Table 1.

Chemical structure of CCR systems and evaluation of the reaction kinetics of the CCR system. All reactions were conducted in PBS containing 10% DMSO.

Tetrazine	Alkyne	k2 at r.t (M−1s−1)	k2 at 37 °C (M−1s−1)	k1 at r.t (h−1)	Dox Peak % within 48 h, at r.t (%)
5	1	0.25± 0.06	1.9± 0.4	0.029± 0.006	60± 6 (48 h)
	2	0.0075± 0.0009	0.021± 0.005	*	90± 5 (20 h)
	3b	0.042± 0.012	0.14± 0.04	0.16±0.04	90 ±3 (16 h)
6	1	0.36± 0.07	2.1 ±0.4	Δ	20±5 (48 h)
	2	0.0078±0.0009	0.025±0.006	*	85±5 (21 h)
	3b	0.065±0.013	0.19±0.04	0.20±0.04	80±5 (18 h)
	3a	0.061±0.013	0.17±0.05	0.21±0.04	80±5 (19 h)
9	3b	0.091±0.013	0.25±0.05	0.22±0.05	78±6 (18 h)

^*not detectable because of the slow second order reaction and fast lactonization reaction. No intermediates were observed.

^Δnot detectable because of Dox decomposition in PBS. (n = 3, p= 0.95).