In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC50 values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.

A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.