Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound collections, coupling protocols were developed reducing the amount of coupling reagent and fluorophore. To enable further chemical derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanalytical applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compound collections with uniform labeling yields.
Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 μM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.
A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.
Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3.
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