片段法合成抗骨质疏松多肽药物特立帕肽

将特立帕肽序列中的34个氨基酸分成4个片段:1-11、 12-16、 17-24和25-34;以Wang Resin（王树脂）为固相载体制得25-34肽树脂;以CTC Resin（2-氯三苯甲基氯树脂）为固相载体制得1-11、 12-16和17-24等3个片段的全保护肽,然后将3个片段的全保护肽按照肽序依次缩合到25-34的肽树脂上,经三氟乙酸切割并脱除侧链保护基得特立帕肽粗品,再经液相色谱纯化得特立帕肽,纯度大于99%,总收率达33%,其结构经MS(ESI)确证。     

Merrifield树脂负载的固相法合成1-氨基-2,4-咪唑二酮

设计了两条新的固相有机合成路线合成了1-氨基-2,4-咪唑二酮化合物4. 一条是由Merrifield树脂负载的羟基苯甲醛1a～1c和氨基脲反应得到缩氨基脲树脂2a～2c, 再在乙醇钠存在下和氯乙酸乙酯成环, 经盐酸切割得到1-氨基-2,4-咪唑二酮; 另一条是将Merrifield树脂用二甲基亚砜氧化氯甲基末端醛化后, 与氨基脲反应得到负载的缩氨基脲6, 经环化、切割得到目标产物4. 这两种方法中用1 mol/L的盐酸代替三氟乙酸作为切割剂, 产物单一、操作简便、可定量反应, 是合成1-氨基-2,4-咪唑二酮化合物的新方法.     

阿基瑞林制备方法的改进研究

阿基瑞林,又称乙酰基六肽-8、六胜肽等。它作为肉毒毒素的安全替代品,具有显著的抗皱活性,是美妆市场上需求潜力很大的抗皱多肽原料。当前阿基瑞林主要是通过固相合成方法得到的,由于所使用的Rink Amide AM树脂单价高、上载量低,造成了阿基瑞林原料价格高昂,不利于其大规模生产及推广应用。本文中使用固-液相结合的方法,首先用高上载量、单价低的2-CTC树脂合成六肽片段,六肽片段再经酰胺化处理得阿基瑞林粗品,粗品经纯化后得纯度95%以上的阿基瑞林,收率59.77%。本方法成本低、收率高,为C端酰胺化多肽的合成提供了一种新思路。     

六胜肽片段的液相合成及组装研究

六胜肽是一种药妆肽,序列为Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2,是相对安全的类肉毒素活性物质.拟用液相方法分别合成片段A[Ac-Glu(Ot Bu)-Glu(Ot Bu)-Met-OH]和片段B[H-Gln(Trt)-Arg-Arg-OH],然后将两个片段在固相载体上进行组装,经切割、纯化得到六胜肽产品,纯度大于98%,片段组装的收率为65%,并对组装反应的条件进行分析和讨论.该方法结合了固相和液相合成的优点,是一种成本低廉、收率较高和周期较短的六胜肽合成方法.     

碳酸酯把手改构Wang树脂及其性能

基于碳酸酯结构易于亲核脱除的原理, 使用氯甲酸氯甲酯改构Wang树脂, 并探究了改构树脂与首位氨基酸的缩合效率. 实验结果表明, 改构后的Wang树脂与20种Fmoc保护氨基酸均能达到70%以上的缩合效率; 且对于带有较大侧链基团的Fmoc保护氨基酸, 通过降低树脂取代度或延长反应时间可提高其连接率. 为了验证改构后的碳酸酯型树脂在裂解时侧链未受到影响, 设计合成了3种模型肽, 并用温和裂解剂3-吡啶甲醛肟铯盐进行裂解. 实验结果表明, 利用改构树脂能得到侧链全保护的肽片段, 可初步应用到长链困难肽的合成中.     

三七水煎液中还原型谷胱甘肽及腺嘌呤的分离与鉴定

三七经水煎提取,阴、阳离子交换树脂分离以及RP-HPLC分离获得多种含肽类化合物组分,采用游离及酸水解后氨基酸组成及含量对照分析的方法预示了提取液中肽类化合物的存在,经氨基酸组成分析、聚丙烯酰胺薄膜双向层析、羧肽酶C-末端序列分析、质谱测定等方法分析,将YN-3H12组分鉴定为还原型谷胱甘肽,YN-3H11组分鉴定为腺嘌呤,本研究方法可为植物中水溶性肽类化合物等活性成分的研究提供参考。     

抗肿瘤环八肽Samoamide A的固相合成及其细胞毒性

采用Fmoc固相合成法,以2-氯三苯甲基氯（CTC）树脂为固相载体,Fmoc保护的氨基酸为原料,合成了环八肽Samoamide A,其结构经¹H NMR, ¹³C NMR和LC MS（EI）确证。研究了反应溶剂、缩合剂、切割条件和pH对Samoamide A收率的影响。结果表明：合成Samoamide A的最佳条件为：60%DCM/DMF为溶剂,O-苯并三氮唑-N,N,N′,N′-四甲基脲四氟硼酸(TBTU)为缩合剂,TFA/EDT/PhOH/H₂O/硫茴香醚为切割试剂(80/2.5/7.5/5/5, V/V/V/V/V), pH 8.0,总收率54.5%。采用MTT法研究了Samoamide A的细胞毒性。结果表明：Samoamide A浓度为50.0 μg·mL^-1时,4T1细胞的存活率为20.79%。     

从国产紫胶树脂中分离鉴定壳脑酸

本文利用紫胶树脂碱水解后,其链状与环状组份性质的差别,首次从国产紫胶树脂中分离得到一种无色透明的立方晶体,经光谱证实为壳脑酸二甲酯。     

用于肽合成的肟铯盐裂解剂的制备及性能

以获得侧链全保护的多肽为总目标,选择接入溴乙酰基把手的Wang树脂作为固相载体,采用多肽固相合成法制备了3种模型肽,将其用于对具有弱碱性和强亲核性的肟盐类裂解剂的研究;设计并制备了5种不同结构的肟铯盐,将其用于模型肽的裂解,并选择综合效果最好的2-吡啶甲醛肟铯盐进行裂解条件的优化.结果表明,肟铯盐类裂解剂可以温和、高效地裂解模型肽,得到侧链全保护的多肽.     

一种有机膦树脂的合成及在锂镁分离中的应用

本文以线性酚醛树脂为母体,其经溴甲基化、三乙氧基磷亲核取代及Arbuzov重排反应,合成一种有机膦树脂。该树脂经红外,电镜扫描,热重等方法进行了表征。另外,通过Gauss软件对产物结构单元类似物的单体进行频率计算,得到其理论计算红外图谱,与产物红外图谱进行比对,表明对酚醛树脂高分子骨架上官能团改造是成功的,合成了一种有机膦树脂。用该树脂对锂镁分离进行了简单的分离实验,结果表明该有机膦树脂对锂镁吸附的分离系数(β)为2.3。     

2‐Methoxy‐4‐methylsulfinylbenzyl: A Backbone Amide Safety‐Catch Protecting Group for the Synthesis and Purification of Difficult Peptide Sequences

The use of 2‐methoxy‐4‐methylsulfinylbenzyl (Mmsb) as a new backbone amide‐protecting group that acts as a safety‐catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid‐mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi‐unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH₄I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the “difficult peptide” sequence H‐(Ala)₁₀‐NH₂ was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac‐(RADA)₄‐NH₂ sequence was chosen to validate this methodology. The improvements in solid‐phase peptide synthesis combined with the enhanced solubility of the quasi‐unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac‐(RADA)₄‐NH₂. To extend the scope of the approach, the challenging Aβ(1‐42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.     

Preparation of a core-shell type MBHA resin and its application for solid-phase peptide synthesis

MBHA (4-methylbenzhydrylamine) resin has been extensively used as a solid support for the synthesis of peptide amides. Herein, we prepared the core-shell-type MBHA resin by benzotriazole-catalyzed amidoalkylation and partial hydrolysis. The core-shell structure of the MBHA resin was confirmed by two-photon microscopy and its synthetic performance in solid-phase peptide synthesis (SPPS) was evaluated.     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

SPED-(styrene-polyethyleneglycol diacrylate-9-decen-1-ol): a novel resin for solid phase peptide synthesis; synthesis and characterization of biologically potent endothelin classes of peptides

Here we present a novel beaded chemically stable, highly permeable hydrophobic/hydrophilic balanced support for solid phase peptide synthesis. The resin (SPED) was prepared by free radical suspension polymerization using monomers styrene and 9-decen-1-ol with amphiphilic cross-linking agent polyethyleneglycol diacrylate (Mn 258). Different cross-linking densities were prepared to check the extent of swelling in different polar and non-polar solvents. The SPED resin was characterized with IR, 13C NMR and surface by SEM. The chemical stability of the support in various peptide synthetic conditions was investigated and monitored by IR spectroscopy. To evaluate the applicability of the new resin in synthetic conditions more challenging peptide sequence of retro-ACP (74-65) was synthesized and compared to commercially available Merrifield resin. The efficiency of SPED was further confirmed by synthesizing biologically important endothelin family of peptides in high yield and purity. The purity of all peptides was checked by RP-HPLC and mass by MALDI-TOF MS.     

Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin

The NY-ESO-1 (A39-A68) peptide hydrazide was prepared through 9-fluorenyl-methoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) from a new 9-fluorenyl-methoxycarbonyl hydrazine 2-chlorotrityl chloride (Fmoc-hydrazine 2CTC) resin. The new resin was ideal for long-term storage and usage in Fmoc SPPS. Besides, the title peptide hydrazide could be transformed nearly quantitatively into the corresponding peptide thioester, which was both isolable and usable directly in native chemical ligation (NCL).     

On-resin native chemical ligation for cyclic peptide synthesis

A novel cysteine derivative, N(alpha)-trityl-S-(9H-xanthen-9-yl)-l-cysteine [Trt-Cys(Xan)-OH] has been introduced for peptide synthesis, specifically for application to a new strategy for the preparation of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i). side-chain anchoring of Fmoc-Asp-OAl via its free beta-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii). stepwise chain elongation of the peptide by standard Fmoc/tBu solid-phase chemistry; (iii). removal of the N-terminal Fmoc group; (iv). coupling of Trt-Cys(Xan)-OH; (v). selective Pd(0)-promoted cleavage of the C-terminal allyl ester; (vi). coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii). selective removal of the N(alpha)-Trt and S-Xan protecting groups under very mild acid conditions; (viii). on-resin cyclization by native chemical ligation in an aqueous milieu; and (ix). final acidolytic cleavage of the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR), and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the desired cyclic product was obtained in 76-86% overall yield with initial purities of approximately 70%, whereas for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native chemical ligation/cyclization methodology appears to have advantages of convenience and specificity, which make it promising for further generalization.     

Identification of a potent peptide deformylase inhibitor from a rationally designed combinatorial library

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).     

A new method for the preparation of 2-chlorotrityl resin and its application to solid-phase peptide synthesis

2-Chlorotritylchloride (2-CTC) resin was prepared efficiently from 1% DVB-crosslinked polystyrene resin and 1-chloro-2-(dichloro(phenyl)methyl)benzene, which was easily obtained from 2-chlorobenzophenone. This 2-CTC resin showed excellent properties as a support for solid-phase peptide synthesis. Four peptide fragments were obtained in high purity using the resin.     

Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography

Chromatographic resins of a family of linear Fc-binding hexamer peptides (HWRGWV, HYFKFD, and HFRRHL) exhibited the ability to selectively adsorb and isolate human IgG (hIgG) from complete mammalian cell culture medium (cMEM). Among them, the HWRGWV resin with a peptide density of 0.08 mequiv./g of resin was able to purify hIgG from cMEM with both purity and yield as high as 95%, comparable to Protein A and A2P agarose gels. The influences of N-terminal acetylation of the HWRGWV resin, ligand density on the resin, initial hIgG concentration, and temperature on IgG isolation were also investigated. The results indicate that these small peptide ligands, especially HWRGWV, offer a potential alternative to the use of Protein A or Protein G for large scale affinity chromatography.