Table 1.

Spectroscopic characteristics (absorption maxima, λ_abs (nm), emission maxima, λ_em (nm), fluorescence lifetimes, τ (ns)) of flexibly linked tetrazine-dyes after click reaction with TCO*-lysine and turn-on ratios measured in PBS, pH 7.4. Click reactions were performed by adding 25 µM TCO*-Lys to 1 µM dye solutions

Tetrazine-dye	λabs (nm)	λem (nm)	τav (ns)a	Turn-on ratiob
Me-Tet-ATTO425	441	478	1.28 (3.93)	15
Me-Tet-ATTO465	455	505	0.21 (2.82)	39
Me-Tet-ATTO488	502	522	1.39 (3.60)	25
Me-Tet-ATTO532	535	553	3.13 (3.00)	7
H-Tet-Cy3	551	564	0.37 (0.44)	2
Me-Tet-5-TAMRA	553	580	1.96 (2.38)	1
Me-Tet-AZ519	478	602	3.29 (4.08)	8
Me-Tet-AZ503	498	629	2.38 (2.79)	8
Me-Tet-ATTO550	555	573	1.95 (3.40)	4
Me-Tet-ATTO565	566	587	2.24 (3.80)	2
Me-Tet-ATTO590	598	621	1.99 (3.70)	3
Me-Tet-ATTO594	604	664	1.85 (3.74)	4
Me-Tet-ATTO620	620	637	1.47 (2.86)	3
Me-Tet-JF646	646	667	2.45 (3.01)	10
Me-Tet-ATTO647N	647	660	3.01 (3.58)	2
H-Tet-Cy5	649	664	0.76 (1.21)	3
H-Tet-SiR	649	669	2.40 (2.71)	4
H-Tet-HMSiR	654	667	2.56 (2.49)	1
Me-Tet-ATTO655	668	676	1.50 (2.12)	13
H-Tet-Cy5B	670	683	1.08 (1.91)	3
Me-Tet-ATTO680	686	694	1.38 (1.91)	6
Me-Tet-ATTO700	707	712	1.28 (1.81)	8

^aThe quality of the fit was judged by the reduced χ² values and the randomness of the weighted residuals. For all fits, χ² values between 0.900 and 1.200 were obtained. Most clicked fluorescent dyes showed nonexponential fluorescence kinetics, which could be best described using a biexponential model. Therefore, average fluorescence lifetimes τ_av = a₁τ₁ + a₂τ₂ are given. The lifetime values in brackets were measured after clicking with TCO*-lysine

^bThe turn-on ratio was determined from the minimum and maximum fluorescence intensity value recorded before and after addition of 25 µM TCO*-Lys to the tetrazine-dyes (Fig. 1)