Bannon Mark S, Ellena Jeffrey F, Gourishankar Aditi S, Marsh Spencer R, Trevisan-Silva Dilza, Sherman Nicholas E, Jourdan L Jane, Gourdie Robert G, Letteri Rachel A
Department of Chemical Engineering, University of Virginia Charlottesville VA 22903 USA
Biomolecular Magnetic Resonance Facility, School of Medicine, University of Virginia Charlottesville VA 22903 USA.
Mol Syst Des Eng. 2024 Sep 13;9(12):1215-1227. doi: 10.1039/d4me00072b. eCollection 2024 Nov 26.
Peptides are naturally potent and selective therapeutics with massive potential; however, low cell membrane permeability limits their clinical implementation, particularly for hydrophilic, anionic peptides with intracellular targets. To overcome this limitation, esterification of anionic carboxylic acids on therapeutic peptides can simultaneously increase hydrophobicity and net charge to facilitate cell internalization, whereafter installed esters can be cleaved hydrolytically to restore activity. To date, however, most esterified therapeutics contain either a single esterification site or multiple esters randomly incorporated on multiple sites. This investigation provides molecular engineering insight into how the number and position of esters installed onto the therapeutic peptide α carboxyl terminus 11 (αCT11, RPRPDDLEI) with 4 esterification sites affect hydrophobicity and the hydrolysis process that reverts the peptide to its original form. After installing methyl esters onto αCT11 using Fischer esterification, we isolated 5 distinct products and used 2D nuclear magnetic resonance spectroscopy, reverse-phase high performance liquid chromatography, and mass spectrometry to determine which residues were esterified in each and the resulting increase in hydrophobicity. We found esterifying the C-terminal isoleucine to impart the largest increase in hydrophobicity. Monitoring ester hydrolysis showed the C-terminal isoleucine ester to be the most hydrolytically stable, followed by the glutamic acid, whereas esters on aspartic acids hydrolyze rapidly. LC-MS revealed the formation of transient intramolecular aspartimides prior to hydrolysis to carboxylic acids. proof-of-concept experiments showed esterifying αCT11 to increase cell migration into a scratch, highlighting the potential of multi-site esterification as a tunable, reversible strategy to enable the delivery of therapeutic peptides.
肽是具有巨大潜力的天然强效且具选择性的治疗剂；然而，低细胞膜通透性限制了它们的临床应用，特别是对于具有细胞内靶点的亲水性阴离子肽。为克服这一限制，治疗性肽上阴离子羧酸的酯化可同时增加疏水性和净电荷以促进细胞内化，随后安装的酯可被水解切割以恢复活性。然而，迄今为止，大多数酯化治疗剂要么包含单个酯化位点，要么在多个位点随机掺入多个酯。本研究提供了分子工程方面的见解，即安装在治疗性肽α羧基末端11（αCT11，RPRPDDLEI）上具有4个酯化位点的酯的数量和位置如何影响疏水性以及使肽恢复其原始形式的水解过程。使用费歇尔酯化法在αCT11上安装甲酯后，我们分离出5种不同的产物，并使用二维核磁共振光谱、反相高效液相色谱和质谱来确定每种产物中哪些残基被酯化以及由此产生的疏水性增加情况。我们发现将C末端异亮氨酸酯化可使疏水性增加最大。监测酯水解表明C末端异亮氨酸酯水解稳定性最高，其次是谷氨酸，而天冬氨酸上的酯水解迅速。液相色谱 - 质谱分析显示在水解为羧酸之前形成了瞬时分子内天冬酰胺。概念验证实验表明将αCT11酯化可增加细胞向划痕处的迁移，突出了多位点酯化作为一种可调节、可逆策略以实现治疗性肽递送的潜力。
