| Oligo-N-substituted glycines (peptoids) are a class of sequence specific peptidomimetics that are notable for their facile synthesis, sequence specificity, and peptide-like architectures. Additionally, due to their proteolytic resistance and enhanced cell permeability, peptoids are considered attractive candidates for therapeutics. In an effort to expand the chemical diversity and biomedical relevance of peptoids, we report here a novel technique to site-specifically functionalize linear peptoid oligomers utilizing the copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction.;Peptoid sequences including azide or alkyne-functionalized side chains were synthesized on solid-phase support and used as substrates for CuAAC. We were successful at conjugating such diverse pendant groups as fluorophores, nucleobases, metallocenes, and steroid ligands at up to six positions along the oligomer scaffold. We have also demonstrated that the triazole linkages formed from the CuAAC reaction are compatible with standard peptoid submonomer addition chemistry. This reactive orthogonality allows for multiple cycles of conjugation and oligomer extension to be performed, facilitating the generation of highly functionalized peptidomimetic oligomers outfitted with multiple heterogeneous pendant groups.;This CuAAC-mediated ligation technique was used to generate novel multivalent bioactive constructs. Estradiol-peptidomimetic conjugates (EPCs) were synthesized via site-specific conjugation of 17alpha-ethynylestradiol to multiple sites on azide-functionalized peptoid scaffolds. Multivalent EPCs were generated in high yield and their efficacy on binding the human estrogen receptor alpha (hERalpha) was evaluated in vitro. It was determined that EPC-hERalpha binding avidities are enhanced when the valency of hormone ligand is increased.;To monitor EPC-mediated activation of the hERalpha in vivo, HEK293 cells were stably transfected with plasmids coding for hERalpha and subsequently treated with EPCs. EPC length and valency influenced both cell uptake and receptor modulation in this system, with highest levels of hERalpha activation coming from divalent 18-mer and trivalent 9-mer EPCs. Non-steroidal peptidomimetic conjugates did not activate the hERalpha when compared to their EPC analogues, indicating that the estradiol pendant groups are primarily responsible for the bioactivity of these constructs. Given intrinsic length and valence "tunability", and the ability to modulate the hERalpha in living systems, EPCs may prove to be beneficial chemical tools in activating specific nuclear, and perhaps extranuclear effects of estrogens. |
