Antibiotic resistance: mono- and dinuclear zinc complexes as metallo-beta-lactamase mimics

Biomimetic systems containing one or two zinc(II) ions supported by phenolate ligands were developed as functional mimics of metallo-beta-lactamase. These complexes were shown to catalytically hydrolyze beta-lactam substrates, such as oxacillin and penicillin G. The dinuclear zinc complex 1, which has a coordinated water molecule, exhibits high beta-lactamase activity, whereas the dinuclear zinc complex 2, which has no water molecules, but labile chloride ligands, shows a much lower activity. The high beta-lactamase activity of complex 1 can be ascribed to the presence of a zinc-bound water molecule that is activated by being hydrogen bonded to acetate substituents. The kinetics of the hydrolysis of oxacillin by complex 1 and the effect of pH on the reaction rates are reported in detail. In addition, the kinetic parameters obtained for the synthetic analogues are compared with those of the natural metallo-beta-lactamase from Bacillus cereus (BcII). To understand the role of the second metal ion in hydrolysis, the syntheses and catalytic activities of two mononuclear complexes (3 and 4) that include coordinated water molecules are described. Interestingly, the mononuclear zinc complexes 3 and 4 also exhibit high activity, supporting the assumption that the second zinc ion is not crucial for the beta-lactamase activity.     

Osmium(II) and ruthenium(II) arene maltolato complexes: rapid hydrolysis and nucleobase binding

Density functional calculations show that aquation of [Os(eta6-arene)(XY)Cl]n+ complexes is more facile for complexes in which XY=an anionic O,O-chelated ligand compared to a neutral N,N-chelated ligand, and the mechanism more dissociative in character. The O,O-chelated XY=maltolato (mal) [M(eta6-p-cym)(mal)Cl] complexes, in which p-cym=p-cymene, M=OsII (1) and RuII (2), were synthesised and the X-ray crystal structures of 1 and 22 H2O determined. Their hydrolysis rates were rapid (too fast to follow by NMR spectroscopy). The aqua adduct of the OsII complex 1 was 1.6 pKa units more acidic than that of the RuII complex 2. Dynamic NMR studies suggested that O,O-chelate ring opening occurs on a millisecond timescale in coordinating proton-donor solvents, and loss of chelated mal in aqueous solution led to the formation of the hydroxo-bridged dimers [(eta6-p-cym)M(mu-OH)3M(eta6-p-cym)]+. The proportion of this dimer in solutions of the OsII complex 1 increased with dilution and it predominated at micromolar concentrations, even in the presence of 0.1 M NaCl (conditions close to those used for cytotoxicity testing). Although 9-ethylguanine (9-EtG) binds rapidly to Os(II) in 1 and more strongly (log K=4.4) than to RuII in 2 (log K=3.9), the OsII adduct [Os(eta6-p-cym)(mal)(9EtG)]+ was unstable with respect to formation of the hydroxo-bridged dimer at micromolar concentrations. Such insights into the aqueous solution chemistry of metal-arene complexes under biologically relevant conditions will aid the rational design of organometallic anticancer agents.     

Intermediate in beta-lactam hydrolysis catalyzed by a dinuclear zinc(II) complex: relevance to the mechanism of metallo-beta-lactamase.

Inactivation of beta-lactam antibiotics by metallo-beta-lactamase enzymes is a well-recognized pathway of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a beta-lactam substrate nitrocefin (1) catalyzed by dinuclear zinc(II) model complexes was investigated in nonaqueous solutions. The initial step involves monodentate coordination of the nitrocefin carboxylate group to the dizinc center. The coordinated substrate is then attacked intramolecularly by the bridging hydroxide to give a novel intermediate (2') characterized by its prominent absorbance maximum at 640 nm, which affords a blue color. The NMR and IR spectroscopic data of 2' are consistent with it being zinc(II)-bound N-deprotonated hydrolyzed nitrocefin that forms from the tetrahedral intermediate upon C-N bond cleavage. Protonation of the leaving group is the rate-limiting step in DMSO solution and occurs after the C-N bond-breaking step. Addition of strong acids results in rapid conversion of 2' into hydrolyzed nitrocefin (3). The latter can be converted back to the blue species (2') upon addition of base. The low pK(a) value for the amino group in hydrolyzed nitrocefin is explained by its involvement in extended conjugation and by coordination to zinc(II). The blue intermediate (2') in the model system resembles well that in the enzymatic system, judging by its optical properties. The greater stability of the intermediate in the model, however, allowed its characterization by (13)C NMR and infrared, as well as electronic, spectroscopy.     

A new fluorescent probe for zinc(II): an 8-hydroxy-5-N,N-dimethylaminosulfonylquinoline-pendant 1,4,7,10-tetraazacyclododecane

A new fluorescent probe for Zn2+, namely, 8-hydroxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (L8), was designed and synthesized (cyclen=1,4,7,10-tetraazacyclododecane). By potentiometric pH, 1H NMR, and UV spectroscopic titrations, the deprotonation constants pKa1-pKa6 of L(8)4 HCl were determined to be <2, <2, <2 (for amino groups of the cyclen and quinoline moieties), 7.19+/-0.05 (for 8-OH of the quinoline moiety), 10.10+/-0.05, and 11.49+/-0.05, respectively, at 25 degrees C with I=0.1 (NaNO3). The results of 1H NMR, potentiometric pH, and UV titrations, as well as single-crystal X-ray diffraction analysis, showed that L8 and Zn2+ form a 1:1 complex [Zn(H-1L8)], in which the 8-OH group of the quinoline ring of L8 is deprotonated and coordinates to Zn2+, in aqueous solution at neutral pH. On addition of one equivalent of Zn2+ and Cd2+, the fluorescence emission of L8 (5 microM) at 512 nm in aqueous solution at pH 7.4 [10 mM HEPES with I=0.1 (NaNO3)] and 25 degrees C increased by factors of 17 and 43, respectively. We found that the cyclen moiety has the unique property of quenching the fluorescence emission of the quinolinol moiety when not complexed with metal cations, but enhancing emission when complexed with Zn2+ or Cd2+. In addition, the Zn2+-L8 complex [Zn(H-1L8)] is much more thermodynamically and kinetically stable (Kd{Zn(H-1L8)}=[Zn2+]free[L8]free/[Zn(H-1L8)]=8 fM at pH 7.4) than the Zn2+ complexes of our previous Zn2+ fluorophores ([Zn(H-1L2)] and [Zn(L3)]). Furthermore, formation of [Zn(H-1L8)] is much faster than those of [Zn(H-1L2)] and [Zn(L3)]. The staining of early-stage apoptotic cells with L8 is also described.     

Density functional computations of alkynylation of ethanimine catalyzed by chiral zinc(II)‐complexes

The alkynylation of ethanimine catalyzed by chiral zinc(II)‐complexes was studied by means of the density functional theory (DFT). All the intermediates and transition states were optimized completely at the B3LYP/6‐31G(d,p) level. Calculation results confirm that the alkynylation of ethanimine is exothermic and the total released energy is about ?13 kJ/mol. The formation of the catalyst–alkynyl complexes M4 is the rate‐determining step for this alkynylation, and the formation of the catalyst–amine complexes M5 is the chirality‐limiting step for this alkynylation. The transition states for the chirality‐limiting step have a H? O? Zn? C? C? N six‐membered ring. The dominant products predicted theoretically for this alkynylation are, respectively, S‐amine for ethanimine anti and R‐amine for ethanimine syn . © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

X-ray and solution structures of Cu(II) GHK and Cu(II) DAHK complexes: influence on their redox properties

The Gly‐His‐Lys (GHK) peptide and the Asp‐Ala‐His‐Lys (DAHK) sequences are naturally occurring high‐affinity copper(II) chelators found in the blood plasma and are hence of biological interest. A structural study of the copper complexes of these peptides was conducted in the solid state and in solution by determining their X‐ray structures, and by using a large range of spectroscopies, including EPR and HYSCORE (hyperfine sub‐level correlation), X‐ray absorption and ¹H and ¹³C NMR spectroscopy. The results indicate that the structures of [Cu^II(DAHK)] in the solid state and in solution are similar and confirm the equatorial coordination sphere of NH₂, two amidyl N and one imidazole N. Additionally, a water molecule is bound apically to Cu^II as revealed by the X‐ray structure. As reported previously in the literature, [Cu^II(GHK)], which exhibits a dimeric structure in the solid state, forms a monomeric complex in solution with three nitrogen ligands: NH₂, amidyl and imidazole. The fourth equatorial site is occupied by a labile oxygen atom from a carboxylate ligand in the solid state. We probe that fourth position and study ternary complexes of [Cu^II(GHK)] with glycine or histidine. The Cu^II exchange reaction between different DAHK peptides is very slow, in contrast to [Cu^II(GHK)], in which the fast exchange was attributed to the presence of a [Cu^II(GHK)₂] complex. The redox properties of [Cu^II(GHK)] and [Cu^II(DAHK)] were investigated by cyclic voltammetry and by measuring the ascorbate oxidation in the presence of molecular oxygen. The measurements indicate that both Cu^II complexes are inert under moderate redox potentials. In contrast to [Cu^II(DAHK)], [Cu^II(GHK)] could be reduced to Cu^I around ?0.62 V (versus AgCl/Ag) with subsequent release of the Cu ion. These complete analyses of structure and redox activity of those complexes gave new insights with biological impact and can serve as models for other more complicated Cu^II–peptide interactions.     

Less symmetrical dicopper(II) complexes as catechol oxidase models--an adjacent thioether group increases catecholase activity

Three new unsymmetrical compartmental dinucleating ligands, 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(1-piperidyl)ethyl}aminomethyl]phenol (HL1), 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(morpholin-4-yl)ethyl}aminomethyl]phenol (HL2), and 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(thiomorpholin-4-yl)ethyl}aminomethyl]phenol (HL3), have been synthesized in order to model the active site of type 3 copper proteins. The dicopper(II) complexes of these ligands give first hints about the influence of a thioether group close to the metal site. The bromophenol-based ligands have one piperazine arm and one other bidentate arm in positions 2 and 6 of the phenolic ring, respectively. With each ligand a dinuclear copper(II) complex was prepared and structurally characterized. The copper ions were found to have square pyramidal environments and a mixture of endogenous phenoxo and exogenous acetate bridging. The influence of a heteroatom in one arm of the ligand on catecholase activity and speciation in solution was studied by UV/Vis spectroscopy, ESI-MS experiments and, DFT calculations.     

Directed synthesis of a heterobimetallic complex based on a novel unsymmetric double-Schiff-base ligand: preparation, characterization, reactivity and structures of hetero- and homobimetallic nickel(II) and zinc(II) complexes

A series of bimetallic zinc(II) and nickel(II) complexes based on the novel dinucleating unsymmetric double-Schiff-base ligand benzoic acid [1-(3-{[2-(bispyridin-2-ylmethylamino)ethylimino]methyl}-2-hydroxy-5-methylphenyl)methylidene]hydrazide (H(2)bpampbh) has been synthesized and structurally characterized. The metal centers reside in two entirely different binding pockets provided by the ligand H(2)bpampbh, a planar tridentate [ONO] and a pentadentate [ON(4)] compartment. The utilized ligand H(2)bpampbh has been synthesized by condensation of the single-Schiff-base proligand Hbpahmb with benzoic acid hydrazide. The reaction of H(2)bpampbh with two equivalents of either zinc(II) or nickel(II) acetate yields the homobimetallic complexes [Zn(2)(bpampbh)(mu,eta(1)-OAc)(eta(1)-OAc)] (ZnZn) and [Ni(2)(bpampbh)(mu-H(2)O)(eta(1)-OAc)(H(2)O)](OAc) (NiNi), respectively. Simultaneous presence of one equivalent zinc(II) and one equivalent nickel(II) acetate results in the directed formation of the heterobimetallic complex [NiZn(bpampbh)(mu,eta(1)-OAc)(eta(1)-OAc)] (NiZn) with a selective binding of the nickel ions in the pentadentate ligand compartment. In addition, two homobimetallic azide-bridged complexes [Ni(2)(bpampbh)(mu,eta(1)-N(3))]ClO(4) (NiNi(N(3))) and [Ni(2)(bpampbh)(mu,eta(1)-N(3))(MeOH)(2)](ClO(4))(0.5)(N(3))(0.5) (NiNi(N(3))(MeOH)(2)) were synthesized. In all complexes, the metal ions residing in the pentadentate compartment adopt a distorted octahedral coordination geometry, whereas the metal centers placed in the tridentate compartment vary in coordination number and geometry from square-planar (NiNi(N(3))) and square-pyramidal (ZnZn and NiZn), to octahedral (NiNi and NiNi(N(3))(MeOH)(2)). In the case of complex NiNi(N(3)) this leads to a mixed-spin homodinuclear nickel(II) complex. All compounds have been characterized by means of mass spectrometry as well as IR and UV/Vis spectroscopies. Magnetic susceptibility measurements show significant zero-field splitting for the nickel-containing complexes (D=2.9 for NiZn, 2.2 for NiNi(N(3)), and 0.8 cm(-1) for NiNi) and additionally a weak antiferromagnetic coupling (J=-1.4 cm(-1)) in case of NiNi. Electrochemical measurements and photometric titrations reveal a strong Lewis acidity of the metal center placed in the tridentate binding compartment towards external donor molecules. A significant superoxide dismutase reactivity against superoxide radicals was found for complex NiNi.     

5-Methylcytosine is Oxidized to the Natural Metabolites of TET Enzymes by a Biomimetic Iron(IV)-Oxo Complex

Ten-eleven-translocation (TET) methyl cytosine dioxygenases play a key role in epigenetics by oxidizing the epigenetic marker 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxy cytosine (5cC). Although much of the metabolism of 5mC has been studied closely, certain aspects—such as discrepancies among the observed catalytic activity of TET enzymes and calculated bond dissociation energies of the different cytosine substrates—remain elusive. Here, it is reported that the DNA base 5mC is oxidized to 5hmC, 5fC, and 5cC by a biomimetic iron(IV)-oxo complex, reminiscent of the activity of TET enzymes. Studies show that 5hmC is preferentially turned over compared with 5mC and 5fC and that this is in line with the calculated bond dissociation energies. The optimized syntheses of d₃-5mC and d₂-5hmC are also reported and in the reaction with the biomimetic iron(IV)-oxo complex these deuterated substrates showed large kinetic isotope effects, confirming the hydrogen abstraction as the rate-limiting step. Taken together, these results shed light on the intrinsic reactivity of the C−H bonds of epigenetic markers and the contribution of the second coordination sphere in TET enzymes.     

The versatile binding mode of transition-state analogue inhibitors of tyrosinase towards dicopper(II) model complexes: experimental and theoretical investigations

We describe 2-mercaptopyridine-N-oxide (HSPNO) as a new and efficient competitive inhibitor of mushroom tyrosinase (K(IC) =3.7 μM). Binding studies of HSPNO and 2-hydroxypyridine-N-oxide (HOPNO) on dinuclear copper(II) complexes [Cu(2)(BPMP)(μ-OH)](ClO(4))(2) (1; HBPMP=2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol) and [Cu(2)(BPEP)(μ-OH)](ClO(4))(2)) (2; HBPEP=2,6-bis{bis[2-(2-pyridyl)ethyl]aminomethyl}-4-methylphenol), known to be functional models for the tyrosinase diphenolase activity, have been performed. A combination of structural data, spectroscopic studies, and DFT calculations evidenced the adaptable binding mode (bridging versus chelating) of HOPNO in relation to the geometry and chelate size of the dicopper center. For comparison, binding studies of HSPNO and kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone) on dinuclear complexes were performed. A theoretical approach has been developed and validated on HOPNO adducts to compare the binding mode on the model complexes. It has been applied for HSPNO and kojic acid. Although results for HSPNO were in line with those obtained with HOPNO, thus reflecting their chemical similarity, we showed that the bridging mode was the most preferential binding mode for kojic acid on both complexes.     

Synthesis,crystal structure and fluorescence properties of two dinuclear zinc(II) complexes incorporating tridentate (NNO) Schiff bases

Reactions of hydrated zinc(II) trifluoroacetate and sodium azide with two tridentate Schiff bases HL¹ (2-((E)-(2-(dimethylamino)ethylimino)methyl)-4-chlorophenol) and HL² (2-((E)-(2-(dimethylamino)ethylimino)methyl)-4-bromophenol) under the same reaction conditions yielded two dinuclear isostructural zinc(II) complexes, [Zn(L¹)(N₃)]₂ (1) and [Zn(L²)(N₃)]₂ (2), respectively. The complexes were characterized systematically by elemental analysis, UV–Vis, FT-IR, and ¹H NMR spectroscopic methods. Single-crystal X-ray diffraction studies reveal that each of the dinuclear complexes consists of two crystallographically independent zinc(II) ions connected by double bridging phenoxides. All zinc(II) ions in 1 and 2 are surrounded by similar donor sets and display distorted square–pyramidal coordination geometries. The ligands and complexes reveal intraligand ¹(π → π*) flourescence. The enhancement of the fluorescence intensities for the complexes compared to the ligands indicates their potential to serve as photoactive materials.     

Metallovesicular catalytic hydrolysis of p-nitrophenyl picolinate catalyzed by zinc(II) complexes of pyridyl ligands in vesicular solution

The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by the Zn(II) complexes of 6-(n-butyloxymethyl)-2-(hydroxymethyl)pyridine and 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine was kinetically investigated by observation of the rates of release of p-nitrophenol in a vesicular solution at different pH values and temperatures. A 1:1 ligand:Zn²⁺ stoichiometry was found to produce the most active species based on a kinetic version of the Job plot analysis. Experimental results also showed that the complex formed from ligand 2 and Zn²⁺ exhibited a more remarkable rate acceleration effect on the hydrolysis of PNPP in vesicular solution than that formed from ligand 1 and Zn²⁺.     

Direct asymmetric aldol condensation catalyzed by aziridine semicarbazide zinc(II) complexes

Previously obtained semicarbazides derived from N-triphenylmethyl-aziridine-2-carbohydrazide were explored as ligands in Zn(II) catalyzed diastereo- and enantioselective direct aldol reactions. Complexes of aziridine-semicarbazides with Zn(II) were efficient catalysts in reactions of acetone and hydroxyacetone with NO₂-substituted aromatic aldehydes in the presence of water.     

A Promising Approach for Controlling the Second Coordination Sphere of Biomimetic Metal Complexes: Encapsulation in a Dynamic Hydrogen-Bonded Capsule

The design of biomimetic models of metalloenzymes needs to take into account many factors and is therefore a challenging task. We propose in this work an original strategy to control the second coordination sphere of a metal centre and its distal environment. A biomimetic complex, reproducing the first coordination sphere, is encapsulated in a self-assembled hydrogen-bonded capsule. The cationic complex is co-encapsulated with its counter-anion or with solvent molecules. The capsule is dynamic, allowing a fast in/out exchange of the co-encapsulated species. It also provides both a hydrogen-bonding site in the second coordination sphere and a source of proton as it can be deprotonated in the presence of the complex, providing a globally neutral host-guest assembly. This simple and broad scope strategy is unprecedented in biomimetic studies. The approach appears to be a very promising method for the stabilisation of reactive species and for the study of their reactivity.     

Density functional computations of alkynylation of nitrones catalyzed by chiral zinc(II)-complexes

The alkynylation of nitrones catalyzed by chiral zinc(II)-complexes was studied by means of the density functional theory (DFT). All the intermediates and transition states were optimized completely at B3LYP/6-31G(d,p) level. Calculation results confirm that this alkynylation of nitrones was endothermic and the total absorbed energy was about 14 kJ/mol. The formation of the M5 complexes was the rate-determining step and the chirality-limiting step for this alkynylation. The transition states TS3-re involve a H(8)–O(2)–Zn(6)–C(9)–C(14)–N(13)–O(12) 7-membered ring, and thus the transition states TS3-si involve a Zn(6)–C(9)–C(14)–N(13)–O(12) 5-membered ring. The dominant reaction is the attack of nitrones from the re-surface of M4. The reactivity of anti-nitrones was stronger than that of syn-nitrones for zinc-catalyzed alkynylation of nitrones. The dominant reaction channel was cata → TS1b → M1b → M2 → M3b → TS2b → M4b → TS3b1-anti-re → M5b1-anti-re → Pro-R. The dominant products predicted theoretically were of R-chirality, (2R)-N-hydroxy-N-methylpent-3-yn-2-amine. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.