Synthesis and Purification of Responsive Peptides for Bottlebrush Construction

• Katherine Nilaj, Chemistry, University of Central Florida

• April Kloxin, Chemical Engineering, University of Delaware
• Rafael Castro, Chemical Engineering, University of Delaware
• Caitlyn DAmbrosio, Chemical Engineering, University of Delaware

Bundlemers have gained high interest in the world of chemical engineering and drug delivery due to their highly customizable self-assembling structure. They are composed of four identical alpha-helix peptides in parallel and anti-parallel configurations. Recently, bundlemers have been proposed to have potential as drug delivery systems because they can trigger hyper-sensitive reactions. Responsive peptides can be conjugated onto a bundlemer to allow them to form larger, bottle-brush assemblies and lengthen the resulting “bristles” for drug delivery systems. Responsive peptides were synthesized through solid phase peptide synthesis (SPPS), a process in which amino acids are added sequentially onto a solid resin support. A functional group is then coupled onto the N-terminus of the completed peptide before it is cleaved from the resin and precipitated into solid form. The peptide is purified through high-performance liquid chromatography (HPLC). Finally, mass spectroscopy was used to characterize the peptide and ensure the synthesis was successful. For bottlebrush construction, the peptide Alkyne-EGKSSGSGSESKST was conjugated onto the bundlemer through copper-catalyzed azide-alkyne cyclo addition. The AF647-AhxWGRGDSGLPETGG peptide lengthened the bristles through a sortase mediated ligation which covalently adds the peptide from the C terminus. After bottlebrush construction, confocal microscopy was used to image the bundlermers. The HPLC results for AF647-AhxWGRGDSGLPETGG showed a major peak where the peptide was collected. The mass spectroscopy of these tubes showed characteristic m/z values (701 and 1400) for the peptide. Confocal microscopy showcased the fluorescence of the AF647–conjugated bundlemer assembly, confirming successful attachment of the peptide. Beyond this project, the constructed bundlemers will be used as biolubricants or drug delivery systems in a hope to replace spherical nanoparticles. Bundlemers offer high retention in the body and deeper tissue penetration due to their architecture. Once cultivated with cells, the bioactive sequences in the peptides will become active for drug delivery or biolubrication.