From supramolecular vanadate receptors to enzyme models of vanadium Haloperoxidase (2005)

V-HPOs are enzymes catalyzing the halogenation of a variety of organic substrates using hydrogen peroxide and halide ions at slightly acidic condition[1-3]. The X-ray structure of V-HPO from Curvularia inaequalis[4, 5] reveals that the positively charged residues at the active site bind orthovanadate (HVO42-) through electrostatic interaction and hydrogen bonds, together with one coordinating bond from the nitrogen (N 2) of His496 to vanadium, which is the only direct bond from protein to metal center. Accordingly the coordination sphere at vanadium is trigonalbipyramidal, resembling the transition state of SN2-type reactions involving phosphates[6]. Apart from that, vanadate and phosphate are also very similar in the tetrahedral ground state. Thus, it is no surprise that vanadate is an inhibitor of various phosphate metabolizing enzymes[7]. Since the structural assignment and reaction mechanism of V-HPOs are still under debate, and no structural model reported so far shows high fidelity concerning the non-covalent binding fashion, we decided to prepare supramolecular models structurally related to the binding mode of HVO42- in the enzyme, and study the influence of the binding sphere of vanadate to its catalytic activity. The vanadate receptor tris-(2-guanidinium-ethyl)aminewas rationally designed and conveniently synthesized from tris-(2-aminoethyl)amine via single step. Its three guanidinium-arms can provide not only positive charges but also several hydrogen bond donor sites. The central nitrogen with pKa=2.48 for its conjugated acid, allows the V-N coordination to occur within a broad pH window. The X-ray structure ofreveals that it is already preorganized in a basket shape before binding vanadate, which verifies our design. NMR titration data from 51V of vanadate and 1H of the ligand give complementary&#13;\nresults. The ligand 1 binds vanadate as 1/1 complex 5 (Scheme 19) at pH 10.21 in&#13;\nwater solution, with Ka = 103 Mol-1. The complex 5 was also detected by ESI-MS.&#13;\nThe absorption at 306 nm observed in UV difference spectrum is an indication of a VN&#13;\nbond formation in agreement with coordination in V-HPO. The time dependent&#13;\nDFT calculation both for enzyme and model system 5 provided in-depth evidence that&#13;\nV-N coordination is responsible for this UV band.&#13;\n5 is the first supramolecular structural model V-HPO. These model studies provided&#13;\nfor the first time evidence that the V-N bond of V-HPO is coordinative and not&#13;\ncovalent as original proposed[8].&#13;\nA series of more rigid novel guanidinium-cryptands containing the key scaffold of 1&#13;\nwere synthesized (Scheme 20), the preorganization was expected to provide higher&#13;\naffinity to vanadate. A general flexible synthetic strategy was developed which allows&#13;\nthe preparation of the guanidinium cryptands with different size and geometry.&#13;\nHowever, spectroscopic studies failed to demonstrate any enhancement of binding&#13;\nconstant for encapsulating vanadate. The association of vanadate most likely occurres&#13;\nnot interior but outside of the cavity even for the largest cage receptor 35. Three pyrene moieties introduced to the terminal-N atoms of 1 not only improved the&#13;\nsolubility of the receptor in unpolar solvent, but also served as UV and fluorescence&#13;\nsensor for vanadate recognition (see Scheme 21). In addition, the - stacking&#13;\ninteraction within pyrenes holds three arms together to further preorganize itself&#13;\nfavoring the binding of vanadate. This preorganization was demonstrated by&#13;\nobserving the pyrene excimer emission, which can be also observed for the neutral&#13;\nanalogue 53. The binding of vanadate to 36 and 53 is coupled with the significant UV and&#13;\nfluorescence response in acetonitrile. Upon the addition of vanadate, the UV bands of&#13;\n36 became broader and red shifted, together with a PET-type fluorescence quenching.&#13;\nTherefore, deduced from the UV, fluorescence titration and additional 51V-NMR data,&#13;\n36 exhibits an association constant &gt;&gt; 3 × 107 mol-1 with pyrovanadate (V2O7&#13;\n4-, V2)&#13;\n(Scheme 22) in acetonitrile, which is at least 4 times magnitude higher than that of 1 to vanadate (HVO4&#13;\n2-, V1). The preference of 36 to bind V2 over V1 was also&#13;\nconfirmed in titration studies with the pyrophosphate and phosphate, the structure&#13;\nanalogue of V2 and V1 respectively. The neutral thiourea receptor 53 shows much lower binding constant and almost no&#13;\npreference to any vanadate or phosphate species mentioned above, revealing the&#13;\nimportance of positive charge on the affinity and selectivity for anion binding.&#13;\nKinetic studies reveal that simple vanadate in acetonitrile is a more efficient&#13;\nfunctional model than in water. The rate acceleration in acetonitrile is thought to be&#13;\noriginated from an enzyme-like hydrophobic environment for the catalytic species.&#13;\nUV and fluorescence titration shows that the supramolecular receptor 36 exhibits high&#13;\naffinity to the peroxovanadate as well, which verifies that vanadium core keeps being&#13;\nbounded in the catalytical cycle. A competitive catalytic bromination experiment was&#13;\ndesigned and successfully demonstrated the kinetic process for the catalytic&#13;\nbromination of 1,3,5-tri-methoxy-benzene (TMB) and monochlorodimedone (MCD)&#13;\nmixture substrates. The addition of 36 to the reaction system significantly enhances&#13;\nthe catalytic efficiency. The rate enhancement by 36 may be reasoned to the&#13;\nincreasing of Lewis acidity of vanadate by forming hydrogen bonds with positively&#13;\ncharged guanidiniums of 36. In addition, the central nitrogen atom of 36 may act as an&#13;\nacid base catalyst, facilitating the protonation of peroxovanadate. All together, 36 is&#13;\nan effective functional model for V-HPO based on the structural fidelity to the&#13;\nsupramolecular binding fashion of the enzyme.