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 resistant, and more readily and economically prepared.

Figure 1

FACS cellular uptake assay of truncated analogs of Tat_49–57 (Fl-ahx-RKKRRQRRR): Tat_49–56 (Fl-ahx-RKKRRQRR), Tat_49–55 (Fl-ahx-RKKRRQR), Tat_50–57 (Fl-ahx-KKRRQRRR), and Tat_51–57 (Fl-ahx-KRRQRRR). Jurkat cells were incubated with varying concentrations (12.5 μM shown) of peptides for 10 min at 23°C.

Figure 2

FACS cellular uptake assay of alanine-substituted analogs of Tat_49–57: A-49 (Fl-ahx-AKKRRQRRR), A-50 (Fl-ahx-RAKRRQRRR), A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR), A-53 (Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55 (Fl-ahx-RKKRRQARR), A-56 (Fl-ahx-RKKRRQRAR), and A-57 (Fl-ahx-RKKRRQRRA). Jurkat cells were incubated with varying concentrations (12.5 μM shown) of peptides for 10 min at 23°C.

Figure 3

FACS cellular uptake assay of d- and retro-isomers of Tat_49–57: d-Tat_49–57 (Fl-ahx-rkkrrqrrr), Tat_57–49 (Fl-ahx-RRRQRRKKR), and d-Tat_57–49 (Fl-ahx-rrrqrrkkr). Jurkat cells were incubated with varying concentrations (12.5 μM shown) of peptides for 15 min at 23°C.

Figure 4

FACS cellular uptake of a series of arginine oligomers and Tat_49–57: R5 (Fl-ahx-RRRRR), R6 (Fl-ahx-RRRRRR), R7 (Fl-ahx-RRRRRRR), R8 (Fl-ahx-RRRRRRRR), R9 (Fl-ahx-RRRRRRRRR), r5 (Fl-ahx-rrrrr), r6 (Fl-ahx-rrrrrr), r7 (Fl-ahx-rrrrrrr), r8 (Fl-ahx-rrrrrrrr), r9 (Fl-ahx-rrrrrrrrr). Jurkat cells were incubated with varying concentrations (12.5 μM shown) of peptides for 15 min at 23°C.

Scheme 1

(a) BrCH₂CO₂H, DIC, DMF. (b) BocNH-X-NH₂, DMF. (c) Fmoc-ahx-CO₂H, DIC, DMF. (d) piperidine, DMF. (e) FITC-NCS, DIEA, DMF. (f) 95:5 TFA/TIS. (g) pyrazole-1-carboxamidine, aq. Na₂CO₃, 50°C. Abbreviations: N-etg, X = (CH₂)₂; N-arg, X = (CH₂)₃; N-btg, X = (CH₂)₄; N-hxg, X = (CH₂)₆; N-ocg, X = (CH₂)₈; N-chg, X = trans-1,4-cyclohexyl.

Figure 5

FACS cellular uptake of polyguanidine peptoids and d-arginine oligomers. Jurkat cells were incubated with varying concentrations (12.5 μM shown) of peptoids and peptides for 4 min at 23°C.

Figure 6

FACS cellular uptake of d-arginine oligomers and polyguanidine peptoids. Jurkat cells were incubated with varying concentrations (12.5 μM shown) of fluorescently labeled peptoids and peptides for 4 min at 23°C.

Figure 7

FACS cellular uptake of d-arginine oligomers and N-hxg peptoids. Jurkat cells were incubated with varying concentrations (6.3 μM shown) of fluorescently labeled peptoids and peptides for 4 min at 23°C.

Figure 8

FACS cellular uptake of d-arginine oligomers and N-chg peptoids. Jurkat cells were incubated with varying concentrations (12.5 μM shown) of fluorescently labeled peptoids and peptides for 4 min at 23°C.