Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea-Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc-based solid-phase peptide synthesis in combination with a tea-bag approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo-CVX-5c. Cyclotides were synthesized in a single-pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose-based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell-based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell-based assays for rapid screening.