Synthesis of Alanine-Containing Peptides

Introduction: Peptides are molecules composed of two or more amino acids linked by peptide bonds. Unique combinations of just twenty canonical amino acids can result in the production of hundreds of thousands of different peptides and proteins, each of which have different structures and thus different functions in the human body. Alanine (A) is one such fundamental amino acid, with important roles such as the facilitation of gluconeogenesis, which is linked to muscle metabolism and energy production. This research focuses on synthesizing short peptides containing alanine, i.e., the peptides VGTA, CAG and ACG. Method: Peptides were synthesized through solid phase peptide synthesis (SPPS), starting with the addition of rink amide resin to an SPPS vessel. The resin acts as the foundation for building the peptide chain. The procedure starts with a deprotection step using 20% piperidine in dimethylformamide (DMF) to remove the Fmoc protecting group from the resin. This allows for the first amino acid to be coupled to the deprotected resin. Prior to the amino acid coupling, the peptide goes through a series of wash steps involving methanol (MeOH) for removing unwanted polar byproducts, dichloromethane (DCM) for removing nonpolar impurities, and DMF for washing out any residual soluble reagents from prior deprotection steps. The coupling reaction utilizes Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU) as the activating agent, N,N’- Diisopropylethylamine (DIPEA) as the base, and DMF as the solvent. The process of deprotection and coupling is repeated until the desired peptide chain is successfully built. The resin is removed through cleavage using trifluoroacetic acid (TFA) for cleaving the peptide from the resin and for removing side chain protecting groups, phenol for stabilizing and protecting amino acid side chains during the process, triisopropylsilane (TIPS) for preventing any undesirable side reactions, and MilliQ water as the solvent. The cleaved peptide is then purified and shell frozen before being placed on the lyophilizer where the peptide undergoes sublimation to remove excess solvents and unbound reagents. Results/Conclusion: Multiple alanine-containing peptides were successfully synthesized and confirmed through mass spectrometry analysis. These peptides will be utilized in future research projects to thermochemical properties and reactivity.