An optimized protocol for the synthesis of peptides containing trans-cyclooctene and bicyclononyne dienophiles as useful multifunctional bioorthogonal probes

The synthetic availability and functional diversity of peptides make these biomacromolecules an attractive modality with properties that, in many aspects, lie between antibodies and small synthetic molecules. Traditional solid phase peptide synthesis (SPPS) has been used for decades for construction of natural and functionalized peptides, which found utility in medicinal chemistry, biochemistry, polymer sciences, chemical biology and other disciplines. Despite the great advances in SPPS, the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene and bicyclononyne, into different peptide sequences. The two groups enable fast, modular and bioorthogonal post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide conjugates, peptide-drug conjugates as well as smart, multifunctional chemical biology probes. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.