Abstract
The rigid conformation of constrained bicyclic peptides provides a number of advantages over larger protein-based ligands, including better chemical stability, enhanced tissue penetration, and a wider field of possible applications. Selective chemical modification strategies are able to extend the scope of applications not only in a therapeutic manner but also for the development of novel tools for protein capturing, bioimaging, and targeted drug delivery. Herein, we report the synthesis of an adamantane-based, symmetrical, tetravalent, sulfhydryl-specific peptide linker. We have developed an in vitro two-step modification strategy that allows the generation of differently functionalized bicyclic peptides. This “tool kit” strategy was applied to cyclize and functionalize a phage-encoded peptide library bearing the sequence CX6CX6C. After phage display against a model target, isolated peptides show strong consensus sequences, indicating target-specific binding. The newly developed symmetric tetravalent linker opens new avenues for the combinatorial selection and functionalization of bicyclic peptide ligands with affinity to virtually any target.
Highlights
We have developed an in vitro two-step modification strategy that allows the generation of differently functionalized bicyclic peptides
The comparatively high nucleophilicity of the sulfhydryl moiety of cysteine enables the selective reaction with certain electrophilic trivalent linker molecules like, e.g., tris(bromomethyl)benzene (TBMB) to generate bicyclic peptides
To amplify the spectrum of potential applications, we suggest to expand the one-step modification method on phage toward a two-step reaction by introducing the tetravalent linker N,N′,N′′,N′′′
Summary
In the second step of the protocol, this fourth position can be functionalized by a broad variety of sulfhydryl-bearing probes, warheads, linkers, tools, or drugs Following this approach, the two-step modification allows the phage display of bicyclic peptide libraries that already bear a functional additive attached to the adamatane-based linker on phage during the selection process against any target. The two-step modification allows the phage display of bicyclic peptide libraries that already bear a functional additive attached to the adamatane-based linker on phage during the selection process against any target This tool kit strategy facilitates the development of novel peptide hybrids for therapeutic and diagnostic applications. The linear peptides were selectively modified by the two-step modification under optimized conditions using NATBA as a linker and FlcSH as an additive. For all of the analyzed sequences, except the negative control, the light and the heavy chain of the rabbit anti-Goat IgG antibody was clearly detected as an indicator of the successful capturing by the respective functionalized peptide
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