Small Molecules as Molecular Probes

小分子作为分子探针

• 批准号: 7593531
• 负责人: Kenneth Kirk
• 金额: $ 31.39万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593531
• 关键词: Acids; Active Sites; Acylation; Amides; Amino Acids; Antibiotic Resistance; Antibiotics; Area; Arsenic; Azides; Bacteria; Benzene; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Biotin; Breast Cancer Cell; Cancer cell line; Carbon; Cell model; Chemicals; Chemistry; Chloride Ion; Chlorides; Cleaved cell; Codon Nucleotides; Communities; Coupling; Cysteine; Development; Enzymatic Biochemistry; Enzymes; Escherichia coli; Esters; Future; Glucosamine; Glycosides; Guanosine Triphosphate; Hydrolysis; In Vitro; Link; Lithium; MCF7 cell; Modification; Molecular Probes; Object Attachment; Peptides; Phenotype; Plant Resins; Polyacrylamide Gel Electrophoresis; Positioning Attribute; Post-Translational Protein Processing; Property; Proteins; Pyruvate Kinase; Reaction; Reagent; Research; Research Design; Research Project Grants; Ribosomal RNA; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptavidin; Succinic Acid; Succinic Acids; Sulfhydryl Compounds; Threonine; Tin; Transferase; Tubulin; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Western Blotting; Work; X-Ray Crystallography; analog; aspartic semialdehyde; base; carbene; design; enolate; epimerization; inhibitor/antagonist; molecular modeling; oxophenylarsine; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; phosphonate; polymerization; ribosomal protein S12; small molecule; tool

Our group continues to develop new mechanistic probes using a rational approach to compound development. Beta-Methylthioaspartic Acid: Beta-methylthioaspartic acid occurs at position 88 in E. coli ribosomal protein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. In bacteria, S12 binds to 16S rRNA in regions associated with the fidelity of codon recognition. This posttranslational modification is highly conserved phylogenetically and thus should be both structurally and functionally important. Research designed to determine the biological function of beta-methylthioaspartic will involve elucidation of the enzymology of this modification. Critical to this work is having available synthetic beta-methylaspartic acid as well as derivatives designed for peptide incorporation. Our contribution to this project involves carrying out the synthesis of these amino acid derivatives. The key installation of the beta-methylthio functionality was accomplished by reaction of the lithium enolate of 2-benzyloxycarbonylamino-succinic acid 1-methyl ester 4-tert-butyl ester with 1-methyldisulfanyl-2,4-dinitro-benzene. Acid hydrolysis cleaved the three protecting groups to give the target compound as a mixture of diastereomers. In order to prepare orthogonally protected material, the same sequence was carried out using the 1-benzyl ester. Careful chromatographic separation provides the orthogonally protected compound. Attempts to selectively remove the benzyl ester to provide the free acid suitable for peptide coupling have as yet been unsuccessful but are continuing. Alternate orthogonal protecting groups will be explored if necessary. Biotin conjugate of 4-aminophenylarsenoxide. An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with 4-aminophenylarsenoxide. Previous studies suggest that phenylarsenoxide reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. The biotin conjugate prepared by our group was used to examine these interactions. In a collaborative project, a human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, -tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of -tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys12 alone or vicinal Cys residues (Cys12 and Cys213) of -tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. In contrast, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed -tubulin and PKM2 as arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins. Future synthetic work will include acylation of 4-aminophenylarsenoxide with propyonyl chloride to afford the propyonyl amide. This will be used for click chemistry with biotin azide, either before or after interaction with cellular protein. Analogues of UDP-Glc-NAc: potential inhibitors of O-linked GlcNAc transferase (OTG). We have prepared the phosphonate analogue of GlcNAc from a key C-allyl glycoside of GlcNAc. The phosphonate proved to be devoid of activity as an inhibitor of OTG transferase. This was surprising, so crystals of key intermediates were prepared. X-ray crystallography ruled out epimerization during the synthesis, confirming the alpha-configuration of the final product. The lack of activity provides further evidence that OTG has strict structural requirements in substrate recognition. Inhibitors of O-GlcNAcAse. The products of OGT-catalyzed transfer of GlcNAc are O-linked conjugates with serine and threonine residues in proteins. We are continuing the syntheses of amino acid and peptide beta-C-linked conjugates of UDP-Glc-NAc. Coupling of a tributyl tin derivative of N,N-diactyl-tribenzyl glucosamine with N-t-Boc methyl aspartate semialdehyde constitutes the key carbon-carbon bond forming step. Subsequent functionalization will include deoxygenation or fluorodeoxygenation to produce methylene, fluoromethylene and difluoromethylene isosteres of O-linked GlcNAc conjugates.

我们的小组继续使用合理的方法来开发新的机械探针。 β-甲基硫代甲酸： β-甲基硫代氨基甲酸发生在大肠杆菌核糖体蛋白S12中的88位，该位置是突变热点，导致抗生素耐药性和抗生素敏感的表型。在细菌中，S12在与密码子识别的忠诚度相关的区域中与16S rRNA结合。这种翻译后的修饰在系统发育上是高度保守的，因此在结构和功能上应很重要。旨在确定β-甲基硫代基本的生物学功能的研究将涉及阐明这种修饰的酶学。这项工作至关重要的是具有可用的合成β-甲基天冬氨酸以及为肽掺入而设计的衍生物。我们对该项目的贡献涉及进行这些氨基酸衍生物的合成。 β-甲基硫硫醇功能的关键安装是通过锂含锂的2-苯甲酰氧甲苄氨基氨基 - 核酸1-甲基酯与1-甲基二甲基酯与1-甲基二硫代苯基-2,4-二硝基-2initro-苯甲烯的反应来完成的。酸水解裂解了三个保护组，使靶化合物作为非对映异构体的混合物。为了制备正交保护材料，使用1-苯苯酯进行相同的序列。仔细的色谱分离提供了正交保护的化合物。尝试选择性去除苄基酯以提供适合肽偶联的游离酸的尝试尚未成功，但仍在继续。如有必要，将探索替代的正交保护组。 4-氨基苯基苯甲苷的生物素结合物。 通过将生物素的五氟酚酯与4-氨基苯基苯甲酰氧化物耦合，合成了砷生物素结合物。先前的研究表明，苯苯二甲酰与蛋白质含量高的蛋白质的紧密间隔的半胱氨酸（Cys）残基反应，具有高Cys含量和可访问的硫酰基（SH）组。我们小组制备的生物素结合物用于检查这些相互作用。在一个协作项目中，将人类乳腺癌细胞系MCF-7视为探索砷结合蛋白和结合机制的细胞模型。将砷结合蛋白从砷生物素处理的MCF-7细胞中洗脱，用聚丙烯酰胺凝胶电泳分离，并通过基质辅助激光解吸电离电离质谱法（MALDI-MS）鉴定。在体外进一步研究了这些蛋白质中的两种， - tubulin和丙酮酸激酶M2（PKM2）的砷结合特性，并评估了这种结合的生物学后果。用蛋白质印迹的结合测定证实了砷以浓度依赖性方式通过砷对 - 微粉蛋白和PKM2的结合。砷结合抑制了小管蛋白聚合，但令人惊讶的是对PKM2活性没有影响。 分子建模表明，通过砷的cys12或牛皮化Cys残基（Cys12和Cys213）的结合通过砷阻断了活性位点以访问GTP，这对于小管蛋白聚合是必不可少的。相反，PKM2的所有CYS残基都远离酶的活性位点。总而言之，这项研究证实了-Tubulin和PKM2是MCF-7细胞中的砷结合蛋白。这种结合的功能后果可能取决于砷结合是否会导致构象变化或阻断靶蛋白的活性位点。 未来的合成工作将包括与丙酰氯的4-氨基苯基苯甲苷酰化，以提供丙酰酰胺。它将用于与细胞蛋白相互作用之前或之后与生物叠氮化物进行点击化学。 UDP-GLC-NAC的类似物：O连接GlcNAC转移酶（OTG）的潜在抑制剂。 我们已经从GlcNAC的关键C-酰基糖苷中制备了GlcNAC的磷酸类似物。膦酸酯被证明没有活性作为OTG转移酶的抑制剂。这是令人惊讶的，因此准备了关键中间体的晶体。 X射线晶体学排除了合成过程中的呈现，证实了最终产物的α-构型。缺乏活动提供了进一步的证据，表明OTG在底物识别中具有严格的结构要求。 O-Glcnacase的抑制剂。 GlcNAC的OGT催化转移的产物与蛋白质中的丝氨酸和苏氨酸残基是O-联合的结合物。我们正在继续使用UDP-GLC-NAC的氨基酸和肽β-C连接的共轭物的合成。 N，N-二辛基 - 三丁嗪葡萄糖胺与N-T-Boc甲基天冬氨酸半二醛的N，N-二酰基 - 三氮嗪的衍生物的偶联构成了关键的碳碳键形成步骤。随后的功能化将包括脱氧或荧光氧化，以产生甲基，荧光甲基和二氟甲基二甲基二氧化物的异氧化物GlcNAC偶联物。