Synthesis and Characterization of Fluorescent Streptavidin Conjugates

Example 10

Preparation of 70 kD amino dextran AF488 scaffold: 10 mg of amino dextran (70,000 MW, 20 amino groups; Thermo Fisher Scientific, Cat. No. D1862) was dissolved in 1.2 ml of dry DMSO containing 1.0 μl of DIEA. 0.9 mg of ALEXA FLUOR® 488 succinimidyl ester lithium salt (643 MF; Thermo Fisher Scientific, Cat. No. A20000) was added to solution and the mixture was stirred for 3.5 hours at ambient temperature. The solution was diluted with 12 mL of ethyl acetate and the resulting suspension was centrifuged. The supernatant was discarded and the solid material was shaken with 10 mL of fresh ethyl acetate and centrifuged. This washing was repeated 3 more times with 10 mL of fresh ethyl acetate and the resulting precipitate was dried in vacuum. The solid was re-dissolved in 0.5 ml of water and solution put in 10 cm Spectra/Por Dialysis membrane (Spectrum Labs, MWCO 12-14,000 flat width 10 mm) clipped from both side. The dialysis membrane was slowly stirred in 1 L of water for 1 week. The water was replaced twice per day. The dialysis membrane was open from one end and solution was lyophilized to give amino dextran ALEXA FLUOR® scaffold. The measured DOL is 9.7 and relative QY is 0.6 (referenced to QY of ALEXA FLUOR® 488).

Attaching thiol linker to 70 kD amino dextran AF488 scaffold: Amino dextran AF488 scaffold (4.5 mg) was dissolved in 0.5 mL of DMSO containing 0.055 μL of N,N-Diisopropylethylamine (DIEA). Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (20 μg) was added to solution and the mixture was kept at ambient temperature overnight and then capped with acetic acid succimidyl ester (1.0 mg, 3 hrs). The solution was diluted with 10 mL of ethyl acetate. The resulting suspension was centrifuged and supernatant discarded. The solid was shaken with 10 mL of fresh ethyl acetate and centrifuged. The washing was repeated 5 more times. The resulting solid was dried in vacuum. The measured DOL is 0.74. This material was re-dissolved in 2 mL of water and 16 mg of DTT was added to solution. The mixture was stirred for 5 min and loaded on G15 SEPHADEX® column, the product was eluted with DE water as green fluorescent solution which was used for conjugation to SMCC modified streptavidin. The determined concentration was 48 μM (by dye adsorption).

Conjugation of amino dextran AF488 scaffold modified with thiol linker to SMCC modified streptavidin: SMCC modified streptavidin (35 μL solution in water) was treated with 1, 2, 3 and 4 equivalents of thiol modified amino dextran AF488 scaffold (48 μM solution in water). The reaction was carried out at ambient temperature for 3 hours and after that reaction mixture was kept overnight at 4° C. overnight. The conjugates are purified on P100 size exclusion column with 10 nM PBS buffer.

Results: As shown in Tables 26 and 27, conjugates made from the scaffold are brighter as compared to conjugates made from single AF488 dye. Also, QY of AF488 fluorophore drops from 0.70 to 0.34 for single dye conjugation in contrast to almost constant QY for labeling with the amino dextran scaffold.