Labeling peptides has been widely practiced in numerous applications in chemistry and biology. For example, fluorescent dyes make peptides both useful probes for in vitro and in vivo imaging and suitable substrates for determining enzyme activity; biotinylation has vast applications including affinity-based protein purification; PEGylation enhances plasma half-life and protects against metabolic degradation; radiolabeling offers in vitro and in vivo molecular imaging agents and targeted radionuclide therapy; and cell permeable peptides allow access to intracellular targets. Thus, peptides bearing such multiple labels may serve as powerful research tools as they become available for diverse biomedical studies. For labeling peptides, several coupling methods have been reported, such as alkyne-azide click chemistry, thiol-maleimide Michael addition, Staudinger ligation, and oxime-hydrazone formation. Despite the versatility of and broad use in traditional organic reactions, Suzuki-Miyaura coupling has been rarely practiced in peptide labeling. In addition, while each of these methods has been efficiently used alone in peptide conjugation, their combination for orthogonal coupling reactions has not been well studied. Therefore, we developed an efficient synthetic strategy for multiple labeling of peptides via various orthogonal coupling reactions, described herein utilizing Suzuki-Miyaura cross-coupling, thiol-maleimide reaction, and alkyne-azide cycloaddition. The potential of these orthogonal chemistries was demonstrated by a peptide with multiple labels of a cell-penetrating peptide,  fluorescent dye, and biotin. The triple-tagged peptide was conjugated via thiol-maleimide addition to a BAK peptide and incubated with HCC95 lung cancer cells. Cellular uptake was confirmed by fluorescent microscopy, and biotin-streptavidin pulldown assay confirmed the interaction between the conjugated BAK peptide and its target protein, Mcl-1.

Triple-tagged peptides utilizing orthogonal chemistries for intracellular targets

ACS Spring Meeting 2021

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