ABSTRACT
We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.
Subject(s)
Click Chemistry/methods , Peptide Library , Peptides/chemistry , Vascular Endothelial Growth Factor A/chemistry , Amino Acid Sequence , Animals , Antibodies/administration & dosage , Antibodies/chemistry , Antibodies/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/immunology , Bacterial Toxins/metabolism , Calorimetry, Differential Scanning , Catalysis , Chromatography, High Pressure Liquid , Circular Dichroism , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/metabolism , Female , HT29 Cells , Humans , Injections, Intraperitoneal , Injections, Intravenous , Ligands , Male , Mass Spectrometry , Mice , Microsomes, Liver/metabolism , Peptides/metabolism , Peptides/pharmacokinetics , Protein Binding , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Special agents for protein capture: Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one-bead-one-compound method for the creation of a peptide library enable the fragment-based assembly of selective high-affinity protein-capture agents. The resulting ligands are water-soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper(I)-catalyzed cycloaddition.
Subject(s)
Peptide Library , Proteins/chemistry , Triazoles/chemistry , Antibodies/chemistry , Catalysis , Copper/chemistry , Ligands , Peptides/chemistry , Protein BindingABSTRACT
The development of a miniaturized sensing platform for the selective detection of chemical odorants could stimulate exciting scientific and technological opportunities. Oligopeptides are robust substrates for the selective recognition of a variety of chemical and biological species. Likewise, semiconducting nanowires are extremely sensitive gas sensors. Here we explore the possibilities and chemistries of linking peptides to silicon nanowire sensors for the selective detection of small molecules. The silica surface of the nanowires is passivated with peptides using amide coupling chemistry. The peptide/nanowire sensors can be designed, through the peptide sequence, to exhibit orthogonal responses to acetic acid and ammonia vapors, and can detect traces of these gases from "chemically camouflaged" mixtures. Through both theory and experiment, we find that this sensing selectivity arises from both acid/base reactivity and from molecular structure. These results provide a model platform for what can be achieved in terms of selective and sensitive "electronic noses."
Subject(s)
Acetic Acid/analysis , Ammonia/analysis , Microfluidic Analytical Techniques/methods , Nanowires/chemistry , Oligopeptides/chemistry , Acetic Acid/chemistry , Ammonia/chemistry , Biosensing Techniques/methods , Models, Chemical , Silicon/chemistryABSTRACT
A general method for the non-oxidative functionalization of single-crystal silicon(111) surfaces is described. The silicon surface is fully acetylenylated using two-step chlorination/alkylation chemistry. A benzoquinone-masked primary amine is attached to this surface via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition ("click" chemistry). The benzoquinone is electrochemically reduced, resulting in quantitative cleavage of the molecule and exposing the amine terminus. Molecules presenting a carboxylic acid have been immobilized to the exposed amine sites. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV), and contact angle goniometry were utilized to characterize and quantitate each step in the functionalization process. This work represents a strategy for providing a general platform that can incorporate organic and biological molecules on Si(111) with minimal oxidation of the silicon surface.
