The attachment of polyethylene glycol (PEG) to peptide or protein drugs is known to enhance residence half-life in serum;[1] however, its effect on the intracellular stability and distribution as well as on the activity of the conjugated biological entity in cells has not been investigated so far. Here, we probe the impact of small to medium-sized phosphoramidate-linked oligo- and polyethylene glycol chains on the stabilization of apoptosis-inducing BH3-peptides against proteolysis in cell lysates as well as on the peptides' intracellular activity.[2] PEG-chains were attached to azido-containing peptides by the chemoselective Staudinger-phosphite reaction,[3] thereby employing inexpensive linear PEG alcohols for the synthesis of phosphites to yield bis-phosphoramidate-linked polymer-peptide conjugates. The chemoselective Staudinger-phosphite reaction of unprotected azido-peptides in solution was essential for the synthesis of pure phosphoramidate-PEG-peptide-conjugates. Analogous transformations on the solid support delivered only inseparable peptide mixtures. The branched bis-phosphoramidate-PEG-peptide-conjugates showed an enhanced stability in Jurkat cell lysate with a half-life of several days when two only medium-sized PEG 2000 chains were attached. Most importantly, when introduced into cells by electroporation, the PEG-modified BH3-peptides displayed a homogeneous intracellular distribution and a specific intracellular apoptosis-inducing activity when compared to the non-modified derivatives, thus pointing towards a great potential of PEG conjugates in intracellular applications.[1] F. M. Veronese, G. Pasut, Drug Discovery Today 2005  , 10, 1451-1458.[2] N. Nischan, A. Chakrabarti, R. A. Serwa, P. H. M. Bov�e-Geurts, R. Brock, C. P. R. Hackenberger, Angew. Chem. Int. Ed 2013 , DOI: 10.1002/ange.201303467[3] a) R. Serwa, P. Majkut, B. Horstmann, J.-M. Swiecicki, M. Gerrits, E. Krause, C. P. R. Hackenberger, Chem. Sci. 2010  , 1, 596-602; b) R. A. Serwa, J.-M. Swiecicki, D. Homann, C. P. R. Hackenberger, J. Pept. Sci. 2010  , 16, 563-567.

Stabilization of peptides for intracellular applications by branched phosphoramidate-linked PEG chains

247th National Meeting (2014)

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