Multiple linkages via sortase-mediated ligation with the example of protein-polymer conjugates

索糖酶是一种存在于革兰氏阳性细菌细胞壁中的酶，它以共价方式将表面蛋白连接到细胞壁。山梨酸酶A在多肽基序LPXTG和低聚甘氨酸之间形成多肽键。这种连接技术被称为排序酶介导的连接(SML)或排序标记，并发展成为基础研究中常用的工具。使用索尔特酶A，蛋白质可以连接到另一个生物分子，一个小的合成分子，一个聚合物或一个表面。因此，底物只需要配备C-端LPXTG和N-端GX序列。SML的一个弱点是形成的键(LPXTGGG)再次代表酶的底物。该反应是可逆的，因此，形成的键可以被酶再次切割。如今，有几种可能性可以改变反应的平衡，使连接能够以近乎定量的产率进行。然而，通过一个分级酶形成连续的键仍然是不可能的，或者只能通过苛刻的策略才能达到。因此，蛋白质链、表面的多层蛋白质以及在C-末端和N-末端修饰的蛋白质都很难获得。在这个项目的过程中，应该开发一种有效的方法来通过分类酶A将几个构建块连接起来，这应该以蛋白质-聚合物共轭化合物为例来说明，并允许获得一种具有两个不同聚合物链连接到一个蛋白质上的连接物。此外，蛋白质低聚体的产生过程应该类似于Merrifield肽的合成。该项目包括合成用于连接到蛋白质的C-端和N-端的聚合物块。在此基础上，利用SML技术合成蛋白质-聚合物偶联物。使用分解酶A的连续连接，其中形成的键不能被酶切割，还没有显示出来。这应该通过利用形成β-发夹结构的氨基酸序列来实现，该结构不再是分类酶A的底物。这种方法将首先通过对一种蛋白质的多次(非控制)连接来演示。为了确保三个或更多构建块的受控连接，必须首先阻止一个蛋白质末端。这一过程将使用掩蔽的低聚糖来开发，并通过连接几个蛋白质块来展示。随后，应将战略转移到将两个聚合物链连接到一个蛋白质上。

Sortases are enzymes occurring in the cell wall of gram-positive bacteria that covalently link surface proteins to the cell wall. Sortase A forms a peptide bond between the peptide motif LPXTG and an oligoglycine. This ligation technique is known as sortase-mediated ligation (SML) or sortagging and developed to a frequently used tool in basic research. Using sortase A, a protein can be ligated to another biomolecule, a small synthetic molecule, a polymer or a surface. Therefore, the substrates only need to be equipped with a C-terminal LPXTG and an N-terminal Gx sequence. A weakness of SML is that a formed bond (LPXTGGG) represents again a substrate for the enzyme. The reaction is reversible and, consequently, a formed bond can be cleaved again by the enzyme. Nowadays, several possibilities allow to shift the equilibrium of the reaction enabling ligations in nearly quantitative yield. However, the formation of consecutive bonds by one sortase is still impossible or can be only reached via demanding strategies. Therefore, chains of proteins, multiple layers of proteins on surfaces, and proteins modified at both C- and N-termini are difficult to access. In the course of this project, an efficient approach should be developed to link several building blocks by means of sortase A. This should be shown with the example of protein-polymer conjugates and give access to a type of conjugates with two different polymer chains linked to a protein. Furthermore, protein oligomers should be generated in a procedure similar to the Merrifield peptide synthesis.The project consists of the synthesis of polymer blocks for the ligation to both C- and N-terminus of a protein. On this basis, protein-polymer conjugates will be synthesized by SML. Consecutive ligations using sortase A, where the formed bonds could not be cleaved by the enzyme, have not been shown yet. This should be reached by utilizing amino acid sequences that form β-hairpin structures which are not a substrate for sortase A anymore. The approach will be demonstrated first with the multiple (uncontrolled) ligation of a protein. To ensure a controlled ligation of three or more building blocks, one protein terminus has to be blocked at first. This procedure will be developed using masked oligoglycines and shown with the ligation of several protein blocks. Subsequently, the strategy should be transferred to the linkage of two polymer chains to one protein.