Development of improved post-solid-phase peptide synthesis sample work-up procedures.

摘要

The technology of solid-phase peptide synthesis has undergone continual development during the past two to three decades. While most of this work focused on solid-phase peptide chain elongation chemistry, this thesis deals with the processes (cleavage, deprotection, sample purification) that occur after synthesis.; The efficacy of a new, low-odor cleavage reagent (Reagent L: trifluoroacetic acid, water, triisopropylsilane, dithiothreitol, 88:5:2:5) for cleavage of peptides from the resin support and removal of side-chain protecting groups was compared with that of traditional reagents (Reagent R: trifluoroacetic acid, thioanisole, ethanedithiol, anisole, 90:5:3:2; and Reagent K: trifluoroacetic acid, water, thioanisole, ethanedithiol, phenol, 82.5:5:2.5:5), which contain extremely malodorous thiols. Experiments were conducted to determine the protection afforded by the various cleavage cocktails to peptides that contain amino acid residues (Trp, Cys, Met) sensitive to alkylation by carbocations formed from protective groups during the cleavage reaction. Purified peptides were exposed to large excesses of alkylating species (e.g., t-butyl and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl ions) in the presence and absence of thiols and other carbocation scavengers to determine the effectiveness of these reagents in suppressing side reactions. Similarly, peptidyl-resins were cleaved and deprotected to determine the level of protection afforded by the cocktails. The rates of alkylation of ethanedithiol (in reagents R and K) and of dithiothreitol (in reagent L) in a t-butyl ion-generating system were compared. Reagent L was found to be as effective as conventional reagents (e.g., reagents R and K), but to have the great advantage of minimal odor.; A new technique that utilizes solid-phase extraction for the post-cleavage work-up and isolation of synthetic peptides was explored, with the aim of avoiding conventionally used, hazardous ether extraction methods. Peptide-containing cleavage cocktails were diluted with water and passed through a solid support, which selectively adsorbs the peptide (and some reagent adducts), allowing trifluoroacetic acid and other reagents to be washed away. The method was evaluated with a wide range of peptides and was found to be comparable in performance to the traditional ether extraction procedures. The two advantages of the solid-phase extraction technique are that it greatly eliminates the volume of ether required, thus reducing hazard and solvent disposal problems, and that it has the potential for automatable simultaneous multiple work-ups. It also produces a product that is at least as pure as that obtained by ether extraction.