类乳腺癌易感基因BRCA1肽的设计、合成及与抑癌基因蛋白RAD51肽段的相互作用

乳腺癌易感基因BRCA1肽对抑制女性乳腺癌病发有积极作用,其与乳腺癌细胞内抑癌基因蛋白RAD51相互作用的研究是乳腺癌药物发现的重要部分.通过Discovery Studio模拟类BRCA1肽与RAD51对接过程,利用R-DOCK评价系统从结果中筛选出4条评分较高的类BRCA1肽,采用固相逐步合成法制得.通过荧光光谱、圆二色光谱等方法研究了其与RAD51肽段(Pep158-180、Pep181-200、Pep241-260)的相互作用,发现4条类BRCA1肽的作用都强于BRCA1肽,这与模拟的结果相一致,其中BRCA1-3与RAD51的两个关键肽段(Pep158-180、Pep241-260)的相互作用明显强于BRCA1肽和其他类肽.该研究结果为乳腺癌药物分子设计提供了依据.     

分子对接法和光谱法研究BRCA1846-871及其五突变肽与RAD51181-200相互作用

影响RAD51蛋白的修复功能,可有效改善肿瘤细胞对化疗药物的耐受性。利用计算机模拟BRCA1~(846-871)关键肽段五个活性位点的突变,通过分子对接法筛选出与RAD51~(181-200)相互作用较强的9条五突变肽。采用固相合成法合成目标多肽,利用反相高效液相色谱(RP-HPLC)技术分离、纯化得到纯度大于93%的纯品肽,电喷雾电离质谱(ESI-MS)技术进行表征。通过荧光光谱法和圆二色光谱法(CD)研究BRCA1~(846-871)及其五突变肽与RAD51~(181-200)的相互作用。荧光谱图显示,除P6外,其余配体均与受体RAD51~(181-200)发生静态猝灭。在室温下,P1 (4.17×10~4 L·mol~(-1))、P4 (7.61×10~4 L·mol~(-1))和P5 (1.19×10~4 L·mol~(-1))与受体RAD51~(181-200)结合较强。圆二色光谱图显示,P1、P4和P5使受体RAD51~(181-200)的α-螺旋结构含量降低非常明显,从60.5%分别下降为35.7%、36.3%和36.4%,BRCA1~(846-871)、P3、P6和P9对受体α-螺旋结构含量影响较小,从60.5%分别下降为56.3%、55.6%、55.6%和54.6%,而P2、P7和P8对受体α-螺旋结构含量几乎不产生影响。综合光谱分析结果可知,配体P4与受体RAD51~(181-200)的相互作用最强。     

计算机模拟HsRAD51蛋白与多肽BRC4分子对接

研究使用ZDOCK算法进行HsRAD51蛋白与多肽BRC4分子对接的准确性.采用Accelrys Discovery Studio 3.5软件计算平台,使用基于快速傅立叶转换的ZDOCK分子对接程序进行蛋白HsRAD51与乳腺易感基因BRCA2的重复基元BRC4的分子对接,然后使用RDOCK程序进行结果优化,最后将通过分子对接得到的几个蛋白-多肽复合物的三级结构与文献报道的通过X射线测得的蛋白HsRAD51与BRC4复合物的准确结构进行叠合比较,结果发现其中一些结构的叠合效果较好,碳链骨架叠合的RMSD最小的为0.034 4nm.推测使用ZDOCK分子对接程序来进行HsRAD51蛋白与多肽的对接,具有较高的准确性.该结果为今后研究HsRAD51蛋白与BRCA2其他重复基元的分子对接以及相互作用提供了重要依据.     

以二肽为连接臂的新型聚乙二醇修饰阿霉素的合成

采用Fmoc固相合成策略，以Wang树脂为载体，Fmoc保护的L-氨基酸为原料，EDC/HOBt为缩合剂，合成了8种聚乙二醇修饰的二肽。以HATU/DIPEA为缩合剂，通过酰化反应将修饰后的多肽连接到阿霉素上，合成了一系列新型阿霉素前药，纯度高于90%，收率高于52%,其结构经¹H NMR和MS（ESI）表征。     

计算机辅助设计新型人脂肪酸合酶抑制剂及其抗肿瘤活性

以Orlistat为先导化合物,利用AutoDock进行计算机模拟对接,在对接结果中选择能量较低的6个结构进行合成与抗肿瘤活性筛选。以orlistat为原料,经2步反应制得苄基(2S,3S,5S)-2-己基-3,5-二羟基十六酸酯(2); 再经4步反应获得(3S,4S)-3-己基-4-［(S)-2-羟基十三烷基］氧杂环丁烷-2-酮(5); 5在EDCI作用下与酸经缩合反应合成了4个计新型人脂肪酸合酶抑制剂（6a~6d）,其中6b~6d为新化合物,其结构经¹H NMR, ¹³C NMR 和HR-MS（ESI）表征。体外初步活性测试表明：(S)-1-［(1S,2S)-3-己基-4-氧代氧杂环丁烷-2-基］十三烷-2-烟酸酯（6a）对MDA-MB-231细胞有较好的抑制作用,其IC₅₀为11.72 μmol·mL^-1,优于Orlistat(21.5 μmol·mL^-1)。     

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%。     

新型1,4-苯并二氮杂?-5-酮衍生物的合成及抗肿瘤活性初探

以邻硝基苯甲酸衍生物为底物,经3步反应并在最后一步使用了乙酰丙酮钼和三氟甲磺酸铜为催化剂,共合成了8个新型的1,4-苯并二氮杂?-5-酮衍生物（4a~4h）,其结构经¹H NMR,¹³C NMR和HR-MS(ESI)表征。并测试了4a~4h对肺癌（A549）、乳腺癌（MDA-MB-231）、宫颈癌（HeLa）等3种肿瘤细胞系的抑制活性。结果表明：目标化合物对所有肿瘤细胞系均有一定的抑制活性。其中9-氯-2-(丙-1-烯-2-基)-4-(2-(噻吩-2-基)乙基)-1,2,3,4-四氢-5H-苯并[e][1,4]二氮杂-5-酮(4e)对A549的IC₅₀可达1.7μM。而阳性对照药苯达莫司汀（Bendamustiune）和伏立诺他（SAHA）对A549没有明显的抑制作用。      

YBa2Cu4O8/La2/3Ca1/3MnO3/YBa2Cu4O8异质结中的超导转变温度

采用对靶溅射技术制备了YBa2Cu4O8/La2/3Ca1/3MnO3/YBa2Cu4O8（Y-124/LCMO/Y-124）异质结, 研究了超导转变温度（TC）随LCMO层厚度（dL）的振荡行为. 当dL＞dLCR时, TC-dL曲线表现出清晰的非单调行为, 而金属-绝缘体转变温度（TMI）仅当dL＞dLCR时才能观测到. Y-124/LCMO/Y-124系统中所存在的这种对中间层的依赖关系, 显示了铁磁和超导耦合间强烈的相互作用.     

新型20(S)-O-取代苯甲酸-7-苯甲酰基喜树碱酯的合成及其抗肿瘤活性

以20(S)-喜树碱为起始原料,对其进行结构修饰,在7-位导入苯甲酰基,在20-位羟基上导入取代苯甲酰基,设计并合成了11个新的20(S）-O-取代苯甲酸-7-苯甲酰基喜树碱酯化合物（4a~4k）,其结构经¹H NMR, IR,MS(ESI)和元素分析表征。采用MTT法初步考察了目标化合物4a～4j对人胃癌细胞（BGC-823）、人乳腺癌细胞（MDA-MB-231）、人肺腺癌细胞（H460）及人肝癌细胞（Bel-7404）的体外抑制活性。结果表明：化合物4a、 4g和4h具有一定的体外抑瘤活性。在药物浓度为10 μmol·L^-1时,4a对MDA-MB-231细胞和H460细胞的抑制率分别为50.42%和54.40%, 4g〗对MDA-MB-231细胞的抑制率为69.91%, 4h对H460细胞抑制率为52.34%。     

抗菌肽IB-367的固相合成与抑菌活性

采用固相合成方法,以Rink Amide树脂为载体,Fmoc保护氨基酸为原料,经苯并三唑-1-四甲基六氟磷酸酯(HBTU)/N,N-二异丙基乙胺(DIEA)缩合,三氟乙酸/苯甲硫醚/乙二硫醇/苯甲醚裂解体系脱除保护基制得IB-367线性肽(4); 4经双氧水氧化制得IB-367一环肽(5); 5经碘乙醇溶液氧化合成抗菌肽IB-367(6),收率34.1%,纯度＞95.0%,其结构经MS(ESI)和氨基酸组成分析确证。抑菌活性研究结果表明：6对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度为5.0 μg·mL^-1。     

β-发夹多肽的全新设计和构象研究

引入跨股氨基酸队的方法进行β-发夹结构的设计,序列[R1G2T3F4W5V6d-P7S8V9N10Y11F12, β2] 中包含二个氨基酸对V6V9和F4Y11,并以d-P7S8作转角来稳定结构.多肽合成采用Fmoc/But固相合成方法.圆二色谱研究显示,β2在202 nm呈现正峰,在217.5 nm处呈负峰,为β转角和β折叠共同贡献的叠加,是典型的β-发夹结构圆二色谱特征.红外光谱研究进一步验证了圆二色谱的结果,表明β2在溶液中主要以β-发夹结构存在.     

β-发夹多肽的全新设计和构象研究

引入跨股氨基酸队的方法进行β-发夹结构的设计,序列[R1G2T3F4W5V6d-p7S8V9N10Y11F12,β2]中包含二个氨基酸对V6V9和F4Y11,并以d-p7S8作转角来稳定结构.多肽合成采用Fmoc/Bu4固相合成方法.圆二色谱研究显示,β2在202 nm呈现正峰,在217.5 nm处呈负峰,为β转角和β折叠共同贡献的叠加,是典型的β-发夹结构圆二色谱特征.红外光谱研究进一步验证了圆二色谱的结果,表明β2在溶液中主要以β-发夹结构存在.     

Gelation capability of cysteine-modified cyclo(L-Lys-L-Lys)s dominated by Fmoc and Trt protecting groups

A series of symmetrical peptidomimetics(3–8) based on cysteine-modified cyclo(L-Lys-L-Lys)s were synthesized, and their gelation capability in organic solvents was dominated by fluorenylmethyloxycarbonyl(Fmoc) and triphenylmethyl(Trt) protecting groups and the exchange of thiol-to-disulfide as well. The peptidomimetics holding Trt(3 and 4) showed no gel performance, while the Fmoc groups promoted 5 and 6 to give rise to thermo-reversible organogels in a number of organic solvents. The self-assembled fibrillar networks were distinctly evidenced in the organogels by transmission electron microscopy(TEM) and scanning electron microscopy(SEM) observations. Fourier transform infrared spectroscopy(FT-IR) and fluorescence analyses revealed that the hydrogen bonding and ?-? stacking play as major driving forces for the self-assembly of these organogelators. A ?-turn secondary structure was deduced for the organogel of 6 by virtue of X-ray diffraction, FT-IR and circular dichroism(CD) measurements, and an interdigitated bilayer structure was also presented.     

Self‐Assembled Peptide‐Based Hydrogels as Scaffolds for Proliferation and Multi‐Differentiation of Mesenchymal Stem Cells

Fluorenyl‐9‐methoxycarbonyl (Fmoc)‐diphenylalanine (Fmoc‐FF) and Fmoc‐arginine‐glycine‐­aspartate (Fmoc‐RGD) peptides self‐assemble to form a 3D network of supramolecular hydrogel (Fmoc‐FF/Fmoc‐RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc‐FF/Fmoc‐RGD hydrogel increase in proliferation and survival compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. Moreover, MSCs encapsulated in Fmoc‐FF/Fmoc‐RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc‐FF/Fmoc‐RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentiation, compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. This study concludes that nanofibers formed by the self‐assembly of Fmoc‐FF and Fmoc‐RGD are suitable for the attachment, proliferation, and multi‐differentiation of MSCs, and can be applied in musculoskeletal tissue engineering.

     

Preparation of cruciferous phytoalexin related metabolites, (−)-dioxibrassinin and (−)-3-cyanomethyl-3-hydroxyoxindole, and determination of their absolute configurations by vibrational circular dichroism (VCD)

Cruciferous phytoalexin related metabolites, (−)-dioxibrassinin (1) and (−)-3-cyanomethyl-3-hydroxyoxindole (2) were prepared from isatin as racemates and were resolved by chiral HPLC. Their absolute configurations were determined by the new chiroptical technique, vibrational circular dichroism (VCD), as well as by the conventional electronic circular dichroism (ECD). It is concluded that the absolute configurations of the naturally occurring (−)-1 and (−)-2 are both S.     

Homodimerization and heteroassociation of 6-O-(2-sulfonato-6-naphthyl)-gamma-cyclodextrin and 6-deoxy-(pyrene-1-carboxamido)-beta-cyclodextrin

6-O-(2-sulfonato-6-naphthyl)-gamma-cyclodextrin (1) and 6-deoxy-(pyrene-1-carboxamido)-beta-cyclodextrin (2) were prepared. Homodimerizations of 1 and 2 and heteroassociation between 1 and 2 were investigated by (1)H NMR, circular dichroism, and fluorescence spectroscopic methods. The compounds 1 and 2 form head-to-head dimers with dimerization constants of 140 +/- 50 and 270 +/- 70 M(-)(1), respectively. We also determined the association constants of 1 with beta-CD as 270 +/- 20 M(-)(1) and 2 with gamma-CD as 100 +/- 30 M(-)(1) from fluorescence and circular dichroism titration data, respectively. The heteroassociation between 1 and 2 was manifested in increased circular dichroism ellipticities of 2, downfield shift of the H-2 proton of the pyrene group of 2, and upfield shift of the H-5 proton of the naphthyl group of 1 upon mixing 1 and 2. The analysis of circular dichroism titration data of 2 with 1 gave the association constant as 9300 +/- 1600 M(-)(1). The NMR and circular dichroism spectra suggested that the naphthyl group of 1 is deeply included into the beta-CD cavity of 2, while the pyrene group of 2 is partially inserted in the gamma-CD cavity of 1 in the complex. The energy-minimized structure from molecular modeling of the complex supports this. We believe that the facile heteroassociation of two cyclodextrin derivatives having different sizes of cavity and pendant group could be utilized as a useful strategy for assembling functionalized CDs for various applications.     

Investigating cyclic sotolon,maple furanone and their dimers in solution using optical rotation,electronic circular dichroism and vibrational circular dichroism

The experimental optical rotation (OR), electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra of (R)-3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolon, 1) and (R)-5-ethyl-3-hydroxy-4-methylfuran-2(5H)-one (maple furanone, 2) taken in chloroform were compared to their spectra calculated with time-dependent density functional theory (TDDFT). Sotolon was shown to exist as a dimer in chloroform while maple furanone remains a monomer. Transition state barriers for the enol/keto tautomerization of sotolon were calculated and found to be high. The VCD method offers promise to ultimately distinguish between the presence of monomers or dimers.     

Unidirectional α-cyclodextrin-based [2]rotaxanes bearing viologen unit on axle

1-(2-Carboxyethyl)-1′-(10-carbazole-9-yl-decyl)-4,4′-bipyridinium dibromide 2 forms a unidirectional [2]pseudorotaxane with α-cyclodextrin (α-CD) in water. Condensation of 2/α-CD [2]pseudorotaxane with 4-amino-1-naphthalenesulfonate or 6-amino-β-CD provided the unidirectional [2]rotaxanes 3 and 4, in which the secondary face of α-CD is oriented toward the viologen moiety. The structures were elucidated from two-dimensional ROESY and circular dichroism spectra.     

Optimized protocol for synthesis of cyclic gramicidin S: starting amino acid is key to high yield

[structures: see text] A simple and highly efficient Fmoc solid-phase protocol for synthesizing the antimicrobial decapeptide gramicidin S and various labeled analogues is presented. When preparing the linear precursor peptides (1a-e), a systematic permutation of the starting amino acid within the cyclic sequence gave different yields between 51% and 93%. Also the subsequent step of cyclization gave widely diverging yields between 26% and 74%, depending again on the starting amino acid. The ease of cyclization was found to correlate with the tendency of the respective linear precursor peptide to assume a preorganized conformation, as observed by circular dichroism. The overall yield is thus critically dependent on the starting amino acid and can be raised from 20% to 70% using (D)Phe. The choice of coupling agent and its counterion was found to play only a marginal role. Irrespective of being able to assume a preorganized conformation, none of the linear precursor peptides exhibited any antimicrobial or hemolytic activity. Using the optimized protocol, which involves only simple Fmoc-couplings and requires no intermittent purification steps, several gramicidin S analogues (3-8) containing 19F-labeled phenylglycine derivatives and/or 15N-labeled amino acids were synthesized for solid-state NMR structure analysis.     

Enantioselective Solid‐Phase Peptide Synthesis Using Traceless Chiral Coupling Reagents and Racemic Amino Acids

The enantioselective condensing reagent 4,6‐dimethoxy‐1,3,5‐triazine (DMT)/strychnine/BF$\rm{{_{4}^{-}}}$ was obtained by treatment of 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) with strychnine tetrafluoroborate. The reagent was useful under typical conditions of solid‐phase peptide synthesis (SPPS) with enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84–94% yield using 4 equiv. of racemic Fmoc‐Ala, Fmoc‐Phe, and/or Fmoc‐Tyr for 1 equiv. of Wang resin loaded with Gly, Ala, Leu, Phe, Glu(^tBu), and/or Pro, respectively. For all three Fmoc‐protected amino acids, the configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the acylated component and identical to that established in condensations proceeding in solution. In all cases, the enantiomer ratios L /D (er) were in a similar range, and varied from 9 : 92 to 2 : 98 for alanine, and from 90 : 10 to 100 : 0 for aromatic amino acids. The synthesis of Ac‐L ‐Lys(Ac)‐D ‐Ala‐D ‐Ala‐OH from racemic Fmoc‐Ala gave an L /D ratio of 10 : 90 for the esterification of Wang resin, and 0 : 100 for the formation of peptide bonds.