Synthesis, Spectral, Electrochemical Analysis and Screening for α -Glucosidase Inhibition of Some Complexes of Copper (II) with Amino acids

Complexes of some metal ions with amino acids can be used as models to study the pharmaco-dynamic effects of drugs or for increasing the biocompatibility and minimize toxic effects of some metal ions. Interac-tions between transitional metal ions and amino acids are very interesting in the biological applications. A series of com- plexes of Cu(II) and amino acid (L), ie. glycine, valine, asparginine and arginine with formula [Cu(L)2]+2 have been synthesized and characterized as mononuclear species on the basis of elemental chemical analysis, infrared spectra, UV-Visisble and cyclicvoltametery measurements. The IR spectra indicated the presence of amino acid coordinated through nitrogen atom and the oxygen from the carboxylic group. The experimental data suggest that the ligands act as bidentate and adopt an octahedral stereochemistry. In this study, we have also focused on the inhibitory activity of these metal complexes on α -glucosidase.


Introduction
Cu(II) is a bio-essential element occurring in multitude of metalloproteins (Bhattacharjee et al 2010). Cu(II) complexes are interesting due to their biological applications and considerable amount of interest in the studies is due to their coordination modes when bound to metals, high pharmacological potentiality and good chelating property. Many metal ions are known to play very important roles in biological processes in the human body (Kaim et al 1996 andXiao-Ming et al 1996). Cu(II) ions are the second and third most abundant transition metals in humans. They are found either at the active sites or as structural components of a good number of enzymes (Cotton et al 1988 andGreenwood et al 1984). Amino acid coordination to metals confirms structural lability (Rombach et al 2002) and amino acid complexes are also of relevance in enzyme inhibition (Kahn et al 1999 andFarkas et al 2002). The interesting property of these compound ions may coordinate to the metal ions in the usual bidentate way through the amine and carboxylic group of the amino acids side chain. Pharmacological and toxicological properties of amino acid complexes are another area that has drawn lot of current attention (Chang et al 2005, Tanase et al 1985, Roberts et al 1983, Dondoni et al 2004and Janiaj et al 2003. Complexes of Cu(II) with amino acids can be used as models to study the pharmaco-dynamic effects of drugs or for increasing the biocompatibility and minimize toxic effects of some metal ions (Grecu et al 1986 andAsmaI et al 2001). Metallotherapy is a very unique therapeutic method to treat many diseases like as diabetes. The goal of diabetes treatment is to control the blood glucose levels, body weight, blood pressure, and cholesterol and triglyceride levels, and prevent the development of complications (Pinhas et al 2007). Several metal ions and their complexes exhibit anti-diabetic effects (Schwarz et al 1959, Rubenstein et al 1962, Coulston et al 1980, Heyliger et al 1985, Sakurai et al 1990and Yoshikawa et al 2000. In addition, some metal ions, such as tungsten (Dominguez et al 2003), vanadium (Heyliger et al 1985 and selenium (Esaki et al 1990) lower high blood glucose levels in the diabetic state. It appears attractive to many researchers to study the relationship between diabetes mellitus and metal ions. Therefore, this attraction we were synthesized the new Cu(II) complexes containing glycine, valine, asparginine and arginine as ligands. The synthesized complexes were characterized by elemental analysis, CV, IR, UV-visible and evaluated their enzymatic inhibition activity.

Synthesis of complexes:
The [Cu(L) 2 ] +2 complexes were prepared from three different salts of copper and amino acids (L-glycine, valine, asparginine and arginine) as ligand. 2 mM of amino acid was added in 20 ml of aqueous solution which containing 2 mM of sodium acetate and allow it to a clear solution with continuous string. Then 1 mM of metal salt in 2 ml of triple distilled water was added drop by drop into this aqueous solution with continuous string for 3 hours. A deep blue colored solution obtained which were transferred into petri dish for crystalization. After few days deep blue colored crystals obtained.
Infrared Spectroscopy: Infrared (IR) spectra were obtained by the KBr method using a Bruker Alfa-T model Fourier transform (FTIR) spectrometer (Bruker Instrument Germany). The spectrometer was equipped with a Glober IR source, KBr beam spillter and detector. For each spectrum, 16 scans were obtained with the resolution of 4 cm -1 . The obtained IR spectra were proceed by mean of the program OPUS 7.0.

UV-VIS spectroscopy:
The UV-visible transmittance spectra of the complexes were recorded at 25°C on a Shimadzu UV-Vis 160 spectrophotometer, in quartz cells at the desired wave length region. 3 mM solution of complexes in DMSO was used in all UV -visible measurements.

Cyclic voltametry:
The cyclicvoltametric measurements were carried out with a Metrohm Instrument (Germany) having an electrochemical cell with a three-electrode system. The reference electrode was an Ag/AgCl 2 . Platinum wire used an as a working electrode, Platinum wire electrode used as an auxiliary electrode. The 3 mg of complex were dissolved in supporting electrolyte 25 ml of 0.01 M solution of KCL solution. The voltamograme, peak position and area were calculated using NOVA 1.9 software.

α-Glucosidase Inhibition :
The determination of α-Glucosidase was adopted from the method (I.P. Tripathi et al 2014). Rat intestinal acetone powder (Sigma chemicals,USA) was sonicated properly in normal saline (100:1 w/v) and after centrifugation at 3000 rpm × 30 mins the supernatant was treated as crude intestinal α-Glucosidase. 50 μl various dilutions in DMSO (0.1mg /ml solution) were mixed and incubated with 50μl of enzyme in a 96-well microplate for 5mins. Reaction mixture was further incubated for another 10 mins with 50 μl substrate (5 mM, pnitrophenyl-α-D-glucopyranoside) prepared in 100 mM phosphate buffer (pH~ 6.8) and release of nitrophenol was read at, 405 nm spectrophotometerically (Multimode SynergyH4 micro plate reader, BioTek instrument, inc. Winoosci, VT, USA). All the samples were run in triplicate and acarbose was taken as standard reference compound. Several dilutions of primary solution (5mg/ml DMSO) were made and assayed accordingly to obtain concentration of the test sample required to inhibit 50% activity (IC50) of the enzyme. Quantification was performed with respect to the standard curve of acarabose (Y = 26.63X + 46.26, R² = 0.958) results were expressed as milligram of acarbose equivalent per ml of extract.
Results and Discussion: Characterization of metal complexes All the complexes are colored, non-hygroscopic and thermally stable solids.

UV-VIS spectroscopy:
The electronic spectra data of the complexes were recorded in 100% DMSO and their assignments were given in Table-3 and one representative ligand field spectra of complex (2) [Cu(val) 2 ] is shown in Fig.-2 and band position are presented in Table-3. The UV-Vis spectra of Cu(II) complexes with the four ligands show absorption bands assigned to a large band around 620 nm (16000 cm-1 ). The presence of the later band supports an octahedral stereochemistry for these complexes (Miessler et al 1999). Two bands were observed in the electronic spectrum of the complex(2), at 631 nm and 819 nm which can be assigned to 2 B 1 g → 2 B 2 g and 2 B 1 g → 2 E 1 g transitions (Miessler et al 1999). The absorption bands of the complexes corresponded to the n→σ*, n→π* and π*→π* transitions of -NH 2 and -COO -, Shifts in these bands and the observed d-d transitions of the compounds, as presented in Table-3, indicated coordination. Characteristic π-π * transitions are observed in the spectrum at 236, 234, 249, 257 nm respectively (Eskander et al 2000 andReddy et al 2000).

Infra Red Spectroscopy:
Infrared studies on coordination compounds of amino acids have shown that the coordination of metal with ligand, making it a useful tool in structural studies (Nakamoto et al 2009).
In the IR spectrum of complex(1), [Cu(gly) 2 ] the spectra exhibited a marked difference between bands belonging to the stretching vibration of υ(N-H) of the amine group in the range between 3448-3383 cm -1 , shifted to higher frequencies by 92-27 cm -1 , suggesting the possibility of the coordination of ligand through the nitrogen atom at the amine group (Nakamato et al 1967, Maracotrigiano et al 1975, Kothar et al 1996. In order to get further information about the coordination behavior of the ligand with metal ion, the N-H stretching vibration at 3119 cm -1 , in the complex was shifted to higher frequencies with the complexes, suggesting that the coordination of the metal ions with the ligand was via the nitrogen atom (Fessenden 1990, Nakamoto et al 2009and Elzahany et al 2008. The infrared spectra of the complex(1) is given in Fig.-1. The absorption band at 1624 cm -1 was attributed to the ν(C=O) stretching vibration in the spectrum. The consecutive bands at 1600 and 1572 cm -1 , in the spectrum of the ligand were assigned to the symmetric and asymmetric bending vibrations of N-H bond. The complexes 1, 2, 3 and 4 spectra, which involves the carboxylic group in the covalent bonding to the metal ion (David et al 2003). In the spectrum of the complexes are shifted to 1578 cm -1 and 1584 cm -1 , which also indicates the involvement of this group in the metal-ligand bond formation. The important absorption at assignment of the complex 1, 2, 3 and 4 are listed in Table-2.
Electrochemical studies of Complexes: Fig-4 shows cyclic voltammogram (CV) scanned cathodically in the potential region between +0.00 and -0.750 V vs Ag/ AgCl in 0.1M sodium perchlorate solution [Cu(II)L 2 ] 2+ system at different pH (isoelectric point of amino acids). In this scan range, the CVs show a single reduction peak at -498.05 mV (B1) in the forward sweep only one oxidation waves A1 at 124.51 mV/s at the scan rate of 0.01 V/s. Voltamogram clearly represents that reduced moiety of Cu(II) doesn't fully oxidized in further sweep.
α-Glucosidase Inhibition: The chemically inert and configurationally stable complexes revealed an astonishing range of interesting biological activi-

ReseaRch PaPeR
ties, such as the inhibition of the enzyme. In this study, we have also focused on the inhibitory activity of these complexes on α-glucosidase to analyze alternative action mechanisms of these metal ions. We have evaluated the α-glucosidase inhibitory activity of these copper complexes. The previous researches have showed the potent alpha-glucosidase inhibitory effects (Bulut et al 2007, Yoshikawa et al 2009, Warra et al 2011, Hiromu et al 2010, Yutaka et al 2010and Tripathi et al 2013. But in our case the synthesized copper complexes with amino acid doesn't show any activity.

Conclusion:
The amino acids complexes of Cu(II) have global interest in scientific community due to their potential pharmacological activities. In this work, we have successfully synthesized the complex of copper (II) using amino acids as ligand. The coordination of the copper (II) with amino acids arises from the shift of the ν s (C=O), ν s (C-N) 1623 and 1584 cm -1 respectively. The assignment of copper (II) complexes with amino acids have corroborated by infrared, electro chemical and electronic spectral measurements. The cylic voltamogram represents only reduction peak at -498.05mV. The broad band is observed at 16,638 cm -1 in the electronic spectrum of the Cu(II) complex assigned to 2 Eg -2 T 2 g transition which is conform the octahedral geometry of all the complexes (Dunn et al 1960)