Procedure E (N-Boc deprotection) The N-Boc protected tetrazine was dissolved in dry ... quantitative N-Boc deprotected product as an ammonium chloride salt.
Table Of Contents Supplementary Figures Computational Figures (S1-S4)
2
Geometry optimization: IEDDA transition state structures Geometry optimization: IEDDA adduct structures Analytical Figures (S7-S25)
4
Fluorescence characterization Solvent dependent elimination pH dependent elimination LC-MS elimination Background fluorescence analysis Materials and Methods Computational Procedures
23 24
Geometry optimization: IEDDA transition state structures Geometry optimization: IEDDA adduct structures General Synthetic Procedures
25
General Analytic Procedures
26
Experimental
28
NMR data
36
DFT Calculations
71
Supplementary References
77
1
Supplementary Figures Computational Figures Geometry optimization: IEDDA transition state structures
Figure S1: ωB97XD/6-31+G(d)-optimized transition states for “head-to-head” (TS1) and “head-to-tail” (TS2) reactions of tetrazines 2, 4 and 5 with model 2-oxy-transcyclooctene. Distances of interest (forming C-C bonds, interactions) are labelled in Å.
Figure S2: ωB97XD/6-31+G(d)-optimized transition states for “head-to-head” (TS1) and “head-to-tail” (TS2) reactions of tetrazines 6 and 7 with model 2-oxy-transcyclooctene. Distances of interest (forming C-C bonds, interactions) are labelled in Å.
2
Geometry optimization: IEDDA adduct structures
Figure S3: ωB97XD/6-311G(d)-optimized geometries of IEDDA adduct tautomers of tetrazine 2 with model 2-oxy-transcyclooctene.
Figure S4: ωB97XD/6-311G(d)-optimized geometries of IEDDA adduct tautomers of tetrazine 5 with model 2-oxy-transcyclooctene. Both neutral (left) and cationic (right) structures are shown. Distances of interest (interactions) are labelled in Å.
3
Analytical Figures Fluorescence characterization
AMC (27)
2-TCO-AMC (28)
DMSO/H2O Max Ex Max Em
= 357 = 438
Max Ex Max Em
= 331 = 390
Phosphate Buffer Max Ex Max Em
= 348 = 444
Figure S5: Normalized excitation (left) and emission (right) spectra of AMC 27 (blue) and 2-TCO-AMC 28 (orange). Excitation wavelength (380nm) is visualised by the vertical red line. Emission wavelength (450) is visualised by the vertical green line.
Excitation ratio Max (Ex28 % / Ex27 %) = 360-fold (380 nm) Emission Δ % Max (Em28 % – Em27 %) = 63% (444 nm)
Figure S6: Excitation ratio (left) and emission “Δ %” (right) spectra of AMC 27 against 2-TCO-AMC 28. Excitation wavelength (380nm) is visualised by the vertical red line. Emission wavelength (450) is visualised by the vertical green line.
4
Solvent dependent elimination
Figure S7: XY-scatterplot of tetrazines 1, 2, 4b-17b, 4, 17 used to determine maximum elimination efficiencies (Eff %) and the elimination rate constants (kelim). Values for all tetrazines were plotted as dots for both DMSO/H2O (1:1, v:v) and PBS (1% DMSO).
Figure S8: Corrected normalized elimination efficiency over time of tetrazines 2, 23, 24, 25 and 1 in DMSO/H2O (1:1, v:v) and PBS (1% DMSO). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
5
Figure S9: Corrected normalized elimination efficiency over time of tetrazines 4b-7b and 4-7 in DMSO/H2O (1:1, v:v) and PBS (1% DMSO). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
6
Figure S10: Corrected normalized elimination efficiency over time of tetrazines 8b-11b and 8-11 in DMSO/H2O (1:1, v:v) and PBS (1% DMSO). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
7
Figure S11: Corrected normalized elimination efficiency over time of tetrazines 12b-15b and 12-15 in DMSO/H2O (1:1, v:v) and PBS (1% DMSO). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
8
Figure S12: Corrected normalized elimination efficiency over time of tetrazines 16b, 17b, 16 and 17 in DMSO/H2O (1:1, v:v) and PBS (1% DMSO). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
9
Figure S13: Elimination rate constants (kelim) were determined for each tetrazine following first order rate equations: ln( [Tetrazine]t ) = -kelim * t – ln ( [Tetrazine]t = 0 ) Slopes were determined by linear approximations (black lines) from which elimination rate constants were extracted.
10
Figure S14: Results in DMSO/H2O depicted as bar graphs ( t = 1, 2, 4, 8, 24, 48, 96, 192 hours) including AMC 27 and 2-TCO-AMC 28 as 100% and 0% control.
11
Figure S15: Results in PBS depicted as bar graphs ( t = 1, 2, 4, 8, 24, 48, 96, 192 hours) including AMC 27 and 2-TCO-AMC 28 as 100% and 0% control.
12
pH dependent elimination
Figure S16: Corrected normalized elimination efficiency over time of tetrazines 1, 2, 3 and 21 in PBS at pH 3 (red), pH 6 (green) and pH 7 (blue). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
13
Figure S17: Corrected normalized elimination efficiency over time of tetrazines 4b-7b and 4-7 in PBS at pH 3 (red), pH 6 (green) and pH 7 (blue). Maximum elimination efficiencies (Eff %) were determined by first-order exponential decay approximations (black lines).
14
Figure S18: Initial elimination rate constants (kelim) were determined for each tetrazine following first order rate equations: ln( [Tetrazine]t ) = -kelim * t – ln ( [Tetrazine]t = 0 ) Slopes were determined by linear approximations (black lines) from which elimination rate constants were extracted.
15
LC-MS elimination
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.84
127273
251894
275786
432062
2076545
AMC
4.84
5
10
11
17
63
A1.1 A1.2 A1.3 A1.4
5.07 5.17 5.78 5.97
760911
1415636 48197 460062 127607
1360994 94104 175828 86275
314425
1372010 140494
1351906 31682 581404 156720
38349 141106
A1.1 A1.2 A1.3 A1.4
5.07 5.17 5.78 5.97
31 0 56 6
55 1 23 6
57 2 18 5
53 4 7 3
10 0 1 4
Ox1 End1 Total Area
5.46 6.35
44773 2445461
101987 2475592
160745 2488032
395501 2544765
90734 642474 3303633
Ox1 End1 Total %
5.46 6.35
0 2 100
0 4 100
0 6 100
0 16 100
3 19 100
Figure S19. Top: UV trace of LC-MS based analysis of tetrazine 1 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
16
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.84
85312
93118
100054
153712
692007
AMC
4.84
5
5
6
9
36
A2.1 A2.2 A2.3
5.75 6.15 6.25
1390177 71215 235009
1434918 64015 221043
1405878 62914 218319
1160525 54213 187858
225901 40381
A2.1 A2.2 A2.3
5.75 6.15 6.25
78 4 13
79 4 12
78 3 12
69 3 11
12 0 2
Ox2 Total Area
6.41
10250 1823344
23017 1810182
123325 1679633
968854 1927143
Ox2 Total %
6.41
1781712
0 100
1 100
1 100
7 100
50 100
Figure S20. Top: UV trace of LC-MS based analysis of tetrazine 2 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
17
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.78
1075768
1179151
1229635
1370626
1956447
AMC
4.78
48
50
51
55
72
A3.1
5.53
1157998
1184392
1168803
955866
57299
A3.1
5.53
52
50
48
38
2
Ox3 Total Area
5.21
17184 2380727
31482 2429920
166147 2492639
718628 2732374
Ox3 Total %
5.21
2233767
0 100
1 100
1 100
7 100
26 100
Figure S21. Top: UV trace of LC-MS based analysis of tetrazine 3 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
18
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.84
1682427
1756349
1759441
1786798
956602
AMC
4.84
75
76
76
77
77
A5.1 A5.2
5.61 5.8
486999 83706
478519 74790
467254 65466
332941 18079
A5.1 A5.2
5.61 5.8
22 4
21 3
20 3
14 1
0 0
Ox5 End5 Total Area
5.3 5.48
12340
22994 2315154
Ox5 End5 Total %
5.3 5.48
2321997
136490 49421 2323729
0 0 100
1 0 100
1 0 100
6 2 100
20 3 100
2253131
251647 33293 1241541
Figure S22. Top: UV trace of LC-MS based analysis of tetrazine 5 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
19
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.84
1740608
1739101
1732605
1717752
1611539
AMC
4.84
70
70
70
72
70
A6.1
6.07
741248
736417
736076
654029
400964
A6.1
6.07
30
30
30
28
17
Ox6 Total area
5.41 2475518
2468680
2371781
295432 2307935
Ox6 Total %
5.41
2481856
0 100
0 100
0 100
0 100
13 100
Figure S23. Top: UV trace of LC-MS based analysis of tetrazine 6 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
20
Component
RT
5 min
30 min
60 min
4 hr
o.n.
Component
RT
5 min
30 min
60 min
4 hr
o.n.
AMC
4.84
2176464
2169864
2165001
2146810
2409702
AMC
4.84
80
80
81
82
87
A7.1
5.54
544542
546954
517814
478398
255158
A7.1
5.54
20
20
19
18
9
Ox7 Total Area
5.03 2716819
2682815
2625208
119192 2784052
Ox7 Total %
5.03
2721006
0 100
0 100
0 100
0 100
4 100
Figure S24. Top: UV trace of LC-MS based analysis of tetrazine 7 (1.0 mM) with TCO-AMC 28 (100µM) in PBS (10% DMSO). Bottom: Table of relevant intermediates (adduct, oxidized adduct) and products (AMC, dead-end adduct) reported as absolute area (left) or % of total (right).
21
Fluorescence background analysis Tetrazine 1 2 3 21 23 24 25
Fluorescence Fluorescence Fluorescence Tetrazine Tetrazine Background % Background % Background %
