Abstract
Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.
Highlights
For more than a decade, polycationic cell penetrating peptides (CPPs) have been well established as molecular transporters for intracellular drug delivery or as probes for bioconjugation [1,2,3,4]
Cell penetrating peptoids (CPPos) library in IRORI MiniKans to expand the amount of peptoids for future phenotypic high throughput screens
The split and mix process of the MiniKans was performed manually due to the size of the “proof of principle” CPPo library, the IRORI system has many advantages
Summary
For more than a decade, polycationic cell penetrating peptides (CPPs) have been well established as molecular transporters for intracellular drug delivery or as probes for bioconjugation [1,2,3,4]. For the discovery of novel targeting CPPos, microscopy based phenotypic high-throughput screening seems to be a potent technique This often requires large and chemically diverse compound libraries. The synthesis of diverse peptoid scaffolds is usually performed by the so called submonomer approach through microwave-assisted amide bond formation and subsequent nucleophilic substitution with primary amines or via heterocyclic halomethyl carboxylic building blocks [25]. The latter approach was used for the synthesis of a one bead-one compound library of peptoids comprising a theoretical diversity of about 16,000 peptoids with a representation of 87% of the theoretical compounds [25]. The CPPo library was applied to HeLa cells for the screening of organelle targeting CPPos
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