Studies of Coupling Kinetics and Correlation of Reaction Conversions to Color Tests for Solid-Phase Peptide Synthesis of AMG 416 by NMR

By using the solution NMR technique and the “cleave and analyze” approach, we have modeled the kinetics of coupling reactions between activated Fmoc-Arg(Pbf)-OH and NH2-Arg(Pbf)-Arg(Pbf)-Ala-Arg(Pbf)-Rink amide-AM resin, determined the reaction rates, and correlated the extent of conversions to the Kaiser and TNBS color test results. Since NMR spectroscopy is a highly specific and quantitative tool with structural elucidation capability, the NMR assay for conversion determination of reaction in solid phase peptide synthesis (SPPS) can be developed by comparing the peaks from the reactant peptide containing the free amino groups with the product peptide containing the newly coupled Fmoc-amino acid moiety in the 1D 1H NMR spectrum. We utilized the NMR assays to measure conversion for each cycle in SPPS to assemble Ac-c(C)arrrar-NH2 and provided an in-process testing (IPT) time limit for the full-scale production. The data showed that after 18 hours of reaction, the conversion for all cycles achieved > 99.9%...