Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
J Org Chem. 2011 Feb 18;76(4):1118-25. doi: 10.1021/jo102329c. Epub 2011 Jan 18.
Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), is also a target for the action of host defense enzymes, such as lysozymes. Many bacteria have developed methods of masking their cell wall from these environmental dangers through the addition of aglycon moieties that prevent recognition or sterically hinder the degradative action of exogenous enzymes that would otherwise prove detrimental to the cell. Peptidoglycan acetyl-transferases (Pat's) and O-acetylpeptidoglycan esterases (Ape's) are the enzymes responsible for the controlled addition and removal of acetate onto the C-6 hydroxyl group of MurNAc residues in peptidoglycan. Studies on Ape1, an O-acetylpeptidoglycan esterase found in Neisseria gonorrheae, have suggested that this enzyme is essential for bacterial viability and thus presents an attractive target for antibacterial design. Previous studies on Ape1 have been hindered by the fact that Ape1's natural substrate is an insoluble polymer. In this paper we outline the design, synthesis, and testing of the water-soluble di- and monosaccharide substrate analogues 1 and 2. Both 1 and 2 serve as substrates of Ape1 with k(cat)/K(M) values of (5.1 ± 1.7) × 10(3) M(-1) s(-1) and (3.1 ± 0.8) × 10(3) M(-1) s(-1), respectively. It was determined that the substitution of the GlcNAc residue in compound 1 with an O-benzyl group in compound 2 did not significantly decrease the enzyme's affinity for the monosaccharide. These findings are important as they demonstrate that the catalytic prowess of Ape1 is not dependent on its binding to a polymeric substrate. This ensures that small molecule transition state/intermediate analogues can also capture the transition state binding energy of Ape1 and potentially serve as potent inhibitors. The synthetic route to compounds 1 and 2 could readily be modified to allow for the installation of a wide variety of functional groups at the MurNAc C-6 position in both the mono- and disaccharide scaffolds. This will serve as a general method for the construction of Ape1 substrates and inhibitors.
肽聚糖是细菌细胞壁的组成部分，对于维持细胞的形状和刚性至关重要。因此，其聚合物结构由交替的 N-乙酰葡萄糖胺（GlcNAc）和 N-乙酰胞壁酸（MurNAc）单元组成，也是宿主防御酶（如溶菌酶）作用的靶标。许多细菌通过添加糖苷配基来掩盖细胞壁免受这些环境危害，这些糖苷配基可以防止识别或阻碍外源性酶的降解作用，否则这些酶会对细胞造成损害。肽聚糖乙酰转移酶（Pat's）和 O-乙酰肽聚糖酯酶（Ape's）是负责在肽聚糖的 MurNAc 残基的 C-6 羟基上加乙酰基和去除乙酰基的酶。对淋病奈瑟氏球菌中发现的 O-乙酰肽聚糖酯酶 Ape1 的研究表明，该酶对于细菌的生存能力至关重要，因此是抗菌设计的一个有吸引力的靶标。以前对 Ape1 的研究受到其天然底物是不溶性聚合物这一事实的阻碍。在本文中，我们概述了水溶性二糖和单糖底物类似物 1 和 2 的设计、合成和测试。1 和 2 都是 Ape1 的底物，k(cat)/K(M) 值分别为（5.1±1.7）×10(3) M(-1) s(-1)和（3.1±0.8）×10(3) M(-1) s(-1)。确定在化合物 1 中的 GlcNAc 残基用 O-苄基取代后，化合物 2 对单糖的酶亲和力没有明显降低。这些发现很重要，因为它们表明 Ape1 的催化能力不依赖于其与聚合物底物的结合。这确保了小分子过渡态/中间体类似物也可以捕获 Ape1 的过渡态结合能，并可能成为有效的抑制剂。化合物 1 和 2 的合成路线可以很容易地进行修改，以便在单糖和二糖骨架的 MurNAc C-6 位置上安装各种官能团。这将作为构建 Ape1 底物和抑制剂的通用方法。
