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Synthesis of structurally modified peptides and peptide-cargo conjugates by novel strategy, their cellular uptake and their application in anti-sense

In order to gain insight into structural requirements for CPPs, we synthesized peptides showing systematically altered structural properties, charge and size, derived from the -helical model peptide KLALK LALKA LKAAL KLA-NH2 (KLA). Moreover, a set of modified ApoE-derived peptides has been synthesized and studied with regard to cellular uptake. Beside the synthesis of intracellularly cleavable disulfide-linked CPP-cargo conjugates, several CPP-PNA conjugates were synthesised by a novel enzyme-mediated ligation strategy, resulting in stable amide bond links between CPP and cargo. This newly developed methodology has been proved to be very useful in the preparation of highly pure and well-characterized conjugates.

Uptake studies using different cell cultures and different analytical tools demonstrated that most of the cargo-CPP conjugates possess a significant intracellular accumulation. However, the intracellular concentration is seemingly strongly influenced not only by the uptake behaviour, but also by the export, which was found to be significant e. g. for acidic peptides, whereas strongly basic and amphipathic peptides are retained inside the cells, probably by binding to intracellular proteins and/or oligonucleotides.

For assessing the influence of novel CPP-conjugations on the biological activity of PNAs, we measured the influences of the pre-treatment of spontaneously beating neonatal rat cardiomyocytes (CM) with PNA-derivatives, complementary to bases 12-23 of the translated region of the nociceptin/orphanin FQ receptor, on the positive chronotropic effect exerted by the neuropeptide nociceptin. In the second case the splice-correction assay developed by Kole et al. was used in collaboration with Lebleu's group. In both assays we achieved anti-sense effects. Corresponding conjugates with scrambled PNA-sequences proved inactive, thus supporting the anticipated anti-sense mechanism.

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