- Email: [email protected]
New insights into coordination chemistry of Monensin A towards divalent metal ions
Journal Pre-proofs Research paper New insights into coordination chemistry of Monensin A towards divalent metal ions Ivayla Pantcheva, Ahmed Nedzhib, Liudmil Antonov, Béla Gyurcsik, Peter Dorkov PII: DOI: Reference:
S0020-1693(19)31787-6 https://doi.org/10.1016/j.ica.2020.119481 ICA 119481
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
17 December 2019 27 January 2020 27 January 2020
Please cite this article as: I. Pantcheva, A. Nedzhib, L. Antonov, B. Gyurcsik, P. Dorkov, New insights into coordination chemistry of Monensin A towards divalent metal ions, Inorganica Chimica Acta (2020), doi: https:// doi.org/10.1016/j.ica.2020.119481
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
New insights into coordination chemistry of Monensin A towards divalent metal ions
Ivayla Pantchevaa,*, Ahmed Nedzhiba, Liudmil Antonovb, Béla Gyurcsikc,*, Peter Dorkovd
aLaboratory
of Biocoordination and Bioanalytical Chemistry, Department of Analytical
Chemistry, Faculty of Chemistry and Pharmacy, “St. Kl. Ohridski” University of Sofia, 1, J. Bourchier blvd., 1164 Sofia, Bulgaria bInstitute
of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences,
Sofia, Bulgaria cDepartment
of Inorganic and Analytical Chemistry, University of Szeged, 7, Dom ter, Szeged,
Hungary dBiovet
Ltd., Research & Development Department, Peshtera, Bulgaria
*Corresponding authors’ e-mail: [email protected], phone number: +359-2-8161446, and e-mail: [email protected], phone number: +36-62-544335.
1
Abstract
Complexation reactions of polyether ionophorous antibiotic Monensin A with divalent metal ions were studied in methanolic solutions by means of synchrotron radiation circular dichroism (SRCD) spectroscopy. Data showed that in the presence of organic base (Et4NOH) Monensin A forms two types of metal complexes depending on metal-to-ligand molar ratio. Complexes of composition [M(Mon)2(H2O)2] (Mon– = Monensinate A anion; M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+) exist mainly in the presence of ligand excess (M2+:Mon‒ ~ 1:10 – 1:2), while the increase of metal(II) ion concentration results in the formation of new [M(Mon)(H2O)]+ species (M2+:Mon‒ ~ 1:1 – 6:1), not detected up to now with these metal ions. Experimental data showed that coordination of divalent metal ions to Monensinate A anion significantly influences the SRCD spectral pattern in different manner. Based on sets of SRCD experiments, species distribution, individual spectra of [M(Mon)2(H2O)2] and [M(Mon)(H2O)]+ as well as their conditional stability constants were calculated. The relative affinity of Monensin A to bind divalent metal ions demonstrates the metal ion selectivity of the ligand.
Keywords: Synchrotron radiation circular dichroism, Monensin A, divalent metal ion, complex formation
2
1. Introduction Monensin A (Monensic acid A, MonH, HL) is a natural antibiotic with a wide application in veterinary medicine, displaying pronounced coccidiostatic and antibacterial properties [1-7]. It belongs to the polyether ionophores (PI) family – membrane-active compounds, able to coordinate metal ions and to transfer them across cell membranes as neutral complex species. Although known as monovalent PI for its affinity to bind monovalent metal ions [8-25], Monensin A reacts also with divalent metal cations to form derivatives of various composition and structure. Divalent metal complexes of Monensin A were characterized by IR, FAB-MS, NMR, EPR spectroscopy and by X-ray diffraction on single crystals [26-31]. Solid state chemistry of divalent Monensin A complexes is already well-known, but there are limitations studying their properties in solution. One of these is the very low solubility of the ligand and its complexes in the most common solvents, including water. Another factor is that there is no general spectroscopic method suitable for elucidation of solution behaviour of these compounds. In our previous paper we demonstrated that UV circular dichroism (CD) spectroscopy is a useful technique to evaluate Monensin A ability to bind ammonium, as well as light and heavy monovalent metal ions. Both the experiment and theoretical simulations have shown that CD spectra of [M(Mon)] complexes (M = Li+, Na+, K+, Rb+, Ag+ and Et4N+) are very sensitive to the captured ions and can be used for their discrimination in spite of the high similarity of the crystal structures of the compounds [32]. Crystallographic studies of the Monensin A complexes with divalent metal ions revealed different coordination mode than observed for monovalent metal ions. The divalent transition metals form bis complexes coordinated through the terminal donor groups of the ligand by macrochelate formation. In this paper we discuss the synchrotron radiation circular dichroism
3
(SRCD) spectral pattern of Monensinate A anion (Mon–) in the presence of divalent metal ions as obtained in methanolic solutions. Following the changes in the SRCD spectra at various metal-toligand molar ratio we observed for the first time the formation of mono complexes, calculated the equilibrium constants and individual spectra of complex species, as well as evaluated the relative affinity of Monensin A to bind divalent metal cations.
2. Experimental 2.1. Materials Sodium Monensinate A (MonNa) was cordially supplied by Biovet Ltd. (Bulgaria). Monensic acid A monohydrate (MonH×H2O), [Co(Mon)2(H2O)2] and [Mn(H2O)2(H2O)2] were prepared as previously described [25, 31]. Isolated compounds were dissolved in methanol for subsequent measurements. Et4NOH, MeOH and metal(II) salts (CaCl2×2H2O, MgCl2×2H2O, CoCl2×6H2O, MnCl2×4H2O, NiCl2×6H2O, Zn(ClO4)2×6H2O, CdCl2) were provided by Fluka and were of analytical grade.
2.2. Physical measurements SRCD spectra were recorded at the SRCD facility at the AU-CD beam line, itself part of the ASTRID2 storage ring at the Institute for Storage Ring Facilities (ISA), University of Aarhus, Denmark [33, 34]. Samples were placed in 0.1045 mm or 0.014 mm cuvettes. All spectra were recorded in 1 nm steps with a dwell time of 2 s per step, in the wavelength range of 170-300 nm. Two accumulations were averaged and solvent (methanol) spectra acquired at identical conditions were subtracted from those of the samples.
4
ESI-MS spectra were recorded on Waters Micromass ZQ 2000 LCMS System in the range from 0 to 2000 m/z.
2.3. Equilibrium studies Methanolic solutions containing MonH×H2O and Et4NOH (1:1) were mixed with an appropriate amount of corresponding metal(II) salt to obtain series of mixtures with metal-toligand molar ratio varying from 1:10 to 6:1 at a final total ligand concentration of cL = 16-20 mM. Complexation of M2+ ions (M2+ = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+ and Cd2+) was monitored following the changes in the SRCD spectral pattern of Monensinate A anion. Competition experiments were performed in two different experimental design sets using each pair of metal ions studied indicated as M1 and M2, respectively: (1) To a methanolic solution of given M1L2 complex, prepared in situ, a methanolic solution of corresponding competitive divalent metal ion (M2) was added to obtain series of reaction mixtures with molar ratio of both metal ions varying from 1:8 to 8:1. Changes in the spectral pattern of the starting complex M1L2 were followed by SRCD. (2) Experiments were performed recording SRCD spectra of solutions, containing M1 or M2 at metal-to-ligand molar ratio of 4:1. Then we constructed mixtures with M1:M2:L at ratio of 4:4:1. The resultant spectrum observed for M1:M2:L system was treated as an accumulative due to the different contribution of both single spectra belonging to M1L+ and M2L+ species, respectively. Thus, the effect of each divalent cation (M1) was estimated by its distribution coefficient in the presence of the corresponding competitive metal(II) ion (M2).
5
3. Results and discussion 3.1. New CD bands and new complex species observed in the solutions containing Monensinate A anion and divalent metal ions Based on our previous experience with monovalent metal complexes of Monensin A [32], our aim was to characterize the solution behaviour of divalent metal complexes [M(Mon)2(H2O)2] (M2+ = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+ and Cd2+), which are structurally similar in crystalline state. We expected that each divalent metal cation will affect differently the SRCD spectral pattern giving opportunity to discriminate them. SRCD spectra of MonH×H2O, [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2], isolated in solid state and subsequently dissolved in methanol, were recorded in 0.1045 mm optical pathlength supporting the above assumption (Fig. 1a; relates to 1 M total ligand concentration and l = 1 cm pathlength if not otherwise specified). At these conditions reliable data were obtained in the range from 187 nm to 300 nm. We also recorded SRCD spectra of Monensinate A anion prepared in situ (Et4NMon) and of mixtures containing metal(II) ion and Et4NMon at molar ratio of 1:2, analogous to that in complexes [M(Mon)2(H2O)2]. Spectra were obtained in a cell with shorter pathlength (0.014 mm, Fig. 1b). The data were cut off at 178 nm (due to high total absorbance below this wavelength) and new bands in far UV region were observed as compared to longer optical pathlength. The good agreement between spectra of divalent metal complexes as dissolved solids or prepared in situ revealed that obtained species distribution in methanol is identical for both systems confirming the reversibility of complex formation.
6
Fig. 1. SRCD spectra of: a) MonH×H2O, [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] isolated in solid state and dissolved in MeOH, 0.1045 mm optical pathlength; b) Monensinate A anion (Mon–) and solutions containing Co2+:Mon– = 1:2 and Mn2+:Mon– = 1:2 compounds prepared in situ, 0.014 mm cuvette; 20 mM total ligand concentration
Crystal structures of [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] complexes are very close to each other (RMSD < 0.1 Å, see later), but their SRCD spectral characteristics are very different, and also are significantly distinct from the spectral pattern of Monensin A either in its acidic or anionic form. For that reason we monitored the effect of other divalent metal ions they might render on the shape, sign(s) and intensity of the SRCD signal as well. 7
The spectral patterns of reaction mixtures prepared at M2+:Mon– molar ratio of 1:2 are summarized in Fig. 2. A considerable variation depending on metal(II) ion is observed. This kind of sensitivity and selectivity of SRCD as indicator of Monensinate A anion ability to bind various metal ions provoked us to follow the spectral changes caused by the presence of changing amounts of divalent metal cations related to the amount of the ligand.
Fig. 2. SRCD spectral patterns of reaction mixtures, containing M2+:Mon– = 1:2 (recorded in l = 0.014 mm cell)
A set of titration-like experiments was performed, with metal(II) ions and Et4NMon at metal-to-ligand molar ratio ranging from 1:10 to 6:1. Co(II) complexes are selected as a representative system throughout the discussion of results, and the data with the other divalent metal ions studied are collected in Supplementary Section. SRCD spectra of Monensinate A anion in the presence of divalent metal ions are shown in Fig. 3 (Co2+) and Fig. S1, respectively. The positive band of Et4NMon at 193 nm gradually increased in intensity with an increasing amount of Co2+ ions and reached a maximum at 191 nm at metal-to-ligand molar ratio of 1:2. At the same time, positive Et4NMon signal in the range of 200-220 nm diminished and finally a negative band of low intensity appeared in this spectral range. 8
Further increase of Co(II) ion concentration led to formation of second type of species with a positive band at 190 nm, and a weak negative signal within 200-220 nm interval (Fig. 3, left). Spectral changes observed at metal deficit were mainly attributed to the formation of complex [Co(Mon)2(H2O)2], which has been already isolated and characterized in solid state. The second type of Co2+-containing Monensin A species is detected for the first time at comparable Co2+:Mon– molar ratio (1:1) and the spectral pattern of the reaction mixture did not significantly change at a further excess of the metal(II) ion (up to 6:1 metal-to-ligand molar ratio). Coordination of Monensinate A anion in this new species is supposed to be very similar to that in [Co(Mon)2(H2O)2] due to their similar spectral pattern, although the spectra differ in shape and intensity (Fig. 3, left).
Fig. 3. Spectra of Et4NMon (black) at deficit (red) and excess (blue) of Co2+ ions (left); spectral changes at selected wavelengths as a function of the metal-to-ligand molar ratio (right) (20 mM total ligand concentration, l = 0.014 mm)
SRCD spectra of solutions containing Monensinate A anion and other divalent metal ions studied under the same reaction conditions also suggested the formation of two complex species depending on molar metal-to-ligand ratio (Fig. S1, left). To approve this on mathematical basis, 9
an extended Multiple Regression Analysis (MRA) was performed on datasets of the studied systems [35]. In principle the rank of the ellipticity data matrix shall provide the number of independent chiral species. However, the significance of eigenvalues depends very much on the quality of the data. Therefore, we have also analyzed the residual CD signal after considering increasing number of species in the system, to decide which eigenvalues are large enough to take them into account (Fig. 4 for Co2+-Mon– system and Fig. S2 for the other studied cations, respectively). This procedure confirmed that including only two species in the calculation (i.e. the free ligand and the ML2 complex) still results in a systematic deviation of the residual ellipticity from the zero line. Thus, minimum three types of species are required for the satisfactory description of the detected spectral changes.
Fig. 4. Five wavelength-dependent residual CD signal curves for the Co2+-Mon– system
We have plotted the trends in the spectral intensities in Figs. 3 and S1, right. For this we selected wavelengths, which allow to monitor the most significant differences. The changes in the 10
slope of the tangent lines (in certain cases even the sign of the slope is changed) at around metalto-ligand molar ratios 1:2 and 1:1 in most of the systems occur. These kind of titration curves cannot be described by the formation of only one complex species. Taking the metal-to-ligand molar ratios into consideration the third species can be assigned to ML+ mono complexes. Thus, SRCD technique allowed us to observe the formation of the coordination compounds with composition ML+ for divalent metal ions for the first time. Moreover, for the first time we evidenced that Monensin A can bind more than half equivalents of divalent metal ions in the presence of increasing metal-to ligand ratio. It is worth mentioning that the above changes are less pronounced for Zn2+ and Ca2+, while for Cd2+ they are ambiguous. The reason for this is most probably due to the close similarity of the CD spectra of ML2 and ML+ complexes of these metal ions. Eventually we cannot exclude the lack of the formation of ML+ species with these metal ions in methanolic solutions. In parallel we applied ESI-MS technique as an independent method to record the spectra of Monensinate A solutions at excess of divalent metal ions (metal-to-ligand molar ratio of 4:1). The data confirmed the existence of ML+ species in gas phase. Representative MS spectra are placed in Fig S3.
3.2 Stability of the Monensin A complexes with divalent metal ions At this state of the research we suggest that Monensinate A anion forms two types of complex species [M(Mon)2(H2O)2] (ML2) and [M(Mon)(H2O)]+ (ML+), respectively, in methanolic solutions according to equilibria I and II (L– = Mon–, and water molecules were omitted for clarity):
M2+ + 2 L-
ML2 (equilibrium I) 11
2 ML+ (equilibrium II)
M2+ + ML2
Using PSEQUAD program [36] we attempted to calculate the conditional stability constants 𝛽′2 and 𝛽′1 and constant K′ characteristic for the relative stability of the mono and bis complexes from the SRCD data. K' is defined by the equation (1). [𝑀𝐿]2
𝐾1′
(1),
𝐾′ = [𝑀] ∙ [𝑀𝐿 ] = 𝐾2′ 2
where [X] is the equilibrium concentration of species X in solution (free ligand L, metal ion M, complexes ML2 and ML+). The K1' and K2' stepwise stability constants can be calculated from the above values following (2):
𝐾1′ = 𝐾′ ∙ 𝛽′2; 𝐾2′ =
𝛽′2 𝐾′
(2)
Taking into account the limitation of the SRCD data, reasonably good fits of the experimental spectra were obtained (Fig S4, Co2+–Mon– system). Allowing all parameters to be refined in the calculation, we could not get unambiguous data for the ML+ species of Cd2+ and for the ML2 species of Ca2+ complexes in accordance with the above discussion. Since we could measure the SRCD spectrum of the free ligand separately and this species exists in all systems at ligand excess, we have repeated the calculations keeping the spectrum of Monensinate A anion constant. The obtained data by these calculations are shown in Table 1.
Table 1. Parameters characterizing the stabilities of the Monensin A complexes with divalent metal ions obtained from SRCD spectroscopic data by PSEQUAD program keeping the CD spectrum of Monensinate A anion constant (the standard deviation of the refined parameter from the fitting procedure is in parenthesis). 12
Mn2+
Co2+
lgβ1'(ML)
4.8(0.2)
lgβ2'(ML2)
7.8(0.3)
Ni2+
Zn2+
5.9(0.5) 16.0(0.3)
Cd2+
6.7(0.6)
Mg2+
Ca2+
0.9(*) 4.9(0.4) 3.3(0.2)
10.2(0.8) 24.5(0.5) 12.1(1.0) 4.8(0.2) 7.4(0.4)
lgK2'
3.0
4.3
8.5
K'
63
40
3.2×107
5.32
4.7(*)
3.9
2.5
1.4*
27.54 1.0×10–3
251
79
Restraining the spectrum of the free ligand did not change the data and the trends significantly. For the two above mentioned ambiguous systems (these values are marked with * because of their too high standard deviations) we have tried to calculate stability constants by further modification of the species matrix and calculation settings. Assuming that the ML+ spectrum does not differ significantly from that measured at metal ion excess (since the solvated metal ion itself is not optically active) the calculation for Cd2+ complexes yielded 3.4(0.3) for lgβ1' and 5.8(0.3) for lgβ2'. For Ca2+ we could not obtain reliable stability constants for both ML+ and ML2 species. Assuming only one complex was formed we could fit the data with slightly higher deviation, and obtained either 4.0(0.4) value for lgβ1' or 4.8(0.4) for lgβ2'. With the exception of the Ca2+–Mon– system the overall fit of the experimental curves significantly improved with the inclusion of both ML+ and ML2 complexes compared to the fitting procedures with only one complex. The species distribution diagram for the Co2+-Mon– system is shown in Fig 5.
13
Fig. 5. Species distribution diagram of the Co2+-Monensinate A system (the Monensin A total concentration was kept within 16-20 mM)
Under the conditions of the measurements the complex formation occurred almost quantitatively. For this reason the high stability determined for Ni(II)-complexes is also ambiguous, but it clearly reflects that Ni2+ ions have very high affinity towards the Monensinate A anion. In spite of the difficulties to obtain reliable stability constants we shall note that the order of the stabilities for the ML2 complexes - Ni2+ > Zn2+ > Co2+ ~ Mg2+ ~ Mn2+ (> Cd2+ > Ca2+) - is in a very good agreement with the metal–oxygen distances observed for carboxylate and terminal hydroxyl oxygens participating in the direct coordination in the crystal structures of these complexes (Table S1). The shorter are the distances in the crystal structures, the higher is the stability in solution. This is also in correlation with the decreasing ionic radii of the metal ions. The stability order may slightly vary depending on which dataset we use for comparison, but the seeming deviations from the above established order could be explained by the deviating M–O bonds. E.g. while Zn2+ is at relatively low distance to carboxylate oxygen, it has longer bonds to hydroxyl OH and water molecule, which may explain its weak binding in competition experiments (vide infra). 14
The trend of the ML2 stability constants agrees well with the Irving-Williams order. Nickel(II) is a borderline metal ion having high affinity for nitrogen and oxygen donor atoms. Although the stabilities may vary by the microenvironment, i.e. by the geometry, quality of the donor atoms, charges, etc., Ni2+ favours the octahedral geometry with oxygen donors [37-40]. The stability of the ML+ complexes - Ni2+ > Zn2+ > Co2+ ~ Mg2+ > Mn2+ (> Ca2+ ~ Cd2+) follows very similar order as observed for the ML2 complexes. It is worth mentioning that the K' constants reflect in most cases the stronger binding of the ligand to the metal ions in ML+ than in ML2 species in spite of the fact the only the electrically neutral ML2 complexes were obtained so far in solid state. This may arise from the steric and electronic repulsion of the bulky and charged ligands in the metal ion coordination sphere. From that point of view Monensin deficit will lead to competition of the various metal ions for the ligand. To check whether this hypothesis is correct, we initiated series of competition experiments using a solution containing given metal(II) ion M1 and Monensinate A anion at metal-to-ligand molar ratio of 1:2 as a starting point. Spectral changes in this reaction mixture were monitored at deficit and excess of competitive divalent metal ions M2. Results revealed that addition of increasing amount of any M2 ion shifted the equilibrium from M1L2 towards M2L+ species and vice versa – the presence of M1 converts M2L2 into corresponding M1L+ species. Thus, starting out from the M1L2 complex of any studied divalent metal ion by addition of a competing metal ion (M2) we could always reach an equilibrium state in which the M2L+ species of the competing metal ion is dominant. These experiments outlined the general tendency that using ML2 as a starting compound, Monensin A is able to coordinate preferably those metal(II) cations which exceed in a competitive medium. In attempt to look more deeply into chemistry of ML+ complexes, we prepared pairs of mixtures containing M1-Mon– or M2-Mon–, both at molar ratio of 4:1. The selected molar ratio
15
ensures the dominant formation of corresponding ML+ complexes and thus, the SRCD spectrum recorded for each solution belongs mainly to single complex species, M1L+ or M2L+, respectively. Then we mixed methanolic solutions of M1, M2 and Mon– at molar ratio of 4:4:1. The spectrum obtained was treated as an additive one resulting from different contributions of individual M1L+ and M2L+ spectra. Exemplary spectra of the studied systems are given in Fig. S5. The distribution coefficients calculated in such way account for divalent metal ion competition, i.e. for Monensin propensity to selectively complex M2+ ions in ML+ species. Contribution of metal ions studied is summarized in Table 2. These data reflect that the relative affinity of Monensinate A anion in the designed systems decreases in the order of Ni2+ > Co2+ ~ Ca2+ > Mn2+ ~ Mg2+ ~ Zn2+ ~ Cd2+.
Table 2. Effect of competition reactions between divalent metal ions on formation of ML+ Contribution (%) of M1-Mon to the resultant SRCD spectrum at M1:M2:Mon = 4:4:1
Competitive M2 Ni
Co
Ca
Mn
Mg
Co
75
Ca
75
55
Mn
90
75
65
Mg
90
75
70
55
Zn
90
75
90
85
55
Cd
95
80
70
75
60
Zn
Cd
60
Comparison of the presented stability for ML2 and ML+ complexes and that obtained for competing systems revealed a drastic change in the position of the Zn2+ ion in the corresponding series. At this stage of research we cannot explain the observed tendency in the behavior of Monensinate A in the presence of divalent metal ions at different conditions. It can be assumed
16
that additional factors as origin of the metal salt’s counterion, solvolysis, etc. may influence the processes studied. To answer this interesting question is a matter of further studies.
3.3. Solid state and solution structure of divalent metal complexes of Monensin Monensic acid A reacts with divalent metal ions in the presence of Et4NOH to form mononuclear complexes of composition [M(Mon)2(H2O)2] (M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+). Their solid state structure was resolved by X-ray diffractometry on single crystals. Data revealed that divalent metal cations are located in a distorted octahedral environment achieved by coordination of two Monensinate A monoanions. Ligands act in a bidentate coordination mode via the carboxylate moiety and the hydroxyl group both located at the opposite ends of the antibiotic molecule. Axial positions in the inner coordination sphere of metal(II) cations are occupied by two water molecules (Fig. 6) [28-31].
O
O
O O O
H H
O
O
O H
H
O
H H
O O
O M2+ O
O H
O O O O
H
O
O O H O H H H O O
Fig. 6. Bidentate coordination mode of Monensin A in complexes [M(Mon)2(H2O)2]
17
To better understand the effect of divalent metal cations on Monensin conformation, we compared crystal structures of [M(Mon)2(H2O)2] using the root-mean-square deviation (RMSD) values [41] calculated by the PyMol alignment procedure (Table 3). Divalent metal complexes consist of two non-equivalent ligand subunits, containing 47 atoms each, A and B, respectively. RMSD values for the comparison of single Monensinate A anions within every [M(Mon)2(H2O)2] complex vary within a range from 0.090 (Co47A-Co47B) to 0.122 (Mg47A-Mg47B) Å units. Each ligand subunit shows higher RMSD value, c.a. of 0.3 Å, as compared to the crystal structure of Monensin A.
Table 3. Root-mean-square deviations (RMSD in Å units) as a measure of similarity between 94 non-hydrogen backbone atoms of the superimposed divalent metal complexes of Monensin A (47 atoms for each ligand). Crystal structures obtained with various divalent cations were compared among each other. Mg94 M(II) Mg47A Mg47B Mg94 Ca47A Ca47B Ca94 Mn47A Mn47B Mn94 Co47A Co47B Co94 Ni47A Ni47B Ni94
Mg47A
Ca94
Mn94
Co94
Ni94
Zn94
Cd94
MonH
Mg47B
Ca47A
Ca47B
Mn47A
Mn47B
Co47A
Co47B
Ni47A
Ni47B
Zn47A
Zn47B
Cd47A
Cd47B
0.122
0.064
0.149
0.050
0.127
0.058
0.124
0.036
0.113
0.073
0.139
0.102
0.147
0.302
0.125
0.084
0.114
0.043
0.100
0.048
0.132
0.046
0.108
0.053
0.124
0.111
0.342
0.201 0.122
2.219
2.197
0.068
0.084
0.143
0.039
0.116
0.064
0.120
0.080
0.129
0.067
0.134
0.074
0.144
0.296
0.129
0.055
0.124
0.076
0.162
0.111
0.127
0.077
0.121
0.056
0.334
2.265 0.111
2.250
0.248
0.229
0.120
0.043
0.109
0.060
0.112
0.052
0.127
0.064
0.149
0.292
0.101
0.044
0.138
0.065
0.107
0.048
0.112
0.083
0.340
0.094 0.090
2.206
2.206
2.240
0.068
0.097
0.038
0.106
0.068
0.139
0.303
0.132
0.061
0.097
0.042
0.109
0.088
0.333
2.183 0.115
2.183
2.221
0.087
0.149
0.114
0.184
0.294
0.111
0.075
0.130
0.136
0.342
0.084
0.179
18
Zn47A
0.110
Zn47B Zn94
0.061
0.140
0.309
0.121
0.087
0.342
0.139
Cd47A
0.128
Cd47B
0.302 0.332
Comparison of structures of divalent metal complexes revealed close similarity between corresponding A and B units, respectively. The RMSD lay within relatively narrow range from 0.036 (Mg47A–Ni47A) to 0.088 (Co47B-Cd47B) Å except Ni47B-Cd47B subunits (0.136 Å). The RMSD values comparing the pairs of opposite ligand subunits show larger variation from 0.097 (Co47A-Ni47B) to 0.184 (Ni47A–Cd47B) Å units, respectively. The observed deviations within the structures of individual divalent metal complexes show close resemblance of single ligand anion conformation, which is not strongly affected by the coordination of corresponding metal cations. On the other hand, comparison of overall structures of [M(Mon)2(H2O)2] (two Monensinate A anions, 94 atoms), classifies complexes into three groups. Group 1 ([Co(Mon)2(H2O)2], [Mn(Mon)2(H2O)2]) can be distinguished from other metal complexes by large RMSD values of ~ 2.2 Å. The members of group 2 ([Mg(Mon)2(H2O)2], [Ni(Mon)2(H2O)2], [Zn(Mon)2(H2O)2]) and group 3 ([Ca(Mon)2(H2O)2], [Cd(Mon)2(H2O)2]) show smaller RMSD values of ~ 0.2 Å between the groups, but the metal-oxygen donor atom distances differ significantly being ~ 2.02-2.04 Å and 2.21-2.26 Å for the two groups, respectively. Values of RMSD within the members of each group are less than 0.1 Å, with the exception of group 3 where it is 0.12. The question arose, whether these characteristics can account for the observations in methanolic solutions by SRCD? One of the greatest achievements of the equilibrium calculations is that instead of the spectra of reaction mixtures, they provide the spectra of the individual 19
complex species. The shapes of the spectra were rather independent on the mode of the calculations as shown in Fig. S6 for the Co2+-Monensin A system. Fig. 7a shows the comparison of the spectra for ML2 complexes of all studied metal ions. It is clear that SRCD can differentiate between the various divalent metal complexes, as their spectral patterns are different from each other. Such discrimination of divalent metal ion complexes of Monensin A observed by SRCD could be assumed to rise from the fine changes in ligand conformation due to the metal ion’s geometry requirements. Various metal ions also affect the SRCD spectral pattern uniquely through the involvement of the electrons of metal ions into the molecular orbitals responsible for the chiral effects as it was also shown in our previous paper for monovalent alkaline metal ions [32].
20
Fig. 7. Individual molar SRCD spectra of ML2 (a) and ML+ (b) complexes obtained from PSEQUAD calculations. Spectra of the ML2 complex of Ca2+ and ML+ complex of Cd2+ could not be reliably determined - see text for more details.
The spectral patterns for the individual ML+ complexes obtained from the calculations by PSEQUAD program are summarized in Fig. 7b. From the similarities between corresponding pairwise ML+ and ML2 SRCD spectra we suggest that the excess of metal(II) ions undergoes formation of positively charged complex species of composition [M(Mon)(H2O)]+ where ligand anion acts in a bidentate coordination manner similarly as in [M(Mon)2(H2O)2] complexes. Thus, the metal ion is most probably not bound in the cavity of the ligand as it happens with the monovalent metal ions. This is also supported by the similarity of the stability order of the ML+ and ML2 complexes. The presence of water molecules in the structure of Monensic acid and corresponding neutral complexes in solution was previously confirmed by NMR studies of diamagnetic compounds [29-30]. The difference between spectra of [M(Mon)2(H2O)2] and [M(Mon)(H2O)]+ species can be detected by SRCD due to the sensitivity of this technique towards fine conformational changes of the ligand upon metal ion complexation. To elucidate the intimate structure of [M(Mon)(H2O)]+, further experiments on their possible isolation, crystallization and characterization have to be performed.
4. Conclusions The ability of Monensin A (MonH) to bind divalent metal cations (M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+) in methanolic solutions was evaluated by means of SRCD spectroscopy. The experimental results revealed that:
21
1). SRCD was proven to be an efficient methodology to discriminate among different divalent metal ions bound in neutral complex species [M(Mon)2(H2O)2] (ML2). 2). Monensin A formed new positively charged complex species of suggested composition [M(Mon)(H2O)]+ (ML+) at comparable or higher metal-to-ligand molar ratio with a similar to the coordination mode of the ligand in ML2. The presence of ML+ was confirmed for the first time using SRCD technique, but further structure elucidation is required. 3) Although close, overall SRCD signals of ML2 and ML+ differ in position and intensity most likely due to fine ligand conformational changes caused by the coordination of divalent metal ions and complemented by charge transfer processes. 4). Equilibrium constants of the most of the processes studied were calculated and individual molar spectra of ML+ and ML2 species were derived. 5). It was found that Monensin A binds preferably those divalent metal cations which exceed in a competitive reaction medium. In addition, metal(II) ion excess increases the coordination ability of the ligand in ML+ species. 6). At comparable but excessive concentration of competitive metal ions (compared to the ligand) Monensin A affinity to bind M(II) as ML+ species decreases in the order of Ni2+ > Co2+ ~ Ca2+ > Mn2+ ~ Mg2+ ~ Zn2+ ~ Cd2+. The above results envisage a more complex role of Monensin A as ionophore in binding and transporting various metal ions, which may have importance in regulating the metal ion homeostasis and the toxic effects of metal ions. Such comparative studies including a range of metal ions is necessary to better understand these processes.
22
Acknowledgements Financial support of TÁMOP-4.2.4.A/2, John von Neumann International Scholarship for senior foreign teachers-researchers, CALIPSOplus (EU Framework Programme for Research and Innovation HORIZON 2020, grant no. 730872) and from the Hungarian National Research, Development and Innovation Office (GINOP-2.3.2-15-2016-00038) is greatly acknowledged. The authors are thankful to Nykola Jones and Søren Vrønning Hoffmann (Aarhus University, Denmark) for the valuable discussion on SRCD technique and data processing procedures. IP is thankful to the National Science Fund, Bulgarian Ministry of Education and Science (contract № KP-06-H29/3).
References 1. Chapman, H.D., Barta, J.R., Blake, D., Gruber, A., Jenkins, M., Smith, N.C., Suo, X., Tomley, F.M. A selective review of advances in coccidiosis research, Adv. Parasitol. 83 (2013) 93-171. https://doi.org/10.1016/B978-0-12-407705-8.00002-1. 2. Chapman, H.D., Jeffers, T.K., Williams, R.B. Forty years of monensin for the control of coccidiosis in poultry, Poult. Sci. 89 (2010) 1788-1801. https://doi.org/10.3382/ps.201000931. 3. Anadón, A., Martinez-Larrañaga, M.R., Castellano, V. Antimicrobials (including coccidiostats) in poultry, J. Vet. Pharmacol. Therap. 32 (SUPPL. 1) (2009) 26-28. 4. Croft, S.L. The current status of antiparasite chemotherapy, Parasitology, 114 (SUPPL.), (1997) S3-S15.
23
5. Rutkowski, J., Brzezinski, B. Structures and properties of naturally occurring polyether antibiotics,
BioMed
Res.
Intern.
(2013)
art.
ID
162513.
http://dx.doi.org/10.1155/2013/162513. 6. Nelson, M.L., Grier, M.C., Barbaro, S.E., Ismail, M.Y. Polyfunctional antibiotics affecting bacterial membrane dynamics, Anti-Infective Agents Med. Chem. 8 (2009) 3-16. https://doi.org/10.2174/187152109787047779. 7. Kevin II, D.A., Meujo, D.A.F., Hamann, M.T. Polyether ionophores: Broad-spectrum and promising biologically active molecules for the control of drug-resistant bacteria and parasites,
Expert
Opin.
Drug
Disc.
4
(2009)
109-146.
https://doi.org/10.1517/17460440802661443. 8. Huczyński, A., Janczak, J., Łowicki, D., Brzezinski, B. Monensin A acid complexes as a model of electrogenic transport of sodium cation, Biochim. Biophys. Acta - Biomembranes 1818 (2012) 2108-2119. https://doi.org/10.1016/j.bbamem.2012.04.017. 9. Huczyński, A., Ratajczak-Sitarz, M., Katrusiak, A., Brzezinski, B. Molecular structure of rubidium six-coordinated dihydrate complex with monensin A, J. Mol. Str. 888 (2008) 224-229. https://doi.org/10.1016/j.molstruc.2007.12.005. 10. Huczyński, A., Ratajczak-Sitarz, M., Katrusiak, A., Brzezinski, B. Molecular structure of the 1: 1 inclusion complex of monensin A sodium salt with acetonitrile, J. Mol. Str. 832 (2007) 84-89. https://doi.org/10.1016/j.molstruc.2006.07.043. 11. Martinek, T., Riddell, F.G., Wilson, C., Weller, C.T. The conformations of monensin-A metal complexes in solution determined by NMR spectroscopy, J. Chem. Soc. Perkin Transact. 2 (2001) 35-41. https://doi.org/10.1039/A907984J.
24
12. Raban, M., Quin III, J., Belguise, A., Durocher, D., Kost, D. Conformationally switchedon polyether ionophores, Chirality 10 (1998) 78-87. https://doi.org/10.1002/chir.13. 13. Wagner-Czauderna, E., Rzeszotarska, J., Orlowska, E., Kalinowski, M.K. Complexing ability of monensin anion for alkali metal cations in binary mixtures of dipolar aprotic solvents, Berichte der Bunsengesellschaft / Physical Chemistry Chemical Physics, 101 (1997) 1154-1157. https://doi.org/10.1002/bbpc.19971010811. 14. Pointud, Y., Bernard, C., Touzain, S., Astier, L., Sabatier, B., Juillard, J. Thermodynamics of complexation of monovalent metal cations by the ioniophore monensin free acid in acetonitrile, J. Solution Chem. 26 (1997) 479-495. https://doi.org/10.1007/BF02767602. 15. Hebrant, M., Pointud, Y., Juillard, J. Thermodynamics of reaction in heterogeneous systems (water-organic phases) between the ionophore monensin and alkali-metal cations, J. Phys. Chem. 95 (1991) 3653-3662. https://doi.org/10.1021/j100162a041. 16. Riddell, F.G., Arumugam, S., Cox, B.G. The monesin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR, Biochim. Biophys. Acta Biomembranes 944 (1988) 279-284. https://doi.org/10.1016/0005-2736(88)90442-7. 17. Cox, B.G., Van Truong, N., Rzeszotarska, J., Schneider, H. Rates and equilibria of alkali metal and silver ion complex formation with monensin in ethanol, JACS 106 (1984) 59655969. https://doi.org/10.1021/ja00332a035. 18. Koryta, J., Du, G., Ruth, W., Vanýsek, P. Transfer of alkali-metal and hydrogen ions across liquid/liquid interfaces mediated by monensin: A voltammetric study at the interface of two immiscible electrolyte solutions, Faraday Discussions of the Chemical Society, 77 (1984) 209-216. https://doi.org/10.1039/DC9847700209.
25
19. Duax, W.L., Smith, G.D., Strong, P.D. Complexation of metal ions by monensin. Crystal and molecular structure of hydrated and anhydrous crystal forms of sodium Monensin JACS 102 (1980) 6725-6729. https://doi.org/10.1021/ja00542a010. 20. Hoogerheide, J.G., Popov, A.I. A study of metal complexes of a naturally occurring macrocyclic
ionophore-monensin,
J.
Solution
Chem.
8
(1979)
83-95.
https://doi.org/10.1007/BF00646810. 21. Hoogerheide, J.G., Popov, A.I. Study of monensin complexes with monovalent metal lons in
anhydrous
methanol
solutions,
J.
Solution
Chem.
7
(1978)
357-372.
https://doi.org/10.1007/BF00662896. 22. Gertenbach, P.G., Popov, A.I. Solution chemistry of monensin and its alkali metal ion complexes. Potentiometric and spectroscopic studies, JACS 97 (1975) 4738-4744. https://doi.org/10.1021/ja00849a041. 23. Steinrauf, L.K., Czerwinski, E.W., Pinkerton, M. Comparison of the monovalent cation complexes of monensin, nigericin, and dianemycin, Biochem. Biophys. Res. Comm. 45 (1971) 1279-1283. https://doi.org/10.1016/0006-291X(71)90156-2. 24. Lutz, W.K., Winkler, F.K., Dunitz, J.D. Crystal structure of the antibiotic monensin similarities and differences betweeen free acid and metal complex, Helv. Chim. Acta 54 (1971) 1103-1108. https://doi.org/10.1002/hlca.19710540419. 25. Pinkerton, M., Steinrauf, L.K. Molecular structure of monovalent metal cation complexes of Monensin, J. Mol. Biol. 49 (1970) 533-546. https://doi.org/10.1016/00222836(70)90279-2. 26. Pantcheva, I.N., Dorkov, P., Atanasov, V.N., Mitewa, M., Shivachev, B.L., Nikolova, R.P., Mayer-Figge, H., Sheldrick, W.S. Crystal structure and properties of the copper(II)
26
complex of sodium monensin A, J. Inorg. Biochem. 103 (2009) 1419-1424. https://doi.org/10.1016/j.jinorgbio.2009.08.007. 27. Dorkov, P., Pantcheva, I.N., Sheldrick, W.S., Mayer-Figge, H., Petrova, R., Mitewa, M. Synthesis, structure and antimicrobial activity of manganese(II) and cobalt(II) complexes of the polyether ionophore antibiotic sodium monensin A, J. Inorg. Biochem. 102 (2008) 26-32. https://doi.org/10.1016/j.jinorgbio.2007.06.033. 28. Pantcheva, I.N., Zhorova, R., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. First solid state alkaline-earth complexes of monensic A acid: X-ray crystal structure of [M(Mon)2(H2O)2] (M = Mg, Ca), spectral properties and cytotoxicity against Grampositive bacteria, BioMetals 2 (2010) 59-70. https://doi.org/10.1007/s10534-009-9269-5. 29. Pantcheva, I.N., Ivanova, J., Zhorova, R., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. Nickel(II) and zinc(II) dimonensinates: crystal structure, spectral properties and bactericidal activity, Inorg. Chim. Acta 363 (2010) 1879-1886. https://doi.org/10.1016/j.ica.2010.02.009. 30. Ivanova, J., Pantcheva, I.N., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. Crystal structures and spectral properties of new Cd(II) and Hg(II) complexes of monensic acid with different coordination modes of the ligand, Centr. Eur. J. Chem. 8 (2010) 852860. https://doi.org/10.2478/s11532-010-0067-2. 31. Pantcheva, I.N., Mitewa, M.Io., Sheldrick, W.S., Oppel, I. M., Zhorova, R., Dorkov, P. First divalent metal complexes of the polyether ionophore monensin A: X-ray structures of [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] and their properties, Curr. Drug Discov. Technol. 5 (2008) 154-161. https://doi.org/10.2174/157016308784746247.
27
32. Nedzhib, A., Kessler, J., Bour, P., Gyurcsik, B., Pantcheva, I., Circular dichroism is sensitive to monovalent cation binding in Monensin complexes, Chirality 28 (2016) 420428. https://doi.org/10.1002/chir.22597. 33. Miles, A.J., Hoffmann, S.V., Tao, Y., Janes, R.W., Wallace, B.A., Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy: New beamlines and new applications in biology, Spectroscopy 21 (2007) 245-255. http://dx.doi.org/10.1155/2007/282713. 34. Miles, A.J., Janes, R.W., Brown, A., Clarke, D.T., Sutherland, J.C., Tao, Y., Wallace, B.A., Hoffmann, S.V., Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines, J. Synchrotron Rad. 15 (2008) 420422. http://dx.doi.org/10.1107/S0909049508009606. 35. Peintler, G., Nagypál, I., Jancsó, A., Epstein, I.R., Kustin, K. Extracting experimental information from large matrixes. 1. A new algorithm for the application of matrix rank analysis, J. Phys. Chem. A 101 (1997) 8013–8020. https://doi.org/10.1021/jp970136s. 36. Zékány, I., Nagypál, I., Peintler, G. PSEQUAD for Chemical Equilibria, update 5.10; Technical Software Distributors: Baltimore, MD, 1991. 37. Driessen, W.L., Everstijn, P.L.A., Dimethyl phthalate, a neutral bidentate oxygen-donor ligand
to
divalent
metal
ions,
Z.
Naturforsch.
B
32
(1977)
1284–1286.
https://doi.org/10.1515/znb-1977-1113. 38. Hancock, R.D., Martell A.E. Ligand design for selective complexation of metal ions in aqueous solutions, Chem. Rev. 89 (1989) 1875-1914.https://doi.org/10.1021/cr00098a011. 39. Rulísek, L., Vondrásek. J. Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins, J. Inorg. Biochem. 71 (1998) 115127. https://doi.org/10.1016/S0162-0134(98)10042-9.
28
40. Marino, T., Toscano, M., Russo, N., Grand, A. Structural and electronic characterization of the complexes obtained by the interaction between bare and hydrated first-row transition-metal ions (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+) and glycine, J. Phys. Chem. B 110 (2006) 24666-24673. https://doi.org/10.1021/jp0645972. 41. Heifets, A., Lilien, R. LigAlign: flexible ligand-based active site alignment and analysis, J. Mol. Graph. Modell 29 (2010) 93-101. http://dx.doi.org/10.1016/j.jmgm.2010.05.005.
29
SRCD spectra of ML+ and ML2
Co2+ Zn2+ Ni2+
Ti
n tio a tr
Mg2+
Mn2+ Ca2+ Cd2+
Co
mp
eti
tio n
30
Discrimination among different M2+ ions in neutral ML2 complexes was achieved by SRCD
The presence of new positively charged complexes ML+ was confirmed for the first time
Excess of M2+ increases the coordination ability of the ligand in ML+ species
Individual spectra of ML+ and ML2 and their stability constants were calculated
Monensin A affinity to bind M(II) as ML+ species was evaluated
31
S0020-1693(19)31787-6 https://doi.org/10.1016/j.ica.2020.119481 ICA 119481
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
17 December 2019 27 January 2020 27 January 2020
Please cite this article as: I. Pantcheva, A. Nedzhib, L. Antonov, B. Gyurcsik, P. Dorkov, New insights into coordination chemistry of Monensin A towards divalent metal ions, Inorganica Chimica Acta (2020), doi: https:// doi.org/10.1016/j.ica.2020.119481
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
New insights into coordination chemistry of Monensin A towards divalent metal ions
Ivayla Pantchevaa,*, Ahmed Nedzhiba, Liudmil Antonovb, Béla Gyurcsikc,*, Peter Dorkovd
aLaboratory
of Biocoordination and Bioanalytical Chemistry, Department of Analytical
Chemistry, Faculty of Chemistry and Pharmacy, “St. Kl. Ohridski” University of Sofia, 1, J. Bourchier blvd., 1164 Sofia, Bulgaria bInstitute
of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences,
Sofia, Bulgaria cDepartment
of Inorganic and Analytical Chemistry, University of Szeged, 7, Dom ter, Szeged,
Hungary dBiovet
Ltd., Research & Development Department, Peshtera, Bulgaria
*Corresponding authors’ e-mail: [email protected], phone number: +359-2-8161446, and e-mail: [email protected], phone number: +36-62-544335.
1
Abstract
Complexation reactions of polyether ionophorous antibiotic Monensin A with divalent metal ions were studied in methanolic solutions by means of synchrotron radiation circular dichroism (SRCD) spectroscopy. Data showed that in the presence of organic base (Et4NOH) Monensin A forms two types of metal complexes depending on metal-to-ligand molar ratio. Complexes of composition [M(Mon)2(H2O)2] (Mon– = Monensinate A anion; M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+) exist mainly in the presence of ligand excess (M2+:Mon‒ ~ 1:10 – 1:2), while the increase of metal(II) ion concentration results in the formation of new [M(Mon)(H2O)]+ species (M2+:Mon‒ ~ 1:1 – 6:1), not detected up to now with these metal ions. Experimental data showed that coordination of divalent metal ions to Monensinate A anion significantly influences the SRCD spectral pattern in different manner. Based on sets of SRCD experiments, species distribution, individual spectra of [M(Mon)2(H2O)2] and [M(Mon)(H2O)]+ as well as their conditional stability constants were calculated. The relative affinity of Monensin A to bind divalent metal ions demonstrates the metal ion selectivity of the ligand.
Keywords: Synchrotron radiation circular dichroism, Monensin A, divalent metal ion, complex formation
2
1. Introduction Monensin A (Monensic acid A, MonH, HL) is a natural antibiotic with a wide application in veterinary medicine, displaying pronounced coccidiostatic and antibacterial properties [1-7]. It belongs to the polyether ionophores (PI) family – membrane-active compounds, able to coordinate metal ions and to transfer them across cell membranes as neutral complex species. Although known as monovalent PI for its affinity to bind monovalent metal ions [8-25], Monensin A reacts also with divalent metal cations to form derivatives of various composition and structure. Divalent metal complexes of Monensin A were characterized by IR, FAB-MS, NMR, EPR spectroscopy and by X-ray diffraction on single crystals [26-31]. Solid state chemistry of divalent Monensin A complexes is already well-known, but there are limitations studying their properties in solution. One of these is the very low solubility of the ligand and its complexes in the most common solvents, including water. Another factor is that there is no general spectroscopic method suitable for elucidation of solution behaviour of these compounds. In our previous paper we demonstrated that UV circular dichroism (CD) spectroscopy is a useful technique to evaluate Monensin A ability to bind ammonium, as well as light and heavy monovalent metal ions. Both the experiment and theoretical simulations have shown that CD spectra of [M(Mon)] complexes (M = Li+, Na+, K+, Rb+, Ag+ and Et4N+) are very sensitive to the captured ions and can be used for their discrimination in spite of the high similarity of the crystal structures of the compounds [32]. Crystallographic studies of the Monensin A complexes with divalent metal ions revealed different coordination mode than observed for monovalent metal ions. The divalent transition metals form bis complexes coordinated through the terminal donor groups of the ligand by macrochelate formation. In this paper we discuss the synchrotron radiation circular dichroism
3
(SRCD) spectral pattern of Monensinate A anion (Mon–) in the presence of divalent metal ions as obtained in methanolic solutions. Following the changes in the SRCD spectra at various metal-toligand molar ratio we observed for the first time the formation of mono complexes, calculated the equilibrium constants and individual spectra of complex species, as well as evaluated the relative affinity of Monensin A to bind divalent metal cations.
2. Experimental 2.1. Materials Sodium Monensinate A (MonNa) was cordially supplied by Biovet Ltd. (Bulgaria). Monensic acid A monohydrate (MonH×H2O), [Co(Mon)2(H2O)2] and [Mn(H2O)2(H2O)2] were prepared as previously described [25, 31]. Isolated compounds were dissolved in methanol for subsequent measurements. Et4NOH, MeOH and metal(II) salts (CaCl2×2H2O, MgCl2×2H2O, CoCl2×6H2O, MnCl2×4H2O, NiCl2×6H2O, Zn(ClO4)2×6H2O, CdCl2) were provided by Fluka and were of analytical grade.
2.2. Physical measurements SRCD spectra were recorded at the SRCD facility at the AU-CD beam line, itself part of the ASTRID2 storage ring at the Institute for Storage Ring Facilities (ISA), University of Aarhus, Denmark [33, 34]. Samples were placed in 0.1045 mm or 0.014 mm cuvettes. All spectra were recorded in 1 nm steps with a dwell time of 2 s per step, in the wavelength range of 170-300 nm. Two accumulations were averaged and solvent (methanol) spectra acquired at identical conditions were subtracted from those of the samples.
4
ESI-MS spectra were recorded on Waters Micromass ZQ 2000 LCMS System in the range from 0 to 2000 m/z.
2.3. Equilibrium studies Methanolic solutions containing MonH×H2O and Et4NOH (1:1) were mixed with an appropriate amount of corresponding metal(II) salt to obtain series of mixtures with metal-toligand molar ratio varying from 1:10 to 6:1 at a final total ligand concentration of cL = 16-20 mM. Complexation of M2+ ions (M2+ = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+ and Cd2+) was monitored following the changes in the SRCD spectral pattern of Monensinate A anion. Competition experiments were performed in two different experimental design sets using each pair of metal ions studied indicated as M1 and M2, respectively: (1) To a methanolic solution of given M1L2 complex, prepared in situ, a methanolic solution of corresponding competitive divalent metal ion (M2) was added to obtain series of reaction mixtures with molar ratio of both metal ions varying from 1:8 to 8:1. Changes in the spectral pattern of the starting complex M1L2 were followed by SRCD. (2) Experiments were performed recording SRCD spectra of solutions, containing M1 or M2 at metal-to-ligand molar ratio of 4:1. Then we constructed mixtures with M1:M2:L at ratio of 4:4:1. The resultant spectrum observed for M1:M2:L system was treated as an accumulative due to the different contribution of both single spectra belonging to M1L+ and M2L+ species, respectively. Thus, the effect of each divalent cation (M1) was estimated by its distribution coefficient in the presence of the corresponding competitive metal(II) ion (M2).
5
3. Results and discussion 3.1. New CD bands and new complex species observed in the solutions containing Monensinate A anion and divalent metal ions Based on our previous experience with monovalent metal complexes of Monensin A [32], our aim was to characterize the solution behaviour of divalent metal complexes [M(Mon)2(H2O)2] (M2+ = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+ and Cd2+), which are structurally similar in crystalline state. We expected that each divalent metal cation will affect differently the SRCD spectral pattern giving opportunity to discriminate them. SRCD spectra of MonH×H2O, [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2], isolated in solid state and subsequently dissolved in methanol, were recorded in 0.1045 mm optical pathlength supporting the above assumption (Fig. 1a; relates to 1 M total ligand concentration and l = 1 cm pathlength if not otherwise specified). At these conditions reliable data were obtained in the range from 187 nm to 300 nm. We also recorded SRCD spectra of Monensinate A anion prepared in situ (Et4NMon) and of mixtures containing metal(II) ion and Et4NMon at molar ratio of 1:2, analogous to that in complexes [M(Mon)2(H2O)2]. Spectra were obtained in a cell with shorter pathlength (0.014 mm, Fig. 1b). The data were cut off at 178 nm (due to high total absorbance below this wavelength) and new bands in far UV region were observed as compared to longer optical pathlength. The good agreement between spectra of divalent metal complexes as dissolved solids or prepared in situ revealed that obtained species distribution in methanol is identical for both systems confirming the reversibility of complex formation.
6
Fig. 1. SRCD spectra of: a) MonH×H2O, [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] isolated in solid state and dissolved in MeOH, 0.1045 mm optical pathlength; b) Monensinate A anion (Mon–) and solutions containing Co2+:Mon– = 1:2 and Mn2+:Mon– = 1:2 compounds prepared in situ, 0.014 mm cuvette; 20 mM total ligand concentration
Crystal structures of [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] complexes are very close to each other (RMSD < 0.1 Å, see later), but their SRCD spectral characteristics are very different, and also are significantly distinct from the spectral pattern of Monensin A either in its acidic or anionic form. For that reason we monitored the effect of other divalent metal ions they might render on the shape, sign(s) and intensity of the SRCD signal as well. 7
The spectral patterns of reaction mixtures prepared at M2+:Mon– molar ratio of 1:2 are summarized in Fig. 2. A considerable variation depending on metal(II) ion is observed. This kind of sensitivity and selectivity of SRCD as indicator of Monensinate A anion ability to bind various metal ions provoked us to follow the spectral changes caused by the presence of changing amounts of divalent metal cations related to the amount of the ligand.
Fig. 2. SRCD spectral patterns of reaction mixtures, containing M2+:Mon– = 1:2 (recorded in l = 0.014 mm cell)
A set of titration-like experiments was performed, with metal(II) ions and Et4NMon at metal-to-ligand molar ratio ranging from 1:10 to 6:1. Co(II) complexes are selected as a representative system throughout the discussion of results, and the data with the other divalent metal ions studied are collected in Supplementary Section. SRCD spectra of Monensinate A anion in the presence of divalent metal ions are shown in Fig. 3 (Co2+) and Fig. S1, respectively. The positive band of Et4NMon at 193 nm gradually increased in intensity with an increasing amount of Co2+ ions and reached a maximum at 191 nm at metal-to-ligand molar ratio of 1:2. At the same time, positive Et4NMon signal in the range of 200-220 nm diminished and finally a negative band of low intensity appeared in this spectral range. 8
Further increase of Co(II) ion concentration led to formation of second type of species with a positive band at 190 nm, and a weak negative signal within 200-220 nm interval (Fig. 3, left). Spectral changes observed at metal deficit were mainly attributed to the formation of complex [Co(Mon)2(H2O)2], which has been already isolated and characterized in solid state. The second type of Co2+-containing Monensin A species is detected for the first time at comparable Co2+:Mon– molar ratio (1:1) and the spectral pattern of the reaction mixture did not significantly change at a further excess of the metal(II) ion (up to 6:1 metal-to-ligand molar ratio). Coordination of Monensinate A anion in this new species is supposed to be very similar to that in [Co(Mon)2(H2O)2] due to their similar spectral pattern, although the spectra differ in shape and intensity (Fig. 3, left).
Fig. 3. Spectra of Et4NMon (black) at deficit (red) and excess (blue) of Co2+ ions (left); spectral changes at selected wavelengths as a function of the metal-to-ligand molar ratio (right) (20 mM total ligand concentration, l = 0.014 mm)
SRCD spectra of solutions containing Monensinate A anion and other divalent metal ions studied under the same reaction conditions also suggested the formation of two complex species depending on molar metal-to-ligand ratio (Fig. S1, left). To approve this on mathematical basis, 9
an extended Multiple Regression Analysis (MRA) was performed on datasets of the studied systems [35]. In principle the rank of the ellipticity data matrix shall provide the number of independent chiral species. However, the significance of eigenvalues depends very much on the quality of the data. Therefore, we have also analyzed the residual CD signal after considering increasing number of species in the system, to decide which eigenvalues are large enough to take them into account (Fig. 4 for Co2+-Mon– system and Fig. S2 for the other studied cations, respectively). This procedure confirmed that including only two species in the calculation (i.e. the free ligand and the ML2 complex) still results in a systematic deviation of the residual ellipticity from the zero line. Thus, minimum three types of species are required for the satisfactory description of the detected spectral changes.
Fig. 4. Five wavelength-dependent residual CD signal curves for the Co2+-Mon– system
We have plotted the trends in the spectral intensities in Figs. 3 and S1, right. For this we selected wavelengths, which allow to monitor the most significant differences. The changes in the 10
slope of the tangent lines (in certain cases even the sign of the slope is changed) at around metalto-ligand molar ratios 1:2 and 1:1 in most of the systems occur. These kind of titration curves cannot be described by the formation of only one complex species. Taking the metal-to-ligand molar ratios into consideration the third species can be assigned to ML+ mono complexes. Thus, SRCD technique allowed us to observe the formation of the coordination compounds with composition ML+ for divalent metal ions for the first time. Moreover, for the first time we evidenced that Monensin A can bind more than half equivalents of divalent metal ions in the presence of increasing metal-to ligand ratio. It is worth mentioning that the above changes are less pronounced for Zn2+ and Ca2+, while for Cd2+ they are ambiguous. The reason for this is most probably due to the close similarity of the CD spectra of ML2 and ML+ complexes of these metal ions. Eventually we cannot exclude the lack of the formation of ML+ species with these metal ions in methanolic solutions. In parallel we applied ESI-MS technique as an independent method to record the spectra of Monensinate A solutions at excess of divalent metal ions (metal-to-ligand molar ratio of 4:1). The data confirmed the existence of ML+ species in gas phase. Representative MS spectra are placed in Fig S3.
3.2 Stability of the Monensin A complexes with divalent metal ions At this state of the research we suggest that Monensinate A anion forms two types of complex species [M(Mon)2(H2O)2] (ML2) and [M(Mon)(H2O)]+ (ML+), respectively, in methanolic solutions according to equilibria I and II (L– = Mon–, and water molecules were omitted for clarity):
M2+ + 2 L-
ML2 (equilibrium I) 11
2 ML+ (equilibrium II)
M2+ + ML2
Using PSEQUAD program [36] we attempted to calculate the conditional stability constants 𝛽′2 and 𝛽′1 and constant K′ characteristic for the relative stability of the mono and bis complexes from the SRCD data. K' is defined by the equation (1). [𝑀𝐿]2
𝐾1′
(1),
𝐾′ = [𝑀] ∙ [𝑀𝐿 ] = 𝐾2′ 2
where [X] is the equilibrium concentration of species X in solution (free ligand L, metal ion M, complexes ML2 and ML+). The K1' and K2' stepwise stability constants can be calculated from the above values following (2):
𝐾1′ = 𝐾′ ∙ 𝛽′2; 𝐾2′ =
𝛽′2 𝐾′
(2)
Taking into account the limitation of the SRCD data, reasonably good fits of the experimental spectra were obtained (Fig S4, Co2+–Mon– system). Allowing all parameters to be refined in the calculation, we could not get unambiguous data for the ML+ species of Cd2+ and for the ML2 species of Ca2+ complexes in accordance with the above discussion. Since we could measure the SRCD spectrum of the free ligand separately and this species exists in all systems at ligand excess, we have repeated the calculations keeping the spectrum of Monensinate A anion constant. The obtained data by these calculations are shown in Table 1.
Table 1. Parameters characterizing the stabilities of the Monensin A complexes with divalent metal ions obtained from SRCD spectroscopic data by PSEQUAD program keeping the CD spectrum of Monensinate A anion constant (the standard deviation of the refined parameter from the fitting procedure is in parenthesis). 12
Mn2+
Co2+
lgβ1'(ML)
4.8(0.2)
lgβ2'(ML2)
7.8(0.3)
Ni2+
Zn2+
5.9(0.5) 16.0(0.3)
Cd2+
6.7(0.6)
Mg2+
Ca2+
0.9(*) 4.9(0.4) 3.3(0.2)
10.2(0.8) 24.5(0.5) 12.1(1.0) 4.8(0.2) 7.4(0.4)
lgK2'
3.0
4.3
8.5
K'
63
40
3.2×107
5.32
4.7(*)
3.9
2.5
1.4*
27.54 1.0×10–3
251
79
Restraining the spectrum of the free ligand did not change the data and the trends significantly. For the two above mentioned ambiguous systems (these values are marked with * because of their too high standard deviations) we have tried to calculate stability constants by further modification of the species matrix and calculation settings. Assuming that the ML+ spectrum does not differ significantly from that measured at metal ion excess (since the solvated metal ion itself is not optically active) the calculation for Cd2+ complexes yielded 3.4(0.3) for lgβ1' and 5.8(0.3) for lgβ2'. For Ca2+ we could not obtain reliable stability constants for both ML+ and ML2 species. Assuming only one complex was formed we could fit the data with slightly higher deviation, and obtained either 4.0(0.4) value for lgβ1' or 4.8(0.4) for lgβ2'. With the exception of the Ca2+–Mon– system the overall fit of the experimental curves significantly improved with the inclusion of both ML+ and ML2 complexes compared to the fitting procedures with only one complex. The species distribution diagram for the Co2+-Mon– system is shown in Fig 5.
13
Fig. 5. Species distribution diagram of the Co2+-Monensinate A system (the Monensin A total concentration was kept within 16-20 mM)
Under the conditions of the measurements the complex formation occurred almost quantitatively. For this reason the high stability determined for Ni(II)-complexes is also ambiguous, but it clearly reflects that Ni2+ ions have very high affinity towards the Monensinate A anion. In spite of the difficulties to obtain reliable stability constants we shall note that the order of the stabilities for the ML2 complexes - Ni2+ > Zn2+ > Co2+ ~ Mg2+ ~ Mn2+ (> Cd2+ > Ca2+) - is in a very good agreement with the metal–oxygen distances observed for carboxylate and terminal hydroxyl oxygens participating in the direct coordination in the crystal structures of these complexes (Table S1). The shorter are the distances in the crystal structures, the higher is the stability in solution. This is also in correlation with the decreasing ionic radii of the metal ions. The stability order may slightly vary depending on which dataset we use for comparison, but the seeming deviations from the above established order could be explained by the deviating M–O bonds. E.g. while Zn2+ is at relatively low distance to carboxylate oxygen, it has longer bonds to hydroxyl OH and water molecule, which may explain its weak binding in competition experiments (vide infra). 14
The trend of the ML2 stability constants agrees well with the Irving-Williams order. Nickel(II) is a borderline metal ion having high affinity for nitrogen and oxygen donor atoms. Although the stabilities may vary by the microenvironment, i.e. by the geometry, quality of the donor atoms, charges, etc., Ni2+ favours the octahedral geometry with oxygen donors [37-40]. The stability of the ML+ complexes - Ni2+ > Zn2+ > Co2+ ~ Mg2+ > Mn2+ (> Ca2+ ~ Cd2+) follows very similar order as observed for the ML2 complexes. It is worth mentioning that the K' constants reflect in most cases the stronger binding of the ligand to the metal ions in ML+ than in ML2 species in spite of the fact the only the electrically neutral ML2 complexes were obtained so far in solid state. This may arise from the steric and electronic repulsion of the bulky and charged ligands in the metal ion coordination sphere. From that point of view Monensin deficit will lead to competition of the various metal ions for the ligand. To check whether this hypothesis is correct, we initiated series of competition experiments using a solution containing given metal(II) ion M1 and Monensinate A anion at metal-to-ligand molar ratio of 1:2 as a starting point. Spectral changes in this reaction mixture were monitored at deficit and excess of competitive divalent metal ions M2. Results revealed that addition of increasing amount of any M2 ion shifted the equilibrium from M1L2 towards M2L+ species and vice versa – the presence of M1 converts M2L2 into corresponding M1L+ species. Thus, starting out from the M1L2 complex of any studied divalent metal ion by addition of a competing metal ion (M2) we could always reach an equilibrium state in which the M2L+ species of the competing metal ion is dominant. These experiments outlined the general tendency that using ML2 as a starting compound, Monensin A is able to coordinate preferably those metal(II) cations which exceed in a competitive medium. In attempt to look more deeply into chemistry of ML+ complexes, we prepared pairs of mixtures containing M1-Mon– or M2-Mon–, both at molar ratio of 4:1. The selected molar ratio
15
ensures the dominant formation of corresponding ML+ complexes and thus, the SRCD spectrum recorded for each solution belongs mainly to single complex species, M1L+ or M2L+, respectively. Then we mixed methanolic solutions of M1, M2 and Mon– at molar ratio of 4:4:1. The spectrum obtained was treated as an additive one resulting from different contributions of individual M1L+ and M2L+ spectra. Exemplary spectra of the studied systems are given in Fig. S5. The distribution coefficients calculated in such way account for divalent metal ion competition, i.e. for Monensin propensity to selectively complex M2+ ions in ML+ species. Contribution of metal ions studied is summarized in Table 2. These data reflect that the relative affinity of Monensinate A anion in the designed systems decreases in the order of Ni2+ > Co2+ ~ Ca2+ > Mn2+ ~ Mg2+ ~ Zn2+ ~ Cd2+.
Table 2. Effect of competition reactions between divalent metal ions on formation of ML+ Contribution (%) of M1-Mon to the resultant SRCD spectrum at M1:M2:Mon = 4:4:1
Competitive M2 Ni
Co
Ca
Mn
Mg
Co
75
Ca
75
55
Mn
90
75
65
Mg
90
75
70
55
Zn
90
75
90
85
55
Cd
95
80
70
75
60
Zn
Cd
60
Comparison of the presented stability for ML2 and ML+ complexes and that obtained for competing systems revealed a drastic change in the position of the Zn2+ ion in the corresponding series. At this stage of research we cannot explain the observed tendency in the behavior of Monensinate A in the presence of divalent metal ions at different conditions. It can be assumed
16
that additional factors as origin of the metal salt’s counterion, solvolysis, etc. may influence the processes studied. To answer this interesting question is a matter of further studies.
3.3. Solid state and solution structure of divalent metal complexes of Monensin Monensic acid A reacts with divalent metal ions in the presence of Et4NOH to form mononuclear complexes of composition [M(Mon)2(H2O)2] (M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+). Their solid state structure was resolved by X-ray diffractometry on single crystals. Data revealed that divalent metal cations are located in a distorted octahedral environment achieved by coordination of two Monensinate A monoanions. Ligands act in a bidentate coordination mode via the carboxylate moiety and the hydroxyl group both located at the opposite ends of the antibiotic molecule. Axial positions in the inner coordination sphere of metal(II) cations are occupied by two water molecules (Fig. 6) [28-31].
O
O
O O O
H H
O
O
O H
H
O
H H
O O
O M2+ O
O H
O O O O
H
O
O O H O H H H O O
Fig. 6. Bidentate coordination mode of Monensin A in complexes [M(Mon)2(H2O)2]
17
To better understand the effect of divalent metal cations on Monensin conformation, we compared crystal structures of [M(Mon)2(H2O)2] using the root-mean-square deviation (RMSD) values [41] calculated by the PyMol alignment procedure (Table 3). Divalent metal complexes consist of two non-equivalent ligand subunits, containing 47 atoms each, A and B, respectively. RMSD values for the comparison of single Monensinate A anions within every [M(Mon)2(H2O)2] complex vary within a range from 0.090 (Co47A-Co47B) to 0.122 (Mg47A-Mg47B) Å units. Each ligand subunit shows higher RMSD value, c.a. of 0.3 Å, as compared to the crystal structure of Monensin A.
Table 3. Root-mean-square deviations (RMSD in Å units) as a measure of similarity between 94 non-hydrogen backbone atoms of the superimposed divalent metal complexes of Monensin A (47 atoms for each ligand). Crystal structures obtained with various divalent cations were compared among each other. Mg94 M(II) Mg47A Mg47B Mg94 Ca47A Ca47B Ca94 Mn47A Mn47B Mn94 Co47A Co47B Co94 Ni47A Ni47B Ni94
Mg47A
Ca94
Mn94
Co94
Ni94
Zn94
Cd94
MonH
Mg47B
Ca47A
Ca47B
Mn47A
Mn47B
Co47A
Co47B
Ni47A
Ni47B
Zn47A
Zn47B
Cd47A
Cd47B
0.122
0.064
0.149
0.050
0.127
0.058
0.124
0.036
0.113
0.073
0.139
0.102
0.147
0.302
0.125
0.084
0.114
0.043
0.100
0.048
0.132
0.046
0.108
0.053
0.124
0.111
0.342
0.201 0.122
2.219
2.197
0.068
0.084
0.143
0.039
0.116
0.064
0.120
0.080
0.129
0.067
0.134
0.074
0.144
0.296
0.129
0.055
0.124
0.076
0.162
0.111
0.127
0.077
0.121
0.056
0.334
2.265 0.111
2.250
0.248
0.229
0.120
0.043
0.109
0.060
0.112
0.052
0.127
0.064
0.149
0.292
0.101
0.044
0.138
0.065
0.107
0.048
0.112
0.083
0.340
0.094 0.090
2.206
2.206
2.240
0.068
0.097
0.038
0.106
0.068
0.139
0.303
0.132
0.061
0.097
0.042
0.109
0.088
0.333
2.183 0.115
2.183
2.221
0.087
0.149
0.114
0.184
0.294
0.111
0.075
0.130
0.136
0.342
0.084
0.179
18
Zn47A
0.110
Zn47B Zn94
0.061
0.140
0.309
0.121
0.087
0.342
0.139
Cd47A
0.128
Cd47B
0.302 0.332
Comparison of structures of divalent metal complexes revealed close similarity between corresponding A and B units, respectively. The RMSD lay within relatively narrow range from 0.036 (Mg47A–Ni47A) to 0.088 (Co47B-Cd47B) Å except Ni47B-Cd47B subunits (0.136 Å). The RMSD values comparing the pairs of opposite ligand subunits show larger variation from 0.097 (Co47A-Ni47B) to 0.184 (Ni47A–Cd47B) Å units, respectively. The observed deviations within the structures of individual divalent metal complexes show close resemblance of single ligand anion conformation, which is not strongly affected by the coordination of corresponding metal cations. On the other hand, comparison of overall structures of [M(Mon)2(H2O)2] (two Monensinate A anions, 94 atoms), classifies complexes into three groups. Group 1 ([Co(Mon)2(H2O)2], [Mn(Mon)2(H2O)2]) can be distinguished from other metal complexes by large RMSD values of ~ 2.2 Å. The members of group 2 ([Mg(Mon)2(H2O)2], [Ni(Mon)2(H2O)2], [Zn(Mon)2(H2O)2]) and group 3 ([Ca(Mon)2(H2O)2], [Cd(Mon)2(H2O)2]) show smaller RMSD values of ~ 0.2 Å between the groups, but the metal-oxygen donor atom distances differ significantly being ~ 2.02-2.04 Å and 2.21-2.26 Å for the two groups, respectively. Values of RMSD within the members of each group are less than 0.1 Å, with the exception of group 3 where it is 0.12. The question arose, whether these characteristics can account for the observations in methanolic solutions by SRCD? One of the greatest achievements of the equilibrium calculations is that instead of the spectra of reaction mixtures, they provide the spectra of the individual 19
complex species. The shapes of the spectra were rather independent on the mode of the calculations as shown in Fig. S6 for the Co2+-Monensin A system. Fig. 7a shows the comparison of the spectra for ML2 complexes of all studied metal ions. It is clear that SRCD can differentiate between the various divalent metal complexes, as their spectral patterns are different from each other. Such discrimination of divalent metal ion complexes of Monensin A observed by SRCD could be assumed to rise from the fine changes in ligand conformation due to the metal ion’s geometry requirements. Various metal ions also affect the SRCD spectral pattern uniquely through the involvement of the electrons of metal ions into the molecular orbitals responsible for the chiral effects as it was also shown in our previous paper for monovalent alkaline metal ions [32].
20
Fig. 7. Individual molar SRCD spectra of ML2 (a) and ML+ (b) complexes obtained from PSEQUAD calculations. Spectra of the ML2 complex of Ca2+ and ML+ complex of Cd2+ could not be reliably determined - see text for more details.
The spectral patterns for the individual ML+ complexes obtained from the calculations by PSEQUAD program are summarized in Fig. 7b. From the similarities between corresponding pairwise ML+ and ML2 SRCD spectra we suggest that the excess of metal(II) ions undergoes formation of positively charged complex species of composition [M(Mon)(H2O)]+ where ligand anion acts in a bidentate coordination manner similarly as in [M(Mon)2(H2O)2] complexes. Thus, the metal ion is most probably not bound in the cavity of the ligand as it happens with the monovalent metal ions. This is also supported by the similarity of the stability order of the ML+ and ML2 complexes. The presence of water molecules in the structure of Monensic acid and corresponding neutral complexes in solution was previously confirmed by NMR studies of diamagnetic compounds [29-30]. The difference between spectra of [M(Mon)2(H2O)2] and [M(Mon)(H2O)]+ species can be detected by SRCD due to the sensitivity of this technique towards fine conformational changes of the ligand upon metal ion complexation. To elucidate the intimate structure of [M(Mon)(H2O)]+, further experiments on their possible isolation, crystallization and characterization have to be performed.
4. Conclusions The ability of Monensin A (MonH) to bind divalent metal cations (M = Co2+, Mn2+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+) in methanolic solutions was evaluated by means of SRCD spectroscopy. The experimental results revealed that:
21
1). SRCD was proven to be an efficient methodology to discriminate among different divalent metal ions bound in neutral complex species [M(Mon)2(H2O)2] (ML2). 2). Monensin A formed new positively charged complex species of suggested composition [M(Mon)(H2O)]+ (ML+) at comparable or higher metal-to-ligand molar ratio with a similar to the coordination mode of the ligand in ML2. The presence of ML+ was confirmed for the first time using SRCD technique, but further structure elucidation is required. 3) Although close, overall SRCD signals of ML2 and ML+ differ in position and intensity most likely due to fine ligand conformational changes caused by the coordination of divalent metal ions and complemented by charge transfer processes. 4). Equilibrium constants of the most of the processes studied were calculated and individual molar spectra of ML+ and ML2 species were derived. 5). It was found that Monensin A binds preferably those divalent metal cations which exceed in a competitive reaction medium. In addition, metal(II) ion excess increases the coordination ability of the ligand in ML+ species. 6). At comparable but excessive concentration of competitive metal ions (compared to the ligand) Monensin A affinity to bind M(II) as ML+ species decreases in the order of Ni2+ > Co2+ ~ Ca2+ > Mn2+ ~ Mg2+ ~ Zn2+ ~ Cd2+. The above results envisage a more complex role of Monensin A as ionophore in binding and transporting various metal ions, which may have importance in regulating the metal ion homeostasis and the toxic effects of metal ions. Such comparative studies including a range of metal ions is necessary to better understand these processes.
22
Acknowledgements Financial support of TÁMOP-4.2.4.A/2, John von Neumann International Scholarship for senior foreign teachers-researchers, CALIPSOplus (EU Framework Programme for Research and Innovation HORIZON 2020, grant no. 730872) and from the Hungarian National Research, Development and Innovation Office (GINOP-2.3.2-15-2016-00038) is greatly acknowledged. The authors are thankful to Nykola Jones and Søren Vrønning Hoffmann (Aarhus University, Denmark) for the valuable discussion on SRCD technique and data processing procedures. IP is thankful to the National Science Fund, Bulgarian Ministry of Education and Science (contract № KP-06-H29/3).
References 1. Chapman, H.D., Barta, J.R., Blake, D., Gruber, A., Jenkins, M., Smith, N.C., Suo, X., Tomley, F.M. A selective review of advances in coccidiosis research, Adv. Parasitol. 83 (2013) 93-171. https://doi.org/10.1016/B978-0-12-407705-8.00002-1. 2. Chapman, H.D., Jeffers, T.K., Williams, R.B. Forty years of monensin for the control of coccidiosis in poultry, Poult. Sci. 89 (2010) 1788-1801. https://doi.org/10.3382/ps.201000931. 3. Anadón, A., Martinez-Larrañaga, M.R., Castellano, V. Antimicrobials (including coccidiostats) in poultry, J. Vet. Pharmacol. Therap. 32 (SUPPL. 1) (2009) 26-28. 4. Croft, S.L. The current status of antiparasite chemotherapy, Parasitology, 114 (SUPPL.), (1997) S3-S15.
23
5. Rutkowski, J., Brzezinski, B. Structures and properties of naturally occurring polyether antibiotics,
BioMed
Res.
Intern.
(2013)
art.
ID
162513.
http://dx.doi.org/10.1155/2013/162513. 6. Nelson, M.L., Grier, M.C., Barbaro, S.E., Ismail, M.Y. Polyfunctional antibiotics affecting bacterial membrane dynamics, Anti-Infective Agents Med. Chem. 8 (2009) 3-16. https://doi.org/10.2174/187152109787047779. 7. Kevin II, D.A., Meujo, D.A.F., Hamann, M.T. Polyether ionophores: Broad-spectrum and promising biologically active molecules for the control of drug-resistant bacteria and parasites,
Expert
Opin.
Drug
Disc.
4
(2009)
109-146.
https://doi.org/10.1517/17460440802661443. 8. Huczyński, A., Janczak, J., Łowicki, D., Brzezinski, B. Monensin A acid complexes as a model of electrogenic transport of sodium cation, Biochim. Biophys. Acta - Biomembranes 1818 (2012) 2108-2119. https://doi.org/10.1016/j.bbamem.2012.04.017. 9. Huczyński, A., Ratajczak-Sitarz, M., Katrusiak, A., Brzezinski, B. Molecular structure of rubidium six-coordinated dihydrate complex with monensin A, J. Mol. Str. 888 (2008) 224-229. https://doi.org/10.1016/j.molstruc.2007.12.005. 10. Huczyński, A., Ratajczak-Sitarz, M., Katrusiak, A., Brzezinski, B. Molecular structure of the 1: 1 inclusion complex of monensin A sodium salt with acetonitrile, J. Mol. Str. 832 (2007) 84-89. https://doi.org/10.1016/j.molstruc.2006.07.043. 11. Martinek, T., Riddell, F.G., Wilson, C., Weller, C.T. The conformations of monensin-A metal complexes in solution determined by NMR spectroscopy, J. Chem. Soc. Perkin Transact. 2 (2001) 35-41. https://doi.org/10.1039/A907984J.
24
12. Raban, M., Quin III, J., Belguise, A., Durocher, D., Kost, D. Conformationally switchedon polyether ionophores, Chirality 10 (1998) 78-87. https://doi.org/10.1002/chir.13. 13. Wagner-Czauderna, E., Rzeszotarska, J., Orlowska, E., Kalinowski, M.K. Complexing ability of monensin anion for alkali metal cations in binary mixtures of dipolar aprotic solvents, Berichte der Bunsengesellschaft / Physical Chemistry Chemical Physics, 101 (1997) 1154-1157. https://doi.org/10.1002/bbpc.19971010811. 14. Pointud, Y., Bernard, C., Touzain, S., Astier, L., Sabatier, B., Juillard, J. Thermodynamics of complexation of monovalent metal cations by the ioniophore monensin free acid in acetonitrile, J. Solution Chem. 26 (1997) 479-495. https://doi.org/10.1007/BF02767602. 15. Hebrant, M., Pointud, Y., Juillard, J. Thermodynamics of reaction in heterogeneous systems (water-organic phases) between the ionophore monensin and alkali-metal cations, J. Phys. Chem. 95 (1991) 3653-3662. https://doi.org/10.1021/j100162a041. 16. Riddell, F.G., Arumugam, S., Cox, B.G. The monesin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR, Biochim. Biophys. Acta Biomembranes 944 (1988) 279-284. https://doi.org/10.1016/0005-2736(88)90442-7. 17. Cox, B.G., Van Truong, N., Rzeszotarska, J., Schneider, H. Rates and equilibria of alkali metal and silver ion complex formation with monensin in ethanol, JACS 106 (1984) 59655969. https://doi.org/10.1021/ja00332a035. 18. Koryta, J., Du, G., Ruth, W., Vanýsek, P. Transfer of alkali-metal and hydrogen ions across liquid/liquid interfaces mediated by monensin: A voltammetric study at the interface of two immiscible electrolyte solutions, Faraday Discussions of the Chemical Society, 77 (1984) 209-216. https://doi.org/10.1039/DC9847700209.
25
19. Duax, W.L., Smith, G.D., Strong, P.D. Complexation of metal ions by monensin. Crystal and molecular structure of hydrated and anhydrous crystal forms of sodium Monensin JACS 102 (1980) 6725-6729. https://doi.org/10.1021/ja00542a010. 20. Hoogerheide, J.G., Popov, A.I. A study of metal complexes of a naturally occurring macrocyclic
ionophore-monensin,
J.
Solution
Chem.
8
(1979)
83-95.
https://doi.org/10.1007/BF00646810. 21. Hoogerheide, J.G., Popov, A.I. Study of monensin complexes with monovalent metal lons in
anhydrous
methanol
solutions,
J.
Solution
Chem.
7
(1978)
357-372.
https://doi.org/10.1007/BF00662896. 22. Gertenbach, P.G., Popov, A.I. Solution chemistry of monensin and its alkali metal ion complexes. Potentiometric and spectroscopic studies, JACS 97 (1975) 4738-4744. https://doi.org/10.1021/ja00849a041. 23. Steinrauf, L.K., Czerwinski, E.W., Pinkerton, M. Comparison of the monovalent cation complexes of monensin, nigericin, and dianemycin, Biochem. Biophys. Res. Comm. 45 (1971) 1279-1283. https://doi.org/10.1016/0006-291X(71)90156-2. 24. Lutz, W.K., Winkler, F.K., Dunitz, J.D. Crystal structure of the antibiotic monensin similarities and differences betweeen free acid and metal complex, Helv. Chim. Acta 54 (1971) 1103-1108. https://doi.org/10.1002/hlca.19710540419. 25. Pinkerton, M., Steinrauf, L.K. Molecular structure of monovalent metal cation complexes of Monensin, J. Mol. Biol. 49 (1970) 533-546. https://doi.org/10.1016/00222836(70)90279-2. 26. Pantcheva, I.N., Dorkov, P., Atanasov, V.N., Mitewa, M., Shivachev, B.L., Nikolova, R.P., Mayer-Figge, H., Sheldrick, W.S. Crystal structure and properties of the copper(II)
26
complex of sodium monensin A, J. Inorg. Biochem. 103 (2009) 1419-1424. https://doi.org/10.1016/j.jinorgbio.2009.08.007. 27. Dorkov, P., Pantcheva, I.N., Sheldrick, W.S., Mayer-Figge, H., Petrova, R., Mitewa, M. Synthesis, structure and antimicrobial activity of manganese(II) and cobalt(II) complexes of the polyether ionophore antibiotic sodium monensin A, J. Inorg. Biochem. 102 (2008) 26-32. https://doi.org/10.1016/j.jinorgbio.2007.06.033. 28. Pantcheva, I.N., Zhorova, R., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. First solid state alkaline-earth complexes of monensic A acid: X-ray crystal structure of [M(Mon)2(H2O)2] (M = Mg, Ca), spectral properties and cytotoxicity against Grampositive bacteria, BioMetals 2 (2010) 59-70. https://doi.org/10.1007/s10534-009-9269-5. 29. Pantcheva, I.N., Ivanova, J., Zhorova, R., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. Nickel(II) and zinc(II) dimonensinates: crystal structure, spectral properties and bactericidal activity, Inorg. Chim. Acta 363 (2010) 1879-1886. https://doi.org/10.1016/j.ica.2010.02.009. 30. Ivanova, J., Pantcheva, I.N., Mitewa, M., Simova, S., Mayer-Figge, H., Sheldrick, W.S. Crystal structures and spectral properties of new Cd(II) and Hg(II) complexes of monensic acid with different coordination modes of the ligand, Centr. Eur. J. Chem. 8 (2010) 852860. https://doi.org/10.2478/s11532-010-0067-2. 31. Pantcheva, I.N., Mitewa, M.Io., Sheldrick, W.S., Oppel, I. M., Zhorova, R., Dorkov, P. First divalent metal complexes of the polyether ionophore monensin A: X-ray structures of [Co(Mon)2(H2O)2] and [Mn(Mon)2(H2O)2] and their properties, Curr. Drug Discov. Technol. 5 (2008) 154-161. https://doi.org/10.2174/157016308784746247.
27
32. Nedzhib, A., Kessler, J., Bour, P., Gyurcsik, B., Pantcheva, I., Circular dichroism is sensitive to monovalent cation binding in Monensin complexes, Chirality 28 (2016) 420428. https://doi.org/10.1002/chir.22597. 33. Miles, A.J., Hoffmann, S.V., Tao, Y., Janes, R.W., Wallace, B.A., Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy: New beamlines and new applications in biology, Spectroscopy 21 (2007) 245-255. http://dx.doi.org/10.1155/2007/282713. 34. Miles, A.J., Janes, R.W., Brown, A., Clarke, D.T., Sutherland, J.C., Tao, Y., Wallace, B.A., Hoffmann, S.V., Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines, J. Synchrotron Rad. 15 (2008) 420422. http://dx.doi.org/10.1107/S0909049508009606. 35. Peintler, G., Nagypál, I., Jancsó, A., Epstein, I.R., Kustin, K. Extracting experimental information from large matrixes. 1. A new algorithm for the application of matrix rank analysis, J. Phys. Chem. A 101 (1997) 8013–8020. https://doi.org/10.1021/jp970136s. 36. Zékány, I., Nagypál, I., Peintler, G. PSEQUAD for Chemical Equilibria, update 5.10; Technical Software Distributors: Baltimore, MD, 1991. 37. Driessen, W.L., Everstijn, P.L.A., Dimethyl phthalate, a neutral bidentate oxygen-donor ligand
to
divalent
metal
ions,
Z.
Naturforsch.
B
32
(1977)
1284–1286.
https://doi.org/10.1515/znb-1977-1113. 38. Hancock, R.D., Martell A.E. Ligand design for selective complexation of metal ions in aqueous solutions, Chem. Rev. 89 (1989) 1875-1914.https://doi.org/10.1021/cr00098a011. 39. Rulísek, L., Vondrásek. J. Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins, J. Inorg. Biochem. 71 (1998) 115127. https://doi.org/10.1016/S0162-0134(98)10042-9.
28
40. Marino, T., Toscano, M., Russo, N., Grand, A. Structural and electronic characterization of the complexes obtained by the interaction between bare and hydrated first-row transition-metal ions (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+) and glycine, J. Phys. Chem. B 110 (2006) 24666-24673. https://doi.org/10.1021/jp0645972. 41. Heifets, A., Lilien, R. LigAlign: flexible ligand-based active site alignment and analysis, J. Mol. Graph. Modell 29 (2010) 93-101. http://dx.doi.org/10.1016/j.jmgm.2010.05.005.
29
SRCD spectra of ML+ and ML2
Co2+ Zn2+ Ni2+
Ti
n tio a tr
Mg2+
Mn2+ Ca2+ Cd2+
Co
mp
eti
tio n
30
Discrimination among different M2+ ions in neutral ML2 complexes was achieved by SRCD
The presence of new positively charged complexes ML+ was confirmed for the first time
Excess of M2+ increases the coordination ability of the ligand in ML+ species
Individual spectra of ML+ and ML2 and their stability constants were calculated
Monensin A affinity to bind M(II) as ML+ species was evaluated
31