ABSTRACT
Unprotected peptidyl phosphoranes 1 with sequence Ac-L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6-12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14-16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.
Subject(s)
Caspase 3/drug effects , Caspase Inhibitors/chemistry , Caspase Inhibitors/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Phosphoranes/chemistry , Phosphoranes/pharmacology , Caspase Inhibitors/chemical synthesis , Molecular Structure , Oligopeptides/chemical synthesis , Phosphoranes/chemical synthesis , Water/chemistrySubject(s)
Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Biomimetic Materials/chemistry , DNA-Binding Proteins/chemistry , Disulfides/chemistry , Peptides, Cyclic/chemistry , Triazoles/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Biomimetic Materials/pharmacology , Click Chemistry , Cyclization , DNA-Binding Proteins/pharmacology , Microbial Sensitivity Tests , Molecular Sequence Data , Peptides, Cyclic/pharmacology , PeptidomimeticsABSTRACT
The difference between site-specific and random immobilisation of the aldo/keto reductase AKR1A1 was explored. AKR1A1 was recombinantly expressed as a thioester by the intein strategy. The thioester was selectively modified with a biotin label by the expressed protein ligation method, and subsequent immobilisation on streptavidin templates was performed. Adsorption of wild-type AKR1A1 to streptavidin templates and of biotinylated AKR1A1 to uncoated templates was used to study randomly immobilised enzymes. Investigation of the kinetic parameters revealed remarkably improved activity for the site-specifically immobilised enzyme, which was comparable to that of the wild-type enzyme in solution and 60-300-fold greater than that of the randomly immobilized enzymes. Furthermore, the enzyme was surprisingly stable. No loss of activity was observed for over a week, and even after 50 days more than 35% of activity was maintained.
Subject(s)
Alcohol Oxidoreductases/chemistry , Enzymes, Immobilized/chemistry , Aldehyde Reductase , Aldo-Keto Reductases , Binding Sites , Biotin/chemistry , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Kinetics , Ligands , Mass Spectrometry , Protein Hydrolysates/chemistry , Trypsin/chemistryABSTRACT
Peptide adhesion on semiconductor surfaces is quantitatively investigated by atomic force microscopy. The selected peptides are shown to cluster at the surface, with the larger, higher, and softer clusters appearing on the surfaces with lower peptide adhesion. Average cluster diameters vary from 40 nm on GaAs (100) to 300 nm on Si (100). Direct adhesion of the peptides to the surface competes with forming molecular aggregates that offer an overall reduced surface contact.
