as well as to examine a synthetic approach to potential C3a-antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open-chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF-labile 4(methoxy)benzyl group (Mob) for β-thiol protection of cysteine; in contrast, the TFA-stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB-MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (<0.1% C3a activity). N-terminal acylation with cationic, arginine-rich sequences like YRRGR- led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGR°CGGLCLAR (P5) and YRRGRAhx°CGGLCLAR (P8), point in the direction of C3a antagonists.     

Ugi四组分缩合反应合成肽核酸单体

目的为了改善肽核酸的理化和生物学性质，对经典肽核酸的结构进行衍生化和修饰。方法采用Ugi四组分缩合反应进行PNA单体的合成并采用Fmoc的保护策略。结果合成了Fmoc保护的PNA单体和关键组分异腈。结论建立了一种简单，灵活多变的合成PNA单体的方法。     

Chemical phosphorylation of the peptides GGXA (X = S,T, Y): an evaluation of different chemical approaches

An evaluation was made of the two methods most commonly used for phosphorylation of hydroxyamino acids in peptides, i.e. the tetrazole-catalysed phosphitylation by di- tert-butyl- N,N-diethylphosphoramidite followed by oxidation and the phosphorylation by dibenzylphosphochlondate. As model system the sequence GGXA (X = S, T, Y) was used which represents a random-coil sequence avoiding the influence on the reaction kinetics of secondary structure formation. In the case of serine- and threonine-containing peptides, both synthetic methods gave comparable yields of the desired phosphopeptides. The phosphorylation of tyrosine was achieved more favorably via the phosphoramidite method. However, phosphotyrosine peptides are most easily obtained by peptide synthesis using Fmoc-Tyr(PO₃Me₂)OH as building block. The dibenzylphosphochloridate method yields the expected phosphopeptides as the only peptide derivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidite method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged derivative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatography, ³¹P- and ¹H-NMR spectroscopy, and ion-spray and electrospray mass spectrometry. Interestingly, even these mild ionisation techniques resulted in partial fragmentation. The observed fragmentation pathways seem to be a diagnostic tool for the identification of phosphorylation sites in peptides. Both the phosphorylated serine and threonine peptide lost phosphoric acid (98 mass units), the tyrosine peptide lost phenyl phosphate (174 mass units). Fragmentation of the dephosphorylated peptides in MS/MS experiments yielded fragments of the masses 69.1u and 83.1u, corresponding to dehydroalanine derived from phosphoserine and α-aminodehydrobutyric acid, derived from phosphothreonine. © Munksgaard 1995.     

Preparation of protected peptide amides using the Fmoc chemical protocol

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH₂ (1) was synthesized on both the acid-labile 4-(2′,4’-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanoll, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Use of the 3-nitro-2-pyridine sulfenyl protecting group to introduce Nε-branching at lysine during solid-phase peptide synthesis I. Application to the synthesis of a peptide template containing two addressable sites

TASPs (template-assembled synthetic peptides) are generated by the covalent attachment of linear peptides to a common peptide backbone, thus generating larger synthetic peptides/proteins with prefolded structure. In this work we present a strategy for the synthesis of a heterotemplate-assembled synthetic peptide containing two addressable sites. This orthogonal protection strategy would allow the selective introduction of different peptide chains via the ε-amino functions of template lysines being protected by either fluorenylmethoxycarbonyl (Fmoc) or 3-nitro-2-pyridine sulfenyl (Npys) groups. The N^α-Boc-N^ε-Npys-l -lysine required for solid-phase peptide synthesis (SPPS) is not readily available at a reasonable cost. To facilitate the more widespread use of this reagent we have compared the two published procedures for synthesizing this protected amino acid and evaluated the suitability of the products for SPPS. Two resin-bound peptides, a tripeptide Ac-G-K-Npys)-G-resin and an octapeptide template Ac-P₁-K₂-K₃-L₄-K_s-K₆-P₇-G₈-resin, were synthesized by SPPS. The ε-amino functions of lysines K₂ & K₆ and K₃ & K₅ of the octapeptide were protected by Fmoc and Npys groups, respectively. Secondly, these peptides were used to evaluate various reagents and reaction conditions for the deprotection of the ε-amino function of lysines bearing the N_ε-Npys protecting group. A procedure for the optimized selective and quantitative deprotection of the Npys group from the ε-amino function of lysine in a resin-bound peptide using 2-mercaptopyridine-N-oxide is described. © Munksgaard 1995.     

Synthesis of disulfide-bridged fragments of ω-conotoxins GVIA and MVIIA

The 3-nitro-2-pyridinesulphenyl (Npys) moiety is finding increasing utility as a protecting-activating group for cysteine, particularly in the synthesis of cyclic and unsymmetrical disulfides using the Boc strategy. This chemistry has been extended to peptides assembled by the Fmoc strategy. N-Terminal Cys(Npys) is introduced via Boc-Cys(Npys)-OPfp. Non-N-terminal Cys(Npys) is incorporated by reacting a resin-bound, fully protected Cys(Acm) peptide with NpysCl. This approach has been applied to the synthesis of four disulfide-bridged fragments of ω-conotoxins GVIA and MVIIA.     

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model

Abstract: Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C‐terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI‐TOF‐MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.     

Synthesis and immunogenic properties of phosphopeptides related to the human insulin receptor

Abstract: Two phosphoserine tetradecapeptides corresponding to sequences 987–1000 (peptide pSer⁹⁹⁴) and 1017–1030 (peptide pSer^1023/1025) from the human insulin receptor involved in the regulation of its activity were successfully synthesized using Fmoc‐based chemistry. Phosphorylation was performed by post‐assembly phosphitylation followed by oxidation. The selective phosphorylation of Ser residues was achieved incorporating into the peptide chain the Ser (Trt) derivative and t‐Bu blocking groups at sites other than those intended to be phosphorylated. The Trt group was selectively removed with dichloroacetic acid while under this condition t‐Bu protecting groups remained unaltered. Following conjugation to keyhole limpet hemocyanin phosphopeptides were used as immunogens to generate sequence‐specific phosphoserine antibodies. Peptide pSer⁹⁹⁴ induced antibodies in New Zealand white rabbits which discriminated between the phosphorylated and nonphosphorylated forms of the peptide, thus representing promising candidates to recognize signaling pathways associated to the regulation of the human insulin receptor.