2-[3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propylamino]-2-(hydroxymethyl)propane-1,3-diol

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

Procedure: N,N-bis [2-Hydroxyethyl]-2-aminoethanesulfonic acid; (1,3-bis[tris(Hydroxymethyl)methylamino]propane); (N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-Hydroxyethyl)amino]ethanesulfonic acid); (N-tris[Hydroxymethyl]methyl-2-aminoethane-sulfonic acid; 2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino) ethanesulfonic acid; 3-[N,N-bis(2-Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid); (3-[N-tris(Hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid); (tris[Hydroxymethyl]aminomethane), (2-amino-2-(hydroxymethyl)-1,3-propanediol); (N-[2-Hydroxyethyl]piperazine-N'-[2-hydroxypropanesulfonic acid]); (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]); (N[2-Hydroxyethyl]piperazine-N'-[3-propane-sulfonic acid]); Triethanolamine, (2,2'2"-Nitrilotriethanol); and mixtures thereof.

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

Procedure: A greased VDX plate and circle siliconized glass cover slides were used. 500 μL of a particular reservoir solution was prepared by admixing ammonium sulfate stock solution, Bis-Tris propane stock solution and Milli Q water in each well. In this example, ammonium sulfate molarity was around 0.5 M, 1 M, 1.5 M or 2 M. The Bis-Tris Propane molarity was 0.1 M throughout, and the Bis-Tris Propane buffer pH was around 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. The resulting 24 conditions were assessed with all of the three protein concentrations as described above, and with storage at ambient temperature or storage at around 27° C., respectively. Around 1 μL of protein solution was admixed with around 1 μL of a particular reservoir solution on a circle siliconized glass cover slide, and the well was sealed with the inverted slide, generating a hanging drop experiment. The plates were stored at ambient temperature. Microscopy of the drops was performed after three days. The conditions were classified into clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals. of the three protein concentrations at ambient temperature at around 27° C. the well was sealed with the inverted slide were stored at ambient temperature containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals

Conditions: temp:37.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of bis-tris propane [2] buffer (70 mL, 50 mM bis-tris propane, pH 8.0, 300 mM NaCl) and a solution of 2,2-dimethoxyethanal X′ (10 mL, 0.5 M in bis-tris propane buffer as described above), an aqueous solution of DERA 02 (10 mL, prepared with analogue method to one described in Example 1) and a solution of acetaldehyde (10 mL, 1.0 M in bis-tris propane buffer as described above) was added. The mixture was stirred for 3 h at 37° C. and pH was regulated at 8.0 with NaOH. After 3 h the proteins in the sample (1 mL) from the reaction mixture (100 mL) were precipitated with acetonitrile (9 mL) and the solution was centrifuged. A sample from the supernatant was collected and analyzed directly by HPLC-MS/MS instrument: After 3 h the proteins in the sample (1 mL) from the reaction mixture (100 mL) were precipitated with acetonitrile (9 mL) A sample from the supernatant was collected

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

Procedure: A greased VDX plate and circle siliconized glass cover slides were used. 500 μL of a particular reservoir solution was prepared by admixing ammonium sulfate stock solution, Bis-Tris propane stock solution and Milli Q water in each well. In this example, ammonium sulfate molarity was around 0.5 M, 1 M, 1.5 M or 2 M. The Bis-Tris Propane molarity was 0.1 M throughout, and the Bis-Tris Propane buffer pH was around 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. The resulting 24 conditions were assessed with all of the three protein concentrations as described above, and with storage at ambient temperature or storage at around 27° C., respectively. Around 1 μL of protein solution was admixed with around 1 μL of a particular reservoir solution on a circle siliconized glass cover slide, and the well was sealed with the inverted slide, generating a hanging drop experiment. The plates were stored at ambient temperature. Microscopy of the drops was performed after three days. The conditions were classified into clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals. of the three protein concentrations at ambient temperature at around 27° C. the well was sealed with the inverted slide were stored at ambient temperature containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals

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

Procedure: The enzymatic reaction can be assayed using labelled (3-14C-) glyceryl glyphosate as a substrate and following the conversion to (3-14C-)glyphosate as a product using a high pressure liquid chromatography system with a radioactive detector. This assay is the most specific for the phosphonate monoester hydrolase enzymes useful for conditional cell lethality. An enzyme assay consists of 40,000 cpm of labelled (3-14C-) glyceryl glyphosate (obtained from NEN Biochemicals), the PEH sample in buffer and buffer to 100 μl final volume. The assay buffer is 10 mM Bis-Tris propane, pH 9.0, plus 100 mM KCl. The reaction is run for 1 hour at 30° C. and then stopped by the addition of 100 μl "quench" which is 100 mM sodium acetate, pH 5.5, in ethanol. The denatured protein is then spun down at 10,000 rpm in a microfuge and the supernatant transferred to an HPLC injection vial. A 100 μl aliquot is then injected onto an Synchropac AX100 anion exchange column 4.6×250 mm) pre-equilibrated in 65 mM potassium phosphate buffer, pH 5.5. The radioactive compounds are eluted isocratically for 15 min at 1 ml/min. The radioactivity of the peaks is measured and integrated by a post-column detector. Typically, the substrate elutes at 4.9 min and the product, glyphosate, at 9.7 min providing baseline separation. The radioactive glyceryl glyphosate has been found to be unstable where stored at -20° C. over a period of 3 months and must first be purified to remove glyphosate and any other contaminant. Alternatively, blank reactions can be included to determine the preexisting glyphosate contamination. The enzymatic reaction stopped by the addition of 100 μl " quench" which The radioactive compounds are eluted isocratically for 15 min at 1 ml/min Typically, the substrate elutes at 4.9 min

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

Procedure: A greased VDX plate and circle siliconized glass cover slides were used. 500 μL of a particular reservoir solution was prepared by admixing ammonium sulfate stock solution, Bis-Tris propane stock solution and Milli Q water in each well. In this example, ammonium sulfate molarity was around 0.5 M, 1 M, 1.5 M or 2 M. The Bis-Tris Propane molarity was 0.1 M throughout, and the Bis-Tris Propane buffer pH was around 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. The resulting 24 conditions were assessed with all of the three protein concentrations as described above, and with storage at ambient temperature or storage at around 27° C., respectively. Around 1 μL of protein solution was admixed with around 1 μL of a particular reservoir solution on a circle siliconized glass cover slide, and the well was sealed with the inverted slide, generating a hanging drop experiment. The plates were stored at ambient temperature. Microscopy of the drops was performed after three days. The conditions were classified into clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals. of the three protein concentrations at ambient temperature at around 27° C. the well was sealed with the inverted slide were stored at ambient temperature containing random precipitation, drops containing crystals and drops containing mixtures of precipitated species and crystals

Conditions: temp:37.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of bis-tris propane [2] buffer (70 mL, 50 mM bis-tris propane, pH 8.0, 300 mM NaCl) and a solution of 2,2-dimethoxyethanal X′ (10 mL, 0.5 M in bis-tris propane buffer as described above), an aqueous solution of DERA 02 (10 mL, prepared with analogue method to one described in Example 1) and a solution of acetaldehyde (10 mL, 1.0 M in bis-tris propane buffer as described above) was added. The mixture was stirred for 3 h at 37° C. and pH was regulated at 8.0 with NaOH. After 3 h the proteins in the sample (1 mL) from the reaction mixture (100 mL) were precipitated with acetonitrile (9 mL) and the solution was centrifuged. A sample from the supernatant was collected and analyzed directly by HPLC-MS/MS instrument: After 3 h the proteins in the sample (1 mL) from the reaction mixture (100 mL) were precipitated with acetonitrile (9 mL) A sample from the supernatant was collected

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

Procedure: N,N-bis [2-Hydroxyethyl]-2-aminoethanesulfonic acid; (1,3-bis[tris(Hydroxymethyl)methylamino]propane); (N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic acid; 2-[bis(2-Hydroxyethyl)amino]ethanesulfonic acid); (N-tris[Hydroxymethyl]methyl-2-aminoethane-sulfonic acid; 2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino) ethanesulfonic acid; 3-[N,N-bis(2-Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid); (3-[N-tris(Hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid); (tris[Hydroxymethyl]aminomethane), (2-amino-2-(hydroxymethyl)-1,3-propanediol); (N-[2-Hydroxyethyl]piperazine-N'-[2-hydroxypropanesulfonic acid]); (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]); (N[2-Hydroxyethyl]piperazine-N'-[3-propane-sulfonic acid]); Triethanolamine, (2,2'2"-Nitrilotriethanol); and mixtures thereof.