All solutions were prepared using ultrapure water from a Milli-Q water plus system with specific conductivity of less than 0.1 µS cm−1 and the chemicals of analytical grade.
Elastase from porcine pancreas, N-Succinyl-Ala-Ala-Ala-p-nitroanilide, HEPES sodium salt, sodium acetate, sodium hydroxide and acetic acid were purchase to Sigma-Aldrich® (St. Louis, Missouri, United States). Sodium chloride and dimethyl sulfoxide (DMSO) were purchase to Merck® (Darmstadt, Germany) and Uvasol® (Darmstadt, Germany), respectively.
A HEPES buffer solution 0.1 M with sodium chloride (NaCl) 0.5 M at pH 7.5 was used to dissolve the reagents. The buffer solution was prepared weighting 14.6 g of sodium chloride and 12.4g of HEPES and dissolving in about 150 mL of water. The pH 7.5 was adjusted with a 1 M NaOH solution, and final volume was adjusted to 500 mL. A sodium acetate buffer (pH 5.5) solution was used to dissolve the enzyme. The buffer solution was prepared weighting 1.64 g of sodium acetate and dissolving in about 50 mL of water. The pH 5.5 was adjusted with a 17.4 M acetic acid solution, and final volume was adjusted to 100 mL.
All the enzyme elastase porcine was reconstituted by stock solution of 1 U mL−1 using 2.5 mg of powder enzyme in 20 mL of sodium acetate buffer (pH 5.5). The stock solution was divided into 14 eppendorfs with a total volume of 1.43 mL per eppendorf. The eppendorfs were frozen.
A solution of N-Succinyl-Ala-Ala-Ala-p-nitroanilide was prepared daily dissolving 11.25 mg of substrate in 10 mL of DMSO.
For enzyme inhibition assays the aqueous solutions of: 1-Butyl-3-methylimidazolium tetrafluoroborate (bmim [BF4]), 1-Butyl-3-methylimidazolium chloride (bmim [Cl]), 1-Ethyl-3-methylimidazolium tetrafluoroborate (emim [BF4]), 1-Ethyl-3-methylimidazolium trifluoromethanesulfonate (emim [TfMs]), 1-Butyl-1-methylpyrrolidinium chloride (bmpyr [Cl]), 1-Butyl-4-methylpyridinium tetrafluoroborate (bmpy (BF4)), Tetrabutylammonium chloride (tba (Cl)), Tetrabutylammonium acetate (tba (Ac)), Tetrabutylphosphonium methanesulfonate (tbph (ms)), 1-Butyl-3-methylimidazoliumacetate (bmim (Ac)), were prepared daily. ILs were purchased from Sigma-Aldrich® and kept in anhydrous environment.
The ionic liquids active pharmaceutical ingredients (ILs-APIs) were synthesized according to [35 (link),36 (link)] and the aqueous solutions of lithium bistriflimide, 1-ethyl-3-methylimidazolium salicylate, benzethonium chloride, sodium salicylate, benzalkonium salicylate, trihexyltetradecylphosphonium docusate, benzethonium salicylate, benzethonium docusate, benzethonium bistriflimide, tributylmethylphosphonium salicylate, and trihexyltetradecylphosphonium chloride.
The chemical structures of all ILs and IL-APIs are presented inSupplementary Materials .
The HNE inhibitor named MeOSuc-Ala-Ala-Pro-Val-chloromethylketone was purchased from BioVision® (San Francisco Bay).
Elastase from porcine pancreas, N-Succinyl-Ala-Ala-Ala-p-nitroanilide, HEPES sodium salt, sodium acetate, sodium hydroxide and acetic acid were purchase to Sigma-Aldrich® (St. Louis, Missouri, United States). Sodium chloride and dimethyl sulfoxide (DMSO) were purchase to Merck® (Darmstadt, Germany) and Uvasol® (Darmstadt, Germany), respectively.
A HEPES buffer solution 0.1 M with sodium chloride (NaCl) 0.5 M at pH 7.5 was used to dissolve the reagents. The buffer solution was prepared weighting 14.6 g of sodium chloride and 12.4g of HEPES and dissolving in about 150 mL of water. The pH 7.5 was adjusted with a 1 M NaOH solution, and final volume was adjusted to 500 mL. A sodium acetate buffer (pH 5.5) solution was used to dissolve the enzyme. The buffer solution was prepared weighting 1.64 g of sodium acetate and dissolving in about 50 mL of water. The pH 5.5 was adjusted with a 17.4 M acetic acid solution, and final volume was adjusted to 100 mL.
All the enzyme elastase porcine was reconstituted by stock solution of 1 U mL−1 using 2.5 mg of powder enzyme in 20 mL of sodium acetate buffer (pH 5.5). The stock solution was divided into 14 eppendorfs with a total volume of 1.43 mL per eppendorf. The eppendorfs were frozen.
A solution of N-Succinyl-Ala-Ala-Ala-p-nitroanilide was prepared daily dissolving 11.25 mg of substrate in 10 mL of DMSO.
For enzyme inhibition assays the aqueous solutions of: 1-Butyl-3-methylimidazolium tetrafluoroborate (bmim [BF4]), 1-Butyl-3-methylimidazolium chloride (bmim [Cl]), 1-Ethyl-3-methylimidazolium tetrafluoroborate (emim [BF4]), 1-Ethyl-3-methylimidazolium trifluoromethanesulfonate (emim [TfMs]), 1-Butyl-1-methylpyrrolidinium chloride (bmpyr [Cl]), 1-Butyl-4-methylpyridinium tetrafluoroborate (bmpy (BF4)), Tetrabutylammonium chloride (tba (Cl)), Tetrabutylammonium acetate (tba (Ac)), Tetrabutylphosphonium methanesulfonate (tbph (ms)), 1-Butyl-3-methylimidazoliumacetate (bmim (Ac)), were prepared daily. ILs were purchased from Sigma-Aldrich® and kept in anhydrous environment.
The ionic liquids active pharmaceutical ingredients (ILs-APIs) were synthesized according to [35 (link),36 (link)] and the aqueous solutions of lithium bistriflimide, 1-ethyl-3-methylimidazolium salicylate, benzethonium chloride, sodium salicylate, benzalkonium salicylate, trihexyltetradecylphosphonium docusate, benzethonium salicylate, benzethonium docusate, benzethonium bistriflimide, tributylmethylphosphonium salicylate, and trihexyltetradecylphosphonium chloride.
The chemical structures of all ILs and IL-APIs are presented in
The HNE inhibitor named MeOSuc-Ala-Ala-Pro-Val-chloromethylketone was purchased from BioVision® (San Francisco Bay).
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