Cyanine 5 (Cy5) dye synthesis

Cy5 with a carboxylic acid as its functional conjugation group (Cy5-COOH) was synthesised on gram-scale using an amended protocol of Korbel et al..^[12]

15.8 g of 2,3,3-trimethylindolenine (100 mmol, 1.2 eq) was added to 15 g of 5-bromovaleric acid (83 mmol, 1 eq) and the reaction was heated, with vigorous stirring, for 20 h at 110°C. The burgundy solid was broken up and washed with 6x 80 mL portions of refluxing ethyl acetate, 2x 80 mL portions of refluxing acetone, and 2x 240 mL portions of refluxing acetone. Filtration of the solid and drying under vacuum afforded 15.4 g (55% yield) of 1 as a pale pink powder. MS[m/z]: MS[calc.]: 260.1645; MS[observed]: 260.1638.

288 mL of acetic anhydride was added to 4 g of 1 (11.8 mmol, 1 eq) and 2.4 g potassium acetate (24.7 mmol, 2.1 eq). The mixture was heated to 110°C and 6.1 g malonaldehydebis(phenylimine) monohydrochloride (23.5 mmol, 2 eq) was added slowly over a period of 45 min (6x 1 g; pink to blue colour change observed). The reaction was maintained at 110°C for an additional hour and then cooled to room temperature. Acetic acid and acetic anhydride were removed by vacuum distillation on the rotary evaporator as their azeotropes with heptane. The crude product was dried over vacuum overnight and used without further purification. To this intermediate 7.79 g of 1,2,3,3-tetramethyl-3H-indolium iodide (25.9 mmol, 2.2 eq), 4.6 g of potassium acetate (47 mmol, 4 eq), 35.2 g of sodium iodide (235.1 mmol, 20 eq) and 288 mL 1-butanol were added and the reaction was kept at 110°C for 2 h. The reaction was then cooled slightly and 1-butanol was removed on the rotary evaporator. The remaining solid was dissolved in CH₂Cl₂ and washed with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to a blue solid. The crude product was purified by flash chromatography using 8% MeOH in CHCl₃ as eluent. The fractions were analysed by ESI-MS and TLC, and appropriate fractions were combined and the solvent removed by rotary evaporation. The solid product was then purified once more by preparative C₁₈-RP-HPLC affording 1.4 g of pure Cy5-COOH (20% yield, >95 % pure). MS[m/z]: MS[calc.]: 469.2855; MS[observed]: 469.8422, ¹H-NMR, (400 MHz, CDCl₃), δ [ppm]: 7.80 (t, 1H, J=13.2 Hz, CH=CH-CH); 7.78 (t, 1H, J=12.8 Hz, CH=CH-CH); 7.40-7.34 (m, 4H, aromatic); 7.25-7.20 (m, 2H, aromatic); 7.10-7.07 (dd, 2H, J₁= 8.0 Hz, J₂= 3.6 Hz, aromatic); 6.76 (t, 1H, J=12.4 Hz, CH-CH=CH); 6.32-6.29 (d, 2H, J=13.6 Hz); 4.04 (t, 2H, J=7.0 Hz, N-CH₂-CH₂); 3.60 (s, 3H, N-CH₃); 2.59 (t, 2H, J=6.8 Hz, CH₂-CH₂-COOH); 1.9-1.82 (m, 4H, CH₂-CH₂-CH₂-CH₂); 1.68 (s, 6H, 2xCH₃); 1.67 (s, 6H, 2xCH₃)