Bioorthogonal Chemistry for Site-Specific Labeling and Surface Immobilization of Proteins

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels–Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol–ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has