Chelating copper ions by aminohydroxamic acids as a potential way to remove them from the body cd.

2. The structure and formation constants of copper complexes

2.1. The structure of macrocycles

Pentameric complex compounds of the 12- metalocrown- 4 type, analogous to crown ethers, have been reinvestigated. The complexes contained copper and different hydroxamic acids: malonomonohydroxamate [5], β-alaninehydroxamate [4], α-glycinehydroxamate and α-phenylalaninehydroxamate [3].



Mass spectrometry allowed us to confirm the exact molecular mass of the complexes, and this allowed us to speculate about their structure. The measurements have been made on a micrOTOF-Q mass spectrometer, Bruker Daltonics, with electrospray being the ionization technique. The quadrupole mass analyser, which lets only the ions with the appropriate mass to charge ratio pass (the MS/MS experiment), was used to evaluate the stability of the investigated structures.

The solution of the ligand has been prepared in a water and ethanol (1:1 volume ratio). The concentration of copper was 10-4M, and the concentration of the appropriate ligand was 2*10-4M. The measurements have been performed in a 50 to 2000 m/z ratio. The results and the proposed structures are shown in the table below:


Tandem mass spectrometry (MS/MS) has shown remarkable stability of the [Cu5Pheha4H-4]2+ and [Cu5Pheha3GlyhaH-4]2+ complexes; the structures (the mass to charge ratios corresponding to the complex structures, to be exact) remained unchanged even in energies as high as 35eV; when exposed to higher energies, the disintegration of the side chain of α-phenylalaninehydroxamic acid occurs, whereas the core of the complex itself remains unchanged. Therefore is seemed interesting to determine the formation constants of the mixed forms of α-glycinehydroxamic and α-phenylalaninehydroxamic acid.

2.2. The determination of the stability constants of the complexes

The formation constants have been calculated based on potentiometric experiments. In these measurements, the electromotive force (EMF) of a cell is measured. It depends on the difference of the electrode potentials, and those depend on the activity of the ions in the solution. The dependence of EMF on ionic activity is given by the formula

where E0 – standard potential of the electrode [mV], E – the measured potential [mV], [H+] – the concentration of hydrogen ions SL – slope factor. In the experiment described below, a Metrhom automatic titration set Has been used. The titrations have been carried out in 0.1M KNO3 with HNO3 having the final concentration of 0,004M. The ligand concentrations were 4mM, and their molar ratio to Cu2+ ions varied from 1:2 to 1:4. The titrations have been carried out in pH 2-11. In general, formation is described by the equation (2); the stability constant for that process is given by (3)


All constants have been calculated using Hyperquad 2006. The protonation constants of ligands and the formation constants of the ligand- copper dimmers are in perfect agreement with the literature data of similar potentiometric measurements carried out in methanol [3]; the formation constants of pentamers in aqueous solutions differ slightly from the values of formation constants in MetOH given in literature [3]. The potentiometric measurements have also shown the existence of a new, not yet shown in any literature mixed complex [Cu5Pheha3GlyhaH-4]2+.





A distribution diagram of Cu(II)/Pheha/Glyha (Cu:Pheha:Glyha = 1:1:1, cCu= 3mM) has been shown below.



LITERATURE

[1] BERTINI I., MESSORI L., VIEZZOLI M.S., Handbook of Metal-Ligand Interactions in Biological Fluids. Bioorganic Chemistry (vol.1 )Copper proteins, New York 1995. [2] CHAN W.Y., GARNICA W.Y., RENNERT O.M., Handbook of Metal-Ligand Interactions in Biological Fluids. Bioinorganic Medicine (vol.2) Genetic trace metal disturbances, New York 1995.
[3] DALLAALLE F., TEGONI M., Speciation and structure of copper(II) complexes with (S)-phenylalanine- and (S)-tryptophanhydroxamic acids in methanol/water solution: a combined potentiometric, spectrophotometric, CD and ESI-MS study; Polyhedron 20 (2001) 2697–2704.
[4] FARKAS E. et al., Synthesis and characterisation of Cu2+, Ni2+ and Zn2+ binding capability of some amino- and imidazole hydroxamic acids: Effects of substitution of side chain amino-N for imidazole-N or hydroxamic-N-H for N-CH3 in metal complexation, Polyhedron 26(2007) 543-554.
[5] GUMIENNA-KONTECKA E. et al., A New Cu(II) [12]metallocrown-4 pentanuclear complex based on a Cu(II)-malonomonohydroxamic acid unit, New J. Chem., 2007, 31, 1798–1805.
[6] KABALT-PENDIAS A., Biochemia pierwiastków śladowych, Wydawnictwo Naukowe PWN, Warszawa 1993.
[7] LIPPARD S.L., BERG J.M., Podstawy chemii bionieorganicznej, Wydawnictwo Naukowe PWN, Warszawa 1998.
[8] SZEMCZUK W., Toksykologia, Wydawnictwo Lekarskie PZWL, Warszawa 2002.