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Bio-Inspired Trinuclear Zinc(II) Complexes as Models for Hydrolytic Metallo-Enzymes: Synthesis and Catalytic Activity in Phosphate Diester Transesterification

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Bio-Inspired Trinuclear Zinc(II) Complexes as Models for Hydrolytic Metallo-Enzymes: Synthesis and Catalytic Activity in Phosphate Diester Transesterification
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MITRA RANJAN ( Author, Primary )
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2008

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Hydrolysis ( jstor )
Hydroxides ( jstor )
Ions ( jstor )
Ligands ( jstor )
Metal ions ( jstor )
Molecules ( jstor )
pH ( jstor )
Phosphates ( jstor )
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Zinc ( jstor )

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University of Florida
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University of Florida
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Copyright Ranjan Mitra. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2017
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1 BIO-INSPIRED TRINUCLEAR ZINC(II) COMP LEXES AS MODELS FOR HYDROLYTIC METALLO-ENZYMES: SYNTHESIS AND CA TALYTIC ACTIVITY IN PHOSPHATE DIESTER TRANSESTERIFICATION By RANJAN MITRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 Copyright 2007 by Ranjan Mitra

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3 To my loving ma and baba

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4 ACKNOWLEDGMENTS First and foremost, I would like to express my sincere gratitude and appreciation to my advisor, Prof. Michael J. Scott for providing me the opportunity to work in his research group, for his expert guidance and ment orship, and for the encouragemen t and support at all levels. His strategic insight and enthusiasm made this research project both interesting and doable. I would also like to thank my other committee members, Prof. David E. Richardson, Prof. Daniel R. Tahlam, Prof. Thomas Lyons and Prof. Angela S. Lindner, for their constructive feedback and insightful comments and words of encouragement. I would also like to take this opportunity to thank Dr. Dave Powell and Dr. Ion Ghiviriga for their guidance and technical assistance. It is a privilege to ha ve such friendly and knowledgeable experts available to us. I am also thankful to Dr. Kathryn Williams for letting me use the potentiometric titrator and for he r help with titration experiments. During my time at the University of Florida, I had the pleasure to meet and work with an exceptional group of coworkers both in terms of th eir talent and their fr iendship. I would like to express my appreciation to th e entire Scott group, past a nd present for their thoughtful discussions, friendship and helping me mature as a chemist. In pa rticular, I would like to thank Dr. Matthew W. Peters for introducing me to my project early in my graduate career, Dr. Kornelia Matloka for being a great lab mate and Dr. Hubert Gill for his help in sharpening my presentation skills. A special note of thanks goes to Candace Zieleniuk, Eric Libra and Ozge Ozbek for proof reading my thesis more thoroug hly than I could have ever done myself. Many other excellent lab mates have crossed my path s over the years: Dr. Ivana Boidarevic, Cooper Dean, Melanie Chandler, Issac Finger, Eric Werner Dr. Ajay K. Shah and Dr. Priya Srinivasan. I would also like to thank Daniel Denevan for his friendship and help with the UV experiments.

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5 All of you have contributed in your own way to a wonderful warm working atmosphere that I will miss for a very long time. Finally, and most importantly, I would like to ex press my sincerest gratitude to my family, ma, baba, didi, jiju, mamon, mummi, papa, shammi and my wife. I consider myself extremely lucky to have such wonderful parents and sister whose innumerable sacrifi ces enabled me to be who I am today and where I am today. There ar e not enough words to describe all the thanks they deserve, but perhaps, three words come close: I love you! To thank my wife Rashmi seems strange to me since she is such an integral part of my existence. Her support, encouragement and companionship over the last ten years, first as a friend and then as a wife, has allowed me to grow as a person. She will always be my greatest love, my truest friend an d my closest companion.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 MULTI-ZINC SYSTEMS IN BIOLOGY AND THEIR SYNTHETIC MODEL COMPLEXES...................................................................................................................... ...14 1.1 Introduction............................................................................................................... ........14 1.2 Phosphate Ester Hydrolysis..............................................................................................14 1.2.1 Phospholipase C.....................................................................................................15 1.2.2 Nuclease P1............................................................................................................17 1.3 Dinuclear Zinc(I I) Complexes..........................................................................................19 1.4 Trinuclear Zinc(II) Complexes.........................................................................................23 2 SYNTHESIS OF TRINUCLEAR LIGAND SYSTEMS USING THE TRIPHENOXYMETHANE PLATFORM.............................................................................33 1.1 Ligand Design.............................................................................................................. .....33 2.2 Synthesis of the Trinuclear Ligand System......................................................................34 2.3 Conclusions................................................................................................................ .......38 2.4 Experimental Section....................................................................................................... .38 2.4.1 General Methods....................................................................................................38 2.4.2 Synthesis................................................................................................................ .39 2.4.3 X-ray Crystallography............................................................................................44 3 SYNTHESIS AND REACTIVITY OF C3-SYMMETRIC TRINUCLEAR ZINC(II) HYDROXIDE CATALYST EFFICIENT AT PHOSPHATE DIESTER TRANSESTERIFICATION...................................................................................................51 3.1 Introduction............................................................................................................... ........51 3.2 Synthesis and Characterization of Tris-Zinc(II) Complex...............................................52 3.3 Solution Equilibria........................................................................................................ ....55 3.3.1 Ligand Protonation Constants................................................................................56 3.3.2 Metal Complexation in Aqueous Solution.............................................................57 3.4 Kinetic Studies............................................................................................................ ......60 3.4.1 Transesterificati on of Hydroxypropylp -nitrophenyl Phosphate (HPNP)..............60 3.4.2 Hydrolysis of Diribonucleoside M onophosphate Diesters (NpN) by Dizinc Complexes....................................................................................................................62

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7 3.5 Mechanism of Catalysis....................................................................................................62 3.6 Conclusions................................................................................................................ .......65 3.7 Experimental Section....................................................................................................... .66 3.7.1 General Methods....................................................................................................66 3.7.2 Synthesis................................................................................................................ .66 3.7.3 Potentiometric Titrations........................................................................................68 3.7.4 Kinetic Measurements............................................................................................69 3.7.5 X-ray Crystallography............................................................................................70 4 SYNTHESIS OF TRINUCLEAR LI GAND SYSTEMS USING MODIFIED TRIPHENOXYMETHANE PLATFORM.............................................................................87 4.1 Introduction............................................................................................................... ........87 4.2 Synthesis of trinuclear ligand system...............................................................................88 4.3 Conclusions................................................................................................................ .......93 4.4 Experimental Section....................................................................................................... .93 4.4.1 General Methods....................................................................................................93 4.4.2 Synthesis................................................................................................................ .94 4.4.3. X-ray Crystallography...........................................................................................97 5 SUMMARY........................................................................................................................ ..103 LIST OF REFERENCES.............................................................................................................105 BIOGRAPHICAL SKETCH.......................................................................................................113

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8 LIST OF TABLES Table page 2-1 Crystal data and struct ure refinement for complex 2-5. ....................................................45 3-1 Selected interatomic distances () and angles () for 3-1 (Me)2CO................................71 3-2 Protonation constants of the ligand 2-5 at 25C; I = 0.1 M KCl........................................71 3-3 Stability constants of the Zn(II) complexes with 2-5 at 25C; I = 0.1 M KCl...................72 3-4 Rate constants for the transesterifica tion of HPNP catalyzed by Zn(II) complexes at 25C........................................................................................................................... .........72 3-5 Crystal data and structure refinement for 3-1. ...................................................................72

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9 LIST OF FIGURES Figure page 1-1 Structure of the active site of three trinuclear phosphatases..............................................26 1-2 Phospholipids catalyzed by PC-PLCBC..............................................................................26 1-3 Mode of action proposed for the hydrolysis of phospholipids in the active site of phospholipase C................................................................................................................ .27 1-4 Proposed mechanism of P1 nuclease.................................................................................27 1-5 Biomimetic zinc complexe s based on calix[4]-arenes.......................................................27 1-6 Biomimetic zinc complexes base d on spacer/compartment techniques............................28 1-7 More biomimetic zinc complexes based on spacer/compartment techniques...................29 1-8 Selected macrocyclic ligands and their zinc complexes....................................................30 1-9 More macrocyclic ligands and their zinc complexes.........................................................30 1-10 Postulated mechanism of phospha te hydrolysis promoted by complex 1-18 ....................30 1-11 Postulated mechanism of phospha te hydrolysis promoted by complex 1-19 ....................31 1-12 Trinuclear zinc complexes base d on spacer/compartment techniques..............................31 1-13 Synthesis of a structural model for the trizinc moiety of nuclease P1 that also features a bridging acetate ligand......................................................................................31 1-14 Schematic representation of po ssible mechanism for HPNP cleavage by 1-4 ..................32 1-15 Multiple stereocenters (*) in 1-4 ........................................................................................32 2-1 Reaction of L1 and L2 with ZnCl2......................................................................................46 2-2 Synthesis of ligand 2-5 .......................................................................................................46 2-3 The X-ray crystal structure of 2-5 ......................................................................................47 2-4 Synthesis of ligand 2-7 .......................................................................................................47 2-5 Synthesis of ligand 2-8 .......................................................................................................48 2-6 Attempted synthesis of trinuclear ligand system containing acyclic amines.....................48 2-7 Synthesis of a cryptand-like molecule by reacting 2-3 with 1,4,7-triazacyclononane......49

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10 2-8 Attempted synthesis of triphenoxymetha ne based tris tacn ligand by reacting 2-13 with 1,4,7-triazacyclo[5.2.1.0.4,10]decane..........................................................................49 2-9 Attempted synthesis of triphenoxymethane based tris tacn liga nd by reacting 2 with diprotected 1,4,7-triazacyclononane..................................................................................50 3-1 Depiction of the so lid-state structure of 3-1 ......................................................................73 3-2 Proton NMR spectra of 2-5 in CD3CN..............................................................................74 3-3 Proton NMR spectra of 3-1 in CD3CN..............................................................................75 3-4 Proton NMR spectra of 2-5 (7.5 mM) in MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O)..................................................................................................................76 3-5 Proton NMR spectra of 3-1 in 1:1 (CD3CN : D2O)...........................................................77 3-6 Proton NMR spectra of th e reaction mixture containing 3-1 (7.5 mM), MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O) after 30 min................................................78 3-7 Proton NMR spectra of th e reaction mixture containing 3-1 (7.5 mM), MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O) after 24h.....................................................79 3-8 Variation of the ratio of free ligand ( 2-5 ) to trinuclear zinc complex ( 3-1 ) as a function of pH................................................................................................................. ...80 3-9 Temperature-dependent 1H NMR spectra of the aromatic region of 3-1 ...........................81 3-10 Potentiometric titration curve of 3-2 with 0 ( ), 1 ( ), 2 ( ) or 3 (*) equiv. Zn(II) in 0.1 M KCl in 1:1 CH3CN/H2O at 25C..............................................................................82 3-11 Species distribution diagram for 3-2 (0.92 mM) in the presence of Zn(II) ions (2.76 mM) as a function of pH in 1:1 CH3CN/H2O at 25C with I = 0.10.................................82 3-12 pH versus rate profile for transester ification of HPNP (2 mM) catalyzed by 3-1 (5 mM) in acetonitrile/80 mM buffer 1:1 (v/v) at 25 C.........................................................83 3-13 Dependence of the observed pseudo-first-orde r rate constant on the concentration of trinuclear zinc(II) catalyst 3-1 at pH 6.6 in 50 % CH3CN/80 mM MES buffer (v/v) at 25 C. [HPNP] = 2 mM......................................................................................................83 3-14 Initial rate as a function of the substrate concentration for the transesterification of HPNP catalyzed by 5 mM of 3-1 in 50 % CH3CN/80 mM MES buffer (v/v) (pH 6.6) at 25 C.............................................................................................................................. .84 3-15 Stack plot of 31P-NMR for the transesterificati on of HPNP (38.56 mM) catalyzed by 3-1 (0.47 mM) at pH 6.7 afte r (b) 1 day (c) 3 days (d) 5 days (e) 8 days..........................84

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11 3-16 Hydrochloride salt of 2-5 ...................................................................................................85 3-17 Structure of ligands mentioned in Table 3-4......................................................................85 3-18 Stack plot of 31P-NMR for the transesterificati on of HPNP (38.56 mM) catalyzed by a mixture of 2-21 (1.41 mM) and Zn(ClO4)2H2O (1.41 mM)at pH 6.7 after (b) 1 day (c) 3 days (d) 5 days (e) 8 days...................................................................................86 3-19 Bronsted plot for the metal-hydroxide (including OH-) promoted hydrolysis of 4nitrophenyl acetate............................................................................................................ .86 4-1 Various trinuclear ligand systems......................................................................................98 4-2 Attempted detert -butylation of 4-2 ...................................................................................98 4-3 Synthesis of ligand 4-3 .......................................................................................................98 4-4 Attempted chloromethylation of 4-3 ..................................................................................99 4-5 Attempted bromomethylation of 4-3 .................................................................................99 4-6 Tris(2-alkoxy-5-nitrophenyl)met hanes reported in literature............................................99 4-7 Synthesis of ligand 4-8 .......................................................................................................99 4-8 Synthesis of ligand 4-9 .....................................................................................................100 4-9 Synthesis of ligand 4-10 ...................................................................................................100 4-10 Schiff base complexes used in literatu re capable of aminope ptidase-like activity..........100 4-11 Synthesis of ligand 4-12 ...................................................................................................101 4-12 Cartoon representation of 4-13 ........................................................................................101 4-13 Synthesis of ligand 4-14 ...................................................................................................101 4-14 Synthesis of ligand 4-16 ...................................................................................................102 4-15 Synthesis of ligand 4-17 ...................................................................................................102 4-16 Depiction of the so lid-state structure of 4-18 ..................................................................102

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIO-INSPIRED TRINUCLEAR ZINC(II) COMP LEXES AS MODELS FOR HYDROLYTIC METALLO-ENZYMES: SYNTHESIS AND CA TALYTIC ACTIVITY IN PHOSPHATE DIESTER TRANSESTERIFICATION By Ranjan Mitra May 2007 Chair: Michael J. Scott Major Department: Chemistry Phosphodiesterase enzymes, such as phospho lipase C and nuclease P1, use three Zn(II) ions in their active si te to catalyze the hydrolytic cleav age of phosphate diester bonds in phospholipids and in nucleotides su ch as single-stranded DNA/RNA, respectively. However, the function of the zinc triad is still not well understood. Functional mimics of phosphoesterases are important for understanding the role of metal ions in the hydrolytic mechanism and for developing artificial nucleases for biochemical and medicinal applications. Inspired by these trinuclear Zn(II) sites in enzymatic systems, a C3-symmetric trinuclear Zn(II) hydroxide complex was synthesized using a triphenoxymethane plat form. This complex induces a 16,900-fold rate enhancement in the catalytic cyclization of the RNA mode l substrate, 2-hydroxypropylp nitrophenyl phosphate (HPNP, pH 6.7, 25C) over the uncatalyzed reaction with multiple turnovers. The observed differences in the pH-rate profile can be attributed to the varying concentration of various trinucle ar zinc species. The trinuclear Zn(II) catalyst exhibits a higher hydrolytic activity compared to its mononuclear an alogue. The reactivity and structural features of this trinuclear Zn(II) complex will be discussed.

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13 Since subtle differences in the mutual arra ngement/orientation of the metal binding arms have a crucial influence on the intrinsic activity of the trinuclear Zn(II) complexes, development of a ligand system wherein the metal binding arms are directly attached to the aromatic ring was undertaken. Extensive exploration aimed at f unctionalization of the para-position of the triphenoxymethane platform led to the successful synthesis of two C3-symmetric triphenoxymethane based Schiff-base ligands. The synthesis of these ligands and their zinc complexes along with the structur al characterization of these co mplexes are presented herein.

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14 CHAPTER 1 MULTI-ZINC SYSTEMS IN BIOLOGY AND THEIR SYNTHETIC MODEL COMPLEXES 1.1 Introduction The phosphodiesters that form the structural backbone of nucleic acids are extremely resistant to hydrolytic cleavage. The estimated ha lf-life of RNA is 110 years and that of DNA is in the range of 10-100 billion years.1,2 As a consequence, nature has to use various enzymes to accelerate the hydrolysis and to enable the pr ocessing of nucleic acids under physiological conditions. Most phosphodiesterases c ontain two or even three divale nt metal ions close to each other, which work synergistically as a single unit.3-6 Phosphodiesterases are of prime importance in the manipulation of DNA and RNA because of th eir involvement in rep lication, transcription, recombination, and DNA repair.7,8 1.2 Phosphate Ester Hydrolysis Phosphohydrolases are responsible for the hydr olytic cleavage of phosphoryl groups, a task of essential importance for the control of metabolic processes in countless biological systems. Many of these enzymes employ metal ions as cofactor. Although different and specific mechanisms are probably involved for individual me tallohydrolases, the metal is believed to play some general roles, including the genera tion of a strong nucleoph ilic hydroxide group at physiological pH by lowering the pKa of water, th e activation and orienta tion of the substrate through metal coordination, as well as the stabi lization of intermedia tes and of the oxyanion leaving group.3,5 Zn(II) appears to be the me tal ion of choice for this purpose since it is a strong Lewis acid capable of undergoing rapid ligand exchange. Additionally, it has the capability to change coordination number and geometry ve ry easily and is free from any undesired redox activity.9,10 These metalloenzymes have a wide struct ural variance and often possess homoor heterodinuclear or even -trinuclear active sites.3-11 The well studied dinuclear zinc phosphatase

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15 includes phosphotriesterase12,13 and alkaline phosphatase.14,15 In addition to zinc, magnesium ions are often present as seen in inositol monophosphatase16,17 and fructose triphosphatase ,18 both of which contain a dinuclear Mg2+ Mg2+ core. Sometimes, nature combines a redox active metal ion (Fe3+ / Fe2+) with a redox inactive ion (Zn2+, Mg2+) in the active site; the best example of this class of enzymes is perhaps the purple acid phosphatase.3,11 Enzymes that incorporate three zinc cente rs are also known such as phospholipase C19-21 and nuclease P122,23 (Figure 1-1). However, the third zinc centers of phospholipase C and nuclease P1 are not directly associated with th e dizinc units. For example, while the ZnZn separation of the principal dizinc unit in phospho lipase C is 3.3 the distances between these zinc centers and the third zinc center are 4.7 and 6.0 .3 Likewise, the corresponding separations in nuclease P1 are 3.2, 4.7 and 5.8 .3 The three catalyti cally active metal ions roughly form a triangle. The chemical identity and arrangement of the ligands in the firs t coordination spheres of the metal ions are remarkably similar, espe cially for phospholipase C and nuclease P1, which differ by only a single ligand (Glu or Asp) on Zn2. As illustrated in Figure 1-1, alkaline phosphatase exhibits a structural similarity to phospholipase C and nuclease P1 in which the third zinc is replaced by magnesium. The magne sium center was originally proposed not to participate directly in the catalytic cycle, but more recent studies suggest that its role is to provide a magnesium hydroxide ligand that acts as a general base to deprotonate a serine residue for nucleophilic attack on the phosphorus atom.15 1.2.1 Phospholipase C The trinuclear zinc-based phospholipase C isolated from Bacillus cereus (PC-PLCBC) catalyzes the hydrolysis of the phosphodiester bo nd in phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup R (Figure 1-2).24-26 In mammals, DAG is an important secondary messenger molecule in the signal trans duction cascade.27 It has not been possible to

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16 isolate and characterize a mammalian PLC, and th ere is much interest in studying the bacterial system as a potential model.28 A mode of action consistent with all avai lable data has been proposed for the PC-PLCBC catalyzed hydrolysis of phospholipids (Figure 1-3)29. The phosphatidylcholine substrate binds at the enzyme active site, thus di splacing the bridging OH moiety and the Zn2-bound water to give an enzyme-substrate complex, which is stabilized by ionic attractions between the three zinc ions and the phosphodiester functionality.29 The solid-state structure of PLCBC bound to a phosphonate substrate analog30 reveals that there are no water molecules in the first coordination sphere of any of the zinc ions. It is thus believed that the primary ro le of the zinc triad is to bind and activate the substrate toward nucleophilic attack through charge neutraliz ation rather than to provide a zinc-bound nucleophile.29 The choline moiety binds in a head group pocket comprised of Glu4, Tyr56, and Phe66 (Figure 1-3).30,31 All three residues are dire ctly involved in substrate binding,32,33 and their specific mutation considerab ly changes the substrate specificity.31 Hydrolysis most probably commences when a prot on is abstracted from an active-site water molecule by a general base.29 Residues that have been postula ted to fulfill this function are Glu4,34 Glu146,30 and Asp55.29 Selected mutagenesis has ruled out Glu433 and Glu14635 and suggests that Asp55 is the general base.33,36 Proton transfer is the rate-limiting step in the catalytic circle.37 The hydroxide thus generated, attack s the zinc-bound phosphor via an inline, associative mechanism that runs over a trigonal bipyram idal transition structure,38 although stereochemical investigations confirming th is are not yet availa ble. Collapse of the pentacoordinate transition struct ure leads directly to two pro ducts, phosphorylcholine (PC) and diacylglycerol (DAG).29

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17 The identity of the general acid n eeded to protonate DAG is still unknown.29 Molecular modeling studies have proposed that Asp55 fulfills this role.38 However, experimental evidence overwhelmingly implies that Asp55 is the general base,33,36 and kinetic data conclusively rule out the possibility that this single residue functi ons as both a general acid and a general base.29 No other residues in the general vicinity are like ly candidates, and the po ssibility that a zinc-bound water serves in this capacity must be considered.29 Product release follows a two-step kinetic mechanism, neither of which is ratedetermining.29 Considering the fact that DAG is a mode rately strong compe titive inhibitor and PC an extremely weak, noncompetitive inhibitor of PC-PLCBC, it is to be expected that PC will be released first.29 This corresponds to the order predic ted by molecular modeling calculations.39 1.2.2 Nuclease P1 Nuclease P1 is a diesterase th at catalyzes the hydrolysis of single-stranded DNA and RNA. The enzyme is a phosphodiesterase cleaving the bond between the 3 hydroxyl and 5 phosphoryl group of adjacent nucleotides, and at the same time acts as a phosphomonoesterase, removing the 3 terminal phosphate group. The final cl eavage products of P1 are solely 5 mononucleotide.3,4,11,40-47 Based on various crystallographic results, seve ral possible reaction mechanisms have been proposed. All of these involve nucleophilic attack by a zinc-ac tivated water molecule and stabilization of the pentacovalent transition st ate by Arg48, but the mechanisms assign different roles to the three zinc ions and the firmly bound water molecules seen in the P1 crystal structure.23 In one mechanism, the more exposed Zn2 activates a nearby water molecule, possibly assisted by an aspartate residue (Asp153) while the dinuclear, co catalytic zinc pair (Zn1 and Zn3)48 plays a more passive role.23 In a second mechanism, the water bridging the buried Zn1 and Zn3 ions is activated and act s as the attacking nucleophile, while Zn2 is

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18 activating and stabilizing the O3 -leaving group.49 In a third mechanism, analogous to the twometal ion mechanism proposed for the 3',5'-exonuclease activity of E. coli DNA polymerase I,50,51 it is assumed that one of the non-bridging p hosphate oxygens binds between Zn1 and Zn3, replacing the bridging water molecule. Romier et al.22 proposed a three-metal-ion mechanism to explain the action of nuclease P1. In this scheme, the scissile phosphate of the substrate sitting between the three zinc ions binds close to Zn2, with its free oxygens replacing two water molecules. The water molecule bridging Zn1 and Zn3 is presumably present as a hydroxide ion due to the lowering of its pKa by the metal ions.3,4 The bridging hydroxide ion acts as the nucleophile attacking the phosphate in-line with the P-O3' bond. Asp45, which also serves as a ligand of Zn1, helps to properly orient the hydro xide for attack. The resulting penta-coordinate transition state is stabilized by Arg48, the att acking hydroxide ion and the leaving O3' occupy apical positions. Zn2 plays a crucial role in act ivating the phosphate and stabilizing the leaving O3'-oxyanion (Figure 1-4). Despite extensive investigations, details of the mechanism of nuclease P1 and other metallohydrolases remain controversial. One crucia l aspect under debate is the identity and the exact binding mode of the nucleophile. Also, the function of a third metal ion in close proximity of a dinuclear metal cluster in these enzymes is yet not fu lly understood. Insight into the structures and mechanisms of action of en zymes is often obtained by studying synthetic analogues, i.e. small molecules that resemble the structural and f unctional sites of the enzymes.52,53 Such studies are important because s ynthetic analogues are more amenable to structural, spectroscopic, and mechanistic studies than are the enzymes themselves. Furthermore, synthetic analogues are also more amenable to fi ne-tuning by systematic substituent effects than are the active sites of their enzy me counterparts, so that it is possible to examine a variety of

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19 factors that influence reactivity. A diverse assortment of ligands have been employed to model various aspects of multinuclear zinc enzymes.2,8,40-46 By using a variety of spacers, two or more simple ligands, each capable of binding one zinc ion, can be linked t ogether by groups ranging from simple aliphatic chains to more complex groups such as calix[n]arenes.54-69 Other approaches have focused on the use of large macr ocycles that encompass two or more metal ions into a single framework.70-78 1.3 Dinuclear Zinc(II) Complexes Calix[4]arenes that are functionalized with Zn(II)-complex 1-1 at the distal positions of the upper rim (Figure 1-5) are capable of catalyt ically cleaving phospha te diesters (an RNA model).44 Extensive kinetic studies have shown that under neutral pH conditions, the dinuclear complex 1-3 increased the rate of transesterif ication of the RNA model substrate 2hydroxypropyl-p-nitrophenyl phosphate (HPNP) by a factor of 23,000 as compared to the uncatalyzed reaction.54,55 The mononuclear catalyst 1-2 is less active than the dinuclear one54,55 by factor of 50 which is attributed to the high degree of cooperation between the two zinc centers. It has been found that a certain confor mational flexibility is essential; attaching the calix-[4]arene together at the lower rim, thus making it rigid, inhibited its catalytic activity.44 The calix[4]-arene is more than just a molecular scaffold since its presence causes a 6-fold increase in the rate as compared to the simple zinc complex 1-1 .54,55 This has been attributed to a lowering in the pKa of a zinc bound water molecule due to the hydrophobic aroma tic surface of the calix[4]arene unit.44 Modification of the model by introdu cing two amino groups on the upper rim of the calix[4]arene (thus making a general base directly available to assist in the deprotonation step) did not improve its performance, which could be due to steric overloading in the vicinity of the active site.56

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20 In an attempt to overcome th e flexibility problem inherent in the spacer strategy, many researchers are employing spacers with a built in donor heteroatom intended to bridge the two metal ions, thus fixing them 3-5 apart from each other. Recently, Richard and Morrow et al. performed extensive kinetic studies on complex 1-5 (Figure 1-6) which catalyzes the hydrolysis of an activated phosphate diester.57 This complex is also capable of hydrolyzing a chemical model for the 5cap of mRNA.58 The dinuclear 1-5 shows enhanced cooperative rate of reaction as compared to the mononuclear 1-6 (Figure 1-6), which can be attr ibuted to a greater ease of formation of an essential zinc-bound hydroxide (pKa 7.2 for 1-5 and 9.2 for 1-6 ).57 The alcohol linker has a very low pKa and is coordinated as an alkoxide to the two zinc ions. In the mononuclear 1-6, the alcohol remains protonated and a zi nc-bound water is deprotonated at pKa 9.2. Although the compartment ligand is symm etric, the solid-state structure of 1-6 demonstrates that the zinc ions have differe nt coordination numbers and geomet ries. It is believed that a -OH bridge is not formed. Although a solid-state structure of th e zinc complex formed from ligand 1-7 is not yet available, a kinetic study has de monstrated that a dinuclear Zn2L unit is responsible for the hydrolytic activity of this ligand.59 In the presence of Zn2+, 1-7 efficiently hydrolyzes uridine 2,3-cyclic monophosphate (a RNA model).59 Komiyama and coworkers, usi ng a related dinuclear complex 1-8 (Figure 1-6) with pyridine arms, studied the cleavage of ribonucleotide di mers NpN by intramolecular transesterification. In contrast to the mononuclear complex, the dinuclear complex efficiently cleaves ApA, for which the authors propose a mechanism comprising Lewis acid activation of the phosphoryl group, the nucleophilic 2A-OH, and the leaving group.60

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21 A completely different tactic based on a sp acer approach developed by Scrimin et al. employs artificial heptapeptides, which have been modified to carry two zinc-complexing pendant ligands.61 An example (species 1-9 ) is illustrated in Figure 1-7. These peptides possess a stable helical conformation (exactly two turns) in solution. The helical twist positions the pendant ligands close enough together so that th ey can cooperatively hydrolyze RNA and even DNA model substrates.62,63 If three of these, zinc-pendantbe aring peptides are linked with each other via a tris(2-aminoethyl)amine platform, an allosteric supramolecular catalyst (not illustrated) is obtained that readily catalyze s the transphosphorylati on of phosphate esters.64 Attempts to synthesize dinuclear zinc com pounds containing a phosphate bridge have been successful with a naphth yridine-based ligand BPAN65,66 (Figure 1-7). This ligand organizes a water and a phosphate molecule to form a phosphate-bridged dinuc lear zinc species 1-10 (Figure 1-7) containing a -hydroxide-bridge with a p K a of 6.8.65,66 This seems to be the first functional phosphatebridged biomimetic complex reported in the literature since it effectively promotes the hydrolysis of phosphodiester, thus mimicking the enzymes nuclease P1.65,66 Kinetic investigations on the hydrolysis of bis( pnitrophenyl)phosphate indicate that the bridging hydroxide in 1-10 acts as the active species, func tioning as a general base to deprotonate a water molecule.65,66 A pthalazine-based ligand (B DPTZ) is also capable of building a discrete bimetallic complex 1-11 with -hydroxide bridges (Figure 1-7).67 If the spacer is a pyrazolate functio nality, a series of linker ligands 1-12 (Figure 1-7) can be employed to obtain discrete bizinc complexes.68 Depending on the length of the linker, either a -OH unit or a bridging H3O2 species can be isolated. The longer linkers 1-12a / c prefer a -OH bridge, whereas the shorter ligand sidearms in 1-12b/d pull the two zinc ions apart, thus inducing incorporation of an additional solven t (water or methanol) to give a H3O2 unit. Extensive

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22 comparative kinetic and spectro scopic studies on the series 1-12 has led to the realization that a -OH unit is a relatively poor nucleophile and either a terminal ZnOH unit or a H3O2 bridge is much more reactive. In 1994, Bencini, Bianchi, and Paoletti reported the synthesis of a m acrocyclic ligand that organizes two Zn2+ ions and two water molecules to form a Zn2( -OH)2 cluster 1-13 (Figure 18).70,71 Unfortunately, this complex is relatively inert, and although 1-13 readily complexes with simple substrates such as 2-hydroxypyridine or cytosine,70,71 no hydrolysis activity was observed. Kimura et al. synthesized a very similar complex 1-14 (eight instead of se ven nitrogen atoms and NH instead of NMe) which prove d capable of cleaving an ac tivated monophosphate ester; however, the hydrolysis product remained stuck to the complex.9 Bencini et al. then turned to a simpler macrocycle 1-15 (Figure 1-8) which is a successful biomimetic complex for the mononucl ear zinc enzyme carbonic anhydrase 72,73 and expanded the ring to make room for two zinc ions (ligand 1-16 ).74 This ligand readily builds a stable dinuclear zinc complex 1-17 (Figure 1-9) with a -hydroxide linkage in aqueous solution.74 Measurement of the p K a of the bound water in 1-17 yielded a value of 7.6.75 In more basic solutions (pH > 8) a second water is bound and deprotonated (complex 1-18 ).75 The bihydroxy species 1-18 (Figure 1-9) promotes the hydrolysis of bis( p -nitrophenyl) phosphate (BNP)75 as well as adenylyl(3'-5')adenosine (ApA).76 Kinetic studies have shown that the hydrolysis occurs via a bimolecular mechanism and that the monohydroxy species is inactive.75 In particular, complex 1-18 is 10 times more active that the mononuclear zinc complex based on ligand 1-15 It has been proposed that the two zinc ions work cooperatively to activate the phosphate diester through a bridging interacti on with both electrophilic Zn2+ centers, which in turn favors the nucleophi lic attack of the Zn-OH func tion at the phosphorus (Figure 1-

PAGE 23

23 10). In support of this mechanism, Bencini et al. succeeded in obtaining a crystal structure of diphenyl phosphate complexed with 1-18 in which the phosphate unit doe s indeed bridge the two metal ions.75 In an attempt to analyze the effect of di fferent coordination environments of the Zn ions, the macrocycle 1-16 was enlarged by introducing additional heteroatoms.77 However, the ability of the resulting dinuclear zinc co mplexes to hydrolyze BNP decreased and the p K a of the active bihydroxy species increased.77 Additionally Bencini et al. have synthe sized a monohydroxide complex by adding an alcohol-pendant to the macrocycle (complex 1-19 in Figure 1-9).78 This compound contains both a Zn-bound alkoxide and a Zn-OH nucleophilic func tion and is thus a biomimetic model for alkaline phosphatases where bot h a deprotonated serine and a Zn-OH function are involved in phosphate ester hydrolysis. Th is complex hydrolyzes both pnitrophenyl acetate (NA) and BNP at an even faster rate than that found for 1-18 .78 NMR tracking experiments identified species 120 (Figure 1-11) as an intermediate step to ward generating the final hydrolysis product 1-22 .78 Compound 1-22 is extremely stable and exceedingly iner t. Release of the hydrolyzed phosphate, thus enabling a catalytic pro cess, could not be realized.78 1.4 Trinuclear Zinc(II) Complexes The difficulty in preorganizing three zinc ions into an environment conducive to intramolecular interaction, which can mimic the active site of trinuclear zinc enzymes, is evident from the scarcity of reported trinuclear zinc complexes. Only a few of these, obtained by introducing a third zinc(II) center to the already known dinuclear zinc (II) systems, have recently been reported.54,79-81 By replacing the O linkage in 1-8 (Figure 1-6) with an am ine linkage, Komiyama and coworkers obtained the trinuclear zinc complex, 1-23 (Figure 1-12). This species is capable of hydrolyzing a whole series of ribonucleotide dimers at pH 7 and 50C.79 The activity of 1-23

PAGE 24

24 shows a considerable dependence on the structure of the substrat e. However, no explanation is given for the reactivity CpA> ApA> GpA> UpA ApG> GpC. Since 1-23 is only 10 times more reactive than 1-8 it is not clear if the greater reactivity is due to the difference in the number of metal ions in the complex or due to the difference in the stru cture of the ligand. In may be more appropriate to compare the reactivity to its dinuclear analog, which has a methyl group, but such a model has not yet b een reported in the literature. A related trinuclear zinc compound wa s recently reported by Bencini et al.80 wherein three bis(2-pyridyl methyl)amine in 1-23 was replaced by three macrocyclic moieties namely 1,4,7,10tetrazacyclododecane ([12]aneN4, 1-24a ) and 1,5,9,12-tetraz acyclotetradecane ([14]aneN4, 1-24b ), Figure 1-12. These trinuclear complexes cleave bis(p-nitrophenyl) phosphate (BNPP) through a bridging in teraction of the substrate with at least two zinc ions and simultaneous nucleophilic attack of a Zn-OH fu nction at phosphorus. In addition, a significant increase of the hydrolysis rate with respect to the mononuclear zinc(II) complex, of [12]ane N4 has been observed.80 A structural model for the trizinc moiety of nuclease P1 that also features a bridging acetate ligand has been obtained by the reacti on of macrocyclic hexaamino triphenolate ligand with a mixture of Zn(OAc)2 and Zn(ClO4)2 as illustrated in Figure 1-13. This complex is capable of cleaving calf thymus DNA.81 Reinhoudt and coworkers enla rged the dinuclear zinc(II) complex 1-3 with the introduction of an additional metal binding arm. Subsequently they obtained the trinuclear zinc(II) complex 1-4 (Figure 1-5), that efficiently catalyzes the cleavage of RNA dinucleotides (3, 5-NpN ) by the cooperative action of th e three zinc(II) centers, with high rate enhancement and significant nucleobas e specificity. The dinuclear complex and the mononuclear reference complex, on the othe r hand, showed only minor activity. The

PAGE 25

25 transesterification of the RNA model substrate (HPNP) by the trinuclear complex induces a rate acceleration of 32,000 times as compared to 23, 000 times for the dinuclear analogue over the uncatalyzed reaction. This trinuclear complex e xhibits a lower substrate binding affinity but a higher catalytic rate compared to its dinuclear analogue. A possible mechanism in which two zinc(II) ions activate the phosphoryl group and the third activates the -hydroxyl group of HPNP was proposed. (Figure 1-14)54 Unfortunately, these calix[4]aren e-based zinc(II) complexes have not yet been structurally characterized. From the analysis of the ligand, pr esence of three distinct stereo centers (*) is evident (Figure 1-15). Therefore, potentially th ere will be up to eight stereoisomers in the reaction mixture after incorporation of three metal centers. Catalysis is dependent on the stereochemistry of the catalyst, and, in this case, it is impossi ble to determine which of the stereoisomers are actually responsible for catal yzing the hydrolysis of the phosphate ester bonds. Perfect synthetic analogues that mimic all as pects of trinuclear zinc enzymes, i.e., structure, function and mechanism, are yet to be obtained. Many of the structural models are completely inactive for catalysis, while in function al model it is frequently difficult to discern the identity of the competent catalysts since the molecu les are often prepared in situ. Thus, to have a better understanding of the catalytic mechanism, it could be advantageous to combine both structural and functional information into a singl e system. With these issues in mind, a simple ligand capable of preorganizing three meta l ions into an environment conducive to intramolecular interaction was designed to give us valuable insight into the chemical processes taking place in the heart of the enzymatic active sites. In the following chapters, the tethering of th ree tridentate amine ligands to Dinger and Scotts triphenoxymethane platform82 will be shown to result in a ligand system (detailed in

PAGE 26

26 chapter 2) with these properties that will then be used to synthesize biomimetic models for enzymes containing three zinc ions (phospholipase C / nuclease P1). Zn1His OH2 O O His Zn2Ser102O His Mg O O Asp Asp H2O Glu Thr H2O H2O Zn1His Zn3Zn2 Glu H2O O H His Asp O O Asp N O His Trp OH2 His His Zn1His Zn3Zn2 Asp H2O O H His Asp O O Asp N O His Trp OH2 His His alkalinephosphatasephospholipaseCnucleaseP1 Figure 1-1. Structure of the active site of thr ee trinuclear phosphatases. Figure adapted from Weston, J. Chem. Rev. 2004 104 699-767. H3C(H2C)n O O O H3C(H2C)n O O P O OR OR=-CH2CH2-NMe3+ -CH2CH2-NH3+ -CH2CH-NH3+CO2 -phosphatidylcholine phosphatidylethanolamine phosphatidylserine PC-PLCBC Figure 1-2. Phospholipids catalyzed by PC-PLCBC. Figure adapted from Weston, J. Chem. Rev. 2004 104 699-767.

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27 Zn3Zn1 Zn2 O H OH2 free enzyme substrate H2O Zn3Zn1 Zn2 O P O NMe3 O O O O + HO O O H Ph Tyr56 Phe66 Glu4 head group pocket Zn3Zn1 Zn2 O P O OR(choline) OR(DAG) O H H O O Asp55 general acid PC enzyme DAG complex DAG free enzyme Figure 1-3. Mode of action proposed for the hydrol ysis of phospholipids in the active site of phospholipase C. Figure adapted from Weston, J. Chem. Rev. 2004 104 699-767. Zn3O Zn1 O O H Asp45 NH NH2 Arg48 Zn2OH2 H2O O O RO P O -O O OR O Zn3O Zn1 O O H Asp45 NH2 Arg48 Zn2O O O P O O RO RO 5'5 '3 '3' O NH Figure 1-4. Proposed mechanism of P1 nuclease. Figure adapted from Bauer-Siebenlist, B. et. al. Inorg. Chem. 2004 43 4189-4202. OR" OR" OR" R R' OR" N N N N N N Zn Zn 1-1 1-2 : R' = R = H 1-3 : R' = H; R = 1-4 : R' = R = R" = CH2CH2OEt N N N Zn Figure 1-5. Biomimetic zinc complexes base d on calix[4]-arenes. Figure adapted from Molenveld, P. et. al. Chem. Soc. Rev. 2000 29 75-86.

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28 N N O N N N N Zn Zn N N O N N N N Zn Zn H H H H Cl OH2 N O N N Zn H H H OH OH2 HN NH OH N NH N HN 1-5 1-6 1-7 1-8 Figure 1-6. Biomimetic zinc complexes ba sed on spacer/compartment techniques. 1-5 & 1-6 are adapted from Iranzo, O. et. al. J. Am. Chem. Soc. 2003 125 1988-1993. 1-7 is adapted from Gajda, T. et.al. Eur. J. Inorg. Chem. 2000 1635-1644. 1-8 is adapted from Yashiro, M. et. al. J. Chem. Soc. Chem. Commun. 1995 1763-1794.

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29 N N NH HN N N O Zn O Zn O P Ph Ph N N N N N N H NN N N N N N N Zn Zn O H O H N N H2O OH2 N N N N NR2 R2N NR 2: N NMe2 NMe2 N NEt2 NEt2 N N N i Pr i Pr N NMe2 Me O H Zn Zn OH O H H Zn Zn BPAN 1-10 BDPTZ 1-11 1-121-12a1-12b 1-12c1-12d -OH bridge H3O2 unit N H H N N H H N N H H N N H O O O O O O O O N N N H H Zn N N N H H Zn 1-9 Figure 1-7. More biomimetic zinc complexe s based on spacer/compartment techniques. 1-9 is adapted from Rossi, P. et. al. J. Am. Chem. Soc. 1999 121 6948-6949. 1-10 is adapted from Kaminskaia, N. V. et. al. Inorg. Chem. 2000 39 3365-3373. 1-11 is adapted from Barrios, A. M. et. al. Inorg. Chem. 2001 40 1060-1064. 1-12 is adapted from Bauer-Siebenlist, B. et. al. Inorg. Chem. 2004 43 4189-4202.

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30 OO HN N H NH NN N N NN N N Zn Zn O O H NN N N NN N O Zn Zn H O O H 1-131-141-15 H H H H Figure 1-8. Selected macrocyclic lig ands and their zinc complexes. 1-13 is adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1995 34 3003-3010. 1-14 is adapted from Kimura, E. et. al. Adv. Inorg. Chem. 1997 44 229-261. 1-15 is adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1996 35 5540-5548. O O HN N H NH O O NH H N HN O O NH NH NH O O NH NH NH Zn Zn O O NH NH NH O O NH NH NH Zn Zn OH OH OH O O NH N NH O O NH NH NH Zn Zn OH O 1-16 1-17 1-18 1-19 pKa = 7.6 pKa > 8 Figure 1-9. More macrocyclic ligan ds and their zinc complexes. 1-16 is adapted Bazzicalupi, C. et. al. Inorg. Chem. 1995 34 5622-5631. 1-17 and 1-18 are adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1997 36 2784-2790. 1-19 is adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1999 38 4115-4122. O P Zn O Zn PhO OPh OH HO Figure 1-10. Postulated mechanism of phosphate hydrolysis promoted by complex 1-18 Figure is adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1997 36 2784-2790.

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31 ZnZn OH O P OR OR -O -O ZnZn OH O P OR -O OZnZn OH O P O OO1-201-211-22 Figure 1-11. Postulated mechanism of phosphate hydrolysis promoted by complex 1-19 Figure is adapted from Bazzicalupi, C. et. al. Inorg. Chem. 1999 38 4115-4122. X N X X NH NH NH N OO O O Zn Cl Zn a b 1-24 N N N Zn X:X:1-23 Figure 1-12. Trinuclear zinc complexes based on spacer/compartment techniques. 1-23 is adapted from Yashiro, M. et. al. Chem. Commun. 1997 83-84. 1-24 is adapted from Bazzicalupi, C. et. al. Dalton. Trans. 2003 3574-3580. NH NH NH NH OH OH NH NH OH NH NH NH NH O O NH NH O ZnZn Zn O O Zn(OAc)2Zn(ClO4)2 Figure 1-13. Synthesis of a structural model for the trizinc moiety of nuclease P1 that also features a bridging acetate ligand. Figur e is adapted from Korupoju, S. R. et. al. Inorg. Chem. 2002 41 4099-4101.

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32 N N N Zn N N N Zn N N N Zn NO2 O P O O O O H HO Figure 1-14. Schematic representation of possible mechanism for HPNP cleavage by 1-4 Figure is adapted from Molenveld, P. J. et. al. J. Org. Chem. 1999 64 3896-3906. OR" OR" OR" OR" R"=CH2CH2OEt N N N Zn N N N Zn N N N Zn * Figure 1-15. Multiple stereocenters (*) in 1-4

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33 CHAPTER 2 SYNTHESIS OF TRINUCLEAR LIGAND SYSTEMS USING THE TRIPHENOXYMETHANE PLATFORM 1.1 Ligand Design Ligands play an important role in modulati ng the ability of the complexed Zn(II) ion to promote the hydrolytic cleavage of phosphate dies ters. The construction of synthetic analogues requires the judicious choice of lig ands and considerable attenti on must be given to the ligand design in order to achieve a coordination environm ent which is similar to that enforced by the unique topology of proteins. The re sidues which bind zinc in protei ns are typically a combination of His(N), Glu(O), Asp(O) or Cys(S), which provide nitrogen, oxygen and sulfur donors, with His being the most commonly encountered.3,11 Histidine coordinates via its imidazole substituent as a neutral donor, while glutamate and aspartat e coordinate via thei r deprotonated anionic carboxylate substituent. A large fraction of all multinuclear biomimetic complexes contain ligands such as amines, pyrid ines and alkoxides rather th an ligands used by nature.40,41 The two structurally relate d trinuclear phosphatases (phospholiase C and nuclease P1) possess an ancillary Zn(II) ion in clos e proximity to a dizinc active site.40,41 All the zincs are pentacoordinate, and each adopts an approximate trigonal-bipyramidal geometry. Each zinc is attached to two nitrogen donors and three oxygen donors. The two zinc ions of the dizinc unit are bridged by the side chain carboxyl group of an aspartate residue and by a -OH linkage.40 The inorganic chemistry of three pre-organized zinc io ns has not been examined in detail because of the lack of an appropriate ligand system capable of orienting three metal ions in close proximity for cooperative activity. Thus synthesis of a C3symmetric ligand system will provide much insight into the little understood r eactivity of three pre-organized zi nc centers and might prove to be a good model for the trinuclear zinc enzymes. With this in mind, a simple ligand capable of preorganizing three metal ions in to an environment conducive to intramolecular interaction was

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34 designed using the C3-symmetric triphenoxymethane as a platform.82 Synthesized by tethering three phenol moieties to a centr al methine carbon, these compounds not only possess all of the properties of calix[4]arenes that make them ideal scaffolds for ligand systems but also possess a useful three-fold symmetry. The ligand was designe d to have an ethyl spa cer between each of the three phenolic groups and a leavi ng group to give the molecule the right balance of rigidity and flexibility needed for efficient binding and hydrolysis of the s ubstrate molecule. This simple synthon can then be treated with a variety of different nitrogen ligand sets to afford many different trinucleating ligands. Rivas et al. found that the reaction of N-methyl piperazine based ligands L1,2 with ZnCl2 affords neutral [(L1,2)ZnCl2] complexes (L1, 2-1 ; L2, 2-2 ) in which the zinc(II) center is tetrahedrally ligated.83 Interestingly, the zinc(II) ion prefers tetrahedral N2Cl2 ligation over trigonal bipyramidal N2Cl2O and N3Cl2 (or N2OCl2) coordination environments (Figure 2-1), despite the fact that chelate e ffects and the possibility of coor dinating an additional group to the zinc(II) center could have provi ded further stability. Encourag ed by this observation, 1-[(2pyridyl)methyl]piperazine, 2-4 was attached to the triphenoxym ethane platform to afford a C3symmetric trinuclear ligand system. Synthesis of various other ligands with acyclic and cyclic amines were attempted, few of which proved to be synthetically inaccessible for a variety of reasons detailed below. The detail s of these failed reactions al ong with the successful synthesis of two new trinuclear ligand systems will be detailed in this chapter. 2.2 Synthesis of the Trinuclear Ligand System The derivatization of the triphenoxymethan e platform is primarily focused on the substitution at its phenolic oxygen. Additions at these oxygens usually involve deprotonation of the phenol with an appropriate base followed by co mbination of the resulting salt with activated reactants such as ethylbromoacetate. The reduction of the resulting ester with lithium aluminium

PAGE 35

35 hydride and further tosylation with p -toluenesulfonyl chloride affo rded the activated platform, 23 .84 The reaction of 2-3 with thirty equivalents of 2-4 resulted in the formation of a trinuclear ligand system, 2-5 (Figure 2-2). The identity of the alkyl group, at the ortho position of the phenol base, was critical in determining the out come of the substitution of the tosylated group with the nucleophilic nitrogen of the amines. Pete rs found that the optimized synthesis of the trinuclear ligand system requires the ortho -methyl platform84. The failure of these substitution reactions to occur with the ditert -butyl platform compounds can be attributed to th e steric clash between the ortho tert -butyl groups (relative to the phenol oxygens) on the platform and the incoming nucleophile. Additionally, solubility of the compounds in polar solvents (e.g. acetonitrile) required for these subs titution reactions also plays an important part in determining the outcome of these reactions. The large hydrophobic platform portion of these compounds dominates their physical properties such as solu bility and polarity, th ereby complicating the purification of these compounds. Fo r this reason, reactions with th ese systems were developed to avoid the very difficult task of separating partia lly substituted platforms from the fully reacted species. All derivatizatio n reactions considered in this thesis were optimized for complete reaction with all three arms of the platform Incomplete reactivity of all three arms disqualified a reaction from furt her consideration. NMR spectra l data and the X-ray crystal structure of 2-5 (Figure 2-3) illustrates that the all up conformation, in which all the phenol oxygens point in the same direction as the cent ral methine hydrogen of the platform, exclusively exists both in solution and in solid state. By maintaining this conf ormation, the three metal binding sites are now preorganized and poised fo r modeling biological tri nuclear active sites. Following the same synthetic strategy another ligand 2-7 containing three N-methyl piperazine groups 2-6 was successfully synthesized (Figure 24). To determine whether there is any

PAGE 36

36 cooperativity between the Zn(II) cen ters with respect to substr ate binding and conversion, the one arm analog of the trinuclear system 2-5 was synthesized, following the method described above for the derivatization of the triphenoxymethane platform (Figure 2-5). A caveat to the optimized conditions of this method arises from the requirement of a ten-fold excess of amine which is usually expensive or difficult to synthe size, but since most of the unreacted amine can be recovered after the reaction, hundreds of milligrams of pr oduct can be obtained within a few weeks starting from phenol and the desired amine. The intrinsic reactivity of the different comple xes is controlled by the nature and structure of the ligand. For example, complexes of triden tate ligands, particularly if characterized by a facial coordination mode, are more reactive than those of tetradentate ligands which can hardly allow binding sites for the substrat e. Thus to have a better unders tanding of the role of ligand in modulating the properties of the metal ion, synthesi s of various ligands with acyclic and cyclic amines were attempted. Reaction of the activated platform 2-3 with thirty equivalents of bis(2-(pyridyl)ethyl)amine 2-13 (Figure 2-6) resulted in the formation of in tractable product mixture. Similar observation has earlier been reported by Peters when attemp ting to make the same trinuclear system by alkylating a primary amine attached to the tri phenoxymethane platform with 2-vinylpyridine.84 An intramolecularly alkylated product was formed before all three arms of the platform became uniformly substituted. Replacing, 2-13 with N,N,N',N'-tetraethyldiethylenetriamine, 2-14 in the above reaction mixture resulted in an insepara ble mixture of mono-, di -, and tri-substituted products (Figure 2-6). The simplest and the most studied example of a cyclic polyamine with a propensity to form facial coordination to metal ions is 1,4,7-triaza cyclononane (tacn, 2-15 ). The coordination

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37 chemistry of this molecule has been comprehens ively investigated, especially by Wieghardt and coworkers.85 The thermodynamic stability of the metal complexes formed by tacn exceeds those of related cyclic triamines due to the favorable entropy effect s arising from the endodentate conformation of the cyclic liga nd limiting rearrangement upon c oordination. Peters found that on reacting the activated platform 2-3 with a huge excess of tacn, in the presence of Na2CO3, a cryptand-like host molecule comp lexed with one equivalent of p -toluenesulfonic acid was obtained.84 However, on using a stronger base (Cs2CO3) and only two equivalents of tacn, the same cryptand-like molecule without th e complexed p-tolu enesulfonic acid, 2-16 was obtained (Figure 2-7). This compound ma y find important application as a proton abstractor, although such work was not undertaken in this thesis. Mono functionalization of the nitrogen atoms of tacn by conventional methods is synthetically more di fficult than tris-functionalization because of the difficulty of separating the diffe rent products formed. A clean r oute to the monosubstitution of tacn was developed by Weisman et. al.86 through the react ion of orthoamide 1,4,7-triazacyclo [5.2.1.04,10]decane, 2-9 with the appropriate alkyl halide.86,87 To exploit this synthetic route for the generation of triphenoxymethan e based tris-tacn ligand, the alc ohol moieties atta ched to the triphenoxymethane platform, 2-17 were first converted into th e corresponding bromo-derivative, 2-18 using triphenoxyphosphine and N-bromosuccinimide in dimethylformamide (Figure 2-8). The reaction of 2-18 with a stoichiometric amount of 2-19 under the same conditions as reported in the literature, resulted in the formation of intractable product mixtur es. The addition of an excess of 2-19 to the reaction mixture had no effect on the outcome, and a similar product mixture was obtained. Another method for accessing the desired triphe noxymethane based tris-tacn ligand system involves reaction of the activated platform 2-3 with diprotected tacn. N,N-bis( tert -

PAGE 38

38 butoxycarbonyl)-1,4,7-triazacyclononane (Boc2-tacn, 2-20 ) was initially used since the Boc protecting group can be easily removed under mild conditions. However, 2-3 did not react with Boc2-tacn under a multitude of substitution conditions resulting in the rete ntion of the starting materials (Figure 2-9). In a sepa rate reaction, reacting N,N-bis( p -tolylsulfonyl),4,7triazacyclononane, 2-21 with 2-3 resulted in the incomplete substitution of the tosyl groups on all the three arms of th e platform (Figure 2-9). In spite of the failure to synthesize the desired tripenoxym ethane based tris-tacn ligand, these failed synthetic pathways uncovered ma ny unique properties of the triphenoxymethane platform, which could be exploited fo r designing future ligand systems. 2.3 Conclusions Extensive exploration of the reac tivity of the activated platform 2-3 led to the successful synthesis of a C3-symmetric trinuclear ligand system, capab le of preorganizing three metal ions into an environment conducive to intramolecular interaction. Synthesis an d hydrolytic activity of a C3-symmetric trinuclear zinc(II) hydroxide ca talyst, prepared usi ng this ligand, will be described in the following chapter. The effect of cooperative action betw een the zinc(II) centers will be demonstrated by comparison of the cataly tic activity of the trinuclear complex with the mononuclear complex. 2.4 Experimental Section 2.4.1 General Methods All reagents and solven ts were of analytical grade a nd were used without purification, unless otherwise noted. All 1H and 13C spectra were recorded on a Varian VXR-300 or Mercury300 spectrometer at 299.95 and 75.4 MHz for the pr oton and carbon channels, respectively. Elemental analyses were performed by the Univ ersity of Florida Spectroscopic services. 1methylpiperazine, 2-6 N,N,N',N'-tetraethyldiethylenetriamine, 2-14 and 4-tert-butyl-2-

PAGE 39

39 methylphenol were obtained from commercial so urces and was used as received. Tris(2-(2toluenesulfonylethoxy)-3-methyl -5-tert-butylphenol)methane, 2-3 ,84 1-((2pyridl)methyl)piperazine, 2-4 ,88 bis(2-pyridylethyl)amine, 2-13 ,89 1,4,7-triazacyclononane, 215 ,90 tris(2-(2-hydroxylethoxy)-3-methyl-5 -tert-butylphenyl)methane, 2-17 ,84 1,4,7triazacyclo[5.2.1.04,10]decane, 2-19 ,91 N,N'-bis( tert -butoxycarbonyl)-1,4,7-triazacyclononane, 22092, N,N'-bis( p -tolylsulfonyl)-1,4,7-triazacyclononane, 2-21 ,90 were synthesized following literature methods. 2.4.2 Synthesis Preparation of tris(2-(2-(4 -((2-pyridyl)methyl)pipera zine)-1-ethoxy)-3-methyl-5tert butylphenyl)methane (2-5). To 0.74 g (0.61 mmol) of 2-3 dissolved in 50 mL dry acetonitrile was added 3.2 g (18.2 mmol) of 1-((2-pyridyl)methyl)piperazine, 2-4 and 0.64 g (6.1 mmol) of Na2CO3, and the mixture was refluxed for five days under an inert atmosphere. The solvent was then removed under vacuum and the remaining material dissolved in diethyl ether. The organic layer was extracted with 0.1 M NaOH (2 X 100 mL), then dried with Na2SO4, filtered, and the solvent removed to afford 0.62 g (91 %) of th e brownish yellow amorphous solid. The excess 1((2-pyridyl)methyl)piperazine was recovered from the aqueous phase by extraction with chloroform and removing the solvent under v acuum. Slow diffusion of pentanes into a concentrated tetrahydrofuran solution of ligand at -30 C afforded crystals suitable for X-ray analysis. 1H NMR (CD3CN): = 1.12 (s, 27H; Ar-C(C H3)3), 2.21 (s, 9H; Ar-C H3), 2.39 (b, 24H, N-C H2C H2-N), 2.57 (t, 3J (H,H) = 6.3 Hz, 6H; Ar-O-CH2C H2N), 3.52 (t, 3J (H,H) = 6.3 Hz, 6H; Ar-O-C H2), 3.56 (s, 6H; Py-C H2), 6.52 (s, 1H; C H ), 6.78 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.07 (d, 4J (H,H) = 2.1 Hz, 3H; ArH ), 7.17 (ddd, 3J (H,H) = 7.7 Hz, 4J (H,H) = 5.0 Hz, 5J (H,H) = 1.2 Hz, 3H; Py), 7.40 (m, 3H; Py), 7.68 (dt, 3J (H,H) = 7.7 Hz, 4J (H,H) = 2.0 Hz, 3H; Py), 8.47 (ddd,

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40 3J (H,H) = 5.4 Hz, 4J (H,H) = 1.7 Hz, 5J (H,H) = 0.9 Hz, 3H; Py). 13C NMR (CD3CN): = 17.1 (ArC H3), 31.8 (Ar-C( C H3)3), 34.8 (ArC (CH3)3), 38.8 ( C -H), 54.2 (Ar-O-CH2CH2-NC H2), 54.6 ( C H2-N-CH2-Py), 58.7 (Ar-O-CH2C H2), 65.2 (NC H2-Py), 71.0 (Ar-OC H2), 123.0 (Py), 123.8 (Py), 126.5 (Ar), 127.0 (Ar), 131.2 (Ar), 137.3 (Ar), 137.5 (Py), 146.4 (Ar), 149.9 (Py), 154.2 (Ar), 160.0 (Py). Anal. Calcd. for 2-5 H2O (C70H101N9O8): C, 73.20; H, 8.86; N, 10.98. Found: C, 73.14; H, 9.16; N, 10.72. ESI FT-ICR MS m/z = 1112.78 [M+H]+. Preparation of tris(2-(2-(4-methylpiperazine)-1-ethoxy)-3-methyl-5tert -butyl phenyl)methane (2-7). To 0.5 g (0.46 mmol) of 2-3 dissolved in 50 ml of dry acetonitrile was added 1.37 g (13.67 mmol) of 1-methyl piperazine, 2-6 and 0.48 g (4.56 mmol) of Na2CO3 and the mixture was refluxed for 5 days under an in ert atmosphere. The solvent was then removed under vacuum and the remaining material dissol ved in diethyl ether. The organic layer was extracted with 0.1 M NaOH (2 X 100 ml), then dried with Na2SO4, filtered, and the solvent removed to afford 0.35 g (88%) of the pale ye llow solid. The excess 1-methylpiperazine was recovered from the aqueous phase by extraction with chloroform and re moving the solvent under vacuum. 1H NMR (CDCl3): = 1.12 (s, 27H; Ar-C(C H3)3), 2.21 (s, 9H; Ar-C H3), 2.25 (s, 9H; NC H3), 2.39 (b, 24H; N-C H2C H2-N), 2.58 (t, 3J (H,H) = 6.6 Hz, 6H; Ar-O-CH2C H2-N), 3.41 (t, 3J (H,H) = 6.6 Hz, 6H; Ar-O-C H2), 6.59 (s, 1H; C H ), 6.82 (d, 4J (H,H) = 2.1 Hz, 3H; ArH ), 6.93 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ). 13C NMR (CDCl3): = 17.1 (ArC H3), 31.6 (Ar-C( C H3)3), 34.3 (ArC (CH3)3), 37.6 ( C -H), 46.2 (NC H3), 53.5 (Ar-O-CH2CH2-NC H2), 55.2 ( C H2-N-CH3), 57.8 (Ar-O-CH2C H2), 69.7 (Ar-OC H2), 125.9, 126.0, 130.0, 136.6, 145.5, 153.4 (Ar). Anal. Calcd. for 2-7 H2O (C55H90N9O4): C, 73.45; H, 10.09; N, 9.34. Found: C, 73.81; H, 10.38; N, 9.17. Preparation of (4tert -butyl-2-methyl-phenoxy)-acetic acid ethyl ester ( 2-9). Following a previously published method for the prep aration of tris(3,5-di-tert-butyl-2-

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41 ethoxycarbonylmethoxyphenyl)methane, in a Schle nk flask under argon 1.0 g (6.1 mmol) of 2methyl-4-tert-butylphenol, 2-8 was dissolved in dry acetone and 1.22 g (7.3 mmol) of ethylbromoacetate and 7.94 g (24.37 mmol) of Cs2CO3 were added. The mixture was refluxed for 12-15 hours and then cooled to room temper ature. The acetone was removed under vacuum and the solids dissolved in diethyl ether. Solid MgSO4 was added and the insoluble salts and drying agent were filtered off. The ether was rem oved from the filterate to give 1.3 g (87%) of the product as a colorless oil, which was pure enough for further modification. 1H NMR (CD3CN): = 1.3 (s, 9H, Ar-C(C H3)3), 1.31 (t, 3J (H,H) = 7.2 Hz, 3H, CO2CH2C H3), 2.31 (s, 3H, Ar-C H3), 4.27 (q, 3J (H,H) = 7.2 Hz, 2H, CO2C H2CH3), 4.62 (s, 2H, Ar-O-C H2CO2Et), 6.65 (d, 3J (H,H) = 8.7 Hz, 1H, ArH ), 7.14 (dd, 3J (H,H) = 8.6 Hz, 4J (H,H) = 2.4 Hz, 1H, ArH ), 7.19 (d, 4J (H,H) = 2.6 Hz, 1H, ArH ). Anal. Calcd. for 2-9 (C15H22O3): C, 71.97; H, 8.86. Found: C, 71.85; H, 8.71. Preparation of 2-(4-tert-butyl -2-methyl-phenoxy)ethanol (2-10) A dry diethylether solution of 2-9 (2 g, 7.99 mmol) was added dropwise ove r 30 min with an addition funnel to a slurry of LiAlH4 (0.61 g, 15.98 mmol) in 100 ml dry diethyl ether cooled to 0C. The mixture was then warmed to room temperature and st irred for 12-15 hours. The excess reductant was destroyed with 1 M HCl (100 ml). The ether laye r was separated and further extracted with 1 M HCl (2 X 100 ml) and brine (100 ml). The ether was then dried with MgSO4. After filtration of the drying agent, the ether was removed to give 1.5 g (90%) of colorless oil. 1H NMR (CD3CN): = 1.4 (s, 9H, Ar-C(C H3)3), 2.39 (s, 3H, Ar-C H3), 3.9 (t, 3J (H,H) = 5.9 Hz, 2H, ArOC H2), 4.3 (t, 3J (H,H) = 5.9 Hz, 2H, ArOCH2C H2), 6.84 (d, 3J (H,H) = 8.3 Hz, 1H, ArH ), 7.28 (dd, 3J (H,H) = 8.4 Hz, 4J (H,H) = 2.5 Hz, 1H, ArH ), 7.32 (d, 4J (H,H) = 2.2 Hz, 1H, ArH ). 13C NMR (CD3CN): = 16.6 (ArC H3), 31.7 (Ar-C( C H3)3), 34.2 (ArC (CH3)3), 42.3 (Ar-OC H2CH2), 68.4 (Ar-O-

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42 CH2C H2), 111.2, 123.4, 126.7, 128.3, 143.9, 154.3 (Ar). Anal. Calcd. for 2-10 (C13H20O2): C, 74.96; H, 9.68. Found: C, 74.49; H, 9.38. Preparation of toluene-4-sulfonic acid-2-( 4-tert-butyl-2-methyl-phenoxy)-ethyl ester. ( 2-11) In a dry flask, 1 g (4.8 mmol) of 2-10 was dissolved in 70 ml of dry pyridine and cooled to 0C in an ice bath. A 1.4 g (7.3 mmol) portion of p -toluenesulfonylchloride was added and the reaction mixture was stirred for 2 hours at 0C and then for 12-15 hours at room temperature. The pyridine was removed under vacuum and the s ticky material dissolved in 100 ml methylene chloride and then extracted with 1 M-HCl (2 X 100 ml). The organic phase was then dried with MgSO4, filtered and the solvent removed under redu ced pressure. The residue was purified by silica gel column (ether/pentane, 9/1) to give the product as colorless oil (1.4 g, 80%).1H NMR (CD3CN): = 1.3 (s, 9H, Ar-C(C H3)3), 2.13 (s, 3H, Ar-C H3), 2.45 (s, 3H, SO2Ar-C H3), 4.14 (m, 2H, ArOC H2), 4.37 (m, 2H, ArOCH2C H2), 6.65 (d, 3J (H,H) = 8.4 Hz, 1H, ArH ), 7.13 (m, 2H, ArH ), 7.34 (d, 3J (H,H) = 8.5 Hz, 2H, SO2ArH ), 7.83 (d, 3J (H,H) = 8.5 Hz, 2H, SO2ArH ).13C NMR (CD3CN): = 16.5 (ArC H3), 21.8 (SO2ArC H3), 31.7 (Ar-C( C H3)3), 34.1 (ArC (CH3)3), 65.7 (Ar-OC H2CH2), 68.6 (Ar-O-CH2C H2), 110.9, 123.4, 126.5, 128.1, 128.2, 130, 133.1, 143.9, 145.1, 154.1 (Ar). Anal. Calcd. for 2-11 (C20H26SO4): C, 66.27; H, 7.23. Found: C, 66.70; H, 7.37. Preparation of 1-(2-(4-t ert-butyl-2-methylphenoxy)ethy l)-4-(pyridine-2-ylmethyl) piperazine. (2-12) To 1 g (2.82 mmol) of 2-11 dissolved in 50 ml dry acetonitrile was added 5 g (28.2 mmol) of 1-((2-pyridyl)methyl)piperazine, 2-4 and 1.2 g (11.3 mmol) of Na2CO3. The resulting mixture was refluxed for five days und er an inert atmosphere. The solvent was then removed under vacuum and the remaining material dissolved in diethyl ether. The organic layer was extracted with 0.1 M NaOH (2 X 100 ml), and then dried with Na2SO4, filtered, and the

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43 solvent removed to afford 0.9 g (90%) of brownish yellow oil. The excess 1-((2pyridyl)methyl)piperazine was recovered from th e aqueous phase by extraction with chloroform and removing the solvent under vacuum. 1H NMR (CDCl3): = 1.28 (s, 9H; Ar-C(C H3)3), 2.20 (s, 3H; Ar-C H3), 2.58 (b, 4H; N-C H2CH2-N), 2.70 (b, 4H; N-CH2C H2-N), 2.86 (t, 3J (H,H) = 6.0 Hz, 2H; Ar-O-CH2C H2-N), 3.67 (s, 2H; Py-C H2), 4.10 (t, 3J (H,H) = 5.7 Hz; 2H; Ar-O-C H2), 6.72 (d, 4J (H,H) = 8.4 Hz, 1H; ArH ), 7.1-7.2 (m, 3H, ArH PyH ), 7.40 (d, 4J (H,H) = 7.8 Hz, 1H; PyH ), 7.64 (dt, 3J (H,H) = 7.5 Hz, 4J (H,H) = 1.8 Hz, 1H, PyH ), 8.55 (d, 3J (H,H) = 4.8 Hz; 1H; PyH ). 13C NMR (CDCl3): = 16.7 (ArC H3), 31.7 (Ar-C( C H3)3), 34.1 (ArC (CH3)3), 53.3 (Ar-O-CH2CH2-NC H2), 53.6 ( C H2-N-CH2-Py), 57.4 (Ar-O-CH2C H2), 64.6 (NC H2-Py), 66.3 (Ar-OC H2), 110.6 (Ar), 122.2 (Py), 123.3 (Ar), 123.5 (Py), 126.1 (Ar), 128.0 (Ar), 136.5 (Py), 143.2 (Ar), 149.4 (Py), 154.7 (Ar), 158.4 (Py). Anal. Calcd. for 2-12 H2O (C23H34N3O1.5): C, 73.37; H, 9.10; N, 11.16. Found: C, 73.49; H, 9.07; N, 11.16. Preparation of 2-16. A solution of 2-3 (1 g, 0.91 mmol) in 50 ml of dry acetonitrile was treated with Cs2CO3 (1.19 g, 3.65 mmol) followed by 1,4,7-triazacyclononane, 2-15 (0.35 g, 2.73 mmol). The resulting mixture was refluxed overn ight. The solvent was then removed under vacuum and the residue dissolved in diethylether and filtered. Removal of the ether from the filtrate afforded 0.62 g (95%) of 2-16 as a white solid. 1H NMR (CDCl3): = 1.21 (s, 27H; ArC(C H3)3), 2.22 (s, 9H; Ar-C H3), 2.74 (b, 12H; N-CH2C H2-N), 3.11 (t, 3J (H,H) = 5.1 Hz, 6H; ArO-CH2C H2-N), 3.90 (t, 3J (H,H) = 5.1 Hz, 6H; Ar-O-C H2), 6.97 (d, 4J (H,H) = 2.7 Hz, 3H; ArH ), 7.11 (s, 1H; C H ), 7.12 (d, 4J (H,H) = 2.7 Hz, 3H; ArH ). Preparation of tris(2-(2-bromoethoxy)-3-methyl-5tert -butyl phenyl)methane (2-18). A solution of tris (2-(2-hydroxyethoxy )-3-methyl-5-tert-butylphenyl)methane, 2-17 (1 g, 1.58 mmol) in 20 ml of dimethylformamide was tr eated with triphenyl phosphine (5.25 g, 20 mmol)

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44 followed by N-bromosuccinamide (3.56 g, 20 mmol) The resulting reaction mixture was stirred at 25C for 12 h during which a white solid precipi tated from the solution. The solid was filtered and washed with cold dimethylformamide to afford 0.91 g (70%) of 2-18 as a white powder. 1H NMR (CDCl3): = 1.19 (s, 27H; Ar-C(C H3)3), 2.29 (s, 9H; Ar-C H3), 3.48-3.52 (m, 6H; Ar-OCH2C H2-Br), 3.59-3.63 (m, 6H; Ar-O-C H2), 6.67 (s, 1H; C H ), 6.96 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.02 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ). 13C NMR (CDCl3): 17.1 (ArC H3), 30.1 (ArC( C H3)3), 31.6 (ArC (CH3)3), 34.4 (Ar-O-CH2C H2-Br), 36.9 ( C -H), 71.8 (Ar-OC H2), 125.7, 126.4, 130.4, 136.1, 146.3, 152.4 (Ar). Anal. Calcd. for 2-13 (C40H55Br3O3): C, 58.34; H, 6.73. Found: C, 58.18; H, 6.89. 2.4.3 X-ray Crystallography Unit cell dimensions and intensity data for all the structures were collected by Prof. Michael J. Scott on a Siemens CCD SMART diff ractometer at 173 K. The data collections nominally covered a hemisphere of reciprocal sp ace, by a combination of th ree sets of exposures; each set had a different angle for the crystal, and each exposure covered 0.3 in The crystal to detector distance was 5.0 cm. The data sets were corrected for absorption using SADABS.14 All the structures were solved by Prof. Michael J. Scott using the Bruker SHELXTL software package for the PC, using the direct methods option of SHELXS. The space groups for the structures were determined from an examina tion of the systematic absences in the data, and the successful solution and refinement of th e structures confirmed these assignments. All hydrogen atoms were assigned idealized locatio ns and were given a thermal parameter equivalent to 1.2 to 1.5 times the thermal parameter of the atom to which it was attached. For the methyl groups, where the location of hydrogen atoms was uncertain, the AFIX 137 card was used to allow the hydrogen atoms to rotate to the maximum area of residu al density, while fixing their geometry. Structural and refinement data for 2-5 is presented in Table. 2-1.

PAGE 45

45 Table 2-1. Crystal data and structure refinement for complex 2-5. 2-5C4H8O Formulaa C74H105N9O4 fw, g/mola 1184.67 Space group R 3c a, 16.7988(5) b, 16.7988(5) c, 102.377(4) 90 90 120 V, 3 25020.1(15) Z 12 Radiation, b 0.71073 mm-1 0.059 R1 a 0.0805 wR2 b 0.2202 a R 1 = (|| Fo| | Fc||) / | Fo|. b wR 2 = [ [ w ( Fo 2 Fc 2)2] / [ w ( Fo 2)2]]1/2, w = 1/[ 2( Fo 2) + [( ap )2 + bp ], where p = [ Fo 2 + 2 Fc 2]/3

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46 N N N N R N Zn N N N H tBu O Cl Cl N Zn N N N R H Cl Cl N Zn N N N Cl Cl H R ZnCl2+ R=COtBu(L1) R=H(L2) R=COtBu( 2-1 ) R=H( 2-2 ) Figure 2-1. Reaction of L1 and L2 with ZnCl2. Figure adapted from Rivas, J. C. M. et. al. Dalton Trans. 2003 3339-3349. O OTs H O N H N N N N NH 33 10Na2CO3,CH3CN Reflux5days 2-32-5 2-4 30 Figure 2-2. Synthesis of ligand 2-5

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47 Figure 2-3.The X-ray crystal structure of 2-5 drawn with 30% ellipsoid s (carbons with arbitrary radii). Hydrogen atoms omitted for clarity. O OTs H O N H N N HN 33 10Na2CO3,CH3CN Reflux5days 2-32-7 2-6 30 Figure 2-4. Synthesis of ligand 2-7

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48 O OTs O N N N N N HN OH O O O OH (i) (ii) (iii) (iv) O 2-82-92-10 2-11 2-12 2-4 Figure 2-5. Synthesis of ligand 2-8 Reagents and conditions: (i) 1.2 BrCH2COOEt, 4 Cs2CO3, acetone, reflux (ii) 2 LiAlH4, Et2O (iii) 1.5 p-CH3C6H4SO2Cl, pyridine (iv) 4 Na2CO3, acetonitrile, 5 days reflux. O OTs H 3 H N N N H N N N 10 Base, CH3CN Reflux 5 days 2-3 2-13 2-14 Figure 2-6. Attempted synthesis of trinuclear ligand system containing acyclic amines.

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49 O OTs H 3 N N N H H H 3 N N N H O O O tBu tBu tBu 4 Cs2CO3, CH3CN Reflux 4 days 2-3 2-15 2-16 Figure 2-7. Synthesis of a crypt and-like molecule by reacting 2-3 with 1,4,7-triazacyclononane. O OH H 3 PPh3NBS O Br H 3 N N N 2-172-18 2-19 Figure 2-8. Attempted synthesis of triphenoxym ethane based tris t acn ligand by reacting 2-13 with 1,4,7-triazacyclo[5.2.1.0.4,10]decane.

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50 O OTs H 3 N N N H N N N S S H O O O O O O O O 10 Base, CH3CN Reflux 5 days 2-3 2-20 2-21 Figure 2-9. Attempted synthesis of triphenoxymethane based tris tacn ligand by reacting 2 with diprotected 1,4,7-triazacyclononane.

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51 CHAPTER 3 SYNTHESIS AND REACTIVITY OF C3-SYMMETRIC TRINUCLEAR ZINC(II) HYDROXIDE CATALYST EFFICIENT AT PHOSPHATE DIESTER TRANSESTERIFICATION 3.1 Introduction Experimental mechanistic studies on zinc en zymes are often hindered by the fact that Zn(II) is spectroscopically silent. The diamagnetic d10 Zn(II) center of the native enzymes offers little in terms of a spectroscopic probe, with neither electronic, ESR, nor 67Zn NMR spectroscopies being useful.93 Although, some information about the structure of active site and nature of intermediates have been acquired by re placing zinc ions in thes e sites with divalent metal ions which have useful spectro scopic probes, such as Co(II) (UV-Vis)94 and Cd(II) (NMR). The extent to which these non-native meta l ions distort the active site and interfere with the function of these enzymes has not been ac curately determined. Thus, parallel to the investigations on enzymatic systems themselves, th e synthesis and mechanis tic studies of smaller inorganic metal complexes (biomimetic systems) pr omises invaluable insight into the chemical processes taking place in the heart of the enzyma tic active sites. Interest in such biomimetic systems is not only restricted to the elucidation of the enzymatic mechanism, but may eventually lead to beneficial applicati ons in biotechnology and medicine.8,95 The chemical properties of the metal cente rs depend on the ligation properties of the chelating sites. Therefore, an appropriate design of the metal binding unit may lead to polynuclear metal complexes with different reac tivity and catalytic properties. Synthetic trinuclear Zn(II) complexes used in biomimetic studies are rare because of the lack of ligand systems capable of orienting three metal ions for cooperative activity.54,79,81,96-99 The trinuclear ligand system 2-5 was thus designed to mimic the protein ligand environment in the trinuclear center of phospholipase C and nuclease P1. The corresponding Zn(II) complex has been

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52 synthesized and exhibits a very hi gh catalytic activity in the transesterification of the RNA model substrate 2-hydroxypropylp -nitrophenyl phosphate (HPNP).100 Determining the basis for this reactivity will add to the paucity of knowledge about th e role of metal ions in the hydrolytic mechanism. The preparation of this complex a nd its catalytic activity in the cleavage of the phosphate diester bond of HPNP will be detailed below. 3.2 Synthesis and Characterizati on of Tris-Zinc(II) Complex The addition of three equivalents of Zn(ClO4)2H2O to a solution of 2-5 in acetone afforded the tris-zinc(II) complex 3-1 (Figure 3-1). Since Zn(II) is diamagnetic, the incorporation of Zn(II) into the trinuclear ligand was followed by 1H NMR spectroscopy using d3-acetonitrile solution of the complex. Three major features in these spectra are used to monitor the incorporation of the metal ions into the ligand. The resonance for the central methane hydrogen of the triphenoxymethane platform appears as a singlet usually be tween 6 and 7 ppm, and, because this proton points up into the substitu ents attached to the phenol oxygens of the platform, its position varies with changes in the number and type of these extensions. Additionally, if or when a sample contains plat forms with varying types or numbers of these extensions, multiple resonances, each one corres ponding to a different molecule, will appear in this region of the 1H NMR spectrum. The symmetric distribution of the derivatized arms on the platform can easily be determined by the number and relative size of the two J4-coupled doublet resonances between 7 and 8 ppm associated with the two platfo rm aromatic hydrogens and the two single resonances associated with ortho -methyl (~ 2 ppm) and para tert -butyl (~ 1 ppm) groups located on each phenol ring on the platform. The 1H NMR spectra of this triphenoxymethane platform substituted with the same moiety on each phenol oxygen will contain exactly two double t peaks for these two aromatic hydr ogens and two singlet peaks for these two alkyl groups. In the spec trum of an asymmetrically subs tituted platform, two sets of

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53 each of these peaks will appear and integrate 2 to 1 if only one arm differs from the other two, while three equivalently weighed but shifted se ts of these peaks will appear if each phenol substituent is unique. The third 1H NMR spectral feature applies to the metal complex of 2-5 The coordination of zinc by the ligand arms fo rms new cyclic structures that result in the splitting and shifting of various peaks, especially those associated with the 1-[(2pyridyl)methyl]piperazine moiety. A fluctuation in the linker moieties connecting these groups to the triphenoxymethane platform and within the coordination sphe re of the metal causes these split peaks to appear as broadened ones in the spectrum. The 1H NMR spectra of 2-5 (Figure 3-2) and its tris-zinc(II) complex, 3-1 (Figure 3-3) in d3-acetonitrile demonstrates these spectral features. Crystals of 3-1 were grown by diffusing pentane into an acetone solution of the complex at 0C. The solid-state structure of 3-1 as determined by X-ray crys tallography, is shown in Figure 3-1 and selected bond lengths and angles are given in Table 3-1. In the solid state, the molecule possesses rigorous C3-symmetry with a distance of 3.43 between the zinc centers. The three zinc cations are assembled into a distorted six-membered ring connected together by three bridging hydroxyl groups, and the angle about the zinc and hydroxides differ significantly [O1Zn1-O1" 109.4(2) and Zn1-O1-Zn1' 127.6(2)]. The metal hydroxide distances are typical for a Zn3(OH)3 core and differ slightly [1.925(4) Zn1-O1; 1.903(4) Zn1-O1"].101-103 The zinc nitrogen separations are asymmetric with bond length of 2.055(5) to the pyridyl nitrogen and 2.143(5) to the piperazine nitrogen. The meta l maintains a highly di storted tetrahedral geometry with bond angles ranging from 81.0(2) [N1-Zn1-N2] to 127.28(19) [N2-Zn1-O1]. Three perchlorate anions balance the charge on the complex.

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54 Similar coordination geometry ha s been observed in only a few N -methylpiperazine and morpholine based ligands.83 Although, the Zn3(OH)3 structural core has been obtained previously from selfassembly reactions with small liga nds, to the best of our knowledge, hydrolytic cleavage of phosphate diester bonds using these complexes has not yet been reported.101-103 Combined with the information obtained from th e solution state NMR spectrum, this structure demonstrates that the three Zn(II) sites in 3-1 are indeed pre-organized and poised for modeling trinuclear Zn(II) hydrolases. Moreover, in 3-1 the metal coordination sphere is not fulfilled by the ligand donors and the Zn(II) i ons may thus be used for s ubstrate binding and activation. 1H NMR spectroscopy suggests th at on the NMR time scale a C3-symmetric core is maintained in solution, under the conditions (pH 6.7, 50% (v/v) CD3CN/D2O, buffer, [ 3-1 ] = 7.5 mM) used for the kinetic experiments described below. Howeve r the species distributio n diagram, Figure 3-11 ( vide infra ), indicates the presence of various trinuclear zinc species in solution at and around pH 6.7. This suggests that the proton exchange reaction between the va rious species is sufficiently fast in 50% (v/v) CD3CN/D2O on the NMR time scale and thus some sort of hydroxide bridged C3-symmetric species was observed.104 All solutions remained cl ear during the time of the kinetic experiments and no peak corresponding to the free ligand was observed. After prolonged periods at pH 6.7, however, peaks correspond ing to the free liga nd do appear in the 1H NMR spectrum indicating that the metals can be slowly leached out of the ligand. After about 24 hours in the absence of HPNP, the system reached chemical equilibrium with the buffer solution containing complex 3-1 and free ligand 2-5 in a ~ 6:1 ratio, but under the catalytic condition, the core of 3-1 is stable with very little free Zn(I I) or free ligand in solution (Figures 3-4 3-5 3-6 and 3-7). Figure 3-8 shows the variat ion of the ratio of free ligand ( 2-5 ) to trinuclear zinc complex ( 3-1 ) as a function of pH. The change in the ligand concentration was followed by

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55 measuring the peak area at 8.44 ppm correspondi ng to a pyridyl proton of the free ligand. The peak area was normalized relative to the peak area of the same proton, at 8.62 ppm, in the trinuclear zinc complex 3-1 A gradual decrease in the ratio of free ligand ( 2-5 ) to trinuclear zinc complex ( 3-1 ) was observed as the pH was raised from pH 5.87 to pH 6.7. On further increasing the pH to 7.01 there is however a slight increase in the ratio. The depletion is most probably due to the formation of Zn(II)-MES buffer complex105 because in the absence of buffer the 50% (v/v) CD3CN/D2O solution of 3-1 is stable for weeks (Figure 3-5). At higher pH, the increase in the ratio may also be due to the instability of the complex at higher pH owing to the formation of insoluble zinc hydroxide. Variable temperature 1H NMR studies were carried out on compound 3-1 in 50% (v/v) CD3CN/D2O at pH 6.7 to see if the co mpound was undergoing any fluxional process in solution. Figure 3-9 show s the temperature dependence of the 1H NMR spectrum of 31 in the aromatic region (peaks corresponding to th e aliphatic region is not shown as they are masked due to the presence of large excess of ME S buffer). There is only a slight sharpening of the peaks as the temperature was increased from 10C to 55C indicati ng that there is no significant fluxional process in 3-1 on the NMR time scale. Th e compound precipitates out of solution below 10C thus restricting the experiment within a span of 45C. 3.3 Solution Equilibria While the X-ray crystallographic results pr ovide structural insi ghts for the various complexes, knowledge of the species distribution in solutions is crucial for understanding any trends in hydrolytic reactivity of artificial metallohydrolases. To investigate the relationship between the pH dependences of th ese reactions and the structures of the a quo-trizinc complexes in solution, the p Ka values of the ligand, 2-5 (Table 3-2), as well as the stability constants of its zinc complexes and the p Ka values of zinc bound water molecules in these complexes (Table 33), were determined by pH titrations.

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56 3.3.1 Ligand Protonation Constants The protonation constants of the ligand was determined by potentiometric titration of 3-2 (1 mM) with 0.1 M KOH, I = 0.1 M (KCl) at 25C. Studies were carried out in 50% (v/v) CH3CN/H2O mixtures due to low water solubility of the ligand and the metal complex in pure water. The pH titration curve is shown in Figur e 3-10. Although the ligand has as many as nine protonation sites (three per side arm), only six measurable deprot onation processes were found in the pH range 2.5 to 11.5. The nitrogen atoms on th e pyridyl groups are less basic than those of the aliphatic amines because the former has more s character than the latter.106 It has also been observed that as the separation between the proton ation sites becomes smalle r or as the charge on the ligand increases, the successive protonation of the less basic nitrogens becomes progressively more difficult till it reaches a level where proton ation is barely accessible in dilute aqueous solutions of low ionic strength.107 If only three protons attach ed to the pyridyl groups are released on dissolving in a 50% (v/v) CH3CN/H2O solution of low ionic strength (0.1 M), the initial hydrogen ion concen tration will be three times that of ligand concentration and hence the theoretical initial pH of the so lution can be computed as follows108: pH = -log [(3 X mmol of ligand)/total volume(ml)] = -log [(3 X 0.046)/50] = 2.56. This value is, in fact very close to the observed value of 2.53. The first three p Kas are thus below 2.5, and were not considered in the equilibrium model for data analysis. In the follo wing three steps, one proton is removed from each arm, and corresponds to the proton attached to the nitrogen of the piperazinium ion closer to the pyridyl group. The final three deprotonations again correspond to th e dissociation of one proton per sidearm, but this time the protons involv ed are attached to the other nitrogen of the piperazinium ion. The titration data were anal yzed for equilibria 3-1 to 3-6. The protonation constants K1-K6 are defined as follows:

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57 H+ + L HL+ K1= [HL+]/([H+][L]) H+ + HL+ H2L2+ K2= [H2L2+]/([H+][HL+]) H+ + H2L2+ H3L3+ K3= [H3L3+]/([H+][H2L2+]) H+ + H3L3+ H4L4+ K4= [H4L4+]/([H+][H3L3+]) H+ + H4L4+ H5L5+ K5= [H5L5+]/([H+][H4L4+]) H+ + H5L5+ H6L6+ K6= [H6L6+]/([H+][H5L5+]) The values of the protonation constants obtai ned are in agreement with the values of various similar amines in literature.109 While comparing the values, how ever, it should be kept in mind that hydrogen bonding with pyr idine nitrogen and various ot her interactions, such as substituent effects where by tertiary amines gain electron density by ali phatic substituents and suffer from a withdrawal of electron density by the picolyl group, have a profound effect on the protonation constant of the ligand. 3.3.2 Metal Complexation in Aqueous Solution Titration of the protonated ligand in the pres ence of 1, 2 and 3 equivalent of Zn(II) were analyzed in batch calculations in which all titrations curves we re simultaneously fitted with one model (Figure 3-11). The accessible pH range has been limited in some of these experiments due to the formation of precipitates. Titrations were stopped as soon as a steady drift was noted in the mV meter reading, indicative of the initiation of th e precipitation process. Formation of insoluble species was observed for pH values higher th an pH 8.2, 8.0 and 7.8 for 1:1, 1:2, and 1:3 (M/L) ratios, respectively. Accordingly, the calculations were carried out using data obtained below their respective pH values. Best fit of the titra tion data could be atta ined using the following equilibrium model: L + 3Zn2+ Zn3L6+ K Zn3L = [Zn3L6+]/([L][Zn2+]3) H+ + Zn3L6+ Zn3LH7+ K Zn3LH = [Zn3LH7+]/([H+][Zn3L6+]) H+ + Zn3LH7+ Zn3LH2 8+ K Zn3LH2 = [Zn3LH2 8+]/[H+][Zn3LH7+]

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58 H+ + Zn3LH2 8+ Zn3LH3 9+ K Zn3LH3 = [Zn3LH3 9+]/[H+][Zn3LH2 8+] Zn3L + H2O Zn3L(OH)5+ + H+ K Zn3LOH = [Zn3L(OH)][H + ]/[Zn3L] Zn3L(OH)5+ + H2O Zn3L(OH)2 4+ + H+ K Zn3L(OH)2 =([Zn3L(OH)2][H+])/[Zn3L(OH)] Zn3L(OH)2 4+ + H2O Zn3L(OH)3 3+ + H+ K Zn3L(OH)3=[Zn3L(OH)3 3+][H+]/[Zn3L(OH)2 4+] The occurrence of mononuclear and dinuclear sp ecies was also considered. Using such an extended equilibrium model for the curve-fitting procedure did not affect the overall standard deviation, which suggest that the formation of th ese species was negligib le. This observation is consistent with the NMR spectral data of 3-1 in 50% (v/v) CH3CN/H2O wherein formation of mononuclear or dinuclear species was not observe d even after two weeks. The results of the fitting allowed the calculation of the stability constants of the trinuclear species [Zn3LH3]9+, [Zn3LH2]8+, [Zn3LH]7+, [Zn3L]6+, [Zn3L(OH)]5+, [Zn3L(OH)2]4+ and [Zn3L(OH)3]3+. As often found in polyamine ligands with a large number of amine donors,109 the mononuclear and dinuclear complexes display a marked tendency to add a third metal to give stable trinuclear Zn(II) species, which are largel y prevalent in 50% (v/v) CH3CN/H2O solutions containing metal and ligand in 3:1 molar ratio, Figure 3-11. Though th e species distribution diagram indicates the presence of various trinuclear zinc species in solution the 1H NMR spectra of 3-1 in 50% (v/v) CD3CN/D2O indicate a highly symmetric structure. This suggests that the proton exchange reaction between the various species is sufficiently fast in 50% (v/v) CD3CN/D2O on the NMR time scale.104 The formation of [Zn3L]6+ species occurs at acidic pH and is followed by the formation of mono-, diand tri-hydroxo comp lexes by successive deprotonation of the coordinated water molecules at sl ightly acidic to alkaline pH va lues. The structure of tri-hydroxo species, [Zn3L(OH)3]3+ should correspond to the st ructure of the cation of 3-1 which was characterized by X-ray crystallo graphy. In this complex, the me tal displays a coordination environment not saturated by the ligand donors. All three zinc binding sites are occupied by

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59 tetrahedral zinc(II) ions coordi nated by the pyridyl nitrogen and the piperazine nitrogen atoms from each arm of the ligand. The remaining two c oordination sites of the zinc ion are filled by bridging hydroxides. The trinuclear Zn(II) species, [Zn3L]6+ shows rather low p Ka values for the formation of mono-, diand tr i-hydroxo trinuclear complexes (p Ka1 = 6.53, p Ka2 = 6.88, p Ka3 = 7.64). The first two p Ka values are quite close to one a nother and differ by only 0.35 units. High tendencies to form hydroxo complexes are generall y related to a metal c oordination sphere not saturated by the ligand donors, wh ich leads to facile deprotona tion of the coordinated water molecule. Moreover, the three Zn(II)-bound wate r molecules could come close enough to each other, to allow direct hyd rogen bonding between three ZnII-OH (or OH2) and /or ZnII-OH-ZnII bridging.97,110-112 The p Ka value for deprotonation of the first water molecule in the [Zn3L]6+ trinuclear complex is very low. This behavior indicates a strong binding of the hydroxide ion in [Zn3L(OH)]5+ and is generally ascribed to a bridgi ng coordination mode of OH between two metal centres.68,75,80 This hypothesis is corroborated by the crystal structure of [Zn3L(OH)3]3+ cation, Figure 3-1, which shows three zinc ions connected to each ot her by three bridging hydroxides. The metal coordination spheres of th e three metal centers are not fulfilled by the ligand donors. Therefore, they would provide effi cient substrate binding and activation. At the same time, facile deprotonati on of metal-bound water molecules occur from slightly acidic to neutral pH, giving rise to ZnOH groups as potential nucleophiles in hydrolytic reactions. These features make the trinuclear Zn(II) complex 3-1 a promising hydrolytic agent. It is not possible to determin e reaction mechanism based solely on crystal structure data. Nevertheless, structural informa tion, when combined with kinetic data, can be very powerful for solving reaction mechanisms.

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60 3.4 Kinetic Studies 3.4.1 Transesterificatio n of Hydroxypropylp -nitrophenyl Phosphate (HPNP) Complex 3-1 is insoluble in pure water and hence CH3CN was added as a cosolvent.113 The catalytic activity of th e trinuclear zinc complex 3-1 towards the transesterification of HPNP was studied in 50% CH3CN / 80 mM aqueous buffer at 25C. The pH dependence for the transesterification of HPNP by 3-1 was studied in the pH range of 6.4 to 7.2 and the optimum pH for the reaction was found to be 6.7 (Figure 3-12). The catalyst precipitat ed out of the reaction mixture above pH 7.4. At pH 6.7, 5 mM of 3-1 induces a 16,900 fold rate enhancement in the catalytic cyclization of the R NA model substrate, HPNP, compar ed to the uncatalyzed reaction, corresponding to a reducti on of the half-life from approximately 308 days ( kuncat = (2.6 0.1) X 10-8 s-1, error limits are given at the 95% c onfidence level unless otherwise stated)54,99 for the uncatalyzed reaction to 26 minutes ( kobs = (4.4 0.1) X 10-4 s-1). The pH rate profile was then compared with the species distribution diagram (Figure 3-11) in order to identify the reactive species. Since considerable concentration of [Zn3LH]7+, [Zn3L]6+, [Zn3L(OH)]5+ and [Zn3L(OH)2]4+ are present in the solution, in the pH ra nge 6.4 to 7.2 thus any or all of these might be the active species catalyzing the transesterification of HPNP. The effect of concentration of 3-1 on the reaction rate was then investigated at pH 6.6. Within the concentration range explored (1.0 to 5.0 mM) the observed ps eudo first order rate constant ( kobs), for the cleavage of HPNP, exhibits a linear dependence on th e concentration of the complex. The second order rate constant was determined as the slope of the linear plot of kobs against catalyst concentration ( k2 = (7.9 0.2) X 10-2 M-1s-1, Figure 3-13). In order to investigat e the catalytic process in more detail, the binding affinity of 3-1 towards HPNP was studied by measuring the rate of transesterification as a function of HPNP concentration, at a fixed catalys t concentration. No saturation kinetics could be observed for 3-1

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61 up to 7 equivalents of HPNP (pH 6.6) indicating a low affinity for the substrate (Figure 3-14). It is not possible to determine kobs when [Zn(ClO4)2] is 0.8 mM at pH 6.7 in the absence of ligand due to precipitation of Zn(II) hydroxide. Ho wever, in the pres ence of 0.4 mM Zn(ClO4)2 the value of kobs was found to be (1.2 0.1) X 10-6 s-1, demonstrating that th e catalytic contribution of free Zn(II) towards the cleavage of HPNP in presence of 3-1 is negligible In order to determine whether 3-1 acts as a catalyst for the transessterification of HPNP, reactions in the presence of 82 equivalents of the substrate were followed by 31P NMR spectroscopy. A progressive increa se in the intensity of signal corresponding to the cyclic phosphate ester with a simultaneous decrease of the HPNP signal was noted as outlined in Figure 3-15. No other signal, including that of inorganic phosphate, wa s observed even after prolonged reaction times, indicating that the cyclic phosphate ester is no t further hydrolyzed by 3-1 In a separate experiment, catalytic turnover was demonstrated by preparing a reaction mixture consisting of 0.1 mM 3-1 2 mM HPNP and 80 mM MES buffe r (pH 6.7). The absorbance at 400 nm produced by the release of p -nitrophenolate ion was monitored over a two day period and more than 10 turnovers of catalyst was noted. During the course of the multiple turnover reaction, the catalyst activity does decrease possi bly due to product inhibition or catalyst decomposition over prolonged periods of time. With ligands adept at forming trimetallic complexes, the orientation and flexibility of the metal binding groups appear to impact substrate binding. Re inhoudt and coworkers investigated the transesterification of HPNP catalyzed by the co-operative action of multiple Zn(II) centers tethered to the upper rim of a semi-flexible calix[4]arene.54,99 The tri-zinc calix[4]arene complex showed saturation kinetics, and the second order rate constant, k2 for the transesterification of HPNP by this catalyst at pH 7 is 2.9 M-1s-1. Strong substrate binding to the Zn(II) complex has

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62 the effect of increasing the second or der rate constant. In the case of 3-1, substrate binding appears to be impeded either by the crowded natu re of the trinuclear cluster or possibly by the presence of bridging hydroxides between the me tal centers, and consequently the value for k2 is low. The bound hydroxides lower the substrate binding affinity of the catalyst by diminishing the Lewis acidity of the Zn(II) centers.68,75,80 Nevertheless, once bound, HP NP is rapidly converted to product by 3-1 Table 3-4 summarizes the s econd order rate constants ( k2, M-1s-1) for the transesterification of HPNP catalyzed by seve ral different Zn(II) co mplexes (Figure 3-17). 3.4.2 Hydrolysis of Diribonucleoside Monophospha te Diesters (NpN) by Dizinc Complexes The use of HPNP to mimic natural phosphoesterases is not fully justifie d, since in contrast to biological phosphoesters, the p -nitrophenolate leaving group doe s not require electrophilic stabilization. Thus to have a more accurate unde rstanding of the possible roles of metal ions in the cleavage of RNA, diribonucleoside monophospha te diesters (NpN), which more closely resemble RNA, was used. The hydrolysis reactions of NpN (N = A, U, G and C) (0.32 M) were carried out in the presence of excess amount of trinuclear zinc complex 3-1 (1.5 mM) in 20 mM HEPES (pH 6.7) at 50C. Although remarkable rate acceleration in the tr ansesterification of HPNP has been observed, no signi ficant activity for the cleavage of the internucleosidic phosphodiester bonds of NpNs has been found. These re sults indicate that stabilization of the leaving group indeed plays an important part in the hydrolytic cleava ge of phosphate diester bonds in nucleotides like RNA and DNA. 3.5 Mechanism of Catalysis In order to have better insight into th e mechanism, phosphodiesterase activity of the trinuclear zinc complex 3-1 was compared with that of mono nuclear zinc complex, prepared in situ by the addition of on e equivalent of Zn(ClO4)2H2O to a solution of 2-21 A progressive increase in the intensity of si gnal corresponding to the cyclic phos phate ester with a simultaneous

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63 decrease of the HPNP signal was noted as outlined in Figure 3-18. No othe r signal, including that of inorganic phosphate, was observed even afte r prolonged reaction times, indicating that the cyclic phosphate ester is not furt her hydrolyzed by the mononuclear zinc complex. The trinuclear Zn(II) catalyst exhibits a higher hydrolytic activity as compared to its mononuclear analogue as can be realized by comparing Figure 3-15 and 3-1 8. In the trinuclear zinc complex, the three Zn(II) ions are kept in close proximity by use of a hydroxide bridging ligand.68,75,80 The bridging hydroxide group presumably shields the electrosta tic interactions between the Zn(II) ions and allows the cations to be drawn relatively close together in a complex of greatly enhanced activity. This high density of positive charge at 3-1 is ideal for providing electrostatic stabilization of the transition state for cleavage of HPNP relative to the reactant state because there is a net increase in negative charge on pr oceeding from the reactant to transition state. A hydroxide (or water) spanning two zinc ions has been detected cr ystallographically in many hydrolases and is often considered as the active nucleophile, but it can be expected to exhibit rather low nucleophilicity if coordinated in a tightly bridging form.3,11,22,23 Thus it has been suggested that upon substrate binding a sh ift of the bridging hydr oxide to a terminal position occurs prior to attack on the coordinated substrate.114,115 As discussed above, in 3-1 three metal ions are held at a short distance, and at the sa me time, the ligand donors poorly saturate the coordination sphere of the metal ions. These structural features would favor a bridging interaction mode of the phosphate ester.116 Substrate interaction with two electrophilic metal centers promotes the nucleophilic attack of a Zn-OH function and thus enhances the rate of hydrolytic process, as found in several synthetic Zn(II) complexes.44,54,110 The -1,3 bridged phosphoester unit has frequently been postulated as the preferred substrate binding mode in various metallo phosphoesterases. A recent crystal structure of native Eschericia Coli alkaline

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64 phosphatase complexed with inorganic phosphate s hows the phosphate ani ons bridging the two Zn(II) ions, which lie 3.94 apart from each other.117-120 Similarly, the X-ray crystal structure of the complex of PLCBC (phospholipase C from Bacillus Cereus ) with a competitive inhibitor (a phospholipid analog) revealed that the phosphate binds to all thre e Zn(II) ions by replacing two of zinc-bound water molecules in the native PLCBC.21,30,121 Although 3-1 efficiently catalyzes the transesterifi cation of HPNP, it is not catalytically active in the hydrolysis of ethyl p -nitrophenyl phosphate de monstrating that the -hydroxyl group of HPNP is essential for hydrolysis. Base d on the above discussion, the high activity in HPNP transesterification observed for our tr inuclear Zn(II) complex may be explained by considering that the phosphate este r would interact with the Zn(II) centers, re sulting in substrate activation. This is then followed by facile intram olecular transesterifica tion of phosphate diester bond, which may either involve the participation of the bridging hydroxide as a general base or the nucleophilic attack by the deprotonated hydroxy group of the substrate. Solvent deuterium isotopic studies needs to be pe rformed to distinguish between a general base and a nucleophilic reaction path. To verify the pr oposed bridging coordination of HP NP to the trinuclear zinc catalyst, various crystallizations involving 3-1 and inert substrates (e.g. diphenyl phosphate, dimethyl phosphate etc.) were attempted. Unfort unately, we were not successful in growing suitable crystals of phosphate bound cata lyst for detailed X-ray analysis. In reactions with activated esters, metal hyd roxides act as typical O-donor nucleophiles, and the reactivity is generally predictable from the p Ka of the metal-bound water.122,123 Buckingham et. al. published a Bronsted plot for the metal-hydroxide (including OH-) promoted hydrolysis of 4-nitrophenyl acetate (npa).123 A good linear correlation ( = 0.40) was observed between log(kMOH) and p KMOH2 for npa reaction. The values of log(kZnOH) corresponding to p Ka1

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65 and p Ka2 (p Ka values for the formation of monoand di-hydroxo trinuclear zinc complex) as predicted by the above Bronsted plot are is cl ose agreement with the experimentally obtained log(kZnOH) values for the hydrolysis of npa catalyzed by 3-1 in 50% CH3CN/80 mM aqueous buffer at 25C, Figure 3-19. This suggests that the monoand di-hydroxo trinuclear zinc species might be the active species catal yzing the hydrolysis of npa, whic h is in agreement with the species distribution diagram of 3-2 in presence of three equivalent of Zn(II) ions, since at pH 6.7 there is considerable concentration of both these species in solution. 3.6 Conclusions Inspired by trinuclear Zn sites in enzymatic systems, a C3-symmetric trinuclear Zn(II) hydroxide complex of 2-5 was synthesized and fully charac terized using NMR spectroscopy and X-ray crystallography. This complex at 5mM concentration induces a 16,900-fold rate enhancement in the catalytic cyclization of the RNA model substrate, 2-hydroxypropyl-pnitrophenyl phosphate (HPNP, pH 6.7, 25C), over the uncatalyzed reaction with multiple catalyst turnovers. 1H NMR spectroscopy suggests th at on the NMR time scale a C3-symmetric core is maintained in solution, under the conditions us ed for the kinetic expe riments (pH 6.7, 50% (v/v) CD3CN/D2O, buffer, [ 3-1 ] = 7.5 mM). However the species di stribution diagram indicates the presence of various trinuclear zinc species in solution at and around pH 6.7. This suggests that the proton exchange reaction between the variou s species is sufficiently fast in 50% (v/v) CD3CN/D2O on the NMR time scale and thus some sort of hydroxide bridged C3-symmetric species was observed. The trinuclear Zn(II) complex exhibits a hi gher hydrolytic activity as compared to its mononuclear analogue. Although 3-1 efficiently catalyzes the tran sesterification of HPNP, it is not catalytically active in the hydrolysis of ethyl p -nitrophenyl phosphate demonstrating that the

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66 -hydroxyl group of HPNP is essential for hydrolys is. Based on the experime ntal observations, it is proposed that substrate, HPNP, is activated due to its interaction with the Zn(II) centers. This is then followed by facile intramolecular tran sesterification of phosphate diester bond, which may either involve the participation of the bridging hydroxide as a general base or the nucleophilic attack by the depr otonated hydroxy group of the substrate. Solvent deuterium isotopic studies needs to be pe rformed to distinguish between a general base and a nucleophilic reaction path. 3.7 Experimental Section 3.7.1 General Methods All reagents and solven ts were of analytical grade a nd were used without purification, unless otherwise noted. Aqueous solu tions were prepared using 18 M Millipore deionized water. 2-Morpholinoethanesulfonic acid (MES), N-(2-hydroxyethyl)piperazine-N-(2ethanesulfonic acid) (HEPES) a nd Zn(II) perchlorate hexahydrate were purchased from SigmaAldrich. The barium salt of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP) was prepared following literature method.100 All 1H, 13C and 31P NMR spectra were recorded on a Varian VXR-300 or Mercury-300 spectrometer at 299.95, 75.4 and 121.42 MHz for the proton, carbon and phosphorus channels, respectively. Elementa l analyses were performed by Complete Analysis Laboratories, Inc. in Parsippany, NJ. The pH meter used for adjustment of buffered solution was calibrated daily. 3.7.2 Synthesis Preparation of [2-5Zn3( -OH)3](ClO4)3 (3-1). ( Caution Although, the perchlorate salt is moderately stable, it is a pot ential hazard and should ther efore be handled with care.) 2-5 (20 mg, 0.018 mmol) was dissolved in 1 mL acetone and added to a solution of Zn(ClO4)2H2O (20.11 mg, 0.054 mmol) in 1 mL of acet one mixed with a few drops of methanol (to dissolve

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67 Zn(ClO4)2H2O completely). Diffusion of pentanes at 0 C into the above solution afforded colorless crystals suitable for X-ray analysis. Isolated yield (17.9 mg, 60%). 1H NMR (CD3CN): = 1.20 (s, 27H; Ar-C(C H3)3), 2.20 (s, 3H; OH ), 2.29 (s, 9H; Ar-C H3), 2.6-3.7 (b, 36H; N-C H2C H2-N & Ar-O-C H2C H2N), 4.17 (s, 6H; Py-C H2), 7.10 (s, 1H; C H ), 7.12 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.20 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.70 (m, 6H; Py), 8.20 (dt, 3J (H,H) = 7.7 Hz, 4J (H,H) = 1.8 Hz, 3H; Py), 8.80 (d, 3J (H,H) = 5.1 Hz, 3H; Py). 13C NMR (CD3CN): = 16.8 (ArC H3), 31.7 (Ar-C( C H3)3), 34.9 (ArC (CH3)3), 35.6 ( C -H), 55.6 (Ar-O-CH2-CH2-NC H2), 55.8 ( C H2-N-CH2-Py), 58.3 (Ar-O-CH2C H2), 63.0 (NC H2-Py), 74.8 (Ar-OC H2), 125.9 (Ar), 126.2 (Py), 126.9 (Ar), 131.5 (Ar), 137.2 (Ar), 143.2 (Py), 147.1 (Ar), 150.5 (Py), 153.4 (Ar), 155.1 (Py). Anal. Calcd. for 3-1 H2O (C70H104N9O20Cl3Zn3): C, 49.63; H, 6.19; N, 7.44; Cl, 6.28. Found: C, 49.70; H, 5.99; N, 7.42; Cl, 6.24. Preparation of hydrochloride salt of Tr is(2-(2-(4-((2-pyridyl)methyl)piperazine)-1ethoxy)-3-methyl-5-tert-butylphenyl)methane (3-2). The HCl salt of of 2-5 (tris(2-(2-(4-((2pyridyl)methyl)piperazine)-1-ethoxy)-3-methyl-5 -tert-butylphenyl)metha ne) (Figure 3-16) was prepared by dissolving it in diethyl ether and ad ding 2 N-HCl in diethyl ether resulting in the formation of a white precipitate, which is thor oughly washed with diet hyl ether and dried under vacuum. The product formed was 2-5HClH2OEt2O. 1H NMR (D2O): = 1.02 (s, 27H; ArC(C H3)3), 2.31 (s, 9H; Ar-C H3), 3.05-4.19 (m, 42H), 6.60 (s, 1H; C H ), 6.64 (s, 3H; ArH ), 7.21 (s, 3H; ArH ), 8.04 (m, 6H; Py), 8.57 (dt, 3J (H,H) = 7.95 Hz, 4J (H,H) = 1.5 Hz, 3H; Py), 8.77 (d, 4J (H,H) = 5.4 Hz, 3H; Py). 13C NMR (D2O): = 16.87 (ArC H3), 31.19 (Ar-C( C H3)3), 34.07 (ArC (CH3)3), 49.11 ( C -H), 50.85 (Ar-O-CH2-CH2-NC H2), 52.04 ( C H2-N-CH2-Py), 56.07 (ArO-CH2C H2), 57.1 (NC H2-Py), 65.52 (Ar-OC H2), 125.61 (Py), 126.49 (Py), 127.23 (Ar), 127.58 (Ar), 128.44 (Ar), 130.87 (Ar), 135.98 (Py) 141.58 (Ar), 147.2 (Py), 152.1 (Ar), 152.37

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68 (Py). Anal. Calcd. for 2-5HClH2OEt2O (C74H124Cl9N9O8): C, 56.01; H, 7.88; N, 7.94. Found: C, 55.75; H, 8.13; N, 8.29. 3.7.3 Potentiometric Titrations Potentiometric titrations we re conducted in 50% (v/v) CH3CN/H2O mixture at an ionic strength of 0.1 M KCl with a Metrohm 702SM titrator equipped with a Metrohm combined pH glass electrode. The electrode system was calibrated before each measurement by titrating known amount of HCl in the solvent mixture wi th a known concentration of KOH. The base solution with a concentration of ca. 0.1 M was made in the above solvent mixture and standardized using potas sium hydrogen phthalate.124 A plot of millivolts (measured) vs pH (calculated) gave a working slope and intercept so that pH could be read as log[H+] directly. The p Kw value in 50% (v/v) CH3CN/H2O was determined to be 15.19 at 25C and is in good agreement with published value.125 The electrode was stored in solvent mixture between measurements. All solutions were prepared with purified CH3CN and freshly boiled 18 M Millipore deionized water cooled in a nitr ogen stream. All solutions were car efully protected from air by a stream of nitrogen gas. The linearity of elect rode response and carbonate contamination of the standardized KOH solution was determined by Gr ans method and was found to be less than 2%.108,109,126,127 The stock solution of Zn(II) (0.02 M) was prepared by dissolving Zn(ClO4)2H2O in 50% (v/v) CH3CN/H2O. This was standardized with EDTA using Eriochrome Black T as indicator. The potentiometric titration of 3-2 (0.92 mM) in the absence and presence of 1, 2 and 3 equivalents of Zn(II) respectively were carried out at 25C with I = 0.1 M (KCl) under nitrogen atmosphere. The equilibrium constants were calculated using the program BEST108. All fit values (as defined in the pr ogram) are smaller than 0.025. Species distribution was calculate d using the program SPE.108 At least two titratio n experiments (of about

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69 100 data point each) were performed for each sy stem. Potentiometric determination of the stability constants of iminodiacetic acid with Pb(II) was initially pe rformed to gain an appreciation of and a feel for the method, th e equipment, and the program. The stepwise constants are denoted by K while th e overall constants are denoted by 3.7.4 Kinetic Measurements UV-Vis spectra and kinetic traces were record ed with a diode array spectrometer equipped with a thermostated multicell cuvette holder (7 cuvettes, 1.0 cm path le ngth) (Hewlett Packard 8453). Solutions for kinetic measurements were made by adding CH3CN (spectrophotometric grade) upto 50% (v/v) to a 80 mM aqueous buffer solution adjusted with NaOH to the desired pH. Buffers (MES, pH 5.6-7.0 and HEPES, pH 7.08.2) were obtained from commercial sources and used without further purification in 18 M Millipore deionized wate r. Stock solutions were freshly prepared before perfor ming the kinetic measurements. In a typical experiment, complex 3-1 (1040 L, 10 mM in CH3CN) was added to a cuvette containing 38 L of CH3CN and 962.4 L of 173.25 mM aqueous buffer solution and thermo stated at 25C. After a couple of minutes of equilibration time, HPNP (41.6 L, 100 mM in water) was injected and an increase in the UV absorption at = 400 nm due to the release of p -nitrophenolate ion was re corded. All solutions remained clear during the time of the kineti c measurements. In the absence of ligand, precipitation of polymeric Zn(II) hydroxide took place. The observed pseudo-first-order rate constants kobs (s-1) were calculated with the extinction coefficient of p -nitrophenolate at = 400 nm by the initial slope method (< 5% conversion). Solutions of p -nitrophenolate in CH3CN/20 mM buffer 1:1 (v/v) were prepared at various pH values. Molar extinction coefficient for p nitrophenolate at 400 nm was then determined from the plot of absorbance against concentration. Correction for the spontaneous hydrolysis of HPNP (less than 0.1%) was accomplished by direct observation of the production of p -nitrophenolate relative to a re ference cell containing no metal

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70 complex. The pseudo-first-order rate constants for the transesterific ation of HPNP in the absence of the catalyst (kuncat, s-1) were measured with a 2.0 mM HPNP solution by the method of initial rates. Each experiment was run in triplicate. Agreement between the calculated initial rates for replicate experiments was within 5%. The kinetics for cleavage of 3',5'-NpN were measured by monitoring the appearance of guanosine, uridine, cytidine and adenosine at = 254, 260, 272 and 260 nm, respectively using a Water 1525EF HPLC equipped with Waters 24 87 UV-vis detector. Aliquots of the reaction mixtures were analyzed on a C18 column (4.6 mm X 150 mm). Typically, an isocratic flow of 90% solvent A (60 mM acetate buffer at pH 4.3) and 10% solvent B (Methanol) was used with a isocratic flow of 2.0 mL/min over 30 min. In a typical e xperiment, 250 L of 6 mM 3-1 was added to a mixture of 500 L of 40 mM buffer solution (pH 6.7), 100 L of 1 M NaNO3 and 10 L of 2 mM p -nitrobenzenesulfonate sodi um salt (internal indicator).128 The mixture was then thermostated at 50C. After a couple of minutes of equilibration time, 140 L of 2.27 mM NpN was injected into the above mixture. Aliquots (75 L) of the reaction mixture were quenched with 75 L of 100 mM acetate buffer (pH 4.23) and analyzed with HPLC. The concentration of the formed nucleoside was determined by means of a calibration curve made with commercially obtained nucleoside (corre lation coefficient > 0.95). 3.7.5 X-ray Crystallography Unit cell dimensions and intensity data for all the structures were collected by Prof. Michael J. Scott on a Siemens CCD SMART diff ractometer at 173 K. The data collections nominally covered a hemisphere of reciprocal sp ace, by a combination of th ree sets of exposures; each set had a different angle for the crystal, and each exposure covered 0.3 in The crystal to detector distance was 5.0 cm. The data sets were corrected for absorption using SADABS.129

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71 All the structures were solved by Prof. Michael J. Scott using the Bruker SHELXTL software package for the PC, using the direct methods option of SHELXS. The space groups for the structures were determined from an examina tion of the systematic absences in the data, and the successful solution and refinement of th e structures confirmed these assignments. All hydrogen atoms were assigned idealized locatio ns and were given a thermal parameter equivalent to 1.2 to 1.5 times the thermal parameter of the atom to which it was attached. For the methyl groups, where the location of hydrogen atoms was uncertain, the AFIX 137 card was used to allow the hydrogen atoms to rotate to the maximum area of residu al density, while fixing their geometry. Structural and refinement data for 3-1 is presented in Table 3-5. Table 3-1. Selected interatomic distances () and angles () for 3-1 (Me)2CO. Bond lengths () Bond angles () Zn1-O1 1.925(4) N1-Zn1-N2 81.0(2) Zn1-O1" 1.903(4) N1-Zn1-O1 111.7(2) Zn1-N1 2.055(5) N1-Zn1-O1" 127.28(19) Zn1-N2 2.143(5) N2-Zn1-O1" 104.3(2) ZnZn 3.435 O1-Zn1-O1" 109.4(2) N2-Zn1-O1 121.01(19) Table 3-2. Protonation constants of the ligand 2-5 at 25C; I = 0.1 M KCl. Species log pKa [LH6]6+ 37.96 3.81 [LH5]5+ 34.15 3.95 [LH4]4+ 30.20 6.10 [LH3]3+ 24.10 7.27 [LH2]2+ 16.83 7.60 [LH]+ 9.23 9.23

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72 Table 3-3. Stability constants of the Zn(II) complexes with 2-5 at 25C; I = 0.1 M KCl. Species log pKa [Zn3LH3]9+ 36.09 4.63 [Zn3LH2]8+ 31.46 5.53 [Zn3LH]7+ 25.93 6.66 [Zn3L]6+ 19.27 6.50 [Zn3L(OH)]5+ 12.77 6.89 [Zn3L(OH)2]4+ 5.88 7.63 [Zn3L(OH)3]3+ -1.75 Table 3-4. Rate constants for the transesterific ation of HPNP catalyzed by Zn(II) complexes at 25C. Catalysta pH k2(M-1s-1) X 102 Ref 3-1 6.7 7.9 This work Zn3( 3-6 ) 7.0 290 37 Zn2( 3-5 ) 7.0 4300 37 Zn2( 3-5 ) 7.4 1700 37 Zn( 3-4 ) 7.0 1.5 37 Zn2( 3-11 ) 7.6 0.58 92 Zn2( 3-10 ) 7.6 0.89 92 Zn2( 3-9 ) 7.6 1.1 92 Zn2( 3-8 ) 7.6 25 92 Zn( 3-7 ) 7.6 0.21 92 aFor structures of these complexes see Figure 3-17 Table 3-5. Crystal data and structure refinement for 3-1. 3-1 (CH3)2CO Formulaa C79H109Cl3N9O21Zn3 fw, g/mola 1823.21 Space group P 3 a, 16.7532(16) b, 16.7532(16) c, 23.629(3) 90 90 120

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73 Table 3-5. Continued. V, 3 5743.4(11) Z 2 Radiation, b 0.71073 mm-1 0.747 R1 a 0.0849 wR2 b 0.2355 a R 1 = (|| Fo| | Fc||) / | Fo|. b wR 2 = [ [ w ( Fo 2 Fc 2)2] / [ w ( Fo 2)2]]1/2, w = 1/[ 2( Fo 2) + [( ap )2 + bp ], where p = [ Fo 2 + 2 Fc 2]/3 N C O H 3 N N Zn O H 3+ Figure 3-1. Depiction of the solid-state structure of 3-1 (a) Side view with hydrogen atoms and perchlorate anions omitted for clarity ( 30% probability ellipsoids, carbon atoms arbitrary radii). Primed and unprimed atoms are related by a 3-fold symmetry axis. (b) Cartoon of the top vi ew. (c) ChemDraw of 3-1 a b c

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74 Figure 3-2. 1H NMR spectra of 2-5 in CD3CN

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75 Figure 3-3. 1H NMR spectra of 3-1 in CD3CN N C O H 3 N N Zn O H 3+

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76 Figure 3-4. 1H NMR spectra of 2-5 (7.5 mM) in MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O) OH N N N 3N O S O O O +MES BufferH

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77 Figure 3-5. 1H NMR spectra of 3-1 in 1:1 (CD3CN : D2O) N C O H 3 N N Zn O H 3+

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78 Figure 3-6. 1H NMR spectra of the reaction mixture containing 3-1 (7.5 mM), MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O) after 30 min N C O H 3 N N Zn O H 3+ N O S O O O +MES BufferH

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79 Figure 3-7. 1H NMR spectra of the reaction mixture containing 3-1 (7.5 mM), MES buffer (22.5 mM) at pH=6.7 in 1:1 (CD3CN : D2O) after 24h. N C O H 3 N N Zn O H 3+ N O S O O O +MES BufferH

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80 0.00 0.05 0.10 0.15 0.20 0.25 0.30 5.806.006.206.406.606.807.007.20 pHRatio of Free Ligand to Trinuclear Zinc Complex Figure 3-8 Variation of the ratio of free ligand ( 2-5 ) to trinuclear zinc complex ( 3-1 ) as a function of pH.

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81 Figure 3-9. Temperature-dependent 1H NMR spectra of the aromatic region of 3-1 The reaction mixture contains 3-1 (7.5 mM), MES buffer (22.5 mM) in 1:1 (CD3CN : D2O) at pH = 6.7.

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82 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0.02.04.06.08.010.012.0 Equivalent of KOH AddedpH Figure 3-10 Potentiometric titration curve of 3-2 with 0 ( ), 1 ( ), 2 ( ) or 3 (*) equiv. Zn(II) in 0.1 M KCl in 1:1 CH3CN/H2O at 25C. Figure 3-11 Species distribution diagram for 3-2 (0.92 mM) in the presence of Zn(II) ions (2.76 mM) as a function of pH in 1:1 CH3CN/H2O at 25C with I = 0.10

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83 1.5 2 2.5 3 3.5 4 4.5 5 6.36.56.76.97.17.3 pHkobs X 104 (s-1) Figure 3-12. pH versus rate profile for transester ification of HPNP (2 mM) catalyzed by 3-1 (5 mM) in acetonitrile/80 mM buffer 1:1 (v/v) at 25 C. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0246 [Cat] (mM)kobs X 104 (s-1) Figure 3-13. Dependence of the obser ved pseudo-first-order rate c onstant on the concentration of trinuclear zinc(II) catalyst 3-1 at pH 6.6 in 50 % CH3CN/80 mM MES buffer (v/v) at 25 C. [HPNP] = 2 mM.

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84 0 2 4 6 8 10 12 14 16 010203040 [HPNP] (mM)Rate X 103 (mM s-1) Figure 3-14. Initial rate as a func tion of the substrate concentrati on for the transesterification of HPNP catalyzed by 5 mM of 3-1 in 50 % CH3CN/80 mM MES buffer (v/v) (pH 6.6) at 25 C. Figure 3-15. Stack plot of 31P-NMR for the transesterificati on of HPNP (38.56 mM) catalyzed by 3-1 (0.47 mM) at pH 6.7 after (b ) 1 day (c) 3 days (d) 5 days (e) 8 days. Spectrum (a) was taken before addition of the catalyst

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85 O N H N N 3 3-2 H H H Cl Cl Cl Figure 3-16. Hydrochloride salt of 2-5 OR" OR" OR" R R' OR" N N N N N N 3-3 3-4 : R' = R = H 3-5 : R' = R = 3-6 : R' = R = R" = CH2CH2OEt N N N N N N H H N N N H H OH N N N H H N N N H H N N N N N H H N N N H H N N N H H H N N N H H N N N H H 3-7 3-8 3-9 3-10 3-11 Figure 3-17. Structure of liga nds mentioned in Table 3-4.

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86 Figure 3-18. Stack plot of 31P-NMR for the transesterificati on of HPNP (38.56 mM) catalyzed by a mixture of 2-21 (1.41 mM) and Zn(ClO4)2H2O (1.41 mM)at pH 6.7 after (b) 1 day (c) 3 days (d) 5 days (e) 8 days. Spec trum (a) was taken before addition of 2-21 and Zn(ClO4)2H2O -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 05101520 pKalog10(kNA) Figure 3-19. Bronsted plot for th e metal-hydroxide (including OH-) promoted hydrolysis of 4nitrophenyl acetate.( Literature Data, [Zn3L(OH)]5+, [Zn3L(OH)2]4+)

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87 CHAPTER 4 SYNTHESIS OF TRINUCLEAR LI GAND SYSTEMS USING MODIFIED TRIPHENOXYMETHANE PLATFORM 4.1 Introduction In the previous chapter, it was shown that the C3-symmetric trinuclear Zn(II) hydroxide complex 3-1 (Figure 4-1) acts as an efficient cataly st for the transesterification of HPNP. However, this trinuclear Zn(II) complex appeared to be inactive for the cleavage of natural substrates, such as RNA dinucleotides, that contain poor leaving groups. The low activity of 3-1 could be due to several factors. First, the binding constant for th e coordination of the substrate is low as is evident from the absence of any saturation effects for 3-1 even at a large excess of the substrate. This can be attributed to the shield ing of the metal ions by non-labile and bulky side arms. Moreover, the bound hydroxides lower the substr ate affinity of the catalyst by diminishing the Lewis acidity of the Zn(II) cente rs. Second, the bridging hydroxide in 3-1 is tightly bound to the zinc ions and therefor e exhibits low nucleophilicity. The construction of a trinuclear ligand system using a calix[4]arene platform to form trinuclear Zn(II) complex 1-4 (Figure 4-1) was reported by Reinhoudt et al .54 The functionalization of the upper rim was proposed to be more appropriate from the perspective of preorganization and steric requirements of th e substrates. The trinuclear Zn(II) complex 1-4 efficiently catalyzes the cleavage of RNA dinucleotides (3',5' -NpN), by the cooperative action of the three Zn(II) centers, with high rate enhancement and significant nucleobase specificity.99 Since subtle differences in the mutual arra ngement/orientation of the metal binding arms have a crucial influence on the intrinsic activity of these trinuclear Zn(II) complexes, development of a ligand system 4-1 (Figure 4-1) by modifying the previously used triphenoxymethane platform 4-2 was undertaken. In contrast to 3-1 the metal binding arms in 4-1

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88 are directly attached to the aromatic moieties si milar to the upper rim de rivative of calix[4]arene 1-4 (Figure 4-1). 4.2 Synthesis of trinuclear ligand system Due to the rigidity of the triphenoxymethane platform, these molecules share many traits with the calix[4]arene macrocyc les and therefore exhibit divers e chemistry characteristic of calixarenes.82 The extension of the platform appears to be easily accessible via the relatively mature derivatization chemistry of phenol and ca lixarene systems. In particular, two methods have been commonly employed for the derivatization of the para -positions. The first approach involves the removal of p tert -butyl groups, by AlCl3 catalyzed transalkylation, in the presence of a suitable acceptor such as toluene or phenol.130,131 This reaction plays a key role in the calixarene chemistry, since a large variety of cal ixarenes with different substituents in the para positions can be obtained by subsequent electrophilic substitution.132 Applying these reaction conditions to 4-2 (Figure 4-2) resulted in the formati on of a pale-yellow solid soluble in nonpolar organic solvents. Our experience has shown th at generally the most diagnostic signal in the 1H and 13C NMR spectra of materials derived from 4-2 is the central methane.82 The absence of this peak in the 1H and 13C NMR spectra of the product suggests decomposition of the tripod under the reaction conditions. The mass spectroscopy analysis i ndicates the formation of a mixture of products, which could not be characte rized individually. Syst ematic variation of solvent and the amount of Lewis acid used di d not have any impact on the outcome of the reaction. Repeated attempts to grow single crysta ls of the product were unsuccessful. The second method for accessing the desired lig and system involves the metal directed synthesis of tris -phenoxides directly from the phenolic precursors following a synthetic scheme originally developed by Casnati and coworkers.133,134 This method involves the activation of the open ortho -position on the phenol by the Grignard reagen t, which then subsequently reacts with

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89 triethylorthoformate in a one -pot synthesis to form symmetr ic triphenoxymethane platform. Because the Grignard reagent activates only the ortho -positions of the phenol, platforms without substituents at the para -position are accessible with this method. The above methodology successfully led to the formation of the detert -butylated triphenoxymethane platform 4-3 (Figure 4-3). Unlike 4-2 the compound 4-3 was, somewhat surprisingl y, extremely soluble in all organic solvents and exhaustive attempts at crys tallization by evaporation were futile. With the para -position of the triphenoxymethane pl atform made available by the detert -butylation, various para -functionalization proced ures were explored. One such procedure is chloromethylation, wh ich is the most widely used method among Friedal Crafts alkylations.132 It was first introduced in the late 1980s by Ungaro and coworkers.135 Chloromethyl alkyl ether in the presence of a Lewis acid such as SnCl4 was used to place a CH2Cl group on the para -position of the calixarene ring. The chloromethyl group is generally used because it can readily be conve rted into various other moieties. However, on reacting 4-3 with chloromethyl n-octyl ether in the presence of SnCl4 resulted in the formation of intractable product mixtures (Figure 4-4). In an attempt to introduce a halomethyl group at the para -position of the triphenoxymethane platform, 4-3 was reacted with paraformaldehyde and HBr-acetic acid mixture.136 However, this resulted in the fo rmation of inseparable polymerized product mixtures (Figure 4-5). Another promising method for para -functionalization of calixaren es is the a pplication of ipso nitration, where by the tert -butyl groups of the calixarenes ar e directly replaced with nitro groups, obviating the necessity of their prior removal in a separate step.137 The nitrocalixarenes provide extremely useful intermediates for the introduction of other functional groups, generally

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90 via the amino calixarenes obtained by reducti on with common reducing agents used in literature.137,138 Using this methodology Bhmer and coworker s successfully synthe sized two tris(2alkoxy-5-aminophenyl)methanes 4-4 4-5 (Figure 4-6).139 Synthesis of two related triphenoxymethane derivatives 4-8 4-9 was performed by slight modification of the above procedure. 4-9 was obtained as shown in Figure 4-7. Ac id catalyzed condensation of 2-hydroxy5-nitrobenzaldehyde with an excess of p -nitrophenol led to triphenylmethane 4-6 in nearly 79 % yield. This was then esterified using methyl iodide in the presence of K2CO3 ( 4-7 95 % yield). Subsequent reduction with hydrazine and Pd-C afforded 4-8 (50 % yield) (F igure 4-7). An alternative reaction sequen ce involving O-alkylation, ipso -nitration followed by reduction lead to the formation of 4-9 (Figure 4-8). 4-8 and 4-9 are valuable intermedia tes for the introduction of useful moieties onto the para -positions. Levine et al found that on refluxing a metha nolic solution containing one equivalent each of aniline, 2-vinylpyridine and glacial acetic acid for eight hours resulted in the formation of a monopyridylated product.140 Using the reported experimental conditions, we obtained a trispyridylethylated derivative of 4-8 in which each arm of the triphenoxymethane platform contains on e pyridyl ethyl group 4-10 (Figure 4-9). Various attempts to attach a second pyridyl ethyl group to each of th e three arms by using trifluoroace tic acid, a stronger acid than acetic acid, were not successf ul and resulted in the formation of the same product as 4-10 (Figure 4-9). Despite the well-known susceptibility of imin e bonds to hydrolysis, metal complexes of Schiff bases are generally re sistant towards hydrolysis.141 In order to model peptide hydrolysis by dimetallic aminopeptidases, the dinuclear Zn(II) Schiff base complex 4-11 (Figure 4-10) has

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91 been prepared and structurally char acterized by Erxleben and coworkers.142 In 4-11 zinc ions are bridged by the deprotonated phenolic oxygen of the ligand and two acetate groups. Complex 411 was shown to promote hydrolysis of glycine ethyl ester under mild conditions.142 Condensation of 4-9 with 5tert -butyl-2-hydroxy-3-methyl benzaldehyde led to the formation of the corresponding Schiff base, 4-12 (Figure 4-11). Addition of three equivalents of Zn(ClO4)2H2O to a solution of 4-12 in chloroform, containing three equivalents of triethylamine, afforded the tris-Zn(II) complex, 4-13, in which three zinc i ons are held together by two Schiff base units (Figure 4-12). Due to the formation of the dimer, the three zinc centers appear to be sterically hindere d for efficient substrate binding. To overcome this caveat bulky groups were introduced in the Schiff base to sh ift the equilibrium towa rds the formation of monomeric trinuclear zinc species. Condensation of 4-7 with 3,5-ditert -butyl-2-hydroxybenzaldehyde led to the formation of the corresponding Schiff base 4-14 (Figure 4-14). Refluxing 4-14 and Zn(O2CCH3)H2O in tetrahydrofuran resulted in the fo rmation of a yellow material. The 1H and 13C NMR specta hints at a C3-symmetric structure. Elemental analysis of th e product is consistent with the formation of a dimer with three zinc ions be ing held together by two Schiff base units. Unfortunately, all attempts to grow X-ray diffrac tion quality single crystals of this zinc compound were not successful. Since several weeks are require d to obtain appreciable am ounts of triphenoxymethanebased ligands, simultaneous exploration of other C3-symmetric tripodal ligands, which could be synthesized easily in fewer step s, was undertaken. Hexasubstitute d benzene derivatives with a 1,3,5and 2,4,6substituted patter n, leading to characte ristically preorganized systems, have recently attracted much attention as buildi ng blocks for supramolecular assemblies or

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92 bioinorganic model compounds.143-145 Such derivatives adopt an overall ababab conformation with the side chains pointing al ternatively to opposite sides, ( a denotes above and b denotes below), of the benzene plane in the most th ermodynamically stable configuration due to the steric repulsion between th e neighboring substituents.146,147 Various tripodal ligands based on 1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene ( 4-15 )145 and 1,3,5-tri(bromomethyl)-benzene148 have been used to model the tri nuclear sites of multi-copper oxidases.145,148 However, Zn(II) complexes of these ligand syst ems have not been reported yet. Thus we decided to synthesize 1, 3,5-tris(4,7-dimethyl-1,4,7-tr iazacyclonon-1-ylmethyl)-2,4,6triethylbenzene ( 4-16 ) and 1,3,5-tris(1,4,7-triaza cyclonon-1-ylmethyl)-2,4,6-triethylbenzene ( 417 ). These are accessible in a simple one-step procedure starting from 4-15 and corresponding amines as illustrated in Fi gure 4-14 and 4-15 respectively. 4-16 and 4-17 provide three tridentate donor sets to coordinate three metal ions. The NMR spectra of 4-16 and 4-17 are in agreement with the C3-symmetry of an ababab conformation in solution. Add ition of three equivalents of Zn(ClO4)2H2O to a solution of 4-17 in acetone afforded the tris-Zn(II) complex 4-18 the solid state structure of which, as determined by X -ray crystallography, is shown in Figure 4-16. As seen in the structure of 4-18 each ligand arm of the platform is coordinated to a single octahedral Zn(II) ion with bond lengths typi cal of TACN-Zn(II) complexe s, and the remaining three coordination sites are occupied by acetonitrile or water molecu les. The coordinated solvent molecules cause the zinc sites to turn outward fr om the center of the platform, but the ability of these groups to rotate inwards for intramolecu lar substrate binding appears possible from primitive computer models of the complex.

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93 It will be interesting to compare the activity of 4-18 with the C3-symmetric trinuclear Zn(II) hydroxide catalyst 3-1 to give us a better insight into the role played by the triphenoxymethane platform in preorganizing three zinc ions for cooperative behavior. 4.3 Conclusions Extensive exploration aimed at the functionalization of the para -positions of the triphenoxymethane platform led to the successful synthesis of two C3-symmetric trinuclear ligand systems 4-12 and 4-14 capable of preorganizing three metal ions into an environment conducive to intramolecular inter action. The extent of exploratory synthesis required for the synthesis of the above ligands was surprising si nce the triphenoxymethane platform apparently appears to be similar to the calixarenes. Apart fr om binding zinc ions, these ligands can also be used to bind other metal ions such as copper. These C3-symmetric ligand systems will provide much insight into the little understood reactivity of three pre-organized copper centers and might prove to be a good model for the tri-copper center of particulate methane mono-oxygenase.149 4.4 Experimental Section 4.4.1 General Methods All reagents and solven ts were of analytical grade a nd were used without purification, unless otherwise noted. All 1H and 13C spectra were recorded on a Varian VXR-300 or Mercury300 spectrometer at 299.95 and 75.4 MHz for the proton and carbon channels, respectively. Elemental analyses were performed by the Univ ersity of Florida Spectroscopic services. 3,5tert butyl-2-hydroxybenzaldehyde and 4tert -butyl-2-methylphenol were obtained from commercial sources and was used as r eceived. 1,4,7-tr iazacyclo[5.2.1.04,10]decane,91 1,4-dimethyl-1,4,7triazacyclononane,150,151 5tert -butyl-2-hydroxy-3-methylbenzaldehyde152 were synthesized following literature methods.

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94 4.4.2 Synthesis Preparation of tris(3tert -butyl-2-hydroxyphenyl)methane (4-3) 4-3 was prepared by a slight modification of the procedure reported by Casnati et al .133,134 To a solution of 2tert butylphenol (22.2 g, 0.15 moles) in a nhydrous diethylether was added dr op wise to an equivalent of ethyl magnesium bromide (19.73 g, 0.15 moles). When the evolution of ethane had ceased, a third of an equivalent of triethylorthofor mate (7.31 g, 0.05 moles) was added. The resulting solution was heated, allowing the ether to evapor ate, and occasionally a small amount of toluene was added to aid mixing. The mixture was heat ed to 100C for 12 hours, during which time the solution became deep blue, purple and then grad ually turned yellow. After allowing the mixture to cool, hydrochloric acid (2 M) was added, and the product extracte d into diethylether. Evaporation of the solvent left a viscous oil, which, upon addition of hexane, precipitated as a red solid. The filterate, which contains the produc t, was concentrated under vacuum to afford a red solid, which after sublimation gave 4-3 in 50 % yield. 1H NMR (CDCl3): = 1.40 (s, 27H; Ar-C(C H3)3), 5.00 (s, 3H; O H ), 5.81 (s, 1H; C H ), 6.75 (dd, 3J (H,H) = 7.8 Hz, 4J (H,H) = 1.5 Hz, 3H; ArH ), 6.88 (t, 3J (H,H) = 7.8 Hz, 3H; ArH ), 7.33 (dd, 3J (H,H) = 7.6 Hz, 4J (H,H) = 1.7 Hz, 3H; ArH ). 13C NMR (CDCl3): = 30.05 (Ar-C( C H3)3), 34.98 (ArC (CH3)3), 41.38 (C H ), 121.34, 126.64, 127.16, 127.27, 138.25, 153.41 (Ar). ESI FT-ICR MS m/z = 483.29 [M+Na]+. Preparation of 4-12 To 0.50 g (1.2 mmol) of 4-7 dissolved in 150 ml of absolute ethanol was added to a slurry of 0.69 g (3.6 mmol) of 3-methyl-5-tert-butyl-2-hydroxybenzaldehyde in 10 ml of ethanol. The reaction was refluxed fo r 12 hours. On cooling the solution to room temperature a bright yellow solid precipitated. Th e solution was filtered and dried to afford the product in an 89 % yield (1 g). 1H NMR (CDCl3): = 1.28 (s, 27H; Ar-C(C H3)3), 2.26 (s, 9H; Ar-C H3), 3.64 (s, 9H; OC H3), 6.59 (s, 1H; C H ), 6.66 (d, 4J (H,H) = 2.7 Hz, 3H; ArH ), 7.03 (d, 4J (H,H) = 2.7 Hz, 3H; ArH ), 7.18 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.24 (d, 4J (H,H) = 2.1 Hz,

PAGE 95

95 3H; ArH ), 8.53 (s, 3H; NC H ).13C NMR (CDCl3): = 15.99 (ArC H3), 16.85 (ArC H3), 31.67 (Ar-C( C H3)3), 34.11 (ArC (CH3)3), 38.69 ( C H), 60.43 (O C H3), 117.97, 121.79, 121.82, 125.56, 126.39, 131.67, 132.41, 137.86, 141.27, 144.42, 155.60, 157.25 (Ar), 162.70 (N C H). Anal. Calcd. For 4-12 (C61H73N3O6): C, 77.59; H, 7.79; N, 4.45. Found: C, 77.63; H, 7.96; N, 4.18. Preparation of 4-14 To 0.50 g (1.2 mmol) of 4-7 dissolved in 150 ml of absolute ethanol was added to a slurry of 0.83 g (3.6 mmol) of 3,5-ditert -butyl-2-hydroxybenzaldehyde in 10 ml of ethanol. The reaction was refluxed for 12 hours. The solution was cooled to room temperature and was concentrated under vacuum. Cooling this concentrated solution to 0C resulted in the precipitation of a pale yellow solid. The solution was filtered and dried to afford the product in 91 % yield (1.15 g). 1H NMR (CDCl3): = 1.27 (s, 27H; Ar-C(C H3)3), 1.42 (s, 27H; ArC(C H3)3) 2.36 (s, 9H; Ar-C H3), 3.61 (s, 9H; OC H3), 6.61 (s, 1H; C H ), 6.69 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.00 (d, 4J (H,H) = 2.1 Hz, 3H; ArH ), 7.15 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 7.38 (d, 4J (H,H) = 2.4 Hz, 3H; ArH ), 8.49 (s, 3H; NC H ).13C NMR (CDCl3): = 16.81 (ArC H3), 29.63 (Ar-C( C H3)3), 31.67 (Ar-C( C H3)3), 34.34 (ArC (CH3)3), 35.22 (ArC (CH3)3), 38.61 ( C H), 60.40 (O C H3), 118.54, 121.32, 121.34, 126.94, 127.86, 132.38, 136.97, 140.64, 144.61, 155.50, 158.20 (Ar), 163.45 (N C H). Anal. Calcd. for 4-14 H2O (C70H93N3O7): C, 77.24; H, 8.61; N, 3.86. Found: C, 77.23; H, 8.85; N, 3.72. Preparation of 1,3,5-tris(1,4,7-triazacycl onon-1-ylmethyl)-2,4,6-triethylbenzene trihydrobromide (4-16). 4-16 was prepared using a slight modification of the procedure reported by Murray et al.148 A solution of 1,3,5-tris(bromome thyl)-2,4,6-triethylbenzene (0.93 g, 2.1 mmol) in dry CH2Cl2 (25 ml) was added drop wise to a stirred solution of 1,4,7triazatriacyclo-[5.2.1.04,10]decane (1 g, 7.2 mmol) in dry CH2Cl2 (25 ml) over one hour. The solution was stirred overnight, resulting in the formation of an off-white precipitate. The

PAGE 96

96 precipitate was then dissolved in deionized water (10 ml) and refluxed for 4 hours. Sodium hydroxide pellets (2 g, 50 mmol) were carefully added in portions to th e resulting solution and refluxing was continued for another 4 hours. Toluen e (50 ml) was then adde d to the solution and the water was distilled off using a Dean-Stark azeotropic apparatus. Th e toluene solution was filtered hot to remove the precipitate of NaBr and NaOH. The solid residue was then extracted twice with hot toluene (100 ml) and the combined toluene fr actions evaporated to dryness under vacuum to yield an oily residue. The crude product was purified by column chromatography (CHCl3/CH3OH, 95/5) to afford light yellow oil. This was then dissolved in water (5 ml) and concentrated HBr (20 ml). To the resulting so lution, absolute ethanol (50 ml) was added drop wise to afford 0.86 g (70 %) of 4-16 as an off-white precipitate. 1H NMR (CDCl3): = 1.04 (t, 3J (H,H) = 7.2 Hz, 9H; CH2C H3), 2.59-2.76 (m, 36H; tacn-C H2), 2.95 (q, 3J (H,H) = 7.2 Hz, 6H; C H2CH3), 3.71 (s, 6H; tacn-N-C H2), 5.74 (s, 6H; N H ). 13C NMR (CDCl3): = 16.06 (CH2C H3), 22.03 ( C H2CH3), 43.37, 42.26, 49.18 (tacnC H2), 52.53 (tacn-NC H2), 131.66, 144.93 (Ar). Anal. Calcd. for 4-16 3HBr3EtOH (C39H84N9O3Br3): C, 48.45; H, 8.75; N, 13.04. Found: C, 48.53; H, 8.27; N, 13.02. Preparation of 1,3,5-tris(4,7-dimethyl1,4,7-triazacyclonon-1-ylmethyl)-2,4,6triethylbenzene trihydrobromide (4-17). A suspension of 1,3,5-tris(bromomethyl)-2,4,6triethylbenzene (0.44 g, 2.5 mmol ), 1,4-dimethyl-1,4,7-triazacy clononane (1.3 g, 8.3 mmol) and solid KOH (0.56 g, 10 mmol) in toluene (20 ml) wa s heated at 80C while stirring for 2 days. The solid material was then filtered off and the solution was dried over MgSO4. After removing the solvent under reduced pressure the residue was dissolved in water (5 ml) and concentrated HBr (20 ml). To the resulting solu tion, absolute ethanol (50 ml) was added drop wise to afford 1.5 g (89 %) of 4-17 as a pale yellow solid. 1H NMR (CDCl3): = 0.97 (t, 3J (H,H) = 7.2 Hz, 9H;

PAGE 97

97 CH2C H3), 2.16 (s, 18H; N-C H3), 2.41-2.61 (m, 36H; dmtacn-C H2), 3.02 (q, 3J (H,H) = 7.5 Hz, 6H; C H2CH3), 3.47 (s, 6H; dmtacn-N-C H2). 13C NMR (CDCl3): = 15.92 (CH2C H3), 22.28 ( C H2CH3), 46.14 (NC H3), 54.82, 55.17, 55.97 (dmtacnC H2), 56.89 (dmtacn-NC H2), 132.20, 144.43 (Ar). Anal. Calcd. for 4-17 HBrH2O (C39H84N9O3Br3): C, 48.45; H, 8.76; N, 13.04. Found: C, 48.28; H, 8.29; N, 13.11. 4.4.3. X-ray Crystallography Unit cell dimensions and intensity data for all the structures were collected by Prof. Michael J. Scott on a Siemens CCD SMART diff ractometer at 173 K. The data collections nominally covered a hemisphere of reciprocal sp ace, by a combination of th ree sets of exposures; each set had a different angle for the crystal, and each exposure covered 0.3 in The crystal to detector distance was 5.0 cm. The data sets were corrected for absorption using SADABS. All the structures were solved by Prof. Michael J. Scott using the Bruker SHELXTL software package for the PC, using the direct methods option of SHELXS. The space groups for the structures were determined from an examina tion of the systematic absences in the data, and the successful solution and refinement of th e structures confirmed these assignments. All hydrogen atoms were assigned idealized locatio ns and were given a thermal parameter equivalent to 1.2 to 1.5 times the thermal parameter of the atom to which it was attached. For the methyl groups, where the location of hydrogen atoms was uncertain, the AFIX 137 card was used to allow the hydrogen atoms to rotate to the maximum area of residu al density, while fixing their geometry.

PAGE 98

98 C O H 3 N C O H 3 N N Zn O H OR" OR" OR" OR" R" = CH2CH2OEt N N N Zn N N N Zn N N N Zn 3-1 1-44-1 =Functional group Figure 4-1. Various trinuc lear ligand systems. OH 4-2 3 10 AlCl3 Toluene H Figure 4-2. Attempted detert -butylation of 4-2 OH 3+3 MgBrEt + HC(OEt)3 Et2O OH H 4-3 3 Figure 4-3. Synthesis of ligand 4-3

PAGE 99

99 + CH3(CH2)7OCH2Cl + SnCl4 CHCl3 OH H 4-3 3 Figure 4-4. Attempted chloromethylation of 4-3 + (HCHO)n+ 33% HBr / CH3COOH OH H 4-3 3 Figure 4-5. Attempted bromomethylation of 4-3 NH2 O CH 3 4-5 4-4 NH2 O CH 3 C3H7 C5H11 Figure 4-6. Tris(2-alkox y-5-nitrophenyl)methanes reported in literature. 4-6 NO2 OH (iii) 4-8 (ii) (i) 3 NO2 O 3 NH2 O 3 NO2 OH NO2 OH O H + 4-7 Figure 4-7. Synthesis of ligand 4-8 Reagents and conditions: (i) H2SO4, 155C (ii) 5 MeI, 5 K2CO3, DMF (iii) Pd/C, H2N-NH2, EtOH

PAGE 100

100 OH 4-2 O 33 NO2 O 3 (iii) NH2 O 3 4-9 (ii) (i) Figure 4-8. Synthesis of ligand 4-9 Reagents and conditi ons: (i) 5 MeI, 5 K2CO3, DMF (ii) 6.6 HNO3, 6.6 TFA, CH2Cl2 (iii) Pd/C, H2N-NH2, EtOH O NH H 3 O NH2 H 3 + 6 CH3COOH Dry MeOH 4-8 4-10 N N 6 Figure 4-9. Synthesis of ligand 4-10 Zn O Zn N N O O N N O O OH HN NH N N Hbdaip (bdaip)[Zn2(O2CMe)2]+4-11 Figure 4-10. Schiff base complexes used in lite rature capable of ami nopeptidase-like activity.

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101 O N OH H 3 O NH2 H 3 OH O H + EtOH Reflux 4-9 4-12 3 Figure 4-11. Synthesis of ligand 4-12 N Zn O N O N Zn O N O N Zn O N O N O N O N O O N O H 3 4-124-13 Figure 4-12. Cartoon representation of 4-13 O N OH H 3 O NH2 H 3 OH O H + EtOH Reflux 4-9 4-14 3 Figure 4-13. Synthesis of ligand 4-14

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102 N N N 4-154-16 Br Br Br +3.4 N N N H H N N N H H N N N H H CH2Cl2 Figure 4-14. Synthesis of ligand 4-16 N N N 4-154-17 Br Br Br +3.4 N N N N N N N N N CH2Cl2 H Figure 4-15. Synthesis of ligand 4-17 Figure 4-16. Depiction of th e solid-state structure of 4-18

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103 CHAPTER 5 SUMMARY Understanding the mode of action of trinuclear zinc hydrolases is a very challenging field of research, which requires a great amount of interdisciplinary c ooperation. Traditional molecular and structural biology lays the f oundation upon which mechanistic investigations follow. Parallel investigations on the enzyma tic systems themselves, the synthesis and mechanistic studies of smaller inorganic trizinc complexes, promise invaluable insight into the chemical processes taking place in the heart of the enzymatic active sites. The inorganic chemistry of three pre-organized zinc ions has not been examined in detail because of the lack of an appropriate ligand system capable of orien ting three metal ions in close proximity for cooperative activity. To address this issue, a simple ligand capabl e of preorganizing three metal ions into an environment conducive to intr amolecular interactions was designed using a C3symmetric triphenoxymethane platform. Synthesi zed by tethering three phenol moieties to a central methine carbon, these compounds not only po ssess all the properties of calix[4]arene that make them ideal scaffolds for ligand system but also possess a useful three-fold symmetry. The corresponding Zn(II) complex was subsequently s ynthesized and fully characterized using NMR spectroscopy and X-ray crystallography. This complex induces a 16,900-fold rate enhancement in the catalytic cyclization of the RNA model substrate, 2-hydroxypropyl-p -nitrophenyl phosphate (HPNP, pH 6.7, 25C), over the uncataly zed reaction with multiple catalyst turnover. The observed differences in the pH-rate profile can be attributed to the varying concentration of various trinuclear zinc species The Zn(II) catalyst exhibits a higher hydrolytic activity as compared to its mononuclear analogue. Si nce subtle differences in the mutual arrangement/orientation of the metal binding arms have a cruc ial influence on the intrinsic activity of these trinuc lear Zn(II) complexes, a modified triphenoxymethane platform wherein

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104 the metal binding arms are directly attached to the aromatic ring was synthesized. The extent of exploratory synthesis required for the synthesis and functiona lization of the platform was surprising since the triphenoxymethane platform apparently appears to be similar to the calixarenes. Derivatization of this modified platform led to the synthesis of two new C3symmetric trinuclear ligand system which were su bsequently used to generate some interesting trinuclear Zn(II) clusters. Interest in such biomim etic systems is not restricted to the elucidation of the enzymatic mechanism, but may eventually l ead to beneficial appli cations in biotechnology and medicine.

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113 BIOGRAPHICAL SKETCH Ranjan Mitra was born in Dhanbad, Indi a on December 25, 1976. He performed his high school studies at De Nobili School, Dhanbad. After which he left the comforts of his home and traveled across the country to join the renowned Hindu College, De lhi to pursue a under graduate degree in chemistry. He then succ essfully qualified in the joint entrance exam of the prestigious Indian Institute of Technology, Delhi to pursue hi s masters in chemistry. During his masters, he worked under the guidance of Professor Debkumar Bandyopadhyay on selective C-Pd bond oxidation using hydrogen peroxide. Af ter the completion of his mast ers degree, Ranjan joined the research group of professor Mi chael J. Scott at the Univers ity of Florida in August 2001. He completed his requirements for the degr ee of Doctor in Philosophy in January 2007.