A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry

Abstract

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis.

Protein-Catalyzed Capture Agents

“…To analyze the peptide sequence using MALDI-TOF/TOF, ring opening of the macrocycle was achieved by treating the glycoamidic ester with ammonium hydroxide. 115…”

“…Selective cleavage of the OPp protecting group and cyclization between the aspartic acid and the N-terminal Ala generated the cyclic peptide. To analyze the peptide sequence using MALDI-TOF/TOF, ring opening of the macrocycle was achieved by treating the glycoamidic ester with ammonium hydroxide …”

ADAMTS4-specific MR probe to assess aortic aneurysms in vivo using synthetic peptide libraries

“…To identify peptides with higher binding affinities, we expanded this technique to cyclic peptides. Sequencing of cyclic peptides can be difficult 42 and often requires special reagents, like photochemical linkers 43 or special protection group strategy, to form cleavable cycles 44 , 45 . In our study, we used a disulfide for ring closure.…”

One-Pot In Vitro Ribosomal Synthesis of Macrocyclic Depsipeptides

“…Despite the successful isolation of CDPs as naturally occurring secondary metabolites, chemical synthesis of CDP libraries leading to the discovery of de novo active CDPs remains a major challenge. In-solution macrolactonization methods had been developed for the chemical synthesis of natural CDPs (Figure a), , and some of these elegant methods have been applied to solid phase peptide synthesis (SPPS) to prepare certain types of CDPs, − allowing for high-throughput affinity screenings, e.g., one-bead-one-compound libraries (Figure b). − However, due to low efficiency of ester bond formation in SPPS, , the achievable level of crude quality and sequence complexity of CDPs are yet limited. To the best of our knowledge, such an approach only afforded a subtle improvement of the parental activity of natural CDPs. ,, Thus, such strategies using SPPS methods are yet insufficient to construct high diversity libraries to discover bioactive de novo CDPs.…”