All about that rate

ACS Chem. Biol. doi:10.1021/cb5006728

Chemoselective ligation reactions, bond-forming reactions that proceed in the presence of diverse, unprotected functional groups, are essential for multiple applications in chemical biology. For reactions that detect or isolate a specific biomolecule, one reagent can be used in large excess. If, however, they are to be used to couple two large molecules, the rate of ligation becomes very important. The high M_w of typical proteins means that, when dissolved, the concentrations are much smaller than for small molecules. High conversions are a prerequisite for effective ligation reactions and, at a typical concentration of 10 mM, this requires a second-order rate constant >1 M⁻¹ s⁻¹ and ideally >10 M⁻¹ s⁻¹. Saito et al. have now evaluated the rates and other important factors in the application of eight well-known ligation reactions. They designed substrates that all contain the typical functionalities present in peptides—thiols, carboxylic acids and amine side chains—and vary only in the pendant groups necessary for ligation. Rate constants were measured in 1:1 mixtures of the components at micromolar concentrations; the reactions were followed using the absorbance of a UV tag after separation by HPLC. In addition to the rate, the stability of both the starting materials and the ligated products was evaluated. The highest rate (734 M⁻¹ s⁻¹) was observed for a thiol-maleimide ligation, but the stability of some reagents could be an issue. The copper-catalyzed azide-alkyne cycloaddition was reasonably fast (3.4 M⁻¹ s⁻¹), though oxidation of thiols and coordination of amines or thiols to copper can pose problems. The strain-promoted version offers a lower rate, but the simple conditions are advantageous. The potassium acyltrifluoroborate ligation offers a good reaction rate (22 M⁻¹ s⁻¹) as well as good stability of both starting materials and products. By studying these reactions using realistic substrates and under realistic conditions, Saito et al. have produced a primer that should be valuable to those wishing to apply them.