Rapid Determination of Ionization Constants (pk a ) by UV Spectroscopy Using 96-well Microtiter Plates

Transcription

1 Supporting Information Rapid Determination of Ionization Constants (pk a ) by UV Spectroscopy Using 96-well Microtiter Plates Carlos H. Ríos Martínez and Christophe Dardonville * Instituto de Química Médica, IQM-CSIC, Juan de la Cierva, E 86 Madrid, Spain. Table of contents: ) Preparation of buffer solutions S ) Experimental procedure S5 ) Data analysis S7 ) Spectra and pk a determination plots for compounds 8 S8 S

2 ) Preparation of buffer solutions The of the buffers was measured with a glass electrode (HANNA HI- meter) at ºC. All the solutions were prepared with distilled water. The. M standard solution of HCl was titrated with K CO (C =.8 M). The. M standard solution of NaOH was sonicated during min and titrated with potassium phthalate (C =. M). The ionic strength of each buffer solution was calculated using equation and the amount of KCl needed to adjust the ionic strength to. M was calculated for each buffer (Table S). () where c i is the molar concentration of ion i, z i is the charge number of that ion, and the sum is taken over all ions in the solution. a) Acetic acid/sodium acetate buffers (AcOH/AcONa) covering the range. to 5.. ml of. M sodium acetate solution was prepared by dissolving sodium acetate (8. mg, mmol) in ml of distilled water. 5 ml of each buffer solution (.,. and 5.) was prepared as follows: 5 ml of. M sodium acetate solution was stirred at ºC. The was adjusted to the required value by adding. M HCl standard solution with a burette. The volume was adjusted to 5 ml with distilled water. Finally, the ionic strength of the buffer solutions was calculated using equation and adjusted to. M by adding KCl (see Table ). S

3 b) Potassium hydrogen phosphate/dipotassium hydrogen phosphate (KH PO /K HPO ) buffers covering the range 6. to 8.. L of 5 M potassium hydrogen phosphate solution was prepared by dissolving KH PO (6.8 g, 5 mmol) in L of distilled water. 5 ml of each buffer solution ( 6. to 8.) was prepared as follows: 5 ml of. M KH PO was stirred at ºC in an erlenmeyer flask. The was adjusted to the required value by adding. M HCl or. M NaOH standard solution with a burette. The volume was adjusted to 5 ml with distilled water. The ionic strength calculated for these buffer solutions (Eq ) were in the range 7 68 M. Hence, the ionic strength was adjusted to. M by adding the necessary quantity of KCl (see Table S). c) Sodium tetraborate decahydrate (Borax)/boronic acid (Na B O 7 /H BO ) buffers covering the range 8. to.8 L of 5 M borax solution was prepared by dissolving borax decahydrate (9.5 g, 5 mmol) in L of distilled water. 5 ml of each buffer solution ( 8. to 9.) was prepared as follows: 5 ml of 5 M borax solution was stirred at ºC. The was adjusted to the required value by adding. M HCl. The volume was adjusted to 5 ml with distilled water. The ionic strength calculated for these buffer solutions were in the range of 5 6 M. Hence, the ionic strength was adjusted to. M by adding the necessary quantity of KCl (see Table S). 5 ml of each buffer solution ( 9. to.8) was prepared using 5 ml of 5 M borax (Na B O 7.H O) stirred at ºC. The was adjusted to the required value by adding. M NaOH. Finally, the ionic strength was adjusted to. M by adding KCl (See Table Sl). S

4 d) Disodium hydrogen phosphate/sodium phosphate (Na HPO /Na PO ) buffers covering the range.9 to. L of 5 M disodium hydrogen phosphate solution was prepared by dissolving Na HPO (7.98 g, 5 mmol) of in L of distilled water. 5 ml of a solution of 5 M Na HPO was stirred at ºC. The was adjusted to the required value by adding. M NaOH. The volume was adjusted to 5 ml with distilled water. The ionic strength calculated for these buffer solutions were in the range M. Hence, the ionic strength was adjusted to. M by adding the necessary quantity of KCl (see Table ). e) Glycine/NaOH buffer (.6) This buffer was commercially available (Fluka 55). Table S. Preparation of buffer solutions of constant ionic strength (I =. M) buffer Target Measured Conc. of salt solution (M) Vol. of salt solution (ml) Vol. of. M* HCl (ml) Vol. of. M** NaOH (ml) Vol. H O (ml) Calculated Ionic strength (M) Conc. of KCl required (M) Mass of KCl (mg) NaOAc/HOAc KH PO /K HPO Borax Na HPO /Na PO * C =.8 M ** C =. M S

5 ) Experimental procedure A small quantity ( mg) of each compound was weighted in an eppendorf tube using a high precision analytical balance (Mettler Toledo XS5). The compound was dissolved in DMSO to a concentration of mm (stock solution). The UV transparent 96-well microplate (Thermo Scientific Nunc) was loaded as shown in Figure S: each line of the plate was loaded with 96 µl of buffer solutions of increasing ( ranging from to.6). Then, µl of the compound stock solutions were added to each well with a micropipette (the resulting analyte solution was premixed with the micropipette). The final concentration of the analyte compound in each well was µm. One blank solution was prepared for each buffer by adding µl of DMSO to 96 µl of the corresponding buffer solution (i.e., free of analyte compounds) in the well. The 96-well plate was loaded into the UV-spectrophotometer (THERMO Multiskan Spectrum apparatus), incubated at ºC and shaken for min before the reading was performed. UV-spectra scans were recorded between nm and nm ( nm resolution). It should be noted that the number and range of buffer solutions needed to determine the pk a (e.g., every., or units) was adjusted depending on the compound tested. In general, a first screening with buffers ranging from to should give an approximate pk a value which can be refined when repeating the experiment using buffers within ± units of the pk a value. S5

6 Figure S. Example of loading of the 96-wells microtiter plate Buffer ( 96 µl) Buf f er : Compound ( µl) Compound ( µl) Compound ( µl) Compound ( µl) Compound 5 ( µl) Compound 6 ( µl) Compound 7 ( µl) Blank: DMSO ( µl) S6

7 ) Data Analysis In the present study we have used a similar data analysis as described by Tomsho et al. for the determination of ionization constants by spectral analysis. The raw UV-spectra scans were imported to the Excel program and processed as follows: ) UV-spectra of the analyte compounds were corrected by subtracting the UVspectra of the blank solutions; ) The raw scans were normalized (Abs nm = ); ) The spectral difference between the acid spectra and the spectra at every other was plotted; ) The wavelengths of maximum positive and negative absorbance were determined graphically from the spectral difference plot; 5) The total absorbance difference at the chosen wavelengths (that is: the sum of the absolute values of the maximum positive and negative absorbance in the spectral difference plot) was plotted against the. 6) These data were imported to the Prism program and the pk a values were worked out by non linear regression using equation : total = ε ΗΑ ε Α [ ( pka ) ] + ( pka ) * [ S t] Eq. ε HA and ε A - are the extinction coefficients of the acid and base forms of the compound, respectively (i.e., the minima and maxima of the absorbance difference curve, respectively), and [S t ] is the total compound concentration. S7

8 ) Spectra and pk a determination plots for compounds 8 o -Methylbenzimidazole (). -Methylbenzimidazole () Methylbenzimidazole () Methylbenzimidazole () 68 nm 78 nm 6 8 Difference. -Methylbenzimidazole () 6 8 o 5-Nitro-H-imidazole () 5-Nitro-H-imidazole () Nitro-H-imidazole () Nitro-H-imidazole () 9 nm 5 nm Difference. 5-Nitro-H-imidazole () 6 8 S8

9 o Clonidine ().5. Clonidine () Clonidine (). - nm Clonidine () nm 5 5 Difference Clonidine () 6 8 o Oxybis(,-phenylene) dicarbamimidate (5).5. (5) (5) - (5) 5 nm 9 nm Difference (5) 6 8 S9

10 o Compound 6a (6a) (6a) - - (6a) 58 nm 8 nm Difference.5. (6a) 6 8 o Compound 6b (6b) (6b) (6b) (6b) - 58 nm nm Difference 6 8 S

11 o Compound 6c 5 - (6c) (6c) - - (6c) 6 nm nm Difference.5. (6c) 6 8 o Compound 6d (6d) (6d) (6d) 6 nm - - nm Difference.5. (6d) 6 8 S

12 o Compound 7a (7a) (7a) - - (7a) 6 nm nm Difference. (7a) 6 8 o Compound 7b (7b) (7b). - (7b) 6 nm 6 nm Difference.5. (7b) 6 8 S

13 o Compound 7c (7c) (7c) - - (7c) 6 nm 6 nm Difference. (7c) 6 8 o Compound 7d (7d) (7d) nm (7d) 6 nm Difference. (7d) 6 8 S

14 o Compound 8a (8a) (8a) - - (8a) 6 nm 9 nm 5 5 Difference (8a) 6 8 o Compound 8b (8b) (8b) - - nm (8b) Spectral difference plot 7 nm 5 5 Difference (8b) 6 8 S

15 o Compound 8d (8d) (8d) - - (8d) 96 nm 58 nm 5 5 Difference. (8d) 6 8 References. Tomsho, J. W.; Pal, A.; Hall, D. G.; Benkovic, S. J. Ring Structure and Aromatic Substituent Effects on the pka of the Benzoxaborole Pharmacophore. ACS Med. Chem. Lett.,, 8-5. S5