Center for Peptide-Based Antibiotics, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, DK-2100, Denmark; Department of Chemistry, Khalifa University, Abu Dhabi 127788, United Arab Emirates.
Center for Peptide-Based Antibiotics, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, DK-2100, Denmark.
Bioorg Chem. 2023 Dec;141:106876. doi: 10.1016/j.bioorg.2023.106876. Epub 2023 Sep 19.
Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc-RPRPPR) of the AMP oncocin (i.e., VDKPPYLPRPRPPRRIYNR-NH) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N-terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Arg → Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans-4-hydroxy-l-proline (Hyp) or 4,4-difluoro-l-proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l-Arg → d-Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Pro → Dfp modifications proved to possess the best activity profile: MICs of 1-2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC above 1280 µg/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure-activity studies aimed at optimization of Pro-rich AMPs.
抗菌肽 (AMPs) 通常具有胍基官能团，因此需要强大的合成程序来促进对具有非天然精氨酸 (Arg = R) 同系物的类似物的访问。最初，采用 AMP oncocin 的树脂结合的 Arg/Pro 丰富的芴甲氧羰基保护片段 (Fmoc-RPRPPR)（即 VDKPPYLPRPRPPRRIYNR-NH），在商业胍基化试剂的比较树脂评估中，与最近研究的双 Boc 保护的三唑基试剂 1H-三唑-1-[N,N'-双(叔丁氧基羰基)]-甲脒进行了头对头比较，该试剂通过无色谱程序合成。该试剂被发现能够在固相肽合成 (SPPS) 中定量转化显示同精氨酸 (Har) 残基和/或 N 端胍基的肽。使用 SPPS 获得了带有单个或多个 Arg → Har 修饰的 18 肽 oncocin 的类似物。此外，通过掺入单个或多个反式-4-羟基-l-脯氨酸 (Hyp) 或 4,4-二氟-l-脯氨酸 (Dfp) 残基来探索 oncocin 中脯氨酸 (Pro) 残基的取代对亲脂性的影响，这两种残基都影响亲脂性。所得的肽文库针对革兰氏阴性和革兰氏阳性细菌进行了测试。最小抑菌浓度 (MIC) 的分析表明，在位置 4、5 和 12（最初是 Pro 残基）进行修饰的类似物保留或略有提高了抗菌活性。接下来，在 C 末端部分具有两个稳定的 l-Arg → d-Arg 取代的 oncocin 类似物通过在位置 4、5 和 12 处用 Dfp 或 Hyp 三重取代 Pro 进一步修饰。显示三个 Pro → Dfp 修饰的类似物被证明具有最佳的活性谱：对大肠杆菌和肺炎克雷伯菌的 MIC 为 1-2 µg/mL，在 800 µg/mL 时低于 1%的溶血，在 HepG2 细胞中的 IC 大于 1280 µg/mL。因此，双氟代 Pro 残基的掺入似乎构成了针对富含 Pro 的 AMP 进行结构-活性研究的优化的新型工具。
