Hydrolysis of 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue™) succinimidyl ester under acidic and basic conditions

The highly sensitive technique of microchip capillary electrophoresis (μCE) with laser-induced fluorescence (LIF) detection is under development for future in situ spaceflight missions to search for the organic chemical signatures of life. One fluorescent probe that enables this technology for amine, amino acid, and dipeptide analysis is 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue™, PB) succinimidyl ester. Of particular importance is the hydrolysis of PB succinimidyl ester, which precludes long-term aqueous storage during spaceflight and therefore has a significant impact on instrument design and operation. As such, it is important to characterize the chemical stability of this dye to hydrolysis prior to spaceflight. Here, we study the hydrolysis kinetics of the PB succinimidyl ester at pH values between 3 and 10.5 using μCE-LIF. The PB succinimidyl ester has the longest lifetime at pH 4 (7.3 ± 0.1 h), with dramatically shorter half-lives in the basic pH regime. This work represents a first step in the full characterization of this fluorescent probe for spaceflight applications.

► Hydrolysis kinetics of succinimidyl ester studied. ► Rapid hydrolysis (t1/2 ∼ min) at basic pH. ► Slowest hydrolysis at pH 4 (∼7 h).