KAT Ligation for Rapid and Facile Covalent Attachment of Biomolecules to Surfaces

Abstract

The efficient and bioorthogonal chemical ligation reaction between potassium acyltrifluoroborates (KATs) and hydroxylamines (HAs) was used for the surface functionalization of a self-assembled monolayer (SAM) with biomolecules. An alkane thioether molecule with one terminal KAT group (S-KAT) was synthesized and adsorbed onto a gold surface, placing a KAT group on the top of the monolayer (KAT-SAM). As an initial test case, an aqueous solution of a hydroxylamine (HA) derivative of poly(ethylene glycol) (PEG) (HA-PEG) was added to this KAT-SAM at room temperature to perform the surface KAT ligation. Quartz crystal microbalance with dissipation (QCM-D) monitoring confirmed the rapid attachment of the PEG moiety onto the SAM. By surface characterization methods such as contact angle and ellipsometry, the attachment of PEG layer was confirmed, and covalent amide-bond formation was established by X-ray photoelectron spectroscopy (XPS). In a proof-of-concept study, the applicability of this surface KAT ligation for the attachment of biomolecules to surfaces was tested using a model protein, green fluorescent protein (GFP). A GFP was chemically modified with an HA linker to synthesize HA-GFP and added to the KAT-SAM under aqueous dilute conditions. A rapid attachment of the GFP on the surface was observed in real time by QCM-D. Despite the fact that such biomolecules have a variety of unprotected functional groups within their structures, the surface KAT ligation proceeded rapidly in a chemoselective manner. Our results demonstrate the versatility of the KAT ligation for the covalent attachment of a variety of water-soluble molecules onto SAM surfaces under dilute and biocompatible conditions to form stable, natural amide bonds.