title: Acid-labile traceless click linker for protein transduction.
abstract: Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with  implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system.
authors.affiliation: Pharmaceutical Biotechnology, Center for System-based Drug Research, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: Kevin
authors.initials: K
authors.lastname: Maier
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D002949:Citraconic Anhydrides / Q000737:chemistry* / Q000378:metabolism; D057930:Click Chemistry; D003600:Cytosol / Q000378:metabolism; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D001616:beta-Galactosidase / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D001386:Azides; D002949:Citraconic Anhydrides; D004337:Drug Carriers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase