Previously reported mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes of 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) with different heterocyclic spacers (triazine, pyridine) of various lengths (bi- and tripyridine) or an azacrown-pendant have been tested for the hydrolysis of bis(4-nitrophenyl)phosphate (BNPP) under physiological conditions (pH 7-9, 25 degrees C). All Zn(II) complexes promote the hydrolysis of BNPP under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The hydrolysis kinetics in buffered solutions (0.05 M Bis/Tris, TRIS, HEPES, or CHES, I=0.1 M, NaCl) at 25 degrees C were determined by the initial slope method (product conversion<5%). Comparison of the second-order pH-independent rate constants (kBNPP, M(-1) s(-1)) for the mononuclear complexes ZnL1, ZnL3, and ZnL6, which are 6.1x10 (-5), 5.1x10(-5), and 5.7x10(-5), respectively, indicate that the heterocyclic moiety improves the rate of hydrolysis up to six times over the parent Zn([12]aneN4) complex (kBNPP=1.1x10(-5) M(-1) s(-1)). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile. For dinuclear complexes Zn2L2, Zn2L4, and Zn2L5, the rate of reaction is defined by the degree of cooperation between the metal centers, which is determined by the spacer length. Zn2L2 and Zn2L4 possessing shorter spacers are able to hydrolyze BNPP 1 to 2 orders of magnitudes faster than Zn2L5. The second-order rate constants k of Zn2L4 and Zn2L2 at pH 7, 8, and 9 are significantly higher than those of previously reported related complexes. The high BNPP hydrolytic activity may be related to pi-stacking and hydrophobic interactions between the aromatic spacer moieties and the substrate. Complexes Zn2L4 and Zn2L2 show hydrolytic activity at pH 7 and 8, which allows for the hydrolysis of activated phosphate esters under physiological conditions.

New 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) ligands with different heterocyclic spacers (triazine and pyridine) of various lengths (bi- and tripyridine) or an azacrown pendant and their mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes have been synthesized and characterized. The pKa values of water molecules coordinated to the complexed metal ions were determined by potentiometric pH titrations and vary from 7.7 to 11.2, depending on the metal-ion and ligand properties. The X-ray structure of [Zn2L2]mu-OH(ClO4)3.CH3CN.H2O shows each Zn(II) ion in a tetrahedral geometry, binding to three N atoms of cyclen (the average distance of Zn-N = 2.1 A) and having a mu-OH bridge at the apical site linking the two metal ions (the average distance of Zn-O- = 1.9 A). The distance between the Zn(II) ion and the fourth N atom is 2.6 A. All Zn(II) complexes promote the hydrolysis of 4-nitrophenyl acetate (NA) under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The kinetic studies in buffered solutions (0.05 M Tris, HEPES, or CHES, I = 0.1 M, NaCl) at 25 degrees C in the pH range of 6-11 under pseudo-first-order reaction conditions (excess of the metal complex) were analyzed by applying the method of initial rates. Comparison of the second-order pH-independent rate constants (kNA, M-1 s-1) for the mononuclear complexes ZnL1, ZnL3, and ZnL8, which are 0.39, 0.27, and 0.38, respectively, indicates that the heterocyclic moiety improves the rate of hydrolysis up to 4 times over the parent Zn([12]aneN4) complex (kNA = 0.09 M-1 s-1). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile, which attacks intermolecularly the carbonyl group of the acetate ester. For dinuclear complexes Zn2L2, Zn2L4, Zn2L5, Zn2L6, and Zn2L7, the mechanism of the reaction is defined by the degree of cooperation between the metal centers, determined by the spacer length. For Zn2L7, having the longest triaryl spacer, the two metal centers act independently in the hydrolysis; therefore, the reaction rate is twice as high as the rate of the mononuclear analogue (kNA = 0.78 M-1 s-1). The complexes with a monoaryl spacer show saturation kinetics with the formation of a Michaelis-Menten adduct. Their hydrolysis rates are 40 times higher than that of the Zn[12]aneN4 system (kNA approximately 4 M-1 s-1). Zn2L6 is a hybrid between these two mechanisms; a clear saturation curve is not visible nor are the metal cores completely independent from one another. Some of the Zn(II) complexes show a higher hydrolytic activity under physiological conditions compared to other previously reported complexes of this type.

The anion binding ability of bis-zinc cyclen complexes in buffered aqueous solution was investigated using indicator displacement assays (IDA) as well as luminescent labelled complexes. A high affinity to phosphate anions, such as UTP or pyrophosphate was observed in IDA while there was no observable binding of other anions. The binding affinity, and as a result the selectivity, between different phosphate anions correlates with their overall negative charge and steric demand. Complexes bearing luminescent labels did not respond to the presence of phosphate anions in homogeneous solution, but did if embedded as amphiphiles in small unilamellar vesicle (SUV) membranes. The scope of possible anionic analytes was extended to phosphorylated protein surfaces by using such metal complex-functionalized vesicles bearing oligoethylene glycol residues in an optimized amount on their surface to suppress non-specific interactions. Under physiological conditions these surface-modified vesicles show a selective response and nanomolar affinity for alpha-S1-Casein, which is multiple phosphorylated, while not responding to the corresponding dephosphorylated Casein or BSA. The vesicular luminescent metal complexes do not currently reach the sensitivity and selectivity of reported enzymatic assays or some chemosensors for phosphate anions, but they present a novel type of artificial receptor for molecular recognition. Membrane-embedding of multiple, different receptors and their possible structuring on the vesicular surface is expected to improve affinities and selectivities and may allow the design of artificial antibodies.

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