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 Tat49-57 (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 Tat49-57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alaninesubstituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49-57 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 Tat49-57 in cellular uptake. In contrast, a 9-mer of L-arginine (R9) was 20-fold more efficient than Tat49-57 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 Tat49-57 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 Tat49-57 and even to r9. Overall, a transporter has been developed that is superior to Tat49-57, protease resistent, and more readily and economically prepared.

[reaction: see text]. Short oligomers of arginine function as remarkably efficient molecular transporters of drugs and probe molecules into cells and tissue. Currently, these compounds are prepared on resin through a unidirectional solid-phase synthesis. To extend the utility of these compounds for therapeutic and research applications, a scalable solution-phase synthesis of Arg8 (1) has been developed on the basis of a segment doubling strategy that proceeds in 13 steps and 28% overall yield from 4, including a novel one-step perdeprotection-perguanidinylation reaction.

[reaction: see text] Nine fluorescently labeled structurally varied polyguanidino dendrimers based on diamino acid monomeric units were individually synthesized in an efficient, scalable sequence using a trifluoroacetamide protecting group-perguanidinylation strategy. While the dendrimers varied significantly in their ability to enter a human lymphocyte cell line, the best transporters out-performed an oligoarginine reference standard.

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A regiocontrolled synthesis of 3,4-disubstituted pyrrole-2-carboxaldehydes was completed in two steps from acyclic starting materials. A Barton-Zard pyrrole synthesis between N-methoxy-N-methyl-2-isocyanoacetamide and alpha-nitroalkenes or beta-nitroacetates provided N-methoxy-N-methyl pyrrole-2-carboxamides (pyrrole Weinreb amides), which were converted into the corresponding pyrrole-2-carboxaldehydes by treatment with lithium aluminum hydride. A regioselective oxidation of the pyrrole-2-carboxaldehydes gave the corresponding 3,4-disubstituted 3-pyrrolin-2-ones.