ESI Mass Spectral Data Research Articles

Metallothionein-3 and carbonic anhydrase metalation properties with Zn(II) and Cd(II) change as a result of protein-protein interactions.

Metallothioneins (MT) are regulators of the metals Zn(II) and Cu(I) and act as antioxidants in many organisms, including in humans. Isoform 3 (MT3) is expressed constitutively in central nervous tissue and has been shown to have additional biological functions, including the inhibition of neuronal growth, the regulation of apoptosis, and cytoskeleton modulation. To facilitate these functions, protein-protein interactions likely occur. These interactions may then impact the metalation status of the MT and the recipient metalloprotein. Using electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report that the interaction between the zinc metalloenzyme, carbonic anhydrase (CA), and MT3, impacts the metalation profiles of both apo-MT3 and apo-CA with Cd(II) and Zn(II). We observe two phases in the metalation of the apo-CA, the first of which is associated with an increased binding affinity of apo-CA for Cd/Zn(II) and the second pathway is associated with apo-CA metalated without a change in binding affinity. The weak interactions that result in this change of binding affinity are not detectable as a protein complex in the ESI-mass spectral data or in the circular dichroism spectra. These unusual metalation properties of apo-CA in the presence of apo-MT3 are evidence of the effects of protein-protein interactions. With adjustment to take into account the interaction of both proteins, we report the complete Cd(II) and Zn(II) binding constants of MT3 under physiological conditions, as well as the pH dependence of these binding pathways.

Cu(I) binds to Zn7-MT2 via two parallel pathways.

Metallothionein proteins are essential for Cu(I) and Zn(II) homeostasis as well as heavy metal detoxification. The metallation properties of MT2 are of great interest due to their wide patterns of expression and correlation with multiple diseases including cancers, neurological disorders, and respiratory diseases. Use of isotopically pure 63Cu(I) and 68Zn(II) eliminates the complexity of the Cu, Zn-MT2 mass spectral peaks due to significant overlap of naturally abundant isotopes. This allows for the resolution of the precise Cu(I) and Zn(II) stoichiometries when both Cu(I) and Zn(II) are bound to MT2 at physiological pH as expected in vivo. Exact Cu: Zn ratios were determined from mass spectral simulations carried out for every point in the titration. We report that Cu(I) metallation of Zn7-MT2 can only be understood in terms of two pathways occurring in parallel with pathway ① resulting in Cu5Zn5-MT2 and Cu9Zn3-MT2. Pathway ② results in Cu6Zn4-MT2 and Cu10Zn2-MT2, which are the major products of the reaction. From the electrospray ionization (ESI)-mass spectral data we report a series of formation constants (KF) for species starting from Zn7-MT2 up to Cu11Zn2-MT2. Room temperature phosphorescence and circular dichroism (CD) spectra were measured in parallel with the ESI-mass spectrometry data allowing for the assignment of specific species to specific spectral bands. Through analysis of the CD spectral bands, we propose that Cu(I) binds to the β domain first to form a Cu5Zn1 cluster or Cu6 cluster with emission at 670 and 750nm, respectively, leaving the Zn4 cluster in the α domain.

Database Creator for Mass Analysis of Peptides and Proteins, DC-MAPP: A Standalone Tool for Simplifying Manual Analysis of Mass Spectral Data to Identify Peptide/Protein Sequences.

Proteomic studies typically involve the use of different types of software for annotating experimental tandem mass spectrometric data (MS/MS) and thereby simplifying the process of peptide and protein identification. For such annotations, these softwares calculate the m/z values of the peptide/protein precursor and fragment ions, for which a database of protein sequences must be provided as an input file. The calculated m/z values are stored as another database, which the user usually cannot view. Database Creator for Mass Analysis of Peptides and Proteins (DC-MAPP) is a novel standalone software that can create custom databases for "viewing" the calculated m/z values of precursor and fragment ions, prior to the database search. It contains three modules. Peptide/Protein sequences as per user's choice can be entered as input to the first module for creating a custom database. In the second module, m/z values must be queried-in, which are searched within the custom database to identify protein/peptide sequences. The third module is suited for peptide mass fingerprinting, which can be used to analyze both ESI and MALDI mass spectral data. The feature of "viewing" the custom database can be helpful not only for better understanding the search engine processes, but also for designing multiple reaction monitoring (MRM) methods. Post-translational modifications and protein isoforms can also be analyzed. Since, DC-MAPP relies on the protein/peptide "sequences" for creating custom databases, it may not be applicable for the searches involving spectral libraries. Python language was used for implementation, and the graphical user interface was built with Page/Tcl, making this tool more user-friendly. It is freely available at https://vit.ac.in/DC-MAPP/.

Metallothionein-3: 63 Cu(I) binds to human 68 Zn7 -βα MT3 with no preference for Cu4 -β cluster formation.

Human metallothioneins (MTs) are involved in binding the essential elements, Cu(I) and Zn(II), and the toxic element, Cd(II), in metal-thiolate clusters using 20 reduced cysteines. The brain-specific MT3 binds a mixture of Cu(I) and Zn(II) in vivo. Its metallation properties are critically important because of potential connections between Cu, Zn and neurodegenerative diseases. We report that the use of isotopically pure 63 Cu(I) and 68 Zn(II) greatly enhances the element resolution in the ESI-mass spectral data revealing species with differing Cu:Zn ratios but the same total number of metals. Room temperature phosphorescence and circular dichroism spectral data measured in parallel with ESI-mass spectral data identified the presence of specific Cu(I)-thiolate clusters in the presence of Zn(II). A series of Cu(I)-thiolate clusters form following Cu(I) addition to apo MT3: the two main clusters that form are a Cu6 cluster in the β domain followed by a Cu4 cluster in the α domain. 63 Cu(I) addition to 68 Zn7 -MT3 results in multiple species, including clustered Cu5 Zn5 -MT3 and Cu9 Zn3 -MT3. We assign the domain location of the metals for Cu5 Zn5 -MT3 as a Cu5 Zn1 -β cluster and a Zn4 -α cluster and for Cu9 Zn3 -MT3 as a Cu6 -β cluster and a Cu3 Zn3 -α cluster. While many reports of the average MT3 metal content exist, determining the exact Cu,Zn stoichiometry has proven very difficult even with native ESI-MS. The work in this paper solves the ambiguity introduced by the overlap of the naturally abundant Cu(I) and Zn(II) isotopes. Contrary to other reports, there is no indication of a major fraction of Cu4 -β-Znn -α-MT3 forming.

Enhancing the student experience: the role of the Midwifery Practice Coordinator (MPC)

In this paper, a new dual optical behavior Cu2+ selective Schiff base (L) was developed by reacting thiophene carbaldehyde with diamino malononitrile. The sensing ability of L was studied with different metal ions by visual test, UV–Vis and spectrofluorometric methods in MeOH:H2O (8:2, v/v) media. Accordingly, the visual test was confirmed by changing initial yellow to colorless upon interaction with Cu2+ while for other it did not respond in natural light while the same solution when it observed under UV light, it exhibits emission color (cyan) only with Cu2+. Spectrophotometrically, L alone exhibits a strong absorption band at 420 nm, upon interaction with Cu2+, it exhibits an additional new peak at 290 nm while spectrofluorimetrically, a new emission peak was observed at 360 nm while other metal ions it does not exhibits this optical behavior. The spectral titration data reveals that the LOD limit down to of 6.21 × 10−6 and 1.45 × 10−8 M by UV–vis and fluorescence respectively which is lower than the WHO guidelines (0.02 μM) in drinking water. The formation 1:1 binding stoichiometry of host-guest complexation between L and Cu2+ is studied by using Jobs plot and the binding capacity is estimated by Benesi-Hilderbrand method (Ka = 7.31 × 104 M-1). The coordination behavior of L with Cu2+ is well supported by FTIR, 1H NMR, 13C NMR, ESI mass spectral data and theoretical calculations using Gaussian 09 W program.

Dual optical properties of new schiff base based on bisthiophene for sensing of Cu2+ in protic media

In this paper, a new dual optical behavior Cu2+ selective Schiff base (L) was developed by reacting thiophene carbaldehyde with diamino malononitrile. The sensing ability of L was studied with different metal ions by visual test, UV–Vis and spectrofluorometric methods in MeOH:H2O (8:2, v/v) media. Accordingly, the visual test was confirmed by changing initial yellow to colorless upon interaction with Cu2+ while for other it did not respond in natural light while the same solution when it observed under UV light, it exhibits emission color (cyan) only with Cu2+. Spectrophotometrically, L alone exhibits a strong absorption band at 420 nm, upon interaction with Cu2+, it exhibits an additional new peak at 290 nm while spectrofluorimetrically, a new emission peak was observed at 360 nm while other metal ions it does not exhibits this optical behavior. The spectral titration data reveals that the LOD limit down to of 6.21 × 10−6 and 1.45 × 10−8 M by UV–vis and fluorescence respectively which is lower than the WHO guidelines (0.02 μM) in drinking water. The formation 1:1 binding stoichiometry of host-guest complexation between L and Cu2+ is studied by using Jobs plot and the binding capacity is estimated by Benesi-Hilderbrand method (Ka = 7.31 × 104 M-1). The coordination behavior of L with Cu2+ is well supported by FTIR, 1H NMR, 13C NMR, ESI mass spectral data and theoretical calculations using Gaussian 09 W program.

Synthesis, Characterization and Antimicrobial Evaluation of Cobalt(III) Complexes of 4-(2-Substituted Phenylimino)-2-(4-Substituted Phenyl)-4H-chromen-3-ol

A series of eight Co(III) complexes [CoL1-8(H2O)2Cl] (I-1 to I-8) incorporating 4-(2-substituted phenylimino)-2-(4-substituted phenyl)-4H-chromen-3-ol, as a tridentate imino flavone ligands (L1 to L8, 2-sub. = NH2, SH, 4-sub. = OMe, OH, Cl, NMe2) have been synthesized, characterized and the geometry of the complexes were optimized by DFT. The chemical structure of synthesized imino flavone ligands and their complexes were characterized by elemental analysis, 1H NMR, 13C NMR, UV-visible, IR, ESI-mass spectral data, conductometric and magnetic measurements. The synthesized compounds have been screened for their in vitro antibacterial activities against bacteria Vibrio cholerae, Salmonella typhi, Staphylococcus aureus, Escherichia coli and antifungal activities against fungi Candida albicans and Aspergillus flavus by paper disc diffusion method. The complexes I-3, I-4, I-7 and I-8 showed good antimicrobial activities against pathogens.

Highly selective CHEF-type chemosensor for lutetium (III) recognition in semi-aqueous media.

A simple phosphoryl quinolone (L) based sensor has been synthesized for the selective recognition of Lu3+ by spectrofluorimetric method. In methanol-water (1:1, v/v), the ligand L exhibits a weak emission peak at 400 nm upon excitation at 280 nm. Upon interaction with various f-metal and other selected metals from s, p, and d-block elements, the fluorescence of L is selectively enhanced in the presence of Lu3+ due to the chelation enhanced fluorescence (CHEF) effects. The quantum yield (φ) of L (φ = 0.063) is enhanced to φ = 0.118 upon chelation with Lu3+ ion. From the titration experiment, the limit of detection (LOD) of sensor L to recognize Lu3+ is estimated down to 24.2 nM, which is much lower than the WHO guidelines (76 μM) in drinking water. The formation of host-guest complexation between L and Lu3+ in 2:1 binding stoichiometry is studied by Job's method and the binding constant is estimated by band fit analysis (logKf = 5.1). Further, the coordination behaviour between L and Lu3+ is well supported by FT-IR, 1H NMR, 13C NMR, 31P NMR, ESI mass spectral data and the theoretical results.

SYNTHESIS OF BENZOXAZEPINE DERIVATIVES FROM PYRAZOLE-CHALCONE VIA A SIMPLE AND CONVENIENT PROTOCOL USING BASIC ALUMINA AS SOLID SUPPORT

In the present study a series of novel benzoxazepine (5a-h) derivatives were synthesized by the thermal cyclization reaction of various pyrazole-chalcones (3a-h) with 2-aminophenol, by conventional heating and microwave irradiation (180 W) in solvent-free conditions in short reaction times (9–12 min), giving high yields of products (80–88%). The homogeneity of all the newly synthesized compounds has been checked by TLC. Their IR, NMR, ESI-mass spectral data and elemental analysis are in accord with the assigned structure. The title compounds were evaluated for their antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli. Compounds 3c, 3h, 5b, 5c, 5g and 5h were found to show good antibacterial activity when compared with that of standard drug Ampicillin. Furthermore, the same library of compounds were evaluated for antifungal activity against Aspergillus nigerzeae, Penicillium italicum and Fusarium oxysporum using Grieseofulvin as standard drug. The results of the above studies show that the compounds 3b, 3c, 3e, 3h, 5a, 5c, 5g and 5h showed good antifungal activity against all the tested organisms.

Unravelling the mechanistic details of metal binding to mammalian metallothioneins from stoichiometric, kinetic, and binding affinity data.

Metallothioneins (MTs) are small, cysteine-rich proteins, found throughout Nature. Their ability to bind a number of different metals with a range of stoichiometric ratios means that this protein family is critically important for essential metal (Zn2+ and Cu+) homeostasis, metal storage, metal donation to nascent metalloenzymes as well as heavy metal detoxification. With its 20 cysteines, metallothionein is also considered to protect cells against oxidative stress. MT has been studied by a large number of researchers over the last 6 decades using a variety of spectroscopic techniques. The lack of distinguishing chromophores for the multitude of binding sites has made the evaluation of stoichiometric properties for different metals challenging. Initially, only 113Cd-NMR spectroscopy could provide strong evidence for the proposed cluster formation of Cd-MT. The extraordinary development of electrospray ionization mass spectrometry (ESI-MS), where all coexisting species in solution are observed, revolutionized MT research. Prior to the use of ESI-MS data, a range of "magic numbers" representing metal-to-MT molar ratios were reported from optical spectroscopic studies. The availability of ESI mass spectral data led to (i) the confirmation of cluster formation, (ii) a conceptual understanding of the cooperativity involved in multiple metal binding events, (iii) the presence of domain specificity between regions of the protein and (iv) mechanistic details involving both binding affinities and rate constants. The kinetic experiments identified the presence of multiple individual binding sites, each with a unique rate constant and an analogous binding affinity. The almost linear trend in rate constants as a function of bound As3+ provided a unique insight that became a critical step in the complete understanding of the mechanistic details of the metalation of MT. To fully define the biological function of this sulfur-rich protein it is necessary to determine kinetic rate constants and binding affinities for the essential metals. Recently, Zn2+ competition experiments between both of the isolated fragments (α and β) and the full-length protein (βα-MT 1a) as well as Zn2+ competition between βα-MT 1a and carbonic anhydrase were reported. From these data, the trend in binding affinities and the values of the Kf of the 7 bimolecular reactions involved in metalation were determined. From the analysis of ESI-MS data for Cu+ binding to βα-MT 1a at different pH-values, a trend in the 20 binding affinities for the complete metalation mechanism was reported. This review details a personal view of the historical development of the determination of stoichiometry for metal binding, the structure of the binding sites, the rates of the metalation reactions and the underlying binding affinities for each metalation step. We have attempted to summarize the experimental developments that led to the publication in May 2017 of the experimental determination of the 20 binding constants for the 20 sequential bimolecular reactions for Cu+ binding to the 20 Cys of apoMT as a function of pH that show the appearance and disappearance of clusters. We report both published data and in a series of tables an assembly of stoichiometries, and equilibrium constants for Zn2+ and Cu+ for many different metallothioneins.

Selective cysteine modification of metal-free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI-MS.

Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (α with 11 Cys and β with 9), was analyzed in its metal-free form by selective, covalent Cys modification coupled with ESI-MS. The modification profiles of the isolated β- and α-fragments reacted with p-benzoquinone (Bq), N-ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI-mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.

Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase.

Mammalian metallothioneins (MTs) bind up to seven Zn(2+) using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7-MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal β-domain [(Zn3Cys9)(3-)] and C-terminal α-domain [(Zn4Cys11)(3-)]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn-βαMT1A (human metallothionein 1A isoform; n=0-7). The apo-CA is shown to compete effectively only with Zn2-3-βMT and Zn4-αMT. Detailed modelling of the ESI mass spectral data have revealed the zinc-binding affinities of each of the zinc-binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities [log(KF)] of the isolated β-domain were: 12.2, 11.7 and 11.4 and the four isolated α-domain affinities were: 13.5, 13.2, 12.7 and 12.6. These data provide guidance in identification of the location of the strongest-bound and weakest-bound zinc in the intact two-domain Zn7βαMT. The β-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn7-βαMT. The α-domain with the highest affinity binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apo-peptide bundle. We propose that stabilization of the intact Zn6-MT and Zn7-MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.

Metalation kinetics of the human α-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein.

Mammalian metallothioneins are cysteine rich metal-binding proteins comprising, when fully metalated, two metal-binding domains: the α-domain binds with M4(II)S(Cys)11 stoichiometry and the β domain binds as M3(II)S(Cys)9 stoichiometry. While the fully metalated species have been widely studied, the metalation of the apoprotein is poorly understood. Key to a description of the metalation pathway(s) is the initial conformation of the apoprotein and the arrangement of the metal-coordinating cysteines prior to metalation. We report the effect of the ill-defined, globular structure of apoMT on metalation rates. In order to overcome the experimental limitations inherent in structural determinations of a fluxional protein we used a detailed analysis of the apo-α-metallothionein conformation based on the differential rate of cysteine modification with benzoquinone. The ESI mass spectral data show the presence of two distinct conformational families: one a folded conformational family at neutral pH and a second an unfolded family of conformations at low pH. The Cd(II) metalation properties of these two conformationally distinct families were studied using stopped-flow kinetics. Surprisingly, the unfolded apoprotein metalated significantly slower than the folded apoprotein, a result interpreted as being due to the longer time required to fold into the cluster structure when the fourth Cd(II) binds. These results provide the first evidence for the role of the structure of the apo-αMT in the metalation reaction pathways and show that cysteine modification coupled with ESI-MS can be used to probe structure in cysteine-rich proteins.

The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A

The small, cysteine-rich metallothionein family of proteins is currently considered to play a critical role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These experiments revealed the relative affinities of zinc to all species in solution. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calculated equilibrium zinc binding constants of each of the 7 zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The 8 equilibrium constants connecting the 10 active species in competition for the zinc were modeled by fitting the KF values of the 8 competitive bimolecular reactions to the ESI-mass spectral data. These modeled K values are shown to be experimentally connected to the metalation efficiency of the carbonic anhydrase. The series of 7 metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.

<i>In vitro</i> Antimicrobial Activity of a Semi-synthetic Derivative of Embelin

One of the strategies for the development of new drugs involves semi-synthesis of natural products. In the present study, the antimicrobial activity of embelin (1) and its semi-synthetic derivative 5-(p-tolylamino)-2-hydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione (2) were evaluated against 21 bacterial and 4 fungal pathogens using the disc diffusion method. Embelin (1) was isolated from the fruits of Embelia schimperi Vatke by column chromatography over silica gel while its derivative (2) was synthesized in good yield (98.3%; w/w) by using a one-step condensation reaction after treating embelin with p-toluidine. The structures of these compounds were determined on the basis of 1H, 13C NMR, DEPT-135 and ESI-mass spectral data. Both 1 and 2 showed broad spectrum antibacterial activity against Gram-positive and Gram-negative bacteria at a concentration of 200 µg/ml. Among the tested bacteria, some strains of Escherichia coli, Salmonella typhi, Staphylococcus aureus and Vibreo cholera were found to be highly susceptible to the tested compounds with activity ranging between 75 and 94% of that of the standard drug ciprofloxacin. Similarly the tested compounds displayed good activity against four pathogenic fungal strains when with their effects were compared with that of griseofuvin.Keywords: Embelia schimperi, embelin, 5-(p-tolylamino)-2-hydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione, antimicrobial, disk diffusion.

Substituted thieno[2,3-b]thiophenes and related congeners: Synthesis, β-glucuronidase inhibition activity, crystal structure, and POM analyses

A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The chemical structures of these compounds were deduced from elemental analyses, 1H NMR, 13C NMR, and ESI-mass spectral data. The enzyme inhibition potential of these compounds was evaluated, in vitro, against β-glucuronidase, xanthine oxidase, and α-chymotrypsin enzymes. The cytotoxicity was evaluated by a cell viability assay utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Among the compounds tested, compound 3 was the most potent β-glucuronidase inhibitor with an IC50 value of 0.9±0.0138μM; it was much more active than the standard, d-saccharic acid 1,4-lactone (IC50=45.75±2.16μM). Compound 12, on the other hand, was the most potent as a xanthine oxidase inhibitor with an IC50 of 14.4±1.2μM. With the characterization of their mechanism of action and with further testing, these compounds could be useful candidates as anticancer drugs. In addition, the newly synthesized compounds were subjected to POM analyses to get insights about their degree of their toxicity.

Phenyl carbohydrazone conjugated 2-oxoindoline as a new scaffold that augments the DNA and BSA binding affinity and anti-proliferative activity of a 1,10-phenanthroline based copper(II) complex

A new type of copper(II) complex, [CuL(phen)2](NO3) (CuIP), where L ((E)-N′-(2-oxoindolin-3-ylidene)benzohydrazide) is a N donor ligand and phen is the N, N-donor heterocyclic 1,10-phenanthroline, has been synthesized. The phenyl carbohydrazone conjugated isatin-based ligand L and CuIP were characterized by elemental analysis, infrared, UV–Vis, 1H and 13C NMR and ESI-mass spectral data, as well as single-crystal X-ray diffraction. The interaction of calf thymus DNA (CT DNA) with L and CuIP has been investigated by absorption, fluorescence and viscosity titration methods. The complex CuIP displays better binding affinity than the ligand L. The observed DNA binding constant (Kb=4.15(±0.18)×105M−1) and binding site size (s=0.19), viscosity data together with molecular docking studies of CuIP suggest groove binding and/or a partial intercalative mode of binding to CT DNA. In addition, CuIP shows good binding propensity to the bovine serum albumin (BSA) protein, giving a KBSA value of 1.25(±0.24)×106M−1. In addition, the docking studies on DNA and human serum albumin (HSA) CuIP interactions are consistent with the consequence of binding experiments. The in vitro anti-proliferative study establishes the anticancer potency of the CuIP against the human cervical (HeLa) and breast (MCF7) cancer cells; noncancer breast epithelial (MCF10a) cells have also been investigated. CuIP shows better cytotoxicity and sensitivity towards cancer cells over noncancer ones than L under identical conditions, with the appearance of apoptotic bodies.

Synthesis of copper(II) complexes of isatin thiosemicarbazone derivatives: In vitro anti-cancer, DNA binding, and cleavage activities

Six new Cu(II) complexes of thiosemicarbazone Schiff base with isatin moiety were synthesized through the reaction of Cu(II) with (Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (CuL1), (Z)-2-(5-methyl-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (CuL2), (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (CuL3), (Z)-N-methyl-2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (CuL4), (Z)-N-methyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (CuL5), and (Z)-N-ethyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (CuL6). The structures of the Schiff bases and their copper complexes were characterized based on the elemental analysis, and on the infrared, UV–Vis, 1H and 13C NMR and ESI-mass spectral data. The structures of the CuL2 and CuL3 complexes were further characterized by single-crystal X-ray diffraction. The interaction of these complexes with calf thymus (CT)-DNA exhibited high intrinsic binding constant (Kb=1.6×105–14.6×105M−1), which reflected intercalative activity of these complexes toward CT-DNA. This result was also confirmed by the viscosity data. Electrophoresis studies revealed that the CuL1 and CuL2 complexes were able to cleave the DNA via oxidative pathway, whereas CuL3, CuL4, CuL5, and CuL6 induced DNA cleavage via oxidative and hydrolytic pathways. The in vitro anti-proliferative study establishes the anticancer potency of these compounds against the human colorectal carcinoma cell line.

Synthesis, Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N-(pyrimidin-2-yl)benzenesulfonamide derivatives, 3a-3i and 4a-4i, was synthesized from pyrimidin-2-amines, 2a-2i, with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT-IR, (1) H- and (13) C-NMR, and ESI mass-spectral data. In vitro anti-amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC50 values were calculated by using the double dilution method. The results were compared with the IC50 value of the standard drug 'metronidazole'. The selected compounds were tested for their cytotoxic activities by cell-viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.

Fluorescence Turn on Sensor for Sulfate Ion in Aqueous Medium Using Tripodal-Cu2+ Ensemble

We report the selective recognition of sulfate anion in aqueous medium at biological pH 7.2 over the other interfering anions based on naphthoic acid bearing tripodal ligand by applying fluorescence turn off -on mechanism. The carboxylic acid groups in the ligand enhance the solubility in water and enable it to form complex with copper salt. Thus formed L-Cu2+ ensemble quench the fluorescence of the parent ligand and in turn recognize sulfate anion via revival of fluorescence intensity. The 1:2 stoichiometry was confirmed by ESI mass spectral data and Job's plot. The average binding constant was found to be 6.2×10(8) M(-2).