EPR study of complex formation between copper (II) ions
and sympathomimetic amines in aqueous solution
E. A. PREOTEASA(1), O. G. DULIU(2) and V. V. GRECU(2)(1) Institute of Atomic Physics, IFIN, Laboratory 8
Magurele, P.O. Box MG-6, RO-76900 Bucharest, Romania
(2) University of Bucharest, Department of Atomic and Nuclear Physics Magurele, P.O. Box MG-11, RO-76900 Bucharest, Romania
(ricevuto il 13 Marzo 1996; approvato il 7 Aprile 1997)
Summary. — The complex formation between sympathomimetic amines (SA):
adrena-line (AD), noradrenaadrena-line (NA), dopamine (DA), ephedrine (ED) and p-tyramine (pTA), and Cu(II) ion in aqueous solution has been studied by X-band EPR at room temperature. Excepting pTA, all investigated SA yielded two types of complexes in different pH domains. All complexes presented a gllD g» relationship, consistent
with a ligand field having a distorted octahedral symmetry, i.e. hexacoordination of Cu(II). The covalence coefficient calculated from the isotropic g and A values has shown strong ionic sigma-type ligand bonds. A structural model with the Cu(II) ion bound by four catecholic O(hydroxy) atoms for the low pH complexes of AD, NA, and DA is proposed. For the high pH complexes of the former compounds as well as for both ED complexes, we suppose Cu(II) bound by two N (amino) and two O (hydroxy) atoms. The spectra are consistent to water binding on the longitudinal octahedron axis in all compounds excepting the high pH complex of ED, where OH2ions are
bound. Possible implications for the SA-cell receptors interactions are discussed. PACS 87.15.By – Structure, bonding, conformation, configuration and isomerism of biomolecules.
PACS 87.64.Hd – EPR and NMR spectroscopy.
1. – Introduction
Sympathomimetic amines (SA) are biological molecules with strong pharmacologi-cal action, involved in various physiopathologipharmacologi-cal processes. Some of them, like adrenaline (AD), noradrenaline (NA) and ephedrine (ED), are common drugs in medicine. Best known among the SA are catecholamines (CA), namely AD, NA and dopamine (DA). They play an important role in cardiomiopathies, probably in association with free radicals mechanisms [1], and impairments of their metabolism are incriminated in schizophrenia and Parkinson’s disease [2]. Together with insulin, CA
are regulators of lipolysis and miscontrol of this process occurs in conditions such as aging, stress, obesity and diabetes [3, 4].
The SA act as chemical messengers, that is, molecules which include drugs, hormones, and neurotransmitters and which interact very specifically with the so-called cell receptors, inducing biological responses even at very low concentrations (A10211g Oml) [5]. The SA activate the alpha- and beta-adrenergic as well as the dopaminergic receptors and compete with histamine for its binding site. It has been suggested that a divalent metal ion mediates the interaction of some SA with receptors [5]. Other specific interactions of the SA involve also metal ions. Thus CA, which are substrates for copper oxidases and particularly for the ceruloplasmin, bind to the active sites of Cu(II) ions in the enzymes [6]. Noteworthy, in experimental copper-deficient animals, the CA show an accelerated turn-over and an alterated distribution [7, 8].
In the same time, the SA form stable coordination complexes with divalent copper. These provide convenient model systems for the mechanisms underlying the biological activity of the SA. The complexes of AD and NA with the Cu(II) ion in aqueous solution have been investigated by spectrophotometric and electrochemistry methods [9-11]. Studies of Cu(II) chelates of the SA and related compounds done so far by Electron Paramagnetic Resonance (EPR) spectroscopy are not very numerous. They include the investigation of the interaction between AD and NA and the free and ceruloplasmin-bound copper ion [6], the study of the complex formation between Cu(II) and three catecholamines (AD, NA, and DA) as well as their precursor amino acids (Phe, Tyr, and Dopa) [12], detailed investigations of the copper-phenylalanine in monocrystal [13] and of the copper-catechol in solution [14]. Later, the complexes of Cu(II) with the semiquinone radical anion forms of AD, NA, DA, Dopa, and catechol were studied [15].
In the present paper we report the investigation of the Cu(II)-SA complex formation in aqueous solution at various pH values by X-band EPR spectroscopy at room temperature. The interactions with divalent copper were followed comparatively for three representative CA on the one hand—AD, NA, and DA—and for two noncatecholic SA, i.e. ED and p-tyramine (pTA), on the other.
2. – Materials and methods
Adrenaline bitartrate (BDH), noradrenaline HCl (Calbiochem), dopamine HCl (Calbiochem), ephedrine HCl (NBC) and p-tyramine HCl (NBC) all of B grade or better purity have been used. Fresh 1.25–3.75 31021M solutions of the SA were mixed with equal volumes of a 5 –12.5 31022M solution of Cu
2SO4 (Merck). No buffers were used and the pH between 4 and 12 was adjusted with drops of KOH and H2SO4 solutions. The samples were introduced into glass capillaries of 0.5 mm inner diamter; the liquid column height was of 2 cm. The EPR spectra were recorded at room temperature with a X-band JEOL ME-3X spectrometer equipped with a TE011 resonant cavity. A modulation field of 100 kHz frequency and 0.63 mT amplitude has been used. The magnetic field has been calibrated using the diphenyl-picryl-hydrazyl (DPPH) line (g 42.0036). Spectra subtraction has been done only in the case of some Cu(II)-ED spectra recorded at different pH values.
3. – Results and discussion
The EPR spectra (see fig. 1) showed that all the investigated SA, excepting pTA, yielded coordination complexes with the Cu(II) ion. However, while AN, NA, DA showed spectroscopically similar chelating behaviour, the properties of ED complexes were different. Moreover, the three CA on the one hand and ED on the other formed two distinct types of copper complexes towards weakly acid and, respectively basic pH values. We termed these complex by C8 and C9, respectively. The C8 species began to appear in weakly acid and neutral environment but developed fully only at higher pH values, and C9 attained appreciable levels only at strong alkalinity of the solution. This
Fig. 1. – The EPR spectra of the Cu(II) complexes with sympathomimetic amines. The line shows the position of DPPH.
TABLE I. – The spin Hamiltonian parameters evaluated from the computer simulation of the ESR spectra of copper complexes formed by investigated SA (*) as well as the corresponding
chemical bond parameters for various pH domains.
Complex pH range A0 (1024cm21) gll (1024cm21) g» (6 0.007) g0 (6 0.007) a2 (6 0.05) x (%) C8(CA) C9(CA) C8(ED) C9(ED) 4.5–8.5 D 6.0 6.5–12 D7.5 69 6 4 88 6 6 77 6 5 95 6 3 2.194 2.169 2.157 2.152 2.101 2.054 2.054 2.025 2.166 2.135 2.128 2.119 0.86 0.90 0.81 0.92 72 80 62 84
suggests that the ligand groups had to deprotonate before binding of the Cu(II) ion. Deprotonation is typical for hydroxy and amino groups when the pH increases. In fact nuclear polarographic [9], optical [10, 11] and magnetic resonance [16] studies on deprotonation in AD, NA and Dopa showed that the phenolic hydroxy groups are more acidic than the ammonium side chain. That is, oxygen ionizes first at lower pH and the amino deprotonates at higher alkalinity, enabling the formation of C8 and C9 complexes, respectively.
A direct estimate of the spin Hamiltonian parameters (see table I) was possible even without a spectral fit because all EPR spectra showed a well-resolved hyperfine structure at gll due to the I 43O2 nuclear spin of both
63
Cu and 65Cu (fig. 1). The spectroscopic data clearly evidenced the four mentioned types of Cu(II) complexes: C8(CA) and C8(ED) towards lower pH and, respectively, C9(CA) and C9(ED) towards highly basic pH (where CA 4 AN, NA, DA). The spin Hamiltonian parameters were equal within experimental errors for AN, NA, and DA both for the low- and high-pH complexes, while they were different for the corresponding species formed by the ED.
The experimental values of the main components of the g tensor satisfied a gllD g» relationship for all types of complexes. This was consistent with a distorted elongated octahedral symmetry of the ligand field. The last might be predominantly tetragonal (D4 h) or slightly rhombic (D2 h), but owing to our limited precision in the determination of spin Hamiltonian parameters we cannot establish unambiguously the punctual symmetry group of the Cu(II) ion coordination. Admitting D4 h symmetry, we used a molecular orbital approach locating the copper unpaired electron in a B *1 gground state; from the isotropic A0 and g0 values of the solution EPR spectra, we obtained an approximation of the covalency coefficient a2 of the s bonds between copper and ligands [17-19]: a2 4 K021
g
NA 0N P 1 g02 2.00h
, (1)where P 42gbbNadx22 y2Nr23Ndx22 y2b 40.36 cm21; K04 0.4310.02 is the free-ion
Fermi contact term.
This means that the odd electron is located on the Cu(II) with a probability a2and on each of the six ligands with a probability ( 1 O6)(12a2), respectively. In other words,
a2equals 0.5 for completely covalent bonds and 1 for completely ionic bonds, it provides an intuitive estimation of the percent of ionic character of the s bonds between Cu(II)
ion and SA ligand groups in the coordination complexes x 4 a 2 2 0.5 0.5 . ( 2 )
The values of the last equation (table I) suggests a ionic character for the system of coordinative bonds in all the four types of complexes. The ionic character was stronger in the species formed towards more alkaline pH and somewhat weaker for those favoured in a less basic environment, especially for C8(ED). The ligand groups’ basicities or negative charges increase monotonously with the percent of ionic character of the bonds. Thus both for the CA and the ED complexes the overall basicity of ligands was higher for the species formed at more alkaline pH, but the most significant basicity difference between the ligand groups involved was shown in the complexes of ephedrine, namely C9(ED) D C9(ED).
The EPR spectrum of the C9(ED) clearly presents a partially resolved superhyperfine structure with five components due to the interaction of Cu(II) with two equivalent nitrogen atoms (I 43O2). The same feature emerged with less pregnancy in the spectra of the Cu(II) complexes formed by AD, NA, and DA at alkaline pH in solutions tenfold more diluted [12]. However, at those low
concentrations the signal-to-noise ratio was unfavourable for the clear observation of superhyperfine components. On the other hand, now this structure was not resolved any longer for the CA complexes, probably due to broadening associated with enhanced interactions between the paramagnetic centers at the higher concentrations used.
Specific structural models for the complexes have been proposed. In all complexes, four of the six ligand atoms of copper ion are contributed by two SA molecules, in a coplanar array at the corners of a square or a rhomb normal to the octahedron’s axis. In the C8(CA) species the cupric ion is appearently bound to four oxygen atoms from catecholic hydroxy groups, while two N (amino) and two O (hydroxy) atoms bind to the metal in the C9(CA), C8(ED) and C9(ED). The interaction with two nitrogens is in fact visible in the partly resolved superhyperfine structure in the spectrum of C9(ED). Concurrently, on the longitudinal axis of the octahedron, two water molecules attach in the C8(CA), C9(CA) and C8(ED) complexes. The same oxygen and nitrogen atoms are involved in both complexes of the ED, but in the C9(ED) species the more ionic character of the bonds and the strongest overall basicity of ligands suggest that on the longitudinal axis OH2 ions are bond instead of water molecules. In fact ephedrine complexes similar to C9(ED) have been proposed for long [20]. The proposed molecular structures of the complexes are presented in fig. 2. In the C9 species formed by dopamine each DA molecule contributes with a catecholic oxygen and with a nitrogen from the side chain. This has to adopt a strongly bent conformation to allow its nitrogen to reach the right position in the appropriate octahedron’s corner. Similar structures do not occur with AD and NA. Therefore, the side chain of DA must have a flexibility much higher than in AD and NA, where the hydroxy group might produce a steric hindrance.
In support of our model, other Cu(II) chelates showing comparable EPR parameters have similar structural features. Thus the components of the g tensor in the C8(CA) complexes are reasonably close to those of the hexacoordinated complexes of copper with the formic [21] and malonic [22] acids, where the organic ligands contribute four oxygen ligand atoms while other two are provided by water molecules. In their turn, the C9(CA) and C8(ED) complexes showed some similarity in the
Fig. 2. – The proposed structural models of the low and high pH Cu(II) complexes with the investigated sympathomimetic amines. The net electric charges of the complexes are as follows: C8(CA) 4 0; C9(CA) 4 2 4 for AD and NA; C9(CA) 4 2 2 for DA; C8(ED) 4 0; C9(ED) 4 22.
spectroscopic parameters with the Cu(II)-acetylglycylglycyl-L-hystidine [23] and with Cu(II)-dimethylglyoxime in water [24], where both O an N atoms are involved in the binding of copper. In our model the C9(ED) complex shows the same feature and its spin Hamiltonian parameters are in fact comparable to those of Cu(II)-acetyl-glycyl-L-hystidine [23], Cu(II)-bis(dimethylglyoximate) [25], and Cu(II)-diethanola-mine in water [26]. Moreover the EPR parameters of C9(ED) are similar to those of the Cu(II)-(trietanolamine)2(OH2)2[27], which binds also two hydroxy ions, as postulated for the high pH complex of ED.
Some X-ray crystallographic data support our molecular model, where similar distances between the ligand atoms involved in the C8 and C9 complexes of AD and NA are expected. In fact, in these two CA the distances between the catecholic oxygens are close to those between the side chain hydroxy oxygen and amino nitrogen [28, 29].
Therefore, the geometry of copper coordination undergoes little change when passing from the C8 to the C9 complexes.
In previous studies on Cu(II)-CA complexes, copper coordination either with six [9-11] or with four ligands [6] has been proposed. Our model based on EPR data clearly favours the first viewpoint, i.e. hexacoordination of the copper ion with distorted octahedral symmetry, and excludes more complicated structures suggested before, as for example that of a complex involving 4 CA molecules and 4 Cu(II) ions [10]. In the last, spin exchange would be expected to take place between the neighbouring cupric ions, but our spectra did not show such a feature at all.
Considering the possible biological implications, we note first that the pTA which does not form complexes with the Cu(II) ion acts indirectly on the adrenergic receptors, inducing only the release of NA from the storage vesicles of the adrenergic nerve terminations [5]. By contrast, all the other studies SA which form complexes interact directly with the receptors. The predominantly ionic character of the bonds in the Cu(II)-SA complexes is consistent with the hypothesis of a divalent metal ion involved in the interaction of the SA and the receptor protein. Indeed, such bonds are easily dissociable and therefore allow a reversible binding of the amines to the receptor. We note that the binding of water molecules or OH2 ions might take place also in a divalent metal ion-mediated SA-receptor interaction. The high flexibility of the DA molecules suggested by our results might explain why the dopamine is highly active against the structurally different adrenergic and dopaminergic receptor proteins. At the same time, the hypothesis that the OH group on the ethylaminic chain of AD and NA plays the role of a conformational barrier, might relate to the fact that these two CA act preferentially against the adrenergic and less against the dopaminergic receptors.
4. – Concluding remarks
Our results show that EPR spectroscopy yields a relevant insight on the complex formation between the sympathomimetic amines and the cupric ion in aqueous solution. They suggest also that a more accurate analysis of the spectra could help searching further molecular details of these complexes.
* * *
The authors gratefully thank Profs. I. URSU and C. IONESCU-TIRGOVISTE (Bucharest) for encouragement and valuable discussions.
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