大环多胺脂质体介导的核酸转染

以大环多胺为亲水部分构建的脂质体可以有效用于核酸转染。大环多胺中带正电荷的氮原子可通过静电吸引和氢键等方式与核酸(DNA/RNA)结合。当前用于构建脂质体并进行核酸转染的大环多胺主要涉及到1,4,7-三氮杂环壬烷(tacn)、1,4,7,10-四氮杂环十二烷(cyclen)、1,4,8,11-四氮杂环十四烷(cyclam)等类型,其中基于cyclen的脂质体研究较多。亲水的大环多胺头部、疏水基团尾部及连接基团的结构对脂质体的转染效率有很大影响。一些大环多胺的金属配合物除了可以作为基因转染载体,在标记、示踪等方面也有特殊作用。本文综述了基于大环多胺的两亲性分子在核酸载体方面的应用,对其构效关系进行了讨论,并对相关领域的发展做了展望。     

基于低分子量聚乙烯亚胺和油酸的负电性基因载体

构建负电性的基因载体、发展基于低分子量聚乙烯亚胺(PEI)的基因载体对基因传递研究具有重要意义. 本文基于低分子量聚乙烯亚胺(2 kDa)和油酸构建了负电性的基因载体. 它通过混合聚乙烯亚胺(2 kDa)、dsDNA和油酸胶束而自发形成. 该基因载体在血清中很稳定,细胞毒性非常低,可包封80%以上DNA. 通过1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇2000)]铵盐(DSPE-PEG)对其表面进行修饰,发现多达90%的基因可被细胞摄取.     

基于低分子量聚乙烯亚胺和油酸的负电性基因载体(英文)

构建负电性的基因载体、发展基于低分子量聚乙烯亚胺(PEI)的基因载体对基因传递研究具有重要意义.本文基于低分子量聚乙烯亚胺(2 kDa)和油酸构建了负电性的基因载体.它通过混合聚乙烯亚胺(2 kDa)、dsDNA和油酸胶束而自发形成.该基因载体在血清中很稳定,细胞毒性非常低,可包封80%以上DNA.通过1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇2000)]铵盐(DSPE-PEG)对其表面进行修饰,发现多达90%的基因可被细胞摄取.     

聚谷氨酸为骨架的梳状基因载体的合成及生物学性能评价

以聚谷氨酸为骨架, 用低分子量聚乙烯亚胺胺解聚谷氨酸苄酯, 得到聚谷氨酸-g-聚乙烯亚胺, 用异佛尔酮二异氰酸酯将聚乙二醇单甲醚偶联到聚谷氨酸-g-聚乙烯亚胺上, 合成了梳状聚阳离子基因载体聚谷氨酸-g-(聚乙烯亚胺-b-聚乙二醇). 利用核磁共振氢谱、 激光粒度分析仪、 Zeta电位仪和凝胶电泳对聚阳离子载体及其与质粒脱氧核糖核酸(pDNA)形成的复合物进行了表征. 通过噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白质粒pEGFP-C1及荧光素酶质粒pGL3体外转染实验考察了载体的细胞毒性及基因转染效率. 结果表明, 当聚乙烯亚胺中N原子和DNA中P原子的摩尔比(N/P)大于5时, 载体能很好地包裹DNA, 载体与DNA形成的复合物粒径约为130 nm, Zeta电位约为28 mV; 通过MTT实验和体外质粒转染实验显示出载体在测量范围内具有极低的细胞毒性和较高的转染效率.     

聚乙烯亚胺对人血清白蛋白的构象和结合能力的影响

为了解基因载体材料聚乙烯亚胺（PEI）细胞毒性的分子作用机理,本文应用吸收光谱、荧光光谱、圆二色谱、动态光散射和zeta-电位测定分析平均相对分子量为1.8和25 kDa的PEI（记为PEI1.8k和PEI25k）对人血清白蛋白（HSA）构象的影响,同时以8-苯氨基萘-1-磺酸镁（ANS）和槲皮素为模型化合物,了解PEI对HSA结合能力的影响及机理。结果发现,PEI与HSA结合形成静态复合物,导致HSA流体动力学直径变小和分子内环境疏水性增强。PEI1.8k和低浓度的PEI25k引起HSA的a-螺旋结构增加,但是高浓度的PEI25k对HSA二级结构具有稳定作用。PEI对HSA结合能力的影响主要归因于PEI的竞争结合和PEI与HSA结合引起的蛋白质构象变化。PEI的竞争结合降低了HSA对ANS和槲皮素的结合效率,但是蛋白质的构象变化增强了HSA与ANS和槲皮素的结合能力。PEI与HSA的相互作用具有明显的分子尺寸效应,增加PEI的相对分子量可以增强对HSA构象和结合能力的影响。     

通过甲基丙烯酰胺侧基迈克尔加成制备阳离子聚碳酸酯

针对高分子量的聚乙烯亚胺(PEI)作为基因载体效率高但毒性大,而低分子量的聚乙烯亚胺虽毒性低但对基因的结合能力差、转染效率低的矛盾,设计合成了主链可生物降解而侧链带有胺类化合物的阳离子聚碳酸酯.首先,2-氨基-1,3-丙二醇和甲基丙烯酰氯反应制得2-(甲基丙烯酰胺基)-1,3-丙二醇,再用氯甲酸乙酯在室温下用三乙胺作碱...     

氨基修饰β-环糊精衍生物水相仿生催化不对称Michael加成反应

在不对称 Michael加成反应中,有机小分子如伯胺、吡咯烷类衍生物、(硫)脲类、手性方酰胺、联萘类、奎宁类、手性膦、离子液体和肽类等是目前使用的主要催化剂,如果能避免或少量使用有机溶剂,则更符合“绿色化学”的环境友好发展方向.β-环糊精的内腔疏水,而外部亲水,可以类似酶分子结合有机反应物,在水相体系进行催化反应.当β-环糊精分子上连接催化部位或结合部位时,能产生更优异的包结底物和诱导对映选择性的能力.目前基于β-环糊精衍生物构筑人工类酶催化剂用于不对称 Michael加成反应的报道较少.本文通过亲核取代反应将氨基类有机小分子与单(6-O-p-甲苯磺酰基)-β-环糊精结合,得到9个氨基修饰β-环糊精衍生物CD-1–CD-9(收率在24.2%–64.9%,分子结构通过1H NMR,13C NMR和 ESI-MS表征确认),并用于室温水相体系不对称Michael加成的仿生催化反应,以期获得较好的催化反应活性和对映选择性.通过设计不同β-环糊精衍生物的修饰基团结构、改变反应介质pH值和反应底物结构,分析了Michael加成反应体系产物产率和对映选择性的变化,采用2D-1H ROESY NMR、紫外吸收光谱、红外光谱和和量子化学计算,分析了β-环糊精衍生物和反应底物分子的包结状态,探究了反应过程机理.结果显示,在该水相体系中进行的不对称Michael加成反应产物产率和对映体过量值(ee值)受修饰基团结构、反应介质pH值和底物结构影响较大.当反应介质pH值低于6.0时,由于氨基分子被质子化而失去催化活性;当 pH值为7.5时,获得中等水平的对映选择性,通过量子化学在 ONIOM (B3LYP/6-31G(d)：PM3)水平上的优化计算发现,底物分子与β-环糊精衍生物的包结可以出现两种形式：当底物分子的活性部位接近β-环糊精衍生物小口端的修饰基团时,产生分子内催化,诱导反应产生较好的对映选择性;当底物分子的活性部位远离β-环糊精衍生物小口端的修饰基团时,产生分子间催化,几乎没有对映选择性,而这两种情况同时存在.当底物分子以较大的空间位阻与β-环糊精疏水性空腔结合时,产生较好的对映选择性,邻位取代的2-硝基-β-硝基苯乙烯比对位取代的4-硝基-β-硝基苯乙烯 ee值更高,通过量子化学优化计算证实空间位阻效应.应用2-金刚烷酮与β-环糊精衍生物空腔形成竞争性的包结反应实验,产物产率和ee值都下降,说明β-环糊精衍生物的疏水性空腔是产生不对称诱导和催化活性不可或缺的部分,底物分子与β-环糊精衍生物的包结过程通过2D-1H ROESY NMR和紫外吸收图谱获得确认.其中L-2-氨甲基吡咯烷修饰β-环糊精 CD-1表现出较好的反应对映选择性,在溶剂(pH =7.5,0.5 mol/L CH3COONa-HCl)2 mL,环己酮2 mmol,2-硝基-β-硝基苯乙烯0.2 mmol,CD-1用量0.04 mmol,25°C反应96.0 h的条件下,环己酮与2-硝基-β-硝基苯乙烯 Michael加成产物的 ee值达71%,产率为47%.该反应过程在β-环糊精衍生物的疏水性空腔内进行,修饰基团L-2-氨甲基吡咯烷与环己酮形成烯胺的催化反应.     

氨基修饰β-环糊精衍生物水相仿生催化不对称Michael加成反应（英文）

在不对称Michael加成反应中,有机小分子如伯胺、吡咯烷类衍生物、(硫)脲类、手性方酰胺、联萘类、奎宁类、手性膦、离子液体和肽类等是目前使用的主要催化剂,如果能避免或少量使用有机溶剂,则更符合"绿色化学"的环境友好发展方向.β-环糊精的内腔疏水,而外部亲水,可以类似酶分子结合有机反应物,在水相体系进行催化反应.当β-环糊精分子上连接催化部位或结合部位时,能产生更优异的包结底物和诱导对映选择性的能力.目前基于β-环糊精衍生物构筑人工类酶催化剂用于不对称Michael加成反应的报道较少.本文通过亲核取代反应将氨基类有机小分子与单(6-O-p-甲苯磺酰基)-β-环糊精结合,得到9个氨基修饰β-环糊精衍生物CD-1–CD-9(收率在24.2%–64.9%,分子结构通过~1H NMR,~(13)CNMR和ESI-MS表征确认),并用于室温水相体系不对称Michael加成的仿生催化反应,以期获得较好的催化反应活性和对映选择性.通过设计不同β-环糊精衍生物的修饰基团结构、改变反应介质pH值和反应底物结构,分析了Michael加成反应体系产物产率和对映选择性的变化,采用2D-~1HROESY NMR、紫外吸收光谱、红外光谱和和量子化学计算,分析了β-环糊精衍生物和反应底物分子的包结状态,探究了反应过程机理.结果显示,在该水相体系中进行的不对称Michael加成反应产物产率和对映体过量值(ee值)受修饰基团结构、反应介质pH值和底物结构影响较大.当反应介质pH值低于6.0时,由于氨基分子被质子化而失去催化活性;当pH值为7.5时,获得中等水平的对映选择性,通过量子化学在ONIOM(B3LYP/6-31G(d):PM3)水平上的优化计算发现,底物分子与β-环糊精衍生物的包结可以出现两种形式:当底物分子的活性部位接近β-环糊精衍生物小口端的修饰基团时,产生分子内催化,诱导反应产生较好的对映选择性;当底物分子的活性部位远离β-环糊精衍生物小口端的修饰基团时,产生分子间催化,几乎没有对映选择性,而这两种情况同时存在.当底物分子以较大的空间位阻与β-环糊精疏水性空腔结合时,产生较好的对映选择性,邻位取代的2-硝基-β-硝基苯乙烯比对位取代的4-硝基-β-硝基苯乙烯ee值更高,通过量子化学优化计算证实空间位阻效应.应用2-金刚烷酮与β-环糊精衍生物空腔形成竞争性的包结反应实验,产物产率和ee值都下降,说明β-环糊精衍生物的疏水性空腔是产生不对称诱导和催化活性不可或缺的部分,底物分子与β-环糊精衍生物的包结过程通过2D-~1HROESYNMR和紫外吸收图谱获得确认.其中L-2-氨甲基吡咯烷修饰β-环糊精CD-1表现出较好的反应对映选择性,在溶剂(pH=7.5,0.5mol/LCH_3COONa-HCl)2mL,环己酮2mmol,2-硝基-β-硝基苯乙烯0.2mmol,CD-1用量0.04mmol,25°C反应96.0h的条件下,环己酮与2-硝基-β-硝基苯乙烯Michael加成产物的ee值达71%,产率为47%.该反应过程在β-环糊精衍生物的疏水性空腔内进行,修饰基团L-2-氨甲基吡咯烷与环己酮形成烯胺的催化反应.     

主客体组装构建环糊精修饰基因微载体的研究

合成了二茂铁接枝聚乙烯亚胺( PEI-Fc),利用二茂铁与β-环糊精的主客体嵌套作用制备了环糊精修饰聚乙烯亚胺,核磁测定结果显示,每条PEI-Fc链上通过主客体作用嵌套的CD平均为26个.这种基于弱相互作用力的β-环糊精修饰聚乙烯亚胺能有效诱导DNA分子的缔合,在N/P值达到3以上时,可形成表面为正电荷、粒径为150 ～ 250 nm的球形粒子.在含10％胎牛血清的DMEM体外细胞培养基中,由于培养基中的蛋白质能够在粒子表面发生静电吸附,PEI-Fc/CD/DNA基因微载体显示出良好的稳定性.HEK293细胞培养结果显示,以表达绿色荧光蛋白的质粒pEGFP为模型,以N/P值为10的PEI/DNA组装体作为对照,N/P值为3、5和10的PEI-Fc/CD/DNA组装体的转染效率均达到对照组的2～3倍,这种基于主客体组装构建的环糊精修饰基因微载体显著提高了基因转染效率.     

刚棒-线团分子的自组装研究进展

超分子化学领域的自组装研究是近年来研究的热点,对这种由一种或多种结构单元自发聚集而成具有一定尺寸和结构的过程研究已经取得了重大进展.以亲水基团和亲脂基团为主要构成单元的两亲性分子在自组装领域中的表现优异于其他分子,其亲水的刚性棒状基团和疏水的柔性线团基团通过不同方法共同构成了各种类型的刚柔两亲性分子,而在水溶液中自组装...     

Hydrophobic surface induced activation of Pseudomonas cepacia lipase immobilized into mesoporous silica

Lipase from Pseudomonas cepacia (PCL) was successfully immobilized into siliceous mesocellular foams (MCFs) with various hydrophobic/hydrophilic surfaces. The catalytic performances of immobilized PCL were investigated using the transesterification reaction and hydrolytic reaction as model reactions. The specific activity of immobilized PCL greatly increased with enhanced surface hydrophobicity of MCFs, mainly because of lipase activation via hydrophobic interaction between alkyl groups in MCFs and the surface loop (so-called "lid") of PCL. Conformational changes of immobilized PCL were further investigated using time-resolved fluorescence spectroscopy with Trp as an intrinsic probe. When the immobilized PCL was suspended in phosphate buffer, short-lived τ(1) shortened and the fractional contribution of τ(1) significantly increased with the increasing level of surface hydrophobicity of MCFs. These results revealed that Trp(s) of the immobilized PCL were surrounded by a hydrophilic microenvironment because of the fact that the opened "lid" permitted the diffusion of water to the active site cleft. However, for the immobilized PCL suspended in n-hexane, long-lived τ(3) increased with the increase of surface hydrophobicity of MCFs. The reduced interaction between Trp(s) and the surrounding protein matrix was due to intercalation of n-hexane into the active site cleft when the lipase was in open conformation. The above results demonstrated that PCL immobilized into MCF with hydrophobic surfaces were in an activated open conformation.     

Intrinsic interaction mode of an inhalation anesthetic with globular proteins: a comparative study on ligand recognition

Interaction mode of an inhalation anesthetic halothane with water-soluble globular proteins, myoglobin (Mgb) and lysozyme (Lys), was studied by differential scanning calorimetry (DSC) and viscometry, and the results were compared with those of bovine serum albumin (BSA). The anesthetic sensitivity was markedly different among the proteins: Mgb was destabilized, Lys was slightly destabilized, and BSA was conversely stabilized. Further, the interaction mode was quite different from those of specific binders for the proteins. The anesthetic sensitivity was highly correlated with the hydrophilicity on the protein surface (Mgb?<?Lys?<?BSA) and the rigidity of the protein structure (BSA????Mgb?<?Lys). We showed that the anesthetic sensitivity among globular proteins can be roughly classified into four categories, and proteins with small hydrophilicity and soft structure are suitable as model proteins of anesthesia. By contrast, the binding of the specific binders was characterized by the lower effective concentrations. The molar ratio of the binder to the protein at the effective concentration was well consistent with the binding number determined from the X-ray structural analysis. Moreover, the interaction mode of the binder was not necessarily in accord with the mode expected from the change in the protein structure. Considering the above facts, we can systematically interpret the effect of an anesthetic on globular proteins by four factors: (1) hydrophobicity of an anesthetic, (2) hydrophilicity of a protein surface, (3) rigidity of a protein structure, and (4) molar ratio of an anesthetic to a protein at the effective concentration.     

Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (M?bius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the M?bius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.     

Potentiometric and Spectroscopic Characterization of Copolypeptide-Surfactant Complexes Formed by a Cooperative Binding System

The binding behavior of an anionic surfactant, sodium dodecyl sulfate (SDS), to a series of L-lysine-containing copolypeptides in aqueous solutions was investigated in relation to the conformational change of copolypeptide-surfactant complexes with the use of potentiometric and spectroscopic techniques. The present results of CD spectra and the binding isotherm of SDS by copolypeptides of opposite charges can lead us to conclude that SDS binds cooperatively to the positively charged side groups of a series of copolypeptides used in this work, resulting in the formation of a micelle-like cluster due to an additional hydrophobic interaction among bound SDS ions. Solid-state properties of the stoichiometric copolypeptide-SDS complexes were also examined by using CD and FT-IR spectroscopies; (Lys, Tyr) (1:1) and (4:1) systems adopt a beta-pleated sheet conformation, while (Lys, Trp) (4:1) and (Lys, Phe) (1:1) systems adopt an alpha-helical conformation. Based on the results of FT-IR spectra, in all cases surfactant alkyl chains of SDS in the solid complexes were in an extended conformation. Copyright 2000 Academic Press.     

Behavior and characteristics of biogenic amines, ornithine and lysine derivatized with the o-phthalaldehyde-ethanethiol-fluorenylmethyl chloroformate reagent

The stability and characteristics of the ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Diah), spermidine (Spd) and spermine (Spm) derivatives obtained with the o-phthalaldehyde (OPA)-ethanethiol (ET)-fluorenylmethyl chloroformate (FMOC) reagent has been investigated. The stoichiometry of the introduced, two-step derivatization process has been followed by photodiode array (DAD) and fluorescence (FL) detections, simultaneously, while the composition of derivatives was confirmed by on-line HPLC-electrospray ionization (ESI) MS measurements. Depending on the composition of the OPA reagents, in addition to the secondary amino group-containing Spd and Spm, under common aqueous conditions also Orn and Lys do react with FMOC resulting in derivatives of various compositions. Applying the OPA-ET reagent of increasing methanol (Met) content (38-80%, v/v) the formation of the FMOC group containing Orn and Lys derivatives could be considerably decreased. Optimum elution condition (18 min, including equilibration) was developed for the simultaneous quantitation of Orn, Lys, Put, Cad, Diah, Spd and Spm, in the presence of the rest of protein amino acids. The practical utility of the method was demonstrated by the analysis of mouse tissues. Average reproducibility of quantitations, characterized with the relative standard deviation percentages of fluorescence intensities and UV responses, in order of listing, proved to be 2.1% and 2.1%, respectively.     

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor,hGRF(1-29)-NH2

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.     

Noncovalent interactions and copper(II) coordination in systems containing l-aspartic acid and triamines

Interactions between l-aspartic acids (Asp) and polyamines (PA): 3,3-tri (1,7-diamino-4-azaheptane) or spermidine (Spd, 1,8-diamino-4-azaoctane) are investigated in metal-free systems as well as between Cu(II) ions in ternary systems with Asp and 3,3-tri or Spd. The composition and stability constants of the complexes formed have been determined by a potentiometric method, while the centres of interactions in the ligands have been identified by NMR, UV–Vis, IR, and EPR spectroscopy. The centres are the potential sites of metal ion coordination. In the Asp/PA systems, formation of molecular complexes (Asp)H_x(PA) was observed. Comparison of the log K_e of the adducts showed that the stability of the adducts significantly depends on the steric factor contributed by the length of PA. In the (Asp)H₃(PA) species, an inversion effect was observed where one of the amine groups (deprotonated) of 3,3-tri or Spd becomes a negative reaction centre and reacts with the protonated amino group of Asp. Therefore, depending on pH, the amino group of the PA can act as a positive or negative reaction centre. In the ternary systems of Cu(II)/Asp/PA the heteroligand-protonated complexes and molecular complexes are formed. In the molecular complexes ML?L′, where L = Asp and L′ = PA, the metallation involves oxygen atoms from the carboxyl groups and the amino group of the amino acid, while the fully protonated PA is located outside the inner coordination sphere and reacts with the anchoring binary complex CuH(Asp) or Cu(Asp). Introduction of metal ions into the Asp/3,3-tri system was found to change the character of the interaction and in the Cu(Asp)H₂(3,3-tri) complex, the oxygen atoms from the Asp carboxyl groups do not take part in coordination.     

Heparin coating of poly(ethylene terephthalate) decreases hydrophobicity, monocyte/leukocyte interaction and tissue interaction

Woven poly(ethylene terephthalate) (PET) is widely used in implantable medical devices. Upon implantation, fibrinogen interacts with the PET and changes conformation, such that the fibrinogen P2 epitope may become exposed. This allows inflammatory cells to interact with the material. In this study we have coated PET with heparin and show that this decreases PET hydrophobicity and the presence of the fibrinogen P2 epitope on the material surface. In addition, we show that heparin-induced reduction of PET hydrophobicity correlates with decreased exposure of the fibrinogen P2 epitope and reduced adhesion of monocytes. Reduction of PET hydrophobicity was furthermore associated with reduced PMN elastase production and decreased interaction between PET and embryonic chicken tissue. We conclude that the heparin coating-induced decrease in PET hydrophobicity is associated with decreased interaction between PET and inflammatory cells. Independent of this interaction, the hydrophobic nature of the heparin coating is related to tissue interaction as demonstrated by a reduction in adhesion, growth and spreading of tissue on PET. The combination of these properties makes heparin coating a candidate for improving biocompatibility of PET.     

Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.