4.1. Chemistry

All reactions described were performed under an N₂ atmosphere and at ambient temperature, unless stated otherwise. All reagents and solvents were purchased from Sigma-Aldrich (Diegem, Belgium), Fisher Scientific (Merelbeke, Belgium), TCI Europe (Zwijndrecht, Belgium) or Apollo Scientific (Bredbury, Stockport, UK), and used as received. NMR solvents were acquired from Eurisotop (Saint-Aubin, France). ESI-HRMS spectra were measured with a Waters LCT Premier XE Mass spectrometer calibrated using leu-enkephalin as an external standard. LC-MS analyses were carried out on a Waters AutoPurification System equipped with PDA and ESI-MS detection and using a Waters CORTECS C18 Column (4.6 × 100 mm, 2.7 μm) and a water/acetonitrile/formic acid linear gradient system at a flow rate of 1.44 mL min⁻¹.

General procedure 1: Copper mediated azide-alkyne cycloaddition. To a solution of the azido-linker modifies MTEP analogues [19] (1.0 eq.) in dimethylformamide (0.1 M) was added the alkyne-modified BODIPY-red (1.5 eq.), sodium ascorbate (1.0 eq., 0.5 M), CuSO₄ (0.2 eq., 0.05 M), triethylamine (3.0 eq.) and a catalytic amount of tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine. The reaction mixture was stirred overnight at room temperature in the dark under an argon atmosphere. The solvent was evaporated under reduced pressure and the residue was partioned between water and CH₂Cl₂. The organic fraction was washed with brine and dried over Na₂SO₄. The crude compound was purified by silica gel chromatography (NH₄OH/MeOH/CH₂Cl₂, 1:5:94 v/v/v) to give the final compounds as blue solids.

N-(17-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo [1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15-pentaoxaheptadecyl)-2-((5-((2-methylthiazol-4-yl)ethynyl)pyridin-3-yl)oxy)acetamide (4a)

Blue solid, 48%. LC-HRMS: t_R = 7.99 min (10–100% MeCN, 15 min run), purity 95.4%. HRMS (ESI) m/z: calculated for C₅₄H₆₃BF₂N₈O₉S [M + 2H]²⁺ 524.2244; found 524.2223. Calculated for C₅₄H₆₂BFN₈O₉S [M − F + H]²⁺ 514.2219; found 514.2209.

N-(2-(2-(2-(2-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)-ethyl)-2-(3-((2-methylthiazol-4-yl)ethynyl)phenoxy)acetamide (4b)

Blue solid, 52%. LC-HRMS: t_R = 7.62 min (10–100% MeCN, 15 min run), purity 96.8%. HRMS (ESI) m/z: calculated for C₅₁H₅₆BF₂N₇O₇S [M + 2H]²⁺ 479.7006; found 479.6974. Calculated for C₅₁H₅₅BF₂N₇O₇S [M + H]⁺ 958.3939; found 958.3953.

N-(5-((12-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)dodecyl)(methyl)amino)pentyl)-2-(3-((2-methylthiazol-4-yl)ethynyl)phenoxy)acetamide (4c)

Blue solid, 46%. LC-HRMS: t_R = 9.53 min (10–100% MeCN, 15 min run), purity 85.4%. HRMS (ESI) m/z: calculated for C₆₁H₇₇BF₂N₈O₄S [M + 2H]²⁺ 533.2919; found 533.2914. Calculated for C₆₁H₇₆BFN₈O₄S [M − F + H]²⁺ 523.2893; found 523.2885.

N-(5-((12-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)dodecyl)(methyl)amino)pentyl)-2-((5-((2-methylthiazol-4-yl)ethynyl)pyridin-3-yl)oxy)acetamide (4d)

Blue solid, 39%. LC-HRMS: t_R = 6.86 min (10–100% MeCN, 15 min run), purity 90.8%. HRMS (ESI) m/z: calculated for C₆₀H₇₆BF₂N₉O₄S [M + 2H]²⁺ 533.7895; found 533.7896. Calculated for C₆₀H₇₅BFN₉O₄S [M − F + H]²⁺ 523.7870; found 523.7874.

N-(5-((8-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)octyl)(methyl)amino)pentyl)-2-((5-((2-methylthiazol-4-yl)ethynyl)pyridin-3-yl)oxy)acetamide (4e)

Blue solid, 37%. LC-HRMS: t_R = 5.93 min (10–100% MeCN, 15 min run), purity 82.5%. HRMS (ESI) m/z: calculated for C₅₆H₆₈BF₂N₉O₄S [M + 2H]²⁺ 505.7582; found 505.7570. Calculated for C₅₆H₆₇BFN₉O₄S [M − F + H]²⁺ 495.7557; found 495.7557.

N-(6-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ⁴,5λ⁴-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)hexyl)-2-((5-((2-methylthiazol-4-yl)ethynyl)pyridin-3-yl)oxy)acetamide (4f)

Blue solid, 45%. LC-HRMS: t_R= 7.32 min (10–100% MeCN, 15 min run), purity 81.5 %. HRMS (ESI) m/z: calculated for C₄₈H₅₁BF₂N₈O₄S [M + 2H]²⁺ 442.1902; found 442.1881. Calculated for C₄₈H₅₀BF₂N₈O₄S [M + H]⁺ 883.3731; found 883.3779.