2-[bis(2-hydroxyethyl)amino]ethanol;hydrochloride

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

Procedure: To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger and equipped with internal thermometer and mechanical stirrer at 250 rpm was added 300 mL 100 mM pH 8.0 triethanolamine-HCl, 0.6 mL 1 M MgSO4, 3.0 g of SEQ ID NO:20 catalyst as prepared in Example 3, 0.1 g NADP-Na2, to give a pale yellow solution. After stirring for 10 minutes, 100 g 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-ketopropyl]benzoic acid methyl ester was added portion-wise over ˜5 minutes. To the slurry was added 500 mL (390 g) of IPA and 100 mL (85 g) toluene. The progress of the reaction was monitored by achiral HPLC as described in Example 4 and 6. The procedure as described in Example 9 was used for sampling and sample prep. To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger equipped with internal thermometer and mechanical stirrer at 250 rpm to give a pale yellow solution for sampling

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

Procedure: To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger and equipped with internal thermometer and mechanical stirrer at 250 rpm was added 300 mL 100 mM pH 8.0 triethanolamine-HCl, 0.6 mL 1 M MgSO4, 3.0 g of SEQ ID NO:20 catalyst as prepared in Example 3, 0.1 g NADP-Na2, to give a pale yellow solution. After stirring for 10 minutes, 100 g 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-ketopropyl]benzoic acid methyl ester was added portion-wise over ˜5 minutes. To the slurry was added 500 mL (390 g) of IPA and 100 mL (85 g) toluene. The progress of the reaction was monitored by achiral HPLC as described in Example 4 and 6. The procedure as described in Example 9 was used for sampling and sample prep. To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger equipped with internal thermometer and mechanical stirrer at 250 rpm to give a pale yellow solution for sampling

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

Procedure: To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger and equipped with internal thermometer and mechanical stirrer at 250 rpm was added 300 mL 100 mM pH 8.0 triethanolamine-HCl, 0.6 mL 1 M MgSO4, 3.0 g of SEQ ID NO:20 catalyst as prepared in Example 3, 0.1 g NADP-Na2, to give a pale yellow solution. After stirring for 10 minutes, 100 g 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-ketopropyl]benzoic acid methyl ester was added portion-wise over ˜5 minutes. To the slurry was added 500 mL (390 g) of IPA and 100 mL (85 g) toluene. The progress of the reaction was monitored by achiral HPLC as described in Example 4 and 6. The procedure as described in Example 9 was used for sampling and sample prep. To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger equipped with internal thermometer and mechanical stirrer at 250 rpm to give a pale yellow solution for sampling

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

Procedure: A suspension of 3.7 g of the sulfochloride of the formula (7) in 15 g of triethanolamine and 3 g of water is stirred at 150° for one hour. The excess solvent is substantially removed in vacuo at 120°-150° C., and 50 ml of isopropanol are added to the viscous residue. The precipitated product is filtered off with suction, washed repeatedly with isopropanol and taken up in approx. 40 ml of water. The cloudy solution is clarified by filtration with 0.2 g of active charcoal through a pressure filter. This gives an aqueous solution of the bistriethanolamine salt of the formula ##STR16## which still contains triethanolamine hydrochloride. The excess solvent is substantially removed in vacuo at 120°-150° C. 50 ml of isopropanol are added to the viscous residue The precipitated product is filtered off with suction washed repeatedly with isopropanol The cloudy solution is clarified by filtration with 0.2 g of active charcoal through a pressure filter

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

Procedure: A suspension of 3.7 g of the sulfochloride of the formula (7) in 15 g of triethanolamine and 3 g of water is stirred at 150° for one hour. The excess solvent is substantially removed in vacuo at 120°-150° C., and 50 ml of isopropanol are added to the viscous residue. The precipitated product is filtered off with suction, washed repeatedly with isopropanol and taken up in approx. 40 ml of water. The cloudy solution is clarified by filtration with 0.2 g of active charcoal through a pressure filter. This gives an aqueous solution of the bistriethanolamine salt of the formula ##STR16## which still contains triethanolamine hydrochloride. The excess solvent is substantially removed in vacuo at 120°-150° C. 50 ml of isopropanol are added to the viscous residue The precipitated product is filtered off with suction washed repeatedly with isopropanol The cloudy solution is clarified by filtration with 0.2 g of active charcoal through a pressure filter

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

Procedure: Portions of tissues (200-300 mg) were weighed and homogenized in 500 μl ice-cold 0.5 M perchloric acid using 6 mm ceramic beads on a tissue lyzer. Homogenates were centrifuged (10 min at 10,000), and resultant supernatants were neutralized (pH ˜7.4) with 5 M KOH plus 0.5 M triethanolamine hydrochloride with the help of pH paper and centrifuged (10 min at 10,000 g). Most of the procedures were performed at room temperature unlike suggested in the paper. A portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit to access total radioactivity (2-[3H]DG+2-[3H]DG-6-P). Another aliquot (200 μl) was mixed with 200 μl of 0.3 N Ba(OH)2 and 0.3 N ZnSO4. This mixture was centrifuged, and the supernatant was used to determine unphosphorylated 2-[3H]DG levels by using a portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit. Quench corrections were not made at this time. Tissue 2-[3H]DG-6-P was calculated as the difference between total and 2-[3H]DG values. (10 min at 10,000) with the help of pH paper and centrifuged (10 min at 10,000 g) were performed at room temperature A portion (200 μl) of the neutralized extract this radioactive mix Another aliquot (200 μl) was mixed with 200 μl of 0.3 N Ba(OH)2 and 0.3 N ZnSO4 neutralized extract this radioactive mix

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

Procedure: Modified J5 (8 mg, 0.05 μmol) in 5 mM bistris-acetate buffer, pH 5.8 (15 mL), containing NaCl (50 mM), and EDTA (1 mM) is mixed at 0° C. with a 5-fold molar excess of complex 7 (8 mg of gelonin) in 100 mM sodium phosphate buffer, pH 7.0 (11 mL), containing EDTA (1 mM) and then with 0.5 M triethanolamine-HCl buffer, pH 8.0, (0.15 mL) to give a final pH of 7.0. The mixture is incubated at 0° C. for 2 hours and then freshly prepared N-ethylmaleimide (1 mM) in ethanol (0.26 mL) is added to block any remaining free sulfhydryl groups. After 30 minutes at 0° C., the solution is maintained on ice while concentrating to 13 mL using an immersible ultrafiltration unit (Millipore Corporation, CX-10 filter). The mixture is then applied to a column (95 cm×2.6 cm) of Sephacryl S-300 equilibrated at 4° C. with 5 mM sodium phosphate buffer, pH 7.0, containing NaCl (15 mM), and NaN3 (0.4 mM). Gel filtration separates the conjugate and native J5 (Mr =160,000) from non-cross-linked gelonin [Mr =30,500; Thorpe et al., (1981) Euro. J. Biochem. 116:447-454], and from some high molecular weight aggregates. The major peak, corresponding to a molecular weight range of 160,000 to 220,000 and shown by polyacrylamide/sodium dodecyl sulfate gel electrophoresis to contain both native J5 and cross-linked conjugate 8 is pooled and passed through a column (5 mL bed volume) of carboxymethyl cellulose (Whatman CM-52) equilibrated in the same buffer. The column is washed with 1 column volume of buffer and the eluants combined. Under there precise conditions of ionic strength and pH, native J5 is bound by the column while cross-linked complexes of type 8 pass through without retention. Monoclonal antibodies are heterogeneous with respect to charge [Cowan et al. (1973) FEBS Lett. 30:343-346]: the sample of J5 used in these experiments is, prior to modification, passed through a carboxymethyl cellulose column under identical conditions. Only the J5 that bound to the column is used in cross-linking experiments. It is eluted from the column with buffer containing 1.0 M NaCl (yield 66%).

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

Procedure: 100 parts of triethanolamine were dissolved in 500 parts of water. 100 parts of concentrated hydrochloric acid were added to the solution to give a triethanolamine hydrochloride solution. This solution was added dropwise to a solution of 50 parts of C. I. Solubilized Sulphur Black 1 in 500 parts of boiling water under stirring. A crystallization began immediately. The crystal thus formed was separated by filtration, washed with water and dried to give a water-insoluble black pigment of the following formula:

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

Procedure: To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger and equipped with internal thermometer and mechanical stirrer at 250 rpm was added 300 mL 100 mM pH 8.0 triethanolamine-HCl, 0.6 mL 1 M MgSO4, 3.0 g of SEQ ID NO:20 catalyst as prepared in Example 3, 0.1 g NADP-Naz, to give a pale yellow solution. After stirring for 10 minutes, 100 g 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-ketopropyl]benzoic acid methyl ester was added portion-wise over ˜5 minutes. To the slurry was added 500 mL (390 g) of IPA and 100 mL (85 g) toluene. The progress of the reaction was monitored by achiral HPLC as described in Example 4 and 6. The procedure as described in Example 9 was used for sampling and sample prep. To a jacketed 2-L 3-neck flask under nitrogen with jacket temperature set at 51° C. via an external heat exchanger equipped with internal thermometer and mechanical stirrer at 250 rpm to give a pale yellow solution for sampling

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

Procedure: Modified J5 (8 mg, 0.05 μmol) in 5 mM bistris-acetate buffer, pH 5.8 (15 mL), containing NaCl (50 mM), and EDTA (1 mM) is mixed at 0° C. with a 5-fold molar excess of complex 7 (8 mg of gelonin) in 100 mM sodium phosphate buffer, pH 7.0 (11 mL), containing EDTA (1 mM) and then with 0.5 M triethanolamine-HCl buffer, pH 8.0, (0.15 mL) to give a final pH of 7.0. The mixture is incubated at 0° C. for 2 hours and then freshly prepared N-ethylmaleimide (1 mM) in ethanol (0.26 mL) is added to block any remaining free sulfhydryl groups. After 30 minutes at 0° C., the solution is maintained on ice while concentrating to 13 mL using an immersible ultrafiltration unit (Millipore Corporation, CX-10 filter). The mixture is then applied to a column (95 cm×2.6 cm) of Sephacryl S-300 equilibrated at 4° C. with 5 mM sodium phosphate buffer, pH 7.0, containing NaCl (15 mM), and NaN3 (0.4 mM). Gel filtration separates the conjugate and native J5 (Mr =160,000) from non-cross-linked gelonin [Mr =30,500; Thorpe et al., (1981) Euro. J. Biochem. 116:447-454], and from some high molecular weight aggregates. The major peak, corresponding to a molecular weight range of 160,000 to 220,000 and shown by polyacrylamide/sodium dodecyl sulfate gel electrophoresis to contain both native J5 and cross-linked conjugate 8 is pooled and passed through a column (5 mL bed volume) of carboxymethyl cellulose (Whatman CM-52) equilibrated in the same buffer. The column is washed with 1 column volume of buffer and the eluants combined. Under these precise conditions of ionic strength and pH, native J5 is bound by the column while cross-linked complexes of type 8 pass through without retention. Monoclonal antibodies are heterogeneous with respect to charge [Cowan et al. (1973) FEBS Lett. 30:343-346]: the sample of J5 used in these experiments is, prior to modification, passed through a carboxymethyl cellulose column under identical conditions. Only the J5 that bound to the column is used in cross-linking experiments. It is eluted from the column with buffer containing 1.0 M NaCl (yield 66%).

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

Procedure: 100 g of butanediol diglycidyl ether diacrylate were reacted with 5.75 g of chloroformate of the formula III and 3.3 g of triethanolamine at 50° C. for 2 hours. The triethanolammonium chloride formed was then separated off via a pressure filter. (Viscosity: 880 mPas).

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

Procedure: 100 g of a polyether acrylate (Laromer® LR 8812) were admixed with 5.75 g of chloroformate of the formula III and 3.3 g of triethanolamine at room temperature. 300 ml of ethyl acetate were then added. After 2 hours, the triethanolammonium chloride formed was washed out using distilled water in a separating funnel. After removal of the aqueous phase, the ethyl acetate was distilled off. (Viscosity of the end product: 928 mPas).