Obtaining homogenous aspartyl-containing peptides via Fmoc/tBu chemistry is often an insurmountable obstacle. A generic solution for this issue utilising an optimised side-chain protection strategy that minimises aspartimide formation would therefore be most desirable. To this end, we developed the following new derivatives: Fmoc-Asp(OEpe)-OH (Epe = 3-ethyl-3-pentyl), Fmoc-Asp(OPhp)-OH (Php = 4-n-propyl-4-heptyl) and Fmoc-Asp(OBno)-OH (Bno = 5-n-butyl-5-nonyl). We have compared their effectiveness against that of Fmoc-Asp(OtBu)-OH and Fmoc-Asp(OMpe)-OH in the well-established scorpion toxin II model peptide variants H-Val-Lys-Asp-Asn/Arg-Tyr-Ile-OH by treatments of the peptidyl resins with the Fmoc removal reagents containing piperidine and DBU at both room and elevated temperatures. The new derivatives proved to be extremely effective in minimising aspartimide by-products in each application.
In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β-trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp-Gly motif. The N(α)-Fmoc aspartic acid β-tri-(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP-2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation.
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