Synthesis and Characterization of Pyridine Compounds for Amperometric Measurements of Leukocyte Esterase

We introduce a new class of substrates (compounds I-III) for leukocyte esterase (LE) that react with LE yielding anodic current in direct proportion to LE activity. The kinetic constants K_m and k_cat for the enzymatic reactions were determined by amperometry at a glassy carbon electrode. The binding affinity of I-III for LE was two orders of magnitude better than that of existing optical LE substrates. The specificity constant k_cat /K_m was equal to 2.7, 3.8, and 5.8×10⁵  m^-1  s^-1 for compounds containing the pyridine (I), methoxypyridine (II), and (methoxycarbonyl)pyridine (III), respectively, thus showing an increase in catalytic efficiency in this order. Compound III had the lowest octanol/water partition coefficient (log p=0.33) along with the highest topological surface area (tPSA=222 Ų ) and the best aqueous solubility (4.0 mg mL^-1 ). The average enzymatic activity of LE released from a single leukocyte was equal to 4.5 nU when measured with compound III.