The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters

Certain proteins contain subunits that enable their active translocation ac ross the plasma membrane into cells. In the specific case of HIV-1, this su bunit is the basic domain Tat(49-57) (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to deve lop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49-57 were prepared and their cellular uptake in to Jurkat cells was determined by flow cytometry. All truncated and alanine -substituted analogues exhibited diminished cellular uptake, suggesting tha t the cationic residues of Tat(49-57) play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of severa l cationic amino acids (histidine, lysine, and ornithine) are less effectiv e than Taf(49-57) in cellular uptake. In contrast, a 9-mer of L-arginine (R 9) was 20-fold more efficient than Tat(49-57) at cellular uptake as determi ned by Michaelis-Menton kinetic analysis. The D-arginine oligomer (r9) exhi bited an even greater uptake rate enhancement (>100-fold). Collectively, th ese studies suggest that the guanidinium groups of Tat(49-57) play a greate r role in facilitating cellular uptake than either charge or backbone struc ture. Based on this analysis, we designed and synthesized a class of polygu anidine peptoid derivatives. Remarkably, the subset of peptoid analogues co ntaining a six-methylene spacer between the guanidine head group and backbo ne (N-hxg), exhibited significantly enhanced cellular uptake compared to Ta t(49-57) and even to r9. Overall, a transporter has been developed that is superior to Tat(49-57), protease resistent, and more readily and economical ly prepared.