Experimental Procedures - Chemical Synthesis

9 Chemical Synthesis

9.3 Experimental Procedures

9.3.1 Modified Nucleosides 2-Azido-adenosine (14)

Compound 14 was prepared as previously published.^[111] Assignments are in agreement with literature.^[118]

Rf 0.17 (CH2Cl2/MeOH = 10:1).

m.p. 120-122 °C (CH2Cl2).

1H NMR (DMSO-d6, 400 MHz) Azidomethin form (70%):  = 8.29 (s, 1H, 8-H); 7.62 (br, 2H, 6-NH2); 5.97 (d, 1H, ³JHH = 5.5 Hz, 1’-H); 5.53 (d, 1H, ³JHH = 6.1 Hz, 2’-OH); 5.22 (d, 1H, ³JHH

= 5.2 Hz, 3’-OH); 5.14 (t, 1H, ³JHH = 5.6 Hz, 5’-OH); 4.60-4.53 (m, 1H, 2’-H); 4.13 (dd, 1H,

3JHH = 7.6 Hz, 4.0 Hz, 3’-H); 3.94 (dd, 1H, ³JHH = 7.0 Hz, 3.9 Hz, 4’-H); 3.75-3.49 (m, 2H, 5’-CH2). Tetrazol form (30%): δ = 9.43 (br, 2H, 6-NH2); 8.57 (s, 1H, 8-H); 5.80 (d, 1H, ³JHH = 6.1 Hz, 1’-H); 5.44 (d, 1H, ³JHH = 6.2 Hz, 2’-OH); 5.19 (d, 1H, ³JHH = 4.8 Hz, 3’-OH); 5.07 (t, 1H, ³JHH = 5.7 Hz, 5’-OH); 4.60-4.53 (m, 1H, 2’-H); 4.19 (dd, 1H, ³JHH = 9.0 Hz, 4.6 Hz, 3’-H);

3.99 (dd, 1H, ³JHH = 7.3 Hz, 3.8 Hz, 4’-H); 3.75-3.49 (m, 2H, 5’-CH2).

13C NMR (DMSO-d6, 100 MHz) Azidomethin form  = 156.6 (6-C); 155.5 (2-C); 150.5 (4-C);

139.6 (8-C); 116.9 (5-C); 87.3 (1’-C); 85.7 (4’-C); 73.4 (2’-C); 70.5 (3’-C); 61.5 (5’-C);

Tetrazol form  = 153.7 (6-C); 152.2 (2-C); 142.8 (8-C); 141.0 (4-C); 111.5 (5-C); 87.4 (1’-C);

85.4 (4’-C); 73.6 (2’-C); 70.2 (3’-C); 61.2 (5’-C).

HR MS (m/z) [M+H]⁺ (C10H13O4 N8+) found 309.10387, calc. 309.10543, dev. 5.0 ppm.

2-Iodo-adenosine (15)

Compound 15 was prepared as previously published.^{[110, 190]} Assignments are in agreement with literature.

Rf 0.23 (CH2Cl2/MeOH = 10:1).

m.p. > 180 °C decomp.

1H NMR (DMSO-d6, 400 MHz)  = 8.29 (s, 1H, 8-H); 7.71 (br, 2H, 6-NH2); 5.80 (d, 1H, ³JHH

= 6.1 Hz, 1’-H); 5.45 (d, 1H, ³JHH = 6.2 Hz, 2’-OH); 5.19 (d, 1H, ³JHH = 4.9 Hz, 3’-OH); 5.03 (dd, 1H, ³JHH = 6.3 Hz, 5.1 Hz, 5’-OH); 4.52 (dt, ³JHH = 6.2 Hz, 5.1 Hz, 1H, 2’-H); 4.11 (dt, ³JHH

= 4.9, 3.1 Hz, 1H, 3’-H); 3.94 (dt, ³JHH = 3.7 Hz, 3.6 Hz, 1H, 4’-H); 3.64 (dt, ²JHH = 11.9 Hz,

3JHH = 4.6 Hz, 1H, 5’-Ha); 3.54 (ddd, ²JHH = 11.9 Hz, ³JHH = 6.4 Hz, 4.0 Hz, 1H, 5’-Hb).

13C NMR (DMSO-d6, 100 MHz)  = 155.9 (6-C); 149.8 (4-C); 139.4 (8-C); 120.8 (2-C);

119.0 (5-C); 87.2 (1’-C); 85.8 (4’-C); 73.6 (2’-C); 70.5 (3’-C); 61.4 (5’-C)

HR MS (m/z) [M+H]⁺ (C10H13IN5O4+) found 393.99830, calc. 394.00068, dev. 6.0 ppm.

2-Vinyl-adenosine (16)

Compound 16 was prepared as previously described.^[120]

2-Iodo-adenosine (15, 100 mg, 0.25 mmol, 1.0 eq.) and Pd(PPh3)4 (30 mg, 0.03 mmol, 0.1 eq.) were dissolved in degassed NMP (2 mL) under inert atmosphere and tributyl(vinyl)tin (223 μL, 0.76 mmol, 3.0 eq.) was added subsequently. The mixture was stirred at 90 °C for two days. After cooling, the catalyst was filtered off and the solvent was removed under reduced pressure. Then, the crude product was purified by column chromatography on silica using CH2Cl2/MeOH (10:1) elutant. The product was isolated as white solid (69 mg, 0.24 mmol, 92%).

Rf 0.14 (CH2Cl2/MeOH = 10:1).

m.p. 165-168 °C (MeOH).

1H NMR (DMSO-d6, 400 MHz)  = 8.33 (s, 1H, 8-H); 7.29 (s, 2H, 6-NH2); 6.61 (dd, 1H,³JHH

= 17.3 Hz, 10.4 Hz, -CH=CH2); 6.37 (dd, 1H, ³JHH = 17.3 Hz, ²JHH = 2.3 Hz, -CH=CH2-trans);

5.89 (d, 1H, ³JHH = 6.4 Hz, 1’-H); 5.55 (dd, 1H,³JHH = 10.4 Hz, ²JHH = 2.3 Hz, -CH=CH2-cis);

5.43 (d, 1H, ³JHH = 6.0 Hz, 2’-OH); 5.41-5.36 (m, 1H, 5’-OH); 5.22-5.16 (m, 1H, 3’-OH);

4.69-4.63 (m, 1H, 2’-H); 4.18-4.14 (m, 1H, 3’-H); 3.98 (dd, 1H, ³JHH = 6.4 Hz, 3.4 Hz, 4’-H);

3.71-3.66 (m, 1H, 5’-CH2-a); 3.63-3.48 (m, 1H, 5’-CH2-b).

13C NMR (DMSO-d6, 100 MHz)  = 157.8 (2-C); 155.8 (6-C); 149.7 (4-C); 140.4 (8-C);

137.1 (-CH=CH2); 121.3 (-CH=CH2); 118.6 (5-C); 87.8 (1’-C); 86.0 (4’-C); 73.3 (2’-C); 70.9 (3’-C); 61.8 (5’-C).

HR MS (m/z) [M+H]⁺ (C12H16O4N5+) found 294.11911, calc. 294.11968, dev. 1.9 ppm.

Chemical Synthesis 6-Chloro-9-(β-D-ribofuranosyl)purine (17)

Compound 17 was prepared as previously published.^[112] Assignments are in agreement with literature.

Rf 0.29 (CH2Cl2/MeOH = 10:1).

m.p. >165 °C decomp.

1H NMR (DMSO-d6, 400 MHz)  = 8.95 (s, 1H, 8-H); 8.82 (s, 1H, 2-H); 6.04 (d, ³JHH = 5.2 Hz, 1H, 1’-H); 5.56 (d, ³JHH = 5.8 Hz, 1H, 2’-OH); 5.24 (d, ³JHH = 5.2 Hz, 1H, 3’-OH); 5.09 (t, ³JHH = 5.5 Hz, 1H, 5’-OH); 4.59 (dt, ³JHH = 5.7 Hz, 5.3 Hz, 1H, 2’-H); 4.20 (dt, ³JHH = 4.7 Hz, 4.6 Hz, 1H, 3’-H); 3.99 (dt, ³JHH = 4.0 Hz, 4.0 Hz, 1H, 4’-H); 3.70 (ddd, ²JHH =12.0 Hz, ³JHH = 5.3 Hz, 4.0 Hz, 1H, 5’-Ha); 3.59 (ddd, ²JHH =12.0 Hz, ³JHH = 5.7 Hz, 4.0 Hz, 1H, 5’-Hb).

13C NMR (DMSO-d6, 100 MHz)  = 151.8 (2-C); 151.6 (4-C); 149.3 (6-C); 145.8 (8-C);

131.4 (5-C); 88.1 (1’-C); 85.7 (4’-C); 73.9 (2’-C); 70.0 (3’-C); 61.0 (5’-C).

HR MS (m/z) [M+H]⁺ (C10H12O4N4Cl⁺) found 287.05326, calc. 287.05416, dev. 3.1 ppm.

6-N-(6-Azidohexyl)adenosine (18)

Compound 18 was prepared as previously described.^[124]

A mixture of 6-chloro-9-(β-D-ribofuranosyl)purine (17, 500 mg, 1.86 mmol, 1.0 eq.) and 6-azidohexane-1-amine^[122] (1.26 g, 8.88 mmol, 4.8 eq.) in absolute EtOH (9 mL) were stirred at 90 °C for three hours. After cooling, the mixture was allowed to crystallise at -18 °C. The precipitate was filtered off, washed with cold EtOH and dried in vacuo. The product was isolated as a white solid (553 mg, 1.41 mmol, 75%).

Rf 0.31 (CH2Cl2/MeOH = 10:1).

m.p. 92-94 °C (EtOH).

1H NMR (DMSO-d6, 400 MHz)  = 8.33 (s, 1H, 8-H); 8.19 (s, 1H, 2-H); 7.86 (br, 1H, 6-NH);

5.88 (d, 1H, ³JHH = 5.9 Hz, 1’-H); 5.41 (br, 2H, 2’-OH, 5’-OH); 5.16 (br, 1H, 3’-OH); 4.61 (m, 1H, 2’-H); 4.14 (dd, 1H, ³JHH = 6.6 Hz, 3.7 Hz, 3’-H); 3.96 (dd, 1H, ³JHH = 6.6 Hz, 3.4 Hz, 4’-H); 3.67 (ddd, ²JHH =12.2 Hz, ³JHH = 5.7 Hz, 7.2 Hz, 1H, 5’-CH2-a); 3.55 (ddd, 1H, ²JHH

=12.2 Hz, ³JHH = 6.6 Hz, 3.4 Hz, 1H, 5’-CH2-b); 3.47 (m, 1H, -CH2NH-); 3.30 (t, ³JHH = 6.9 Hz, -CH2N3); 1.59 (m, 2H, -CH2-CH2NH-); 1.53 (m, 2H, -CH2-CH2N3); 1.38-1.28 (m, 4H, -CH2-CH2-).

13C NMR (DMSO-d6, 100 MHz)  = 154.7 (6-C); 152.4 (2-C); 148.2 (4-C); 139.6 (8-C);

119.7 (5-C); 88.0 (1’-C); 85.9 (4’-C); 73.5 (2’-C); 70.7 (3’-C); 61.7 (5’-C); 50.6 (-CH2-N3); 39.8 (-CH2NH-); 28.9 (-CH2-CH2NH-); 28.2 (-CH2-CH2N3); 25.9 (-CH2-CH2-).

HR MS (m/z) [M+H]⁺ (C16H25O4N8+) found 393.19947, calc. 393.19933, dev. 0.4 ppm.

6-N-(5-Hexenyl)adenosine (19)

A mixture of 6-chloro-9-(β-D-ribofuranosyl)purine (17, 500 mg, 1.86 mmol, 1.0 eq.) and 5-hexene-1-amine^[123] (881 mg, 8.88 mmol, 4.8 eq.) in absolute EtOH (5 mL) were stirred at 90 °C for three hours. After cooling, the mixture was allowed to crystallise at -18 °C. The precipitate was filtered off, washed with cold EtOH and dried in vacuo. The product was isolated as a white solid (323 mg, 0.92 mmol, 49%).

Rf 0.37 (CH2Cl2/MeOH = 10:1).

m.p. 123-124 °C (EtOH).

1H NMR (DMSO-d6, 400 MHz)  = 8.33 (s, 1H, 8-H); 8.20 (s, 1H, 2-H); 7.88 (br, 1H, 6-NH);

5.88 (d, 1H, ³JHH = 6.2 Hz, 1’-H); 5.79 (ddt, 1H, ³JHH = 16.9 Hz, 10.2 Hz, 6.7 Hz, -CH=CH2);

5.41 (br, 2H, 2’-OH, 5’-OH); 5.16 (br, 1H, 3’-OH); 5.00 (d, 1H, ³JHH = 17.2 Hz, trans-CH=CH2); 4.93 (d, 1H, ³JHH = 10.2 Hz, cis-CH=CH2); 4.64-4.59 (m, 1H, 2’-H); 4.21-4.11 (m, 1H, 3’-H); 3.96 (dd, ³JHH = 3.2 Hz, 6.2 Hz, 4’-H); 3.70-3.64 (m, 1H, 5’-CH2-a); 3.60-3.51 (m, 1H, 5’-CH2-b); 3.51-3.41 (m, 2H, -CH2NH-); 2.05 (q, 2H, ³JHH = 7.1 Hz, -CH2-CH=CH2); 1.60 (p, 2H, ³JHH = 7.2 Hz, -CH2-CH2NH-); 1.40 (p, 2H, ³JHH = 7.6 Hz, -CH2-).

13C NMR (DMSO-d6, 100 MHz)  = 154.7 (6-C); 152.3 (2-C); 148.2 (4-C), 139.6 (8-C); 138.7 (-CH=CH2); 119.7 (5-C); 114.8 (-CH=CH2); 87.9 (1’-C); 85.9 (4’-C); 73.4 (2’-C);

70.7 (3’-C); 61.7 (5’-C); 39.7 (-CH2NH-); 32.9 (-CH2-CH=CH2); 28.6 (-CH2-CH2NH-); 25.7 (-CH2-).

HR MS (m/z) [M+H]⁺ (C16H24N5O4+) found 350.18149, calc. 350.18228, dev. 2.2 ppm.

Chemical Synthesis 2-(5-Trifluoroacetamido-pent-1-yn-1-yl)adenosine (58)

Compound 58 was prepared as previously described.^[145]

Trifluoro‐N‐(pent‐4‐ynyl)‐acetamide^[191] (0.59 mg, 3.30 mmol, 1.3 eq.) was dissolved in DMF (12.5 mL) and degassed subsequently. Next, a flask was charged with 2-iodo-adenosine (15, 1.00 g, 2.54 mmol, 1.0 eq.), CuI (0.10 g, 0.51 mmol, 0.2 eq.) and Pd(PPh3)4 (0.29 g, 0.25 mmol, 0.1 eq.) and evacuated and flushed with N2 for three times. The dissolved amine was transferred to the flask und dry NEt3 (3.5 mL, 25.1 mmol, 10.0 eq.) was added to the mixture. The reaction mixture was stirred for 16 hours at room temperature and under N2. After evaporation, the crude product was purified by column chromatography on silica using CH2Cl2/MeOH (10:1) as elutant. After removing the solvents under reduced pressure, the resulting yellow oil was overlaid with hexane and precipitated overnight. The product was isolated as a beige solid (0.99 mg, 2.23 mmol, 88%).

Rf 0.19 (CH2Cl2/MeOH = 10:1).

m.p. 134-136 °C (Hexane).

1H NMR (DMSO-d6, 600 MHz)  = 9.50 (t, 1H, ³JHH = 5.6 Hz, NHCOCF3); 8.41 (s, 1H, 8-H);

7.44 (br, 2H, 6-NH2); 5.85 (d, 1H, ³JHH = 6.1 Hz, 1‘-H); 5.45 (d, 1H, ³JHH = 6.2 Hz, 2‘-OH);

5.25 (dd, 1H, ³JHH = 6.8 Hz, 4.8 Hz, 5‘-OH); 5.19 (d, 1H, ³JHH = 4.8 Hz, 3‘-OH); 4.53 (dt, 1H,

3JHH = 5.9 Hz, 5.2 Hz, 2‘-H); 4.12 (dt, 1H, ³JHH = 4.8 Hz, 3.1 Hz, 3‘-H); 3.95 (dt, 1H, ³JHH = 3.6 Hz, 3.4 Hz, 4‘-H); 3.66 (dt, 1H, ²JHH = 12.1 Hz, ³JHH = 4.2 Hz, 5‘-CH2-a); 3.55 (ddd, ²JHH = 12.0 Hz, ³JHH = 6.9 Hz, 3.7 Hz, 5‘-CH2-b); 3.32 (td, 2H, ³JHH = 6.7 Hz, 6.6 Hz, -CH2-CH2-NHCOCF3); 2.45 (t, 2H, ³JHH = 7.1 Hz, -C≡C-CH2-CH2-); 1.77 (p, 2H, ³JHH = 7.1 Hz, -CH2-CH2-CH2-).

13C NMR (DMSO-d6, 150 MHz)  = 156.4 (q, ²JCF = 36.0 Hz, NHCOCF3); 155.9 (6-C); 149.4 (4-C); 145.6 (2-C); 140.4 (8-C); 118.7 (5-C); 116.9 (q, ¹JCF = 288.0 Hz, NHCOCF3); 87.4 (1‘-C); 85.8 (4‘-C); 84.6 (-C≡C-CH2-); 81.3 (-C≡C-CH2-); 73.7 (2‘-C); 70.5 (3‘-C); 61.5 (5‘-C);

38.5 (CH2-CH2-NHCOCF3); 27.1 (-CH2-CH2-CH2-); 15.8 (-C≡C-CH2-CH2-).

19F NMR (DMSO-d6, 376 MHz)  = -74.3 (NHCOCF3).

HR MS (m/z) [M+H]⁺ (C17H20F3N6O5+) found 445.14204, calc. 445.14418, dev. 4.8 ppm.

6-N-(6-Aminohexyl)adenosine (65)

Compound 65 was prepared as previously published.^[145] Assignments are in agreement with literature.

Rf 0.20t (CH2Cl2/MeOH = 10:1).

m.p. 172-174 °C (EtOH).

1H NMR (DMSO-d6, 400 MHz)  = 8.32 (s, 1H, 8-H); 8.19 (s, 1H, 2-H); 7.86 (br, 1H, 6-NH);

5.87 (d, 1H, ³JHH = 6.2 Hz, 1’-H); 5.44 (br, 2H, 2‘-OH, 5‘-OH) 5.19 (br, 1H, 3‘-OH); 4.60 (t, 1H,

3JHH =5.6 Hz 2’-H); 4.14 (dd, 1H, ³JHH = 5.0 Hz, 3.0 Hz, 3’-H); 3.96 (dt, 1H, ³JHH = 3.5 Hz, 3.5 Hz, 4’-H); 3.67 (dd, ²JHH =12.1 Hz, ³JHH = 7.2 Hz, 1H, 5’-Ha); 3.55 (dd, 1H, ²JHH =12.4 Hz,

3JHH = 3.6 Hz, Hz, 5’-Hb); 3.47 (m, 2H, 1‘‘-H); 2.51 (br, 2H, 6‘‘-H); 1.58 (t, 2H,³JHH = 7.1 Hz, 2‘‘-H); 1.35-1.27 (m, 6H, 3‘‘-H, 4‘‘-H, 5‘‘-H).

13C NMR (DMSO-d6, 100 MHz)  = 155.2 (6-C); 152.9 (2-C); 148.7 (4-C); 140.1 (8-C);

120.2 (5-C); 88.5 (1’-C); 86.4 (4’-C); 74.0 (2’-C); 71.2 (3’-C); 62.2 (5’-C); 42.1 (6‘‘-C); 40.7 (1‘‘-C); 33.8 (5‘‘-C); 29.6 (2‘‘-C); 26.9 (4‘‘-C); 26.7 (3‘‘-C).

HR MS (m/z) [M+H]⁺ (C16H27O4N6+) found 367.20709, calc. 367.20883, dev. 4.7 ppm.

6-N-(6-Trifluoroacetamidohexyl)adenosine (66)

Compound 66 was prepared as previously published.^[145] Assignments are in agreement with literature.

Rf 0.63 (CH2Cl2/MeOH = 5:1).

m.p. 147-150°C (MeOH).

1H NMR (DMSO-d6, 400 MHz)  =9.38 (s, 1H, NHCOCF3); 8.33 (s, 1H, 8-H); 8.19 (s, 1H, 2-H); 7.86 (br, 1H, 6-NH); 5.87 (d, 1H, ³JHH = 6.2 Hz, 1’-H); 5.41 (d, 2H,³JHH = 6.4 Hz, 2‘-OH, 5‘-OH) 5.19 (d, 1H, ³JHH = 4.6 Hz, 3‘-OH); 4.61 (dd, 1H, ³JHH =11.7 Hz, 5.9 Hz 2’-H); 4.14 (dd, 1H, ³JHH = 7.8 Hz, 4.4 Hz, 3’-H); 3.96 (d, 1H, ³JHH = 3.5 Hz, 4’-H); 3.67 (dt, ²JHH =12.1 Hz,

3JHH = 4.1 Hz, 1H, 5’-Ha); 3.55 (ddd, 1H, ²JHH = 12.4 Hz, ³JHH = 7.3 Hz, 3.7 Hz, 5’-Hb); 3.46

Chemical Synthesis

(m, 2H, 1‘‘-H); 3.16 (dt, 2H,³JHH = 10.6 Hz, 5.3 Hz, 6‘‘-H); 1.58 (p, 2H,³JHH = 7.0 Hz, 2‘‘-H);

1.48 (p, 2H,³JHH = 6.9 Hz, 5‘‘-H); 1.35-1.27 (m, 4H, 3‘‘-H, 4‘‘-H).

13C NMR (DMSO-d6, 100 MHz)  = 156.6 (q, ²JCF = 36.0 Hz, NHCOCF3); 154.8 (6-C); 152.8 (2-C); 148.7 (4-C); 140.1 (8-C); 120.2 (5-C) 116.4 (q, ¹JCF = 288.3 Hz, NHCOCF3); 88.4 (1’-C); 86.4 (4’-C); 73.9 (2’-C); 71.1 (3’-C); 62.2 (5’-C); 42.1 (6‘‘-C); 39.6 (1‘‘-C); 39.1 (6‘‘-C);

29.4 (2‘‘-C); 28.7 (5‘‘-C); 26.4 (2C, 3‘‘-C, 4‘‘-C).

19F NMR (DMSO-d6, 376 MHz) δ = -74.4 (s, NHCOCF3).

HR MS (m/z) [M+H]⁺ (C18H26N6O5F3+) found 463.18958, calc. 463.19113, dev. 3.3 ppm.

2-(3-Trifluoroacetamido-prop-1-yn-1-yl)adenosine (71)

Trifluoro‐N‐(prop‐2‐ynyl)‐acetamide^[157] (0.77 g, 5.10 mmol, 2.0 eq.) was dissolved in DMF (40 mL) and degassed subsequently. Next, a flask was charged with 2-iodo-adenosine (15, 1.00 g, 2.54 mmol, 1.0 eq.), CuI (0.10 g, 0.51 mmol, 0.2 eq.) and Pd(PPh3)4 (0.29 g, 0.25 mmol, 0.1 eq.) and evacuated and flushed with N2 for three times. The dissolved amine was transferred to the flask und dry NEt3 (3.5 mL, 25.1 mmol, 10.0 eq.) was added to the mixture. The reaction mixture was stirred for 16 hours at room temperature and under N2. After evaporation, the crude product was purified by column chromatography on silica using CH2Cl2/MeOH (10:1) as elutant. After removing the solvents under reduced pressure, the product was isolated as a beige solid (0.82 mg, 1.97 mmol, 78%).

Rf 0.29 (CH2Cl2/MeOH = 10:1).

m.p. 119-111 °C (MeOH).

1H NMR (DMSO-d6, 400 MHz)  = 10.12 (t, 1H, ³JHH = 5.6 Hz, NHCOCF3); 8.49 (s, 1H, 8-H); 7.50 (br, 2H, 6-NH2); 5.87 (d, 1H, ³JHH = 5.7 Hz, 1‘-H); 5.45 (d, 1H, ³JHH = 6.1 Hz, 2‘-OH); 5.17 (d, 1H, ³JHH = 4.9 Hz, 3‘-OH); 5.14 (t, 1H, ³JHH = 5.3 Hz, 5‘-OH); 4.50 (dt, 1H,

3JHH = 5.8 Hz, 5.6 Hz, 2‘-H); 4.26 (d, 2H, ³JHH = 5.5 Hz, -C≡C-CH2-NHCOCF3); 4.12 (dt, 1H,

3JHH = 4.4 Hz, 4.1 Hz, 3‘-H); 3.94 (dd, 1H, ³JHH = 5.6 Hz, 4.5 Hz, 4‘-H); 3.66 (dt, 1H, ²JHH = 12.2 Hz, ³JHH = 4.3 Hz, 5‘-CH2-a); 3.55 (ddd, ²JHH = 12.0 Hz, ³JHH = 6.4 Hz, 3.7 Hz, 5‘-CH2-b).

13C NMR (DMSO-d6, 150 MHz)  = 156.2 (q, ²JCF = 36.7 Hz, -NHCOCF3); 155.9 (6-C);

150.0 (4-C); 145.0 (2-C); 141.2 (8-C); 119.4 (5-C); 115.8 (q, ¹JCF = 288.1 Hz, NHCOCF3);

87.6 (1‘-C); 85.7 (4‘-C); 82.4 (-C≡C-CH2-); 79.6 (-C≡C-CH2-); 73.9 (2‘-C); 70.4 (3‘-C); 61.4 (5‘-C); 29.1 (-C≡C-CH2-NHCOCF3).

19F NMR (DMSO-d6, 376 MHz)  = -74.3 (NHCOCF3).

HR MS (m/z) [M+H]⁺ (C15H16F3N6O5+) found 417.11138, calc. 417.11288, dev. 3.6 ppm.

6-(2-Propynyl)adenosine (77)

Compound 77^[67] was prepared as previously described.

A mixture of 6-chloro-9-(β-D-ribofuranosyl)purine (17, 540 mg, 1.89 mmol, 1.0 eq.) and prop-2-yn-1-amine (0.36 mL, 5.7 mmol, 3.0 eq.) and DIPEA (0.96 mL, 5.7 mmol, 3.0 eq.) in absolute EtOH (9 mL) were stirred at 90 °C for 12 hours. After cooling, the mixture was allowed to crystallise at -18 °C. The precipitate was filtered off, washed with cold EtOH and dried in vacuo. The product was isolated as a white solid (380 mg, 1.25 mmol, 66%).

Rf 0.29 (CH2Cl2/MeOH = 10:1).

m.p. 166-167 °C (EtOH).

1H NMR: (DMSO-d6, 400 MHz) δ = 8.39 (s, 1H, 8-H); 8.28 (s, 1H, 2-H); 8.21 (s, 1H, 6-NH);

5.91 (d, 1H, ³JHH = 6.0 Hz, 1’-H); 5.44 (d, 1H, ³JHH = 6.1 Hz, 2’-OH); 5.34 (dd, 1H, ³JHH = 7.0 Hz, 4.6 Hz, 5’-OH); 5.17 (d, 1H, ³JHH = 4.6 Hz, 3’-OH); 4.61 (dt, 1H, ³JHH = 5.8 Hz, 5.7 Hz, 2’-H); 4.28 (s, 2H, -CH2-C CH); 4.16 (td, 1H, ³JHH = 4.8 Hz, 3.1 Hz, 3’-H); 3.97 (dt, 1H, ³JHH = 3.5 Hz, 3.3 Hz, 4’-H); 3.68 (dt, 1H, ²JHH = 12.1 Hz,³JHH = 4.2 Hz, 5’-CH2-a); 3.61-3.53 (ddd, 1H, ²JHH = 12.0 Hz, ³JHH = 6.9 Hz, 3.7 Hz, 5’-CH2-b); 3.02 (t, 1H,⁴JHH = 2.4 Hz, -CH2-C CH).

13C NMR: (DMSO-d6, 100 MHz) δ = 154.0 (6-C); 152.3 (2-C); 148.8 (4-C); 140.2 (8-C);

119.9 (5-C); 88.0 (1‘-C); 85.9 (4‘-C); 81.8 (-CH2C CH); 73.6 (2‘-C), 72.4 (-CH2-C CH); 70.6 (3‘-C); 61.6 (5‘-CH2); 29.2 (-CH2C CH).

HR MS (m/z) [M+H]⁺ (C13H16N5O4+) found 306.11909, calc. 306.11968, dev. 1.9 ppm.

8-Bromoadenosine (79)

Adenosine (5.00 g, 18.7 mmol, 1.0 eq.) was suspended in NaOAc buffer solution (110 mL, 0.5 M, pH 4). Then, bromine (2.2 mL, 42.7 mmol, 2.3 eq.) in water (185 mL) was added dropwise. The mixture was stirred at room temperature for 16 hours. Afterwards, the reaction was quenched with a NaHSO3 solution (5 N, 30 mL) and the pH was adjusted to 7 with a

Chemical Synthesis

NaOH solution (5 N. The precipitate formed was filtered off, washed with water (10 mL) and acetone (10 mL), and dried in vacuo, yielding a white solid (4.21 g, 12.2 mmol, 65%).

Rf 0.29 (CH2Cl2/MeOH = 10:1).

m.p. decomposition at 189-191 ^oC.

1H NMR (DMSO-d6, 400 MHz) δ = 8.12 (s, 1H, 2-H); 7.55 (br, 2H, 6-NH2); 5.84 (d, 1H, ³JHH

= 6.8 Hz, 1’-H); 5.50 (br, 3H, 2’-OH, 3’-OH, 5’-OH); 5.09 (dd, 1H, ³JHH= 6.9 Hz, 5.2 Hz, 2’-H);

4.21 (dd, 1H, ³JHH = 5.2 Hz, 2.4 Hz, 3’-H); 3.99 (td, 1H, ³JHH = 4.0 Hz, 2.3 Hz, 4’-H); 3.69 (dd, 1H, ²JHH= 12.1 Hz, ³JHH= 3.9 Hz, 5’-CH2-a); 3.53 (dd, 1H, ²JHH= 12.2 Hz, ³JHH= 4.1 Hz, 5’-CH2-b).

13C NMR (DMSO-d6, 100 MHz) δ = 155.2 (6-C); 152.4 (2-C); 149.9 (4-C); 127.2 (8-C);

119.7 (5-C); 90.5 (1‘-C); 86.8 (4‘-C); 71.2 (2‘-C); 70.9 (3‘-C); 62.2 (5‘-C).

HR MS (m/z) [M+H]⁺ (C10H13BrN5O4+) found 346.01280, calc. 346.01454, dev. 5.0 ppm.

8-(2-Propynylamino)-adenosine (80)

A mixture of 8-bromoadenosine (78, 0.87 g, 2.51 mmol, 1.0 eq.), propargylamine (0.48 mL, 7.54 mmol, 3.1 eq.) and CaCO3 (0.75 g, 7.54 mmol, 3.1 eq.) in absolute EtOH (9 mL) were stirred at 90 °C for four days. The salts were filtered off, and the solvents were removed under reduced pressure after cooling to room temperature. The crude compound was purified by column chromatography using silica gel and CH2Cl2:MeOH (10:1) as elutant. The product was obtained as a viscous brown oil (0.54 g, 1.69 mmol, 67%).

Rf 0.14 (CH2Cl2/MeOH = 10:1).

1H NMR (DMSO-d6, 400 MHz) δ = 7.91 (1H, s, 2-H); 7.38 (t, 1H, ³JHH= 5.8 Hz, -NH-CH2-);

6.58 (br, 2H, 6-NH2); 5.87 (br, 1H, 5’-OH); 5.87 (d, 1H, ³JHH=7.3 Hz, 1’-H); 5.24 (br, 1H, 3’-OH); 5.14 (br, 1H, 2’-3’-OH); 4.68 (t, 1H, ³JHH= 6.3 Hz, 2’-H); 4.20-4.04 (m, 1H, 3’-H); 4.13 (dt, 2H,³JHH= 6.3 Hz, ⁴JHH= 2.9 Hz, -NH-CH2-C≡CH); 3.97 (dt, 1H, ³JHH= 4.9 Hz, 2.4 Hz, 4’-H);

3.70-3.55 (m, 2H, 5’-CH2); 3.08 (t, 1H, ⁴JHH= 2.4 Hz, -CH2-C CH).

13C NMR (DMSO-d6, 100 MHz) δ =153.1 (6-C); 151.1 (8-C); 150.2 (4-C); 149.3 (2-C); 117.3 (5-C); 87.0 (1‘-C); 86.3 (4‘-C); 82.2 (-CH2-C CH); 73.6 (-CH2-C CH ); 71.4 (2C, 2‘-C, 3’-C);

62.2 (5’-C); 32.0 (-CH2-C CH).

HR MS (m/z) [M+H]⁺ (C13H17N6O4+) found 321.12923, calc. 321.13058, dev. 4.2 ppm.

9.3.2 Modified Adenosine Monophosphates General Procedure

In a typical reaction^[59], the respective nucleoside (50-200 mg, 1.0 eq.) and with or without N,N,N′,N′-tetramethyl-1,8-naphthalenediamine (1.5 eq.) were co-evaporated twice with dry MeCN and pre-dried overnight in vacuo. After dissolving in dry PO(OMe)3 (5 mL), the solutions were cooled to 0 °C. Dry POCl3 (1.2 eq.) was added and the mixture was stirred for 2 to 5 hours at 0 °C until complete conversion monitored by TLC. Then, the reaction was quenched by the addition of 0.1 M TEAB solution (20 mL) and left to stir for another 30 min.

Afterwards, the mixture was extracted with EtOAc (3x 10 mL) and the aqueous phase was concentrated under reduced pressure. The crude product was purified by ion exchange chromatography (0.1 M - 1.0 M TEAB buffer) and by RP-HPLC (50 mM TEAA buffer, MeCN) or only by RP-MPLC (50 mM TEAA buffer, MeCN). After lyophilisation, the products were obtained as their triethylammonium salts. Yields ranged between 10-66% (by UV detection).

General Method for the Deprotection of Trifluoroacetamido-modified Monophosphates (60, 68, 73)

In a typical reaction^[146], available amounts of protected monophosphate (1.0 eq, 10 mM) were dissolved in 10% aqueous NH3 and stirred at room temperature until complete conversion is indicated by TLC. (In case of compound 60: Stirring should proceed for maximal one hour, because longer reaction times lead to a decomposition product with the same mass). All solvents were removed under reduced pressure and the crude product was purified by MPLC (50 mM TEAB buffer, MeCN). After lyophilisation, the respective free amine was stored in the freezer until use.

2-Azido-AMP (20)

Compound 20^[111] was obtained as a light yellow salt (30%).

1H NMR (D2O, 400 MHz) Azidomethin form (60%):  = 8.35 (s, 1H, 8-H); 6.06 (d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.82-4.80 (m, 1H, 2’-H); 4.59-4.51 (m, 1H, 3’-H); 4.41-4.35 (m, 1H, 4’-H); 4.15 (dt, 1H, ³JHH = 6.0 Hz, 2.9 Hz, 5’-CH2). Tetrazol form (40%): δ = 8.61 (s, 1H, 8-H); 6.22 (d, 1H, ³JHH = 5.4 Hz, 1’-H); 4.82-4.80 (m, 1H, 2’-H); 4.59-4.51 (m, 1H, 3’-H); 4.43 (t, 1H, ³JHH = 3.2 Hz, 4’-H); 4.20-4.17 (m, 2H, 5’-CH2).

Chemical Synthesis

13C NMR (D2O, 150 MHz) Azidomethin form  = 156.9 (6-C); 156.2 (2-C); 150.5 (4-C);

139.5 (8-C); 116.0 (5-C); 87.1 (1’-C); 83.9 (4’-C); 74.1 (2’-C); 70.4 (3’-C); 64.5 (5’-C);

Tetrazol form δ = 153.6 (6-C); 152.4 (2-C); 142.9 (8-C); 141.4 (4-C); 111.6 (5-C); 87.2 (1’-C);

84.0 (4’-C); 74.3 (2’-C); 70.3 (3’-C); 64.3 (5’-C).

31P NMR (D2O, 162 MHz)  = 0.45 (ROPO3

2-tetrazol), 0.42 (ROPO3

2-azidomethin).

HR MS (m/z) [M-H]^- (C10H13N8O7P^-) found 387.05551, calc. 387.05611, dev. 1.6 ppm.

2-Vinyl-AMP (21)

Compound 21 was obtained as a white salt (35%).

1H NMR (D2O, 400 MHz)  = 8.41 (s, 1H, 8-H); 6.63 (dd, 1H,³JHH = 17.3 Hz, 10.7 Hz, -CH=CH2); 6.33 (dd, 1H, ³JHH = 17.3 Hz, ²JHH = 1.3 Hz, -CH=CH2-trans); 6.12 (d, 1H, ³JHH = 5.5 Hz, 1’-H); 5.72 (dd, 1H,³JHH = 10.7 Hz, ²JHH = 1.2 Hz, -CH=CH2-cis); 4.78-4.74 (m, 1H, 2’-H); 4.53 (dd, 1H,³JHH = 5.1 Hz, 3.9 Hz, 3’-H); 4.39 (dd, 1H,³JHH = 3.9 Hz, 3.9 Hz, 4’-H); 4.23-4.05 (m, 2H, 5’-CH2).

13C NMR (D2O, 100 MHz)  = 158.6 (2-C); 154.7 (6-C); 149.6 (4-C); 140.2 (8-C); 134.2 (-CH=CH2); 123.9 (-CH=CH2); 117.3 (5-C); 86.9 (1’-C); 83.8 (4’-C); 74.3 (2’-C); 70.3 (3’-C);

64.3 (5’-C).

31P NMR (D2O, 162 MHz)  = 1.2 (ROPO32-).

HR MS (m/z) [M-H]^- (C12H15N5O7P^-) found 372.07135, calc. 372.07146, dev. 0.3 ppm.

6-N-(6-Azidohexyl)-AMP (22)

Compound 22 was obtained as a white salt (66%).

1H NMR (D2O, 400 MHz)  = 8.43 (s, 1H, 8-H); 8.18 (s, 1H, 2-H); 6.10 (d, 1H, ³JHH = 5.8 Hz, 1’-H); 4.75 (t, 1H,³JHH = 5.5 Hz, 2’-H); 4.50 (dd, 1H, ³JHH = 5.1 Hz, 3.6 Hz, 3’-H); 4.41-4.36 (m, 1H, 4’-H); 4.17-4.08 (m, 2H, 5’-CH2); 3.58-3.41 (br, 2H, -CH2NH-); 3.27 (t, ³JHH = 6.8 Hz,

-CH2N3); 1.70-1.61 (m, 2H, -CH2-CH2NH-); 1.59-1.51 (m, 2H, -CH2-CH2N3); 1.43-1.33 (m, 4H,

Compound 23 was obtained as a white salt (61%):

1H NMR (D2O, 400 MHz)  = 8.47 (s, 1H, 8-H); 8.22 (s, 1H, 2-H); 6.12 (d, 1H, ³JHH = 6.0 Hz,

Compound 59 was obtained as a white salt (66%).

1H NMR (D2O, 600 MHz)  = 8.39 (s, 1H, 8-H); 5.93 (d, 1H, ³JHH = 5.3 Hz, 1‘-H); 4.62 (d, 1H, ³JHH = 5.2 Hz, 2‘-H); 4.41 (t, 1H, ³JHH = 4.5 Hz, 3‘-H); 4.28 (br, 1H, 4‘-H); 4.06-3.98 (m,

Chemical Synthesis

2H, 5‘-H); 3.41 (t, 2H, ³JHH = 6.9 Hz, CH2-CH2-NHCOCF3); 2.44 (t, 2H, ³JHH = 7.0 Hz, -C≡C-CH2-CH2-); 1.82 (p, 2H, ³JHH = 7.0 Hz, -CH2-CH2-CH2-).

13C NMR (D2O, 150 MHz)  = 158.7 (q, ²JCF = 37.1 Hz, NHCOCF3); 154.8 (6-C); 148.8 (4-C); 145.8 (2-C); 140.2 (8-C); 117.6 (5-C); 116.9 (q, ¹JCF = 286.2 Hz, -NHCOCF3); 88.3 (-C≡C-CH2-); 87.0 (1‘-C); 84.1 (4‘-C); 79.0 (-C≡C-CH2-); 74.7 (2‘-C); 70.4 (3‘-C); 64.1 (5‘-C);

39.0 (CH2-CH2-NHCOCF3); 26.3 (-CH2-CH2-CH2-); 15.9 (-C≡C-CH2-CH2-).

19F NMR (D2O, 376 MHz)  = -73.5 (NHCOCF3).

31P NMR (D2O, 162 MHz)  = -0.8 (ROPO32-).

HR MS (m/z) [M-H]^- (C17H19F3N6O8P^-) found 523.09677, calc. 523.09596, dev. 1.5 ppm.

2-DIBAC-AMP (61)

2-(5-Aminopent-1-yn-1-yl)-AMP (60, 50 µmol, 1.0 eq.) was dissolved in NaHCO3 solution (100 mM, pH 8.7, 10 mL) and DIBAC-NHS (30.2 mg, 75 µmol, 1.5 eq.) in DMF (4 mL) was added. After stirring at room temperature for four hours, the mixture was evaporated, re-dissolved in water (20 mL) and extracted with CH2Cl2 (2x 10 mL). The aqueous phase contained the crude product which was further purified by HPLC (50 mM TEAA buffer, MeCN). After lyophilisation, the product was obtained as a beige salt (27 µmol, 54%).

1H NMR (DMSO-d6, 600 MHz)  = 8.50 (s, 1H, 8-H); 7.84 (t, 1H, ³JHH = 5.6 Hz, NHCO);

7.68 (dd, 1H, ³JHH = 7.7 Hz, ⁴JHH = 1.4 Hz, arom. H); 7.63 (dd, 1H, ³JHH = 7.5 Hz, ⁴JHH = 1.3 Hz, arom. H); 7.50 (dd, 1H, ³JHH = 7.0 Hz, ⁴JHH = 2.0 Hz, arom. H); 7.48 (dd, 1H, ³JHH = 7.5 Hz, ⁴JHH = 2.0 Hz, arom. H); 7.45 (td, 1H, ³JHH = 7.4 Hz, ⁴JHH = 1.4 Hz, arom. H); 7.38 (td, 1H, ³JHH = 7.4 Hz, ⁴JHH = 1.7 Hz, arom. H); 7.38 (br, 2H, 6-NH2); 7.34 (td, 1H, ³JHH = 7.5 Hz,

4JHH = 1.4 Hz, arom. H); 7.29 (dd, 1H, ³JHH = 7.5 Hz, ⁴JHH = 1.5 Hz, arom. H); 5.88 (d, 1H,

3JHH = 6.1 Hz, 1‘-H); 5.03 (d, 1H, ²JHH = 14.1 Hz, -N-CH2-Ph-a); 4.59 (dd, 1H, ³JHH = 5.5 Hz, 5.4 Hz, 2‘-H); 4.19 (dd, 1H, ³JHH = 4.8 Hz, 2.9 Hz, 3‘-H); 4.04 (dt, 1H, ³JHH = 3.5 Hz, 3.5 Hz, 4‘-H); 3.84 (dd, 2H, ³JHH = 6.4 Hz, 3.6 Hz, 5‘-CH2); 3.60 (d, 1H, ²JHH = 14.0 Hz, -N-CH2-Ph-b);

3.05 (dt, 2H, ³JHH = 6.6 Hz, 6.5 Hz, -CH2-CH2-NHCO-); 2.59 (dt, 2H, ²JHH = 15.8 Hz, ³JHH = 7.5 Hz, -NHCO-CH2-CH2-CON-a); 2.34 (t, 2H, ³JHH = 7.1 Hz, -C≡C-CH2-CH2-); 2.24 (dt, 2H,

2JHH = 15.3 Hz, ³JHH = 7.6 Hz, -NHCO-CH2-CH2-CON-a); 2.02 (dt, 2H, ²JHH = 15.4 Hz, ³JHH = 7.0 Hz, -NHCO-CH2-CH2-CONH-b); 1.78 (dt, 2H, ²JHH = 16.5 Hz, ³JHH = 6.9 Hz, -NHCO-CH2-CH2-CON-b); 1.57 (p, 2H, ³JHH = 7.1 Hz, -CH2-CH2-CH2-).

13C NMR (DMSO-d6, 150 MHz)  = 171.1, 171.1 (2C, CON, CONH); 155.7 (6-C); 151.6 (arom. Cq-N); 149.9 (4-C); 148.4 (arom. -CH2-Cq); 145.8 (2-C); 139.9 (8-C); 132.5, 129.6, 128.9, 128.1, 128.0, 127.7, 126.8, 125.2 (8C, arom. CH); 122.5, 121.4 (2C, arom.

Cq-C≡C-Cq); 118.1 (5-C); 114.2, 108.2 (2C, Cq-C≡C-Cq); 86.4 (1‘-C); 84.8 (C≡C-CH2); 84.3 (4‘-C); 81.4 (C≡C-CH2); 74.2 (2‘-C); 71.1 (3‘-C); 64.1 (5‘-C); 54.9 (N-CH2-Ph); 37.8 (-CH2-CH2-NHCO); 30.4 (-NHCO-CH2-CH2-CON-); 29.7 (-NHCO-CH2-CH2-CON-); 27.8 (C≡C-CH2); 15.8 (CH2-CH2-CH2).

31P NMR (DMSO-d6, 162 MHz)  = -0.8 (ROPO32-).

HR MS (m/z) [M-H]^- (C34H33N7O9P^-) found 714.20885, calc. 714.20829, dev. 0.8 ppm.

2-Cyclopropenyl-AMP (62)

2-(5-Aminopent-1-yn-1-yl)-AMP (60, 50 µmol, 1.0 eq.) was dissolved in NaHCO3 solution (100 mM, pH 8.7, 10 mL) and (2-methylcycloprop-2-en-1-yl)methyl (4-nitrophenyl) carbonate (CPNP, 20% in dioxane, 31.1 mg, 25 µmol, 0.5 eq.) was added. The mixture was stirred at room temperature for three days and each day a new portion of CPNP (0.5 eq.) was added.

Then, the mixture was evaporated, re-dissolved in water (10 mL) and extracted with EtOAc (2x 5 mL). The aqueous phase contained the crude product which was further purified by HPLC (50 mM TEAA buffer, MeCN). After lyophilisation, the product was obtained as a white salt (16 µmol, 32%).

1H NMR (D2O, 600 MHz)  = 8.56 (s, 1H, 8-H); 6.53 (s, 1H, CH=C-CH3); 6.09 (d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.74 (t, 1H, ³JHH = 5.3 Hz, 2’-H); 4.51 (t, 1H, ³JHH = 4.5 Hz, 3‘-H); 4.39 (br, 1H, 4‘-H), 4.14-4.06 (m, 2H, 5‘-CH2); 3.87 (d, 2H, ²JHH = 22.9 Hz, O-CH2-CH); 3.33 (t, 2H, ³JHH = 7.0 Hz, -CH2-CH2-NHCO); 2.55 (t, 2H, ³JHH = 6.8 Hz, -C≡C-CH2-CH2-); 2.03 (s, 3H, -CH=C-CH3); 1.86 (p, 2H, ³JHH = 6.8 Hz, CH2-CH2-CH2); 1.47 (s, 1H, O-CH2-CH-).

13C NMR (D2O, 150 MHz)  = 159.1 (CO); 155.4 (6-C); 149.3 (4-C); 146.3 (2-C); 140.5 (8-C); 120.5 (CH=C-CH3); 118.1 (5-C); 101.1 (CH=C-CH3); 89.4 (-C≡C-CH2-); 87.0 (1’-C);

84.5 (4’-C); 79.1 (-C≡C-CH2-); 74.8 (2’-C); 72.7 (O-CH2-CH); 70.6 (3’-C); 64.1 (5’-C); 40.0 (-CH2-CH2-NHCO); 27.4 (-C≡C-CH2-); 16.4 (-CH2-CH2-CH2-); 16.1 (O-CH2-CH); 10.8 (-CH=C-CH3).

31P NMR (D2O, 162 MHz) δ = 1.8 (ROPO32-).

HR MS (m/z) [M-H]^- (C21H26N6O9P^-) found 537.15040, calc. 537.15044, dev. 0.0 ppm.

Chemical Synthesis 2-TMR-AMP (64)

2-(5-aminopent-1-yn-1-yl)-AMP (60, 100 µmol, 1.0 eq.) was dissolved in NaHCO3 solution (100 mM, pH 8.7, 20 mL) and TMR-NHS (63, 79 mg, 150 µmol, 1.5 eq.) in DMF (5 mL) was added. After stirring at room temperature for 16 hours, the mixture was evaporated and the crude product was purified by column chromatography on silica using iPrOH/H2O/NH3 (6:1:1

 3:1:1) as elutant. Then, the product was further purified by HPLC (50 mM TEAB buffer, MeCN). After lyophilisation, the product was obtained as a dark purple salt (34 µmol, 34%).

1H NMR (DMSO-d6, 600 MHz)  = 9.00 (t, 1H, NHCO); 8.47 (s+d, 2H, ⁴JHH = 1.7 Hz, 8-H, 6**-H); 8.27 (dd, 1H, ³JHH = 8.1 Hz,⁴JHH = 1.7 Hz, 4**-H); 7.38 (br, 2H, 6-NH2); 7.30 (d, 1H,

3JHH = 8.0 Hz,3**-H); 6.52 (d, 2H, ³JHH = 8.7 Hz,1*-H, 8*-H); 6.50 (d, 2H, ⁴JHH = 2.1 Hz, 4*-H, 5*-H); 6.48 (dd, 2H, ³JHH = 8.8 Hz,⁴JHH = 2.5 Hz, 2*-H, 7*-H); 5.89 (d, 1H, ³JHH = 6.0 Hz, 1’-H); 4.57 (t, 1H, ³JHH = 5.4 Hz, 2’-H); 4.19 (dd, 1H, ³JHH = 4.9 Hz, 3.0 Hz, 3’-H); 4.04 (dt, 1H,

3JHH = 3.7 Hz, 3.0 Hz, 4’-H); 3.88-3.83 (m, 2H, 5’-CH2); 3.48-3.42 (br, 2H, -CH2-NHCO); 2.94 (s, 12H, -N(CH3)2); 2.53 (t, 2H, ³JHH = 7.1 Hz, -C≡C-CH2-); 1.86 (p, 2H, ³JHH = 7.1 Hz, -CH2-).

13C NMR (DMSO-d6, 151 MHz)  = 168.4 (-COO^-); 164.8 (-NHCO-); 155.7 (6-C); 154.8 (2**-C); 152.1, 152.0 (4C, 3*-C, 4a*-C, 10a*-C, 6*-C); 149.8 (4-C): 145.8 (2-C); 139.9 (8-C);

136.1 (5**-C); 134.5 (4**-C); 128.5 (2C, 1*-C, 8*-C); 126.9 (1**-C); 124.2 (3**-C); 123.3 (6**-C); 118.1 (5-C); 109.0 (2C, 2*-C, 7*-C); 105.6 (2C, 8a*-C, 9a*-C); 98.0 (2C, 4*-C, 5*-C);

86.5 (1’-C); 84.9 (9*-C); 84.8 (-C≡C-CH2-); 84.2 (4’-C); 81.5 (-C≡C-CH2-); 74.1 (2’-C); 71.1 (3’-C); 64.2 (5’-CH2); 45.4 (4C, -N(CH3)2); 38.8 (-CH2-NHCO-); 27.8 (-CH2-); 16.0 (-C≡C-CH2-).

31P NMR (DMSO-d6, 162 MHz)  = -0.1 (ROPO32-).

HR MS (m/z) [M-H]^- (C40H40N8O11P^-) found 839.25363, calc. 839.25596, dev. 2.8 ppm.

6-N-(6-Trifluoroacetamidohexyl)-AMP (67)

Compound 67^[124] was obtained as a white salt (66%).

1H NMR (D2O, 400 MHz) δ = 8.36 (s, 1H, 8-H); 8.09 (s, 1H, 2-H); 6.04 (d, 1H, ³JHH = 5.7 Hz, 1’-H); 4.69 (t, 1H, ³JHH =5.4 Hz 2’-H); 4.47 (dd, 1H, ³JHH = 5.1 Hz, 3.7 Hz, 3’-H); 4.38-4.35 (m, 1H, 4’-H); 4.16-4.09 (m, 2H, 5’-H); 3.38 (br, 2H, -CH2-NH); 3.23 (t, 2H,³JHH = 7.0 Hz, -CONH-CH2-); 1.57 (p, 2H,³JHH = 7.1 Hz, -CH2-CH2-NH); 1.49 (p, 2H,³JHH = 7.1 Hz, -CONH-CH2-CH2-); 1.37-1.25 (m, 4H, -CH2-CH2-).

13C NMR (D2O, 100 MHz) δ = 158.7 (q, ²JCF = 37.1 Hz, -NHCOCF3); 154.0 (6-C); 152.4 (2-C); 147.6 (4-C); 139.0 (8-C); 118.6 (5-C), 116.0 (q, ¹JCF = 285.9 Hz, -NHCOCF3); 87.0 (1’-C); 84.0 (4’-C); 74.5 (2’-C); 70.5 (3’-C); 64.5 (5’-C); 46.7 (3C, N(CH2CH3)3); 40.7 (1‘‘-C);

39.7 (6‘‘-C); 28.3 (2‘‘-C); 27.7 (5‘‘-C); 25.7 (2C, 3‘‘-C, 4‘‘-C); 8.3 (3C, N(CH2CH3)3).

19F NMR (D2O, 376 MHz) δ = -75.8 (s, NHCOCF3).

31P NMR (D2O, 162 MHz)  = 0.42 (ROPO32-).

HR MS (m/z) [M-H]^- (C18H26F3N6O8P^-) found 541.14494, calc. 541.14291, dev. 3.8 ppm.

6-TMR-AMP (69)

6-((6-aminohexyl)amino)-AMP (68, 100 µmol, 1.0 eq.) was dissolved in NaHCO3 solution (100 mM, pH 8.7, 20 mL) and TMR-NHS (63, 79 mg, 150 µmol, 1.5 eq.) in DMF (5 mL) was added. After stirring at room temperature for 16 hours, the mixture was evaporated and the crude product was purified by column chromatography on silica using iPrOH/H2O/NH3 (6:1:1

 3:1:1) as elutant. Then, the product was further purified by RP-HPLC (50 mM TEAB buffer, MeCN). After lyophilisation, the product was obtained as a dark purple salt (35 µmol, 35%).

1H NMR (D2O, 600 MHz)  = 8.33 (s, 1H, 8-H); 8.23 (s, 1H, 6**-H); 7.98 (s, 1H, 2-H); 7.90 (d, 1H, ³JHH = 7.7 Hz, 4**-H); 7.26 (d, 1H, ³JHH = 7.8 Hz, 3**-H); 7.00 (2d, 2H, ³JHH = 10.8 Hz, 1*-H, 8*-H); 6.66 (2s, 2H, 2*-H, 7*-H); 6.39 (2s, 2H, 4*-H, 5*-H); 5.93 (d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.60 (t, 1H, ³JHH = 4.8 Hz, 2’-H); 4.42 (t, 1H, ³JHH = 4.8 Hz, 3’-H); 4.28 (s, 1H, 4’-H);

4.03 (s, 2H, 5’-CH2); 3.39 (br, 2H, -CONH-CH2-); 3.34 (br, 2H, -CH2-NH-); 3.09 (s, 12H, -N(CH3)2); 1.62-1.54 (m, 4H, -CONH-CH2-CH2-,-CH2-CH2-NH-); 1.42-1.34 (m, 4H, -CH2-CH2-).

Chemical Synthesis

Compound 72 was obtained as a white salt (10%).

1H NMR (D2O, 600 MHz)  = 8.48 (s, 1H, 8-H); 5.99 (d, 1H, ³JHH = 5.4 Hz, 1‘-H); 4.68 (t, 1H,

evaporated and the crude product was purified by RP-HPLC (50 mM TEAA buffer, MeCN).

After lyophilisation, the product was obtained as a dark red salt (8 µmol, 16%).

1H NMR (CD3OD, 600 MHz)  = 8.58 (s, 1H, 8-H); 7.42 (s, 1H, 8*-H); 7.01 (d, ³JHH = 4.0 Hz, 1H, 1*-H); 6.37 (d, ³JHH = 4.0 Hz, 1H, 2*-H); 6.20 (s, 1H, 6*-H); 6.07 (d, ³JHH = 5.9 Hz, 1H, 1’-H); 4.64 (t, ³JHH = 5.4 Hz, 1H, 2’-H); 4.39 (dd, ³JHH = 5.1 Hz, 3.0 Hz, 1H, 3’-H); 4.23 (s, 3H, 4’-H, -C≡C-CH2-NHCO-); 4.17-4.01 (m, 2H, 5’-CH2); 3.25 (t, ³JHH = 7.6 Hz, 2H, 3**-H); 2.67 (t,

3JHH = 7.6 Hz, 2H, 2**-H); 2.51 (s, 3H, 5*-CH3); 2.27 (s, 3H, 7*-CH3).

13C NMR (CD3OD, 150 MHz)  = 172.9 (1**-C); 159.7 (5*-C); 157.0 (3*-C); 149.4 (4-C);

144.3 (7*-C); 140.4 (8-C); 135.0 (7*a-C); 133.5 (1*a-C); 128.6 (1*-C); 124.5 (8*-C); 119.8 (6*-C); 118.2 (5-C); 116.5 (2*-C); 87.5 (1’-C); 84.6 (4’-C); 81.3 (-C≡C-CH2-); 81.1 (-C≡C-CH2-); 75.0 (2’-C); 70.9 (3’-C); 64.3 (5’-C); 24.0 (-C≡C-CH2-NHCOCF3); 34.4 (2**-C);

28.5 (-C≡C-CH2-); 24.0 (3**-C); 13.4 (5-CH3); 9.8 (7-CH3).

19F NMR (CD3OD, 376 MHz)  = -146.0 (d, ²JFF = 32.8 Hz, BF); -146.1 (d, ²JFF = 32.9 Hz, BF).

31P NMR (CD3OD, 162 MHz)  = 0.9 (ROPO32-).

HR MS (m/z) [M-H]^- (C27H29BF2N8O8P^-) found 673.19468, calc. 673.19173, dev. 4.4 ppm.

6-N-(6-BODIPY-amidohexyl)-AMP (75)

6-((6-aminohexyl)amino)-AMP (68, 220 µmol, 1.0 eq.) was dissolved in DMF (1 mL), BODIPY Fl-NHS (128 mg, 330 µmol, 1.5 eq.) in DMF (5 mL) and DIPEA (120 µL, 660 µmol, 3.0 eq.) was added. After stirring at room temperature for 16 hours, the mixture was evaporated and the crude product was purified by RP-HPLC (50 mM TEAA buffer, MeCN).

After lyophilisation, the product was obtained as a dark red salt (72 µmol, 33%).

1H NMR (CD3OD, 600 MHz)  = 8.49 (s, 1H, 8-H); 8.22 (s, 1H, 2-H); 7.39 (s, 1H, 8*-H); 6.99 (d, ³JHH = 4.0 Hz, 1H, 1*-H); 6.31 (d, ³JHH = 4.0 Hz, 1H, 2*-H); 6.18 (s, 1H, 6*-H);

6.09 (d, ³JHH = 6.2 Hz, 1H, 1’-H); 4.67 (t, ³JHH = 5.6 Hz, 1H, 2’-H); 4.40 (dd, ³JHH = 5.0 Hz, 2.9 Hz, 1H, 3’-H); 4.23 (t, ³JHH = 2.7 Hz, 1H, 4’-H); 4.14-4.00 (m, 2H, 5’-CH2); 3.67-3.43 (br, 2H, -CH2-NH); 3.26-3.14 (m, 3H, 3**-H, CONH-CH2-); 2.60 (t, ³JHH = 7.5 Hz, 2H, 2**-H); 2.49

Chemical Synthesis

(s, 3H, 5*-CH3); 2.24 (s, 3H, 7*-CH3); 1.67 (p, ³JHH = 7.4 Hz, 2H, -CH2-CH2-NH); 1.54-1.37 (m, 4H, CONH-CH2-CH2-); 1.38-1.32 (m, 4H,-CH2-CH2-).

13C NMR (CD3OD, 150 MHz)  = 174.5 (CONH); 161.3 (5*-C); 158.5 (3*-C); 155.6 (6-C);

153.9 (2-C); 149.9 (4-C); 145.8 (7*-C); 140.5 (8-C); 136.5 (7*a-C); 134.9 (1*a-C); 129.6 (1*-C); 125.7 (8*-C); 121.3 (6*-C); 120.4 (5-C); 117.7 (2*-C); 88.8 (1’-C); 86.0 (4’-C); 76.3 (2’-C); 72.5 (3’-C); 65.9 (5’-C); 41.5 (-CH2NH-); 40.4 (CONH-CH2); 36.0 (2**-C); 30.8, 30.4 (-CONH-CH2-CH2, -CH2-CH2-NH-); 27.7, 27.6 (-CH2-CH2-); 25.7 (3**-C); 14.9 (5*-CH3); 11.2 (7*-CH3).

19F NMR (CD3OD, 376 MHz)  = -145.9 (d, ²JFF = 32.7 Hz, BF); -146.1 (d, ²JFF = 32.8 Hz, BF).

31P NMR (CD3OD, 162 MHz)  = 0.9 (ROPO32-).

HR MS (m/z) [M-H]^- (C30H39BF2N8O8P^-) found 719.26680, calc. 719.27004, dev. 4.5 ppm.

6-(2-Propynyl)-AMP (81)

Compound 81^[57] was obtained as a white salt (28%).

1H NMR: (D2O, 400 MHz) δ = 8.53 (s, 1H, 8-H); 8.27 (s, 1H, 2-H); 6.13 (d, 1H, ³JHH = 5.7 Hz, 1’-H); 4.79 (below H2O, 1H, 2’-H); 4.53 (dd, 1H, ³JHH = 5.1 Hz, 3.7 Hz, 3’-H); 4.40 (dd, 1H, ³JHH = 3.7 Hz, 1.9 Hz, 4’-H); 4.34 (br, 2H, -CH2C CH); 4.16-4.07 (m, 2H, 5’-CH2); 2.68 (t, 1H, ⁴JHH = 2.5 Hz, -CH2C CH).

13C NMR: (D2O, 100 MHz) δ = 153.9 (6-C); 152.7 (2-C); 148.5 (4-C); 140.0 (8-C); 119.1 (5-C); 87.0 (1’-C); 84.5 (4’-C); 80.5 (-CH2C CH); 74.5 (2’-C); 72.0 (CH2-C CH); 70.6 (3’-C);

64.1 (5’-C); 30.3 (-CH2C CH).

31P NMR: (D2O, 162 MHz) δ = 2.0 (s, 1P, ROPO32-).

HR MS (m/z) [M-H]^- (C13H15N5O7P^-) found 384.07037, calc. 384.07146, dev. 2.8 ppm.

8-Propynylamino-AMP (82)

Compound 82^[57] was obtained as a white salt (38% by UV detection).

1H NMR: (D2O, 400 MHz) δ = 8.04 (s, 1H, 2-H); 6.03 (d, 1H, ³JHH = 7.7 Hz, 1’-H); 4.71 (dd, 1H, ³JHH = 7.7 Hz, 5.5 Hz, 23’-H); 4.46 (dd, 1H, ³JHH = 6.0 Hz, 2.4 Hz, 3’-H); 4.38-4.33 (m, 1H, 4’-H); 4.28 (s, 2H, -CH2C CH); 4.24-4.11 (m, 2H, 5’-CH2); 2.64 (t, 1H, ⁴JHH = 2.4 Hz, -CH2C CH).

13C NMR: (D2O, 100 MHz) δ = 151.5 (6-C); 151.3 (8-C); 149.5 (4-C); 148.7 (2-C); 116.2 (5-C); 86.6 (1’-C); 84.5 (4’-C); 81.1 (-CH2C CH); 71.8 (-CH2-C CH); 70.9 (2’-C); 70.3 (3’-C);

64.7 (5’-C); 31.9 (-CH2C CH).

31P NMR: (D2O, 162 MHz) δ = 0.3 (s, 1P, ROPO32-).

HR MS (m/z) [M-H]^- (C13H16N6O7P^-) found 399.08121, calc. 399.08236, dev. 2.9 ppm.

9.3.3 Modified Nicotinamide Adenine Dinucleotides General Procedure

For a typical reaction^[59], -NMN (10.0 mg, 30 µmol, 1.5 eq.) and CDI (16.2 mg, 50 µmol, 1.7 eq.) were dried in vacuo for three hours and subsequently dissolved in dry DMF (0.1 mL) and dry NEt3 (8 µL, 60 µmol, 2.0 eq.) was added. The mixture was stirring for four hours at room temperature and turned red. Then, dry MeOH (0.1 mL) was added and the mixture was co-evaporated three times with dry DMF (3x 1.0 mL). The respective pre-dried AMP analogues (20 µmol, 1.0 eq.) was dissolved in dry DMF (1.0 mL) and added to the activated

-NMN. The mixture was stirred at room temperature for four days and the colour turned yellow. Then, the reaction was quenched by the addition of 0.1 M TEAB solution (2 mL) and all solvents were removed under reduced pressure. The crude product was purified by ion exchange chromatography (0.1 M - 1.0 M TEAB buffer) and by RP-HPLC (50 mM TEAB buffer, MeCN). After lyophilisation, the product was obtained as their triethylammonium salts.

Yields ranged between 10-58% (by UV detection).

6-(2-Propynyl)-NAD⁺ (2)

Compound 2^[57] was obtained as a light yellow salt (13%).

1H NMR: (D2O, 400 MHz) δ = 9.35 (s, 1H, 2‘‘‘-H); 9.17 (d, 1H, ³JHH = 6.2 Hz, 6‘‘‘-H); 8.87 (dt, 1H, ³JHH = 8.1 Hz, ⁴JHH = 1.5 Hz, 4‘‘‘-H); 8.47 (s, 1H, 8-H); 8.27 (s, 1H, 2-H); 8.24 (dd, 1H,

3JHH = 8.1 Hz, 6.3 Hz, 5‘‘‘-H); 6.11 (d, 1H, ³JHH = 5.6 Hz, 1‘‘-H); 6.09 (d, 1H, ³JHH = 6.0 Hz, 1’-H); 4.83 (t, 1H, ³JHH = 5.6 Hz, 2’-H); 4.59 (t, 1H,³JHH = 2.5 Hz, 4’’-H); 4.56 (dd, 1H,³JHH = 5.2 Hz, 3.7 Hz, 3’-H); 4.53 (t, 1H,³JHH = 5.3 Hz, 2’’-H); 4.48 (dd, 1H,³JHH = 5.1 Hz, 2.7 Hz,

Chemical Synthesis

3’’-H); 4.40-4.37 (m, 1H, 4’-H); 4.38 (br, 2H, -CH2C CH); 4.35-4.22 (m, 4H, 5’- CH2, 5’’-CH2);

2.73 (t, 1H, ⁴JHH = 2.5 Hz, -CH2C CH).

31P NMR (D2O, 162 MHz)  = -11.2 (d, 1P, ²JPP = 20.5 Hz; R-PO3-O-PO3-R’); -11.5 (d, 1P,

2JPP = 20.9 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C24H28N7O14P2-) found 700.11900, calc. 700.11750, dev. 2.1 ppm.

8-Propynylamino-NAD⁺(4)

Compound 4^[57] was obtained as a yellow salt (10%).

1H NMR: (D2O, 400 MHz) δ = 9.41 (s, 1H, 2‘‘‘-H); 9.25 (d, 1H, ³JHH = 6.3 Hz, 6‘‘‘-H); 8.91 (d, 1H, ³JHH = 8.1 Hz, 4‘‘‘-H); 8.29 (dd, 1H, ³JHH = 8.0 Hz, 6.3 Hz, 5‘‘‘-H); 8.11 (s, 1H, 2-H); 6.16 (d, 1H, ³JHH = 5.1 Hz, 1‘‘-H); 5.96 (d, 1H, ³JHH = 7.1 Hz, 1’-H); 4.79 (below H2O, 2’-H); 4.63 (s, 1H,4’’-H); 4.56 (t, 1H,³JHH = 4.9 Hz, 2’’H); 4.534.44 (m, 3H, 3’H, 3’’H, 4’H); 4.36 (br, 2H, -CH2C CH); 4.36-4.22 (m, 4H, 5’- CH2, 5’’-CH2); 2.66 (t, 1H, ⁴JHH = 2.4 Hz, -CH2C CH)

31P NMR (D2O, 162 MHz)  = -11.5 (s, 2P, R-PO3-O-PO3-R’.

HR MS (m/z) [M-H]^- (C24H29N8O14P2-) found 715.13085, calc. 715.12839, dev. 3.4 ppm.

2-Azido-NAD⁺(5)

Compound 5^[109] was obtained as a light yellow salt (27%).

1H NMR (D2O, 400 MHz) Azidomethin form (70%):  = 9.35 (s, 1H, 2‘‘‘-H); 9.16 (d, 1H, ³JHH

= 6.4 Hz, 6‘‘‘-H); 8.87 (dt, 1H, ³JHH = 8.1 Hz, ⁴JHH = 1.5 Hz, 4‘‘‘-H); 8.29 (s, 1H, 8-H); 8.22 (dd, 1H, ³JHH = 8.1 Hz, 6.3 Hz, 5‘‘‘-H); 6.09 (d, 1H, ³JHH = 5.7 Hz, 1‘‘-H); 5.95 (d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.82 (m, 1H, 2’-H); 4.59-4.53 (m, 2H, 4’’-H, 3’H); 4.49 (t, 1H, ³JHH = 4.5 Hz, 2’’-H); 4.45 (dd, ³JHH = 5.2 Hz, 2.7 Hz, 3’’-H); 4.40-4.35 (m, 1H, 4’-H); 4.43-4.19 (m, 3H, 4’’-H, 5’-CH2, 5’’- CH2). Tetrazol form (40%): δ = 9.37 (s, 1H, 2‘‘‘-H); 9.22 (d, 1H, ³JHH = 6.4 Hz, 6‘‘‘-H); 8.91 (dt, 1H, ³JHH = 8.2 Hz, ⁴JHH = 1.5 Hz, 4‘‘‘-H); 8.56 (s, 1H, 8-H); 8.29 (dd, 1H, ³JHH = 8.1 Hz, 6.3 Hz, 5‘‘‘-H); 6.15 (d, 1H, ³JHH = 5.7 Hz, 1‘‘-H); 6.08 (d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.82 (m,

1H, 2’-H); 4.59-4.53 (m, 2H, 4’’-H, 3’H); 4.49 (t, 1H, ³JHH = 4.5 Hz, 2’’-H); 4.45 (dd, ³JHH = 5.2 Hz, 2.7 Hz, 3’’-H); 4.40-4.35 (m, 1H, 4’-H); 4.43-4.19 (m, 3H, 4’’-H, 5’-CH2, 5’’- CH2).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 20.7 Hz; R-PO3-O-PO3-R’); -11.5 (d, 1P,

2JPP = 20.7 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C23H28N7O14P2-) found 688.11926, calc. 688.11750, dev. 2.6 ppm.

2-Vinyl-NAD⁺ (6)

Compound 6 was obtained as a light yellow salt (15%).

1H NMR (D2O, 400 MHz)  = 9.30 (s, 1H, 2‘‘‘-H); 9.07 (d, 1H, ³JHH = 6.3 Hz, 6‘‘‘-H); 8.83 (d, 1H, ³JHH = 8.0 Hz, 4‘‘‘-H); 8.43 (s, 1H, 8-H); 8.19 (dd, 1H, ³JHH = 8.1 Hz, 6.3 Hz, 5‘‘‘-H); 6.66 (dd, 1H,³JHH = 17.3 Hz, 10.7 Hz, -CH=CH2); 6.36 (dd, 1H, ³JHH = 17.3 Hz, ²JHH = 1.3 Hz, -CH=CH2-trans); 6.07 (d, 1H, ³JHH = 6.0 Hz, 1‘‘-H); 6.03 (d, 1H, ³JHH = 5.2 Hz, 1’-H); 5.74 (dd, 1H,³JHH = 10.7 Hz, ²JHH = 1.2 Hz, -CH=CH2-cis); 4.84 (t, 1H, ³JHH = 5.7 Hz, 2’-H); 4.57 (dd, 1H,³JHH = 5.2 Hz, 3.7 Hz, 3’-H); 4.53 (t, 1H,³JHH = 2.5 Hz, 4’’-H); 4.46 (t, 1H,³JHH = 5.1 Hz, 2’’-H); 4.43 (dd, 1H,³JHH = 5.1 Hz, 2.5 Hz, 3’’-H); 4.43-4.40 (m, 1H, 4’-H); 4.31 (ddd, 2H, ²JHH

= 11.9 Hz,³JHH = 4.6 Hz, 2.6 Hz, 5’’-CH2); 4.27-4.20 (m, 2H, 5’- CH2).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 20.7 Hz; R-PO3-O-PO3-R’); -11.7 (d, 1P,

2JPP = 20.7 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C23H28N7O14P2-) found 688.11926, calc. 688.11750, dev. 2.6 ppm.

6-N-(6-Azidohexyl)-NAD⁺ (7)

Compound 7 was obtained as a light yellow salt (58%).

1H NMR (D2O, 400 MHz)  = 9.34 (d, 1H, ⁴JHH = 1.6 Hz, 2‘‘‘-H); 9.17 (dt, 1H, ³JHH = 6.3 Hz,

4JHH = 1.3 Hz, 6‘‘‘-H); 8.84 (dt, 1H, ³JHH = 8.1 Hz, ⁴JHH = 1.3 Hz, 4‘‘‘-H); 8.39 (s, 1H, 8-H); 8.21 (dd, 1H, ³JHH = 8.1 Hz, 6.2 Hz, 5‘‘‘-H); 8.14 (s, 1H, 2-H); 6.08 (d, 1H, ³JHH = 5.5 Hz, 1’’-H);

6.04 (d, 1H, ³JHH = 6.0 Hz, 1’-H); 4.77 (s, 1H, 2’-H); 4.56 (q, 1H,³JHH = 2.6 Hz, 4‘‘-H);

4.54-Chemical Synthesis

4.50 (m, 1H, 3‘-H); 4.52 (t, 1H, ³JHH = 5.1 Hz, 2‘‘-H); 4.46 (dd, 1H, ³JHH = 5.1 Hz, 2.7 Hz, 3‘‘-H); 4.42-4.39 (m, 1H, 4‘-H); 4.42-4.35 (m, 1H, 5‘‘-CH2-a); 4.32-4.18 (m, 3H, 5‘-CH2, 5‘‘-CH2-b); 3.60-3.44 (br, 2H, -CH2NH-); 3.30 (t, ³JHH = 6.8 Hz, -CH2N3); 1.68 (p, 2H, ³JHH = 7.0 Hz, -CH2-CH2NH-); 1.59 (p, 2H, ³JHH = 6.8 Hz, -CH2-CH2N3); 1.50-1.36 (m, 4H, -CH2-CH2-).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 22.3 Hz; R-PO3-O-PO3-R’); -11.6 (d, 1P,

2JPP = 22.3 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-NEt3-H]^- (C27H37O14N10P2-) found 787.19603, calc. 787.19605, dev.

0.1 ppm.

6-N-(5-Hexenyl)-NAD⁺ (8)

Compound 8 was obtained as a light yellow salt (23%).

1H NMR (D2O, 400 MHz)  = 9.33 (s, 1H, 2‘‘‘-H); 9.16 (d, 1H, ³JHH = 6.3 Hz, 6‘‘‘-H); 8.83 (d, 1H, ³JHH = 8.1 Hz, 4‘‘‘-H); 8.40 (s, 1H, 8-H); 8.20 (dd, 1H, ³JHH = 8.1 Hz, 6.3 Hz, 5‘‘‘-H); 8.18 (s, 1H, 2-H); 6.07 (d, 1H, ³JHH = 6.0 Hz, 1‘‘-H); 6.05 (d, 1H, ³JHH = 6.5 Hz, 1’-H); 5.94 (ddt, 1H,

3JHH = 17.3 Hz, 10.1 Hz, 6.7 Hz, -CH=CH2); 5.09 (d, 1H, ³JHH = 17.7 Hz, -CH=CH2); 5.02 (d, 1H, ³JHH = 10.1 Hz, -CH=CH2); 2’-H below water; 4.58-55 (m, 1H, 4’’-H); 4.55-4.53 (m, 1H, 3’-H); 4.52 (t, 1H, ³JHH = 5.4 Hz, 2’’-H); 4.46 (dd, 1H,³JHH = 5.0 Hz, 2.7 Hz, 3’’-H); 4.43-4.39 (m, 1H, 4’-H); 4.43-4.36 (m, 1H, 5’’-CH2-a); 4.32-4.20 (m, 2H, 5’’-CH2-b); 4.29-4.20 (m, 2H, 5’-CH2); 3.62-3.52 (m, 2H, -CH2NH-); 2.15 (q, 2H, ³JHH = 6.8 Hz, -CH2-CH=CH2); 1.73 (p, 2H,

3JHH = 7.2 Hz, -CH2-CH2NH-); 1.54 (p, 2H, ³JHH = 7.6 Hz, -CH2-).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 21.8 Hz; R-PO3-O-PO3-R’); -11.7 (d, 1P,

2JPP = 21.9 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C27H36N7O14P2-) found 744.17988, calc. 744.18010, dev. 3.0 ppm.

2-DIBAC-NAD⁺ (9)

Compound 9 was obtained as a beige salt (38%).

1H NMR (D2O, 400 MHz)  = 9.29 (s, 1H, 2‘‘‘-H); 9.05 (dd, 1H, ³JHH = 5.2 Hz,⁴JHH = 1.4 Hz, 6‘‘‘-H); 8.86 (dt, 1H, ³JHH = 8.2 Hz, ⁴JHH = 1.6 Hz, 4‘‘‘-H); 8.46 (2s, 1H, 8-H); 8.20 (dd, 1H, ³JHH

= 8.3 Hz, 6.3 Hz, 5‘‘‘-H); 7.50 (d, 1H, ³JHH = 7.6 Hz, arom. CH); 7.41-7.15 (m, 6H, arom. CH);

7.08 (2d, 1H, ³JHH = 7.5 Hz, arom. CH); 5.98 (d, 1H, ³JHH = 4.5 Hz, 1‘‘-H); 5.91 (2d, 1H, ³JHH = 5.4 Hz, 1’-H); 4.89 (d, 1H, ²JHH = 14.4 Hz, N-CH2-Ph-a); 4.67 (2t, 1H, ³JHH = 5.4 Hz, 3.6 Hz, 2’-H); 4.54-4.50 (m, 1H, 4’’-H); 4.50-4.46 (m, 1H, 3’-H); 4.46-4.40 (m, 2H, 2‘‘-H, 3’’-H); 4.40-4.33 (m, 1H, 4’-H); 4.31-4.18 (m, 4H, 5‘‘-CH2, 5‘-CH2); 3.48 (d, 1H, ²JHH = 14.6 Hz, N-CH2-Ph-b); 3.11 (t, 2H, ³JHH = 6.3 Hz, -CH2-CH2-NHCO); 2.48 (dd, 1H, ²JHH = 15.6 Hz, ³JHH

= 8.0 Hz, -NHCO-CH2-CH2-CON-a); 2.28 (t, 2H, ³JHH = 7.4 Hz, -C≡C-CH2-CH2-); 2.25-2.13 (m, 2H, -CONH-CH2-CH2-CON-); 2.02 (dd, 1H, ²JHH = 15.7 Hz, ³JHH = 7.6 Hz, -NHCO-CH2-CH2-CON-b); 1.56 (p, 2H, ³JHH = 7.7 Hz, CH2-CH2-CH2).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 20.3 Hz; R-PO3-O-PO3-R’); -11.6 (d, 1P,

2JPP = 20.7 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C45H46N9O16P2-) found 1030.25028, calc. 1030.25432, dev. 3.9 ppm.

2-Cyclopropenyl-NAD⁺ (10)

Compound 10 was obtained as a light yellow salt (35%).

1H NMR (D2O, 600 MHz)  = 9.33 (s, 1H, 2‘‘‘-H); 9.05 (dd, 1H, ³JHH = 6.3 Hz,⁴JHH = 1.4 Hz, 6‘‘‘-H); 8.92 (dt, 1H, ³JHH = 8.1 Hz, ⁴JHH = 1.5 Hz, 4‘‘‘-H); 8.48 (s, 1H, 8-H); 8.23 (dd, 1H, ³JHH

= 8.2 Hz, 6.2 Hz, 5‘‘‘-H); 6.55 (s, 1H, CH=C-CH3); 6.00 (d, 1H, ³JHH = 5.1 Hz, 1‘‘-H); 5.97 (d, 1H, ³JHH = 5.9 Hz, 1’-H); 4.54 (dd, 1H, ³JHH = 5.3 Hz, 3.6 Hz, 2’-H); 4.50 (t, 1H, ³JHH = 2.5 Hz, 4‘‘-H); 4.44 (t, 1H, ³JHH = 5.1 Hz, 2‘‘-H); 4.42 (dd, 1H, ³JHH = 5.3 Hz, 2.4 Hz, 3‘‘-H); 4.43-4.40 (m, 1H, 3‘-H); 4.32-4.17 (m, 5H, 4‘-H, 5‘‘-CH2, 5‘-CH2); 3.89 (d, 2H, ²JHH = 11.6 Hz, O-CH2 -CH); 3.32 (t, 2H, ³JHH = 7.0 Hz, CH2-CH2-NHCO); 2.54 (t, 2H, ³JHH = 6.8 Hz, C≡C-CH2-CH2-);

2.05 (s, 3H, CH=C-CH3); 1.85 (p, 2H, ³JHH = 6.6 Hz, CH2-CH2-CH2); 1.50 (s, 1H, O-CH2-CH-).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 20.6 Hz; R-PO3-O-PO3-R’); -11.6 (d, 1P,

2JPP = 20.7 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C32H39N8O16P2-) found 853.19314, calc. 853.19647, dev. 3.9 ppm.

Chemical Synthesis 2-TMR-NAD⁺ (11)

Compound 11 was obtained as a dark purple salt (42%).

1H NMR (D2O, 400 MHz) = 9.34 (s, 1H, 2‘‘‘-H); 9.09 (dd, 1H, ³JHH = 6.3 Hz, 6‘‘‘-H); 8.92 (d, 1H, ³JHH = 8.1 Hz, 4‘‘‘-H); 8.44 (s, 1H, 8-H); 8.32 (s, 1H, 6**-H); 8.25 (t, 1H, ³JHH = 7.2 Hz, 5‘‘‘-H); 8.09 (d, 1H, ³JHH = 8.2 Hz, 4**-H); 7.54 (d, 1H, ³JHH = 8.1 Hz, 3**-H); 7.02 (2d, 2H,

3JHH = 10.1 Hz, 1*-H, 8*-H); 6.79 (2d, 2H, ³JHH = 10.2 Hz, 2*-H, 7*-H); 6.48 (2s, 2H, 4*-H, 5*-H); 6.00 (d, 1H, ³JHH = 5.2 Hz, 1‘‘-H); 5.87 (2d, 1H, ³JHH = 5.6 Hz, 1’-H); 4.69 (t, 1H, ³JHH = 5.5 Hz, 2’-H); 4.52 (t, 1H, ³JHH = 4.3 Hz, 3’-H); 4.51-4.48 (m, 1H, 4’’-H); 4.46 (t, 1H,³JHH = 5.2 Hz, 2‘‘-H); 4.43 (dd, 1H,³JHH = 4.4 Hz, 2.7 Hz, 3‘‘-H); 4.37-4.34 (m, 1H, 4’-H); 4.33-4.19 (m, 4H, 5‘‘-CH2, 5‘-CH2); 3.52 (t, 2H, ³JHH = 6.2 Hz, -CH2-CH2-NHCO-); 3.17 (s, 12H, N(CH3)2); 2.43 (t, 2H, ³JHH = 5.8 Hz, -C≡C-CH2-CH2-); 1.90 (p, 2H, ³JHH = 5.9 Hz, -CH2-CH2-CH2-).

31P NMR (D2O, 162 MHz)  = -11.3 (d, 1P, ²JPP = 20.4 Hz; R-PO3-O-PO3-R’); -11.6 (d, 1P,

2JPP = 20.6 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C51H53N10O18P2-) found 1155.29687, calc. 1155.30200, dev. 4.4 ppm.

6-TMR-NAD⁺(12)

Compound 12 was obtained as a dark purple salt (50%).

1H NMR (D2O, 400 MHz)  = 9.35 (s, 1H, 2‘‘‘-H); 9.19 (dd, 1H, ³JHH = 6.3 Hz, 6‘‘‘-H); 8.84 (d, 1H, ³JHH = 8.1 Hz, 4‘‘‘-H); 8.35 (s, 1H, 8-H); 8.30 (s, 1H, 2-H); 8.22 (t, 1H, ³JHH = 7.2 Hz, 5’’’-H); 8.02 (s, 1H, 6**-H); 7.97 (d, 1H, ³JHH = 7.8 Hz, 4**-H); 7.33 (d, 1H, ³JHH = 7.8 Hz, 3**-H); 7.07 (2d, 2H, ³JHH = 8.1 Hz, 2*-H, 7*-H); 6.74 (2s, 2H, 2*-H, 7*-H); 6.45 (2s, 2H, 4*-H,

5*-H); 6.05 (d, 1H, ³JHH = 5.3 Hz, 1’’-H); 5.97 (d, 1H, ³JHH = 5.8 Hz, 1’-H); 4.72 (t, 1H, ³JHH = 5.0 Hz, 2’-H); 4.51 (t, 1H, ³JHH = 4.5 Hz, 2’’-H); 4.53-4.49 (m, 2H, 3’-H, 4’’-H); 4.45 (dd, 1H,

3JHH = 4.3 Hz, 2.7 Hz, 3‘‘-H); 4.39-4.35 (m, 2H, 4’-H, 5‘‘-CH2a); 4.30-4.20 (m, 3H, 5‘‘-CH2b, 5‘-CH2); 3.49-3.36 (br, 4H, CONH-CH2, -CH2-NH); 3.12 (s, 12H, N(CH3)2); 1.67-1.53 (m, 4H, CONH-CH2-CH2, -CH2-CH2-NH); 1.44-1.31 (m, 4H, -CH2-CH2-).

31P NMR (D2O, 162 MHz)  = -11.2 (d, 1P, ²JPP = 20.2 Hz; R-PO3-O-PO3-R’); -11.6 (d, 1P,

2JPP = 20.7 Hz; R-PO3-O-PO3-R’).

HR MS (m/z) [M-H]^- (C52H59N10O18P2-) found 1173.34842, calc. 1173.34895, dev. 0.5 ppm.

6-BODIPY Fl-NAD⁺(76)

Compound 76 adsorbed strongly on anion exchange material and was inseparable from AMP on HPLC.

HR MS (m/z) [M-H]^- (C41H53BF2N10O15P2-) found 1036.32112, calc. 1036.32408, dev.

2.3 ppm.

9.3.4 Cyclooctynes and Tetrazines

1-Fluorocyclooct-2-yne-1-carboxylic acid (MFCO, 24)

Compound 24 was prepared as previously described.^{[126, 192]}

Rf 0.34 (CH2Cl2/MeOH/NH3 = 10:1:0.1).

1H NMR (CDCl3, 400 MHz)  = 10.26 (br, 1H, 1-COOH); 2.50-2.23 (m, 4H), 2.15-1.81, (m, 4H), 1.82-1.62 (m, 1H), 1.53-1.44 (m, 1H).

13C NMR (CDCl3, 100 MHz)  = 172.8 (d, ²JCF = 29.3 Hz, CF-COOH); 109.5 (d, ³JCF = 10.4 Hz, 3-C); 91.7 (d, ¹JCF = 186.9 Hz, 1-C) 86.3 (d, ²JCF = 32.3 Hz, 2-C); 46.4 (d, ¹JCF = 25.0 Hz, 8-C); 33.9 (5-C); 29.2 (6-C); 25.6 (7-C); 20.7 (4-C).

19F NMR (CDCl3, 376 MHz)  = -146.7 (CFCO2H).

Chemical Synthesis

HR MS (m/z) [M+H]⁺ (C9H12FO2+) found 171.08158, calc. 171.08093, dev. 3.8 ppm.

Pentafluorophenyl 1-fluorocyclooct-2-yne-1-carboxylate (MFCO PFP ester)

Compound was prepared as previously described.^[131]

Rf 0.40 (CH2Cl2/MeOH/NH3 = 10:1:0.1).

1H NMR (CDCl3, 600 MHz)  = 2.60-2.30 (m, 4H), 2.13-1.89 (m, 4H), 1.86-1.76 (m, 1H), 1.58-1.49 (m, 1H).

13C NMR (CDCl3, 150 MHz) δ = 164.9 (d, ²JCF = 31.3 Hz, CF-CO); 141.1 (dd, ¹JCF = 253.2 Hz, ²JCF = 12.6 Hz, o-CF); 140.0 (dt, ¹JCF = 254.2 Hz, ²JCF = 13.6 Hz, p-CF); 138.1 (dt, ¹JCF = 252.6 Hz, ²JCF = 13.8 Hz, m-CF); 124.7 (t, ²JCF = 14.4, O-Cq); 110.6 (d, ³JCF = 10.0 Hz, 3-C);

91.6 (d, ¹JCF = 189.4 Hz, 1-C); 85.4 (d, ²JCF = 31.8 Hz, 2-C); 46.7 (d, ³JCF = 24.8 Hz, 8-C);

33.9 (5-C); 29.2 (6-C); 25.5 (7-C); 20.7 (4-C).

19F NMR (CDCl3, 376 MHz)  = -146.4 (s, 1F, CFCO2CH3); -152.4 (d, 2F, ³JFF = 17.0 Hz, o-CF); -156.7 (t, 1F, ³JFF = 21.7 Hz, p-CF); -161.7 (dd, 2F, ³JFF = 21.7 Hz, 17.2 Hz, m-CF).

N-(4-aminobutyl)-1-fluorocyclooct-2-yne-1-carboxamide (25)

Compound 25 was prepared as previously described and the crude material was used without further purification.^[131]

Rf 0.30 (CH2Cl2/MeOH/NH3 = 10:1:0.1).

1H NMR (CD3OD, 400 MHz)  = 3.30-3.21 (m, 2H, 1’-H); 2.36-2.27 (m, 2H, 4’-H); 1.69-1.57 (m, 4H, 2’-H, 3’-H); 2.47-1.33 (m, 10H, 4-H, 5-H, 6-H, 7-H, 8-H).

13C NMR (CD3OD, 100 MHz)  = 170.9 (d, ²JCF = 24.8 Hz, CF-CO2NH); 109.9 (d, ³JCF = 8.9 Hz, 3-C); 95.1 (d, ¹JCF = 187.5 Hz, 1-C) 88.4 (d, ²JCF = 31.7 Hz, 2-C); 47.7 (d, ¹JCF = 25.0 Hz, 8-C); 40.2 (4’-C); 40.0 (1’-C); 35.0 (5-C); 30.1 (6-C); 28.5 (3’-C); 28.1 (2’-C); 26.8 (7-C); 21.0 (4-C).

19F NMR (CD3OD, 376 MHz)  = -147.7 (CFCO2NH).

HR MS (m/z) [M+H]⁺ (C13H22FN2O⁺) found 241.17175, calc. 241.17107, dev. 2.8 ppm.

N-[(1R,8S,9s )-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (BCN, 26)

This compound was commercially obtained from Sigma Aldrich and characterised for easier assignments of conjugation products.

Rf 0.37 (CH2Cl2/MeOH = 5:1).

1H NMR (CDCl3, 100 MHz)  = 5.34 (s, 1H, CONH); 4.15 (d, 2H, ³JHH = 8.1 Hz, 10-H); 3.62 (s, 4H, 4’-H, 5’-H); 3.57 (t, 2H, ³JHH = 5.1 Hz, 2’-H); 3.53 (t, 2H, ³JHH = 5.2 Hz, 7’-H); 3.38 (q, 2H, ³JHH = 5.3 Hz, 1’-H); 2.89 (t, 2H, ³JHH = 5.2 Hz, 8’-H); 2.38-2.12 (m, 6H, 2-Ha, 3-H, 6-H, 7-Ha); 1.65 (s, 2H, NH2); 1.60 (m, 2H, 2-Hb, 7-Hb); 1.36 (p, 1H, ³JHH = 8.6 Hz, 9-H); 0.94 (t, 2H, ³JHH = 8.8 Hz, 1-H, 8-H).

13C NMR (CDCl3, 100 MHz)  = 157.0 (CO); 99.0 (4-C, 5-C); 73.5 (7’-C); 70.5 (2’-C); 70.4, 70.3 (4’-C, 5’-C); 62.9 (10-C); 41.9 (8’-C); 41.0 (1’-C); 29.2 (2-C, 7-C); 21.6 (3-C, 6-C); 20.3 (1-C, 8-C); 18.0 (9-C).

HR MS (m/z) [M+H]⁺ (C17H29N2O4+) found 325.21210, calc. 325.21218, dev. 0.3 pm.

N-(6-Aminohexanoyl)-5,6-dihydro-11,12-didehydrodibenzo[b,f]-azocine (ADIBO, 27)

Compound 27 was prepared as previously published.^{[127, 133]}

Rf 0.39t (CH2Cl2:MeOH = 5:1).

1H NMR (CDCl3, 400 MHz)  = 7.54 (d, 1H, ³JHH = 7.3 Hz, 7-H); 7.33-7.26 (m, 3H, CHarom);

7.26 – 7.18 (m, 2H, CHarom); 7.18-7.12 (m, 2H, CHarom); 6.64 (br, 2H, 6‘-NH2); 5.02 (d, 1H,

2JHH = 13.9 Hz, 6-Ha); 3.57 (d, 1H, ²JHH = 13.8 Hz, 6-Hb); 2.64-2.52 (m, 2H, 6‘-H), 2.07 (dt,1H, ²JHH = 15.0, ³JHH = 7.1 Hz, 2‘-Ha); 1.77 (dt,1H, ²JHH = 15.0, ³JHH =7.1 Hz, 2‘-Hb); 1.31 (p, 1H, ³JHH = 7.6 Hz, 5‘-H), 1.28-1.14 (m, 2H, 3‘-H); 1.05-0.80 (m, 2H, 4‘-H).

13C NMR (CDCl3, 100 MHz)  = 173.6 (1‘-C); 151.6 (4a-C); 148.0 (6a-C); 132.2 (7-C);

129.0, 128.6, 128.3, 128.3, 127.8, 127.1, 125.5 (CHarom); 123.1 (10a-C); 122.5 (12a-C); 115.0 (12-C); 107.9 (11-C); 55.5 (6-C); 39.4 (6‘-C); 34.4 (2‘-C); 26.8 (5‘-C); 25.4 (4‘-C); 24.4 (3‘-C).

Chemical Synthesis HR MS (m/z) [M+H]⁺ (C22H22N2O⁺) found 319.18011, calc. 319.18049, dev. 1.2 ppm.

5-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoic acid (DIBAC, 50)

Compound 50 was prepared as previously published.^[148]

Rf 0.29 (50:1 CH2Cl2/MeOH).

1H NMR (DMSO-d6, 400 MHz)  = 11.98 (br, 1H, COOH); 7.65 (d, 1H, ³JHH = 7.4 Hz, 4-H);

7.62 (d, 1H, ³JHH = 7.2 Hz, 7-H); 7.59-7.42 (m, 3H, 1-H, 2-H, 3-H); 7.38, 7.34 (2t, 2H, ³JHH = 7.2 Hz, 8-H, 9-H); 7.29 (d, 1H, ³JHH = 7.2 Hz, 10-H); 5.03 (d, 1H, ²JHH = 14.0 Hz, 6-CH2-a);

3.62 (d, 1H, ²JHH = 14.0 Hz, 6-CH2-b); 2.59 (dt, 1H, ²JHH = 16.7 Hz,³JHH = 6.8 Hz, 2‘-CH2-a);

2.30 (dt, 1H, ²JHH = 17.2 Hz,³JHH = 6.9 Hz, 3‘-CH2-a); 2.19 (dt, 1H, ²JHH = 16.9 Hz,³JHH = 6.4 Hz, 3‘-CH2-b); 1.78 (dt, 1H, ²JHH = 16.3 Hz,³JHH = 6.2 Hz, 2‘-CH2-b).

13C NMR (DMSO-d6, 100 MHz)  = 173.5 (COOH); 170.7 (CON); 151.4 (4a-C); 148.4 (6a-C); 132.4 (7-C); 129.6 (4-C); 128.9, 128.2, 128.0, 127.7, 126.8, 125.1 (arom. CH); 122.5 (10a-C); 121.6 (12a-C); 114.3 (12-C); 108.0 (11-C); 55.0 (6-CH2); 29.3, 29.0 (2’-C, 3’-C).

HR MS (m/z) [M+H]⁺ (C19H16NO3+) found 306.10988, calc. 306.11247 dev. 8.5 ppm.

DIBAC NHS ester (51)

Compound 51 was prepared as previously published.^[147]

Rf 0.59 (50:1 CH2Cl2/MeOH).

1H NMR (CDCl3, 400 MHz)  = 7.69 (d, 1H, ³JHH = 7.5 Hz, 4-H); 7.44-7.38 (m, 4H, arom.

CH); 7.36, 7.31 (2t, 2H, ³JHH = 7.6 Hz, 8-H, 9-H); 7.25 (d, 1H, ³JHH = 7.5 Hz, 10-H); 5.18 (d, 1H, ²JHH = 13.9 Hz, 6-CH2-a); 3.69 (d, 1H, ²JHH = 13.8 Hz, 6-CH2-b); 2.97 (dt, 1H, ²JHH = 17.3 Hz,³JHH = 7.6 Hz, 2‘-CH2-a); 2.82 (dt, 1H, ²JHH = 17.1 Hz,³JHH = 7.7 Hz, 3‘-CH2-a); 2.78 (s, 4H, CH2-CONO); 2.64 (ddd, 1H, ²JHH = 17.4 Hz,³JHH = 7.7 Hz, 5.4 Hz, 2‘-CH2-b); 2.08 (ddd, 1H, ²JHH = 16.8 Hz,³JHH = 7.9 Hz, 5.4 Hz, 3‘-CH2-b).

13C NMR (CDCl3, 100 MHz)  = 170.4 (COON); 169.0 (CH2CON); 168.4 (CON); 151.2 (4a-C); 148.0 (6a-C); 132.4 (7-C); 129.2 (4-C); 128.8, 128.5 (2C), 128.0, 127.4, 125.7 (arom.

CH); 123.3 (10a-C); 122.9 (12a-C); 115.2 (12-C); 107.7 (11-C); 55.7 (6-CH2); 29.4 (2’-C);

26.6 (3’-C); 25.6 (2C, CH2CONO).

(4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (Tz, 28)

Compound 28 was prepared similar to previously published.^[128]

To a mixture of 4-(aminomethyl)benzonitrile hydrochloride (2.50 g, 14.8 mmol, 1 eq.) and formamidine acetate (7.72 g, 74.1 mmol, 5 eq.) was added hydrazine monohydrate (80%

solution, 18.5 mL, 304 mmol, 20 eq.) and the resulting solution was heated at 80 °C for 30 min under a nitrogen atmosphere. After cooling to room temperature, an aqueous solution of NaNO2 (5.11 g in 7.4 mL, 74.1 mmol, 5 eq.) was added to the reaction mixture. While stirring at 0 °C, 1 M HCl solution was added slowly until no more gas evolution could be observed and the solution reached a pH of 3. During the addition, the solution turned bright pink and large amounts of nitrogen gases evolved.

Subsequently, the solution was saturated with solid NaCl and afterwards with solid NaHCO3, followed by quick extractions of the aqueous phase with CH2Cl2 until no more pink colour is extracted into the organic layer (20x 150 mL). TFA (2 mL) is added to the organic layer and the solvents were removed under reduced pressure. The crude product was purified by MPLC on RP18 cartridges (30 g) in five separate injections using a gradient with H2O (0.1% TFA) and MeCN (0.1% TFA) from 0 to 25% in 60 min. All fractions containing the product were combined, evaporated and re-dissolved in H2O (15 mL). The mixture was loaded again on the RP18 cartridge, washed with H2O (0.1% HCl, 200 mL) and eluted with H2O:MeCN (1:1). The fractions were combined, evaporated and dried in vacuo.

The product was isolated as a pink solid in its hydrochloride salt (346 mg, 1.55 mmol, 10%).

Rf 0.31 (CH2Cl2/MeOH = 5:1).

m.p. >211 °C gas evolution and decomp.

1H NMR (D2O, 400 MHz)  = 10.24 (s, 1H, CH-Tetrazine); 8.49 (d, 2H, ³JHH = 8.5 Hz, m-Ph); 7.74 (d, 2H, ³JHH = 8.4 Hz, o-Ph); 4.37 (s, 2H, Ph-CH2-NH2).

Chemical Synthesis

13C NMR (D2O, 100 MHz)  = 166.1 (1C, Cq-Tetrazine); 157.5 (1C, CH-Tetrazine); 137.6 (1C, Cq-p-Ph); 131.9 (1C, Cq-CH2-NH2); 129.8 (2C, o-Ph); 128.9 (2C, m-Ph); 42.7 (1C, Ph-CH2-NH2).

HR MS (m/z) [M+H]⁺ (C9H10N5+) found 188.09343, calc. 188.09307, dev. 1.9 ppm.

9.3.5 Dyes

4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene 3-propio-nic acid (BODIPY® Fl, 32)

Compound 32 was prepared by Anke Gerull as previously described.^[140]

Rf 0.51 (CH2Cl2/MeOH = 10:1).

m.p. 184-186 °C.

1H NMR (CDCl3, 400 MHz)  = 7.09 (s, 1H, 8-H); 6.88 (d, 1H, ³JHH = 4.0 Hz, 1-H); 6.29 (d, 1H, ³JHH = 4.0 Hz, 2-H); 6.12 (s, 1H, 6-H); 3.30 (t, 2H, ³JHH = 7.6 Hz, 3’-H); 2.83 (t, 2H, ³JHH = 7.6 Hz, 2’-H); 2.57 (s, 3H, 5-CH3); 2.25 (s, 3H, 7-CH3).

13C NMR (CDCl3, 100 MHz)  = 176.8 (1’-C); 160.9 (5-C); 156.7 (3-C); 144.1 (7-C); 135.5 (7a-C); 133.5 (8a-C); 128.1 (1-C); 124.0 (8-C); 120.7 (6-C); 116.8 (2-C); 33.0 (2’-C); 23.8 (3’-C); 15.1 (5-CH3); 11.5 (7-CH3).

19F NMR (CDCl3, 376 MHz)  = -145.3 (d, ²JFF = 33.1 Hz, BF); -145.5 (d, ²JFF = 33.2 Hz, BF).

HR MS (m/z) [M-H]^- (C14H14BF2N2O2-) found 291.11302, calc. 291.11244, dev. 2.0 ppm.

BODIPY® Fl NHS ester (70)

BOPDIPY Fl acid (32, 292 mg, 1.00 mmol, 1.0 eq.), N,N′-disuccinimidyl carbonate (512 mg, 2.00 mmol, 2.0 eq.) were dissolved in dry DMF (25 mL). Then, 4-(dimethylamino)pyridine in THF (0.5 M, 4.4 mL, 2.20 mmol, 2.2 eq.) was added. After stirring for two hours at room temperature in the dark, the solution was diluted with CH2Cl2 (300 mL) and washed with NaCl solution (1:1=water:brine, 3x100 mL). The organic phase was dried over Na2SO4 and the

solvents were removed under reduced pressure. The crude was pre-purified by short column chromatography on silica using CH2Cl2 as elutant.

The product was isolated as dark green solid (377 mg, 0.97 mmol, 97%).

Rf 0.80 (CH2Cl2/MeOH = 20:1).

1H NMR (CDCl3, 400 MHz)  = 7.09 (s, 1H, 8-H); 6.89 (d, 1H, ³JHH = 3.9 Hz, 1-H); 6.34 (d, 1H, ³JHH = 4.0 Hz, 2-H); 6.12 (s, 1H, 6-H); 3.38 (t, 2H, ³JHH = 7.4 Hz, 3’-H); 3.08 (t, 2H, ³JHH = 7.3 Hz, 2’-H); 2.83 (s, 4H, CH2CONO); 2.57 (s, 3H, 5-CH3); 2.25 (s, 3H, 7-CH3).

13C NMR (CDCl3, 150 MHz)  = 168.0 (CH2CONO); 162.7 (1’-C); 161.4 (5-C); 154.8 (3-C);

144.5 (7-C); 135.7 (7a-C); 133.4 (8a-C); 128.0 (1-C); 124.2 (8-C); 120.9 (6-C); 116.9 (2-C);

30.5 (2’-C); 25.7 (CH2CONO); 23.5 (3’-C); 15.2 (5-CH3); 11.5 (7-CH3).

19F NMR (CDCl3, 376 MHz)  = -145.1 (d, ²JFF = 33.1 Hz, BF); -145.3 (d, ²JFF = 33.1 Hz, BF).

4-Dimethylamino-2-hydroxy-1‘,5‘(4’)-dicarboxy-benzophenone (37)

Compounds 37 was prepared as previously described.^[142]

3-Dimethylaminophenol (6.02 g, 43.8 mmol, 1.0 eq.) in toluene (130 mL) was stirred at 60 °C and benzene-1,2,4-tricarboxylic anhydride (10.08 g, 52.5 mmol, 1.2 eq.) was added.

After stirring for 24 hours at 120 °C, the cooled mixture was filtered off and washed with toluene (3x 25 mL). The obtained purple crystals were dissolved in MeOH (150 mL) and stirred at 70 °C for 10 min. Acetic acid (84 mL) was added and the solvent was removed under reduced pressure. The obtained solid was again dissolved in MeOH (120 ml), stirred at 70 °C for 2 hours and crystallised overnight at 4 °C. The precipitate was filtered off and washed with cold MeOH (25 mL) to give pure 5’-isomer (37 b, 2.20 g, 6.68 mmol, 15%) as a purple solid. The mother liquid was evaporated to dryness, refluxed for one hour with acetic acid (70 mL), and stored at room temperature overnight. Crystals were filtered off and dried in vacuo at 120 °C to give a mixture of isomers. This was re-crystallised twice from acetic acid to give pure 4’-isomer as a purple crystalline solid (37 a,0.45 g, 1.37 mmol, 3%

)

4’-Isomer (37 a)

1H NMR (DMSO-d6, 400 MHz)  = 13.18 (s, 2H, COOH); 12.39 (s, 1H, 2-OH); 8.14 (dd, 1H,

3JHH = 8.1 Hz,⁴JHH = 1.7 Hz, 5’-H); 8.06 (d, 1H, ³JHH = 8.1 Hz, 6’-H); 7.83 (d, 1H, ⁴JHH =

Chemical Synthesis

1.6 Hz, 3’-H); 6.86 (d, 1H, ³JHH = 9.1 Hz, 6-H); 6.23 (dd, 1H, ⁴JHH = 9.2 Hz,⁴JHH = 2.5 Hz, 5-H); 6.12 (d, 1H, ⁴JHH = 2.5 Hz, 3-H); 3.01 (s, 6H, 4-N(CH3)2).

13C NMR (DMSO-d6, 100 MHz)  = 197.3 (CO); 166.5 (1’-COOH); 166.1 (4’-COOH); 164.4 (2-C); 155.9 (4-C); 140.1 (2‘-C); 133.9 (6-C); 133.7, 133.6 (1’-C, 4’-C); 130.4 (6‘-C); 130.2 (5‘-C); 128.2 (3‘-C); 109.4 (1-C); 104.5 (5-C); 97.1 (3-C); 39.6 (4-N(CH3)2).

5’-Isomer (37 b)

1H NMR (DMSO-d6, 400 MHz)  = 13.36 (s, 2H, COOH); 12.36 (s, 1H, 2-OH); 8.49 (d, 1H,

4JHH = 1.7 Hz, 6’-H); 8.20 (dd, 1H, ³JHH = 7.9 Hz,⁴JHH = 1.7 Hz, 4’-H); 7.52 (d, 1H, ³JHH = 7.9 Hz, 3’-H); 6.81 (d, 1H, ³JHH = 9.1 Hz, 6-H); 6.21 (dd, 1H, ⁴JHH = 9.1 Hz,⁴JHH = 2.5 Hz, 5-H); 6.11 (d, 1H, ⁴JHH = 2.5 Hz, 3-H); 3.01 (s, 6H, 4-N(CH3)2).

13C NMR (DMSO-d6, 100 MHz)  = 197.6 (CO); 166.1 (2C, COOH); 164.2 (2-C); 155.8 (4-C); 143.7 (2‘-C); 133.8 (6-C); 132.6 (4‘-C); 131.6 (5‘-C); 130.7 (6‘-C); 130.0 (1‘-C); 128.2 (3‘-C); 109.5 (1-C); 104.5 (5-C); 97.0 (3-C); 39.6 (4-N(CH3)2).

5’-Carboxy-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate (TMR, 33)

Compound 33 was prepared as previously described.^[142]

5’-Benzophenone isomer (2.00 g, 6.07 mmol, 1.0 eq.) and 3-dimethylaminophenol (1.09 g, 7.94 mmol, 1.3 eq.) were dissolved in DMF (50 mL). Trimethylsilyl-polyphosphate (5.73 g, 29.8 mmol 5.0 eq.) in CHCl3 (20 mL) was added and the mixture was stirred at 65 °C for 3 hours. After removing the solvents under reduced pressure, the solid was treated with aqueous NaOH (5%, 50 mL) overnight at room temperature. After dilution with water

Im Dokument Synthesis and Characterisation of NAD<sup>+</sup> Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation (Seite 85-131)