Journal of Solution Chemistry

Mixed Ternary Complexes of Sparfloxacin with Some Biologically Important Amino

Acids
--Manuscript Draft--

Manuscript Number: JOSL-D-20-00150

Full Title: Mixed Ternary Complexes of Sparfloxacin with Some Biologically Important Amino
Acids

Article Type: Original Research

Keywords: Mixed complexes; Amino acids; Binding constant; Sparfloxacin

Abstract: The potentiometric measurements for the interaction of sparfloxacin (SPFX) and metal
ions Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with Amino acids (AA) aspartic acid, alanine,
valine, glutamic acid, threonine, methionine, arginine, phenylalanine were studied. The
formation constant values for the 1:1 binary complexes in addition to the ternary ones
in 1:1:1, in 10% (v/v) ethanol-water mixture, I = 0.1 mol L-1 KNO3 and at 25 oC. The
values are refined using HYPERQUAD computer program. The binding constants of
M(II)-SPFX binary complexes with bovine serum albumin using absorption titration
experiments were determined.

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1
2
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5 Mixed Ternary Complexes of Sparfloxacin with Some Biologically Important
6
7 Amino Acids
8
9
10 R. M. Kamel*, W. H. Hegazy, R. S. Ali and A. A. El-Sayed
11
12
13 Chemistry Department, Faculty of Science, Suez University, Suez 43518, Egypt
14
15
16 Abstract
17
18
19 The potentiometric measurements for the interaction of sparfloxacin (SPFX) and
20
21 metal ions Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with Amino acids (AA) aspartic
22
23 acid, alanine, valine, glutamic acid, threonine, methionine, arginine, phenylalanine
24
25 were studied. The formation constant values for the 1:1 binary complexes in
26
27 addition to the ternary ones in 1:1:1, in 10% (v/v) ethanol-water mixture, I = 0.1
28
29
30
mol L-1 KNO3 and at 25 oC. The values are refined using HYPERQUAD computer
31
32
program. The binding constants of M(II)-SPFX binary complexes with bovine
33
34 serum albumin using absorption titration experiments were determined.
35
36
37 Keywords: Mixed complexes; Amino acids; Binding constant; Sparfloxacin.
38
39
40 *Corresponding author: rashamoka@yahoo.com
41
42
43 1. Introduction
44
45
46 Metals have vital function in human body, also metal complexes of drugs
47
48 play an important role in drug metabolism than drug alone [1]. Mixed ligand
49
50 complexes formed between two different types of bioligands may be considered as
51
52 models for metal-enzyme interactions. The study of ternary complexes with
53
54 biological important molecules such as amino acids and pharmaceutical drugs has
55
56 much interest in various biological and analytical processes [2].
57
58
59
60 1
61
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4
5
Sparfloxacin is a third generation quinolone antimicrobial drug and is
6
7 mainly used for the treatment of acute exacerbations of chronic bronchitis and
8
9 community-acquired pneumonia [3] due to its good bioavailability and its long
10
11 half-life permitting once-daily dosing, which may contribute to improved
12
13 adherence to therapy.
14
15
16 In the present paper the result of the potentiometric measurements for the
17
18 interaction of sparfloxacin and metal ions Cu (II), Cd (II), Co (II), Zn (II) or Ni (II)
19
20 with amino acids, alanine, valine, glutamic acid, aspartic acid, threonine,
21
22
23
methionine, arginine and phenylalanine have been evaluated in 10 % ethanol –
24
25 water mixture. The formation of various 1:1:1 ternary complex species are inferred
26
27 from the potentiometric pH titration curves. Initial estimates of the stability
28
29 constants of the resulting species have been refined with the HYPERQUAD
30
31 computer program [4]. Furthermore, the formation constants values of the different
32
33 1:1 M (II)–sparfloxacin or amino acids have been determined under identical
34
35 conditions. This is made with the aim to compare the stability of the formed 1:1:1
36
37 ternary complexes with of the corresponding 1:1 binary metal complexes.
38
39
40
41
2. Experimental
42
43 2.1 Materials and Solutions
44
45
46
47
Amino succinic acid C4H7NO2 (Aspartic acid), 2-amino-propanoic acid
48
49 C3H7NO2 (Alanine), 2-amino-3-methylbutanoic acid C5H11NO2 (Valine), 2-
50
51 Aminopentanedioic acid C5H9NO4 (Glutamic acid), (2S,3R)-2-amino-3-
52
53 hydroxybutanoic acid C4H9NO3 (Threonine), 2-amino-4-methyl thiobutanoic acid
54
55 C5H11NO2S (Methionine), 2-amino 5-guanidino pentanoic acid C6H14N4O2
56
57 (Arginine) and 2-Amino-3-phenylpropanoic acid C9H11NO2 (Phenylalanine) were
58
59
60 2
61
62
63
64
65
 1
2
3
4
5
of ≥ 99.0%, purchased from Sigma chemical Co. and were used without further
6
7 purification.
8
9
10 5-amino-1-cyclopropyl-7-[(3R,5S)3,5-dimethyl piperazine-1-yl]-6.8-
11
12 difluoro-4-oxo-quinoline-3-carboxylic acid (C19H22F2N4O3) (Sparfloxacin) (SPFX)
13
14 99.0% was purchased from Sigma chemical Co. and was used without purification
15
16 [Scheme 1].
17
18
19 <Scheme 1>
20
21
22 For a stock solution of 1.0 x 10-2 molL-1 of SPFX, 0.3924 g of solid ligand
23
24 was dissolved in 100 cm3 ethanol while 10-2 molL-1 stock solutions of amino acids
25
26 were prepared using bi-distilled water.
27
28
29 Metal salts Co(NO3)2.6H2O, Ni(NO3)2.6H2O, Cu(NO3)2.6H2O,
30
31 Zn(NO3)2.6H2O, and Cd(NO3)2 were supplied by Sigma Chemicals Company.
32
33
34 Stock solutions (1.0 x10-2 molL-1) of metal salts were prepared by dissolving
35
36 precisely weighed amount of the salt in bi-distilled water. The concentrations of
37
38
39
the metal ion stock solutions were determined complexometrically by
40
41 ethylenediamine tetracetic acid dissodium salt (Na2EDTA) using suitable
42
43 indicators [5].
44
45
46 Tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer solution was
47
48 prepared using bi-distilled water. All experiments involving the interaction of
49
50 M(II)-SPFX complexes with bovine serum albumin were carried out in Tris-HCl
51
52 buffer solution (5.0 mM Tris-HCl, pH 7.4).
53
54
55 The direct method for determining albumin concentrations is utilizing a
56
57 neutral buffered solution of bromocresol (BC) as the dye binding indicator. At pH
58
59
60 3
61
62
63
64
65
 1
2
3
4
5
4.2, bromocresol reacts with bovine serum albumin to form an intense blue
6
7 complex. The absorbance of the albumin-BC complex is measured
8
9 spectrophotometrically at 670 nm and is proportional to the bovine serum albumin
10
11 concentration. Add 200 mL of bromocresol (BC) to 10 μg bovine serum albumin
12
13 and 5 minute incubation at room temperature and measure absorbance at 670 nm.
14
15
16 2.2 Instrumentation
17
18
19 Potentiometric-pH measurements were performed of the solutions in a
20
21 double-walled glass vessel at (25.0 ± 0.1) oC using Fisher pH meter model 325
22
23 MP, and a magnetic stirrer was used. The electrode used was calibrated using
24
25 standard buffer solutions.
26
27
28 To transform the pH values read for each sample, H(R), into real pH, pH* (-
29
30 logaH), the following equation was used:
31
32
33 pH* = pH(R)-δ (1)
34
35
36 according to the notation of Bates et al. The value of δ (± 0.002) have also been given
37
38
39
by Bates et al. [6] for 10% (v/v) ethanol/water mixtures.
40
41
42
A Shimadzu-1601PC UV-Visible automatic recording spectrophotometer
43
44 with 1 cm quartz cell was used for the absorbance and spectral measurements.
45
46 2.3 Procedure for Potentiometric Measurements
47
48
49 The stability constants of binary metal ion-sparfloxacin (SPFX) and amino
50
51
52
acid (AA) were determined by titration of 25.0 mL of aqueous solution (4.0 x10-3
53
54
mol L-1 HNO3 + 1.0 x10-2 molL-1 KNO3 + 1.0 x10-3 molL-1 of sparfloxacin (SPFX),
55
56 or amino acids in the absence or in the presence of metal ion with standard
57
58 carbonate free KOH solution at t = 25.0  0.1C. Titrations were carried out using
59
60 4
61
62
63
64
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 1
2
3
4
5
1:1 molar ratio of concentrations of metal ion to sparfloxacin (SPFX) or amino
6
7 acids.
8
9 For the ternary systems studied in 10 % (v/v) ethanol–water mixture
10
11
12
solutions containing metal ion: sparfloxacin (SPFX) (ligand 1): amino acids
13
14 (Aspartic acid, Threonine, Phenylalanine, D-Alanine, Glutamic acid, Valine,
15
16 Arginine and Methionine) (ligand 2) in 1:1:1 molar concentration ratios were
17
18 potentiometrically titrated with standard carbonate free KOH solution at I = 0.1
19
20 molL-1 KNO3, t = 25.0  0.1C. The solutions titrated can be presented according to
21
22 the following scheme:
23
24
25 (a) 4.0 x10-3 mol L-1 HNO3 + 1.0 x10-3 mol L-1 SPFX (ligand 1).
26
27
28 (b) 4.0 x10-3 mol L-1 HNO3 + 1.0 x10-3 mol L-1 amino acids (Aspartic acid,
29
30 Threonine, Phenylalanine, D-Alanine, Glutamic acid, Valine, Arginine and
31
32 Methionine) (ligand 2).
33
34
35
(c) Solution (a) + 1.0 x10-3 mol L-1 M (II).
36
37 (d) Solution (b) + 1.0 x10-3 mol L-1 M (II).
38
39
40 (g) 4.0 x10-3 molL-1 HNO3 + 1.0 x10-3 molL-1 SPFX + 1.0 x10-3 molL-1 amino
41
42 acids + 1.0 x10-3 molL-1 M (II).
43
44
45 A constant ionic strength was obtained with 0.1 molL-1 KNO3 and the total
46
47 volume was kept constant at 25.0 mL.
48
49 The calculation of the stability constants of binary complexes having 1:1
50
51
52 molar ratios of metal ion-Amino acid or metal ion-SPFX consider the equilibrium
53
54 M + HL ML + H+ (2)
55
56 M
57 So K can be calculated using the following equations [7]:
58 ML
59
60 5
61
62
63
64
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 1
2
3
4
5
6
7
8
9
10
11
12
13
14 Where CL = the total molar concentration of the ligand under investigation, CM =
15
16
17
the total molar concentration of the metal ion and Ka = the dissociation constant
18
19 value of the ligand under investigation.
20
21 For the calculation of the stability constants of the ternary complexes having
22
23
24
1:1:1 molar ratios of M(II)-SPFX-AA which take place according to stepwise
25
26 mechanism, consider the equilibrium
27
28 ML + HA MLA + H+ (5)
29
30 M
31 So K MLA be calculated using the following equations [7]:
32
33
34
35
36
37
38
39
40
41
42
43
44 Where CA = the total molar concentration of the secondary ligand, CM = the total
45
46 molar concentration of the metal ion and Ka (A) = the dissociation constant value
47
48
49
of the secondary ligand.
50
51 For the calculation of the stability constants of the monoprotonated ternary
52
53 complexes, consider the equilibrium, where ligand A acquires two values of
54
55 dissociation constants Ka, K2a.
56
57
58 M + A + HL M(HL)A (8)
59
60 6
61
62
63
64
65
 1
2
3
4 M
5
So K M(HL)A be calculated using the following equations [7]:
6
7 M TM - [L] XA
8 K (9)
9 M(HL)A = [L]3 . XA 2
10
11
12
13 6 TM - (2 mTM - 2[H+] + 2[OH-]
14 [L] = (10)
15 AL . XA / XL) + (XA / XL)
16
17
18
19
XA = [H+]2 / Ka K2a + ([H+] / K2a) + 1 (11)
20
21
22 A = (3[H+]2 / Ka K2a + ([H+] / K2a) - 1 (12)
23
24
25 [H+] [H+]
26 L = XL = +1
27 Ka Ka
28
29
30 Where TM = the total molar concentration of the metal ion, Ka = the first
31
32 dissociation constant value of the deprotonated ligand and K2a = the second
33
34
35
dissociation constant value of the deprotonated ligand.
36
37 Initial estimates of the formation constants of the resulting species have been
38
39 refined with the HYPERQUAD computer program [4].
40
41
42 2.4 Procedure for Spectrophotometric Measurements
43
44
45 The UV/Vis absorption were conducted at room temperature and allowed to
46
47 stand for 10 min. The measurements were conducted in 1% (v/v) ethanol water
48
49 mixture according to the following scheme:
50
51
52 (a) 1.0 x10-4 molL-1 of SPFX
53
54
55 (b) 1.0 x10-4 molL-1 SPFX + 1.0 x 10-4 molL-1 M (II).
56
57
58
(c) 1.0 x10-4 molL-1 SPFX + 1.0 x10-4 molL-1 Amino Acids + 1.0 x 10-4 molL-1
59
60 7
61
62
63
64
65
 1
2
3
4
5
M(II).
6
7 The free ligand solutions were scanned from 195 to 400 nm against 1% (v/v)
8
9 ethanol water mixture as a blank. For the binary and ternary metal ion complexes
10
11
12
the same procedure was operated.
13
14 2.5 Evaluation of bovine serum albumin binding constant
15
16
17 Absorption titration experiments were performed with constant
18
19 concentrations of the compounds ([M2+] =1.0 x10-4 molL-1 and [SPFX] = 1.0 x10-4
20
21 molL-1) with varying concentration of bovine serum albumin (0-80 μM). While
22
23 measuring the absorption spectra, an equal amount of bovine serum albumin was
24
25 added to both the test solution and the reference solution to eliminate the
26
27
28
absorbance of bovine serum albumin itself. The values of the intrinsic binding
29
30 constants Kb of bovine serum albumin with metal complexes were calculated by
31
32 regression analysis using the equation: [8, 9].
33
34
35
[Albumin] / (εa – εf) = [Albumin] / (εb – εf) + 1/ [Kb (εb – εf)] (13)
36
37 Where [albumin] is the concentration of bovine serum albumin. While εa, εf and εb
38
39 are Aobs / [Complex], molar absorbitivity of the free metal complex and molar
40
41 absorbitivity of the bound metal complex to bovine serum albumin, respectively
42
43
44
and Kb is the equilibrium binding constant. In the plots of [Albumin] / (εa- εf)
45
46 versus [Albumin], Kb is given by the ratio of slope to the intercept.
47
48 3. Result and Discussion
49
50
51 3.1 Metal (II) Complexes of Sparfloxacin with Amino Acids.
52
53
54 Sparfloxacin (SPFX) is a member of the fluoroquinolone family. The acid-
55
56 base properties of Sparfloxacin in 10 % (v/v) ethanol-water mixture and in ionic
57
58
59
strength (0.1 molL-1 KNO3) indicate that the dissociation constant of SPFX pKa1
60 8
61
62
63
64
65
 1
2
3
4
5
corresponding to the ionization of carboxylic group was found to be 6.30 ±0.02
6
7 whereas the value of pKa2 for the nitrogen of piperazine proton was 8.50 ±0.02 [10]
8
9
10 Although the pKa values of the amino acids have already been reported.
11
12 These values are determined for the sake of uniformity in experimental conditions.
13
14 The acidity constants of amino acids determined at 25 oC of arginine (pKa2 = 8.91
15
16 ± 0.02), aspartic acid (pKa2 = 9.77 ± 0.02), methionine (pKa2 = 9.37 ± 0.02), D-
17
18 alanine (pKa2 = 9.54 ± 0.02), glutamic acid (pKa2 = 9.59 ± 0.02), valine (pKa2 =
19
20 9.49 ± 0.02), phenylalanine (pKa2 = 9.11 ± 0.02) and threonine (pKa2 = 8.97 ±
21
22
23
0.02). The pKa values of amino acids agree well with those reported in the
24
25 literature [11].
26
27
28 In continuation of our previous work on ternary complexes containing
29
30 biologically important ligands [10], the stoichiometrics and stability constants of
31
32 the mixed ligand complexes of metal (II) ion of Cu (II), Cd (II), Co (II), Zn (II) or
33
34 Ni (II) with amino acids (AA) arginine, aspartic acid, methionine, D-alanine,
35
36 glutamic acid, valine, phenylalanine and threonine were calculated from the
37
38 titration graphs in which the metal to ligand ratio was 1:1 are listed in Table 1 and
39
40 the results were found to agree well with those reported in the literature [11]. The
41
42
43 conditions for the measurements were the same as for the acidity constants. The
44
45 potentiometric titration curve of some M(II)–SPFX or M(II)–AA systems are
46
47 shown in Figures [1-3].
48
49
50 <Table 1>
51
52
53 <Figure 1>
54
55
56 <Figure 2>
57
58
59 <Figure 3>
60 9
61
62
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 1
2
3
4
5
It is characteristic of aliphatic amino acids (alanine and valine) that they do
6
7 not contain any donor group in the side chain, and thus their protonation and
8
9 complexation equilibria are very similar to those of glycine. Analogous to glycine,
10
11 aliphatic amino acids generally act as bidentate ligands via the coordination of
12
13 amino-N and carboxylate-O donors.
14
15
16 Valine is also a bidentate ligand, but the larger distance between the donor
17
18 groups results in an increased basicity of the carboxylate and amino groups as
19
20 compared to the situation in alanine. On the other hand, valine forms higher
21
22
23
stability constant complexes compared to stability constants of alanine complexes
24
25 [12].
26
27
28 Methionine contains a largely aliphatic side chain that includes a thioether (-
29
30 S-) group. Sulfur atoms are soft bases and interact most favorably with soft metal
31
32 ions [1]. This softness that furnishes the basis for interaction of thioethers with
33
34 metal ions. The ether sulfur will interact best with soft metal ions, marginally with
35
36 borderline metal ions, and not at all with hard metal ions [13]. Most borderline
37
38 metal ions consistently form slightly weak complexes with methionine than with
39
40 alanine. The thioether group in methionine does not contribute importantly in
41
42
43 complex stability, it chelate borderline metal ions primarily as substituted
44
45 glycinates.
46
47
48 Threonine contains aliphatic hydroxyl groups, it can be thought of as a
49
50 hydroxylated version of valine with a hydroxyl group in place of one of the valine
51
52 methyl groups. The hydroxyl groups on threonine make it much more hydrophilic
53
54 and reactive than valine [14]. As indicated in Table 4 a weak chelation is indicated
55
56 to metal ions Co(II), Ni(II), Zn(II) and Cd(II), whereas little chelation is evident
57
58 with Cu(II), which infers a stronger alcohol chelation to Cu(II) [11].
59
60 10
61
62
63
64
65
 1
2
3
4
5
Arginine has relatively long side chains that terminate with groups that are
6
7 positively charged at neutral pH, it is capped by a guanidinium group. The
8
9 guanidino side chain of arginine, like guanidine itself, is only very weakly acidic,
10
11 the loss of the first proton can be ascribed to the carboxyl group and the pKCOOH
12
13 changes in accordance with the electron-withdrawing effect of the ammonium
14
15 group(s) and the electron-repelling effect of the alkyl chain.
16
17
18 Glutamic acid and aspartic acid can release totally three protons, two protons
19
20 from carboxyl groups and one from amine group. Glutamic acid and aspartic acid
21
22
23
chelate metal ions weakly via the amino nitrogen and carbonyl oxygen. A stronger
24
25 chelation occurs upon amide nitrogen bound hydrogen by some metal ions such as
26
27 Cu2+ [15].
28
29
30 In contrast to increasing of basicity from Asp to Glu, show the stability of
31
32 regarding complexes a decreasing trend to compare the stability constants of two
33
34 species M(Glu) and M(Asp). It could easily distinguish that those constants of
35
36 M(Asp) are generally larger than those of the corresponding M(Glu) species.
37
38
39 Phenylalanine is purely hydrophobic, behaves practically as bifunctionaly
40
41 acids. The weak basicity of phenylalanine does not provide a basis for its
42
43
44
coordination, hence this ligand is capable only of normal bidentate (N, O) –
45
46 coordination [11].
47
48
49 The stability constants for the ternary systems were computed from the
50
51 titrations in which the concentrations of M(II) : SPFX : AA were kept in the ratio
52
53 1: 1 : 1, listed in Table 2. The experimental data show that the formation of the
54
55 ternary complex M-SPFX-AA.
56
57
58 There following equilibria were used
59
60 11
61
62
63
64
65
 1
2
3
4
5 [M-SPFX] + H2-AA [M-SPFX-AA]+ 2H+ (14)
6
7
8 AA = amino acids (alanine, valine, glutamic acid, aspartic acid, threonine,
9
10 methionine, arginine and phenylalanine).
11
12
13 <Table 2>
14
15
16 To the author's knowledge no data for the ternary complexes containing
17
18 SPFX with the amino acids alanine, valine, glutamic acid, aspartic acid, threonine,
19
20 methionine, arginine and phenylalanine are available in the literature for
21
22
23
comparison.
24
25
26
To calculate the initial estimates of the formation constants of the ternary
27
28 complexes of Cu (II), Cd (II), Co (II), Zn (II) or Ni (II) with sparfloxacin (SPFX)
29
30 and amino acids (AA) alanine, valine, glutamic acid, aspartic acid, threonine,
31
32 methionine, arginine and phenylalanine, the following equilibria were used:
33
34
35 [Mp(SPFX)q (AA)r]
36 K M(SPFX)(AA) = (15)
37
38 [MP(SPFX)q] [AA]r
39
40
41 Which refers to the addition of AA to the binary complex MP(SPFX)q
42
43
44 The overall complexation reaction involving protonation is
45
46
47 p M + q SPFX + r AA + s H
48
Mp (SPFX)q (AA)r (H)s (16)
49
50
51 Mp (SPFX)q (AA)r (H)s
52 pqrs = (17)
53 [M]p [(SPFX)]q [(AA)]r [(H)]s
54
55
56
57 In which SPFX = Sparfloxacin, AA = Amino Acids ligand alanine, valine,
58
59 glutamic acid, aspartic acid, threonine, methionine, arginine and phenylalanine and
60 12
61
62
63
64
65
 1
2
3
4
5
M = Co(II), Zn(II), Cd(II), Ni(II) and Cu(II) 10% ethanol-water mixture solvent.
6
7
8 The data collected in the pH range 3.0 – 11.0 were used for the calculations
9
10 and refinements. The formation constants of all the ternary complexes studied are
11
12 given in Table 2.
13
14
15 The formation constants for M(II)-SPFX-arginine and M(II)-SPFX-
16
17 phenylalanine in 1:1:1 ratio are calculated based on that the titration curve lies
18
19 between the corresponding binary curves for M(II)-SPFX and M(II)-AA, i.e. the
20
21 formed complex species is of monoprotonated type M(II)(SPFX)(H-AA) where the
22
23 arginine or phenyl alanine reacts as a secondary ligand in its protonated form.
24
25
26 The ternary systems of M(II)-SPFX-Asp and M(II)-SPFX-Glu formed is of
27
28
29
step wise type where glutamic acid or aspartic acid act as a secondary ligand, while
30
31 sparfloxacin act as a primary ligand.
32
33
34 In comparison the stability constants values of M(II)-SPFX-Asp and M(II)-
35
36 SPFX-Asp, it is noticed that the ternary system of M(II)-SPFX-Asp is more stable
37
38 than M(II)-SPFX-Glu
39
40
41 The formation constants of M(II)-SPFX-Asp, follow the order according to
42
43 the metal ion:
44
45
46 Cu (II) > Ni (II) > Co (II) > Zn (II) > Cd(II)
47
48
49 The formation constants of M(II)-SPFX-Glu, follow the order according to
50
51 the metal ion:
52
53
54 Cu (II) > Co (II) > Zn (II) > Ni (II) > Cd(II).
55
56
57
58
59
60 13
61
62
63
64
65
 1
2
3
4
5
The low stability constant of all ternary systems of Cd(II)-SPFX-AA when
6
7 compared with other ternary complexes under study, this may be due to it is larger
8
9 ionic radius (0.95 Å) than other metal Cu(II), Co(II), Ni(II) or Zn(II).
10
11
12 The observed different orders may be attributed to different types of
13
14 interactions depending on metal ion or different geometrical behavior during
15
16 formation of binary and ternary complexes in solution.
17
18
19 Some ΔlogKM values (Table 3) are positives for some ternary complexes
20
21 studied, with the higher values for the formation constants of ternary complexes
22
23 compared with the binary systems being attributed to the inter-ligand interactions
24
25
26
or some cooperativity between the coordinated ligands such as H-bond formation.
27
28
29
<Table 3>
30
31
32
The behavior described above, is better observed in the distribution species
33
34 diagrams as a function of pH of these systems (Fig. 4).
35
36
37 The binary complex of Ni(II)-SPFX starts forming from pH close to 4.5,
38
39 binding the maximum concentration of Ni ions (20%) at pH 5.80, while Cu(II)-
40
41 SPFX complex starts forming from pH close to 3.0, binding the maximum
42
43 concentration of Cu(II) ions (30%) at pH 5. While for The binary complex of
44
45 Ni(II)-Glu binding the maximum concentration of Ni ions (10%) at pH 6.0, while
46
47 Cu(II)-Asp complex, binding the maximum concentration of Cu(II) ions (60%). It
48
49
50
can be seen that in the pH range 3-9 the M(II)-SPFX-AA complexes appears.
51
52
53
<Figure 4>
54
55
56
3.2 Spectroscopic confirmation for the formation of ternary complexes of M(II)-
57
58 Sparfloxacin-amino acids
59
60 14
61
62
63
64
65
 1
2
3
4
5
Figure 5 show the absorption spectra for some ternary complexes of the type
6
7 M(II)-SPFX-Amino Acids. The absorption spectra of the ternary systems under
8
9 study are quite different from of binary M(II)-SPFX complex, emphasizing the
10
11 formation of ternary complexes of the type M(II)-SPFX-Amino Acids.
12
13
14 <Figure 5>
15
16
17 In comparison of ternary complex M(II)-SPFX-Asp and M(II)-SPFX-Glu
18
19 with the binary M(II)-SPFX complex, a marked hypochromic effect is observed in
20
21 the 290 nm band. The interaction of the M(II)-SPFX with aspartic acid or glutamic
22
23 acid cause bathochromic shift of 7 nm in the near UV absorption maximum, owing
24
25
26
to the perturbation of the complexed chromophore system upon binding to aspartic
27
28 acid or glutamic acid [16].
29
30
31 The same behavior is noticed for all ternary systems under study with all
32
33 metal ions Co(II), Zn(II), Cd(II), Ni(II) or Cu(II).
34
35
36 Absorption spectra of Co(II)-SPFX complexes in presence of bovine serum
37
38 albumin are shown in Figure 6. In absence of bovine serum albumin, the spectrum
39
40 is characterized by the peak around 292 nm. As the concentration of bovine serum
41
42 albumin was increased the intensity of this band exhibited slightly bathochromism
43
44 shift and hypochromism for Zn(II)-SPFX, Ni(II)-SPFX, Cd(II)-SPFX complexes,
45
46
47
or exhibited hyperchromism, for Cu(II)-SPFX and Co(II)-SPFX complexes,
48
49 indicates that the complexes bind to bovine serum albumin.
50
51
52 <Figure 6>
53
54
55 The binding strength of the complexes, the intrinsic binding constants Kb of
56
57 complexes with bovine serum albumin were determined from the decay of the
58
59 absorbance monitored for complexes. The intrinsic stoichiometric constants for
60 15
61
62
63
64
65
 1
2
3
4
5
Zn(II) Cu(II), Ni(II) and Cd(II) binding to serum albumin are 7.28 ± 0.60, 11.03 ±
6
7 0.76, 10.1 ± 2.3, 14.4 ± 1.8 respectively [17-18].
8
9
10 The intrinsic binding constant Kb of the complexes with bovine serum
11
12 albumin was evaluated from the Eq. (13) [Figure 7]. The binding constants (Kb) of
13
14 the complexes were listed in Table 4.
15
16
17 <Figure 7>
18
19
20 The order of the binding strength of M(II)-SPFX complexes were obtained as:
21
22
23 Co(II)-SPFX > Cd(II)-SPFX > Zn(II)-SPFX > Ni(II)-SPFX > Cu(II)-SPFX
24
25
26 By comparing the binding constants (Kb) of the complexes and sparfloxacin,
27
28 it is noticed that sparfloxacin have high degree of binding bovine serum albumin
29
30 than M(II)-SPFX complexes.
31
32
33 <Table 4>
34
35
36 4. Conclusion
37
38
39 The formation constants for M(II)-SPFX-AA in 1:1:1 ratio are calculated.
40
41
42
M(II)-SPFX-arginine and M(II)-SPFX-phenylalanine form mono protonated
43
44 complexes M(II)(SPFX)(H-AA) where the arginine or phenylalanine reacts as a
45
46 secondary ligand in its protonated form. While aspartic acid and glutamic acid
47
48 forms step wise ternary complexes where glutamic acid or aspartic acid act as a
49
50 secondary ligand, while sparfloxacin act as a primary ligand. The low stability
51
52 constant of all ternary systems of Cd(II)-SPFX-AA when compared with other
53
54 ternary complexes under study. By comparing the binding constants (Kb) of the
55
56 complexes and sparfloxacin, it is noticed that sparfloxacin have high degree of
57
58
59
binding bovine serum albumin than M(II)-SPFX complexes.
60 16
61
62
63
64
65
 1
2
3
4
5 5. References
6
7
8 [1] Pearson R. G., Hard and Soft Acids and Bases, Dowden, Hutchinson, and Ross,
9
10 Stroudsberg, Pennsylvania (1973).
11
12
13 [2] Bilgic A. D., Karaderi S. Erdogan G., Ternary complex formation of isoniazid
14
15 with some transition metals and amino acids, Trakya Univ J Nat Sci, 14(1):1 -
16
17 14 (2013).
18
19
20 [3] Stephanos J. J., Journal of Inorganic Biochemistry 62, 155-169 (1996).
21
22
23 [4] Gans P., Sabatini A., Vacca, A., Investigation of equilibria in solution.
24
25 Determination of equilibrium constants with the HYPERQUAD suite of programs,
26
27 Talanta 43, 1739-1753 (1996).
28
29
30 [5] Welcher F.J., The Analytical Uses of Ethylene diaminetetraacetic acid, D. Von.
31
32 Nostrand Co.,Inc., Princeton, (1965).
33
34 [6] Bates R. B., Paabo M., Robinson R. A., Interpretation of pH Measurements in
35
36 Alcohol-Water Solvents, J. Phys. Chem. 67, 1833-1845 (1963).
37
38
39 [7] Khan B. T., Raju M., Zakeeruddin S. M., J. Chem. Soc. Dalton Trans 67-71
40
41 (1988).
42
43 [8] Bologni L., Sabatini A., Vacca A.: Complex formation equilibria between 2-
44
45
46 amino-2(hydroxymethyl)-1,3,-propanediol (tris, tham) and nickel(II), copper(II),
47
48 zinc(II) and hydrogen ions in aqueous solutions, Inorg. Chim. Acta 69, 71-75
49
50 (1983).
51
52
53
[9] Wolfe A., Shimer G.H., Meehan T.: Polycyclic aromatic hydrocarbons
54
55 physically intercalate into duplex regions of denatured DNA, Biochem. 26, 6392-
56
57 6396 (1987).
58
59
60 17
61
62
63
64
65
 1
2
3
4
5
[10] Kamel R. M., Hegazy W. H., Ali R. S., El-Sayed A. A., Sparfloxacin Metal
6
7 Complexes with Nucleosides: Potentiometric and Spectral Studies, J. Solution
8
9 Chem 46:1680–1697 (2017).
10
11
12
[11] Sigel H., Metal ions in biological system, Marcel Dekker, INC., New York,
13
14 Vol. 8 (1979).
15
16
17 [12] SóVagó I., Kiss T., Gergely A., Critical survey of the stability constants of
18
19 complexes of aliphatic amino acids, Pure & App. Chem. 65(5) 1029 (1993).
20
21
22 [13] Martin R. B., Metal Ions in Biological Systems Marcel Dekker, INC., New
23
24 York, Vol 9 (1979).
25
26
27 [14] Berg J. M., Tymoczko J. L., Stryer L., The Molecular Design of Life-Protein
28
29 Structure and Function, Biochemistry, 5th Edition, (2003).
30
31
32 [15] Sajadi S. A. A., Natural Science 2, 85-90 (2010).
33
34
35 [16] Yegorova A. V., Scripinets Y. V., Duerkop A., Karasyov A. A., Antonovich
36
37 V. P., Wolfbeis O. S.. J. Anal. Chim. Acta 584, 260-267 (2007).
38
39
40 [17] Masuokal J., Saltman P. Zinc(I1) and Copper(I1) Binding to Serum Albumin.
41
42 J. Biological Chemist 269(41), 25557-25561(1994).
43
44
45 [18] Guthans S. L., Morgan W. T. The interaction of zinc, nickel and cadmium
46
47
48
with serum albumin and histidine-rich glycoprotein assessed by equilibrium
49
50 dialysis and immunoadsorbent chromatography, Archives of Biochemistry and
51
52 Biophysics 218 (1) 320-328(1982).
53
54
55
56
57
58
59
60 18
61
62
63
64
65
 1
2
3
4
5
6
7
8
9
10
11 Figure Captions
12
13
14 Fig. 1: pH against volume of 0.042 molL-1 KOH for Co (II) + Glutamic acid +
15
16 Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and
17
18 at 25.0 oC.
19
20 Fig. 2: pH against volume of 0.042 molL-1 KOH for Co (II) + Methionine +
21
22
23
Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and
24
25
at 25 oC.
26
27 Fig. 3: pH against volume of 0.042 molL-1 KOH for Cu (II) + Glutamic acid +
28
29 Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and
30
31 at 25 oC.
32
33
34 Fig. 4 Electronic absorption spectra of Cd (II)-SPFX complex with amino acids
35
36 (AA) under study, [Cd2+] = 1×10-⁴ molL-1, [SPFX] = 1×10-⁴ molL-1 and [AA] =
37
38 1×10-⁴ molL-1.
39
40
41 Fig. 5: Electronic absorption spectra of Co(II)-SPFX complex in Tris-HCl buffer
42
43 (pH 7.4) in the absence and presence of increasing amount of Bovine serum
44
45
46
albumin, [Co2+] = 1 × 10-⁴ molL-1 and [SPFX] = 1 × 10-⁴ molL-1.
47
48
49
Fig. 6: Plot of [Albumin] / (εa – εf) vs [Albumin] for absorption titration of Bovine
50
51 serum albumin and SPFX or Cu (II)-SPFX complex in Tris-HCl buffer (pH 7.4) (at
52
53 λabs = 299 nm).
54
55
56 Table Captions
57
58
59
60 19
61
62
63
64
65
 1
2
3
4
5
Table 1: Formation constants for M (II) + Amino Acids (AA) (1:1) binary
6
7 complexes, in 10% (v/v) ethanol water mixture, I = 0.1 molL-1 KNO3 and at 25.0 ±
8
9 0.1oC.
10
11
12 Table 2: Formation constants for M (II) + Sparfloxacin + Amino Acids (1:1:1)
13
14 ternary complexes, in 10% (v/v) ethanol water mixture, I = 0.1 molL-1 KNO3 and at
15
16 25.0 ± 0.1oC.
17
18
19 Table 3: ΔLog10KM values for the 1:1:1 M (II) + SPFX + Amino Acid (AA)
20
21 Ternary Complexes as Determined by Potentiometric Titrations in 10% (v/v)
22
23 ethanol water mixture, I = 0.1 molL-1 KNO3 and at 25.0 ± 0.1oC.
24
25
26 Table 4: Binding constants of Bovine serum albumin with SPFX and M(II)-SPFX
27
28
29
complexes at 25.0 oC in Tris-HCl buffer (pH 7.4).
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 20
61
62
63
64
65
Figure Click here to access/download;Figure;List of Figure.doc

12

10

8
pH

2
0 1 2 3 4 5 6 7 8 9

V (mL)

Fig. 1: pH against volume of 0.042 molL-1 KOH for Co (II) + Glutamic acid +
Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and at
25 oC.
(a) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid ()
(b) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid + 1 x10-3 molL-1 Co (II) ()
(c) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX (▲)
(d) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX + 1 x10-3 molL-1 Co (II) (◊)
(e) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid +1 x10-3 molL-1 SPFX + 1
x10-3 molL-1 Co (II) (┼)
 12

10

8
pH

2
0 2 4 6 8

V (mL)

Fig. 2: pH against volume of 0.042 molL-1 KOH for Co (II) + Methionine +

Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and at
25 oC.
(a) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Methionine ()
(b) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Methionine + 1 x10-3 molL-1 Co (II) ()
(c) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX (▲)
(d) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX + 1 x10-3 molL-1 Co (II) (◊)
(e) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Methionine + 1 x10-3 molL-1 SPFX + 1
x10-3 molL-1 Co (II) (┼).
 12

10

8
pH

2
0 1 2 3 4 5 6 7 8 9

V (mL)

Fig. 3: pH against volume of 0.042 molL-1 KOH for Cu (II) + Glutamic acid +
Sparfloxacin system at 0.1 molL-1 KNOз in 10 % (v/v) ethanol water mixture and at
25 oC.
(a) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid ()
(b) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid + 1 x10-3 molL-1 Co (II) ()
(c) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX (▲)
(d) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 SPFX + 1 x10-3 molL-1 Co (II) (◊)
(e) 4 x10-3 molL-1 HNOз + 1 x10-3 molL-1 Glutamic acid + 1 x10-3 molL-1 SPFX + 1
x10-3 molL-1 Co (II) (┼).
Fig. 4: Distribution diagrams as a function of pH for the system M(II)-SPFX-AA in
the ratio 1:1:1 at 25.0 oC and at I = 0.1 molL-1 KNO3. Where M(II) =Cu2+, Ni2+ and
AA = Aspartic acid, Glutamic acid.
 1.2
Cd(II)-SPFX
Cd(II)-SPFX-Ala
Cd(II)-SPFX-Arg
Cd(II)-SPFX-Asp

Absorbance (a.u.)
0.9
Cd(II)-SPFX-Glu
Cd(II)-SPFX-Met
Cd(II)-SPFX-phe
0.6 Cd(II)-SPFX-Thr
Cd(II)-SPFX-Val

0.3

250 300 350 400

Wavelength (nm)

Fig. 5: Electronic absorption spectra of Cd (II)-SPFX complex with amino acids

(AA) under study, [Cd2+] = 1×10-⁴ molL-1, [SPFX] = 1×10-⁴ molL-1 and [AA] =
1×10-⁴ molL-1.
 1.2
Co(II)-SPFX
1.0
50  M Albumin
30  M Albumin

Absorbance (a.u)
0.8
20  M Albumin
10  M Albumin
0.6
5  M Albumin
0  M Albumin
0.4

0.2

0.0
250 300 350 400

Wavelength (nm)

Fig. 6: Electronic absorption spectra of Co(II)-SPFX complex in Tris-HCl buffer (pH

7.4) in the absence and presence of increasing amount of Bovine serum albumin,
[Co2+] = 1 × 10-⁴ molL-1 and [SPFX] = 1 × 10-⁴ molL-1.
 -8
1.0x10 SPFX only

-9
8.0x10

[Albumin] / (a-f)
-9
6.0x10

-9
4.0x10

-9
2.0x10

0.0
0.0 1.0x10
-5
2.0x10
-5 -5
3.0x10 4.0x10
-5
5.0x10
-5

-1
[Albumin] (molL )

-9
9.0x10

Cu(II)-SPFX
-9
8.0x10

-9
[Albumin] / (a-f)

7.0x10

-9
6.0x10

-9
5.0x10

-9
4.0x10

-9
3.0x10

-6 -5 -5 -5 -5 -5
5.0x10 1.0x10 1.5x10 2.0x10 2.5x10 3.0x10
-1
[Albumin] (molL )

Fig. 7: Plot of [Albumin] / (εa – εf) vs [Albumin] for absorption titration of bovine
serum albumin and SPFX or Cu (II)-SPFX complex in Tris-HCl buffer (pH 7.4) (at
λabs = 299 nm).
 NH2 O O
F
OH
H3C
N N
HN F

CH3

Scheme 1: Chemical structure of sparfloxacin (SPFX)

Table Click here to access/download;Table;List of Table.doc

Table 1: Formation constants for M (II) + Amino Acids (AA) and overall
formation constants for M(II) + Sparfloxacin (SPPFX) (1:1) binary complexes, in
10% (v/v) ethanol water mixture, I = 0.1 molL-1 KNO3 and at 25.0 ± 0.1oC.

Metal ion M(II) Cu(II) Ni(II) Co(II) Zn(II) Cd(II)

log10K 8.92 ± 0.03 7.65 ± 0.02 6.41 ± 0.02 6.31± 0.04 4.72± 0.03
M (II) (Aspartic acid)

log10K
M (II) (Glutamic acid) 8.04 ± 0.04 6.17± 0.02 5.30± 0.03 5.26± 0.03 4.59± 0.02

log10K
M (II) (Alanine) 7.61 ± 0.03 5.44 ± 0.03 4.27 ± 0.02 4.96 ± 0.02 4.04± 0.03

log10K
M (II) (Arginine) 6.16 ± 0.04 4.38 ± 0.03 3.45 ± 0.03 4.41 ± 0.02 3.30 ± 0.03

log10K 6.88 ± 0.03 4.61 ± 0.03 3.64± 0.02 4.82 ± 0.02 3.70± 0.02
M (II) (Methionine)

log10K 7.23± 0.02 4.69 ± 0.04 3.86 ± 0.04 4.83 ± 0.03 3.53± 0.03
M (II) (Phenylalanine)

log10K 8.22± 0.02 5.24± 0.03 4.61± 0.02 5.39 ± 0.02 3.91± 0.04
M (II) (Valine)

log10K 8.11± 0.02 5.67± 0.03 4.41± 0.04 4.99 ± 0.04 4.09 ± 0.03
M (II) (Theronine)

log 11.94 ± 0.02 9.31 ± 0.02 9.21 ± 0.03 9.41 ± 0.02 8.62 ± 0.02
M(II)(SPFX)

± refers to three times standard deviation (3s)

 Table 2: Formation constants for M (II) + Sparfloxacin + Amino Acids (1:1:1)
ternary complexes, in 10% (v/v) ethanol water mixture, I = 0.1 molL-1 KNO3 and at
25.0 ± 0.1oC.

Metal ion
Cu(II) Ni(II) Co(II) Zn(II) Cd(II)
M(II)
log10K
M(II)-SPFX- Asp 9.39 ± 0.03 a 8.36 ± 0.03 a 8.21± 0.03 a 7.96± 0.03 a 7.55± 0.03 a

log10K 8.50± 0.03 a 7.00± 0.03a 7.42± 0.03 a 7.34± 0.03 a 6.66± 0.03 a
M(II)-SPFX-Glu

log10K
4.38 ± 0.03 a 4.49 ± 0.03 a 4.66 ± 0.03 a 4.66 ± 0.03 a
M(II)-SPFX-Ala 6.37 ± 0.03 a
14.24±0.03b 14.24±0.03b 10.68±0.03b 15.04±0.03b
log10K
M(II)-SPFX-Arg 15.48±0.03b 16.88±0.03b 13.76±0.03b 18.67±0.03b 14.02±0.03b

log10K
M(II)-SPFX-Met 7.42 ± 0.03 a -- -- 23.01±0.03b --

log10K b
5.17 ± 0.03 a 4.72 ± 0.03a 5.09 ± 0.03 a 4.14 ± 0.03 a
M(II)-SPFX-Phe 12.64±0.03
16.77±0.03b 15.37±0.03b 15.31±0.03b 17.76±0.03b

log10K
5.47 ± 0.03 a
M(II)-SPFX-Val 6.88 ± 0.03 a 5.54 ± 0.03a 13.06±0.03b 4.51 ± 0.03 a
15.39±0.03b

log10K
5.93 ± 0.03 a
M(II)-SPFX-Thr -- -- -- 17.61±0.03b
20.35±0.03b
± refers to three times standard deviation (3s)
a
log formation constant of normal ternary complex
b
log formation constant of protonated complex.
Table 3: ΔLog10KM values for the 1:1:1 M (II) + SPFX + Amino Acid (AA)
Ternary Complexes as Determined by Potentiometric Titrations in 10% (v/v)
ethanol water mixture, I = 0.1 molL-1 KNO3 and at 25.0 ± 0.1oC.

M(II) ΔLog10KM

AA Cu(II) Ni(II) Co(II) Zn(II) Cd(II)

Asp + 0.47 + 0.71 +1.8 +1.65 +2.83

Glu + 0.46 + 0.83 +2.12 +2.08 +2.07

Ala - 1.24 - 1.06 +0.22 -0.30 +0.62

Arg -- -- -- -- --

Met + 0.54 -- -- -- --

Phe -- + 0.56 + 0.86 +0.26 +0.61

Val - 1.34 + 0.30 -- +0.08 +0.60

Thr -2.18 -- -- -- --
M(II)(SPFX) M(II)
Log10KM =Log10K - 10
Log K
M(II)(SPFX)(AA) M(II)(AA)

Table 4: Binding constants of bovine serum albumin with SPFX and M(II)-SPFX
complexes at 25.0 oC in Tris-HCl buffer (pH 7.4).
 System K (M-1) R

Co(II)-SPFX 6.01 x104 ± 0.2 0.9445

Ni(II)-SPFX 1.21 x104 ± 0.3 0.9865

Zn(II)-SPFX 1.82 x104 ± 0.3 0.9749

Cu(II)-SPFX 1.17 x104 ± 0.2 0.9898

Cd(II)-SPFX 5.08 x104 ± 0.3 0.9831

SPFX only 2.06 x105 ± 0.3 0.9893

R: correlation coefficients
± refers to three times standard deviation (3s)