Stabilization of peptides against proteolysis through disulfide-bridged conjugation with synthetic aromatics

Abstract

Peptides have been promising molecular scaffolds for the development of potential therapeutics with high affinity and specificity to biomacromolecules. However, their inherent proteolytic instability significantly hampers their biological applications. Strategies that can stabilize peptides against proteolytic digestion on the basis of noncovalent interactions-without extensive manipulation of the sequence or use of unnatural residues-are greatly desired. In this work, we developed a general, convenient, and efficient strategy for the stabilization of peptides against proteolysis, which involves noncovalent π-π interactions between aromatic amino acid residues in peptides and synthetic electron-deficient aromatics (NDI), together with the implication of steric hindrance (from the bulky NDI moiety), and the enhancement of peptide α-helicity. This strategy is complementary in concept to the conventional well-established covalent approaches for peptide stabilization, and is thus promising for being utilized, in combination with the latter ones, to circumvent the problem of proteolytic instability of peptides. We envisioned that this study should provide invaluable guidelines to the design and synthesis of organic molecule-peptide hybrids with significantly improved proteolytic resistance, and benefit the development of peptide therapeutics and probes.

Emerging Methods and Design Principles for Cell‐Penetrant Peptides

“…Them ost common complement to fluorescence microscopy is flow cytometry. [22,113,152] This provides the high throughput and quantitation that microscopy lacks,b ut flow cytometry does not provide even aq ualitative analysis of subcellular localization. Together,f luorescence microscopy and flow cytometry can be used to quantitatively measure cell uptake,a nd qualitatively assess endosomal release and cytosolic localization.…”

“…Using careful confocal microscopy and colocalization studies, one can get a relative measure of degree of endosomal escape, but these techniques cannot absolutely quantitate the amount of endosomally trapped material or the amount of material that has escaped into the cytosol. The most common complement to fluorescence microscopy is flow cytometry . This provides the high throughput and quantitation that microscopy lacks, but flow cytometry does not provide even a qualitative analysis of subcellular localization.…”

Neue Methoden und Designprinzipien für zellgängige Peptide

“…Beispielsweise wurde gezeigt, dass die Fixierung der Zellen vor der mikroskopischen Analyse Artefakte hervorbringt, einschließlich der Umverteilung farbstoffmarkierter Peptide. [22,113,152] Diese bringt den hohen Durchsatz und die Quantifizierungsmçglichkeiten mit sich, die der Mikroskopie fehlen, allerdings kann Durchflusszytometrie nicht einmal eine qualitative Analyse der subzellulären Lokalisierung liefern. Erstens erfordert sie die Anbindung eines großen, hydrophoben Farbstoffs an das interessierende Molekül.…”

“…Durch die sorgfältige Anwendung von konfokaler Mikroskopie und Kolokalisationsstudien ist es mçglich, ein relatives Maß fürd ie endosomale Freisetzung zu erlangen, jedoch ermçglichen diese Te chniken keine absolute Quantifizierung der Menge an endosomal eingeschlossenem Material oder der Menge des in das Zytosol ausgetretenen Materials.D ie gebräuchlichste Ergänzung zur Fluoreszenzmikroskopie ist die Durchflusszytometrie. [22,113,152] Diese bringt den hohen Durchsatz und die Quantifizierungsmçglichkeiten mit sich, die der Mikroskopie fehlen, allerdings kann Durchflusszytometrie nicht einmal eine qualitative Analyse der subzellulären Lokalisierung liefern. In Kombination kçnnen Fluoreszenzmikroskopie und Durchflusszytometrie zur quantitativen Messung der Zellaufnahme sowie zur qualitativen Erfassung der endosomalen Freisetzung und der zytosolischen Lokalisierung verwendet werden.…”

N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides

“…26 Additionally, disulphide linkage of an aromatic moiety that can form π–π stacking interactions with sidechains was reported to increase proteolytic stability toward chymotrypsin. 27 Finally, we have investigated appending short PEG chains as well as bulky groups to the N-terminus of unstructured peptides with only limited success. 15b,c …”