Development of orthogonal peptide bioconjugation methodology for application in targeting GLP-1R

Bioconjugation is a powerful tool for precise modification of biomolecules such as peptides and proteins. Peptide bioconjugation has been a topic of interest for a long time now. Here, we have explored the potential of some of the robust and popular peptide bioconjugation strategies such as maleimide-thiol chemistry, Cu-catalyzed azide-alkyne cycloaddition (CuAAC/click chemistry), native chemical ligation (NCL) and Suzuki-Miyaura cross coupling. The orthogonality of these bioconjugation reactions were utilized to develop an efficient methodology for labeling a peptide with multiple tags such as a cell penetrating K9 peptide, a biotin, and a fluorescein. Its application was demonstrated in a biological system by performing a cell penetration experiment. The peptide was visualized by fliorescence to confirm the cell penetration and it was pulled out from the cell lysate using streptavidin beads by virtue of biotin-streptavidin interaction. Having demonstrated the orthogonality, the bioconjugation tools were further employed to engineer glucagon-like peptide-1 (GLP-1) in order to target its receptor. Using native chemical ligation (NCL) and click chemistry, a facile, yet efficient strategy for the synthesis of dimeric constructs of full length GLP-1 and an N-terminus GLP-1 analog was developed. Also, maleimide-thiol chemistry was employed to modify a high affinity GLP-1 analog, EM2198. To minimize non-specific uptake of EM2198-based PET tracers, differently charged and uncharged amino acid-containing hydrophilic tails were conjugated to the C-terminus of EM2198. While positively charged and negatively charged tails did not minimize the non-specific uptake, peptide 37 with an uncharged penta-Ser tail inhibited non-specific uptake by liver, lungs, and spleen but not by the kidneys. Lastly, the site specific cleavage of GLP-1 peptide bonds by NEP 24.11 was shielded by the introduction of side-chain to side-chain lactam bridges at specific positions. Also, as emphasized by their lower potencies, the lactam bridge near the N-terminal random coil of GLP-1 appeared to be less tolerated than the one distant from the N-terminus. These peptides showed promise as PET tracers for beta cell mass (BCM) imaging as revealed by the in vivo and ex vivo PET/CT imaging experiments, however high renal uptake was also observed.