High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.     

A new method for forensic DNA analysis of the blood meal in chagas disease vectors demonstrated using Triatoma infestans from Chuquisaca, Bolivia

Background

Feeding patterns of the vector are important in the epidemiology of Chagas disease, the leading cause of heart disease in Latin America. Chagas disease is caused by the parasite, Trypanasoma cruzi, which is transmitted by blood feeding insects. Historically, feeding behaviours of haematophagous insects have been investigated using serological reactions, which have detection limits in terms of both taxonomic resolution, and quantity and quality of the blood meal. They are labor intensive, require technical expertise, need fresh or frozen samples and antibodies often are either not available commercially or the resources for synthesis and purification are not available. We describe an assay to identify vertebrate blood meal sources, and the parasite T. cruzi using species-specific PCR assays from insect vectors and use the method to provide information regarding three questions: (1) Do domestic and peri-domestic (chicken coop and animal corral) habitats vary in the blood meals detected in the vectors? (2) What is the pattern of multiple blood meals? (3) Does the rate of T. cruzi infection vary among habitats and is it associated with specific blood meal types?

Methodology/Principal Findings

Assays based on the polymerase chain reaction were evaluated for identification of the blood meal source in the heamatophagous Chagas disease vector Triatoma infestans. We evaluate a technique to identify 11 potential vertebrate food sources from the complex mixture extracted from the vector''s abdomen. We tested the assay on 81 T. infestans specimens collected from the Andean highlands in the department of Chuquisaca, located in central Bolivia, one of the regions in South America where sylvatic T. infestans have been reported. This area is suggested to be the geographic origin of T. infestans and has very high human infection rates that may be related to sylvatic vector populations.

Conclusion/Significance

The results of the assays revealed that a high percentage of insects collected in human dwellings had fed on peri-domestic animals. In contrast, one insect from a chicken coop but no bugs from corrals tested positive for human blood. Forty-eight percent of insects tested positive for more than one vertebrate species. T. cruzi infection was detected in 42% of the specimens. From the epidemiological point of view, the results reveal an overall pattern of movement from peri-domestic structures to human habitations for T. infestans in this region of Bolivia as well as the important role of pigs, dogs, chickens and guinea pigs in the dynamics of T. cruzi infection.     

Trypanosoma cruzi: Synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts

The parasite Trypanasoma cruzi is responsible for Chagas disease and its triatomine vector, Rhodnius prolixus, has a symbiotic relationship with the soil bacterium, Rhodococcus rhodnii.R. rhodnii that was previously genetically engineered to produce the anti-microbial peptide, cecropin A was co-infected with T. cruzi into R. prolixus resulting in clearance of the infectious T. cruzi in 65% of the vectors. Similar anti-microbial peptides have been isolated elsewhere and were studied for differential toxicity against T. cruzi and R. rhodnii. Of the six anti-microbial peptides tested, apidaecin, magainin II, melittin, and cecropin A were deemed potential candidates for the Chagas paratransgenic system as they were capable of killing T.cruzi at concentrations that exhibit little or no toxic effects on R. rhodnii. Subsequent treatments of T. cruzi with these peptides in pair-wise combinations resulted in synergistic killing, indicating that improvement of the 65% parasite clearance seen in previous experiments may be possible utilizing combinations of different anti-microbial peptides.     

Metal-based new sulfonamides: Design, synthesis, antibacterial, antifungal, and cytotoxic properties

Cobalt(II), copper(II), nickel(II) and zinc(II) metal complexes with 5-chlorosalicyladehyde derived Schiff base sulfonamides have been synthesized and characterized. Structure and bonding nature of all the synthesized compounds have been deduced from physical, analytical, and spectral (IR, (1)H NMR, (13)C NMR, Mass, electronic) data. An octahedral geometry has been proposed for all the metal complexes. The ligands and their metal complexes have been screened for their in vitro antibacterial, antifungal, and cytotoxic properties and results are reported.     

Differences in structure, physiological stability, electrochemistry, cytotoxicity, DNA and protein binding properties between two Ru(III) complexes

A novel Ru(III) complex, mer-[RuCl(3)(CH(3)CN)(dpq)] (1), has been synthesized and characterized by X-ray diffraction, where dpq=dipyrido[3,2-d:2',3'-f]quinoxaline. Its chemical and biological properties have been intensively compared with those of mer-[RuCl(3)(DMSO)(dpq)] (DMSO=dimethyl sulfoxide) (2). It has been found that the stability in buffered solutions and the reduction potential for the Ru(III)/Ru(II) couple can be modulated by changing the small molecule bonded to the Ru(III) center. Interactions of 1 with DNA have been investigated by DNA melting experiments, DNA competitive binding with EB (ethidium bromide), plasmid DNA cleavage experiments and viscosity measurements. The interaction of 1 and 2 with BSA (bovine serum albumin) has also been studied using fluorescent quenching method. The experimental results show that 1 exerts higher affinity towards DNA and BSA than 2 does. The cytotoxicity of 1 has been evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, and 2 shows slightly higher anticancer potency than 1 does against all the cell lines screened. Attempts are made to clarify the possible antitumor mechanisms of these two complexes by analyzing the experimental results presented.     

Thermal behaviour,phase diagram,and spectroscopic properties of rac-indobufen and its enantiomers

The binary phase diagram of the enantiomers of indobufen, 1 (Ibustrin), an antithrombotic drug, has been investigated by differential scanning calorimetry (DSC); 1 is a racemic compound (racemate) with melting point lower than that of the enantiomers. Its thermal behaviour (DSC) has been examined and is discussed in comparison with other physical methods (IR spectroscopy and X-ray powder diffraction). Absolute configuration has been assigned to the enantiomers by ¹H-NMR correlations. © 1995 Wiley-Liss, Inc.     

Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.     

Synthesis, spectroscopic, cytotoxic, and DNA binding studies of binuclear 2,2'-bipyridine-platinum(II) and -palladium(II) complexes of meso-alpha,alpha'-diaminoadipic and meso-alpha,alpha'-diaminosuberic acids.

Four new binuclear complexes of formula [M2(bipy)2(BAA)]Cl2 (where M is Pt(II) or Pd(II), bipy is 2,2'-bipyridine, and BAA is a dianion of meso-alpha-alpha'-diaminoadipic acid (DAA) or meso-alpha,alpha'-diaminosuberic acid (DSA) have been synthesized. These complexes have been characterized by chemical analysis and ultraviolet-visible, infrared, and 1H NMR spectroscopy. The mode of binding of ligands in these complexes has been ascertained by infrared and detailed 1H NMR spectroscopy. These complexes are 1:2 electrolyte in conductivity water. They have also been tested against P388 lymphocytic leukemia cells and their target is DNA molecules. [Pt2(bipy)2(DSA)]Cl2, [Pd2(bipy)2(DSA)Cl2, and [Pd2(bipy)2(DAA)]Cl2 show I.D.50 values comparable or lower than cis-diamminedichloroplatinum(II) and [Pt(bipy)(Ala)]Cl. In addition, binding studies of [Pt2(bipy)2(DSA)]Cl2 and [Pd2(bipy)2(DAA)]Cl2 to calf thymus DNA have been carried out and the mode of binding seems to be hydrogen bonding, as suggested earlier for analogous mononuclear amino acid-DNA complexes.     

Sphingosine kinase,sphingosine-1-phosphate,and apoptosis

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.     

Synthesis,molecular structure,solution equilibrium,and antiproliferative activity of thioxotriazoline and thioxotriazole complexes of copper II and palladium II

Preparations of copper(II) and palladium(II) complexes of 4-amino-5-methylthio-3-(2-pyridyl)-1,2,4-triazole (L(1)) and the copper(II) complex of 1,4-dihydro-4-amino-3-(2-pyridyl)-5-thioxo-1,2,4-triazole (HL) are described. These complexes have been characterized by means of spectroscopy and microanalysis. Molecular structures of HL (1), [CuCl(2)(H(2)L)]Cl.2H(2)O (2a), cis-[CuCl(2)(L(1))] (3), and cis-[PdCl(2)(L(1))] (4) have been determined by single-crystal X-ray diffraction. The HL ligand acts as a N,S bidentate through the thioxo moiety and the exo-amino group whilst the ligand L(1) forms N,N coordination complexes through the pyridine and triazole nitrogen atoms. Speciation in solution of the systems Cu/HL and Cu/L(1) have been determined by means of potentiometry and spectrophotometry as well as for the Cu/L(1)/A (HA=glycine) system in order to determine species present at physiological pH. Antiproliferative activity of these complexes and their ligands was evaluated, using the AlamarBlue Assay, on normal human fibroblasts (HF) and human fibrosarcoma tumor (HT1080) cells. The copper compounds cis-[CuCl(2)(H(2)L)]Cl and cis-[CuCl(2)(L(1))] exerted significant antiproliferative activity of both normal and neoplastic cells; although dose-response experiments revealed that the HT1080 cell line was more sensitive to the tested drugs than normal fibroblasts.     

Synthesis, characterization, antibacterial and cytotoxic study of platinum (IV) complexes

Platinum (IV) complexes [Pt (L)2Cl2] [where, L= benzyl-N-thiohydrazide (L1), (benzyl-N-thio)-1,3-propanediamine (L2), benzaldehyde-benzyl-N-thiohydrazone (L3) and salicylaldehyde-benzyl-N-thiohydrazone (L4)] have been synthesized. The thiohydrazide, thiodiamine and thiohydrazones can exist as thione-thiol tautomer and coordinate as a bidentate N-S ligand. The ligands were found to act in monobasic bidentate fashion. Analytical data reveal that metal to ligand stoichiometry is 1:2. The complexes have been characterized by elemental analysis, IR, mass, electronic and 1H NMR spectroscopic studies. In vitro antibacterial and cytotoxic studies have been carried out for some complexes. Various kinetic and thermodynamic parameters like order of reaction (n), activation energy (Ea), apparent activation entropy (S#) and heat of reaction (DeltaH) have also been carried out for some complexes.     

Expression,purification, and characterization of subunit E,an essential subunit of the vacuolar ATPase

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).     

Fluorine-, pyrene-, and nitroxide-labeled sphingomyelin: semi-synthesis and thermotropic properties

A rapid, high-yield method has been developed for the N-acylation of sphingosine-1-phosphocholine (SPC) to obtain a series of sphingomyelin (SM) derivatives bearing different reporter groups in the N-acyl chain. The procedure utilizes a fatty acid activated as the N-hydroxysuccinimide ester. A 1:1 molar mixture of the activated fatty acid and SPC is refluxed in 5% aqueous NaHCO3-ethanol 9:1 (v/v) for 2-3 hr. After acidification, the precipitated SM is purified by column chromatography over silica gel. This procedure offers significant advantages over those reported for the synthesis of well-defined SM: i) only the amino (not the hydroxyl) group is acylated; ii) only one equivalent of fatty acid is required; and iii) the time necessary for the reaction to go to completion is short. The transition temperature and enthalpy of each SM derivative has been measured by differential scanning calorimetry and compared to its unlabeled analog.     

Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding

Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.     

Metal-based new sulfonamides: Design,synthesis, antibacterial,antifungal, and cytotoxic properties

Cobalt(II), copper(II), nickel(II) and zinc(II) metal complexes with 5-chlorosalicyladehyde derived Schiff base sulfonamides have been synthesized and characterized. Structure and bonding nature of all the synthesized compounds have been deduced from physical, analytical, and spectral (IR, ¹H NMR, ¹³C NMR, Mass, electronic) data. An octahedral geometry has been proposed for all the metal complexes. The ligands and their metal complexes have been screened for their in vitro antibacterial, antifungal, and cytotoxic properties and results are reported.     

Genetic susceptibility, biomarker respones, and cancer

A large number of studies have reported associations between polymorphisms of xenobiotic-metabolizing enzymes (XMEs) and various cancers. However, the carcinogenic exposures behind such findings have usually been unclear. Information on susceptibility to specific carcinogens could better be obtained by examining situations where the exposure and the endpoint studied are nearer in time, i.e., by studying biomarkers of carcinogen exposure and early (genotoxic) effect in exposed humans. For example, analyses of DNA adducts and cytogenetic endpoints have indicated an increased susceptibility of glutathione S-transferase M1 (GSTM1) null genotype to genotoxicity of tobacco smoking, supporting the view that the associations of the GSTM1 null genotype with bladder and lung cancer are partly related to smoking. In vitro genotoxicity studies with human cells offer an experimental tool that can be used, within the limits of the cell systems, to predict individual sensitivity and genotype-carcinogen interactions. In vitro sensitivity to the genotoxicity of 1,2:3,4-diepoxybutane, an epoxide metabolite of 1,3-butadiene has clearly been shown to depend on GSTT1 genotype, which has also been implicated to modify, along with GSTM1 genotype, the in vitro genotoxicity of 1,2-epoxy-3-butene, another epoxide metabolite of 1,3-butadiene. These genotypes appear to modulate the excretion of 1,3-butadiene-specific mercapturic acids, and influence genotoxicity biomarker levels in 1,3-butadiene-exposed workers. The excretion of specific mercapturic acids (PHEMA) in workers exposed to styrene has clearly been shown to depend on GSTM1 genotype, and GSTT1 genotype seems to modulate the excretion of one PHEMA diastereoisomer. These genotypes have also been implicated to modulate the in vitro genotoxicity of styrene. In general, the genetic polymorphisms potentially important for biomarker response largely depend on the exposing agent, biological material examined, and ethnicity of the population under study. Individual exposure level may vary a lot, and a reliable estimate of the exposure is essential for correct interpretation of genotype-exposure interaction. Besides XME polymorphisms, any polymorphisms that affect cellular response to DNA damage could, in principle, modify individual sensitivity to genotoxins. For instance, those concerning DNA repair proteins are presently being studied by many laboratories.     

Inhibition of human dimethylarginine dimethylaminohydrolase-1 by S-nitroso-L-homocysteine and hydrogen peroxide. Analysis, quantification, and implications for hyperhomocysteinemia

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys(274) and His(173) are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (0.088 M(-1) s(-1)), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M(-1) s(-1)). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.     

Purification, crystallization, and X-ray diffraction studies of lactotransferrin from buffalo colostrum.

Lactotransferrin is an iron-binding protein. It has been purified from buffalo colostrum. The purified lactotransferrin has been crystallized in 10% ethanol solution. The crystals are orthorhombic and the space group is P2(1)2(1)2(1) with unit cell dimensions a = 161.70 A, b = 155.75 A, c = 113.48 A. The asymmetric unit contains three molecules of the protein with a solvent content of about 59%. The crystals were stable in the X-ray beam and diffract beyond 3.5 A resolution. The native data have been collected and the structure determination is in progress.     

Abruquinone A,B,D,E,F and G from the Root of Abrus precatorius

Eight flavonoids have been isolated from the root of Abrus precatorius L. Among them, six isoflavanquinones, designated (3R)-abruquinone A, B, D and E, (3S)-abruquinone F and G, are characterized by chemical and spectral means including 1H-1H COSY, 1H-13C COSY and CD methods.     

Synthesis, electrochemistry, geometric and electronic structure of oxo-molybdenum compounds involved in an oxygen atom transferring system

The oxygen atom transfer reactivity of Tp( *)MoO(2)(SPh) (1) (where Tp( *)=hydrotris-(3,5-dimethylpyrazol-1-yl)borate) with trimethyl phosphine (PMe(3)) has been investigated. The reaction proceed through a diamagnetic phosphoryl intermediate complex, Tp( *)MoO(SPh)(OPMe(3)) (2), which has been isolated and characterized by IR, NMR, UV-visible spectroscopy, and mass spectrometry. The molecular structure of 2 has been determined by X-ray crystallography. The complex crystallizes in monoclinic (P2(1)/n) space group, a=19.81 (1)A, b=11.1 (4)A, c=18.416 (5)A, beta=121.2 (3) degrees , V=3463.8 (25)A(3) with Z=4. In acetonitrile, complex 2 exchanges its phosphoryl ligand with a solvent molecule resulting in Tp( *)MoO(SPh)(MeCN) (3), which has been isolated and also characterized spectroscopically and by X-ray crystallography. Compound 3 crystallizes in triclinic (P1 ) space group, a=10.159 (6)A, b=18.563 (5)A, c=7.986 (3)A, alpha=96.22 (3) degrees , beta=121.2 (3) degrees , gamma=84.64 (3) degrees , V=1452.4 (11)A(3) with Z=2. The electronic structures of the complexes have been investigated by density functional theory and the redox chemistry has been investigated by cyclic and differential pulse voltammetry. In acetonitrile, complex 2 spontaneously transforms to complex, 3 at a rate of 5.6x10(-4)s(-1).