Design and Synthesis of DNA-Encoded Libraries of Unnatural Peptides to Discover Specific Chemical Probes

项目概述包括环系统在内的编码肽的Vast文库可以使用生物学方法，如核糖体表达系统来合成，并可以通过亲和力选择来筛选，以产生高亲和力的蛋白质配体。然而，上述生物学方法的范围通常仅限于蛋白质来源的氨基酸及其相近的类似物。非天然部分的加入将允许对选定的蛋白质类别进行更特定的靶向，潜在地导致化合物具有更高的蛋白分解稳定性和细胞渗透性。拟议项目的目的是开发合成方法，通过开发化学而不是生物的多肽库合成技术来实现这一目标。这些技术缺乏通过生物机械进行鉴定的要求，因此可以包含更广泛的构件。在第二条线索中，将开发混合方法，允许将选定蛋白质类别的非自然弹头合并到生物展示的多肽库中。编码多肽库的化学合成DELS是一种合成和筛选大量分子的技术。每个DEL都是这些分子的集合，这些分子由连接到DNA寡核苷酸的有机部分组成。“基因”(DNA标签)是显示的“表型”(有机小分子)的可扩增识别条形码。类肽是N-烷基化的甘氨酸的寡聚体；类肽单体是一种氨基酸，其侧链连接到骨架的氮上，而不是α碳。类肽作为药物提供了一定的优势，包括。由于叔胺对蛋白质降解的抵抗力，由于没有用于氢键的酰胺NH而改善了相对于多肽的细胞渗透性，以及侧链的多样性，因为理论上它们可以从任何给定的伯胺合成。类肽通常是通过固体支撑的亚单体方法合成的，首先是溴乙酸，然后是伯胺。这是一个问题，因为-卤代羰基是一种强大的电泳剂，所以会使DNA烷化，所以这种方法与DEL合成是不兼容的。初步工作将集中在类肽单体的DNA外合成以及通往这些构建单元的多条路线的优化。因此，优化的条件将应用于各种胺(以提供一系列类肽单体)，并阐明反应的范围。同时，类肽单体的DNA偶联将在胶束条件下建立。这将通过测试上述合成的类肽单体在DNA上的偶联来实现偶联的范围。这将在进一步的工作中发展到一系列含有类肽的文库的合成，从由一系列合成单体组成的小的线性类肽文库开始。这一技术的扩展将导致整合允许控制类肽构象的构建块，发展为包括构象锁和环状结构。混合生物和化学合成生物技术允许合成比DELs更大的文库，将文库大小的限制推到近1013。这条链上的相关工作将围绕着将非自然基序纳入来自核糖体表达的环肽文库。这些不自然的基序，例如单一的溴苯丙氨酸残基，将允许伙伴之间的偶联，例如铃木偶联的硼酸盐，与某些目标蛋白结合的已知基序。这项工作将涉及所述多肽文库的合成，特定结合基序的偶联，以及随后对各种环多肽文库的测试。

Project SummaryVast libraries of encoded peptides, including cyclic systems, can be synthesised using biological methods, such as ribosomal expression systems, and can be screened by affinity selection to produce high affinity protein ligands. However, the scope of the aforementioned biological methods is usually limited to proteinogenic amino acids and their close analogues. The incorporation of unnatural moieties would allow for the more specific targeting of selected proteins classes, potentially leading to compounds with greater proteolytic stability and cell permeability. The aim of the proposed project is to develop synthetic approaches to realise this goal by developing chemical, rather than biological, techniques for peptide library synthesis. These techniques lack the requirement of identification by biological machinery and can, therefore, incorporate a far wider range of building blocks. In a second strand, hybrid methodology will be developed which allows the incorporation of unnatural warheads for selected protein classes into biologically displayed peptide libraries.Chemical Synthesis of Encoded Peptide LibrariesDELs are a technology for the synthesis and screening of a large number of molecules. Each DEL is a collection of these molecules, which are comprised of an organic moiety linked to a DNA oligonucleotide. The "genotype" (the DNA tag) serves as an amplifiable identification bar code for the displayed "phenotype" (the small organic molecule).Peptoids are oligomers of N-alkylated glycines; a peptoid monomer is an amino acid with the side chain connected to the nitrogen of the backbone, rather than the alpha carbon. Peptoids offer certain advantages to peptides as drugs, incl. resistance to proteolytic degradation due to the tertiary amide, improved cell-permeability relative to peptides due to the absence of the amide NH for H-bonding, and side chain diversity as they can be theoretically synthesised from any given primary amine.Peptoids are most often synthesised via a solid-supported submonomer method, using bromoacetic acid and then a primary amine. This is an issue, as an ?-halo carbonyl is a powerful electrophile, so will alkylate the DNA, so this method is incompatible with DEL synthesis.Initial work will focus on the off-DNA synthesis of peptoid monomers and the optimisation of multiple routes towards these building blocks. Consequently, optimised conditions will be applied to a variety of amines (to afford a range of peptoid monomers) and the scope of the reactions elucidated. Concurrently, the on-DNA coupling of peptoid monomers will be established using micellar conditions. This will be developed to realise the scope of the couplings by testing the coupling of the aforementioned synthesised peptoid monomers on-DNA.This will develop, in further work, to the synthesis of a range of peptoid-containing libraries, beginning with a small linear peptoid library, comprised of a range of the synthesised monomers. Extension of this will lead to the incorporation of building blocks that allow for the control of peptoid conformation, developing into the inclusion of conformational locks and cyclic structures.Hybrid Biological and Chemical SynthesisBiological techniques allow for the synthesis of even larger libraries than DELs, pushing the limit of library size to near 1013. The associated work in this strand will revolve around the incorporation of unnatural motifs into cyclic peptide libraries from ribosomal expression. These unnatural motifs, such as a single bromophenylalanine residue, will allow for the coupling of partners, e.g. boronates for Suzuku coupling, with known motifs that bind to certain target proteins. The work will involve the synthesis of said peptide libraries, the coupling of the specific binding motifs, and the subsequent testing of the diverse cyclic peptide libraries.