Facile de Novo Sequencing of Tetrazine‐Cyclized Peptides through UV‐Induced Ring‐Opening and Cleavage from the Solid Phase

Abstract

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities.The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UVcleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

De Novo Sequencing of Synthetic Bis-cysteine Peptide Macrocycles Enabled by “Chemical Linearization” of Compound Mixtures

“…The results presented here establish that chemical linearization of synthetic macrocyclic peptides can be applied to compound mixtures, which enables their de novo sequencing with fidelity comparable to that of linear peptides using established software. This is a significant advance and suggests that a variety of other chemical linearization methods used with individual macrocycles may be similarly effective for this purpose. – In addition, the work extends the chemical linearization approach to Cys-based, thioether-linked macrocycles of the type used in phage and mRNA display for novel ligand discovery, which are of emerging significance as therapeutics and comprise a larger number of varied positions than typical synthetic libraries where chemical linearization has been employed. – …”

“…However, prior work has focused on individual substrates only, and based on these precedents, it is not obvious whether an extension to complex mixtures would be feasible. Additionally, prior work has focused largely on “head-to-tail” macrocycles of small ring size (<8 amino acids), – and there remains a significant gap with respect to thioether-linked macrocycles of relevance to ligand discovery. – …”

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries

“…In order to screen cyclic peptide libraries with an MS/MS based decoding workflow, a ring opening step prior to mass spectral analysis is required. Several linearization strategies have been described in the literature, including oxidative diol cleavage, photocleavage of tetrazine, oxazolidinone cleavage, and disulfide opening . Most of these studies described the linearization strategy without performing affinity selection discovery experiments.…”

Modulating Secondary Structure Motifs Through Photo‐Labile Peptide Staples

“…Also, light‐responsive peptide staples to inhibit PPIs have been reported [56,57] . For instance, a tetrazine photocage (Figure 1B) was employed to first covalently staple and secondly, photochemically unstaple a short peptide to create a library of peptides that could be used for protein target screening due to tetrazines ability to react with target proteins [57,58] .…”

“…[56,57] For instance, a tetrazine photocage (Figure 1B) was employed to first covalently staple and secondly, photochemically unstaple a short peptide to create a library of peptides that could be used for protein target screening due to tetrazines ability to react with target proteins. [57,58] In contrast, azobenzene photoswitches are frequently used to reversibly control PPIs by stabilizing or destabilizing the peptide's 3D structure, depending on the configuration of the photoswitch. [24,56,59,60] However, due to the larger structural change, the biological effect of photochemical uncaging is often higher than the one observed after photoswitching.…”