Design of the elusive proteinaceous oxygen donor copper site suggests a promising future for copper for MRI contrast agents.

We report the preparation and spectroscopic characterization of a highly elusive copper site bound exclusively to oxygen donor atoms within a protein scaffold. Despite copper generally being considered unsuitable for use in MRI contrast agents, which in the clinic are largely Gd(III) based, the designed copper coiled coil displays relaxivity values equal to, or superior than, those of the Gd(III) analog at clinical field strengths. The creation of this new-to-biology proteinaceous CuOx-binding site demonstrates the power of the de novo peptide design approach to access chemistry for abiological applications, such as for the development of MRI contrast agents.

A Single-Pot Template Reaction Towards a Manganese-Based T1 Contrast Agent.

Manganese-based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium-based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single-pot template synthetic strategy to develop a MnCA, MnLMe , and studied the most important physicochemical properties in vitro. MnLMe displays optimized r1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r1b =21.1 mM-1 s-1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (Ka =4.2×103  M-1 ). In vivo studies show that MnLMe is cleared intact into the bladder through renal excretion and has a prolonged blood half-life compared to the commercial GdCA Magnevist. MnLMe shows great promise as a novel MRI contrast agent.

Differentially selective chemosensor with fluorescence off-on responses on Cu(2+) and Zn(2+) ions in aqueous media and applications in pyrophosphate sensing, live cell imaging, and cytotoxicity.

A new benzoyl hydrazone based chemosensor R is synthesized by Schiff base condensation of 2,6-diformyl-4-methylphenol and phenyl carbohydrazide and acts as a highly selective fluorescence sensor for Cu(2+) and Zn(2+) ions in aqueous media. The reaction of R with CuCl2 or ZnCl2 forms the corresponding dimeric dicopper(II) [Cu2(R)(CH3O)(NO3)]2(CH3O)2 (R-Cu(2+)) and dizinc(II) [Zn2(R)2](NO3)2 (R-Zn(2+)) complexes, which are characterized, as R, by conventional techniques including single-crystal X-ray analysis. Electronic absorption and fluorescence titration studies of R with different metal cations in a CH3CN/0.02 M HEPES buffer medium (pH = 7.3) show a highly selective binding affinity only toward Cu(2+)and Zn(2+) ions even in the presence of other commonly coexisting ions such as Na(+), K(+), Mg(2+), Ca(2+), Mn(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cd(2+), and Hg(2+). Quantification of the fluorescence titration analysis shows that the chemosensor R can indicate the presence of Cu(2+)and Zn(2+) even at very low concentrations of 17.3 and 16.5 ppb, respectively. R-Zn(2+) acts as a selective metal-based fluorescent sensor for inorganic pyrophosphate ion (PPi) even in the presence of other common anions such as F(-), Cl(-), Br(-), I(-), CH3COO(-), CO3(2-), HCO3(-), N3(-), SO4(2-), PPi, AMP, ADP, and ATP in an aqueous medium. The propensity of R as a bioimaging fluorescent probe to detect Cu(2+) and Zn(2+) ions in human cervical HeLa cancer cell lines and their cytotoxicity against human cervical (HeLa), breast cancer (MCF7), and noncancer breast epithelial (MCF10a) cells have also been investigated. R-Cu(2+) shows better cytotoxicity and sensitivity toward cancer cells over noncancer cells than R and R-Zn(2+) under identical conditions, with the appearance of apoptotic bodies.

Naphthalene carbohydrazone based dizinc(II) chemosensor for a pyrophosphate ion and its DNA assessment application in polymerase chain reaction products.

A new naphthalene carbohydrazone based dizinc(II) complex has been synthesized and investigated to act as a highly selective fluorescence and visual sensor for a pyrophosphate ion with a quite low detection limit of 155 ppb; this has also been used to detect the pyrophosphate ion released from polymerase-chain-reaction.

ATP-responsive Mn(II)-based T1 contrast agent for MRI.

A novel diacetylpyridylcarbohydrazide-DAPyCOHz-based manganese(II) chelate with dipicolylamine/zinc(II) (DPA/Zn2+) arms (MnLDPA-Zn2) was developed for adenosine triphosphate (ATP) responsive magnetic resonance imaging (MRI) T1 contrast applications. Compound 2 shows enhanced relaxivity (r1 = 11.52 mM-1 s-1) upon selective ATP binding over other phosphates.

A series of oxyimine-based macrocyclic dinuclear zinc(II) complexes enhances phosphate ester hydrolysis, DNA binding, DNA hydrolysis, and lactate dehydrogenase inhibition and induces apoptosis.

A symmetrical macrocyclic dizinc(II) complex (1) has been synthesized by using the ligand (L(1)) [µ-11,24-dimethyl-4,7,16,19-tetraoxa-3,8,15,20-tetraazatricyclo-[20.3.1.1(10,13)] heptacosa-1(25),2,7,9,11,13(27),14,20,22(26),23-decaene-26,27-diol]. A series of unsymmetrical macrocyclic dizinc(II) complexes (2-6) has been synthesized by Schiff base condensation of bicompartmental mononuclear complex [ZnL] [µ-3,16-dimethyl-8,11-dioxa-7,12-diazadicyclo-[1.1(14,18)] heptacosa-1,3,5(20),6,12,14,16,18(19)-octacaene-19,20-diolato)zinc(II)] with various diamines like 1,2-diamino ethane (L(2)), 1,3-diamino propane (L(3)), 1,4-diamino butane (L(4)), 1,2-diamino benzene (L(5)), and 1,8-diamino naphthalene (L(6)). The ligand L(1) and all the zinc(II) complexes were structurally characterized. To corroborate the consequence of the aromatic moiety in comparison to the aliphatic moiety present in the macrocyclic ring on the phosphate ester hydrolysis, DNA binding and cleavage properties have been studied. The observed first order rate constant values for the hydrolysis of 4-nitrophenyl phosphate ester reaction are in the range from 2.73 × 10(-2) to 9.86 × 10(-2) s(-1).The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence, and circular dichroism spectroscopy. The DNA binding constant values of the complexes were found in the range from 1.80 × 10(5) to 9.50 × 10(5) M(-1), and the binding affinities are in the following order: 6 > 5 > 1 > 2 > 3 > 4. All the dizinc(II) complexes 1-6 effectively promoted the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 6 under physiological conditions give the observed rate constant (k(obs)) of 4.42 ± 0.2 h(-1), which shows a 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. The comparison of the dizinc(II) complexes 1-6 with the monozinc(II) complex [ZnL] indicates that the DNA cleavage acceleration promoted by 1-6 are due to the efficient cooperative catalysis of the two close proximate zinc(II) cation centers. The ligand L(1), dizinc(II) complexes 1, 3, and 6 showed cytotoxicity in human hepatoma HepG2 cancer cells, giving IC(50) values of 117, 37.1, 16.5, and 8.32 µM, respectively. The results demonstrated that 6, a dizinc(II) complex with potent antiproliferative activity, is able to induce caspase-dependent apoptosis in human cancer cells. Cytotoxicity of the complexes was further confirmed by the lactate dehydrogenase enzyme level in HepG2 cell lysate and content media.

Naphthalimide-phenanthroimidazole incorporated new fluorescent sensor for "turn-on" Cu2+ detection in living cancer cells.

In recent years, fluorescent sensors have emerged as attractive imaging probes due to their distinct responses toward bio-relevant metal ions. However, the bioimaging application main barrier is the 'turn-off' response toward paramagnetic metal ions such as Cu2+ under physiological conditions. Herein, we report a new sensor (2-methyl(4-bromo-N-ethylpiperazinyl-1,8-naphthalimido)-4-(1H-phenanthro[9,10-d]imidazole-2-yl) phenol)NPP with multifunctional (Naphthalimide, Piperazine, Phenanthroimidazole) units for fluorescent and colourimetric detection of Cu2+ in an aqueous medium. Both absorption and fluorescence spectral titration strategies were used to monitor the Cu2+-sensing property of NPP. The NPP displays a weak emission at ca. 455 nm, which remarkably enhances (⁓3.2-fold) upon selective binding of Cu2+ over a range of metal ions, including other paramagnetic metal ions (Mn2+, Fe3+, Co2+). The stoichiometry, binding constant (Ka) and the LOD (limit of detection) of NPP toward Cu2+ ions were found to be 1:1, 5.0 (± 0.2) × 104 M-1 and 6.5 (± 0.4) × 10-7 M, respectively. We have also used NPP as a fluorescent probe to detect Cu2+ in live (human cervical HeLa) cancer cells. The emission intensity of NPP was almost recovered in HeLa cells by incubating 'in situ' the derived Cu2+ complex (NPP-Cu2+) in the presence of a benchmark chelating agent such as EDTA (ethylenediaminetetraacetate). The fluorescent emission of NPP was reverted significantly in each cycle upon sequencial addition of Cu2+ and EDTA to the NPP solution. Overall, NPP is a novel, simple, economic and portable sensor that can detect Cu2+ in biological and environmental scenarios.

A Single-Pot Template Reaction Towards a Manganese-Based T1 Contrast Agent.

Manganese-based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium-based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single-pot template synthetic strategy to develop a MnCA, MnLMe, and studied the most important physicochemical properties in vitro. MnLMe displays optimized r 1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r 1 b=21.1 mM-1 s-1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (K a=4.2×103 M-1). In vivo studies show that MnLMe is cleared intact into the bladder through renal excretion and has a prolonged blood half-life compared to the commercial GdCA Magnevist. MnLMe shows great promise as a novel MRI contrast agent.

pH dependent binding in de novo hetero bimetallic coiled coils.

Herein the first example of a bimetallic coiled coil featuring a lanthanide binding site is reported, opening opportunities to exploit the attractive NMR and photophysical properties of the lanthanides in multi metallo protein design. In our efforts to fully characterise the system we identified for the first time that lanthanide binding to such sites is pH dependent, with optimal binding at neutral pH, and that the double AsnAsp site is more versatile in this regard than the single Asp site. Our second site featured the structural HgCys3 site, the chemistry of which was essentially unaltered by the presence of the lanthanide site. In fact, both metal binding sites within the hetero bimetallic coiled coil displayed the same properties as their mononuclear single binding site controls, and operated independently of each other. Finally, pH can be used as an external trigger to control the binding of Hg(ii) and Tb(iii) to the two distinct sites within this coiled coil, and offers the opportunity to "activate" metal binding sites within complex multi metallo and multi-functional designs.

The phenanthroimidazole-based dizinc(II) complex as a fluorescent probe for the pyrophosphate ion as generated in polymerase chain reactions and pyrosequencing.

A highly selective and sensitive phenanthroimidazole tagged Mannich base type dizinc(II) fluorescent probe (R-Zn²âº) has been developed for the pyrophosphate ion (PPi) with a very low limit of detection (LOD) of 0.25 ppm; this also assesses PPi from DNA polymerization chain reaction (PCR).

Synthesis, DNA binding, cellular DNA lesion and cytotoxicity of a series of new benzimidazole-based Schiff base copper(II) complexes.

A series of new benzimidazole containing compounds 2-((1-R-1-H-benzimidazol-2-yl)phenyl-imino)naphthol HL(1-3) (R = methyl, ethyl or propyl, respectively) have been synthesized by Schiff base condensation of 2-(1-R-1-H-benzo[d]imidazol-2-yl)aniline and 2-hydroxy-1-naphthaldehyde. The reactions of HL(1-3) with Cu(NO3)2·2.5H2O led to the corresponding copper(II) complexes [Cu(L)(NO3)] 1-3. All the compounds were characterized by conventional analytical techniques and, for 1 and 3, also by single-crystal X-ray analysis. The interactions of complexes 1-3 with calf thymus DNA were studied by absorption and fluorescence spectroscopic techniques and the calculated binding constants (K(b)) are in the range of 3.5 × 10(5) M(-1)-3.2 × 10(5) M(-1). Complexes 1-3 effectively bind DNA through an intercalative mode, as proved by molecular docking studies. The binding affinity of the complexes decreases with the size increase of the N-alkyl substituent, in the order of 1 > 2 > 3, which is also in accord with the calculated LUMO(complex) energies. They show substantial in vitro cytotoxic effect against human lung (A-549), breast (MDA-MB-231) and cervical (HeLa) cancer cell lines. Complex 1 exhibits a significant inhibitory effect on the proliferation of the A-549 cancer cells. The antiproliferative efficacy of 1 has also been analysed by a DNA fragmentation assay, fluorescence activated cell sorting (FACS) and nuclear morphology using a fluorescence microscope. The possible mode for the apoptosis pathway of 1 has also been evaluated by a reactive oxygen species (ROS) generation study.

Intracellular detection of Cu(2+) and S(2-) ions through a quinazoline functionalized benzimidazole-based new fluorogenic differential chemosensor.

A new quinazoline functionalized benzimidazole-based fluorogenic chemosensor H3L is synthesized and fully characterized by conventional techniques including single crystal X-ray analysis. It acts as a highly selective colorimetric and fluorescence sensor for Cu(2+) ions in DMF/0.02 M HEPES (1 : 1, v/v, pH = 7.4) medium. Reaction of H3L with CuCl2 forms a mononuclear copper(ii) [Cu(Cl)(H2L)(H2O)] (H2L-Cu(2+)) complex which is characterized by conventional techniques and quantum chemical calculations. Electronic absorption and fluorescence titration studies of H3L with different metal cations show a distinctive recognition only towards Cu(2+) ions even in the presence of other commonly coexisting ions such as Li(+), Na(+), K(+), Mg(2+), Ca(2+), Fe(2+), Fe(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+) and Hg(2+). Moreover, H2L-Cu(2+) acts as a metal based highly selective and sensitive chemosensor for S(2-) ions even in the presence of other commonly coexisting anions such as F(-), Cl(-), Br(-), I(-), SO4(2-), SCN(-), AcO(-), H2PO4(-), PO4(3-), NO3(-), ClO4(-), NO2(-), HSO4(-), HSO4(2-), S2O3(2-), S2O8(2-), CN(-), CO3(2-) and HCO3(-) in DMF/0.02 M HEPES (1 : 1, v/v, pH = 7.4) medium. Quantification analysis indicates that these receptors, H3L and H2L-Cu(2+), can detect the presence of Cu(2+) and S(2-) ions at very low concentrations of 1.6 × 10(-9) M and 5.2 × 10(-6) M, respectively. The propensity of H3L as a bio-imaging fluorescent probe for detection of Cu(2+) ions and sequential detection of S(2-) ions by H2L-Cu(2+) in Dalton lymphoma (DL) cancer cells is also shown.

DNA targeting polyaza macrobicyclic dizinc(II) complexes promoting high in vitro caspase dependent anti-proliferative activity against human carcinoma cancer cells.

A series of macrobicyclic dizinc(II) complexes [Zn(2)L(1-2)B](ClO(4))(4) (1-6) have been synthesized and characterized (L(1-2) are polyaza macrobicyclic binucleating ligands, and B is the N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)). The DNA and protein binding, DNA hydrolysis and anticancer activity of these complexes were investigated. The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence and CD spectroscopy. The DNA binding constant values of the complexes were found to range from 2.80 × 10(5) to 5.25 × 10(5) M(-1), and the binding affinities are in the following order: 3 > 6 > 2 > 5 > 1 > 4. All the dizinc(II) complexes 1-6 are found to effectively promote the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 3 and 6 under physiological conditions give observed rate constants (k(obs)) of 5.56 ± 0.1 and 5.12 ± 0.2 h(-1), respectively, showing a 10(7)-fold rate acceleration over the uncatalyzed reaction of dsDNA. Remarkably, the macrobicyclic dizinc(II) complexes 1-6 bind and cleave bovine serum albumin (BSA), and effectively promote the caspase-3 and caspase-9 dependent deaths of HeLa and BeWo cancer cells. The cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme levels in cancer cell lysate and content media.

Naphthylhydrazone based selective and sensitive chemosensors for Cu2+ and their application in bioimaging.

Two new hydroxynaphthyl-hydrazone based fluorogenic chemosensors R1 and R2 have been synthesized by Schiff base condensation of Tris(4-formylphenyl)amine with 1-hydroxynaphthalene-2-hydrazide and 1-hydroxynaphthalene-2-carbohydrazone, respectively. They are examined as highly selective and sensitive receptors for Cu2+ ions in aqueous medium. Electronic absorption as well as fluorescence titration studies of receptors R1 and R2 with different metal cations in H2O/CH3CN medium showed highly selective and very rapid (<2 min) binding affinity towards Cu2+ ions even in the presence of other commonly coexisting metal ions such as Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+. Quantification of the fluorescence titration analysis indicated that these newly synthesized receptors (R1 and R2) can indicate the presence of Cu2+ ions even at very low concentrations of 598 and 676 ppt, respectively. In addition, the propensity of these receptors as bio-imaging fluorescent probes to detect Cu2+ ions in human cervical HeLa cancer cell lines and their cytotoxicity against HeLa cells have been investigated.

Phosphatase-like activity, DNA binding, DNA hydrolysis, anticancer and lactate dehydrogenase inhibition activity promoting by a new bis-phenanthroline dicopper(II) complex.

A new bis-phenanthroline dicopper(II) complex has been synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the dinuclear Cu(II) complex [Cu(2)(µ-CH(3)COO)(µ-H(2)O)(µ-OH)(phen)(2)](2+) (phen = 1,10-phenanthroline) (1) was determined by single crystal X-ray diffraction technique. The coordination environment around each Cu(II) ion in complex 1 can be described as slightly distorted square pyramidal geometry. The distance between the CuCu centers in the complex is found to be 2.987 Å. The electronic, redox, phosphate hydrolysis, DNA binding and DNA cleavage have been studied. The antiproliferative effect of complex 1 was confirmed by the lactate dehydrogenase (LDH) enzyme level in MCF-7 cancer cell lysate and content media. The dicopper(II) complex inhibited the LDH enzyme as well as the growth of the human breast cancer MCF7 cell line at an IC(50) value of 0.011 µg ml(-1). The results strongly suggest that complex 1 is a good cancer therapeutic agent. Electrochemical studies of complex 1 showed an irreversible, followed by a quasi-reversible, one electron reduction processes between -0.20 to -0.8 V. Michaelis-Menten kinetic parameters for the hydrolysis of 4-nitrophenyl phosphate by complex 1 are k(cat) = 3.56 × 10(-2) s(-1) and K(M) = 4.3 × 10(-2) M. Complex 1 shows good binding propensity to calf thymus DNA, with a binding constant value of 1.3 (±0.13) × 10(5) M(-1) (s = 2.1). The size of the binding site and viscosity data suggest a DNA intercalative binding nature of the complex. Complex 1 shows efficient hydrolytic cleavage of supercoiled pBR322-DNA in the dark and in the absence of any external reagents, as demonstrated by the T4 ligase experiment. The pseudo-Michaelis-Menten kinetic parameters for DNA hydrolysis by complex 1 are k(cat) = 1.27 ± 0.4 h(-1) and K(M) = 7.7 × 10(-2) M.

Structural, electrochemical, phosphate-hydrolysis, DNA binding and cleavage studies of new macrocyclic binuclear nickel(II) complexes.

New macrocyclic binuclear nickel(ii) complexes have been synthesized by using the bicompartmental mononuclear complex [NiL] [3,30-((1E,7E)-3,6-dioxa-2,7-diazaocta-1,7-diene-1,8-diyl)bis(3-formyl-5-methyl-2-diolato)nickel(II)] with various diamines like 1,2-bis(aminooxy)ethane (L(1)), 1,2-diamino ethane (L(2)), 1,3-diamino propane (L(3)), 1,4-diamino butane (L(4)), 1,2-diamino benzene (L(5)), and 1,8-diamino naphthalene (L(6)). The complexes were characterized by elemental analysis and spectroscopic methods. The molecular structures of the symmetrical binuclear complex [Ni(2)L(1)(H(2)O)(4)](ClO(4))(2) (1) and unsymmetrical binuclear complex [Ni(2)L(3)(H(2)O)(4)](ClO(4))(2).(H(2)O)(4) (3) were determined by single-crystal X-ray diffraction. The geometry around both the nickel(II) ions in each molecule is a slightly distorted octahedral. The distance between the Ni...Ni centers for complex 1 is 3.039 A and for complex 3 is 3.059 A. The influence of the coordination geometry and the ring size of the binucleating ligands on the electronic, redox, phosphate hydrolysis, DNA binding and cleavage properties have been studied. Electrochemical studies of the complexes show two quasi-reversible one electron reduction processes between -0.49 to -1.69 V. The reduction potential of the binuclear Ni(II) complexes shifts towards anodically upon increasing the macrocyclic ring size. The observed first order rate constant values for the hydrolysis of 4-nitrophenyl phosphate reaction are in the range from 8.69 x 10(-3) to 1.85 x 10(-2) s(-1). The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range from 1.4 x 10(4) to 17.5 x 10(4) M(-1). The absorption, fluorescence and CD spectral data suggests that the complexes are strongly interacting with DNA. These complexes display hydrolytic cleavage of supercoiled pBR322DNA in the presence of H(2)O(2) at pH 7.2 and 37 degrees C. The hydrolytic cleavage of DNA by the complexes is supported by the evidence from free radical quenching and T4 ligase ligation. The pseudo-Michaelis-Menten kinetic parameters k(cat) = 1.27 +/- 0.4 h(-1) and K(M) = 7.7 x 10(-2) M for naphthalene diimine containing macrocyclic binuclear nickel(II) complex, (6) were obtained.