Synthesis and Biological Evaluation of Well-Defined M6P(n)-Modified Glycopeptides for Targeted Protein Degradation.

Lysosome-targeting chimeras (LYTACs) is an emerging therapeutic strategy to deplete circulating and cell-surface proteins. It harnesses endocytic receptors, such as the cation-independent mannose-6-phosphate receptor (CI-M6PR), to direct a protein of interest for lysosomal degradation. We have developed a synthetic methodology to prepare multifunctional M6PR-targeting glycopeptides of varying valency using common building blocks. Peptides were prepared having one to six 4-methyltrityl (Mtt)-protected lysine moieties that could selectively be removed to give amines that were coupled with mannose-6-phosphate (M6P), mannose-6-phosphonate (M6Pn) and mannosyl building blocks having an α-configured anomeric 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)acetic acid. The hydroxyls, phosphate and phosphonate of the building blocks were protected as p-methylbenzyl, which exhibited excellent stability during synthesis but could be removed under mild conditions without affecting sensitive functionalities. The glycopeptides have an azido lysine moiety for functionalization by strain-promoted azide-alkyne cycloaddition (SPAAC). Cetuximab (Ctx) was functionalized with bicyclo[6.1.0]non-4-yne for conjugation to M6P and M6Pn containing peptides via SPAAC. The resulting mAbs were examined for cellular uptake of soluble Epidermal Growth Factor Receptor (EGFR). Only mABs modified by glycopeptides carrying three or six M6P or M6Pn residues significantly increased uptake. Phosphonates were more efficient in removing cell surface EGFR by Ctx modified by tri- or hexavalent M6P or M6Pn ligand.