Mark S. Bannon, Jeffrey F. Ellena, Aditi S. Gourishankar, Spencer R. Marsh, Dilza Trevisan-Silva, Nicholas E. Sherman, L. Jane Jourdan, Robert G. Gourdie and Rachel A. Letteri
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引用次数: 0
Abstract
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. In vitro 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.
期刊介绍:
Molecular Systems Design & Engineering provides a hub for cutting-edge research into how understanding of molecular properties, behaviour and interactions can be used to design and assemble better materials, systems, and processes to achieve specific functions. These may have applications of technological significance and help address global challenges.