ABSTRACT The goal of this supplement to R01 GM132460 will be to support the toolkit being developed under the parent award through acquisition of a preparative HPLC instrument. The goal of the parent R01 is to improve the practicality, generality, tunability and scope of tetrazine ligation. We will develop a new generation of catalysts and reagents for tetrazine cross coupling and will apply these tools to chemical glycobiology applications. We will continue to develop novel tetrazines that can serve a dual role in enabling protein purification followed by subsequent site-selective bioorthogonal chemistry, and we propose to develop efficient catalytic methods for ‘turning on’ rapid bioorthogonal chemistry in biological context. Mechanistic inquiry will continue to guide our discovery efforts as will continued industrial collaboration with the chemical biology group at Pfizer. Efforts to commercialize the tool molecules will be continued. The success of this proposal is critically dependent on small molecule purification which is greatly facilitated by preparative high performance liquid chromatography (prep-HPLC). Currently, the Fox group does not have access to a prep-HPLC instrument. The proposed instrument will greatly accelerate research and maximize the impact of this R01-funded program. All aims of this grant will derive tremendous benefit from the proposed prep-HPLC.