2.5. Reaction Kinetics of Tetrazines

Reaction kinetics were determined using pseudo-first-order measurements with an excess of TCO in PBS (pH = 7.4) at 37.0 ± 0.1 °C.⁴⁷ Measurements were performed in sextuplicates using a SX20 stopped flow photometer (Applied Photophysics, U.K.). Data analysis was performed by nonlinear fit using Prism 6 (Graphpad) to determine the observed rate constants, which were converted into second order rate constants by division through TCO concentration.

Measurements of reference compounds HELIOS 347Me and HELIOS 388Me⁴⁸ with PEG₄-TCO (see SI, Figure S1) were performed in the fluorescence mode by following the increase in fluorescence above 400 nm. Therefore, the SX20 was equipped with a 360 nm LED light source and a photomultiplier type R374 in combination with a 400 nm long-pass filter as detector. Measurements of Tz containing polymers were performed in the absorbance mode using a 530 nm LED light source and following the decrease of absorbance at this wavelength.