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

Abstract

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat_49–57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat_49–57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat_49–57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat_49–57 in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat_49–57 at cellular uptake as determined by Michaelis–Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat_49–57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat_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 economically prepared.