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Modeling Nonheme Iron Enzymes with the Monoanionic Heteroscorpionate Ligand Bdippza By Laura Cunningham A Thesis Submitted to the Department of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree of Bachelor of Arts in Chemistry/Biology Under the sponsorship of Dr. Suzanne E. Sherman Sarasota, FL May, 2013
ii Acknowledgements I would like to thank Dr. Sherman for sponsoring my thesis and providing me with support and advice over the past three semesters. I would like to thank my other committee members, Dr. Scudder and Dr. Clore, both of whom have helped shape my research interests during my time at New College. I would also like to thank Dr. Roberts for assisting me in findi ng v arious items around the lab; I would like to thank Colleen Swessel for purchasing my chemicals for me. Thank you to my family for always supporting me and providing me with encouragement over the past four years, especially as I went through the thesis pr ocess. I would like to thank my roommates Elena Korallis, Daphne Hudson, and Jillian True. Living with them has made the thesis process much more bearable. I would also like to thank Elena for coming to the lab with me at night and on the weekends. Additio nally I would like to thank Jehan Sinclair and Ashley Parks for accompanying me on late night Starbucks excursions and providing me with support. Finally, I would like to thank the New College Foundation and the Council of Academic Affairs for providing me with funding to carry out my thesis project.
iii Modeling Nonheme Iron Enzymes with the Monoanionic Heteroscorpionate Ligand Bdippza Laura Cunningham New College of Florida, 2013 Abstract The N 2 O heteroscorpionate ligand bis 3,5 diisoprop ylpyrazol 1 ylacetate (bdippza) has been synthesized for use in modeling the active site of nonheme iron enzymes. The active sites of nonheme iron enzymes consist of two histidine residues and a carboxylate residue. The ligand Hbdippza can serve as a good mimic of that coordination environment due to the presence of two pyrazole groups and a carboxylate arm. The pale green crystalline bis complex Fe(bdippza) 2 was isolated after treating bdippza with [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] and the synthesis was found to be r eplicable The yield of the reaction was 6.24%. Formation of a bis complex was confirmed by FT IR spectroscopy and elemental analysis. The relevance of the complex for future synthetic models is also discussed. _______________________________________ S uzanne Sherman Natural Sciences Division
iv Table of Contents ACKNOWLEDGEMENTS ii ABSTRACT iii LIST OF FIGURES v LIST OF SCHEMES vi CHAPTER 1: INTRODUCTION 1 Extradiol Dioxygenases 2 Biphenyl Metabolism 3 Enzyme Str ucture of BphC 8 Working Towards a Potential Mechanism 11 Alpha keto Dependent Enzymes 14 AlkB and ABH2: Homologous DNA D emethylases 14 A Potential Catalytic Mechanism 18 Rieske Dioxygenases 20 Naphthalene Metabolism 21 Naphthalene Dioxygenase: Structure and Reactivity 24 Possible Catalytic Mechanism of the Rieske Dioxygenases 28 N 2 O Tripodal Ligands 30 Bdippza: A New Ligand for Modeling Nonheme Iron E nzymes 37 CHAPTER 2: EXPERIMENTAL 39 Genera l Considerations 39 Preparation of Ligands 39 Preparation of Iron Complexes 42 CHAPTER 3: RESULTS AND DISCUSSION 43 Ligand Synthesis: Analysis of Reaction Mechanism and Spectra 43 Iron Complexes: Syn thesis and Spectroscopic Data 50 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS 55 APPENDIX A: SPECTRA 58 REFERENCES 66
v List of Figures Figure 1 : P. pseudoalcaligenes KF707 bph gene cluster and the enzymes encoded by each gene ..5 Figure 2: Gene clusters of P. putid a KF715, Rhodococcus M5, and Rhodococcus RHA1 and the enzymes they encode ...6 Figure 3: Regulation of the biphenyl metabolism genes in P. pseudoalcaligenes KF707 ...7 Figure 4: N and C terminal domains of BphC 9 Figu re 5: Substrate free BphC in a square pyramidal geometry 10 Figure 6: BphC bound to the substrate, DHBP in an octahedral geometry ...10 Figure 7: BphC with both substrate and NO bound ...10 Figure 8: Active site of homoproto catech uate 2,3 dioxygenase with 4 nitrocatechol and O 2 bound to the iron center 12 Figure 9: Active site of homoproto catechuate 2,3 dioxygenase with 4 nitrocatechol and O 2 forming an alkylperoxo species 13 Figure 10: S tructure of taurine ...14 Figure 11: Structure of penicillin N ...14 Figure 12: Stabilization of 1 meG by H131 and W69 and location of Aspartate 135 in AlkB from E. coli ............................................... ................................................................16 Figure 13: Active site of Mn substituted ABH2 16 Figure 14: 1 meA mutated dsDNA bound to Mn substituted AlkB ..17 Figure 15: 1 meA mutated dsDNA bound to Mn substitute d ABH2 17 Figure 16: Rieske cluster ...21 Figure 17: a.) Structure of naphthalene dioxygenase showing the subunits positioned beneath the subunits. b.) The top view of naphthalene dioxygenase showing the mononuclear iron c enter and Rieske cluster ...25 Figure 18: The subunit of NDO. The blue ribbon is the active site domain, and the magenta ribbon is the Rieske domain .25 Figure 19: Route of electron transfer between the Rieske center an d active site, which are connected by Asp205, in naphthalene dioxygenase ..26 Figure 20: a.) Structure of the active site of NDO with O 2 bound. b.) Structure of the active site of NDO with indole and O 2 bound at the active site 27 Fi gure 21: The model complex [Fe(OTf) 2 ( Me,H Pytacn)] is a stereo selective alkane hydroxy lating catalyst 29 Figure 22: Common ligands used in modeling Fe(II) nonheme enzymes .30 Figure 23: A series of N 2 O heteroscorpionate ligan ds used in the synthesis of metal ligand complexes capable of producing the extradiol product of 3,5 di tert butylcatechol (H 2 dtbc) ..31 Figure 24: The substrate 3,5 di tert butylcatechol and the observed products. The product s include the quinone product resulting from auto oxidation, two extradiol products, and two intradiol products ..32 Figure 25: A series of ligands for modeling extradiol dioxygenases .32
vi Figure 26: A series of N 2 O n ligands used i n ferric and ferrous complexes modeling the active site of the keto dependent enzymes .34 Figure 27: General structure of the bis (pyrazol 1 yl)acetate ligands ...35 Figure 28: The bis complex [Fe(bdmpza) 2 ] ...35 Fig ure 29: The dimer complex [Fe(bd t bpza)Cl] 2 ...35 Figure 30: Proposed structure of the complex [Fe(bd t bpza)(O2CC(O) Ph)] ...36 Figure 31: Crystal structure of the ferric complex [NEt 4 ][Fe(bdmpza)Cl 3 37 Figure 32: Structure of Hbdippza ..37 Figure 33: 1 H NMR spectrum of Hbdippza in CDCl 3 ...48 Figure 34: 13 C NMR spectrum of Hbdippza in CDCl 3 .49 Figure 35: Solid state FT IR spectrum of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] .51 Figure 36: UV Vis spectr um of product identified as [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in acetonitrile ..52 Figure 37: Solid state FT IR spectrum of Fe(bdippza) 2 53 Figure 38: 1 H NMR spectrum of 3,5 diisopropylpyrazole in CDCl 3 .57 Figure 39: 1 H NMR spectrum of bdippzm in CDCl 3 .58 Figure 40: 1 H NMR spectrum of Hbdippza in CDCl 3 ...59 Figure 41: 13 C NMR spectrum of Hbdippza in CDCl 3 ..60 Figure 42: Solid state FT IR spectrum of Hbdippza .61 Figure 43: Solid state FT IR spectrum of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] ..62 Figure 44: UV Vis spectrum of product identified as [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in acetonitrile ..63 Figure 45: Solid state FT IR spectrum of Fe(bdippza) 2 64 List of Schemes Scheme 1: General reaction scheme of the extradiol dioxygenases....2 Scheme 2: Biphenyl Degradation Pathway.4 Scheme 3: Reaction carried out by BphC....8 Scheme 4: Proposed mechanism of ex tradiol dioxygenases..11 Scheme 5: Alkenyl migration in the extradiol dioxygenases.13 Scheme 6: Acyl migration in the intradiol dioxygenases..13 Scheme 7: The general reaction scheme of the keto dependent enzymes..1 4 Scheme 8: Reaction schemes of the enzymes AlkB and ABH2. Both enzymes demethylate methylated adenine and cytosine bases.15 Scheme 9: Proposed mechanism for the keto dependent class of dioxygenases ... 18 Scheme 10: Reactions of [(Tp iPr2 )F e II (O 2 CC(O)R)] (left) with O 2 19 Scheme 11: Hydroxylation of the substrate by a high valent oxo species.20 Scheme 12: General reaction scheme of the Rieske dioxygenases.20 Scheme 13: Upper pathway of naphthalene degradation ...22 Scheme 14: Lower pathway of naphthalene degradation..23 Scheme 15: Reaction of naphthalene dioxygenase: Naphthalene is converted to cis 1,2 dihydroxy 1,2 dihydronaphthalene24 Scheme 16: Proposed catalytic mechanism for the Rieske dioxygenases .28
vii Scheme 17: Mechanism for formation of the intradiol product .33 Scheme 18: Proposed reaction mechanism for synthesis of 3,5 diisopropylpyrazole from 2,6 dimethyl 3,5 heptanedione and hydrazine .. 44 Scheme 19: Proposed mechanism for the synthesis of bdippzm from dippz 45 Scheme 20: Proposed mechanism for the synthesis of bdippza from bdippzm 47 Scheme 21: Reaction scheme for the synthesis of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] ....50
1 Chapter 1: Introduction Nonheme iron(II) enzymes are oxygenases that contain a mononuclear iron coordinated to two histidines and one carboxylate amino acid, glutamate or aspartate. These enzymes typical ly fall into one of six classes and are categorized by their re actions. The classes include the extradiol catechol dioxygenases, the alpha keto dependent enzymes, the pterin dependent hydroxylases, and the Rieske dioxygenases. The enzymes isopenicillin N synthase, and 1 aminocyclopropa ne 1 carboxylate oxidase (ACCO) e ach fall into their own category. Although the overall reaction differs between the different classes, each of the enzymes implements the use of molecular oxygen to carry out oxidative chemistry. The biological functions of these enzymes vary widely and i nclude the degradation of aromatic compounds such as biphenyl and naphthalene in soil bacteria 1, 2 demethylation of DNA in both humans and E. coli 3, 4 synthesis of the neurotransmitters dopamin e and serotonin in nerve cells 5, 6 synthesis of anthocyanins in pl ants 7 and degradation of lign in by soil bacteria 8 The field of bioinorganic chemistry has continually implemented the use of synthetic model complexes to probe the reactivity of metal containing enzymes such as the nonheme iron enzymes. These metal complexes have been instrumental in elucidating the mechanisms of the various classes of nonheme iron enzymes by trapping unstable intermediates and high oxidation states of iron. Synthetic models of nonheme iron enzymes, particularly of the extradiol dio xygenase s keto dependent enzymes, and the Rieske dioxygenases, can also lead to the production of greener oxidative catalysts and
2 possible bioremediation tools. The purpose of this thesis project is to synthesize a n ew iron complex as a synthetic model of these enzymes. The following sections outline the expression and structure of three different nonheme iron enzymes, one extradiol dioxygenase, one keto dependent dioxygenase, and one Rieske dioxygenase, a s well as the proposed mechan isms for each of these classes. These three classes of nonheme iron enzymes showcase the importance of model complexes in elucidating the mechanism s of these enzymes and the variety of oxidative reactions these enzymes can carry out. 1.1 Extradiol Dioxygenases Extradiol catechol dioxygenases are found predominantly in soil bacteria and are a part of the pathways for the metabolism of various aromatic compounds such as biphenyl, catechol, and naphthalene, into useable energy sources for the bacteria. The general reaction scheme involves the meta cleavage of a 1,2 dihydroxylated aromatic compound (Scheme 1). Chemists and biochemists have sought to gain a better understanding of the mechanisms of these enzym es, particularly the role of the iron center in catalysis. Both enzyme crystallography and model complexes have assisted in the elucidation of the mechanism. Model complexes are particularly useful for characterizing the oxidation states of iron throughout the catalytic cycle. Active model Scheme 1: General reaction scheme of the extradiol dioxygenases
3 complexes could serve as potential sensors for detecting chlorinated biphenyls and catechols in waterways. 1.1.1 Biphenyl Metabolism Researchers have sought to understand the genetic underpinnings of biphenyl metabolism by soi l bacteria because many of these bacteria are also capable of metabolizing polychlorinated biphenyls (PCB). These compounds have been linked to cancer 9 and complications during cognitive development in children who received breast milk con taminated with PCBs 10 Many areas around the United States remain contaminated with PCBs, despite the fact that their use was banned in the U.S. in 1976 under the Toxic Substances Control Act. Although natural sources of PCB exist, such as coal tar, crude oil and natural gas 11 most PCBs originate from industrial sources during the manufacturing of transformers, capacitors, and electric motors. Areas that still remain contaminated by PCBs include the Great Lakes and Lake Long Martin Alabama 12, 13 where PCBs have b ecome present in the fish populations. An important goal of researchers is to implement soil bacteria that contain extradiol dioxygenases in the cleanup of areas contaminated by the chlorinated derivatives of biphenyl and catechol. In order to utilize the soil bacteria's ability to degrade PCBs, researches have investigated the genetic basis for this metabolism. The metabolic pathway and enzymes involved (Scheme 2) are relatively conserved throughout various species of soil bacteria, including Sphingomonas aromaticivorans F199 14 P. putida KF715 15 P. pseudoalcaligenes KF707 16 and Rhodococcus strains M5
4 and RHA1 17, 18 all of which are capable of degrading biphenyl and its chlorinated derivatives. Scheme 2 shows the metabolism of biphenyl to 2 hydroxypenta 2,4 dienoic acid (Scheme 2 (V)) and benzoic acid (Scheme 2 (VI)). It is a fo ur step process requiring four enzymes, the third of which is BphC, an extradiol dioxygenase. These enzymes are encoded by a total of seven genes 19, 20, 18 1.1.1.a Gene Organization Although the same enzymes are implemented in biphenyl metabolism by the v arious species of bacteria, the gene organization differs. In P. pseudoalcaligenes KF707, the seven above mentioned genes are accompanied by several other genes, including regulatory, in the chromosomal gene cluster shown in Figure 1 19 Scheme 2: Biphenyl Degradation Pathway (From ref. 18) The compounds are (I) biphenyl, (II) 2,3 dihydroxy 1 phenylcyclohexa 4,6 diene (III) 2,3 dihydroxybiphenyl (2,3DHBP), (IV) 2 hydroxy 6 oxo 6 phenylhexa 2,4 dienoic acid (HOPD), (V ) 2 hydroxypenta 2,4 dienoic acid and (VI) benzoic acid. The enzymes are (B phA) biphenyl dioxygenase, (BphB) dihydrodiol dehydrogenase, (BphC) 2,3 DHBP dioxygenase, and (BphD) HOPD hydrolase.
5 BphR1 is a GntR regulatory protein, orf3 is an open reading frame with as of yet no known function with respect to biphenyl metabolism, and bphX0, bphX1, bphX2, and bphX3 encode glutathione S transferase, 2 hydroxypenta 2,4 dienoate hydratase acetaldehyde dehydrogenase and 4 hydroxy 2 oxovalerate aldolase respectively 19 The enzymes encoded by BphX1 3 are responsible for the conversion of 2 hydroxypenta 2,4 die noic acid to citric acid cycle intermediates, and the putative function of the glutathione S transferase, bphX0 is dehalogenation of the biphenyl m etabolism products 21 The gene cluster consists of six transcription initiation sites, one upstream of each of the following genes, bphR1, bphA1, bphX1, and bphX0, and two upstream of bphD. 19 The organization in P. putida KF715 (Figure 2) is similar to that of P. pseudoalcalig enes KF707, but P. putida KF715 lacks the bphX genes inserted between bphC and bphD. 15 However, P. putida KF715 also contains a GntR type regulator protein, orf0 16 indicating possible similarities in the regulatory mechanism of the gene clusters. Both Rho dococcus M5 and Rhodococcus RHA1 contain the regulatory genes bphS and bphT, which encode a transmembrane receptor like histidine kinase and a Figure 1: P. pseudoalcaligenes KF707 bph gene cluster and the enzymes encoded by each gene (From ref. 16)
6 cognate response element, respecti vely 22 While the Rhodococcus M5 gene cluster is chromosomal, the biphenyl meta bolism genes of Rhodococcus RHA1 are located on the plasmids pRHL1 and pRHL2. The gene cluster shown in Figure 2 is located on pRHL1, while another cluster, bphDEF, which encodes HOPDA hydrolase, 2 hydroxypenta 2,4 dienoate hydratase and 4 hydroxy 2 oxovalerate aldolase respectively, is located on pRHL2 23 1.1.1.b Regulation of the Pathway More information remains to be elucidated regarding the regulation of the biphenyl metabolism genes; however, at least one regulatory aspect has been discovered for P. pseudoalcaligenes KF707, P. putida KF715, Rhodococcus M5, and Rhodococcus RHA1. The genes in both P. pseudoalcaligenes KF707 and P. putida KF715 are regulated by GntR type re gulator protein 16 and those in Rhodococcus RHA1 and Rhodococcus M5 are regulated by a two component regulator y system. 25, 22 Figure 2: Gene clusters of P. putida KF715 Rhodococcus M5, and Rhodococcus RHA1 and the enzymes they encode (From ref. 24)
7 One of the more complex regulatory pathways is that of P. pseudoalcaligenes KF707 (Figure 3). In addition to the GntR regulatory protein, referred to as bphR1 another regulatory protein has been discovered, bphR2, whose location within the genome is still unknown. BphR2 is thought to belong to the LysR family of regulatory proteins and is required for transcription initiation of the biphenyl metabolism genes. The transcription of bphR2 is increased in the presence of biphenyl, and the gene product of bphR2 binds to the promoter region of both bphR1 and the bphA1A2orf3A3A4BC gene segment, leading to the production of the Gnt R protein and the compound HOPD, shown in Scheme 2 (IV) and in Figure 3. 16 HOPD then binds to the GntR protein, resulting in the upregulation of bphR1. The GntR protein also binds upstream of bphX0, bphX1, and bphD leading to transcription of those genes 19 Figure 3: Regulation of the biphenyl metabolism genes in P. pseudoalcaligenes KF707 (From ref. 16)
8 The two component regulatory system in Rhodococcus strains M5 and RHA1 consists of a receptor like histidine kinase, encoded by bphS and a cognate response element, encoded by bphT. The histidine kinase functions as a signal transmitter and the cognate response element receives the signal from the transmitter and produces a cha nge in gene expression 22, 26 Analysis of bphS has shown seven possible transmembrane segments within the amino acid sequence, as well as four conserved sequence blocks, H, N, G1, and G2, typically found in receptor like histidine kinases The H sequence contains a histidine phosphorylation site, the G sequences are nucleotide binding sites, and the N sequence is still o f unknown function 22 BphT has two domains, a receiver module, which contains an aspartate resid ue that becomes phosphorylated by bphS, and an effector domain, which conta ins three DNA binding regions 27, 22 It is still unknown, however, how these regulatory genes are intertwined with expression of the other biphenyl metabolism genes. 1.1.2 Enzyme St ructure of BphC The enzyme 2,3 dihydroxybiphenyl 1,2 dioxygenase (BphC), which catalyzes the conversion of 2,3 dihydroxybiphenyl (2,3DHBP) to 2 hydroxy 6 oxo 6 phenylhexa 2,4 dienoic acid (HOPD ) ( Scheme 3), contains a nonheme iron(II) prosthetic group which utilizes molecular Scheme 3: Reaction carried out by BphC. (From ref. 28)
9 oxygen to cleave the hydroxyphenyl ring adjacent to one of the hydroxyl groups. The enzyme exists as a homooctamer, and each subunit consists of an N terminal domain and C ter minal domain, in which the active site remains buried (Figure 4) 28 Crystallographic data on BphC shows the iron atom located at the active site is coordinated to two histidines, His145 and His 209, one carboxylate amino acid, Glu 260, and two water molecules in a square pyramidal geometry (Figure 5). The equatorial ligands are His 145, Glu 260, and the two water molecules, and the axial ligand is His 209. Upon substrate binding, the hydroxyl groups of the substrate displace the water molecules, and t he iron coordination sphere changes to an octahedral geometry with an open e quatorial position (Figures 6). 1 In the substrate bound form of the enzyme the equatorial ligands are His 145, Glu 260, and one of the hydroxyl groups of the substrate (OH(2)), and the axial ligands are the second hydroxyl group of the substrate (OH(3)) Figure 4: N and C terminal domains of BphC. The C terminal domain is colored white and yellow, and the N terminal domain is colored red and blue. The active site is located between the white and yellow portion of the C terminal domain (From ref. 28)
10 and His 209. The open equatorial position is the tentative molecular oxygen binding position, and has been shown to be the binding position of nitric ox ide (NO) (Figure 7) 1 The substrate, DHBP, is stabilized in the active site by Tyr 249, the N of His 194, and a hydrogen bonding network involving His 194, His 193, and Asp 170 29 Tyr 249 is hydrogen bonded to OH(2), and OH(3) is hydrogen bonded to a water molecule and the N of His 194. The hydrogen bonding network falls into place with the binding of DHBP, which causes His 194 to shift forward by 1.1 . It has been postulated that this network is responsible for the deprotonat ion of the axial OH(3), and therefore plays an important role in catalysis, as discussed below 1 Upon binding of NO, Figure 5: Substrate free BphC in a square pyramidal geometry (From ref. 1) Figure 6: BphC bound to the substrate, DHBP in an octahedral geometry (From ref. 1) Figu re 7: BphC with both substrate and NO bound. The NO fills the open equatorial position in the iron coordination sphere. (From ref. 1)
11 Val 147 shifts away from the active site in order to accommodate the NO binding at the iron atom. The NO is stabilized through hydrophobic interactions with the surrounding amino acids, His 145, Val 147, Phe 186, Ala 197, His 194, and His 209 1 1.1.3 Working Towards a Potential Mechanism The catalytic mech anism for the extradiol dioxygenases has yet to be fully elucidated, however, several intermediates are characterized and a proposed mechanism set in place (Scheme 4 ). The first step involves the binding of the substrate, catechol or one of its derivatives, and displacement of two water molecules 1 Electronic absorption spectroscopy and ultraviolet resonance Raman spectroscopy of BphC confirmed that the substrate bind s in th e monoanionic form 30 and it has been proposed that a nearby histidine acts as a base to remove the first hydroxyl proton. After the substrate binds, dioxygen binds to the iron resulting in the removal of the hydrogen from the second hydroxyl group by another histidine (Scheme 4 (C) 31 Once oxygen binds, it has been Scheme 4: Proposed mechanism of extradiol dioxygenases (Adapted from ref. 32)
12 proposed that an Fe(III) su peroxide complex forms (Scheme 4 (C)) as a result of a one electron transfer from iron to O 2 ; however, this step has not been veri fied spectroscopically 32 Another single electron transfer from the catechol substrat e to the iron results in the formation of a semiquino n atoiro n( II) superoxide spec ies (Scheme 4 (D)), whose existence is supported through experiments with a cyclopropyl radical trap and x ray crystallography. Using the enzyme (2,3 dihydroxyphenyl)propionate 1,2 dioxygenase (MhpB) and the substrates cis and trans 2 (2,3 dihydroxyphenyl)cyclopropane 1 carboxylic acid it was shown that the cyclopropyl ring underwent cis trans isomerization (Spence, 1996), which past experiments using rhodium(III) and iridium(III) synthetic complexes have shown is indicative of a nearby radical and radical indu ced cyclopropyl ring opening 34, 35 X ray crystallography of 4 nitrocat e chol bound to the active site of homoprotocatechuate 2,3 dioxygenase in the presence of molecular oxygen reveals molecular oxygen bound side on to the iron with unsymmetrical Fe O bond distances and a slight puckering of the substrate (Figure 8). The puckering of the ring of the substrate indicates loss of aromaticity and the acquisition of radical character, and the unsymmetrical Fe O bond distances indicate superoxide character for the oxygen species 31 Figure 8: Active site of homoproto catechuate 2, 3 dioxygenase with 4 nitrocatechol and O 2 bound to the iron center. The O 2 is bound side on with unsymmetrical Fe O bond lengths. (From ref. 31)
13 Nucleophilic attack of the catechol ring by the superoxide species generates an alkylperoxo species (Scheme 4 (E)), which has been characterized in homoproto catechuate 2,3 dioxygenase with the substrate 4 nitrocatechol through x ray crystallography in a low O 2 atmosphere (Figure 9) 31 The alkylperoxo species undergoes a Criegee rearrangement to for m an oxolactone ring (S cheme 4 (F)), whose existence has been proven through the use of 18 O 2 and H 2 18 O labeling 36 Some debate still exists, however, as to the exact mechanism for the rearrangment from the alkylperoxo species to the lactone intermediate. It has been proposed that the coordination position of the peroxo group, axial or equatorial, determines whether alkenyl migration (Scheme 5) which results in the extradiol product, or acyl migration (Scheme 6) which leads to the ortho (intradiol product) rather than meta cleavage product, oc curs, respectively 37 Figure 9 shows that the peroxo is bound in the axial position. The final pro duct is formed by attack of the hydroxyl group to the ester carbonyl. Figure 9: Active site of homoproto catechuate 2, 3 dioxygenase with 4 nitrocatechol and O 2 forming an alkylperoxo species (From ref. 31) Scheme 5: Alkenyl migration in the extradiol dioxygenases (From ref. 32) Scheme 6: Acyl migration in the intradiol dioxygenases (From ref. 32)
14 1.2 Alpha keto Dependent Enzymes The alpha keto dependent enzymes are found in a wide variety of organisms including bacteria 38 pl ants 7 and animals 39 and function on a diverse set of substrates, which range from taur ine 40 (Figure 10) to penici llin N 38 (Figure 11) As indicated by the name of these enzymes, ketoglutarate is a required cosubstrate and becomes decarboxylated to form succinate. In the general reaction scheme, one atom of dioxygen becomes incorporated into the substrate and th e other into succinate (Scheme 7 ). In some enzymes, however, a side product is oxidized rather than the substrate; two such enzymes are AlkB and ABH2. 1.2.1 AlkB and ABH2: Homologous DNA demethylases The enzyme AlkB, which is found in E. coli, and its human homologue ABH2 demethylate the DNA bases adenine and cytosine after the y become erroneously methylated. S N 2 methylating agents such as methyl methane sulfonate or meth yl halides (Elder, 1998) lead to the formation of 1 methyladenine (1meA) and 3 methylcytosine (3 Figure 10: Structure of taurine Figure 11: Structure of penicillin N Scheme 7: The general reaction scheme of the keto dependent e nzymes (From ref. 32)
15 meC). The DNA base becomes demethylated by oxidation of the methyl group to produce formalde hyde as a side product (Sch eme 8 ). In general, this type of DNA methylation is deleterious to the body and can lead to genetic mutations or poss ibly cell death due to the inability of the methylated bases to hydrogen bond to their complementary bases in the DNA strand, preventing DNA replication 42, In the case of tumor cells, however, this type of DNA methylation can be beneficial. Lee et al fou nd that the ABH2 gene is upregulated in tumor cells by the transcription factor TP53, tumor protein 53, leading to propag ation of tumore cells 43 Blocking the ability of ABH2 to demethylate DNA, however, could lead to the destruction of tumor cells. The preference for 1 meA and 3 meC mutations in both AlkB and ABH2 is a result of negatively charged amino acid residues near the substrate binding site, which hydrogen bond to the exocyclic amines of the adenine and cytosine. In AlkB, the amino acid is As p 135 is hydrogen bonded 44 (Figure 12), and in ABH2 Tyr 122, Glu 175, Asp Scheme 8: Reaction schemes of the enzymes AlkB and ABH2. Both enzymes demethylate methylated adenine and cytosine bases (From ref. 39)
16 174, and a water molecule are hydrogen bonded 45 (Figure 13) In guanine and thymine, the exocyclic amine is replaced by a carbonyl functional group, wh ose interactions with the carboxylate group causes destabilization of the substrate. While AlkB has similar reactivity towards both 1 meA and 3 meC, ABH2 is four times more reactive towards 1 meA than 3 meC as a result of greater stabilization a t the act ive site 46 Both enzymes also contain aromatic residues, which form pi stacking interactions with the methylated base to create further stabili zation. In Alk B these include Trp 69 and His 131 (Figure 12), and in ABH2 they include Phe 124 and His 171 44, 45 (Figure 13 ) Figure 12: Stabilization of 1 meG by H131 and W69 and location of Aspartate 135 in AlkB from E. coli (From ref. 44) Figure 13: Active site of Mn substituted ABH2. The dashed lines indicated hydrogen bonding, and the methylated base is in pink. (From ref. 45)
17 Another way in which AlkB and ABH2 differ is the preference for single stranded DNA versus double stranded DNA; AlkB is more reactive towards ssDNA and A BH2 towards dsDNA 47 In AlkB, this preference arises from binding to only one strand of the DNA, and has been shown to occur even when dsDNA is used as the substrate (Figure 14). The base flipping method implemented by both AlkB and ABH2 results in conformational changes within the DNA strand, which in dsDNA disrupts the hydrogen bonding between the base pairs and distorts the DNA helix, creating a large energy bar rier for the enzyme 45 In ABH2, however, this barrier is overcome by binding to both strands of the DNA and filling the hole created by the flipped base with a phenylalanine residue in order to maintain pi stacking interactions and limit the distortion in the DN A helix 45 (Figure 1 5) Figure 14 : 1 meA mutated dsDNA bound to Mn substituted AlkB (From ref. 45) Figure 15 : 1 meA mutated dsDNA bound to Mn substituted ABH2 (From ref. 45)
18 1.2.2 A Potential Catalytic Mechanism The first step in the mechanism for the keto dependent enzymes is the binding of ketoglutarate to the non heme i ron center, resulting in the displacement of two water molecules. B inding of the substrate results in the loss of the third water molecule, leaving an open coordination site for O 2 as shown in Scheme 9 48 Dioxygen binds to form an Fe (III) superoxo species ; although this intermediate has not been detected directly through spectroscopic means, indirect evidence of its existence has been provided through the use of the model compound [(Tp iPr2 )Fe II (O 2 CC(O)R)], where R was either CH 3 ( 1 ) or Ph ( 2 ). Scheme 10 shows the results of reacting [(Tp iPr2 )Fe II (O 2 CC(O)R)] with O 2 under varying conditions. Each of the outcomes was indicative of the Fe(III) su peroxo species 49 Scheme 9: Proposed mechanism for the keto dependent class of dioxygenases (From ref. 49)
19 After formation of the superoxo species, the oxygen attacks the R group containing carbon of ketoglutarate to form an Fe(IV) peroxo species 48 Although this intermediate has not been proven through spectroscopic means, its existence has been validated th rough a combination of computational and experimental methods. This was accomplished by first synthesizing a complex of 4 hydroxyphenylpyruvate dioxygenase 4 hydroxyphenylpyruvate and NO, and then using computational methods to study the Scheme 10 : Reactions of [(Tp iPr2 )Fe II (O 2 CC(O)R)] (left) with O 2 Reaction of 1 with O 2 at 40¡C resulted in the formation of a peroxo species, whose formati on could be inhibited by 2,4,6 t ri tert butylphenol Species 4 was formed by carrying out the reaction at 25¡C. (From ref. 49)
20 same complex wit h O 2 rather than NO. The calculation led to the prediction of a low spin ( S = 1) Fe(IV) peroxide bridge whose existence is a result of charge donation from iron to O 2 and donation from t he keto ligand 50 After formation of the Fe(IV) peroxo species, th e keto cosubstrate is decarboxylated, leading to the putative formation of an Fe(II) peracid adduct. Heterolytic cleavage of the peroxo bridge produces a high spin Fe(IV) oxo species, whose existence was proven in TauD, an keto dedpendent enzyme found in E. coli, by Mssbauer an d EPR spectroscopy 51 The high spin oxo species is capable of abstracting a hydrogen from the substrate to produce a radical which recombines with the coordinated hydroxyl radical to produce a hydrox ylated substrate 52 (Scheme 11 ) 1.3 Rieske Dioxygenases Similar to the extradiol dioxygenases, R ieske dioxygenases are found predominantly in soil bacteria and are responsible for the degradation of aromatic compounds such as naphthalene, an thranilate, and phthalate. The R ieske dioxygenases Scheme 12: General reaction scheme of the Rieske dioxygenases (From ref. 61) Scheme 11: Hydroxylation of the substrate by a high valent oxo species (From ref. 52)
21 differ from the other groups of nonheme iro n enzymes by the presen ce of a R ieske iron sulfur cluster (Figure 16), which functions as an electron shuttle to the active site. The general reaction involves the cis dihydroxylation of aromatic compounds by O 2 a nd the cofactor NADPH (Scheme 12 ). 1.3.1 Naphthalene Metabolism Although manufactured gas production (M GP) ended in the 1950's 53 approximately 45,000 former MGP and other coal tar sites remain contaminated by polycyclic aromatic hydrocarbons (PAH) 54 such as naphthalene 55, 56 a compound which can lead to adverse health effects including methemoglobinemia, hemolysis, and cataract formation upo n prolonged exposure 57 In order to prevent the flow of naphthalene and other PAH into waterways and underground water sources, these compounds must be removed from the MGP and coal tar sites. One method of removal is with the use of soil Figure 16: Rieske cluster
22 bacteria. A variety of species of soil bacteria are capable of degrading naphthalene along with other PAH. These include strains of Pseudomonas putida 58 Pseudomonas stutzer 59 and Comamonas testosteroni 60 These bacteria utilize naphthalene as an energy source by first converting naph thalene to sal icylate (Scheme 13 ) and then converting salicylate to tricarboxylic acid (TCA ) cycle intermediates (Scheme 14 ). In both P. putida and P. stutzeri the nah genes encode the enzymes responsible for the conversion of naphthalene to salicylate, and the sal genes encode the enzymes which convert salicylate to TCA interme diates 58 The nah genes and sal genes are located on two sep arate operons and are regulated by a LysR type regulatory protein, nahR loc ated on a third operon 62, 63, 64 The protein nahR is required for transcription of the nah and sal operons, and it is postulated that salicylate, which upregulates the expression o f these operons, binds to the nahR gene product and incre ases the rate of transcription 65 Scheme 13: Upper pathway of naphthalene degradation (From ref. 59) The compounds are naphthalene (1), cis 1,2 dihydroxy 1,2 di hydronaphthal ne (2), 1,2 dihydroxynaphthalene (3), 2 hydroxy 2 H chromene 2 carboxylic acid (4), trans o hydroxybenzylidenepyruvic acid (5), salicylaldehyde (6), and sali cylic acid (7) The enzymes are naphthalene dioxygenase (NahAaAbAcAd), cis naphthalene dihydrodiol dehydroge nase (NahB), 1,2 dihydroxynaphthalene dioxygenase (NahC), 2 hydroxy 2 H chromene 2 carboxylate isomerase (NahD), trans o hydroxybenzylidene pyruvate hydratase aldolase (NahE), and salicylaldehyde dehydrogenase (NahF).
23 Another gene has been discovered in P. Putida on the NAH7 plasmid, n ahY that may code for a membrane receptor protein important in the process of chemotaxis towards naphthalene. The gene is located downstream of the sal operon. The gene product contains features similar to chemotaxis proteins from E. coli and Salmonella t yphimurium such as two membrane spanning regions, and a C terminal cytoplasmic region containing two domains with glutamine re sidues. 66 Although the degradation pathway is conserved in P. Putida G7 and P. stutzeri strain AN10, the location and organization of the operons are not. In P. Putida G7, both operons are located on the plasm id NAH7, 62 while in P. stutzeri strain AN10, the operons are located o n the chromosomal DNA. 59 The organization of the first operon, na hAaAbAcAdB FCED is conserved be tween the two species. 67, 59 The organization of the second operon, however, differs slightly. In P. Putida G7, the gene sequence of the second operon is nahGHINLJK 68 but is nahGTHINLOMKJ in P. stutzeri strain AN10 63 These differences may be attributed to the presence of a defective class II transposon, Tn4655, on the NAH7 plasmid, which becomes active in the presence of the Tn4653 Scheme 14: Lower pathway of naphthalene degradation (From ref. 63) The compounds are salicylate (1), catechol (2), 2 hydroxymuconic semialdehyde (3), 2 hydroxyhexa 2,4 diene 1,6 dioate (4), 2 oxohexa 3 3n3 1,6 dioate (5), 2 oxopent 4 enoate (6), 4 hydroxy 2 oxovalerate ( 7), pyruvate (8) acetaldehyde (9), and acetyl CoA (10) The enzymes are NahG, salicylate hydroxylase; NahH, catechol 2,3 dioxygenase; NahI, hydroxymuconic semialdehyde dehydrogenase; NahJ, 4 oxalocrotonate isomerase; NahK, 4 oxalocrotonate decarboxylase; NahL, 2 oxopent 4 enoate hydratase; NahM, 2 oxo 4 hydroxypentanoate aldolase; NahN, hydroxymuconic semialdehyde hydrolase; NahO, acetaldehyde dehydrogenase ; NahW, salicylate hydroxylase.
24 transposase and would allow the transfer of plasmid genes to chromosomal ge nes and vice versa 69 1.3.2 Naphthalene Dioxygenase: Structure and Reaction Naphthalene dioxygenase, the first enzyme in the naphthalene catbolism pathway, is a rieske dioxygenase and catalyzes the conversion of naphthalene to cis 1,2 dihydroxy 1,2 dihydronaphthal e ne (Sch em e 15 ), in a two electron stereospecific reaction. The electrons are supplied by NADPH or NADH. The enzyme has two types of subunits, and 70 that form an 3 3 hexamer in which the subunits are positioned above the subunits, giving a shape roughly that of a mus hroom 71 (Figure 17) The subunit has not been found to have any catalytic function but is merely structural 70 The subunit however, contains both the active site and a Rieske center (Figure 18), which functions to transfer electrons to the active site. NADPH Scheme 15: Reaction of naphthalene dioxygenase: Naphthalene is converted to cis 1,2 dihydroxy 1,2 dihydronaphthalene.
25 Figure 17: a.) Structure of naphthalene dioxygenase showing the subunits positioned beneath the subunits. b.) The top view of naphthalene dioxygenase showing the mononuclear iron center and rieske cluster. (From ref. 71) Figure 18: The subunit of NDO. The blue ribbon is the active site domain, and the magenta ribbon is the rieske domain (From ref. 71)
26 Two iron atoms bridged by two sulfur atoms typify Rieske iron sulfur clusters, which take on the shape of a rhombus 72 (Figure s 16 and 19) One of the iron atoms is also coordinated to two cysteines and the other to two histidines. In naphthalene dioxygenase the two cysteines are Cys 81 and Cys 101, and the histidine residues are His 83 and His 104. Within an individual subunit, the active site and Rieske center are too far apart for electron transfer to occur. The acti ve site and Rieske center of adjacent subunits are 12 away, however, near enough for electron transfer. The two sites are linked by hydrogen bonding to Asp 205 71 (Figure 19 ) Similar to the active sites of the extradiol dioxygenases and the keto depe ndent dioxygenases, the active site of naphthalene dioxygenase consists of two histidines, His Figure 19: Route of electron transfer between the Rieske center and active site, which are connected by Asp205, in naphthalene dioxygenase. (From ref. 71)
27 213 and His 208, a carboxylate residue, Asp 362, and a water molecule (Figure 19). Unlike the other two classes, the carboxylate coordinates to the iron through both oxygen atoms rather than one. The geometry of the active site in the absence of a substrate or O 2 is distorted trigonal bipyram idal 71 and binding of the substrate causes no significant changes in conformation, particularly around t he active site 73 Crystal structures by Karlsson et al. have shown that the dioxygen molecule binds side on (Figure 20a). A structure was also solved with indole, another substrate of naphthalene dioxygenase, held 4 from the active site in a hydrophobic cle ft 74 (Figure 20 b) The side on binding of oxygen explains the stereospecificity of the reaction, since both oxygen molecules will add to the same face of the molecule. Figure 20: a.) Structure of the active site of NDO with O 2 bound. b.) Structure of the active site of NDO with indole and O 2 bound at the active site. (From ref. 74) a.) b .)
28 1.3.3 Possible Catalytic Mechanism of the Rieske Dioxygenases The catalytic mechanism of the Rieske dioxygenases has yet to be definitively proven. However, progress has been made in elucidating the intermediate species, allowing for the pr oposal of a mechani sm (Scheme 16 ). The reaction begins with the one electron reduction of the Rieske cluster from an electron source such as NADPH, followed by binding of the substrate 75 O 2 then binds in a side on manner to produce an Fe( III) hydroperoxo species which either oxidizes the substrate directly or becomes cleaved, forming the high valent oxo species cis HO Fe(V)=O. After the substrate is oxidized by either the Fe(III) hydroperoxo species or the cis HO Fe(V)=O species, the Fe(II I) center becomes reduced to Fe(II) and the product is released. Scheme 16: Proposed catalytic mechanism for the Rieske dioxygenases. The scheme shows the redox states of both the iron center and the Rieske center. (From ref. 75)
29 It was found that the cis diol product could also be produced by using hydrogen peroxide as the oxidizing agent, a process referred to as the peroxide shunt 75 and most mechanistic studies ha ve implemented this process rather than using O 2 This was proven through the use of 18 O labeling studies, in which 18 O appeared in the cis diol when H 2 18 O 2 was used as the oxygen source 75 The peroxide shunt was implemented in proving the existence of the Fe(III) hydroperoxo species in benzoate 1,2 dioxygenase. EPR and Mssbauer spectroscopy of benzoate 1,2 dioxygenase exposed to hydrogen peroxide showed the mononuclear iron center was not r educed to F e(II) but rather remained at a higher oxidation state throughout the ca talytic cycle 76 The use of x ray crystallography also lent credence to the existence of the hydroperoxo intermediate; crystal structu res of naphthalene dioxygenase 71 and car bazole 1, 9a dioxygenase 77 both revealed that dioxygen binds perpendicular to the mononuclear iron center with asymmetric Fe O bond lengths, indicative of a peroxo species. 18 O labeling has also been a method of determining whether the Fe(III) hydroperoxo species oxidizes the substrate or if heterolytic cleavage occurs, leading to formation of the high vale nt oxo species cis HO Fe(V)=O. Although the high valent oxo species has not been directly observed in the dioxygenase enzymes themselves, it has been spectroscopically identified using the model complex [Fe(OTf) 2 ( Me,H Pytacn)] (Figure 21) and variable tempe ratur e mass spectrometry 78 Despite this progress, these Figure 21: The model complex [Fe(OTf) 2 ( Me,H Pytacn)] is a stereo selective alkane hydroxy lating catalyst (From ref. 78)
30 finding do not fully support the presence of the Fe(V)=O species in the biological enzymes. 1.4 N 2 O Tripodal Ligands As the above sections show, synthetic model complexes of the active sites of nonheme iron enzymes can assist in the elucidation of the mechanisms of these enzymes. This usefulness has resulted in a proliferation of research in this area. An impor tant aspect of synthesizing model complexes is ligand synthesis. The conserved facial triad of two histidines and one carboxylate found at the active sites of nonheme iron enzymes allow for the use of a common set of ligands (Figure 22) to model the active sites of these Figure 22: Common ligands used in modeling Fe(II) nonheme enzymes. (From r ef. 32)
31 enzymes. As shown in Figure 22, most of the commonly used ligands result in an iron complex with three nitrogen residues (N 3 ) coordinated rather then two nitrogens and one oxygen, as found in the enzymes. The N,N,O heteroscorpionate class o f ligands, however, retains the N 2 O facial coordination, thereby more closely mimicking the electronics of the enzyme active sites. Several new N 2 O facially coordinating ligands have been synthesized for use in model complexes of the extradiol dioxygenases and keto dependent dioxygenases, and their reactivity with molecular oxygen examined spectroscopically. One such complex is [Fe II (L)(Hdtbc)], in which L is one of the three ligands in Figure 23 and Hdtbc is monoanionic 3,5 di tert butylcatechol (Figure 24). These Fe II complexes react with molecular oxygen to form the blue purple Fe III complexes, [Fe III (L)(dtbc)]. UV Vis spectroscopy confirmed the oxidation of the iron by the appearance of the charge transfer bands characteristic of catecholate to Fe(III) transitions 79 The above mentioned Fe III complexes, [Fe III (L)(dtbc)] were synthesized and reacted with O 2 to produce a combination of extradiol, intradiol and quinone products Figure 23: A series of N 2 O he teroscor pionate ligands used in the synthesis of metal ligand complexes capable of producing the extradiol product of 3,5 di tert butylcatechol (H 2 dtbc) (From ref. 80)
32 (Figure 24). The greatest percentage of extradiol pr oduct was produced with the least sterically hindered ligand, L1 A ddition of one equivalent of the proton donor [Et 3 NH]BF 4 to a solu tion of [Fe III (L2)(dtbc)] in dichloromethane resulted in increased production of the extradiol product compared to the solution of [Fe III (L2)(dtbc)] with no proton donor Quinone production also a ppeared to be related to sterics with less s tericically hindered ligands resulting in less auto oxidation 80 Another series of ligands, which implemented the design of the TPA ligands, was synthesi zed for use in metal complexes as models of the extradiol dioxygenases, and 3,5 di tert butylcatechol used as the catechol substrate. Unlike other TPA molecules, this ligand consisted of only one pyridine substituent, a benzyl group, and a substituted phen ol group (Figure 25). Reacting the complex [Fe III (L)(dtbc)(CH 3 OH)] with O 2 produced different ratios of extradiol to intradiol products; the electron releasing R groups, methyl and Substrate Figure 24: The substrate 3,5 di tert butylcatechol and the observed products. The products include the quinone product resulting from auto oxidation, two extradiol products, and two intradiol products. (From ref. 80) Figure 25: A series of ligands for modeling extradiol dioxygenases (From ref. 81)
33 tert butyl, produced greater amounts of the intradiol product, and the more electron withdrawing group, NO 2 produced a greater amount of extradiol t han intradiol product 81 The reason for the varying production of the intradiol and extradiol product for L1 L4 lies in the shifting Lewis acidity of the Fe(III) center. The electr on releasing groups funnel electron density onto the iron, increasing the likelihood of O 2 attack on the substrate rather t han the iron 82 This corresponds to the postulated mechanism for the i ntradiol dioxygenases (Scheme 17 ), in which the O 2 directly att acks the catechol substr ate 83 The electron withdrawing group, NO 2 removes electron density from the Fe(III) center, increasing the favorability of O 2 attack at the iron center 82 and resulting in the extradiol product. Caradonna et al rec ently synthesized a series of N 2 O n ( n = 1, 2, 3) ligands (Figure 26 ) to investigate the effects of increased numbers of carboxylate ligands o n the reduction potential and O 2 sensitivity o f bot h ferric and ferrous complexes. It was found Scheme 17: Mechanism for formation of the intradiol product (From ref. 83)
34 that increasing t he number of carboxylate groups on the ferric complexes decreased the reductio n potential and increased the O 2 sensitivity for the ferrous complexes 84 As discussed previously, for the ke to dependent enzymes, it has been shown that the cosubstrate, ketoglutarate, must bind before O 2 can react at the iron center 85 The results of this experiment suggest that cosubstrate binding is required prior to O 2 binding in order to decrease the redu ction potential of the iron and assist in stabilizing the high valent iron intermediate 84 Burzlaff et al. has designed a series of bis(pyrazol 1 yl)acetate ligands (Figure 27 ) for the synthesis of both ferrous and ferric iron complexes in order to model the keto dependent enzymes. Initial attempts to synthesize ferrous complexes using the lig ands bdmpza and bd t bpza, in combination with FeCl 2 resulted in a bis complex, [Fe(bdmpza) 2 ] (Figure 28 ), and a dimer complex, [Fe(bd t bpza)Cl] 2 (Figure 29 ), respectively 86 The reaction of bpza with FeCl 2 also resulted in the formation o f a bis complex 87 Burzlaff et al postulated that bpza and bdmpza lacked sufficient steric bulk to prevent the coordination of two ligands to a single iron atom and so resulted in th e bis complexes 87 Figure 26: A series of N 2 O n ligands used in ferric and ferrous complexes modeling the active site of the keto dependent enzymes (From ref. 84)
35 In order to split the dimer species, [Fe(bd t bpza) Cl] 2 was treated with thallium benzoyl formate, a 2 oxocarboxylate compound similar to the cosubstrate keto glutarate. The UV/Vis absorption of the resulting purple complex, [Fe(bdtbpza) (O2CC(O)Ph)] (Figure 30 ), showed a ligand to metal charge transfer (LMCT) band at 544 nm 88 which matched the LMCT band for similar complexes 89, 90 The complex, Figure 27: General structure of the bis (pyrazol 1 yl)acetate ligands (From ref. 88) R = H, bpza R = Me, bdmpza R = t butyl, bd t bpza Figure 28: The bis complex [Fe(bdmpza) 2 ] (From ref. 86) Figure 29: The dimer complex [Fe(bd t bpza)Cl] 2 (From ref. 87)
36 however, was not reactive with O 2 and exposure of the complex to air did not result in the production of benzoic acid, the expected product of the reaction 88 Using the ligands bpza and bdmpza, Burzlaff et al. was able to successfully synthesize the mononuclear high spin ferric complexes, [NEt 4 ][Fe(bpza)Cl 3 ] and [NEt 4 ][Fe (bdmpza)Cl 3 ] and gain crystallographic evidence of the latter 87 (Figure 31) Attempts to synthesize a ferric complex using the ligand bd t bpza, however, were not successful 88 Figure 30: Proposed structure of the complex [Fe( bd t bpza )(O2CC(O) Ph)] (From ref. 88)
37 1.5 Bdippza: A new ligand for modeling nonheme iron enzymes The ligand bis 3,5 diisopropylpyrazol 1 ylacetate (bdippza) (Figure 32 ) was previously synthesized in the Sherman lab for use in complexes modeling the active site of RuBisCo, and several zinc and magnesium complexes were synthesized and characterized by x ray crystallography and NMR spectrosco py 91 Two goals of the project were to synthesize tetrahedral zinc and magnesium complexes A zinc bis complex was first synthesized using the zinc precursor, Zn(OTf) 2 but treating Hbdippza with ZnCl 2 resulted in a dimer complex. X ray crystallography of the dimer complex showed each zinc ion to be tetrahedrally coordinated, and NMR spectroscopy of the dimer comple x indicated that the compound might be a monomer in solution but form a dimer upon crystal formation. Less success Figure 31: Crystal structure of the ferric complex [NEt 4 ][Fe( bdmpza)Cl 3 ]. (From ref. 88) Figure 32: Structure of H bdippza
38 was achieved with magnesium; a bis magnesium complex was formed with the use of both tri flate and chloride s alts To study both steric and electronic effects, Hbdippza is used in this work to model the active sites of nonheme iron enzymes. The goal of this project is to synthesize a mononuclear octahedral iron complex with open coordination sites to which mole cular oxygen and potential substrates can bind. As discussed in the previous section a ferrous complex is not required for reactivity with O 2 and production of an extradiol product. The active models produced by both Palaniandavar et al. 81 and Gebbink et a l. 80 were ferric complexes capable of producing the extradiol product. Therefore, this project focused on ferric complexes as a means of producing an active model of nonheme iron enzymes. Ferric complexes are generally less air sensitive and can be handled without the use of a glovebox or Schlenk line, decreasing the difficulty of their synthesis. The work by Burzlaff et al. as shown in Figure 31, indicates steric bulk is unnecessary for the formation of mononuclear ferric complexes. I n fact too much steri c bulk may prevent the formation of these complexes as was shown by the unsuccessful attempts to synthesize a ferric complex using bd t bpza 88 The ligand bdippza contains isopropyl groups, which offer less steric hindrance than the tert butyl groups and sho uld successfully form mononuclear complexes with ferric iron. The additional electron releasing abilities of bdippza compared to bdmpza and bpza could weaken the Fe Cl bond s allowing for greater reactivity at the iron center.
39 Chapter 2: Experimental 2.1 General Chemical reagents were purchased from either Acros or Sigma Aldrich and used as purchased unless otherwise noted. Dry ice was pu rchased from Publix and used the same day. Carbon and hydrogen NMR spectra were taken with a Bruker AC 250 MHz NMR spec trometer. IR spectra were taken with an Avatar 320 FT IR spectrometer, and a Meltemp or Meltemp II melting point apparatus was used for finding melting points of synthesized compounds. UV Vis spectra were taken on a Car y14, and elemental analysis performed by Atlantic Microlabs, Inc., Norcross, GA. All reactions were carried out u nder N 2 using a Schlenk line unless otherwise noted. Dry THF was obtained by distilling THF over potassium and benzophenone. 2.2 Preparation of Ligands 2.2.1 3,5 Diisopropylpyrazole : Synthesis of 3,5 d iisopropylpyrazole was adapted from litera ture procedures 92 2,6 Dimethyl 3,5 heptanedione (5 g, 32 mmol) was dissolved in ethanol (40 mL), and hydrazine monohydrate (2.9 mL, 60 mmol) added dropwise via syringe th rough a septum into the ethanol solution with stirring under nitrogen. After refluxing for one hour, the resulting solution was washed with an aqueous solution of concentrated sodium chloride (50 mL) and extracted twice with diethyl ether (25 mL). The orga nic layer was dried over anhydrous magnesium sulfate, gravity filtered, and concentrated in vacuo resulting in a white solid. Yield: 4.121 g (85%); mp: 82.6 84.0¡C
40 1 H NMR (CDCl 3 250 MHz): = 1.21 (d, 12H, CH 3 ), 2.95 (sep, 2H, CH), 5.84 (s, 1H, H pz ) Toxic ity n ote : Hydrazine monohydrate is a corrosive and potentially carcinogenic liquid. Adequate precautions should be taken to avoid inhalation and skin contact. The aqueous layer should be combined with bleach in order to neutralize any remaining hydra zine. Additionally, any glassware the hydrazine was in contact with should also be rinsed with bleach in the hood. 2.2.2 Bis 3,5 diisopropylpyrazol 1 ylmethane (bdippzm) : Synthesis of b is 3,5 diisopropylpyrazol 1 ylmethane was adapted from li terature proc edures 86 3,5 Diisopropylpyrazole (3.16 g, 20.7 mmol), benzyltriethylammonium chloride (TEBA) (0.5 g, 2.0 mmol), anhydrous potassium carbonate (11.25 g, 81.5 mmol), and potassium hydroxide (4.5 g, 80.0 mmol) were combined with dichloromethane (125 mL) and refluxed for sixteen hours with stirring under nitrogen. After the remaining salts were removed by gravity filtration, the filtrate was concentrated in vacuo, and the resulting yellow solid washed with water (50 mL) and extracted with pentane (30 mL). The organic p hase was dried over anhydrous magnesium sulfate, gravity filtered, and concentrated in vacuo. The resulting yellow solid was washed with water over vacuum fil tration to give a white solid. Crude y ield: 3.162 g (96%); mp: 49.5 51.8¡C
41 1 H NMR (CDCl 3 250 MHz) : = 0.88 ( pentane) 1.1 (d, 12H, CH 3 ), 1.3 (d, 12H, CH 3 ), 1.56 (H 2 O), 2.18 (acetone), 2.9 (sept., 2H, CH), 3.4 (sept., 2H, CH), 5.85 (s, 2H, CH pz ), 6.24 (s, 2H, CH 2 ) 2.2.3 Bis 3,5 diisopropylpyrazol 1 ylacetic acid (Hbdippza) : Synthesis of b is 3, 5 diisop ropylpyrazol 1 ylacetic acid was adapted from literature procedu res 87, 93 A solution of bdippzm (1.819 g, 5.75 mmol) dissolved in dry THF (50 mL) was cooled to 70¡C while stirred under nitrogen. Then, 1.6 M N butyllithium in hexanes (5.4 mL, 8.63 mmol) w as added dropwise to the solution. After stirring for 1 hour, the reaction flask was warmed to 0¡C, and gaseous carbon dioxide was bubbled into the reaction flask for 1.5 hours. The solution was concentrated in vacuo, and the resulting yellow solid washed with water and the suspension acidified to pH 1 with 6 M HCl. The aqueous layer was extracted twice with diethyl ether (15 mL), and the organic layer dried over anhydrous magnesium sulfate. The magnesium sulfate was gravity filtered and the solution concen trated in vacuo. The resulting solid was washed with pentane over vacuum filtration to yield a white solid. Yield: 1.106 g (53%); mp: 165.8 167.8 ¡C 1 H NMR (CDCl 3 250 MHz): = 0.98 (m, 12H, CH 3 ), 1.2 (d, 12H, CH 3 ), 2.9 (sept, 2H, CH), 3.1 (sept, 2H, CH), 4.45 (broad s, 1H, NH pz ), 9 (s, 2H, CH pz ), 6.9 (s, 1H, CH 2 ) 13 C NMR (CDCl 3 250 MHz): = 22.429 (CH 3 ), 22.59 (CH 3 ), 22.736 (CH 3 ), 23.352 (CH 3 ), 25.229 (CH), 27.657 (CH), 71 (CH), 101.090 (CH pz ), 152.627 (CH pz ), 1 59 (CH pz ), 165.1 (COOH) IR: = 2953.08 2859.68 cm 1 (C H stretch), 1731 cm 1 (C=O stretch), 1548.11 cm 1
42 (C=N stretch) 2.3 Preparation of Iron Complexes 2.3.1 Tetraethylammonium( oxo)bis[trichloroferrate(III), [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] Synthesis of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] was adapted from literature procedur es 94 A solution of sodium methoxide was prepared by comb in ing metallic sodium (0.6 g, 26.1 mmol) with methanol (125 mL) and added dropwise over 2.5 hrs to a solution of FeCl 3 6H 2 O (7.1 g, 26.3 mmol) in me thanol (175 mL). The solution was stirred an additional hour after the sodium methoxide solution was fully added followed by the addition of Et 4 NCl (4.75 g, 28.7 mmol) and ten more minutes of stirring. The methanol was then removed in vacuo resulting in a thick slurry. The slurry was combined with acetonitrile (100 mL) and after stirring for 15 minutes, the solution was filtered through a celite pad. The celite pad was washed with acetonitrile (40 mL), and the resulting solution concentrated in vacuo, produ cing a thick brown oil. The brown oil was dissolved in acetonitrile (10 mL) and chloroform (150 mL) adde d dropwise over several minutes. As the chloroform was added a dark oil again separate from the rest of the liquid. Using a separatory funnel, the dark oil was separated from the chloroform and dissolved in acetonitrile (30 mL) and further diluted in THF (175 mL). The solution was placed in a 20¡C freezer and after 72 hours filtered The resulting brown orange solid was washed with THF over vacuum. Yield : 3.122 g (20%) ; mp: 104.9 106.5 ¡C ; eff : 2.17 B (Lit. MP: 257 258¡C) 95 IR: = 854.90 cm 1 ( Fe O Fe stretch ), 2980.39 cm 1 (C H stretch ) UV Vis (0.07 mM in CH 3 CN) : = 317.00 nm ( 12,471 cm 1 M 1 ), 350.84 nm (sh, 7507 cm 1 M 1 )
43 2.3.2 Fe(bdippza) 2 The synthesis of Fe(bdippza) 2 was adapted from li terature procedures for the synthesis of [Net 4 ][Fe(bdippza)Cl] 87 A solution of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] (300 mg, 0.50 mmol) and Hbdippza (360 mg, 1.00 mmol) in acetonitrile (20 mL) was stirred for 15 minutes un der an atmosphere of nitrogen, filtered, and the filtrate stored at 20¡C for 48 hours. The solution was allowed to warm to room temperature for approximately 12 hours, and then returned to 20¡C for an additional 24 hours, after which the solution was fil tered. The resulting product was a pale gree n crystalline solid. Yield: 24 m g ( 6.25%) ; mp: 306.4 311 ¡C Elemental Analysis: Anal. Calcd. for C 4 0 H 62 FeN 8 O 4 : C (62.01%), H (8.065%), N (14.46 %); Found: C (61.94%), H (7.94 %), N (14.52%) IR: = 2956.97 2863.57 cm 1 (C H stretch ), 1649 cm 1 (C=O stretch ) Chapter 3: Results and Discussion In order to synthesize iron complexes, the ligand Hbdippza was first synthesized in a three step process, with an overall yield of 43% and the iron compou nd [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] also synthesized The reaction of Hbdippza and [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] resulted in a bis iron complex. 3.1 Ligand Synthesis: Analysis of Reaction Mechanisms and Spectra 3.1.1 3,5 D iisopropylpyrazole (dippz) : Previous synthe ses of dippz in the Sherman lab achieved a produc t yield of 53% 91 Dippz has now been synthes ized at a higher yield of 85% from commercially purchased 2,6 dimethyl 3,5 heptanedio ne and hydrazine monohydrate with ethanol implemented as
44 solvent The hydrazine monoh ydrate was implemented as a nucleophile in the attack of either of the two carbonyls of 2,6 dimethyl 3,5 heptandione through an Ad N 2 step followed by proton transfers at the nitrogen and oxygen (Scheme 18 ) After these steps are repeated for the other nitrogen at the neighboring carbonyl and a five membered ring has formed, two E2 steps are carried out to produce the aromatic pyrazole. The product was extracted into diethyl ether from aqueous sodium chlorid e, which contained any excess hydrazine. The excess hydrazine was neutralized with bleach. 1 H NMR spectroscopy showed that the white product remaining after removal of the solvents was the product 3,5 diisopropylpyrazole (Figure 38 in Appendix A) T he spec trum is identical to the 1 H NMR spectrum previously taken of this c ompound in the Sherman lab 91 A doublet at 1.21 ppm corresponds to the two sets of methyls of the isopropyl group, which are in equivalent environments, and thus appear as one doublet integ rating for twelve hydrogens. The septet corresponds t o the protons located on the central carbons of the two isopropyl groups, and the singlet at 5.84 ppm corresponds to t he methine hydrogen of the pyrazole ring. The hydrogen bonded to one of the nitrogens of the pyrazole ring was not visible in the 1 H NMR spectrum due to the quick relaxation time, which results in peak broadening to the extent that the peak becomes undetectable.
45 3.1.2 Bis 3,5 diisopropylpyrazol 1 ylmethane (bdippzm): The intermediate, bdippzm, was synthesized from dippz with a product yield of 96% a 26% increase from the previously reported yield of 76% Dichloromethane (DCM) acted as both the solvent and reactant, and the phase transfer catalyst, TEBA, was implemented to transfer hydroxide ions in to the organic phase to deprotonate the dippz. Potassium carbonate was implemented as a mild drying reagent to prevent hydrolysis of any carbon nitrogen bonds by water molecules present with the sodium hydroxide, which accumulates more water molecules when crushed and lowers the product yield. The yield of this reaction can also be lowered if the sodium hydroxide pellets are not sufficiently dissolved, a problem which can be avoided by heating the reaction vessel with a PEG Scheme 18 : Proposed reaction mechanism for synthesis of 3,5 diisopropylpyrazole from 2,6 dimethyl 3,5 heptanedione and hydrazine. Proton transfer st eps omitted. (From ref. 91 )
46 bath. The solid formed after rem oval of the DCM was washed with water to remove the water soluble impurity, diethylbenzylamine ( DEBA ) formed from the reaction of TEBA with sodium hydro xide However, washing the solid with too much water can result in a decreased yield. By extracting th e aqueous phase with pentane a second time some of the desired product can be recovered if too much water is used. A final water wash is required to remove any remaining DEBA. The likely mechanism (Schem e 19 ) for this reaction consists of an S N 2 with the unprotonated nitrogen of 3,5 diisopropylpyrazole acting as a nucleophile and a chlorine atom of dichloromethane acting as the leaving group. Potassium hydroxide was used to deprotona te the other nitrogen Another molecule of 3,5 diisopropyl pyrazole attacks the newly attached carbon, and potassium h ydroxide deprotonates the nitrogen to form the Scheme 19: Proposed mechanism for the synthesis of bdippzm from dippz. (From ref. 91 )
47 product b is 3,5 diisopropylpyrazol 1 ylmethane In the 1 H NMR spectrum (Figure 39 Appendix A) t wo doublets at 0.95 and 1.05 ppm each integrate for twelve protons and correspond t o the methyls of the isopropyl groups Septets at 2.81 and 3.3 ppm each integrat ing for two protons, correspond to protons of the central carbons of the isopropyl groups. Singlets at 5.76 and 6.18 ppm each integrati ng for two protons, correspond to the pyrazole methine hydrogens and the methylene hydrogens respectively. The peaks and integration correspond to the previously reported spectrum 91 3.1.3 B is 3,5 diisopropylpyrazol 1 ylacetic acid: The final reaction in the synthesis of b is 3,5 diisopropylpyrazol 1 ylacetic acid requires very dry reaction conditions due to the use of N butyllithium, a very stro ng base implemented to deprotonate the central carbon connecting the two pyrazole groups To create the driest reaction conditions possible, all glassware and needles were oven dried, the dry ice used to generate CO 2 was rubbed with a paper towel, and the THF was dried over potassium The reaction must be run at a low temperature of 78 ¡C in order to prevent N butyllithium from deprot onating the THF or other carbon atoms present in bdippzm. The highest yield for this reaction remains 70% and was previou sly achieved by Kriegel. The lower yield of 53% reported here is most likely a result of the presence of more water B etter drying techniques c ould increase the yield further, including removing water from the reactant bdippzm.
48 The presu m ed reaction mechanism (Scheme 20 ) involves deprotonation of the methylene carbon bridging the two pyrazole rings by N butyllithium. The anionic carbon produced then attacks CO 2 gas to form a carboxylate, which is complexed to the lithium metal. After rotary evaporatio n the crude product was washed with water and acidified in order to protonate the carboxylate group and precipitate it out of the aqueous solution Extracting with diethyl ether dissolved the produ ct in the organic phase, and a wash with pentane succeeded in removing any remaining starting material. 1 H (Figur e 33 ) and 13 C NMR (Figure 34 ) spectroscopy confirmed the formation of hbdippza and are in agreement with previously r eported spectra 91 A multiplet at 0.98 ppm and a doublet at 1.2 ppm each integrate for twelve protons and cor respond to the isopropyl methyl s. The septets at 2.9 ppm and 3.1 ppm correspond to the protons of the central carbon of the isopropyl groups, and the two singlets at 5.9 ppm and 6.9 ppm correspond to the methine proton of t he pyrazole ring and the proton of the central bridging carbon respectively. The multiplet at 0.98 ppm was first observed by Kriegel and predicted to result from hind ered rotat ion of the methyl groups due to the carboxylate group. This ro tational hindrance results in a shift difference between the two methyl groups coupling separately to the proton of the central carbon of the isopropyl group, Scheme 20: Proposed mechanism for the synthesis of bdippza f rom bdippzm. (From ref. 91 )
49 resul ting in a two double ts rather than one doublet. In the 13 C NMR spectrum, the strange splitting pattern is again observed. Peaks at 22.4 ppm, 22.6 ppm, 22.7 ppm, and 23.4 ppm correspond to the isopropyl methyl groups, and the peaks at 25.2 ppm and 27.7 ppm correspond to the central carbons of the isopropyl groups The methyls of the isopropyl groups appear as four peaks on the spectrum rather than th e expected two peaks, again indicating a barrier to rotation. Figure 33: 1 H NMR spectrum of Hbdippza in CDCl 3 1, 1' 2, 2 3 4 3 4 2' 2 1 1 1' 1'
50 3.2 Iron Complexes: Synthesis and Spectroscopic Data 3.2.1 Tetraethylammonium( oxo)bis[trichloroferrate(III)], [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] In order to synthesize ferric complexes, the iron compound [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] was synthesized as a source of ferric iron. The co mpound was synthesized from Fe(III)Cl 3 hexahydrate and tetraethylammonium chloride and sodium m ethoxide was used to deprotonate the water molecules (Scheme 21 ) The sodium methoxide solution is 1, 1' 2 3 4 5 6 7 8 8 1' 1' 3 2 1 1 4 5 6 7 Figure 34: 13 C NMR spectrum of Hbdippza in CDCl 3
51 added drop wise in order to prevent the formation of (NEt 4 )(FeCl 4 ) which has been shown to form when Fe(III)Cl 3 hexahydrate is added to the sodium methoxide solution 94 Excess tetraethylammonium chloride is removed by extraction into the chloroform phase, and the pro duct precipitated by the addition of THF. The data acquired for this product indicates that the resulting product was not pure, but may have also contained the contaminant (NEt 4 )(FeCl 4 ) or another iron salt Previous synthesi s of this compound reported a melting point of 257 258 ¡C 95 which is significantly higher than the melting point 104.9 106.5 ¡C obtained here The IR spectrum (Figure 35 ) of the compound contains a sharp peak at 854.9 cm 1 which is attributed to the Fe O Fe stretch, indicating that at least some amount of the desi red product has been synthesized. Literature values for this stretch range from 855 cm 1 for [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] 96 to 879 cm 1 for [NMe 4 ] 2 [C1 3 FeOFeC1 3 ] 95 The two low frequency Fe Cl stretches which range from 365 318 cm 1 94 were not detected on the FT IR spectrum However, the use of a KBr pellet may have allowed for the detection of those frequencies. These stretches may have given more insight into whether or not the contaminating compound was (NEt 4 )(FeCl 4 ) which has only one IR active mode in that 2 Fe(III)Cl 3 6 H 2 O + 2 NaOMe Et 4 NCl MeOH 2 NaCl + [ Et 4 N ] 2 Scheme 21: Reaction scheme for the synthesis of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] (Picture of [Cl 3 FeOFeCl 3 ] taken from ref. 96)
52 r egion. The use of Raman spectroscopy may also have assisted in verifying the composition of the product. Although the UV Vis data are similar to literature values, some discrepancies exist between the two. Literature molar absorptivity values indica te that maximum absorbance is located at 293 nm ; 94 the UV Vis spectrum for this product (Figure 36 ), however, shows a maximum at 317 nm whi ch should be a shoulder according to the li terature 94 The spectrum shown in Figure 3 6 appears to have red shifted relative to the literature values and may be a result of the presence of (NEt 4 )(FeCl 4 ). The related compound (NMe 4 )(FeCl 4 ) has a maximum a t 360 nm and a shoulder at 312 nm 95 Figure 35: Solid state FT IR spectrum of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ]
53 3.2.2 Fe(bdippza) 2 The intended product of the reaction of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] with Hbdippza was [NEt 4 ][Fe(bdippza)Cl 3 ]. T he isolated product however, was a bis ferrous complex rather than a mononuclear ferric complex Although an x ray crystal structure is needed to fully support this conclusion, the elemental analysis of the resulting product is a close match to the calcul ated percentages for a bis complex (See section 2.3.2) The FT IR spectra (Figure 37 ) shows a carbonyl stretch at 1649 cm 1 differing from the carbonyl stretch of the ligand which lies at 1731 cm 1 Figure 36: UV Vis spectrum of product identified as [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in acetonitrile
54 The source of the Fe(II) is unclear. One possibility is that the oxo bridged starting material contained some Fe(II). S ome of the sodium used for generating sodium methoxide may not have fully react ed with the methanol and instead reduced the iron chlorid e. Although it is unclear how a ferrous complex formed, a new ferrous complex has now been synthesized using the ligan d Hbdippza. The formation of a ferrous bis complex is not suprising given that a bis ferrous complex was also synthesized using the more s terically hindered ligand bd t b pza 87 Crystals of Fe(bdippza) 2 did not readily precipitate from solution, but rather required the evaporation of the solvent in order to precipitate from solution. The low Figure 37: Solid state FT IR spectrum of Fe(bdippza) 2
55 yield may be a result of the low concentration of ferrous iron present in the iron precursor. Additionally increa sing the amount of bdippza in the preparation to four times that of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] could also increase the yield, since there are four ligand molecules to each molecule of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in the product In addition to the pale green crystals wh ich precipitated from the solution, a mixture of yellow crystals and orange solid was also isolated. It is postulated that the yellow crystals were iron complexes and the orange solid an iron salt, either FeCl 3 or [Net 4 ][FeCl 4 ]. Attempts to separate the tw o solids by recrystallization from methanol were unsuccessful and resulted in a red brown slurry FT IR showed the compound contained an IR stretch at 1773 cm 1 indicat ing the presence of unbound ligand. Chapter 4: Conclusions and Future Directions A potentially new ferrous iron complex has been synthesized using the N 2 O heteroscorpionate ligand bdippza. Elemental analysis and FT IR spectra suppor t the existence of an air stable octahedral bis complex Many questions remain unanswered as to how the compound was produced The reaction scheme described in section 2.3.2 should be repeated with newly synthesized and pure [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in order to determine if the same results can be obtained. A different synthetic method could also be attempted to synthesize [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in o rder to achieve a purer compound. One such method uses s odium trimethylsilanolate rather than sodium methoxide as the base and (Et 4 N)(FeCl 4 ) as the iron source 96
56 Differen t ferrous and ferric iron precursor compounds should also be impl emented in order to synthesize mononuclear ferric complex es and mononuclear ferrous complex es Such iron compounds include anhydrous Fe(III)Cl 3 and Fe(II)Cl 2 Fe(III)(NO 3 ) 3 9 H 2 O, and Fe(OTf) 2 2 MeCN Burzlaff et al. was able to use Fe(II)Cl 2 to synthesiz e a ferrous dimer complex using the ligand bd t bpza, indicating that a similar complex c ould be synthesized using bdippa 86 Fe(III)Cl 3 could also be used to synthesize a ferric complex and could serve as a comparison to using [NEt 4 ] 2 [ Cl 3 FeOFeCl 3 ] The compounds Fe(III)(NO 3 ) 3 9 H 2 O, and Fe(OTf) 2 2 MeCN were used by Gebbink et al to synthesize mononuclear ferric and f errous compounds as models of the extradiol dioxygenases, respectively 80 Because the ligands (Figure 23) used were similar to Hbdippza, Fe(III)(NO 3 ) 3 9 H 2 O, and Fe(OTf) 2 2 MeCN should have similar reactivity with Hbdippza. Additionally, the use of a substrate compound such as an keto glutara te derivative or catechol derivative, could prevent the formation of a bi s or dimer complex. Burzlaff et al. was a able to split the dimer species [Fe(bd t bpza)Cl] 2 by treating the complex with thallium benzoyl formate. The addition of a catechol derivative such as te trachlorocatechol, or an keto gluta rate derivative, such as sodium benzoylformate, during the synthesis of either ferric or ferrous complexes could lead to the formation of a mononuclear complex.
58 Appendix A: Spectra Figure 38 : 1 H NMR spectrum of 3,5 diisopropylpyrazole in CDCl 3
59 Figure 39 : 1 H NMR spectrum of bdippzm in CDCl 3
60 Figure 40 : 1 H NMR spectrum of Hbdippza in CDCl 3
61 Figure 41 : 13 C NMR spectrum of Hbdippza in CDCl 3
62 Figure 42: Solid state FT IR spectrum of Hbdippza
63 Figure 43: Solid state FT IR spectrum of [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ]
64 Figure 44: UV Vis spectrum of product identified as [NEt 4 ] 2 [Cl 3 FeOFeCl 3 ] in acetonitrile
65 % Transmittance Figure 45: Solid state FT IR spectrum of Fe(bdippza) 2
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