PREPARATION OF FUNCTIONALLY ACTIVE CELL-PERMEABLE PEPTIDES BY SINGLE-STEP LIGATION OF 2 PEPTIDE MODULES

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS), Her ein, we describe a facile method that expedites synthesis of biologica lly active, cell-permeable peptides by site-specific ligation of two f ree peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modul es is produced by ligation of the COOH-terminal aldehyde on the MPS an d the NH2-terminal 1,2-amino thiol moiety on the functional sequence, This thiazolidine ligation approach is performed with stoichiometric a mounts of fully unprotected MPS and functional peptide in an aqueous b uffered solution, eliminating the need for additional chemical manipul ation and purification prior to use in bioassays, Two different MPSs w ere interchangeably combined with two different functional sequences t o generate two sets of hybrid peptides. One set of hybrid peptides, ca rrying the cytoplasmic cell adhesion regulatory domain of the human in tegrin beta(3), inhibited adhesion of human erythroleukemia cells to f ibrinogen-coated surfaces, A second set of hybrid peptides, carrying t he nuclear localization sequence of the transcription factor NF-kappa B, inhibited nuclear import of transcription factors NF-kappa B, activ ator protein 1, and nuclear factor of activated T cells in agonist-sti mulated Jurkat T lymphocytes. In each assay, these nonamide bond hybri ds were found to be functionally comparable to peptides prepared by th e conventional method. Cumulatively, this new ligation approach provid es an easy and rapid method for engineering of functional, cell-permea ble peptides and demonstrates the potential for synthesis of cell-perm eable peptide libraries designed to block intracellular protein-protei n interactions.