Cyanine5 alkyne

Final concentrations of 1 mM DAz-2 (Cayman Chemical), 1 mM Cyanine5 alkyne (Lumiprobe), 2 mM copper(II)-TBTA complex (Lumiprobe) and 4 mM ascorbic acid made in situ, were added sequentially in 15 mM PBS buffer mixed with 30% (vol/vol) acetonitrile. The solution was mixed at RT overnight and then quenched with 20 mM ethylenediaminetetraacetic acid (EDTA) and mixed at RT for 2 hr.

Cy5-B12 was synthesized through Huisgen/Sharpless ‘Click’ Chemistry^{47 ,48 (link)}. Cu(I) (1mg, 0.005mmol) and Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (3.5mg, 0.006mmol) were dissolved in 0.5mL DMF/H2O (4:1 v/v). Once color change occurred, the previously synthesized B12-Azide (3mg, 0.002mmol)^{49 (link)} and Cyanine-5 alkyne (0.5mg, 0.0007mmol) (Lumiprobe) was dissolved in the solution and allowed to stir at room temperature overnight protected from light. This was purified through RP-HPLC on a Shimadzu Prominence HPLC using a C18 column (Eclipse XDB-C18 5 μm, 4.6 × 150mm). Solvents: A: 0.1% TFA water and B: Acetonitrile. Method: B%: 20-72% over 18min. t_R: 4.7min. Yield: 94%. LC-MS analysis (Shimadzu LCMS-8040, Method: 0.1% Formic acid and 35% methanol water at 0.2mL/min, DL temp: 150°C, heat block temp: 400°C.): expected m/z: 2059 observed: 1031 [M+2H]⁺², 1042 [M+Na+2H]⁺², and 1050 [M+K+2H]⁺². Emission and excitation were 645 and 682nm, respectively using a Varian Cary UV Spectrophotometer and Agilent Cary Eclipse Fluorescence Spectrophotometer, solvent H₂O. See Figure 1 for more information.