(2,5-dioxopyrrolidin-1-yl) 5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl)pentanoate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Para-hydroxyphenylpropionyl biocytin (HPPB) was prepared by mixing a solution of p-hydroxyphenylpropionic acid-N-hydroxysuccinimide ester (50 mg [0.2 mMol]/2 ml dimethyl sulfoxide) with biocytin (70.75 mg [0.2 mMol]/2 ml 0.1M NaHCO3) overnight at room temperature (RT). Biotin-tyramine (BT) was prepared by mixing a solution of tyramine (40 mg [0.3 mMol]/1 ml dimethyl sulfoxide) with biotin-N-hydroxysuccinimide ester (100 mg [0.3 mMol]/1 ml dimethyl sulfoxide) overnight at RT. The solutions of HPPB and BT were used as is. The calculated concentrations were 26 mg/ml for HPPB and 55 mg/ml for BT. The calculated concentrations

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Amine functionalized polymer of formula (Vc) is dissolved at 10 mg/mL in anhydrous DMF and divided into two portions. NHS-biotin (0.9 mg in 90 μL, 88 equivalents) (Pierce, 20217) and NHS-LC-LC-biotin (Pierce, 21343) at 10 mg/mL (1.5 mg in 150 μL, 88 equivalents) are dissolved in anhydrous DMF. The NHS-biotin and NHS-LC-LC-biotin solutions are immediately added to the two portions of polymer solution and allowed to mix on a shaker overnight in the dark. to mix on a shaker overnight in the dark

Conditions: temp:20.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.30 g (5 mmol) of product 2b are suspended in 40 ml of distilled DMF and 1.4 ml triethylamine are added under stirring at 20° C. Subsequently, 1.71 g (5 mmol) of biotin-N-hydroxysuccinimide ester 3 are added and stirring is continued for another 3 h at 20° C. Subsequently, the solvent is removed on a rotation evaporator (oil pump vacuum, water bath 50° C.) and the concentrated residue is digested overnight with saturated NaHCO3 solution. Product 4b is drawn off, washed with water and dried in the exsiccator. stirring Subsequently, the solvent is removed on a rotation evaporator (oil pump vacuum, water bath 50° C.) the concentrated residue is digested overnight with saturated NaHCO3 solution washed with water dried in the exsiccator

Conditions: temp:20.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1.30 g (5 mmol) of product 2b are suspended in 40 ml of distilled DMF and 1.4 ml triethylamine are added under stirring at 20° C. Subsequently, 1.71 g (5 mmol) of biotin-N-hydroxysuccinimide ester 3 are added and stirring is continued for another 3 h at 20° C. Subsequently, the solvent is removed on a rotation evaporator (oil pump vacuum, water bath 50° C.) and the concentrated residue is digested overnight with saturated NaHCO3 solution. Product 4b is drawn off, washed with water and dried in the exsiccator. stirring Subsequently, the solvent is removed on a rotation evaporator (oil pump vacuum, water bath 50° C.) the concentrated residue is digested overnight with saturated NaHCO3 solution washed with water dried in the exsiccator

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1,4-Diaminocyclohexane (37.6 mg, 0.33 mmol) was dissolved in dry DMF (3 mL) under nitrogen. Solution of NHS-Biotin (102.3 mg, 0.3 mmol) in dry DMF (3 mL) was added to stirred solution of diamine over a 15 min period. Reaction mixture was stirred for 16 h at ambient temperature. Dilution of reaction mixture with dry Et2O (50 mL) produced white precipitate. Precipitated 19b was filtered, washed with EtOAc (3×20 mL) for removing of NHS and dried in high vacuum. Reaction mixture produced white precipitate Precipitated 19b was filtered washed with EtOAc (3×20 mL) for removing of NHS dried in high vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Each 3-mg portion of the 32-clone monoclonal antibodies prepared in (1) was mixed with 10 mg of biotinyl N-hydroxysuccinimide ester (product of Zymed) and the reaction was carried out in 0.1 M sodium hydrogen carbonate (pH 8) with stirring at room temperature for 3 hours. The reaction mixture was dialyzed overnight against 5 L of PBS at 4° C. to give the biotin-labeled monoclonal antibody. The reaction mixture was dialyzed overnight against 5 L of PBS at 4° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1,4-Diaminocyclohexane (37.6 mg, 0.33 mmol) was dissolved in dry DMF (3 mL) under nitrogen. Solution of NHS-Biotin (102.3 mg, 0.3 mmol) in dry DMF (3 mL) was added to stirred solution of diamine over a 15 min period. Reaction mixture was stirred for 16 h at ambient temperature. Dilution of reaction mixture with dry Et2O (50 mL) produced white precipitate. Precipitated 19b was filtered, washed with EtOAc (3×20 mL) for removing of NHS and dried in high vacuum. Reaction mixture produced white precipitate Precipitated 19b was filtered washed with EtOAc (3×20 mL) for removing of NHS dried in high vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Each 3-mg portion of the 32-clone monoclonal antibodies prepared in (1) was mixed with 10 mg of biotinyl N-hydroxysuccinimide ester (product of Zymed) and the reaction was carried out in 0.1 M sodium hydrogen carbonate (pH 8) with stirring at room temperature for 3 hours. The reaction mixture was dialyzed overnight against 5 L of PBS at 4° C. to give the biotin-labeled monoclonal antibody. The reaction mixture was dialyzed overnight against 5 L of PBS at 4° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Amine functionalized polymer of formula (Vc) is dissolved at 10 mg/mL in anhydrous DMF and divided into two portions. NHS-biotin (0.9 mg in 90 μL, 88 equivalents) (Pierce, 20217) and NHS-LC-LC-biotin (Pierce, 21343) at 10 mg/mL (1.5 mg in 150 μL, 88 equivalents) are dissolved in anhydrous DMF. The NHS-biotin and NHS-LC-LC-biotin solutions are immediately added to the two portions of polymer solution and allowed to mix on a shaker overnight in the dark. Excess reactant is removed by washing the solution using Amicon Ultra-4 mL 10 kD MWCO filter cartridges in a series of wash steps: First, the cartridge is first filled approximately halfway with water, and the reaction solution (by pipet) subsequently added directly into the water. Next, the cartridge is filled with water until it is full. The solution is mixed by pipetting up and down. Then, the cartridge is centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water 1 drop of 1M HCl is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume was reduced to 250 μL. Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water, mixed and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. This final step is repeated for a total of 5 washes. to mix on a shaker overnight in the dark Excess reactant is removed by washing the solution filter cartridges in a series of wash steps First, the cartridge is first filled approximately halfway with water the reaction solution (by pipet) subsequently added directly into the water Next, the cartridge is filled with water until it The solution is mixed The cartridge is then refilled with water 1 drop of 1M HCl is added the solution is mixed centrifuged at 2400 rpm for 30 minutes Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added the solution is mixed centrifuged at 2400 rpm for 30 minutes The cartridge is then refilled with water mixed and centrifuged at 2400 rpm for 30 minutes

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1,4-Diaminocyclohexane (37.6 mg, 0.33 mmol) was dissolved in dry DMF (3 mL) under nitrogen. Solution of NHS-Biotin (102.3 mg, 0.3 mmol) in dry DMF (3 mL) was added to stirred solution of diamine over a 15 min period. Reaction mixture was stirred for 16 h at ambient temperature. Dilution of reaction mixture with dry Et2O (50 mL) produced white precipitate. Precipitated 19b was filtered, washed with EtOAc (3×20 mL) for removing of NHS and dried in high vacuum. Reaction mixture produced white precipitate Precipitated 19b was filtered washed with EtOAc (3×20 mL) for removing of NHS dried in high vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Amine functionalized polymer of formula (Vc) is dissolved at 10 mg/mL in anhydrous DMF and divided into two portions. NHS-biotin (0.9 mg in 90 μL, 88 equivalents) (Pierce, 20217) and NHS-LC-LC-biotin (Pierce, 21343) at 10 mg/mL (1.5 mg in 150 μL, 88 equivalents) are dissolved in anhydrous DMF. The NHS-biotin and NHS-LC-LC-biotin solutions are immediately added to the two portions of polymer solution and allowed to mix on a shaker overnight in the dark. Excess reactant is removed by washing the solution using Amicon Ultra-4 mL 10 kD MWCO filter cartridges in a series of wash steps: First, the cartridge is first filled approximately halfway with water, and the reaction solution (by pipet) subsequently added directly into the water. Next, the cartridge is filled with water until it is full. The solution is mixed by pipetting up and down. Then, the cartridge is centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water 1 drop of 1M HCl is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume was reduced to 2504. Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water, mixed and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 25 μL. This final step is repeated for a total of 5 washes. to mix on a shaker overnight in the dark Excess reactant is removed by washing the solution filter cartridges in a series of wash steps First, the cartridge is first filled approximately halfway with water the reaction solution (by pipet) subsequently added directly into the water Next, the cartridge is filled with water until it The solution is mixed The cartridge is then refilled with water 1 drop of 1M HCl is added the solution is mixed centrifuged at 2400 rpm for 30 minutes Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added the solution is mixed centrifuged at 2400 rpm for 30 minutes The cartridge is then refilled with water mixed and centrifuged at 2400 rpm for 30 minutes

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Amine functionalized polymer of formula (Vc) is dissolved at 10mg/mL in anhydrous DMF and divided into two portions. NHS-biotin (0.9 mg in 90 μL, 88 equivalents) (Pierce, 20217) and NHS-LC-LC-biotin (Pierce, 21343) at 10 mg/mL (1.5 mg in 150 μL, 88 equivalents) are dissolved in anhydrous DMF. The NHS-biotin and NHS-LC-LC-biotin solutions are immediately added to the two portions of polymer solution and allowed to mix on a shaker overnight in the dark. Excess reactant is removed by washing the solution using Amicon Ultra-4 mL 10 kD MWCO filter cartridges in a series of wash steps: First, the cartridge is first filled approximately halfway with water, and the reaction solution (by pipet) subsequently added directly into the water. Next, the cartridge is filled with water until it is full. The solution is mixed by pipetting up and down. Then, the cartridge is centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water 1 drop of 1M HCl is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume was reduced to 250 μL. Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added; the solution is mixed, and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. The cartridge is then refilled with water, mixed and centrifuged at 2400 rpm for 30 minutes, or until the volume is reduced to 250 μL. This final step is repeated for a total of 5 washes. to mix on a shaker overnight in the dark Excess reactant is removed by washing the solution filter cartridges in a series of wash steps First, the cartridge is first filled approximately halfway with water the reaction solution (by pipet) subsequently added directly into the water Next, the cartridge is filled with water until it The solution is mixed The cartridge is then refilled with water 1 drop of 1M HCl is added the solution is mixed centrifuged at 2400 rpm for 30 minutes Next, the cartridge is refilled with water, 1 drop of 1M NaOH is added the solution is mixed centrifuged at 2400 rpm for 30 minutes The cartridge is then refilled with water mixed and centrifuged at 2400 rpm for 30 minutes