The Synthesis of Biotinylated-NAB2 for Biolayer Interferometry Studies with E3 Ligase Nedd4

• Tessa Marion, Biochemistry, University of Delaware

• Zhihao Zhuang, , University of Delaware

Ubiquitin (Ub) is a 76-amino acid peptide that is necessary for regulating many cellular processes. Ubiquitination occurs through a three-enzyme cascade of Ub-activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub ligases (E3). The attachment of Ubiquitin to a protein is responsible for signaling for DNA repair, protein degradation, and cell cycle regulation, to name a few.  In disease-affected neurons, there are Lewy bodies that contain and express an E3 ligase, Nedd4. This ligase is responsible for the addition of ubiquitin to target proteins after ubiquitination. Because of Nedd4’s vital role in ubiquitination, it holds great research interest. To isolate Nedd4, small-molecule binding is needed. N-aryl benzimidazole (NAB) compounds show successful binding to Nedd4 ligase by surface plasmon resonance. An analog compound of NAB, NAB2, has shown the best binding affinity and efficiency. To better study this binding between Nedd4 and NAB2, biolayer interferometry studies using a biotinylated-NAB2 can be used. The nine-step synthesis of biotinylated-NAB2 is outlined below.

Beginning with Methyl-4-fluoro-3-nitrobenzoate, an SNAr reaction occurs to attach 2-5-dimethylaniline, followed by a nitro group reduction and benzimidazole cyclization. From there, a hydrolysis reaction converts the methoxy group to a hydroxyl group to allow for an amide bond formation, shown in intermediate 9. The nitro group is then reduced to an amine to allow for a second amide bond formation with 5-bromovaleric acid. Using the bromovaleryl linker, an azido group is formed through Sn2 chemistry. Under basic conditions and using propargylamine, D-biotin forms an alkyne functional group. Through copper (I) click chemistry, the azido intermediate and alkyne functional group form the final biotinylated-NAB2. The biotinylated-NAB2 can be used in streptavidin tip binding. Then, an affinity flow-through of prepared and purified Nedd4 is expected to produce a large signal output for the interaction.