虚拟原子探针随机采样法用于氨基酸结构描述及其构效关系研究

考虑药物与蛋白质受体的3类非键作用模式, 利用8类虚拟原子探针和Monte Carlo随机采样技术, 得到了一套新的氨基酸侧链表面静电、立体及疏水势能场(ASSPF)参数. 在此基础上对苦味二肽和血管舒缓激五肽进行了结构表征和QSAR研究, 所建模型复相关系数R2和留一法交互检验复相关系数QLOOCV2分别为0.8457, 0.851和0.7688, 0.7952, 同时分析了肽链不同位置上氨基酸侧链对活性的影响, 取得较好的结果.     

Performance of columns packed with the new shell particles,Kinetex-C18

The performance of the new Kinetex-C₁₈ column was investigated. Packed with a new brand of porous shell particles, this column has an outstanding efficiency. Once corrected for the contribution of the instrument extra column volume, the minimum values of the reduced plate heights for a number of low molecular weight compounds (e.g., anthracene and naphtho[2,3-a]pyrene) were between 1.0 and 1.3, breaking the legendary record set 3 years ago by Halo-C₁₈ packed columns. The liquid-solid mass transfer of proteins (e.g., insulin and lyzozyme) is exceptionally fast on Kinetex-C₁₈ much faster than on the Halo-C₁₈ column. The different contributions of dispersion and mass transfer resistances to the column efficiency were determined and discussed. The possible reasons for this extremely high column efficiency are discussed.     

Assay of bradykinin-related peptides in human body fluids using capillary electrophoresis with laser-induced fluorescence detection

A CE with LIF detection was developed for separation and determination of bradykinin (BK)-related peptides, such as BK, kallidin (Kal), and neurokinin A (NKA). BK-related peptides were derivatized with FITC prior to CE-LIF analysis. Sodium borate 10 mmol/L at pH 9.5 was selected as derivatization media in order to get the high efficiency. Three peptides were baseline-separated within 10 min by using 110 mmol/L sodium borate-sodium hydroxide solution at pH 10.0 as the running buffer. Concentration detection limits (S/N = 3) for BK, Kal, and NKA were 0.08, 0.5, and 0.2 nmol/L, respectively. Meanwhile we have also developed a simple, quick, and sensitive large-volume sample stacking (LVSS) technique for CE-LIF detection of BK, Kal, and NKA. By using this stacking technique, the detection limits (S/N = 3) for BK, Kal, and NKA were 0.02, 0.05, and 0.04 nmol/L, respectively. This method has been applied to the assay of human saliva and cerebrospinal fluid with satisfactory results.     

Surface-enhanced Raman studies of bradykinin using colloidal gold

In this paper, bradykinin (BK), an endogenous peptide hormone, which is involved in a number of physiological and pathophysiological processes was deposited onto the colloidal Au nanoparticles. The surface-enhanced Raman spectroscopy (SERS) was used to determine the adsorption mode of BK under different environmental conditions, including: excitation wavelengths (514.5 nm and 785.0 nm), pH of aqueous sol solutions (from pH = 3 to pH = 11), and size of the colloidal nanoparticles (10, 20, and 50 nm). The metal surface plasmon of the colloidal suspended Au nanoparticles was examined by ultraviolet-visible (UV–vis) spectroscopy. The results showed that the C-terminal part of BK plays a crucial role in the adsorption process onto the colloidal suspended Au particles. The Phe^5/8 and Arg⁹ residues of BK mainly participate in the interactions with the colloidal Au nanoparticles. At acidic pH of the solution (pH = 3), the BK COO⁻ terminal group through the both oxygen atoms strongly binds to the Au nanoparticles. The Phe⁵/Phe⁸ rings adopt tilted orientation with respect to the colloidal Au nanoparticles with diameters of 10 and 20 nm. As the particle size increases to 50 nm, the flat orientation of the Phe ring(s) with respect to the Au nanoparticles is observed.     

QSAR based modeling on a series of α-hydroxy amides as a novel class of bradykinin B1 selective antagonists

G-protein coupled receptors like Bradykinin (BK) B₁ represent a potential treatment route for chronic pain and inflammation. Quantitative structure activity relationship has been performed on a series of α-hydroxy amides as a novel class of bradykinin B₁ selective antagonists, using different physicochemical parameters along with appropriate indicator variables. It has been found that physicochemical parameters such as connectivity indices ³χ, ⁴χ and ⁵χ, molecular weight, molar refractivity, density along with indicator variables are significantly correlated with activity. In this paper best results were obtained by using multiple regression analysis. Different models were generated with high values of R² and low values of PRESS/SSY ratio. The significant equations were statistically tested by using leave one out (LOO) technique and cross validation methods.     

2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C₁₈ “transition” SPE columns. A PLRP C₁₈ column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.     

缓激肽多肽片段间非共价作用的质谱研究

以缓激肽(R¹P²P³G⁴F⁵S⁶P⁷F⁸R⁹)分子作为研究模型, 用电喷雾质谱研究缓激肽分子碎片片段之间的非共价相互作用, 探讨了影响气相多肽分子构象稳定的氢键作用. 合成了与缓激肽分子在位置1断裂形成的碎片一致的RPPGFS和PFR多肽序列, 与在位置2断裂形成碎片一致的RPPGF和SPFR多肽, 以及N端或者C端去掉精氨酸的相应碎片多肽. 实验结果表明, 上述两个断裂位置产生的碎片多肽分别进行反应后, 都能发生非共价作用. 在断裂方式1下, PFR多肽在去掉C端的精氨酸R后, 与其他大多数多肽不发生非共价结合, 表明PFR中的R在缓激肽气相分子的构象中发挥重要的作用. 而在断裂方式2下, 去掉N端或者C端精氨酸的多肽之间都存在非共价结合, 即C端带有丝氨酸的SPF或SPFR多肽碎片仍然可以与N端碎片发生氢键结合, 表明丝氨酸很可能处于转角的位置. 通过对碰撞诱导解离(CID)的碰撞能量分析, 发现多肽RPPGFS和PFR, 以及多肽RPPGF和SPFR之间氢键结合较强, 而同时去掉N端和C端精氨酸得到的多肽之间的氢键结合较弱. 质谱滴定法定量测得的RPPGFS和PFR的结合常数为3.53×10³, 与RPPGF和SPFR的结合常数(3.16×10³)相接近,它们均大于去除精氨酸的PPGF和SPF的结合常数(1.25×10³). 质谱滴定实验结果进一步确认了碰撞诱导解离的分析结果, 表明缓激肽分子两端的精氨酸之间的氢键作用是气相缓激肽分子构象稳定的重要因素之一.     

The Influence of Stop-Flow on Band Broadening of Peptides in Micro-Liquid Chromatography

Stop-flow techniques are occasionally needed in combinations of LC-NMR and LC-MS. During the interval when there is no flow on the column, axial diffusion of components not yet eluted can be expected to take place. In this paper the size of the band broadening which is caused by diffusion during stop-flow has been determined for two peptides on reversed-phase packed micro columns. Within a temperature range of 20–40 °C, stop-flow could be extended to 30 minutes for peptides having k values in the range of 0.7–5.1 with little increase in band width on 1.0 mm i.d. columns at isocratic conditions. Stop-flow for 6 h at 20 °C increased the peak width of bradykinin and leucine-enkephalin by 25% and 60%, respectively, depending on the secondary interactions of the peptides. The peak broadening increased with increasing temperature (from 20 to 40 °C), as expected, and the impact was significant at stop-flow times larger than 2 h. Stop-flow during gradient elution resulted in less increase in peak width than isocratic elution due to the peak compression obtained when re-entering the gradient. At 20 °C the effective diffusion coefficients of leucine-enkephalin and bradykinin were determined to 6.5 × 10⁻⁷cm ²/s and 5.5 × 10⁻⁷ cm²/s, respectively, on the packed micro column.     

Rationally designed non-peptides: Variously substituted piperazine libraries for the discovery of bradykinin antagonists and other G-protein-coupled receptor ligands

Summary Molecular modeling studies of potent decapeptide bradykinin antagonists suggested the de novo design of peptide mimetics based on a 1,2,3,4-tetrasubstituted 1,4-piperazin-6-one scaffold. These de novo-designed antagonists exhibited only modest potency (IC₅₀ 55 M) on a cloned human B₂ receptor and antagonist activity in an in vitro human-cell functional assay. The success of these structures led to the creation of prototype libraries based on variously substituted 1,4-piperazine scaffolds, which allowed a rapid and general search of pharmacophores attached to a piperazine scaffold. The parent piperazinedione structures and fully reduced piperazine libraries differ from recently reported diketopiperazine libraries in the use of diverse nonnatural amino acids, on-resin-submonomer synthesis to provide more diverse N-substituted structures, and the adaptation of simultaneous ring closure and resin cleavage to drive the formation of highly hindered amide bonds. Using this chemistry, a rationally directed non-peptide library of approximately 2500 N,N-disubstituted piperazines and piperazinediones was synthesized and screened for ligand affinity on bradykinin, neurokinin, and opioid receptors. A number of lead structures were identified. Notably, a bradykinin antagonist lead, CP-2458, with good receptor selectivity and antagonist activity in human-cell assays was identified and is undergoing optimization by traditional and combinatorial methods.Abbreviations BK bradykinin - Boc tert-butoxycarbonyl - Cbz carboxybenzyl - DMF dimethylformamide - DMSO dimethylsulfoxide - Fmoc fluorenylmethoxycarbonyl - HBTU O-benzotriazol-l-yl-N,N,N,N-tetramethyluronium hexafluorophosphate - HPLC high-performance liquid chromatography - MALDI-MS matrix assisted laser desorption ionization-mass spectrometry - PyBroP bromo-tris-pyrrolydino-phosphonium hexafluorophosphate - RDDA rationally directed diverse analogs - TFA trifluoroacetic acid