The effects of buffer, pH, and temperature upon SPAAC reaction rates†

Abstract

This study investigates the effects of buffer type, pH, and temperature on the kinetics of strain-promoted alkyne–azide cycloaddition (SPAAC) reactions. Using 3-azido-L-alanine and 1-azido-1-deoxy-β-D-glucopyranoside as model azides and sulfo DBCO-amine as the alkyne, we examined reaction rates in a series of buffers, including PBS, HEPES, MES, borate buffer, and cell culture media (DMEM and RPMI), with pH values ranging from 5 to 10 and temperatures of 25 and 37 °C. Absorbance spectrophotometric data revealed that PBS (pH 7) exhibited among the lowest rate constants (0.32–0.85 M⁻¹ s⁻¹), whereas HEPES (pH 7) had the highest (0.55–1.22 M⁻¹ s⁻¹). Additionally, reactions in DMEM were faster than in RPMI (0.59–0.97 vs. 0.27–0.77 M⁻¹ s⁻¹). We observed that higher pH values generally increased reaction rates, except in HEPES buffer. Notably, 1-azido-1-deoxy-β-D-glucopyranoside reacted faster than 3-azido-L-alanine, highlighting the importance of considering the electron-donating capacity of azides in the optimisation of SPAAC reactions. Additional experiments with DBCO-modified antibodies (DBCO-trastuzumab and DBCO-PEG5-trastuzumab) corroborated the trends related to buffer and azide selection. The presence of a PEG linker notably enhanced reaction rates (0.18–0.37 M⁻¹ s⁻¹) by 31 ± 16%. This study offers useful insights into the factors affecting SPAAC kinetics, facilitating the development of optimised bioconjugation strategies.