Binary and ternary complexes involving ethambutol and bio-relevant ligands such as amino acids, peptides and DNA constituents: speciation studies and theoretical approach

Abstract The interaction of Cu(II) with ethambutol (EMB) was investigated. The complexes formed are the 1:1 complex, Cu(EMB) and the deprotonated forms, Cu(EMB)H-1 and Cu(EMB)H-2. The pKa values of the induced ionization of the hydroxyl group of EMB upon coordination are 6.76 and 7.93. The formation equilibria of the ternary complexes Cu(EMB)L, involving the secondary ligand such as amino acids, peptides or DNA constituents (L), were investigated. Ternary complexes with amino acids showed Cu(EMB)L and the protonated form Cu(EMB)LH. The pKa value of the protonated complexes are 5.75–9.09. Peptides form Cu(EMB)L and the deprotonated form Cu(EMB)LH-1. DNA constituents form Cu(EMB)L and Cu(EMB)L2. The thymine complex is more stable than that of uridine. Uridine-5’-monophosphate forms a stronger complex than uridine; inosine-5’-monophosphate (IMP) forms in addition the protonated form of the 1:1 complex. It forms a more stable complex with Cu(EMB)2+ than inosine. The speciation diagrams were evaluated. Density functional theory (DFT) calculations were performed. The calculations included investigation of molecular electrostatic potential surfaces. A molecular docking analysis of ethambutol and its complex with mycobacterium tuberculosis (PDB ID: 5UHG) is investigated.


Introduction
Ethambutol (EMB) was reported to inhibit the growth of both mycobacterium tuberculosis and mycobacterium smegmatis by blocking the synthesis of arabinogalactan.Several lines of evidence suggest that EMB exerts its toxic effect on mycobacteria by inhibiting embABC-encoded proteins [1-5] and mutations in embABC also appear to play a key role in the development of EMB resistance in both M. tuberculosis and M. smegmatis [3].The Cu(II) complex is involved in the mode of action of this drug [5], however, significant toxicity resulted.Neurological effects altered liver and heart function, and morphological changes in the myocardium were noted.Buyske [6] correlated cardiac toxicity with the ability of ethambutol to deplete the copper content of heart tissue.Paralleling the decrease in copper was reduced cytochrome C oxidase activity.
The formation equilibria of drug complexes with metal ions commonly existing in biological fluids help to elucidate the mechanism of actions of drugs.Complex formation equilibria of Cu(II) with ethambutol and ternary complexes involving ligands of biological significance such as amino acids, peptides, and DNA constituents support the biological effect of ethambutol.The results of this study will give information regarding the behavior of ethambutol in biological system.In conjunction with our previous work on metal complexes of biological significance [7][8][9][10][11][12][13][14], the present investigation describes the formation equilibria of binary and ternary complexes involving Cu(II), ethambutol, and amino acids, peptides, or DNA constituents.The equilibrium geometries of ethambutol and its complex were studied by Density Functional Theory (DFT) calculations.The molecular electrostatic potential of ethambutol and its complex are determined, and molecular docking was achieved for ethambutol and its complex to investigate their interaction with mycobacterium tuberculosis (PDB ID: 5UHG).

Procedure and measuring techniques
Protonation constants of the ligands were estimated using potentiometric titration of the ligands (EMB, amino acids, peptides, or DNA constituents) solution (1.25 � 10 −3 M).The stability constants of the binary complexes of ethambutol and amino acids were determined potentiometrically by titrating mixtures of Cu 2þ (1.25 � 10 −3 M) and EMB�2HCl (1.25 � 10 −3 M) or amino acids (2.5 � 10 −3 M).The stability constants of the ternary complexes were evaluated from potentiometric titration of solution mixtures containing Cu 2þ (1.25 � 10 −3 M), EMB (1.25 � 10 −3 M), and amino acids or peptides (1.2 � 10 −3 M) or DNA constituents (2.5 � 10 −3 M).The acid dissociation constants of ethambutol and the formation constants of its Cu(II) complexes were estimated in dioxane-water solutions of different compositions.The volume of titrated solution was 40 ml and the ionic strength was 0.1 M by NaNO 3 .
Potentiometric titrations were performed using a Metrohm 686 titroprocessor equipped with a 665 dosimat (Switzerland-Herisaue).A Metrohm glass-calomel combined electrode and a thermometric probe were used.The titroprocessor and electrode were calibrated with standard buffer solutions and prepared according to NBS specifications [16].The temperature of the sample solutions was maintained at 25.0 ± 0.1 � C by circulating thermostated water through the titration vessel jacket.The ionic strength was adjusted to 0.1 mol�dm −3 with sodium nitrate.All potentiometric measurements were carried out under a nitrogen atmosphere.A standard 0.05 M NaOH solution was used as a titrant.The pK w values in dioxane-H 2 O solutions were determined as described previously [17,18].For this purpose, various amounts of standard NaOH solution were added to a 0.10 mol�dm −3 NaNO 3 solution; [OH -] was calculated from the amount of base added and [H þ ] was calculated from the pH value.The values obtained in this way for log [OH][H] (log K w ) are −14.23,−14.50, −14.92, −15.12, and −15.63 for 25.0, 37.5, 50.0, 62.5, and 75.0%dioxane in H 2 O, respectively.
The general equilibrium for the ternary complexes of amino acids can be written as follows (charges are omitted for simplicity): where l, p, q, and r are the stoichiometric coefficients corresponding to Cu(II), EMB, amino acid, and proton, respectively.The overall formation constants are defined as , where EMB, L, and H stand for ethambutol, amino acid, and proton, respectively (charges are omitted for simplicity).
The general equilibrium for the ternary complexes of peptides or DNA constituents can be written as follows: where l, q, and r are the stoichiometric coefficients corresponding to Cu(EMB), peptide or DNA constituent, and proton, respectively.The formation constants are defined as The formation constants were evaluated using the computer program MINIQUAD-75.The stoichiometry and stability constants of the complexes formed were determined by trying various composition models.The model selected was that which gave the best statistical fit and was chemically consistent with the magnitude of various residuals, as described elsewhere [19].Tables 1-4 list the stability constants together with their standard deviations derived from the MINIQUAD output.The concentration distribution diagrams were obtained using SPECIES [20].

Computational details
Gaussian 09 software [21] was used to perform the DFT calculations.The structure of EMB, [Cu(EMB)H 11 and [Cu(EMB)(GlyGly)H -1 ] were optimized at the B3LYP functional with the 6-311 G � basis set for C, H, O and N atoms and LANL2DZ basis set [22] for copper.Several researchers used the LANL2DZ as a basis set in DFT calculations of systems that included metal atoms [23,24].The Gauss View 5.0, which is supported by Gaussian Inc. [21], was used for generating the input files that correspond to the DFT calculations.The Gauss View 5.0 software is used to visualize and analyze data obtained from Gaussian09 output results.Frequency calculations were performed to identify the most stable structures of the synthesized compounds.The absence of     LANL2DZ basis set for copper of the optimized geometry.The definition, description, and calculation of V(r) were reported by Politzer and Murray in 2002 [25].

Molecular docking
The two-dimensional (2D) chemical structures of EMB, [Cu(EMB)H -2 ], and [Cu(EMB)(GLY)] þ were obtained from the output of the DFT calculations and converted to pdb files using Gauss View 5.0.The mycobacterium tuberculosis (PDB ID: 5UHG) was retrieved from the RCSB protein data bank (http://www.rcsb.org/pdb)for molecular docking study.Protein and grid preparation were made using the Auto Dock Tool 4.2 [26].The missing atoms were repaired and polar hydrogen atoms were added to the 5UHG receptor structure, then the Kollman charge was computed and added, accordingly.The 5UHG was loaded into Auto Dock Tool 4.2, creating a PDBQT file that contains a protein structure with hydrogens in all polar residues.The docking site on the protein target was defined by establishing a grid box with the dimensions of x: 60 y: 60 z: 60 Ð, with a grid spacing of 0.5 Ð, centered on X: 320.32,Y: 330.99,Z: 212.04 Ð.The interactions of complexes and ligands with 5UHG, including hydrogen bonds and hydrophobic interactions, were analyzed using Discovery Studio 4.0 client [27].

Acid-base equilibria of ethambutol dihydrochloride
The acid-base equilibria of (EMB�2HCl) are given in Scheme 1. Ethambutol is used in the fully protonated form, H 2 L 2þ , containing two protonated imino groups.Examination of Scheme 1. Acid-base equilibria of ethambutol dihydrochloride.
the potentiometric equilibrium titration curve shows the presence of two buffer regions.The first one shows deprotonation of the more acidic protonated imino group (pK a1 ¼ 6.32 at 25 � C) yielding HL þ .The second buffer region indicates the deprotonation of the second, less acidic, protonated imino group giving the species L (pK a2 ¼ 9.16 at 25 � C).

Binary complexes involving copper(II) and ethambutol
The potentiometric titration curve for the Cu(II)-(EMB) system is significantly lower than the EMB�2HCl titration curve.This corresponds to the formation of a complex through the release of proton.The potentiometric data are fitted well assuming the formation of [Cu(EMB)] (1100), [Cu(EMB)H -1 ] (110-1), and [Cu(EMB)H -2 ] (110-2).The formation constants of the complexes amount to log b ¼ 10.03, 3.27, and −4.66 for species of stoichiometric coefficients (1100), (110-1) and (110-2), respectively.The Cu(II)-(EMB) (1100) complex undergoes an induced ionization under complex formation through the release of proton of the two hydroxyl groups present in ethambutol to form (110-1) species with a pK a value of 6.76 as calculated by Eq. (3) [28].
The deprotonated complex (110-1) undergoes a second induced ionization for the second hydroxyl group of ethambutol to form the species (110-2) with pK a value of 7.93 as calculated by equation 4 [28]: The complex formation equilibria are shown in Scheme 2. The concentration distribution diagram of Cu(II)-(EMB) is shown in Figure 1.The (1100) complex dominates (89%) at pH � 5.5.The hydroxo-species (110-1) and (110-2) start to form at pH 5 and 7, respectively, and prevail with a concentration percentage of 50% at pH 6.8 (for 110-1) and pH 8.5 (for 110-2).From the biological point of view, 1100 and 110-1 dominate in the physiological pH range.These species are active as they have one or two labilecoordinated water molecules that can be substituted by another ligand from the biological fluid.

Binary complexes with amino acids
The acid-dissociation constants of amino acids and the formation constants of their binary complexes with Cu(II) were used in the determination of the formation constants of the ternary complexes.The corresponding data of amino acids were taken from published work [14] or redetermined, if not available, under the same experimental condition used to study the ternary complexes.

Ternary complex formation equilibria
The ternary complex is formed either through a simultaneous mechanism or a stepwise mechanism depending on the coordination potential of ethambutol and other ligands.The formation constant of Cu(II) complexes with amino acids is higher than those with peptides or DNA constituents.This may be due to the high basicity of the amino acid amino group and amino acids are coordinated as bi-or tridentate.The formation constants of 1:1 copper(II) complexes with ethambutol and amino acids are of the same order of magnitude (Table 1).Consequently, the coordination of ethambutol and amino acids will proceed simultaneously.The potentiometric titration data of the Cu(II) ternary complexes involving EMB and amino acids (HL) are best fitted assuming the formation of the species (1110) and (1111).The pK a values of the protonated complex (1111) are calculated using Eq. ( 5) [29].
The pK a value of the protonated complex is 5.75-9.09.This means the protonated species ( 1111) is formed by binding with carboxylate oxygen, leaving the amino group susceptible to protonation.The pK a of the protonated complex of lysine is exceptionally high, 9.09.This may be explained on the premise that one of the uncoordinated amino groups in the protonated complex is far from the coordination center.
The stability constant values, log b 1110 , of the simple amino acid complexes are compared.Proline has the highest value.This may be due to the highest basicity of the amino group as reflected by its highest pK a value [30].
The concentration distribution diagram of Cu(II)(EMB)(Gly) (as a representative of a-amino acids) given in Figure 2 indicates that the protonated complex (1111) is formed in the acidic pH range and prevails with a formation degree of 24% at pH � 5.2.The protonated complex undergoes deprotonation forming (1110), reaching the maximum concentration of 52% at pH ¼ 7.5.Thus, the deprotonated species dominates in the physiological pH range.
Serine and threonine contain only two dissociable protons in the measurable pH range (-NH 3 þ and -COOH).The alcoholic hydroxyl group is so weakly acidic (pK a < 14)  The latter complex is formed through induced ionization of the b-alcohol group as reported for serine and threonine complexes [31][32][33].The speciation diagram for the ternary copper(II) complex of serine is depicted in Figure S1.Species (1110) predominates with a maximum degree of formation of 58% at pH � 7.3 and (1111) is predominant at pH � 5.2 with a formation degree of 35%.Species (111-1) prevail at pH 9 with a formation degree of 24%.Lysine forms a ternary complex of considerably higher stability constant than those of simple a-amino acids.Lysine has an extra terminal amino group, coordinating bidentate either by the two amino groups (N, N) or glycine-like through the a-amino and carboxylate groups (N, O).The stability constant value of the deprotonated Cu(II)-(EMB)-lysine complex is log b 1110 ¼ 17.28 and the stability constant value of its protonated complex amounts is b 1111 ¼ 26.37.The pK a value of the protonated complex is 9.09 (pK a ¼ log b 1111 -log b 1110 ).
The protonated complex of lysine is acidified by (10.44-9.09),1.35 log units.The acidification upon complex formation was reported previously [12,34,35].The protonated group corresponds to the terminal amino group and lysine coordinates glycinelike at a relatively low pH.This can be explained on the premise that lysine is bound by amino and carboxylate groups, leaving the other amino group susceptible to protonation.The stability constant of the deprotonated complex is higher than simple amino acids such as glycine.This may indicate that the deprotonated complex is formed by the coordination of the two amino groups after deprotonation of the protonated complex.Thus, the coordination sites are pH-dependent and the coordination sites shift to the two amino groups with increasing pH.The concentration distribution diagram of Cu(II)-(EMB)-lysine (Figure S2) indicates that the protonated species (1111) starts to form at low pH and reaches its maximum concentration of 45.5% at physiological pH (�7.3).The deprotonated species (1110) dominates at pH ¼ 9 with the maximum degree of formation (8%).
Histidine has three coordination sites, the carboxylate oxygen, imidazole nitrogen, and amino group.Two are involved in coordination with the Cu-EMB complex.The stability constant of ternary His complex is log b 1110 ¼ 17.54 and that for its protonated form is log b 1111 ¼ 24.37.The pK a value of the protonated complex is 6.83 (pK a ¼ log b 1111 -log b 1110 ).This value is in fair agreement with that of imidazole nitrogen.Histamine has two coordination sites, the imidazole nitrogen and amino group.The stability constant of histidine complex (log b 1110 ¼ 17.54) is in fair agreement with that of histamine (log b 1110 ¼ 16.70), but higher than those of amino acids.This indicates that histidine interacts with the Cu-EMB complex in the same way as histamine does through the amino group and imidazole nitrogen.

Ternary copper(II) complex formation equilibria involving ethambutol and peptides or DNA constituents
The formation constant of the 1:1 copper(II) complex with ethambutol is higher than those with peptides or DNA constituents.The formation constant of Cu-EMB is log b 110 ¼ 10.03, while those of peptides and DNA constituents are much lower [36].Consequently, the coordination of peptides or DNA constituents will proceed by a stepwise mechanism.Cu(II) will coordinate first with EMB, then with peptides or DNA constituents, as given in Eqs. ( 6) and ( 7 The stability constants were determined using the titration data after the formation of the Cu-EMB complex (110).

Peptide complexes
Analysis of potentiometric data of the ternary complex of peptides reveals the formation of [(Cu-EMB)(L)] (110) and [(Cu-EMB)(LH -1 )] (11-1), Table 2.The peptide forms complex (110) by coordination through the amino and carbonyl groups.On increasing the pH, the coordination sites switch from carbonyl oxygen to amide nitrogen.Such changes in coordination centers are well documented [37][38][39].The amide group undergoes deprotonation and [(Cu-EMB)(LH -1 )] (11-1) is formed according to Scheme 3. The pK H value of peptide hydrogen is calculated by Eq. ( 8): The pK H for the glycinamide complex is lower than those of other peptides.This signifies the more bulky substituent group on the peptide opposes the structural change in going from protonated to a deprotonated complex.The pK H values of aspargine and glutamine complexes are higher than the others, probably because the formation of six-and seven-membered chelate rings are more strained and less favored, which hinders the proposed structural change.Therefore, in the physiological condition (pH 7.4), small peptides such as glycinamide and glycylglycine would preferably coordinate in the deprotonated form.The speciation diagram of the glycylglycine complex (as a representative of peptides) is given in Figure S3.The ternary complex [(Cu(EMB)L] (110) started to form at pH � 4.1 and with increasing pH, its concentration increases, reaching maximum of 44% at pH � 7.1.Furthermore, an increase in pH is accompanied by a decrease in (110) complex concentration and an increase of [(Cu(EMB)LH -1 ] (11-1) concentration.In the physiological pH range, both complex (110) and the deprotonated form exist.

DNA complexes
The accepted model for DNA complexes is consistent with the formation of 1:1 and 1:2 complexes (Table 3).The pyrimidines uracil, uridine, thymidine, and uridine-5 0monophosphate have basic nitrogen N(3)-C4(O) groups.They coordinate in the deprotonated form as monoanion through N3 and do not form protonated complexes as reported [40].The thymine complex is more stable than that of uridine, from the higher basicity of the N3 coordination site of thymine, as reflected by high pK a .This is explained due to the inductive effect of the extra electron-donating methyl group.Uridine-5 0 -monophosphate forms a stronger complex than uridine, attributed to the triply negatively charged uridine-5 0 -monophosphate.
The purines, inosine, and inosine-5 0 -monophosphate are protonated at N(7) and form [N(1)H-N(7)H] monocations.In the present investigation, the pK a of N(1)H was only estimated as the pK a value of N(7)H is too low to be determined by potentiometric measurements.The purinic ligands as inosine have lower pK a values than the pyrimidinic ligands (uridine, uracil, thymine, and thymidine), due to the anionic form of the purines having a higher number of resonance forms due to the presence of two condensed rings.In the experimental condition of the present study, N(1) is protonated and the Cu-EMP complex is attached to N(7).The structural change from N(7) binding to N(1) binding upon increasing pH was reported [8,41].Consequently, it is assumed that N(1) acts as a binding site in the ternary complex of inosine and inosine-5 0 -monophosphate at high pH values.Inosine-5 0 -monophosphate forms in addition to the 1:1 and 1:2 complexes, protonated form of the 1:1 complex (111).The pK a of the protonated species (log b 111 -log b 110 ) is 3.89, corresponding to the phosphate group.Acidification of the P-OH group is due to complex formation [35].Inosine-5 0monophosphate forms a more stable complex with Cu(EMB) 2þ than inosine, attributed to the triply negatively charged 5 0 -IMP ion.
The concentration distribution curve of (Cu(EMB)(IMP) (as a representative of DNA species) is given in Figure S4.The species (111) starts to form at low pH values and reaches its maximum percentage of 74% at pH � 5.8.The (110) species dominate in the physiological pH range with a maximum concentration of 68% at pH � 7.4.Species (120) prevails at higher pH values.

Solvent effect
It has been reported that aqueous media is not a good model for biological media.The biological media shows lipophilic character.A lower polarity was found in the enzyme active site and protein side chain.Consequently, the biological medium is better represented by nonaqueous media [42][43][44][45][46].The dielectric constants in such sites are reported to be in the range of 30-70.Hence dioxane-water mixture is expected to be a good model of the biological environment.The results of the effect of dioxane percentage on the formation constant of Cu(II)-EMB complexes are given in Table 4. Also, the pK a values of ethambutol were determined in dioxane-water solutions and the results are given in Table 5.

Solvent effects on formation of Cu(II)-EMB
The stability of compounds containing O-H increases with increasing the organic content of the solvent, due to a decrease in the dielectric constant of the bulk solvents [47][48][49].As the dielectric constant decreases, the ion-ion interaction involving the proton and the anion oxygen donor of the ligand increases to a greater extent than the ion-dipole interaction between the proton and the solvent molecule.The effect of solvent on pK 11-1 of [Cu-(EMB)] (110) and pK 11-2 of [Cu-(EMB)H -1 )] (11-1) was studied in dioxane-water mixtures of different compositions.Increasing the dioxane percentage affected the polarization of the O-H group by retarding proton release, which leads to an increase in the pK 11-1 value.In the same way, the increase of dioxane content retards the release of the second proton belonging to the second (O-H) group, which causes the increase of pK 11-2 as shown in Table 5.The formation constant of Cu(II)-EMB (110) increases upon the addition of dioxane to an aqueous solution of the corresponding species.This can be explained as a result of increasing the electrostatic forces between Cu(II) and the electronegative amino nitrogen of ethambutol.This leads to an increase in complex stability.

Investigation of the equilibrium geometries of EMB, [Cu(EMB)H
112.3 (109.4) a a the corresponding calculated value of the free GlyA.The bonds connected to ethambutol nitrogen atoms in the [Cu(EMB)H -2 ] complex are elongated.The N9-C13 and N7-C11 bond lengths in free ethambutol are 1.491 and 1.476 Ð, respectively.They are elongated to 1.514 and 1.512 Ð, respectively, due to the coordination of N to Cu(II).However, bonds connected to O atoms are shortened upon complex formation.The C24-O35 and C21-O36 bond lengths in uncoordinated EMB are 1.459 and 1.445 Ð, shortened to 1.431 and 1.435 Ð, respectively.This may be due to OH deprotonation and ionic interaction between the negatively charged O35 and O36 to the electropositive Cu 2þ In [Cu(EMB)H -2 ], the bonds between ethambutol nitrogen atoms and Cu 2þ are 2.118 Ð for N9-Cu37 and 2.086 Ð for N7-Cu37.The bonds between the deprotonated oxygen atoms of ethambutol and Cu(II) are 1.912 Ð for O36-Cu37 and 1.911 Ð for O35-Cu37.The Cu 2þ -O -bonds are shorter than the Cu 2þ -N, from the ionic interaction in Cu 2þ -O -bonds shortening the length.
In the [Cu(EMB)(Gly)] þ complex, the Cu39-O48 bond (1.920 Ð) is shorter than the Cu39-N40 bond (2.042 Ð), due to electrostatic interaction between Cu 2þ and O -of glycinate in the complex.The bonds between Cu 2þ and N atoms of ethambutol in the mixed-ligand complex are nearly the same length as those in [Cu(EMB)H -2 ].The bonds attached to nitrogen atoms N7 and N9 as N7-C11, N7-C4, N9-C13, and N9-C1 are elongated in the mixed-ligand complex due to the coordination of N7 and N9 to Cu 2þ (Table 3).
The bond angles around Cu(II) in [Cu(EMB)(Gly)] þ as N40-Cu39-O48, N40-Cu39-N7, O48-Cu39-N9 and N9-Cu39-N7 are 84.07 � , 101.09 � , 89.28 � and 85.53 � , respectively.They show deviation from the regular square planar geometry bond angles due to some distortion in the mixed-ligand complex.The bond angles attached to ethambutol nitrogen donor atoms C13-N9-C1 and C11-N7-C4 decrease in the complex, from the coordination of N7 and N9 of ethambutol to Cu(II).The decrease in bond angles N7-C4-C1 and N9-C1-C4 is more significant in the complex, from the coordination of both N7 and N9 squeezing these angles.The optimized geometry of [Cu(EMB)(Gly)] þ showed the formation of a hydrogen bond between O37 and H42 (Figure 4).The formation of this hydrogen bond affects the geometry around the Cu ion of these complexes and supports the distortion from square-planar geometry.Also, a hydrogen bond is formed between O35 and H10 of the EMB in the formation of [Cu(EMB)(Gly)] þ (Figure 5).The optimized geometry of the free EMB ligand showed the presence of a hydrogen bond between N9 and H38 (Figure 3).

Molecular docking analysis
Molecular docking analysis was used to understand the interactions of EMB, [Cu(EMB)H -2 ], and [Cu(EMB)(Gly)] 1 with biological targets like protein molecules.EMB, [Cu(EMB)H -2 ] and [Cu(EMB)(Gly)] 1 were docked into the mycobacterium tuberculosis (PDB ID: 5uhg) receptor.According to the docking results the binding energy and inhibition constant (Ki) that corresponds to the interaction of EMB, [Cu(EMB)H -2 ] and [Cu(EMB)(Gly)] 1 with 5uhg are listed in Table 11.The interaction of EMB with 5uhg is due to conventional hydrogen bonding and pi-alkyl (hydrophobic) as shown in Figure 5.The interaction of [Cu(EMB)H -2 ] and [Cu(EMB)(Gly)] 1 with 5uhg are due to conventional hydrogen bonding, pi-alkyl (hydrophobic), carbon-hydrogen bonding, and pi-cation (electrostatic) as shown.Based on the binding energy values listed in Table 11, the strongest interaction with 1SC7 was observed for [Cu(EMB)(Gly)] 1 .
a M, L and H are stoichiometric coefficients corresponding to Cu(EMB), peptide and H þ , respectively.b Standard deviations are given in parentheses.c pK H of induced ionization of peptide hydrogn (log b 110 -log b 11-1 ).d Sum of square of residuals.
it does not dissociate in the measurable pH range.Analysis of the potentiometric results indicates the formation of Cu(EMB)(L)(H), Cu(EMB)(L), and Cu(EMB)(LH -1 ).
1 and [Cu(EMB)(GlyGly)H -1 ] were carried out by Gaussian 09 at the B3LYP level of theory using the basis set LANL2DZ for Cu and standard basis set 6-311 G � for C, H, N and O atoms.The optimized structures of the ligand and the complexes are given in Figure3.The computational parameters for the optimized molecular geometry as bond angles and bond lengths are determined and given in Tables 5-9.

Table 1 .
Formation constants of the binary and ternary complexes involving Cu(II), (EMB), and amino acids systems at 25 � C and 0.1 M NaNO 3 .
c Sum of the square of residuals.dTaken from ref.14.

Table 2 .
Stability constants of the ternary complexes of Cu(EMB)-peptide complexes at 25 � C and 0.1 M NaNO 3 .

Table 3 .
The stability constants of Cu(EMB)-DNA constituents at 25 � C and 0.1 M NaNO 3 .
a Standard deviations are given in parentheses.b Sum of the square of residuals.

Table 4 .
Effects of dioxane on the formation constants of Cu(II)-EMB complex at 25 � C and 0.1 M NaNO 3 .pK a of coordinated ethambutol hydroxyl groups (pK 11-2 or pK 11-2 ).
a Standard deviations are given in parentheses.b c Sum of the square of residuals.

Table 5 .
Selected geometrical parameters for EMB, [Cu(EMB)H -2 ] and [Cu(EMB)(Gly)] þ according to B3LYP level of theory using the bases set LANL2DZ for Cu and standard basis set 6-311 G � for C, H, N, and O atoms.
a the corresponding calculated value of the free ligand (EMB).

Table 11 .
The docking interaction calculations of the active sites of the receptor of mycobacterium tuberculosis (PDB ID: 5UHG).