Iraninan Biology Society Journal of Cellular and Molecular Research (Iranian Journal of Biology) 2383-2738 35 4 2022 12 22 Improving Reaction Yields of Binding Amino Acids to Wang Resin and Peptide Labeling with (5/6)-Carboxyfluorescein Improving Reaction Yields of Binding Amino Acids to Wang Resin and Peptide Labeling with (5/6)-Carboxyfluorescein 508 520 2001 FA Reihaneh Khosravi Bioprocess engineering department, institute of industrial and environmental biotechnology, national institute of genetic engineering and biotechnology (NIGEB) 0009-0000-2200-7607 Morteza Daliri Animal biotechnology department, institute of agricultural biotechnology, national institute of genetic engineering and biotechnology (NIGEB) Zahra Azizi Department of Molecular Medicine, Tehran University of Medical Sciences, Tehran, Iran Mohammad Hosein Sanati Medical genetics department, institute of medical biotechnology, national institute of genetic engineering and biotechnology (NIGEB) Maryam Mohaghegh Medical genetics department, institute of medical biotechnology, national institute of genetic engineering and biotechnology (NIGEB) Journal Article 2019 07 15 The most common method for peptide synthesis is to synthetize it on a solid resin. Among the variety of resins, Wang resin is one of the highly use ones. In this study, we introduced improved protocols for anchoring an amino acid on the Wang resin, a famous resin for peptide synthesis, and N-terminally labeling peptide with (5/6)-carboxyfluorescein, a fluorescent dye. The peptide tetra arginine, RRRR, was synthetized on Wang resin with solid phase peptide synthesis (SPPS) method and the dye was attached to its N-terminus. The attachment yield of the first arginine residue (Fmoc-Arg(pbf)-OH) were quantified by liberating the Fmoc groups from the amino acid/resin complex following measure its UV absorption at 278 nm. The labeling was monitored by Ninhydrin test that reveals presence of primary, unbound N-terminally amines. In anchoring the first arginine residue on the resin, DIC (N,N’-Diisopropyl carbodiimide) works more efficiently than HBTU (N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate), only if the incubation time increases to 17 hours; while, HBTU undeniably enhances the efficiency of the fluorescence labeling compared to DIC. However, in most available protocols, DIC was recommended for labeling the peptides with (5/6)-carboxyfluorescein and the recommended incubation time for attaching the first amino acid on the Wang resin is usually far shorter than 17 hours. The most common method for peptide synthesis is to synthetize it on a solid resin. Among the variety of resins, Wang resin is one of the highly use ones. In this study, we introduced improved protocols for anchoring an amino acid on the Wang resin, a famous resin for peptide synthesis, and N-terminally labeling peptide with (5/6)-carboxyfluorescein, a fluorescent dye. The peptide tetra arginine, RRRR, was synthetized on Wang resin with solid phase peptide synthesis (SPPS) method and the dye was attached to its N-terminus. The attachment yield of the first arginine residue (Fmoc-Arg(pbf)-OH) were quantified by liberating the Fmoc groups from the amino acid/resin complex following measure its UV absorption at 278 nm. The labeling was monitored by Ninhydrin test that reveals presence of primary, unbound N-terminally amines. In anchoring the first arginine residue on the resin, DIC (N,N’-Diisopropyl carbodiimide) works more efficiently than HBTU (N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate), only if the incubation time increases to 17 hours; while, HBTU undeniably enhances the efficiency of the fluorescence labeling compared to DIC. However, in most available protocols, DIC was recommended for labeling the peptides with (5/6)-carboxyfluorescein and the recommended incubation time for attaching the first amino acid on the Wang resin is usually far shorter than 17 hours. https://cell.ijbio.ir/article_2001_d0fb963ff976f9c37fc81fe03c21ea7b.pdf