HPMA as a Scaffold for the Modular Assembly of Functional Peptide Polymers by Native Chemical Ligation

Abstract

Synthetic peptides are valuable tools in fundamental and applied biomedical research. On one hand, these molecules provide highly efficient access to competitive inhibitors of molecular interactions and enzyme substrates by rational design. On the other hand, peptides may serve as powerful vectors to mediate cellular uptake of molecules that otherwise enter cells only poorly. The coupling of both such functionalities provides access to molecules interfering with molecular processes inside the cell. However, the combination of several functionalities on one synthetic peptide may be compromised by problems associated with the synthesis of long peptides. Native chemical ligation enables the chemoselective coupling of fully deprotected functional building blocks. However, peptide thioesters are still not accessible by standard solid-phase peptide synthesis. Here, we demonstrate the cofunctionalization of a thioester-activated N-hydroxypropyl methacrylamide (HPMA) copolymer (28,500 Da) with the cell-penetrating peptide (CPP) nonaarginine and a bioactive peptide as independent building blocks by native chemical ligation. Nonaarginine was employed as a cell-penetrating peptide (CPP), a fluorescein-labeled analogue of a pro-apoptotic peptide as a biofunctional cargo. Incorporation of the fluorescein label enabled the highly sensitive quantification of the coupling stoichiometry by fluorescence correlation spectroscopy (FCS) using 0.4 pmol/12 ng of labeled construct. A construct only bearing the functional cargo peptide required cellular import by electroporation in order to show activity. In contrast, a construct combining all functionalities was active upon incubation of cells, validating the modular nature of the approach.

Coupling to Polymeric Scaffolds Stabilizes Biofunctional Peptides for Intracellular Applications

“…Following our procedure published previously (Ruttekolk et al, 2008), the BH3 peptide, containing an uncapped N-terminal cysteine residue, was coupled to an active ester-activated HPMA polymer by native chemical ligation (Scheme 1). A conjugate containing only fluoresceinlabeled cysteine-lysyl moieties and a conjugate carrying a mutated BH3 peptide, in which three residues were replaced for alanines, were used as negative controls.…”

“…Our previous analyses had shown that coupling of peptides to an HPMA polymer by native chemical ligation yielded bioactive conjugates by a modular design (Ruttekolk et al, 2008). These initial analyses had already indicated that the polymer-coupled peptides had a higher activity per peptide moiety.…”

“…The determination of stoichiometry proceeded as described previously (Ruttekolk et al, 2008) using FCS. FCS measurements were performed in 384-well plates (175 m, low-base design; Molecular Machines & Industries GmbH, Eching, Germany).…”

“…In a previous study, we have shown that coupling of peptides containing an N-terminal cysteine residue to thioesterpreactivated HPMA polymers is a convenient and robust method for the production of polymer-coupled peptides via native chemical ligation (Dawson et al, 1994;Tam et al, 2001;Ruttekolk et al, 2008). Conjugates containing an apoptosis-enhancing peptide, loaded into cells by electroporation, had a greatly increased biological activity in comparison with the free peptide.…”

A Peptide‐Functionalized Polymer as a Minimal Scaffold Protein To Enhance Cluster Formation in Early T Cell Signal Transduction

“…A thioesteractivated HPMA copolymer (28.5 kDa) served as the linear scaffold for the coupling of peptides. [17] We have previously shown that for a dextran backbone functionalized with apoptosisinducing BidBH3 peptides, a multivalent display of peptides yielded increased activity, even inside the cell. [18] However, it has not been explored whether such a molecular design can also act as a scaffold for promoting protein cluster formation in cellular signaling.…”