Template Synthesis of Co(II), Ni(II) and Cu(II) Complexes Derived From Oxamide Ligand and the Reactivity of Cu(II) Complex Towards Human Serum Albumin

A new oxamide ligand 2,2'-(oxalydimino)bis(diacetic acid)[C10H11O10N2],[L] has been synthesized by the condensation reaction of Iminodiacetic acid and Diethyloxalate. This ligand [L] was further allowed to interact with triethylene-tetraamine metal complexes [C16H26N6O8M]Cl2 (where M=Co11, Ni11 and Cu11) to yield the new N4 macrocyclic complexes 3, 3', 6, 6' tetraazadodeca 1-1' diimino N N tetraacetic acid M) chloride ([C16H26N6O8Co]Cl2, [C16H26N6O8Ni]Cl2 and [C16H26N6O8Cu]Cl2). These complexes were characterized by elemental analyses, i.r., n.m.r., e.p.r, and u.v.-vis spectroscopy. All the complexes show square planar geometry and are ionic in nature. The kinetic studies of the Cu(lI) complex were ascertained spectrophotometrically by observing the absorbance changes in presence of protein Human Serum Albumin (HSA) in phosphate buffer at different pH's at 300C. The absorbance changes were monitored at 278 nm (λmax of HSA) with respect to time and pseudo-first-order rate constants, kobs, were obtained from the slope of the straight line using the least squares regression method. The electrochemical behaviour of the Cu(ll) complex was monitored by cyclic voltammetry in a phosphate buffer. The Ep values-0.730 and-0.560 V respectively, were obtained at the scan rate of 0.1 Vs-1. The interaction of the Cu(II) complex with the HSA was studied at the same scan rate, which reveals weak binding as the E0 values do not shift considerably. The cyclic voltammogram of the Cu(II) complex bound to HSA was recorded at different pH's also (6.5 to 7.4). The pH-rate profile data reveals that the reactions are pH dependent.


INTRODUCTION
Transition metal ions Cu(ll), Ni(ll) and Zn(ll) bind to imidazole side chain of surface exposed histidines of proteins/1-3/. However, the binding to protein active site domain is stereospecific/4/. Much emphasis has been laid on the design of compounds with increased affinity for a molecular target due to wide applications Template Synthesis f Co(ll), Ni(ll) and Cu(ll) Complexes Derived From Oxamide Ligand and the Reactivity f Cu(ll) in pharmaceutical industries /5-8/. Protein-ligand complexes were designed to maximize the interactions between a protein and a small molecule or, alternatively, alter the chemical properties viz. solubility without disrupting binding. The same approach has been employed to design compounds with decreased affinity for HSA. Compounds having reduced affinity for serum albumin can prove as potent drugs with significantly lower dosage levels and improved in vivo tolerance/9/. The Human Serum Albumin (HSA) are the major soluble protein constituents of the circulatory system, hence involved in the transport, distribution and metabolism of many endogenous ligands such as fatty acids, bilirubin, amino acids, metals etc and numerous pharmaceuticals as well/10-13/. The main function of this protein is as carrier and recent report on HSA reveals the binding studies of Penicillins to HSA. [14,15]. HSA's have six different lysine residues and one of these groups, Lys-199 is the most probable binding site for compounds/16/ (Fig. 1).
To study the specific interactions of HSA-metal complex having reduced albumin binding, herein we report the design and synthesis of novel metal complexes derived from oxamidediiminodiacetic and triethylene tetramine. Cyclic voltammetry and kinetic experiments were performed in phosphate buffer at different pH's to ascertain the binding mode of the Cu(ll) complex of oxamidediiminodiacetic acid to HSA.
In our earlier work/17/, we have studied the effect of the Co(ll) complex of  Iminodiacetic acid (5 gm) in water (10 cm3) was added to diethyloxalate (1.35 gm) in a 2:1 molar ratio. The above solution was refluxed for ca. 6 h and then conc. HC1 (6cm3) was added dropwise with constant stirring. The resulting solution was refluxed for ca. 48 h and allowed to cool overnight in refrigerator. A white amorphous product was obtained, filtered, washed thoroughly with Et20 and dried in vacuo (3.4 g, Yield 68%), M.p. 80+ 3C. Anal. Calc. for CoH20oN2 Found%:C, 37.71; H, 3.  exchange interaction is negligible. In the present case, G appears to be greater than 4, which shows that exchange interactions are absent in the complex. The presence of gl > g_ in the spectrum of the Cu(ll) complex is an authentic evidence for square planar geometry around the copper(ll) atom/21/.

Electronic spectra
The

Redox behaviour
The redox behaviour of the Cu(ll) complex was studied by cyclic voltammetric measurement in phosphate buffer. The cyclic voltammogram of Cu(II) complex exhibits one quasi-reversible redox couple corresponding to the Cun/Cu with Ep values -0.730 and-0.560V respectively at a scan rate of 0.1 Vs [Fig.3a]. For this couple, the difference between cathodic and anodic potential AEp is of the order 128 mV and the lpa/Ipc value is less than one. At different scan rates [Fig. 3b], there is no major change in Ep and values, clearly indicating that Ep is independent of scan rate. For a reversible/quasi-reversible wave Ep is independent of scan rate and Ip is proportional to v /2/31, 32/. On interaction of the Cu(II) complex with HSA, there is a slight shift in Ep values of-0.799 and -0.541 V respectively, at the same scan rate [Fig. 4a], suggesting the binding of HSA with the Cu(II) complex but as the shifts in formal potential of the complex are not so significant (Fig. 4b), therefore it is inferred that the Cu(II) complex exhibits reduced binding for serum albumin.  x 10 tool dm-) at the different time intervals at 30C After the addition of HSA at the different time intervals (Fig. 6), there is a steep decrease in absorption intensity indicative of hypochromicity; however, no band shift has been observed. The spectral evidence support the binding of HSA to the Cu(II) complex but as the absorbance spectra does not record any shift in the wavelength, the reduced binding or low binding affinity for HSA is concluded. The rational design of compounds with reduced albumin binding has been limited due to lack of binding data to albumin active sites. These results are important in understanding the t,000 .. Template S)mthesis of Co(ll), Ni(ll) and Cu(ll) Complexes Derived From ()xamide Ligand and the Reactivity of Cu(ll) structure affinity relationship of metal complex-albumin interaction/9/.
The rate constants Rob were determined using the least square regression method (Fig. 7, 8). The   We have also studied the reaction kinetics of the Cu(II) complex in the presence of HSA at different pH values and consequent changes in absorption spectra were observed. There is a negligible change in the absorbance spectra in 6.0 7.4 pH range. At pH 7.4 the absorption spectra does not show any shift although a high maxima is observed indicating that the interaction between the complex and serum albumin is weak.
However as the pH values decreases, the intensity of the absorption peak at 278 nm (kmax of HSA) decreases, indicating that the coordination of the Cu(II) complex to serum protein may take place through imidazole side chains of surface exposed histidine of protein/33/.
The plots of kobs versus HSA at different pH's give a straight line suggesting that all the reactions are of pseudo-first order type (Fig. 9). l.." 583 7,0 7, Fig. 9: pH-rate profiles of Cu(ll) complex bound with HSA.