Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein |
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Authors: | Dr. Julien Tailhades Ashish Sethi Dr. Emma J. Petrie Prof. Dr. Paul R. Gooley Prof. Dr. Ross A. Bathgate Prof. Dr. John D. Wade Dr. Mohammed A. Hossain |
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Affiliation: | 1. Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia;2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia;3. The School of Chemistry, The University of Melbourne, Victoria, Australia |
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Abstract: | The inhibition of the G protein‐coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41‐residue and three‐disulfide cross‐bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for Nα‐deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca2+ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. |
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Keywords: | insulin-like peptide native chemical ligation NMR spectroscopy relaxin peptides |
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