A bicyclic peptide scaffold was chemically adapted to generate diarylethene‐based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype molecule—sunflower trypsin inhibitor‐1 (SFTI‐1)—we obtained light‐controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20‐fold by irradiation. The inhibitory potency as well as resistance to proteolytic degradation were systematically studied on a series of 17 SFTI‐1 analogues. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme–inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin‐based hydrogel and an antimicrobial peptide BP100‐RW. Finally, our design principles of diarylethene photoswitches are shown to apply also for the development of other serine protease inhibitors.
Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo.
Ein bizyklisches Peptid wurde mit einem Diarylethen modifiziert, um lichtschaltbare Inhibitoren der Serinprotease Bos taurus Trypsin 1 (T1) zu erzeugen. Ausgehend von dem Sonnenblumen‐Trypsin‐Inhibitor‐1 (SFTI‐1) als Prototyp, erhielten wir lichtschaltbare Inhibitoren von T1 mit Ki im niedrigen nanomolaren Bereich, deren Aktivität durch Bestrahlung mit Licht um das 20‐Fache moduliert werden konnte. Die inhibitorische Potenz sowie die Resistenz gegen proteolytischen Abbau wurden systematisch an einer Reihe von 17 SFTI‐1‐Analoga untersucht. Die Isomerisierung des Photoschalters beeinflusst das Netzwerk aus Wasserstoffbrücken, das zur Stabilisierung der Struktur der Inhibitoren dient, und möglicherweise auch die Bindungsdynamik zwischen Enzym und Inhibitor. Die Machbarkeit einer zeitlichen und räumlichen Manipulation der Enzymaktivität wurde durch kontrollierten Verdau eines Hydrogels auf Gelatinebasis und eines antimikrobiellen Peptids BP100‐RW bewiesen. Die angewendeten Designprinzipien für Diarylethen‐Photoschalter stellen eine Grundlage für die Entwicklung anderer Serinprotease‐Inhibitoren dar.
The new efficient, convenient protocol for the synthesis of heteroannelated 3-cyanopyridines and pyrimidines starting from diverse aminoheterocycles and 3,3-dimethoxy-2-formylpropionitrile sodium salt was elaborated. The advantages and improvements of the procedure compared to previously known methods were shown. The scope and limitations of the method were determined. The impact of the structural features on regioselectivity was discussed. The preparativity, scalability and application scope of the elaborated protocol was demonstrated by the synthesis of five heteroannelated 3-cyanopyridines in up to 100 g quantities.
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