Successful synthesis of increasingly complex therapeutic peptides using faster protocols can be attained with the use of highly reactive coupling reagents. The coupling reagent COMU has been demonstrated to be highly efficient in the synthesis of a variety of difficult standard peptides1,2 making it extremely valuable for use in fast synthesis protocols. However, the high reactivity of COMU leads to decreased stability in the presence of solvents like DMF, making it problematic for use over multiple days on automated peptide synthesizers without preparing the reagents fresh.
Herein we performed preliminary tests of the ability to, in essence, make COMU in situ in the reaction vessel by separately adding HDMC3 or HDMA3 with Oxyma Pure4. In this way, the coupling efficiency of COMU might be achieved without losing reactivity over the course of a longer synthesis over several days, or multiple syntheses. The use of an automated peptide synthesizer with independent induction heating and online cleaving capabilities facilitated the screening of different reaction conditions including several reagent combinations and multiple temperatures simultaneously.
Several challenging sequences were chosen for these experiments including the highly hydrophobic Jung-Redemann (JR) 10-mer peptide, which mimics hydrophobic moieties used in the development of peptide therapeutics, and Aib-Leu enkephalin, where Aib replaces both glycines in the sequence that simulate the synthesis of sterically hindered peptides. Novel reagent combinations resulted in comparable crude purities with higher stability in DMF. Additionally, introducing heat to the deprotection and coupling steps improved overall purity results, for example JR-10mer resulted in a crude purity of >60% using a fast, automated protocol (2 min deprotections and 3 min couplings), up from <20% crude purity at room temperature.
JR-10mer: H-WFTTLISTIM-NH2
Aib Enkephalin: H-Tyr-Aib-Aib-Phe-Leu-NH2