Kinase experiment:

Horseradish peroxidase (HRP) is thiolated with 2-iminothiolane and conjugated to mc-MMAE to generate the HRP-MMAE reporter enzyme-drug conjugate. Briefly, a thiolation reaction mixture containing 0.2 mM HRP (8 mg/mL) and 50 mM 2-iminothiolane in 25 mM sodium borate decahydrate (Na2B4O7o10H2O) buffer (pH 9.0) is incubated for 1 hour at 37℃. Unreacted 2-iminothiolane is removed by passage through a PD-10 desalting column equilibrated in PBS (pH 7.4). Peak fractions are pooled and mc-MMAE is coupled to thiolated HRP (HRP-SH) at a molar ratio of 3:1. The final conjugation reaction mixture contained 80 μM HRP-SH (3.2 mg/mL) in sodium borate buffer [50 mM H3BO3, 50 mM NaCl (pH 8.0); 80% v/v] and 240 μM mc-MMAE in ice-cold CH3CN (20% v/v). After 30 minutes on ice, the reaction is terminated with a 20-fold molar excess of free cysteine (4.8 mM) before PD-10 chromatography. Peak fractions containing HRP-MMAE (exchanged into PBS) are pooled and evaluated for extent of modification using the thiol-reactive dye, Alexa Fluor 594 C5 maleimide[1].

Mc-MMAE is a protective group (maleimidocaproyl)-conjugated monomethyl auristatin E (MMAE), which is a potent tubulin inhibitor. Mc-MMAE is a drug-linker conjugate for ADC. Tubulin, Antibody-drug conjugates (ADC)[1]

Synthesis of maleimidocaproyl-MMAE (mc-MMAE) requires the addition of maleimidocaproic acid to a solution of MMAE in dichloromethane followed by the addition of diethyl cyanophosphonate and diisopropylethylamine[1].

[1]. Sanderson RJ, et al. In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res. 2005 Jan 15;11(2 Pt 1):843-52.