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

Lookup NU author(s): Dr Toni Pringle, Dr James KnightORCiD

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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^-1s^-1), whereas HEPES (pH 7) had the highest (0.55–1.22 M^-1s^-1). Additionally, reactions in DMEM were faster than in RPMI (0.59–0.97 vs. 0.27–0.77 M^-1s^-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^-1s^-1) by 31 ± 16%. This study offers useful insights into the factors affecting SPAAC kinetics, facilitating the development of optimised bioconjugation strategies.

Publication metadata

Author(s): Pringle T, Knight JC

Publication type: Article

Publication status: Published

Journal: Organic & Biomolecular Chemistry

Year: 2025

Volume: 23

Issue: 10

Pages: 2432-2438

Online publication date: 28/01/2025

Acceptance date: 27/01/2025

Date deposited: 27/01/2025

ISSN (print): 1477-0520

ISSN (electronic): 1477-0539

Publisher: Royal Society of Chemistry

URL: https://doi.org/10.1039/D4OB01157K

DOI: 10.1039/D4OB01157K

Data Access Statement: The data supporting this article have been included as part of the ESI.

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