Catalysis of imido group hydrolysis in a maleimide conjugate

Abstract

Maleimides are often used for biomolecular conjugation with thiols. An underappreciated aspect of the imido group in a maleimide conjugate is its susceptibility to spontaneous hydrolysis, resulting in undesirable heterogeneity. Here, a chromophoric maleimide is used to demonstrate that both molybdate and chromate catalyze the hydrolysis of an imido group near neutral pH. Tungstate and 4-(dimethylamino)pyridine are less effective as catalysts. This work reveals a new mode of chemical reactivity for molybdate and chromate, and provides a strategy for decreasing the heterogeneity of bioconjugates derived from maleimides.

Figure 1

Molybdate-catalyzed hydrolysis of imido groups. (A) Raw data for catalysis of the hydrolysis of succinimide 2. (B) Transformed data for the hydrolysis of maleimide 1 (k₂ = 2.5 M⁻¹ min⁻¹) and succinimide 2 (k₂ = 0.28 M⁻¹ min⁻¹).

Figure 2

HPLC analysis of succinimide 2 after incubation for 2 h at room temperature with a putative catalyst (0.10 M) of hydrolysis. The twin peaks at ~40 min correspond to the hydrolysis products; the peak at ~45 min corresponds to intact succinimide 2.

Figure 3

Comparison of molybdate and chromate as catalysts for the hydrolysis of succinimide 2. Assays were performed by HPLC, as shown in Figure 2. Data were fitted to the equation: [hydrolyzed 2]_t = [hydrolyzed 2]_t=∞(1 − e^−kt), where k is the apparent first-order rate constant.

Scheme 1

Route for the synthesis of succinimide 2.

Scheme 2

Putative mechanism for the molybdate- and chromate-catalyzed hydrolysis of succinimide 2.