甲酸铵催化转移氢化还原肽链中的芳香硝基——对氨基苯丙氨酸的间接引入

研究了用甲酸铵催化转移氢化法(AF-CTH)对不同类型肽中的芳香硝基的还原行为, 这些肽类化合物包括促黑激素(MSH: 四肽)、促黄体素释放激素(LHRH: 十肽)和强腓肽(十七肽)的类似物. 用HPLC对还原过程进行了跟踪监测, 结果显示, 除含对氯苯丙氨酸残基的LHRH类似物因发生脱氯副反应不适合用AF-CTH还原外, 其余序列还原过程中均无明显副反应发生, 硝基几乎定量地转化成为相应的氨基, 实现了对氨基苯丙氨酸向肽链的间接引入. 另外发现, 硝基还原所需的时间与肽链长度有关, 肽链越长, 还原所需时间越长, 但与其在序列中的位置关系不明显.     

含有络合功能基的非天然氨基酸的设计、合成及在生物活性肽中的应用

设计合成了一类侧链带有络合基团的非天然氨基酸, 即侧链带有N,N-二羧甲基氨甲基、N,N-二酰胺甲基氨甲基和N,N-二羟乙基氨甲基的苯丙氨酸衍生物, 并将这类非天然氨基酸用于促性腺激素释放激素(LHRH)类似物的固相合成. 高效液相色谱分析结果表明, 粗肽的纯度较好, 易于纯化; 用电喷雾质谱测定了多肽的分子量. 这些非天然氨基酸可作为其它肽类药物合成的构建单元.     

基于多肽的药物递送系统研究进展

药物递送系统是将药物输送到药物作用靶点的系统,理想的递送系统可以提高药物作用效果并降低给药剂量和毒副作用.本文综述了药物递送系统中的多肽类药物递送系统的研究进展.多肽具有易合成、易代谢、免疫原性低、毒副作用低等优点,多肽支链上大量的官能团可以和药物偶联,是药物递送系统的重要发展方向.本文从靶向肽、穿透肽、响应肽和组装肽四个方面介绍了多肽药物递送系统的原理和实例.组装肽可以形成纳米结构,显著提升多肽药物递送系统的稳定性,可以实现长效释放.组装肽体内原位调控进一步增加了多肽药物递送系统的智能型、精准性,展现出巨大的转化潜力.     

系列新LHRH拮抗剂的合成及其生物活性

本文报道了一系列在5位上含有新非天然氨基酸的促黄体素释放激素(LHRH)拮抗剂的合成及其生物活性。这些拮抗剂绝大多数都能表现出较强的抗排卵活性。以生理盐水为赋型剂,其中有些在1μg剂量下能100％抑制大白鼠排卵。     

多种酶体系用于肽类药物体外代谢性质的研究

应用反相高效液相色谱法(RP-HPLC)对肽类药物促黄体激素释放激素(LHRH)、醋酸亮丙瑞林(Leuprorelin Acetate,TAP-144-SR)和新药LXT101在6种酶体系中的体外代谢性质进行了研究.流动相采用梯度或等度洗脱;LHRH、TAP-144-SR的检测波长为216 nm,LXT101的检测波长为225 nm.采用液相色谱-电喷雾质谱(LC-ESI-MS)联用技术对其酶解片段进行了鉴定.LHRH、TAP-144-SR和LXT101的浓度在4.0～400 mg/L范围内与其峰面积呈良好线性关系(r＞0.9990);在糜蛋白酶、胰蛋白酶、胃蛋白酶和胰混合酶中的绝对回收率为95.0%～98.7%;在血浆和肝匀浆中的绝对回收率为60.0%～71.0%;日内相对标准偏差(RSD)＜1.5%,日间RSD＜2.5%;半衰期(t1/2)为LXT-101＞TAP-144-SR＞LHRH;质谱鉴定表明LHRH容易在3-4、5-6位发生断裂,TAP-144-SR容易在3-4位发生断裂;选择的酶体系可用于评价多肽药物的体外代谢性质.     

LHRH脉冲型式的变化对灌流的雄性大鼠垂体细胞LH分泌的影响

本实验应用体外分散垂体细胞动力学培养的方法,观察了LHRH脉冲幅度和频率以及LHRH连续刺激对雄性大鼠垂体前叶细胞LH分泌活动的影响。结果表明,LHRH在1×10~(-10)—1×10~(-6)mol/L范围内,与LH分泌的剂量-效应关系曲线呈线性。LHRH脉冲频率使LH分泌产生双相反应,随着刺激频率的增高,LH基础水平升高,LH/脉冲减少。当LHRH脉冲幅度在1×10~(-9)mol/L或以上,刺激频率在3脉冲/h或以上时,可观察到LH分泌达最高峰并随之逐渐下降至基础水平,即明显的自身激发作用和脱敏作用。增加LHRH的脉冲幅度可减少引起激发作用所需要的脉冲频率,增加高幅度LHRH脉冲的频率可使激发作用更快产生。另外,低浓度的LHRH(1×10~(-10)mol/L)连续刺激细胞也可产生自身激发作用,但需要较长时间的刺激。这些结果提示:垂体前叶LH细胞的分泌型式依赖于LHRH脉冲的幅度和频率,这有助于阐明体内的LH脉冲波动的动力学机理。     

低组胺释放副作用的GnRH拮抗剂的设计合成及活性评价

根据肽类组胺释放剂具有多个碱性氨基酸的结构特点, 将具有羧基结构的基团引入促性腺激素释放激素(GnRH)的不同位置, 合成了一系列新的GnRH类似物, 并进行了大鼠体内抑制睾酮释放的生物活性评价及大鼠腹腔肥大细胞中的促组胺释放副作用评价. 结果表明, 某些带有酸性基团的GnRH类似物不仅组胺释放副作用大大降低, 而且保留了原有的生物活性. 该结果为研发具有低组胺释放副作用的安全型GnRH拮抗剂药物提供了新的构效关系信息.     

合成Exendin-4的体外人血浆稳定性研究

为了解决Ⅱ型糖尿病患者注射用药频繁的问题, 需要比Exendin-4更加稳定的GLP-1的激动剂, 本文的研究目的是找到Exendin-4较易水解的氨基酸位点, 为设计新的长效的Exendin-4模拟肽提供相关信息.     

稀土谷胱甘肽配合物红外和Raman光谱研究

谷胱片肽是广泛存在于体内的重要三肽, 在体内主要以还原型(GSH)存在. GSH不但是清除体内过氧化物的还原剂, 而且与某些金属的配合物具有酶活性. 随着稀土的广泛应用, 其生物效应已引起关注. 了解稀土与GSH的配合物结构和性质, 将有利于了解稀土在体内的代谢和作用. 本文合成了十五种稀土与GSH的固体配合物,用IR和Raman光谱研究了配体在配合物中的变化及与稀土的配合位置.     

EFFECTS OF CHANGES IN PATTERN OF LHRH PULSE ON LH SECRETION BY PERFUSED ANTERIOR PITUITARY CELLS OF MALE RATS

In the present study, a perfusion system of dispersed cells was used to investigate theeffects of LHRH pulse amplitude and frequency, and LHRH continuous stimulation on LHsecretion by anterior pituitary cells of adult male rats. The results have shown that, in therange of LHRH concentrations from 1×10~(-10) to 1 ×10~(-6) mol/L, the dose-response curve of LHsecretion was linear. LHRH pulse frequency generated a biphasic LH response: increasingLHRH pulse frequency increased the basal LH secretion and decreased LH/pulse. When1 ×10~(-9), mol/L or greater LHRH was given at frequencies of 3 pulses/h or higher, it wasobserved that a maximal LH peak was induced and then the LH release declined progres-sively to its LH basal level, i.e. LHRH self-priming effect and LH desensitization occurred.Enhancement of amplitude of LHRH pulses could reduce pulse frequency required for prim-ing. Increases in frequency of LHRH pulses with high amplitude would provoke the prim-ing effect more quickly. In addition, continuou     

Design and synthesis of N-terminal cyclic motilin partial peptides: a novel pure motilin antagonist

Motilin antagonist was designed and synthesized on the basis of the structure-activity relationship analysis of porcine motilin that we reported recently. The drug design was performed on a specific concept to reduce a flexibility of peptide conformation of porcine motilin partial peptide by its cyclization. The cyclic peptide was synthesized using Boc (tert-butyloxycarbonyl) solid phase methodology, followed by cyclization using the azide procedure, and tested for the binding activity to motilin receptor and smooth muscle contractile activity. The cyclic peptides 3, 4, and 5 showed antagonistic property on contraction assay (pA2 [the negative logarithm of molar concentration of antagonist causing a 2-hold shift to the right of the concentration-response curve for motilin]: 4.5, 4.34, and 4.04, respectively, in rabbit duodenum) and no contractile activity even at high concentration.     

A Peptide Stapling Strategy with Built-In Fluorescence by Direct Late-Stage C(sp2)−H Olefination of Tryptophan

Fluorescent stapled peptides are important chemical tools for detecting intracellular distribution, protein–protein interactions, and localization of target proteins. These peptides are usually labeled with bulky sized fluorophores through reactive functional groups, which may alter the physical properties and biological activities of peptides. Herein, a unique strategy is developed for synthesizing new stapled peptides with built-in fluorescence. The stapled peptides were prepared through Rh-catalyzed C(sp²)−H olefination in tryptophan (Trp) residues by using pyridine/pyrimidine as the directing groups under mild conditions. This method displays good regioselectivity and high efficiency. Furthermore, as a proof of concept for its biological applications, stapled peptides without additional fluorophore 9 a and 9 b were constructed for a cell imaging study. These peptides displayed strong binding affinity toward integrin αvβ3 in A549 cells by cell imaging experiments. Notably they demonstrated even better anticancer activity than commercial antagonist cyclic (RGDfK). The method will provide robust tools for the peptide macrocyclization field.     

An evolution-inspired strategy to design disulfide-rich peptides tolerant to extensive sequence manipulation

Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.

A general method was developed to design multicyclic peptides with diverse disulfide frameworks amenable to random peptide library design, enabling the development of new disulfide-rich peptide ligands and therapeutics with structures not derived from natural peptides.     

Ab Initio Design of Potent Anti-MRSA Peptides Based on Database Filtering Technology

To meet the challenge of antibiotic resistance worldwide, a new generation of antimicrobials must be developed. (1) This communication demonstrates ab initio design of potent peptides against methicillin-resistant Staphylococcus aureus (MRSA). Our idea is that the peptide is very likely to be active when the most probable parameters are utilized in each step of the design. We derived the most probable parameters (e.g., amino acid composition, peptide hydrophobic content, and net charge) from the antimicrobial peptide database (2) by developing a database filtering technology (DFT). Different from classic cationic antimicrobial peptides usually with high cationicity, DFTamP1, the first anti-MRSA peptide designed using this technology, is a short peptide with high hydrophobicity but low cationicity. Such a molecular design made the peptide highly potent. Indeed, the peptide caused bacterial surface damage and killed community-associated MRSA USA300 in 60 min. Structural determination of DFTamP1 by NMR spectroscopy revealed a broad hydrophobic surface, providing a basis for its potency against MRSA known to deploy positively charged moieties on the surface as a mechanism for resistance. Our ab initio design combined with database screening (3) led to yet another peptide with enhanced potency. Because of the simple composition, short length, stability to proteases, and membrane targeting, the designed peptides are attractive leads for developing novel anti-MRSA therapeutics. Our database-derived design concept can be applied to the design of peptide mimicries to combat MRSA as well.     

Discovery of antagonist peptides against bacterial helicase-primase interaction in B. stearothermophilus by reverse yeast three-hybrid

Developing small-molecule antagonists against protein-protein interactions will provide powerful tools for mechanistic/functional studies and the discovery of new antibacterials. We have developed a reverse yeast three-hybrid approach that allows high-throughput screening for antagonist peptides against essential protein-protein interactions. We have applied our methodology to the essential bacterial helicase-primase interaction in Bacillus stearothermophilus and isolated a unique antagonist peptide. This peptide binds to the primase, thus excluding the helicase and inhibiting an essential interaction in bacterial DNA replication. We provide proof of principle that our reverse yeast three-hybrid method is a powerful "one-step" screen tool for direct high-throughput antagonist peptide selection against any protein-protein interaction detectable by traditional yeast two-hybrid systems. Such peptides will provide useful "leads" for the development of new antibacterials.     

Sequence Decoding of 1D to 2D Self-Assembling Cyclic Peptides

The inherent ability of peptides to self-assemble with directional and rationally predictable interactions has fostered a plethora of synthetic two-dimensional (2D) supramolecular biomaterials. However, the design of peptides with hierarchical assembly in different dimensions across mesoscopic lengths remains a challenging task. We here describe the structural exploration of a d /l -alternating cyclic octapeptide capable of assembling one-dimensional (1D) nanotubes in water, which subsequently pack laterally to form giant 2D nanosheets up to 500 μm long with a constant 3.2 nm thickness. Specific amino acid mutations allowed the mapping of structure–assembly relationships that determine 2D self-assembly. Nine peptide modifications were studied, revealing key features in the peptide sequence that nanosheets tolerated, while a total of three peptide variants included modifications that compromised their 2D arrangement. These lessons will serve as guide and inspiration for new 2D supramolecular peptide designs.     

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.     

Approaches to Synthetic Vaccines Design of Epitope-Containing Amphiphilic Peptides Matching the Antigenic Structure in the Native Protein

A general procedure for the design of synthetic vaccines with the retained conformational features of protein antigenic determinants is described. This new concept emerges from detailed studies on the relationship between primary sequence and secondary structure formation of synthetic peptides and takes advantage of the amphiphilic nature of epitope-containing peptide segments in the native protein to accomplish structural modifications. These segments, for example amphiphilic helices or β-sheets, are stabilized by the insertion of secondary structure-inducing amino-acid residues on the hydrophobic part of the peptide without affecting the spatial arrangement of functional residues on the hydrophilic side. The availability of amphiphilic peptides with tailor-made conformational properties, e.g. helices, β-sheets, and, moreover, assemblies of these blocks to structures of higher order (‘folding units’), allows the presentation and stabilization of continuous as well as discontinuous epitopes by this approach. This strategy is exemplified for the case of two discontinuous epitopes taken from lysozyme, which are matched to host molecules with adequate conformational features by the help of computer-assisted molecular modelling. The implications of this new concept for the design of synthetic vaccines are discussed with special emphasis to the important role of peptide synthesis and chemical structure modification.