Synthesis and comparative properties of two amide‐generating resin linkers for use in solid phase peptide synthesis
Well‐characterized resins of high purity are critical for effective solid phase peptide synthesis (SPPS). The quality of commercial (4‐methyl)benzhydrylamine‐resin (MBHA‐resin), used for the synthesis of peptide amides, is not consistent and residual ketone functionalities are frequently present. Such ketone or aldehyde impurities lead to the formation of acylation‐resistant deletion peptides in SPPS. To avoid these undesirable side reactions, we have optimized the preparation of two amide‐generating linkers, which, in combination with aminomethyl‐resin prepared directly from polystyrene resin, serve as alternatives to MBHA‐resin for peptide amide synthesis. Then we have explored their comparative properties in SPPS. Use of sonication in reductive amination facilitated the synthesis of both benzhydrylamine (BHA) and MBHA linkers. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.
Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies
We compared the synthesis and structural/conformational details of the (66–97) segments of the second transmembrane helix of AT1, MAS and B2, all of which belong to the class of G-protein-coupled receptors (GPCR). Step-by-step monitoring of the coupling reactions during the growth of these transmembrane peptides revealed that the increase in the level of difficulty started at the 6–10 regions of the sequence. Possibly due to their long and hydrophobic sequences, the final estimated synthesis yields decreased progressively by up to 20–25%. Analytical high pressure liquid chromatography showed that the hydrophobicity indexes of each TM-8, -16, -24 and -32 segments correlated linearly with their retention time. Microscopic measurements of peptide–resin beads indicated that, in general, dichloromethane and dimethylsulfoxide were the best solvents for solvating resin beads in the initial and final stages of the synthesis, respectively. Results from electron paramagnetic resonance experiments with Toac (2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled peptide resins revealed that the level of peptide chain mobility throughout the polymer network was in agreement with their swelling data measured in different solvents. Initial results regarding conformational features determined by circular dichroism (CD) spectra revealed typical α-helicoidally structures for MAS and B2 TM32 fragments when in more than roughly 30% (v/v) trifluoroethanol (TFE). In contrast, the AT1-TM32 segment revealed CD spectra, more representatives of a mixture of other secondary helical conformers, regardless of the amount of TFE. These findings observed in different aspects of these receptors’ fragments support further investigations of GPCR-type macromolecules.
Difficult couplings in stepwise solid phase peptide synthesis : predictable or just a guess ?
Stepwise solid phase peptide synthesis, Fmoc-approach, of 88 peptides varying in length from 4 to 24 amino acid residues was performed using a uniform procedure for coupling, monitoring and deprotection steps. The data of 696 couplings have been incorporated into a computer programme in order to study whether the degree of coupling can be predicted. Parameters studied are the nature of the amino acid to be coupled, the amino acid to be acylated and the length of the growing peptide chain. Using this information, prediction of "good" or "difficult" sequences, that is, peptides that can be synthesized without appreciable repeated couplings or the opposite, seems possible.