MccJ25的酵素修飾及仿生分子合成

Abstract

套索胜肽(lasso peptide)，是一系列如套索一般構型的特殊胜肽家族。由於其結構的特殊性，該種類的胜肽對於溫度、酸鹼度、酵素等環境因素皆具有極高的穩定性，然而此結構亦阻礙了科學家們對套索胜肽進行詳細的研究以及開發泛用的合成方法。在本研究中，我們將利用兩種方式來探索microcinJ25 (MccJ25)—一個標誌性的套索胜肽—的化學空間: 一、結合金屬鍵結(metal coordinate)、固相胜肽合成法(solid phase peptide synthesis, SPPS)及有機化學合成仿生構型；二、結合酵素剪切以及化學修飾的方式對現有的MccJ25構型進行修改。 在方法一中，我們設計了數個模仿套索胜肽，並且可經由化學合成得到的仿生分子 (mimic)。我的論文主要著重在金屬仿生分子 (M-mimic) 的合成，其中含有數個可與金屬鍵結的天然及非天然胺基酸，例如組胺酸及2,6-吡啶二甲酸。我們假設當這些分子被放置於空間中適當的位置時，這些雜環將與中心金屬產生配位鍵結並形成金屬錯合物 (metal complexes)。 在方法二中，我們經由篩選一系列的酵素後，發現嗜熱菌蛋白酶 (Thermolysin) 可在特定位點上，將MccJ25分子從[1]輪烷轉變成[2]輪烷。利用此特性，我們嘗試探索修飾該分子骨架的方式。例如，將有機小分子放入此[2]輪烷的骨架。這項如手術般精確的酵素-化學修飾的手法將使我們得以更加深入地探索及利用套索胜肽。

Lasso peptides are a peptide family with a lariat-like 3D structure. Due to its unique threaded structure, lasso peptides are stable to heat, pH, enzyme digestions, and other kinds of environmental stress. However, the same unique structure has also hindered their detailed characterization and prevented the development of generalized synthetic methods. In this study, we aimed to explore the chemical landscape of microcin J25, the prototypical lasso peptide, by two approaches: 1) synthesize structural mimics of MccJ25 by combining solid phase peptide synthesis and organic synthesis, and 2) engineer the existing MccJ25 scaffold by combining enzymatic cleavage and chemical modifications. In the first approach, we proposed designs that are synthetically accessible to mimic the structure of a threaded peptide. My thesis focuses on the M-mimic, which includes natural and non-natural amino acids, such as histidine and 2,6-pyridinedicarboxylic acid. We hypothesized that if these residues are positioned in the appropriation positions within the peptide, their heterocycles may function as ligands to coordinate a central metal cation and form a complex that imitates the shape of MccJ25. In the second approach, we identified thermolysin from screening a collection of proteases6, and it cleaves MccJ25 site-specifically to convert it from a [1]rotaxane to a [2]rotaxane. We are currently exploring the possibility of modifying this scaffold further. For example, various small molecules can be installed by organic synthesis into the [2]rotaxane. Such a precise chemoenzymatic manipulation is akin to a surgical incision-and-repair process and enabled us to expand the functional group repertoire of lasso peptides.