University of Southern California Dissertations and Theses

The design and synthesis of novel ligands as possible mimics of methane monooxygenase

(USC Thesis Other)

The design and synthesis of novel ligands as possible mimics of methane monooxygenase

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content THE DESIGN AND SYNTHESIS OF NOVEL LIGANDS AS POSSIBLE MIMICS OF METHANE MONOOXYGENASE by Patrick Vagner A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements For the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) December 2003 Copyright 2003 Patrick Vagner Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3133347 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3133347 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATESCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90089-1695 This dissertation, written by P a t r i c k V a g n e r under the direction o f h dissertation committee, and approved by all its members, has been presented to and accepted by the Director o f Graduate and Professional Programs, in partial fulfillment o f the requirements fo r the degree o f DOCTOR OF PHILOSOPHY . Director Date D ecem ber 1 7 , 2003 Dissertation Committee Chair Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents List of Tables................................................................................................................... vii List of Figures................................................... viii Abstract............................................................................................................... xi Chapter 1............................................................................................................................. 1 Introduction....................................................................................................................1 New approach................................................................................................................7 Ligand design................................................................................................................ 8 Reference......................................................................................................................11 Chapter I I ......................................................................................................................... 13 Retro synthetic analysis............................................................................................... 13 Synthesis of precursor A .............................................................................................14 Synthesis of precursor B .............................................................................................16 Synthesis of precursor C .............................................................................................23 Synthesis or precursor D .............................................................................................26 Synthesis of target ligand 3 6...................................................................................... 26 Complex formation with ligand 36............................................................................ 33 Reference......................................................................................................................38 Chapter III........................................................................................................................ 39 Ligand Modification 1................................................................................................. 39 Synthesis of ligand 37................................................................................................. 41 Complex formation with ligand 37............................................................................ 44 ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ligand modification II .......................................... 46 Synthesis of ligand 56................................................... 47 Complex formation with ligand 56...... 52 Ligand modification III............................................................................ 56 Synthesis of ligand 64........ 58 Complex formation with ligand 64......................... .62 Reference......................................................................................................................66 Experimental Section...................................................................................................... 67 General Considerations...............................................................................................67 2,7-Dibromofluorene (2)........ 68 Ethyl-2,7-dibromofluorene-9-carboxylate (3).......................................................... 69 Ethyl-2,7-dibromofluorene-9-methyl-9-carboxylate (4).......................................... 70 3 -Methylpheny lboronic acid (6).................................................................................71 5,5-Dimethy 1-2-m-tolyl-[ 1,3,2]dioxaborinane (7).................................................... 71 3-Bromobenzyl bromide (9)....................................................................................... 72 3-Bromophenyl acetonitrile (10)................................................................................72 2-(3-Bromophenyl)-2-methylpropionitrile (11)........ 73 2-(3-Bromophenyl)-2-methylpropionic acid (12).................................................... 74 Tetrakis(triphenylphospine)palladium.......................................................................75 2,7 -Di-p-tolyl-9H-fluorene (13)................................................................ 75 2-(3-Bromophenyl)-2-methylpropionyl chloride (14)..............................................77 2-[l-(3-Bromophenyl)-l-methylethyl]-4,4-dimethyl-4,5-dihydrooxazole (15).....77 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Compound 16....... 78 Compound 17......... 79 2-(3-Bromophenyl)-2-methylpropionic add tert-hutyl ester (18)...........................79 2-(3-{7-[3-(l -terl-Butoxy carbonyl-1 -methylethyl)phenyl] -9H-fluoren-2- yl}phenyl)-2-methylpropionic acid tert-butyl ester (19)............ 80 2.7 -Bis[3-( 1 -fe/7-butoxycarbonyl-1 -methylethyl)phenyl]-9-methyI-9H-£luorene- 9-carboxylic acid ethyl ester (20)...............................................................................82 2.7-Bis[3 -(1 -carboxyl-1 -methylethyl)phenyl]-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (21)..................................................................................................... 83 2-(3-Chlorophenyl)-2-methylpropionitrile (23)..................... 84 2-(3-Chlorophenyl)-2-methylpropionic acid (24)....................................................85 2-(3-Chlorophenyl)-2-methylpropionic acid tert-butyl ester (25)...........................85 /-Histidine methyl ester dihydrochloride (27)...........................................................86 2.7-Bis[3-(2-imidazol-1 -yl-1,1 -dimethyl-2-oxoethyl)phenyl]-9-methyl-9H- fluorene-9-carboxylic acid ethyl ester (28)...............................................................87 2.7-Bis[3-(l -chlorocarbonylmethylethyl)phenyl]-9-methyl-9H-fluorene-9- carboxylic acid ethyl ester (30)..................................................................................87 2.7-Bis(3-{l-[2-(l H-imidazol-4-yl)-1 -methoxycarbonylethylcarbamoyl] -1 - methylethyl}phenyl)-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (31)....88 l-a N, W,-Ditritylhistidine methyl ester (32)..............................................................88 /-W-Tritylhistidine methyl ester trifluoroacetate (33)..............................................89 2.7-Bis(3-{ l-[2-( 1 -methoxycarbonyl-2-( 1 -trityl- lH-imidazol-4- yl)ethylcarbamoyl] -1 -methylethyl} phenyl)-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (34)......................................................................................................90 2.7 -Bis(3-{1 - [2-( 1 H-imidazol-4-yl)-1 -methoxycarbonylethylcarbamoyl]-1- methylethyl}phenyl)-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (35)....91 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.7-Bis(3-{ l-[l-carboxy-2-(lH-imidazol-4-yl)ethylcarbamoyl]-1 - methylethyl}phenyl)-9-methyl-9H-fluorene-9-carboxylate trisodium salt (36) ....91 Fe(OTf)2(CH3CN)2.............................................................................. 92 2.7 -Bis(3 - {1 -[ 1 -carboxy-2-( 1 -trityl-1 H-imidazol-4-yl)ethylcarbamoyl]-1 - methylethyl}phenyl)-9-methyl-9H-fluorene-9-carboxylic acid dihydrochloride (37)............................................................................................................................... 93 Methyl-2,7-dibromofiuorene-9-carboxy late (38).....................................................93 Methyl-2,7-dibromofluorene-9-methyl-9-carboxylate (39).....................................94 2.7 -Bis[3-( 1 -fcr/-butoxycarbonyl~1 -methylethyl)phenyl]-9-mthyI-9H-fluorene-9- carboxylic acid methyl ester (40).............................................................................. 94 2.7-Bis[3-(1 -carboxyl-1 -methylethyl)phenyl]-9-methyl-9H-fluorene-9-carboxylic acid methyl ester (41)..................................................................................................94 2.7-Bis[3-(l -chlorocarbonylmethylethyl)phenyl]-9-methyl-9H-fluorene-9- carboxylic acid methyl ester (42).............................................................................. 94 2.7-Bis(3 - {1 -[2-( 1 -methoxycarbonyl-2-( 1 -trityl-1 H-imidazol-4- yl)ethylcarbamoyl] -1 -methylethyl} phenyl)-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (43)..................................................................................................... 95 5 -Oxo-5,6,7,8-tetrahydroimidazo[ 1,5 -c] pyrimidine-7-carboxylic acid methyl ester (44)................................................................................................................................95 7 -Methoxycarbonyl-2-methyl-5-oxo-5,6,7,8-tetrahydroimidazo [1,5 -c]pyrimidin-2- ium iodide (45)............................................................................................................ 96 7-Methoxycarbonyl-2-methyl-5-oxo-5,6,7,8-tetrahydroimidazo[l,5-c]pyrimidin-2- ium methylsulfonate (46)............................................................................................96 /-W-Methylhistidine (47)............................................................................................97 /-'W-Methylhistidine methyl ester dihydrochloride (48).................. 97 2.7-Dibromo~9-ethoxycarbonylmethyl-9H-fluorene-9-caboxylic acid ethyl ester (49)...................................... 98 2.7 -Dibromo-9H-fluoren-9-yl-acetic acid (50).........................................................99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.7-Dibromo-9H-fluoren-9-yl-acetic acid ethyl ester (51)..................................... 99 2-(3 - { 7-[3-( 1 -fert-Butoxycarbonyl-1 -methylethy!)-phenyl]-9- ethoxycarbonylmethyl-9H-fluoren-2-yl} phenyl)-2-methylpropionic acid tert-butyl ester (52). ................. .100 2-(3-{7-[3-(l-Carboxy-l-methylethyl)phenyl]-9-ethoxycarbonylmethyl-9H- fluoren-2-yl}phenyl)-2-methylpropionic acid (53)................................................101 {2,7-Bis[3 -(1 -chlorocarbonyl-1 -methylethyl)phenyl]-9H-fluoren-9-yl} actic acid ethyl ester (54)..................................................................... 102 2-(2- { 3 - [9-Ethoxycarbonylmethyl-7-(3 - {1 -[ 1 -raethoxycarbonyl-2-( 1 -methyl-1H- imidazol-4-yl)ethylcarbamoyl]-l-methylethyl}phenyl)-9H-fluoren-2-yl]phenyl}- 2-methylpopionylamino)-3 -3-(1 -methyl-1 H-imidazol-4-yl)propionic acid methyl ester (55).....................................................................................................................103 2-(2- (3 - [9-Carboxymethyl-7-{ 4- [ 1 -hydroxymethyl-2-( 1 -methyl- lH-imidazol-4- yl)ethylcarbamoyl]-1 -methylenenbut-2-enyl} -9H-fluoren-2-yl)phenyl]-2- methylpropionylamino} -3-( 1 -methyl- lH-imidazol-4-yl)propionic acid trisodium salt (56)...................................................................................................................... 104 1.8-Octane-dimesylate (58)...................................................................................... 104 1.8-Octane-ditriflate (59)..........................................................................................105 7-Methoxycarbonyl-2-[8-(7-methoxycarbonyl-5-oxo-5,6,7,8- tetrahydroimidazo[l,5-c]pyrimidin-2-yl)octyl]-5-oxo-5,6,7,8- tetrahydroimidazo[ 1,5-c]pyrimidin-2-ium ditriflate (60)........................................ 106 2-Amino-3 -(1 - {8-[4-(2-amio-2-caboxyethyl)imidazoi-1 -yl] octyl}-1 H-imidazol-4- yl)propionic acid dihydrochloride-ditriflate salt (61)..............................................106 2-Amino-3 -(1 - {8- [4-(2-amio-2-methoxycarbonylethyl)imidazol-1 -yl] octyl} -1H- imidazol-4-yl)propionic acid methyl ester dihydrochloride-ditriflate salt (62) ...107 Compound (63).......................................................................................................... 108 Compound (64).......................................................................................................... 109 Alphabetized Bibliography.........................................................................................110 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Tables Chapter II Table II-1 Conditions used in complex preparation with ligand 36 34 Chapter II Table III-l Conditions used In preparation of complexes with ligand 3 7 ......44 Table III-2 Conditions used in preparation of complexes with ligand 5 6 ..................54 Table III-3 Conditions used in preparation of metal complexes with ligand 64........ 62 Table HI-4 Crude pH titration of fully protonated ligand 64.......................................63 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Chapter I Figure 1-1 Enthalpy of methane oxidation................... 2 Figure 1-2 Oxidation of methane by MMO................................................................... 3 Figure 1-3 The active site of MMO ........ 4 Figure 1-4 Catalytic Cycle of MMO........................................................... 5 Figure 1-5 Model ligands A, B, C, D, E ....................................................................6 Figure 1-6 A schematic of novel approach...................................................................... 7 Figure 1-7 Hydrocarbon backbone.................. 8 Figure 1-8 Attachment of symmetrical arm units........................................................... 9 Figure 1-9 New ligand......................................................................................................10 Figure 1-10 Stereo view of proposed diiron complex............................. .......................10 Chapter II Figure II-1 Retrosynthetic analysis.................................................................................13 Figure II-2 Preparation of compound 4 ......... 14 Figure II-3 Preparation of compound 7 ................................................... 16 Figure II-4 Preparation of compound 1 2 .............................................. 18 Figure II-5 Preparation of test compound 13.......................................... 21 Figure II-6 Preparation of compound 17........................................................... 22 Figure II-7 Preparation of compound 19 .............................................................. 23 Figure II-8 Preparation of compound 2 1 ............................ 25 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure II-9 Preparation of compound 2 5 ............................................. Figure 11-10 Preparation of compound 31 (I)...................................... Figure II-11 Preparation of compound 31 (II) ................................... Figure 11-12 Preparation of compound 31 (III)................................... Figure 11-13 Assignment of nitrogen atoms in histidine..................... Figure 11-14 Preparation of compound 33............................................ Figure 11-15 Preparation of compound 34............................................ Figure 11-16 Decarboxylation tests with compounds 4 and 2 1 .......... Figure 11-17 Preparation of ligand 3 6 .................................................. Chapter III Figure III-l Stereo view of proposed diiron complex with ligand 37 Figure HI-2 Preparation of ligand 37 (I).............................................. Figure IH-3 Preparation of compound 3 9 ............................................ Figure III-4 Preparation of compound 4 1 ............................................ Figure III-5 Preparation of ligand 37 (II)............................................. Figure HI-6 Stereo view of designed diiron complex with ligand 56 Figure HI-7 Preparation of compound 4 7 ............................................ Figure HI-8 Preparation of compound 4 8 ............................................ Figure III-9 Preparation of compound 5 1 ............................................ Figure IH-10 Preparation of compound 5 3 .......................................... Figure III-l 1 Preparation of compound 5 5 .......................................... Reproduced with permission of the copyright owner. Further reproduction prohibited without permission Figure III-12 Stereo view of designed diiron complex with ligand 64....................... 58 Figure III-13 Preparation of compound 5 8..................................................................59 Figure III-14 Preparation of compound 6 2 .......................................... 60 Figure III-15 Preparation of ligand 64............................................................. ...61 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT A series of novel ligands as possible mimics of methane monooxygenase has been designed and synthesized. Emphasis was directed at accurately reproducing the environment of the first coordination sphere of the enzyme’s dinuclear active site. Each ligand contains a rigid aromatic backbone, far removed from the reactive center. A carboxylic acid residue capable of bridging the two metal centers was placed at the backbone’s central point. A positioning arm is located at both ends of the backbone attached to a histidine molecule or a derivative thereof. The positioning arm helps to direct the coordinating atoms of histidine toward the metal centers. The synthetic strategy has been fully worked out with future modifications in mind. Thus, the approach is convergent, where each synthetic piece is completely modifiable, making the ligand highly modular. All reactions have been well optimized and run on gram scales. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter I Introduction Methane, the major component of natural gas, is found all over the world and is predicted to outlast oil reserves significantly.1 Unfortunately, most of methane reserves are far removed from centers of high-energy consumption.2 The costs of compression, transportation, and storage make methane an unattractive source of energy. Conversion of methane to liquids or higher hydrocarbons has been carried out to make the use of methane more economically viable. Direct oxidative conversions into methanol,3 formaldehyde,4 benzene and other aromatics,5 and C 7 propanal have been achieved, albeit in low yields. Current industrial use of methane involves its initial conversion to carbon monoxide and hydrogen (synthesis gas) by either steam reforming or by dry reforming.8 Although its use as an industrial process is limited, partial oxidation has spawned great interest due to its exothermicity (Figure 1-1).7 The synthesis gas is then used in processes, such as methanol synthesis, Fischer-Tropsch synthesis, or ammonia synthesis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CH4 + H2O --------^ CO + 3H2 AHe298 = +206 KJ mol' 1 CH4 + C 0 2 --------► 2CO + 2H2 AH^ 9 8 = +247 KJ mol' 1 CH4 + V 2O 2 --------^ CO + 2 H2 A H ^298 = -38 KJ mol"1 Figure I— 1 Enthalpy of methane oxidation One of the major goals of modem chemistry is to find a system in which a selective oxidation and functionalization of methane could be achieved at ambient conditions, a process that would undoubtedly have a significant impact on the global economy. A possible route toward solving this puzzle is to turn to naturally occurring organisms capable of this reaction in their habitats. Methanotrophic bacteria use methane gas as their sole source of carbon and energy.9 They are capable of oxidizing methane to methanol in buffered aqueous medium, using oxygen as the oxidant at ambient temperature and pressure. This remarkable transformation is catalyzed by methane monooxygenase (MMO). Two classes of MMO have been identified. A copper containing, membrane- bound form referred to as pMMO,1 0 and a soluble form, sMMO that contains non heme iron. The latter is by far more understood to date. The sMMO from Methylococcus capsulatus uses three proteins to carry out the reaction shown in Figure 1-2. A multimeric hydroxylase, MMOH, is a 251 kDa protein that contains a carboxylate-bridged diiron active site in each of its two a subunits. In addition the cluster contains MMOR, a 38.5 kDa protein with FAD and [2Fe-2S] cofactors is 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. responsible for the delivery of necessary reducing equivalents to the active site. The third protein, MMOB, serves to couple oxidation ofNADH to methane hydroxylation. CH4 + 0 2 + NADH + H+ ------------ ► CH3OH + NAD+ + H2 0 Figure I—2 Oxidation of methane by MMO The crystal structure of sMMO’s active site has been obtained and it is schematically illustrated in Figure 1-3.1 1 The reactive center consists of two iron atoms bridged by a glutamate residue and two water molecules. The formal oxidation state of each iron is 2+ , the reduced form of the enzyme. Glutamate and 1 /y histidine residues are bound to each iron, the latter via their % nitrogens and in cis fashion with respect to one another. A water molecule and another glutamate residue take up the remaining sites forming a pseudo-octahedral environment around each metal center. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. : ■ r'- GH»144 Figure I—3 The active site of MMO The sMMO has been extensively studied in-vitro and its catalytic cycle has been elucidated.1 3 Several transient species on the reaction pathway have been spectroscopicaly identified and characterized.1 4 The present consensus on the catalytic transformation is summarized in Figure 1-4. A complex of the reduced form of the enzyme and MMOB reacts with oxygen to form a peroxo diiron (III) species, which proceeds to form the highly reactive diiron (IV) intermediate believed to be responsible for methane oxidation.1 5 In the final stages the diiron (III) centers are reduced by intermolecular electron transfer from MMOR.9 Methanol is discharged from the active site thus completing the catalytic cycle. The mechanism of methane oxidation is, however, not well understood and merely speculative. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NAD+ NADH MMOR 2+ 3+ Fe 4+ Fe Fe +2 H Figure I— 4 Catalytic Cycle of MMO A number of synthetic models have been synthesized in order to reproduce and better understand this intriguing transformation. Diiron complexes have been made with many ligands some of which are illustrated in Figure 1-5. All ligands form dinuclear complexes that have been well characterized by modem techniques. In some cases, oxidation with hydrogen peroxide formed a transient species with spectral characteristics similar to the natural enzyme. Although some complexes were shown to be capable of alkane oxidation, none has yet been able to achieve the oxidation of methane or ethane. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure I—5 Model ligands A,1 6 B,1 7 C,1 8 D,1 9 E.2 0 It is unclear why none of the diiron model complexes synthesized to date have the ability to achieve methane oxidation. However, there are some fundamental points that can be noted about this approach. In all of the above ligands there is little if any homology with the natural environment composing the active site. Most ligands are composed of highly coordinating moieties, such as pyridines, and amines, neither of which is found in the active site of the natural enzyme. This inevitably produces an electronic and steric environment that is substantially different from the 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. natural system. In some cases, the formed complexes are highly fluxional, a property that may be problematic for the oxidation process, and may even lead to ligand oxidation. New approach A new approach to the design of novel complexes has been taken in our group. The goal was to reproduce the environment of the first coordination sphere of the natural active site as closely as possible. All coordinating moieties were tied together to a common scaffold to limit fluxionality and decrease entropic barriers in complex formation (Figure 1-6). A relatively inflexible scaffold (A) was designed to carry the bridging carboxyl moiety (B). Special attention was paid to the distance between the metal centers and the scaffold so that ligand oxidation would be avoided. Two spacer arms 7 Figure I—6 A schematic of novel approach Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were attached to the scaffold moiety to correctly position the remaining atoms (D) for coordination. It was important that the two D groups were separated enough to accommodate two iron atoms but not just one. Ligand design An aromatic hydrocarbon chain, composed of fluorene coupled to two benzene rings in the 2,7 positions, was chosen for the scaffold portion of the ligand. The functionalization of the benzylic hydrogens of fluorene allowed the introduction of the carboxyl moiety. The sp3 geometry at this carbon forced the carboxyl group to point away from the hydrocarbon chain, thus further removing any aromatic hydrogens from the iron centers. Due to the inherent acidity of the second benzylic hydrogen, it was decided to exchange it for a methyl group (Figure 1-7). CCLH Figure I—7 Hydrocarbon backbone The spacer arms were essentially composed of an acetic acid moiety placed strategically on each of the phenyl rings, thus rendering them part of the spacer arms. Using a molecular modeling software,2 1 it was calculated that for the best spacing and directionality, attachment of the acetic acid had to be done at the 3 positions of 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the aromatic rings. It was realized that the newly created benzylic position contained rather acidic hydrogens, which could cause eventual synthetic problems. Their replacement with methyl groups would remove this possibility and further add some rotational restriction around that bond, thus removing some degrees of freedom. The carboxylic acid moiety was to be used as a means of attaching the group, which contained the ultimately metal-coordinating atoms (Figure 1-8). HOX Figure I—8 Attachment of symmetrical arm units The last coordinating atoms, nitrogen and oxygen, were designed to come from a histidine molecule. The nitrogen would be supplied by the imidazole moiety and the oxygen would come from the carboxylic acid portion of histidine. Amide bond formation between each of the terminal carboxyl groups and an a nitrogen of histidine was used as the means of attachment. The final ligand is shown in Figure I- 9. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Na HN Figure I—9 New ligand The design of the ligand relied heavily on a molecular modeling computer program, which allowed, albeit crudely, for the visualization of effects cause by structural modifications. The geometry and structural features of the desired diiron complexes were optimized and are displayed in Figure I-10. Figure I— 10 Stereo view of proposed diiron complex 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reference 1 Cornot-Gandolphe, S. Energy Exploration & Exploitation 1995, 13, 3. 2 Leprince, P.; Valais, M. Energy Sources 1993, 15, 95. 3 Periana, R. A.; Taube, D. J.; Evitt, E. R.; Loffler, D. G.; Wentrcek, P. R.; Voss, G.; Masuda, T. Science 1993, 259, 340. 4 Baldwin, T. R.; Burch, R; Squire, G. D.; Tsaug, S. C. Appl. Catal. 1991, 74, 137. 5 Claridge, I. B.; Green, M. L. H.; Tsang, S. C.; York, A. P. E. Appl. Catal. 1992,89, 103. 6 Green, M. L. H.; Tsang, S. C.; Vernon, P. D. F.; York, A. P. E. Catal. Lett. 1992, 13, 341. 7 Claridge, I. B.; York, A, P. E.; Brungs, A. J.; Marquez-Alvarez, Sloan, J.;Tsang, S. C.; Green, M. L. H. J. Catalysis 1998, 180, 85. 8 Rostrup-Nielsen, I. R. in “Catalysis Science and Technology” (J. R. Anderson and M. Boudart, Eds.), Vol. 5, p. 1. Springer-Verlag, Berlin, 1984. 9 Kopp, D. A.; Gassner, T.; Blazyk, J. L.; Lippard, S. J. Biochemistry 2001, 40, 14932. 1 0 Nguyen, H. H. T.; Elliott, S. J.; Yip, I. H. K.; Chan, S. I. J. Biol. Chem. 1998, 273, 7957. 1 1 Whittington, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2001,123, 827. 1 2 Refer to figure II-4. 5 3 Liu, K. E.; Valentine, A. M.; Wang, d.; Huynh, B. H.; Edmondson, D. E.; Salifoglou, A.; Lippard, S. J. J. Am. Chem. Soc. 1995, 117, 10174. 1 4 Valentine, A. M.; Stahl, s. S.; Lippard, S. J. J. Am. Chem. Soc. 1999,121, 3876. 1 3 Shu, L.; Nesheim, J. C.; KaufFmann, K.; Munck, E.; Lipscomb, J. D.; Que, L., Jr. Science 1997, 275, 515. 1 6 Lee, D.; Krebs, C.; Huynh, B. H.; Hendrich, M. P.; Lippard, S.J. J. Am. Chem. Soc. 2000, 122, 5000. 1 7 White, M. C.; Doyle, A. G.; Jacobsen, E. N. J. Am. Chem. Soc. 2001, 123, 7194. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 Costas, M.; Rohde, J. U.; Stubna, A.; Ho, R. Y. N.; Quaroni, L.; Munck, E.; Que, L., Jr. J. Am. Chem. Soc. 2001, 123, 12931. 5 9 Barrios, A. M.; Lippard, S. J. Inorg. Chem. 2001, 40, 1060. 2 0 He, C.; Lippard, J. S. J. Am. Chem. Soc. 2000, 122, 184. 2 1 PCMODEL, version 7.0; Serena Software: Bloomington, IN, 1998. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter II Retrosynthetic analysis H HN NH D + \ c o 2 e B r ^5 - ^ V B r A C Figure II— 1 Retrosynthetic analysis The ligand synthesis was designed to be convergent in order to render the molecule highly modular and increase the overall yield. This allowed for possible Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n / N" i lj N CO Na A C 0 2h future modifications without major redesign of synthetic methodology. Figure II-1 depicts the original retrosynthetic analysis. The target molecule could be prepared as the trisodium salt in two steps from precursors C and D. Compound C could be synthesized by a direct coupling of precursors A and B. Each of the listed precursors was independently modifiable allowing for a relatively facile synthesis of many similar compounds. This was thought to be an imperative feature since it provided a possible way to tune the reactivity of the desired metal complex. Furthermore, a comparative study of different complexes could, in principle, unveil certain mechanistic details. Synthesis of precursor A Br2, FeCI^ CHCIg 1 2 IDA, THF CICOjEt NaOEt, Et20 4 3 Figure II—2 Preparation of compound 4 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Compound A was prepared from fluorene (1) in three steps (Figure II-2). Bromination of 1 in the presence of catalytic amount of anhydrous ferric chloride gave primarily 2,7-dibromofluorene (2) with 2-bromofluorene as the major impurity. Even though complete bromination did not take place under the conditions employed, the pure product was obtained by recrystallization from a tetrahydrofiiran/ethanol mixture. Treatment of 2 at -78 °C with two equivalents of lithium diisopropylamide in tetrahydrofiiran produced a fluorenyl anion, which after reacting with ethyl chloroformate followed by acidic workup, gave the desired ethyl-2,7- dibromofluorene-9-carboxylate (3).1 Two equivalents of lithium diisopropyl amide were necessary to prevent quenching of the fluorenyl anion by the product, as the product’s pKa drops significantly from that of the starting material. The reaction was forced to go to completion by using slight excess of the acylating agent, which seemed to competitively react with diisopropyl amine. The resulting mixture had to be neutralized at -78 °C to prevent di-acylation, which did not take place at this temperature. Deprotonation of 3 with an alcoholic solution of sodium ethoxide produced a characteristically yellow solution of the acyl-fluorenyl anion. Addition of excess methyl iodide produced ethyl-2,7-dibromofluorene-9-methyl-9-carboxylate (4) as the sole product. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Synthesis of precursor B Component B was designed with the intent to use Suzuki coupling as the reaction of choice for the preparation of precursor C. Initial studies depicted in Figure II-3 were based on boron incorporation in the first step. Reacting 3- bromotoluene (5) with one equivalent of ra-hexyllithium in tetrahydrofiiran converted the starting compound to its lithium salt, which when treated with excess of trimethyl borate and subjected to acidic workup, yielded 3-methylphenylboronic acid (6). To enhance its rigidity the boronic acid was protected with one equivalent of 2,2-dimethylpropane-1,3-diol in methylene chloride to form 5,5-dimethyl-2-m-tolyl- [1,3,2] dioxaborinane (7). 'B r Hex-Li / THF H 0\ ^ C / ° H NBS I — x- E*O M e)3 C H 2CI2 o . B . q c c " H e a t 0 ' % Br h3° B(OH) Figure II—3 Preparation of compound 7 Radical bromination was chosen to functionalize the benzylic carbon of 7. Treatment of 7 with an equivalent of JV-bromosuccinimide in refluxing carbon tetrachloride gave the desired product, 2-(3 -bromomethylphenyl)-5,5- dimethyl[l,3,2]dioxaborinane (8) as the major product. However, NMR showed that 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. some dibromination took place. To test the feasibility of this synthetic pathway, the subsequent reaction with sodium cyanide in dimethyl formamide was conducted using crude material. To our disappointment, only a very low yield of the desired substitution product was obtained. Furthermore, it became clear that the likelihood of a boronic acid withstanding the rigorous conditions necessary to hydrolyze a benzylic nitrile was small. Thus it became evident that an alternate synthetic approach had to be developed. A new approach toward component B is illustrated in Figure II-4. Compound 5 was brominated in the benzylic positions using A-bromosuccinimide in refluxing carbon tetrachloride. A 300W tungsten lamp was used to initiate the reaction. The product mixture resulting from use of one equivalent of A-bromosuccinimide contained on average 15% of starting material and 15% of the dibrominated side product based on qualitative analysis via gas chromatography. The yield could be improved if only 0.5 equivalent of A-bromosuccinimide was used and the starting material recycled after distillation. However, it proved to be more cost efficient to purchase 3-bromobenzyl bromide (9) directly. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Br 5 NBS CCL, Heat, Light c o 2h Br NaCN TBAHS, Et2 0 c o 2h Bu-Li I THF --------V ............. iPt " NaOH, H 2 0 2 r B(OMe)3 EtOH B(OH)2 h3 o Br 13 12 Figure II—4 Preparation of compound 12 ( 7 ' “ Br 10 t-BuOK CH3i, THF 3-Bromophenyl acetonitrile 10 was prepared by reacting 9 with 1.2 equivalents of sodium cyanide in a biphasic, diethyl ether/water mixture, using tetrabutylammonium hydrogen-sulfate as a phase transfer catalyst. Separation of the aqueous layer and drying the organic layer prepared the mixture for the next step without any further purification. Methyl groups were introduced into the benzylic position of 10 to remove the relatively acidic hydrogens. Two equivalents of methyl iodide were mixed with the diethyl ether solution of 10 isolated in the previous step. This mixture was added to a cool suspension of potassium fe/t-butoxide in diethyl ether, which after aqueous 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. workup and distillation afforded the desired product 2-(3-bromophenyl)-2- methylpropionitrile (11). Both basic and acidic conditions were inefficient in hydrolyzing 11. Acidic conditions showed almost no hydrolysis, even after maintaining at a vigorous reflux over a period of one day, and a reaction in a refluxing solution of potassium hydroxide was extremely slow. However, with the addition of hydrogen peroxide the reaction proceeded at an appreciable rate, reaching completion within forty hours to yield 2-(3-bromophenyl)-2-methylpropionic acid (12). Two different approaches were taken to convert 12 to its boronic acid derivative. In the first attempt two equivalents of fert-butyllithium were added to a mixture of the acid in tetrahydrofiiran at -78°C followed by excess of trimethyl borate. In the second attempt the acid was first converted to its sodium salt with sodium hydride. One equivalent of fcrt-butyllithium was added at -78°C followed, once again, by excess trimethyl borate. Neither approach succeeded in giving the desired product. Analysis of the product mixture via GC showed two major components one of which was the starting material and the other 2-methyl-2-phenyl- propanoic acid. The composition of the product mixture was further conformed via NMR. Quenching the metal-halogen exchange reaction with dilute aqueous acid instead of trimethyl borate had no effect on the product distribution, which along with a negative flame test further supported no boron incorporation. Another way to prepare the desired boronic acid involved reacting 12 with bis(pinacolato)diboron in the presence of catalytic amount of palladium chloride.2 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Although this is a good general method for preparing aromatic boronates bearing sensitive functional groups, this approach was never explored due to the relatively high cost of the process. To circumvent the preceding problems attention was focused on conducting the coupling step at the nitrile stage using a Negishi coupling. This approach had several advantages. The aryl-zinc derivative was generated in-situ, and unlike the boronate derivative, the starting material could be purified via distillation. However, functionalization of the fluorene moiety had to be performed after the coupling step in order to differentiate among the resulting three carbonyl groups. A test reaction carried out with 4-bromotoluene and 2 is illustrated in Figure II-5. The zinc reagent was prepared at -78°C in tetrahydrofiiran by treating the starting material with one equivalent of «-hexyllithium followed by the addition of anhydrous zinc bromide. Heating the resulting zinc derivative with 2 and a catalytic amount of tetrakis(triphenylphosphine)palladium gave the desired coupled product, 2,7-di-p-tolyl-9H-fluorene (13). Based on these encouraging results the above reaction protocol was used successfully to couple 11 and 2 to form the corresponding dinitrile species, albeit in low yields. However, hydrolysis of the dinitrile proved to be unsuccessful primarily due to solubility issues. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pd(PPh3 )4 Figure II—5 Preparation of test compound 13 A decision was made to conduct the coupling step after initially protecting 12 as the oxazole derivative, Figure II-6. First, heating a mixture of 12 and thionyl chloride at reflux, followed by solvent removal produced the acid chloride 14. Addition of two equivalents of 2,2-dimethylpropanolamine to 14 at 0° C in methylene chloride produced an amido-alcohol intermediate, which after subsequent treatment with thionyl chloride cyclized to form the desired 2-[ 1 -(3 -bromophenyl)-1 - methylethyl]-4,4-dimethyl-4,5-dihydrooxazole 15.3 Iodolysis and hydrolysis tests were conducted to monitor the conversion of 15 to its zinc-bromide derivative at -78 °C. The resulting derivatives were assayed using gas chromatography. The hydrolysis test showed complete reaction within fifteen minutes. However, the iodolysis mixture contained, in some cases, only 50% of iodine incorporation. This suggested a competitive protonation of the aryl-lithium salt. Running the reaction at -100°C inhibited the undesired side reaction substantially, resulting in conversions greater than 95%. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c o 2h socs2 Br 12 COCI 14 ,C 0 C I H O ^ SOCL Nf-L Aq HX Br 15 BuLi ZnBr, Pd(PPh3)4 DBF 17 16 Figure II—6 Preparation of compound 17 Compound 16 was prepared In the same way as described above for previous coupled derivatives. However, subsequent hydrolysis was unsuccessful. Subjecting starting material 16 to either hydrochloric or sulfuric acids resulted only in opening of the oxazole ring to the corresponding ammonium salt 17, which precipitated from the aqueous solution in nearly quantitative yield. No further hydrolysis took place, 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. even after prolonged heating, suggesting that along with steric crowding, insolubility is greatly responsible for the inherent stability of the ester. Synthesis of precursor C To overcome the aforementioned problems the carboxylic acid was protected as a tert-butyl ester. Although alkyl and phenyl lithium reagents readily attack esters, it was postulated that due to sterics this undesired reaction would be inhibited if the metai-halogen exchange and trans-metallation reactions were run at very low temperatures. Figure II-7 depicts a reaction sequence that was used to test this conjecture. 13 Br COCI 18 Br O r pcxphw. 2 BuLi, ZnBr2 19 Figure II—7 Preparation of compound 19 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Synthesis of the ferr-butyl ester was performed by addition of 14 to a suspension of potassium te/t-butoxide in diethyl ether at -78 °C.4 Aqueous workup and distillation afforded 2-(3-bromophenyl)-2-methylpropionic acid tert-butyl ester (18). Conversion of ester 18 to the corresponding lithium salt was executed at - 100°C to prevent attack on the ester, and improve the overall yield of the zinc reagent. After addition of zinc bromide the solution was allowed to reach room temperature. Iodolysis was used to convert a small aliquot of the zinc reagent to the aryl iodide, which was used to estimate the amount of zinc reagent via GC . The assay showed that the reaction proceeded cleanly and in good yield without any apparent addition to the ester functionality. Gentle reflux of the above reaction mixture in the presence of 2 and a palladium catalyst formed the desired coupled derivative. The reaction was monitored via HPLC, and only one major product was detected. Workup of the mixture gave the crude 2-(3-{7-[3-(l-terf-butoxycarbonyl- 1 -methylethyl)phenyl]-9H-fluoren-2-yl}phenyl)-2-mehtylpropionic acid ferf-butyl ester (19). The success of this transformation had great implications. Since tert-butyl esters are readily hydrolyzed under anhydrous conditions with strong acids, this provided a way to differentiate between the terminal and inner carbonyl groups. The coupling step was performed as above using 4 in place of 2 forming the crude tri ester 20, Figure II-8. After the starting material was removed by vacuum distillation selective hydrolysis of the /ert-butyl esters was conducted using trifluoroacetic acid and triethylsilane in methylene chloride.5 Basic workup of the product mixture 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. followed by acidification of the aqueous phase precipitated the desire precursor C, 2,7-bis[3 -(1 -carboxyl-1 -methylethyl)phenyl]-9-methyl-9H-fluorene-9-carboxylic acid ethyl ester (21). An alternate route to 20 was explored using 2-(3 -chlorophenyl)-2- methylpropionic acid ferf-butyl ester (25) in place of 18. The starting material for the synthesis of 24 was 3-chlorobenzyl chloride (22), which was nearly half the price of the currently used precursor 9. Figure II-9 illustrates the preparation of 25. However, the metal halogen exchange reaction failed at -100 °C, a temperature necessary to avoid undesired side reactions. Based on the observation that no perceptible formation of aryl-lithium took place even after one hour, the use 25 as a possible alternate to 18 was abandoned. Pd(PPh3)4, 4 20 21 Figure II—8 Preparation of compound 21 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cl 22 C l 1) NaCN, TBAHS = ► 2) KOtBu, Mel Cl 23 CN NaOH h2 o2 1 ) soci2 2) KOtBu c o2 h L i BuLi -100' C C l 25 Figure H—9 Preparation of compound 25 Synthesis or precursor D /-Histidine methyl ester dihydrochloride (27) was prepared in one step from /- histidine (26) by refluxing a suspension of the amino acid in methanol after a careful addition of thionyl chloride. The product was crystallized from the reaction mixture with diethyl ether.6 Synthesis of target ligand 36 Initially the coupling step between precursors 21 and 26 was attempted by activating compound 21 with 1,1’-carbonyldiimidazole (Figure 11-10). The corresponding activated diamide 28 proved to be quite water stable; in fact, it was 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. possible to safely isolate it via aqueous workup. However, reaction with 27 as its free base in chloroform was very slow, only noticeable after prolonged heating. In addition, the reaction did not proceed cleanly and was not explored further. HO HO C D 1 CHCI3 D, Et3 N X — CHCI3 31 21 28 Figure II—10 Preparation of compound 31 (I) Subsequently the activation of 21 was attempted with ethyl chloroformate, (Figure 11-11). Formation of the ethyl carbonate 29 proceeded quite cleanly in the presence of base. Derivative 29 was stable enough to be chromatographed in aqueous mobile phase but seemed more reactive than the 28 since reactions with 21 were observable at room temperature. Numerous byproducts were observed, rendering it not useful. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Et CICOjEt, EtjN CHCL D, EtgN X — CHCL 31 21 29 Figure II—11 Preparation of compound 31 (II) Compound 21 was successfully converted to the diacid chloride 30 by dissolution in thionyl chloride (Figure 11-12). NMR confirmed that no inadvertent cleavage of the ethyl ester took place under these conditions, and that the di-acid chloride formed cleanly. After removal of excess solvent the coupling step was successfully performed by addition of 30 to a solution of 27 in chloroform in the presence of triethylamine. The reaction proceeded rapidly and cleanly, giving the product in moderate yields. After a basic workup the desired diamide 31 was obtained 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 30 31 Figure II—12 Preparation of compound 31 (III) One difficulty with this procedure is to prevent both x and % nitrogens from participating in this reaction (Figure 11-13). To verify independently which nitrogen undergoes the desired nucleophilic attack, the x nitrogen was protected as its trityl derivative with a slight modification to a known procedure.7 With this protection any reactivity at the n nitrogen would lead to a highly reactive cation intermediate, which would most likely be very unstable if formed at all and would therefore only facilitate the desired diamide formation. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure II—13 Assignment of nitrogen atoms in histidine The reaction sequence to W-trityl-/-histidine methyl ester (33) is presented in Figure 11-14. Initially compound 27 was converted to its ditrityl derivative 32 with trityl chloride in the presence of triethylamine in chloroform. There was no incorporation of the trityl moiety into the % nitrogen. OMe 2 HCI Tr-CI, NEt CHC13 NHTr V-NTr OMe NH. cf3 c o 2 h 27 32 33 Figure II— 14 Preparation of compound 33 Subsequent treatment of 32 with two equivalents of trifluoroacetic acid cleaved the trityl group on the a nitrogen exclusively forming the desired methyl ester 33 as the ditrifluoroacetate salt. Synthesis of the ditrityl diamide 34 was 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conducted under the same conditions employed for the formation of 31. Addition of 30 to a solution of 33 and triethylamine at 0°C gave the desired compound 34, (Figure 11-15). The conversion seemed to proceed cleanly and with a noticeable improvement in yield. OMe V-N Tr CF3 C02 H 33 COjEt 30 CO,Et N-=vN~Tr CHCI3 34 Figure II—15 Preparation of compound 34 The final transformation of the synthetic procedure involved the removal of the protecting groups. Aqueous acidic conditions were chosen initially to affect the removal of all protecting groups in one step. Although the methyl esters and the trityl groups were facilely removed, hydrolysis of the tertiary ethyl ester proved to be quite difficult and only partial hydrolysis was achieved even after prolonged heating. To circumvent this problem the removal of the protecting groups was conducted in a stepwise manner. The trityl moiety was initially cleaved under anhydrous acidic conditions followed by saponification of the esters. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. One foreseen problem with this treatment was the possibility of decarboxylation as a side reaction (Figure 11-16). Test reactions were performed with two models, compounds 4 and 21. Although saponification of compound 4 in refluxing methanol led to a rapid decarboxylation, compound 21 formed only the carboxylic acid derivative. The difference in reactivity was most likely electronic in nature induced by the substituents in the meta position. This postulate correlates well with Hammett’s a m e ta parameters for bromide and phenyl, 0.39 and 0.06 respectively. MeOH MeOH Figure II—16 Decarboxylation tests with compounds 4 and 21 Based on the above observations the deprotection was conducted in two steps (Figure 11-17). First the trityl groups were removed by dissolution of the ditrityl diamide 34 in 50/50 trifluoroacetic acid/methylene chloride mixture in the presence 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of triethylsilane. After solvent removal and aqueous workup the desired diamide 35 was obtained indirectly. HPLC retention times and NMR confirmed this compound to be identical to one prepared by direct coupling of 21 with histidine methyl ester. The hydrolysis of the three esters in the resulting diamide was conducted by saponification in aqueous sodium hydroxide/ethanol mixture at room temperature. After solvent removal and washing, the target compound was obtained as the trisodium salt (36). Complex formation with ligand 36 The ligand contained five ionization sites, which were designated Hg through H0; fully protonated dication and fully deprotonated trianion respectively. 34 NaOH, THF \ 35 38 Figure II—17 Preparation of ligand 36 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Preparation of a metal complex with 36 was conducted by directly mixing the ligand with a metal ion in appropriate solvent. Ideally, a highly positively charged metal ion such as Fe2 + would replace the sodium ions in 36, and with the addition of water would form a stable dinuclear complex. To facilitate complexation several parameters were varied. These included the type and charge of the metal ion, different forms of the ligand, counter ions, and solvent (Table II-1). Ligand form C o u n ter ion S o lv en t Base Metal h5 cr c h 3 cn None Fe(OTf)2 h5 cr c h 3 cn Et3N Fe(OTf)2 h 5 cr THF Et3N Fe(OTf)2 h5 OTf CH3 CN Et3 N Fe(OTf)2 h 5 OTf THF Et3 N Fe(OTf)2 h5 OTf CH3 CN 2,6-Dtbp Fe(OTf)2 h2 NEt3 H+ THF Et3 N Fe(OTf)2 h 2 NEt3 hf THF Et3 N Zn(OTf)2 H o Bu4N+ CH3 CN None Fe(OTf)2 H o Bu4 N" Acetone None Fe(OTf)2 H o Bu4 N+ THF None Fe(OTf)2 H o Bu4 N+ EtOH None Fe(OTf)2 Ho Bu4N+ H2 0 None Fe(OTf)2 H o Bu4N+ CHCI3 None Fe(OTf)2 Table II-l Conditions used in complex preparation with ligand 36 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Initially, ligand 36 was isolated in the Hs form as a dihydrochloride salt. This form, although highly soluble in water, was practically insoluble in dry, polar organic solvents. Nevertheless, it was believed that a polar, relatively weakly ligating solvent would be the best suited for complex formation. A relatively weakly coordinated iron complex, iron ditriflate,8 was chosen for the reaction. It was found to be soluble in most polar solvents; exceptions being diethyl ether and chlorinated hydrocarbons. It was hoped that the weakly coordinating environment around iron would cause the uptake of the insoluble ligand forming the desired complex. A non ligating base was needed to trap protons, which had to be released upon coordination. The two bases used were triethylamine and 2,5-di-ferf-butylpyridine. The general behavior of the reactions was initially a colorless, heterogeneous mixture, which progressively turned darker. All mixtures turned yellow at first but eventually and invariably all ended as brown suspensions. The addition ofbase seemed only to expedite the aforementioned process by immediately forming a white precipitate upon addition, which darkened over time. To improve solubility the ligand was isolated as a ditriflate salt. This form was soluble in most polar organic solvents. Directly mixing separately prepared solutions of 36-ditriflate and Fe2 + resulted in an immediate precipitation of a white solid. Further stirring or the addition ofbase had no noticeable effect and the final mixtures were similar to those described above. In an attempt to inhibit the undesired precipitation the base was added prior to introduction of the metal. Titration of 36 with triethylamine showed that in most 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. poiar organic solvents three equivalents ofbase could be added before precipitation of the ligand took place. It was hoped that if the ligand remained in solution after the metal was added, a pre-coordination could take place, forming the essential skeleton of the desired complex. Once this process occurred, the removal of the remaining protons would then perhaps not cause precipitation. However, introduction of iron ditriflate solution caused an immediate precipitation. Based on the Irving-Williams series9 it was believed that zinc should have higher affinity for the ligand than iron. On repeating the above experiment with zinc triflate a noticeably different behavior was observed. Upon mixing of the two solutions no precipitate was observed even after prolonged stirring. However, precipitate formation was immediate upon further addition ofbase. Addition of a phase transfer catalyst seemed to have no effect. Final attempts to synthesize a metal complex with 36 were made with the ligand in the Ho form. It was postulated that with a greasy cation the solubility of the molecule would be enhanced. The base of choice for the hydrolysis of 35 was aqueous tetrabutylammonium hydroxide. Ideally, the solid would be free of water and inorganic salts, but attempts to isolate 36 as the tris-tetrabutylammonium salt failed. Drying in high vacuum resulted in the solid decomposition, possibly due to a nucleophilic attack on the tetrabutylammonium ion. It was therefore necessary to form the molecule directly in-situ by treating a suspension of the dihydrochloride salt in a polar solvent with five equivalents of aqueous tetrabutylammonium hydroxide. Invariably, the mixtures turned homogenous right away, but after prolonged stirring 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. some fine solid separated which was filtered off. The resulting light yellow solutions were treated with Fe2 + , either as a solution or solid. In either case immediate precipitation occurred, not unlike what had been observed before. Prolonged stirring caused the solid to acquire the now typical, rusty color. Changing the solvent systems had no positive effect. It is not clear why a well-defined complex did not form with 36. However, there are several plausible explanations. The compound is highly insoluble, especially as Ho, and there is a high possibility that the desired complex is insoluble as well. Furthermore, the 36 has many degrees of freedom, which increase the time necessary to find the most stable form. This energy sampling may be competitive with oligomer formation; a process that would most likely be irreversible. The following chapter focuses on modifying compound 36 to address these puzzling questions. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reference 1 Ranger, M.; Rondeau, D.; Leclerc, M. Macromolecules 1997, 30, 7686. 2 Miyaura, I. N. Chem. Cotnmun. 1996, 2073. 3 Schow, S. R.; Bloom, I. D.; Thompson, A. S.; Winzenberg, K. N.; Smith III, A. B. J. Am. Chem. Soc. 1986, 10S, 2662. 4 Crowtlier, G. P.; Kaiser, E. M.; Woodruff, R. A.; Hauser, C. R. Org. Synth., Collect. 1988, 6, 259. 5 Mehta, A.; Jaouhari, R.; Benson, T. X; Douglas, K. T. Tetrahedron Lett. 1992, 33, 5441. 6 Kelley, J. L.; Miller, C. A.; McLean, E. W. J. Med. Chem. 1977, 20, 721. 7 Papaioannou, D.; Athanassopoulos, €.; Magafa, V.; Karigiaimis, G .; Karamanos, N.; Stavropoulos, G.; Napoli, A.; Sindona, G.; Aksnes, D. Acta Chem. Scand. 1995, 49, 103. 8 Hagen, K. S.; Inorg. Chem. 2000, 39, 5867. 9 Seigel, H.; McCormick, D. B.; Acc. Chem. Res. 1970, 3, 201. 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter III Ligand Modification I There were several problems encountered in the preparation of metal complexes with 36. The multiple ionization sites render the ligand soluble only in water and aqueous organic mixtures. The pH required for the ligand to exist as Ho (trianion form) in water was too high and caused Fe3 + to precipitate as the hydroxide. Deprotonation of the ligand in succession was used to allow sufficient time for complexation, which was assumed to correlate with the degrees of freedom that would need to be overcome to form the complex. Low solubility of the ligand coupled with high affinity of iron to form the insoluble Fe(OH)n (3 'n )+ compromised this approach. In the natural enzyme the imidazole ring of a histidine moiety is coordinated via the 7 t nitrogen. Compound 36 could, in principle, coordinate to a metal center via either of the two nitrogens, where coordination via the n nitrogen or the x nitrogen would form a seven or an eight membered ring, respectively. This fact not only increased the degrees of freedom but also complicated the coordination process by increasing the possible coexistence of fast equilibrating mixtures or oligomer formation. To address some of these issues a decision was made to incorporate a trityl group into the final form of the ligand by attaching it to the x nitrogen of the histidine 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. moiety. There were five major reasons for this modification. The trityl group insured that metal coordination proceeded via the % nitrogen of the imidazole ring. The overall solubility of the ligand in polar organic solvents was expected to increase dramatically, which was important for complexation in non-aqueous medium, and isolation of the ligand free of inorganic salts and other water-soluble species. Furthermore, the phenyl ring system was predicted to be large enough to block possible dimerization of two dinuclear centers, and was expected to enhance the ability of the complex to crystallize (Figure HI-l). Figure HI— 1 Stereo view of proposed diiron complex with ligand 37 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Synthesis of ligand 37 The synthetic modification necessary to prepare 37 simply required that the cleavage of the trityl groups be avoided. Saponification of 34 with four equivalents of sodium hydroxide was conducted in water/tetrahydrofuran mixture. Protonation with excess dilute hydrochloric acid followed by solvent extraction with dichloromethane provided 37 as the dihydrochloride salt (Figure III-2). The hydrolysis of the ethyl ester in 34 was slow and incomplete in some cases, which was circumvented by modifying compound 4 to a methyl ester derivative 39 instead (Figure III-3). Although the methyl group could in principle be introduced at the beginning of the synthesis, it was convenient in this case to simply use Fisher trans-esterification to replace the ethyl group in 3 forming the methyl N ^ /N -T r NaOH, THF/H20 34 37 Figure III—2 Preparation of ligand 37 (I) 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. derivative 38. Methylation of 38 produced the methyl analog of 4, methyl-2,7- dibromofluorene-9-methyl-9-carboxylate (39). Br MeOH NaOEt CO,Me Mel Br Br 3 38 39 Figure lH—3 Preparation of compound 39 From this point, the synthesis of the modified ligand was conducted similarly as for the ligand 36. Coupling of 39 with the zinc derivative of 18 produced the triester 40, which after deprotection gave compound 41, a methyl analog of 21 (Figure III-4). 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Br 18 BuLi, ZnBr, Pd(PPh3 )4, 39 OH 40 CH,CI. OH 41 Figure III—4 Preparation of compound 41 Conversion of 41 to the diacid chloride 42 followed by reaction with 33 produced diamide 43, which was saponified to give the desired ligand 37 (Figure III-5). SQCL 33, N E t 4 1 ------- =► 4 2 ------------ - CH2C!2 ^ .C 0 2Me N^ / N" Tr C02 Me NaOH, THF ^ HCI, CH2CI2 37 N^ C 0 2Me H 43 Figure HI—5 Preparation of ligand 37 (II) 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Complex formation with ligand 37 Initially isolated as the dihydrochloride salt, ligand 37 differed substantially from ligand 36, particularly in solubility. The Ms form of 37 was highly soluble in CH2CI2, which rendered it possible to obtain the ligand dry and free of inorganic salts. Furthermore, it allowed for the ligand to be introduced as a solution in preparation of the metal complex. The attempted reactions with 37 are summarized in Table III-1. Ligand form Counter ion Solvent Base Metal h 5 Cl' CH2CI2 None Fe(OTf>2 h 5 cr THF None Fe(OTf) 2 h 5 cr THF Bu4NOH Fe(OTf) 2 h 5 cr CH3CN Bu4 NOH Fe(OTf) 2 H5 cr CH2CI2 NEt3 Fe(OTf) 2 h 5 cr THF KOfBu Fe(OTf) 2 h 3 N/A THF None Fe(OTf) 2 h 3 N /A THF None Zn(OTf) 2 h 3 N /A CH2CI2 2,6-Dtbp Zn(OTf) 2 h 3 N /A CH2CI2 2,6-Dtbp Fe(OTf) 2 Table HI-1 Conditions used in preparation of complexes with ligand 37 Due to the much-improved solubility of 37 the initial attempts to synthesize a stable metal complex were conducted using the ligand in the di-protonated Hg form. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Direct mixing of iron triflate with 37 displayed interesting behavior. Even though all solutions were light yellow to start, prolonged mixing caused darkening and some precipitation was observed. Since the ligand was folly protonated, and there were no basic species in the solution to scavenge the protons released upon coordination, one would expect no reaction. It was postulated that any release of protons would render the solution acidic enough to promote the cleavage of the trityl group. To avoid trityl group cleavage, a solution of 37 was carefully treated with aqueous tetrabutylammonium hydroxide to trap the free protons. The reaction mixture generally remained homogenous up to addition of two equivalents of base. However, further addition caused the formation of a green precipitate, which turned dark brown upon exposure to air. All attempts to re-crystallize the green solid failed due to lack of solubility. To avoid possible reactions between the base and iron triflate, the ligand was converted to the H3 form in-situ in the absence of any metal. Addition of iron triflate produced a light yellow solution, which progressively turned darker. After two days the solution became dark brown. Conducting the same reaction with zinc triflate produced a clear, homogeneous mixture, which did not change even after two days. All attempts to obtain crystals from either solution have failed thus far. It was suggested that the exclusion of water might be very important for promoting complex formation. Therefore, the ligand was deprotonated in-situ with five equivalents of 2,6-di-ferf-butyi pyridine. Even though this base is not strong enough to form the Ho form of the ligand, the excess base was used as an organic 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. buffer. Addition of zinc triflate produced a light yellow homogeneous solution. Repeating the same reaction with iron triflate produced a yellowish green solution, which over time turned brown. Unfortunately, all crystallization attempts have failed thus far for either solution. Ligand modification II The failure to obtain an isolable metal complex after the first modification was attributed to two fundamental points. The trityl group is quite acid sensitive and may be cleaved by Lewis acids or a radical pathway. In addition, there was NMR evidence that the central carboxyl group, designed to bridge the two metal centers, was difficult to deprotect without partial decarboxylation taking place. To address these problems it was decided to replace the trityl moiety with a methyl group even though a completely new synthetic approach was necessary. Furthermore, in order to prevent decarboxylation a methylene spacer was placed between the central carboxylic acid functional group and the fluorene backbone (Figure III-6 ). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure III—6 Stereo view of designed diiron complex with ligand 56 Synthesis of ligand 56 To prepare the ligand with the desired modifications, compounds 3 and 27 had to be modified. Preparation o f’ W-methyl histidine methyl ester is summarized in Figure III-7.1 /-Histidine methyl ester (27) was treated with 1,1’- carbonyldiimidazole in the presence of one equivalent of triethylamine in acetonitrile. Aqueous basic workup followed by crystallization gave 5-oxo-5,6,7,8- tetrahydroimidazo[ 1,5-c]pyrimidine-7-carboxylic acid methyl ester (44) in moderate yield. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H 9 M eo. Tjf> N CH3 CN o o 2 H C S OMe 27 CHgl CHgCN / NH- r-N H CI “yW o 2 HCI _ x_ h2 o N— OMe 47 ' 45 Figure i n —7 Preparation of compound 47 Treatment of 44 with methyl iodide in refluxing acetonitrile produced the desired iodide salt 45. Hydrolysis of 45 however, did not proceed cleanly, probably due to the competitive oxidation of the iodide anion to triiodide, giving rise to a deep purple solution. This problem was overcome by substituting methyl iodide with dimethyl sulfate (Figure III-8). Alkylation of 44 resulted in the monomethyl sulfate salt 46, which when subjected to mild acidic conditions, cleanly produced the desired /-W-methyl-histidine (47). 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o. OMe O Me?SQ4 H N '^ 'N '^ - MeOSOg- CH3 CN Q N" OMe 44 46 MeOH Figure III—8 Preparation of compound 48 It was necessary to remove all of the sulfate anion to facilitate clean workup. The aqueous solution of 47 was rendered neutral with sodium bicarbonate and water was removed. The zwitterion was extracted with methanol to remove sodium chloride and sodium sulfate. The methanolic solution of 47 was then treated with thionyl chloride followed by a gentle reflux to produce /-"JV-methyl-histidine methyl ester (48). The second modification involved the insertion of a methylene fragment between the carbonyl group and the fluorene moiety of 3 (Figure III-9). Compound 3 was deprotonated using sodium ethoxide in tetrahydrofuran and the resulting anion 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was treated with excess ethyl bromoacetate to give 2,7-dibromo-9- ethoxycarbony!methyl-9H-fluorene-9-caboxylic acid ethyl ester (49). Subjecting 49 to basic hydrolysis in water/ethanol mixture under reflux effected decarboxylation, and upon protonation 2,7-dibromo-9H-fluoren-9-yl-acetic acid (50) was obtained. Esterification of 50 in the presence of a catalytic amount of sulfuric acid in boiling ethanol produced upon cooling the desired 2,7-dibromo-9H- fluoren-9-yl-acetic acid ethyl ester (51). Since the pKa of the benzylic hydrogen is not as low as that of 3, this compound was used directly in the coupling step without further modification. NaOEt 3 49 NaOH, H2 0 EtOH V 51 50 Figure HI—9 Preparation of compound 51 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Coupling of 51 with an alkyl zinc derivative of 18 proceeded similarly as with other derivatives and produced 2-(3-{7-[3-(l-fert-butoxy carbonyl-1 - methylethyl)phenyl]-9-ethoxycarbonylmethyl-9H-fluoren-2-yl}phenyl)-2- methylpropionic acid fert-butyl ester (52), Figure III-10. Selective hydrolysis of 52 produced the desired 2-(3-{7-[3-( 1 -carboxy-1 -methylethyl)phenyl]-9- ethoxycarbonylmethyl-9H-fluoren-2-yl}phenyl)-2-methylpropionic acid (53). Preparation of the modified ligand was finalized by converting 53 to its diacid chloride derivative 54, which was reacted with two equivalents of l-n N- methyl-histidine methyl ester (48) to give 2-(2- {3 -[9-ethoxycarbonylmethyl-7-(3 - {1 - [ 1 -methoxycarbonyi-2-( 1 -methyl-1 H-imidazol-4-yl)-ethylcarbamoyl] -1 - Pd(PPfi3)4, 51 S3 Figure III—10 Preparation of compound 53 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methylethyl} phenyl)-9H-fluoren-2-yl]phenyl} -2-methylpopionylamino)-3-3-(l- methyl- 1 H-imidazol-4-yl)propionic acid methyl ester (55) (Figure III-ll). Hydrolysis of 55 was carried out under basic conditions, as it was confirmed that this method produced a purer form of ligand 56 than under acidic hydrolysis. The workup of the reaction mixture was varied depending on the desired extent of protonation of isolated ligand. Complex formation with ligand 56 As expected, 56 showed similar behavior to 36. Substitution of the trityl moiety with a methyl group dramatically lowered the ligand’s solubility compared to ligand 37. However, isolation of the ligand as the ditriflate salt (H5 form) rendered it 53 55 Figure EH — 11 Preparation of compound 55 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. soluble In various polar organic solvents. Furthermore, this method allowed the ligand to be obtained dry and free of inorganic salts. The ligand was often kept as a solution for the salt was extremely hygroscopic. Attempts to form an isolable metal complex with 56 are summarized in Table HI-2. Some changes were made to the typical procedures for complex formation used thus far. To limit the ligand from forming precipitates upon addition of a metal ion, complexations were conducted at high dilution conditions (0.4 mg/mL). It was postulated that this should minimize oligomer formation. Furthermore, if all species remained in solution, the mixture could be heated to insure a thermodynamic form of the complex was obtained. A change was also made to the form of iron used for complexation. It was postulated that using already preformed dinuclear iron complex, tetraethylammonium (p-oxo)-bis(trichloroferrate),2 would increase the probability of 56 coordinating in the desired fashion. Another benefit was the homology of the oxo-bridge with the natural complex. Separate solutions of tetraethylammonium (p-oxo)-bis(trichloroferrate), and 2,6-di-fer/-butyl pyridine were simultaneously added to a dilute solution of 56 over a period of eight hours. The resulting mixture turned light yellow, and slightly opaque even under dilute conditions. Cooling the reaction mixture caused brown solid to precipitate, which upon warming to room temperature did not re-dissolve. UV/Vis spectroscopy showed no noticeable difference between the starting material and the light yellow solution. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ligand form Counter ion Solvent Base Metal h 5 O Tf CH3CN 2,6-Dtbp [Fe2Cl6 O f H 5 O Tf c h3 cn PS [Fe2Cl6O f H o PPlsf EtOH None [Fe2CI6 o f H o ppisr Acetone None [Fe2Ci6O f H o PPM* c h3 cn None [Fe2Ci6 O f H o PPN+ CH3N0 2 None [Fe2CI60 ]^ H o PPN+ c h 2c i2 None [Fe2Cl6Q f H o PPN" C H C I 3 None [Fe2CI60 ]2 ‘ Hq PPN+ THF None [Fe2C!60 f H o PPN+ EtOH None CuC12.2H2 0 H o PPN+ Acetone None CuCI2.2H2 0 H o PPNf CH3CN None CuCI2.2H20 H o PPN+ c h 3n o 2 None CuCI2.2H2 0 H o PPN+ c h 2c i2 None CuCI2.2H2 0 H o PPN+ CHCI3 None CuCI2 .2H20 H o PPN+ THF None CuCI2.2H2 0 H o PPN+ EtOH None Zn(OAc)2.2H2 0 H o PPN+ Acetone None Zn(OAc)2.2H2 0 H o PPM" CH3 CN None Zn(0Ac)2.2H2 0 H o PPN+ c h 3n o 2 None Zn(0A c)2 .2H20 H o PPN+ c h 2c i2 None Zn(0Ac)2.2H2 0 H o PPN+ CHCI3 None Zn(0Ac)2.2H2 0 H o PPN" THF None Zn(0Ac)2.2H2 0 Table III-2 Conditions used in preparation of complexes with ligand 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The above experiment was repeated using the same conditions except for the order of the addition. Separate solutions of tetraethylammonium (p-oxo)- bis(trichloroferrate), and 56 were added to a dilute solution of 2,6-di-fert-butyl pyridine. This time the mixture remained homogeneous, even after stirring for two days. Attempted crystallization caused the separation of a brown solid, which after drying was practically insoluble, again making it difficult to analyze by UV/Vis. The above reaction was repeated once more using a stronger base to insure complete deprotonation. The reaction was carried out at 50 °C for two days under nitrogen. As before, a large amount of a brown solid formed, which was insoluble in organic solvents. The direct preparation of the Ho form had not been attempted before for any of the ligands due to its inherent insolubility in organic medium. However, it was proposed that the use of a bulky organic cation might solubilize 56-Ho. An aqueous solution of 56 buffered to a pH of 7.5 was mixed with a lukewarm aqueous solution of bis-triphenylphosphoiminium chloride. Immediately a white precipitate formed. After extraction with methylene chloride and solvent removal, an off-white solid was obtained which was soluble in most organic solvents. HPLC verified the presence of the ligand in the Ho form. This form of 56 was used directly in small-scale reactions to study its behavior with Fe3 + , Cu2 " 1 " , and Zn2 + in various solvents. Although most mixtures formed a precipitate either immediately after mixing or within several hours, some samples remained in solution. However, in the presence of Fe3 + all reactions formed precipitates immediately after mixing. Zn2 + 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. formed dear solutions In acetonitrile, nltromethane, methylene chloride, and chloroform. Furthermore, after a few days of standing at ambient temperature, all the solutions started to discolor from water-white to yellow to orange. The reason for this behavior is unclear. Attempts at isolation by crystallization have failed thus far. In the case of Cu2 + , only methylene chloride and chloroform gave clear solutions. This result was quite encouraging, since cupric chloride dihydrate, which is not soluble in these solvents, was taken up into solution forming a homogeneous mixture. However, a control experiment showed that [PPN]C1 alone solubilizes Cu2+ , presumably by forming the CuCV ion. In addition, comparison of the obtained solution and the control via UV/Vis spectroscopy showed no spectral difference, which suggest that the identical reaction took place in both cases. It was quite discouraging that no suitable conditions were found to form a well-defined metal complex with ligand 56, particularly with the Ho form. Even more surprising was the fact that the tetraethylammonium (p-oxo)- bis(trichloroferrate) complex did not seem to aid complexation. It was postulated that the ligand might have too many degrees of freedom. Another possibility, which would be surely discouraging, is that the desired complex may not be the thermodynamic product on the reaction profile. Ligand modification HI The previous modifications suggest that the environment around the imidazole ring might have less influence on stable complex formation than 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. previously anticipated. One major aspect that might have been underestimated in all of our modifications was entropy. It was previously thought that in principle these complexes should self-assemble in water, trapping the aquated ions. The displacement of eight water molecules was to provide the driving force for the complex forming reaction. The question was whether the two aims of the molecule had enough time to come together in the anticipated geometry, prior to precipitating out of the solution via only a partial complexation. To enhance the probability of complexation, the decision was made to tie the two arms together with a hydrocarbon bridge. There were several caveats in this approach. Firstly, it was imperative that the bridge be long enough to accommodate two metal atoms within its cavity. Secondly, the hydrocarbon chain had to be far enough from the reaction centers to not interfere with substrate oxidation. After extensive molecular modeling of the system, a Cs chain was determined to be of the right length and tension. The idea was to bridge the two imidazole rings via their n nitrogen atoms, constructing a flexible ring. This should encourage the molecule to behave as an encapsulating ligand, specific for two metal ions (Figure III-12). 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure III—12 Stereo view of designed diiron complex with ligand 64 Synthesis of ligand 64 The challenge was to synthesize a molecule in which two histidine moieties are connected via an eight-carbon chain. Furthermore, this compound was to be coupled with 54 under high dilution conditions to produce the desired cyclic triester. A direct nucleophilic attack on 1,8 -dibromooctane with 44 showed no reaction. It became clear that a better leaving group was necessary to achieve this transformation. The reaction was repeated with a dimesylate derivative 58 previously synthesized from 1,8-octanediol 57 (Figure III-13). However, the direct combination of 58 with 44 in refluxing acetonitrile seemed to give no reaction since a large amount of starting material was recovered from the reaction mixture after two-days at reflux. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HO OH CH3S 0 2CI, NEt3 THF Ms 57 58 Figure III—13 Preparation of compound 58 It was evident that an even better leaving group than methanesulfonate was necessary in order to achieve this transformation. The approach taken is depicted in Figure IH-14. 1,8-Octanediol was first treated with triflic anhydride and pyridine, equivalents of 44 produced 7-methoxycarbonyl-2-[8-(7-methoxycarbonyl-5-oxo- 5,6,7,8-tetrahydroimidazo[l,5-c]pyrimidin-2-yl)octyl]-5-oxo-5,6,7,8- tetrahydroimidazo [1,5 -c]pyrimidin-2-ium ditriflate (60), which after acid hydrolysis produced 2-amino-3 -(1 - {8- [4-(2-amio-2-caboxyethyl)imidazol-1 -yl] octyl }-lH- imidazol-4-yl)propionic acid as its mixed dihydrochloride-ditrifiate salt (61). Esterification of 61 in methanolic hydrochloric acid formed 2-amino-3-(1 - {8 - [4-(2- amio-2-methoxycarbonylethyl)imidazol-1 -yl]octyl} -1 H-imidazol-4-yl)propionic acid methyl ester as the mixed dihydrochloride-ditrifiate salt (62). producing 1,8 -octane-ditriflate (59).3 Treatment of this derivative with two 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cl C l M eO X OTf- NH+ M e 0 2C A I „ C N H * Cl NH3+ OTf- HOX OTf- .NH+ HCI H MeOH NH+ H O ,C 62 61 Figure lH— 14 Preparation of com pound 62 Coupling of 62 with 54 had to be conducted under highly dilute conditions to insure ring closure (Figure III-15). Both compounds were introduced as solutions in chloroform by a simultaneous addition into a flask containing a large excess of solvent. The reaction was carried out at a final concentration of 3 mM. After solvent removal and a typical workup, the desired cyclic triester 63 was obtained. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The complexity of the NMR spectrum created an aura of skepticism about the success of the ring closure, and confidence was further undermined by the extraordinarily good yield of the reaction. However, only one major peak with a new retention time was observable via HPLC, and high-resolution mass spectroscopy confirmed the molecular weight and precise elemental composition of the compound. Saponification of 63 in aqueous sodium hydroxide/methanol mixture yielded ligand 64. As before, the workup of this reaction depended on the desired counter ion and final form of the ligand. OTf- C l NEt3, 54 CHCI, 63 NaOH MeOH 62 64 Figure HI— 15 Preparation of ligand 64 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Complex formation with ligand 64 Of all of our ligand variations compound 64 was the most insoluble in all of its forms. Initially, isolated as the dihydrochloride salt, this form was only soluble in aqueous mixtures of polar organic solvents. The trisodium salt of the Ho form was soluble in slightly basic water, but had no solubility in organic solvents. The low solubility of this molecule did not seem so surprising as the loss of some degrees of freedom was expected to enhance the molecule’s tendency to form a lattice. Ligand form Counter ion Solvent B ase Metal h5 ci- THF NEt3 CuCI2.2H2 0 h 5 ci- c h 2c i2 NEt3 [Fe2ClsO]^- h5 cr c h3 cn 2,6-Dtbp CuCI2.2H2 0 h 5 cr EtOH 2,6-Dtbp CuCI2.2H2 0 h 5 OTFA" h2 o NaOH CuCI2.2H2 0 h 5 OTFA" h2 o NaOH FeCI3.6H2 0 Hs OTFA" h2 o NaOH FeS04.7H2 0 h 3 N/A c h 3 c n None CuCI2.2H2 0 Ho P P H * c h c i3 None C uCI2.2H20 Ho PPN+ c h3 cn None [Fe2CI6 o f Ho P P f f c h 2 c i2 None Cu(OTf)2 Ho PPN* c h3 cn None Cu(OTf)2 Ho PPN+ c h 2c i2 None Fe(OTf) 2 Table HI-3 Conditions used in preparation of metal complexes with ligand 64 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The synthesis of metal complexes with 64 was conducted using the conditions previously employed for other complexes. The conditions, metal ions, and forms of the ligand are summarized in Table III-3. As its dihydrochloride salt, the ligand was mixed with tetraethylammonium (ju-oxo)-bis(trichloroferrate) in various solvents. All attempts led to formation of a brown precipitate while the solution remained colored. More precipitate formed upon the addition of base. The same experiment when repeated with cupric chloride dihydrate displayed similar behavior with the exception of color. Weighing of the isolated precipitate indicated that the majority of the solid initially introduced had come out of solution. NaOH eq. pH 0 . 2.6 1 3.2 2 4.3 3 5.8 4 7.1 5 8.3 6 1 1 7 12 Table HI-4 Crude pH titration of fully protonated ligand 64 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To improve the ligand’s solubility, compound 64 was isolated as the bis- trifluoroacetate salt. Removal of all volatiles dramatically decreased the ligand’s solubility. Additions of a slight excess of trifluoroacetic acid enhanced the solubility immensely. The dry solid was precipitated with diethyl ether from an acidic solution in acetonitrile. To verify the actual form of the ligand, a crude titration was done to visualize the ligand’s buffered region. To avoid solubility problems, the titration was run in a 50/50 acetonitrile/water mixture. Careful sequential addition of five equivalents of aqueous sodium hydroxide was performed, giving ample time for equilibration between additions. The results of the titration are presented in Table III-4. The initial pH suggests that the ligand is fully protonated and is free of excess trifluoroacetic acid. Furthermore, the dramatic pH increase when the sixth equivalent was added confirms that there are, in fact, five protons in the molecule. Similar titration curves were generated in the presence of Fe2 + , Fe3+ , and Cu2 + . In all cases the initial pH of all mixtures dropped significantly when the ligand was added and, invariably, clear solutions were formed. However, precipitate started to form in all solutions with sequential addition of base. All subsequent attempts to form a metal complex with 64 were conducted with the ligand in the Ho form. As previously described for ligand 56, the solubility of this form in organic solvents increased when the counter ion was bis(triphenylphosphine)imminium. A standard solution of [(PPh3 ]2 N]3[64(Ho)] was prepared in methylene chloride. The solution was stored over molecular sieves to 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. insure complete absence of water. Drying with metal salts, such as magnesium sulfate, was strictly avoided to prevent possible complexation with the drying agent. Preparation of metal complexes was carried out by treating an equivalent of various metal ions with the above-mentioned stock solution. In most cases the presence of precipitate was prevalent. Those samples that remained soluble were compared to the controls (metal ion and [PPN]C1) using UV/Vis spectroscopy. The most encouraging experiment was carried out in the presence of cupric chloride in chloroform. The metal salt, which is insoluble under these conditions, was quickly solubilized, forming an orange solution. However, the same behavior was observed when the reaction was repeated with bis(triphenylphosphine)imminium chloride and CuCl2 alone, excluding 64. The two solutions exhibited the same UV/Vis spectra, suggesting that the color was caused by very similar species. Thus the formation of a Cu2 + complex with 64 remained elusive. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reference 1 Noordham, A.; Maat, L.; Beyerman, H. C, Reel Trav. Chim, Pays-Ras 1978, 97, 293, 3 Armstrong, W. H.; Lippard, S. J. Inorg. Chem. 1985, 24, 981. 3 Solomon, M. F.; Solomon, R. G. J. Am. Chem. Soc. 1979, 15, 4290. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Experimental Section General Considerations All reactions were carried out under nitrogen atmosphere unless otherwise noted. Nitrogen was purified by passage through a glass tower containing a reduced copper catalyst (BASF R3-11) followed by passage through a second tower containing indicating 4A molecular sieves. Standard vacuum and Schlenk line techniques were applied for most reactions. Storage and manipulation of air and/or moisture sensitive compounds was done in a Vaccum Atmospheres Model HE-553-2 glove box equipped with a DriTrain MO40-2 inert gas purifier. Cr(acac)2 was used as an indicator to monitor the oxygen level in the glove box. Benzene, diethyl ether, pentane, and tetrahydrofuran were dried over sodium/benzophenone and distilled prior to use. Methylene chloride was dried over calcium hydride and distilled prior to use. All other solvents were used as received from various suppliers. NMR solvents were used as received from Cambridge Isotopes, Inc. NMR spectra were recorded on Broker AC-F250, AM-360, and AMX-500 MHz FT spectrometers. Chemical shifts are reported in parts per million (6) downfield from TMS via reference to residual protons in the deuterated solvent for *H NMR and the solvent carbon(s) in the °C NMR. Gas chromatography was conducted using a Hewlett Packard Series 6890 GC and a HP-5 (5% diphenyl/95% polydimethylsiloxane) capillary column. High 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. performance liquid chromatography was conducting using a Hewlett Packard Series 1 100 HPLC equipped with a quaternary solvent delivery system and a variable wavelength UV detector. lonizable samples were analyzed using a Waters SymmetrySOO™ C18 3.5 pm, 4.6 x 150 mm column running a mobile phase composed of acetonitrile and a 10 mM phosphate buffer with its pH adjusted to 3.0. All other samples were analyzed using a Waters SymmetryShield™ RPig 3.5 pm, 4.6 x.100 mm column running a mobile phase composed of acetonitrile and water. 2,7-Dibromofluorene (2) A 250-mL round-bottomed flask equipped with a magnetic stir bar and a pressure-equalized addition funnel was charged with fluorene (1) (20.6 g, 123 mmol), anhydrous FeCL, (160 mg, 0.99 mmol), and CHCI3 (70 mL) resulting in a dark blue solution. The flask was wrapped in aluminum foil and the reaction was run in the dark. The flask was placed in an ice bath and bromine (13 mL, 250 mmol) was added slowly via the addition funnel, with monitoring of the rate ofHBr evolution. After the addition was complete, the mixture was kept at 0 °C for additional 10 min followed by stirring at room temperature for 3 hours. The reddish slurry was treated with H2O (100 mL) and enough saturated N aaSaO s solution to destroy the excess bromine. The solid was collected and the organic phase separated and evaporated in vacuo. The solids were combined and recrystallized from THF/EtOH mixture to give 23.8 g (59%) of the title product as a white solid. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lU NMR (360 MHz, CDC13 ): 8 7.62 (s, 2H), 7.54 (d, 2H, J H .H = 7.7 Hz), 7.46 (d, 2H, J h - h = 7.7 Hz), 3.80 (s, 2H). 1 3 C NMR (90 MHz, CDCI3): 8 144.7, 139.6, 130.1, 128.2, 121.1, 120.9, 36.5. Ethyl-2,7-dibromofluorene-9-carboxylate (3) A 500-mL round-bottom flask equipped with an addition funnel and a magnetic stir bar was flame dried under vacuum and flushed with nitrogen. Dry diisopropylamine (15 mL, 110 mmol) was added followed by dry IH F (90 mL). The solution was cooled on ice and 5.35 M w-butyllithium in hexane (18 mL, 96 mmol) was added drop wise. The mixture was stirred at 0 °C for 45 minutes. The ice bath was removed and the mixture was cooled to -78 °C (dry ice/acetone bath). 2,7-dibromofluorene (2) (15 g, 46 mmol) was dissolved in dry THF (150 mL) in a 250-mL round-bottom flask under nitrogen. The resulting solution was transferred via a cannula to the addition funnel and added dropwise to the LDA forming an orange solution. Occasionally, some solid separated. The mixture was stirred for an additional 30 minutes at -78 °C and freshly distilled ethyl chloroformate (6.8 mL, 71 mmol) was added at a moderate rate. The solution was stirred for 10 minutes and 3 M aqueous HC1 (150 mL) was added. The light yellow mixture was allowed to warm to room temperature. The pH was adjusted with HC1 to be acidic to litmus paper, and the solution was extracted with Et20 (1 x 100 mL, 3 x 50 mL). The organic phase was washed with brine, dried with MgSCL, and the solvent was 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. removed in vacuo to yield a white solid. The crude product was recrystallized from hexane to yield 15 g, (82%) of white needles. *H NMR (360 MHz, CDCI3): 6 7.79 (d, 2H, J H -h = 1.4 Hz), 7.55 (d, 2H, J H -h - 8.2 Hz), 7.52 (dd, 2H, J H -h = 8.2 Hz, 1.4 Hz), 4.79 (s, 1H), 4.24 (q, 2H, J H -h = 7.3 Hz), 1.30 (t, 3H, Jh-h = 7.3 Hz). Ethyl-2,7-dibromofluorene-9-methyl-9-carboxylate (4) In a 50-mL round-bottom flask sodium metal (1.0 g, 43 mmol) was dissolved in anhydrous EtOH (10 mL) under nitrogen. The resulting solution of sodium ethoxide was added via cannula to a 250-mL round-bottom flask containing a suspension of 3 (15.1 g, 38 mmol) in dry EtsO (150 m L). The greenish yellow solution was stirred for 15 minutes and methyl iodide (2.7 m L, 43 mmol) was added in one portion. Within a few minutes the formation of sodium iodide precipitate was noticeable and the mixture was allowed to react overnight. The reaction was quenched with water (50 m L ) and transferred to a separatory funnel. The organic phase was washed with brine, dried with MgSCL, and solvent was removed in vacuo to yield a light solid, which was recrystallized from EtOH, yielding 12.8 g, 82%. !H NMR (250 MHz, DMSO-fife): 5 8.30-8.23 (m, 4H,), 8.06 (dd, 2H, JH -h = 8.0 Hz, 2.0 Hz), 4.54 (q, 2H, JH -h = 7.0 Hz), 2.22 (s, 3H), 1.56 (t, 3H, J H -h = 7.0 Hz). 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-Methylphenylboronic acid (6) A flame dried 250 mL Schlenk flask equipped with a stirring bar was charged with 3-Bromotoluene (5) (5.3 g, 31 mmol) and dry THF (60 mL) under nitrogen. The solution was cooled to -78 °C and 2.5 M w-hexyllithium in hexane (14.7 mL, 37 mmol) was added drop wise. The milky white mixture was stirred for 1 hour. The septum was replaced with an addition funnel, which was flushed with nitrogen and charged with tri-methyl borate (9.0 mL, 80 mmol) dissolved in dry THF (30 mL). The mixture was slowly added to the aryl-lithium salt at -78 °C over 30 minutes. The clear, yellow mixture was brought to room temperature and stirred overnight. 10 M aqueous HC1 solution (20 mL) was carefully added to the resulting mixture, and after further stirring for 1 hour the reaction mixture was extracted with Et2 0 (3 x 50 mL). The organic phase was washed with brine, dried with MgSCL, treated with carbon black, and all volatiles were removed in vacuo to yield a white, fluffy solid; 4.2 g, 99%. *H NMR (360 MHz, CDC13 ): 8 8.08-7.78 (m, 2H,), 7.40 (d, 2H, J H .H = 5.0 Hz), 2.47 (s, 3H). 5,5-Dimethyl-2-m-tolyl-[l,3,2]dioxaborinane (7) A dry 100-mL round-bottom flask was charged with 6 (0.96 g, 7.0 mmol) and CCL (50 mL). 2,2-Dimethyl-propane-l,3-diol (0.83 g, 8.0 mmol) was added to the solution and the mixture was stirred for 20 minutes at reflux. All of the diol 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dissolved, and the mixture was cooled to room temperature. Typically the compound was not isolated at this point and was used directly in the next step. 5 H NMR (360 MHz, CDC13 ): 8 7.61-7.49 (m, 2H,), 7.24-7.16 (m, 2H), 3.73 (s, 4H), 2.31 (s, 3H), 0.978 (s, 6H). 3-Bromobenzyl bromide (9) 1000-mL round-bottom flask equipped with a stirring bar and a reflux condenser was charged with, 5 (60 g, 0.35 mol), NBS (36 g, 0.20 mol), and CCI4 (300 mL). The suspension was brought to a reflux in the presence of a 300 W tungsten lamp. A vigorous reaction took place. When the rate of reflux returned to normal the solution was allowed to cool to room temperature and the resulting insoluble succinimide was filtered off. The supernatant was washed with saturated solution ofNaHCCb, dried over MgSCL, and CCI4 was removed in vacuo to give a yellow liquid; a mixture primarily consisting of the starting material and the desired product. Vacuum distillation yielded 5 (20 g, 78% recovery) and 9 (40 g, 80%) as a white viscous solid. 3-Bromophenyl acetonitrile (10) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 9 (50 g, 0.20 mol), Et20 (100 mL), NaCN (12 g, 0.24 mmol), H2O (10 mL), and tetrabutylammonium bisulfate (3.6 g, 10 mmol). An exothermic reaction took place and the biphasic mixture was stirred overnight. The reaction was 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. monitored via GC. Upon completion H20 (100 mL) was added to the yellowish reaction mixture. The phases were separated and the aqueous phase was further extracted with Et2 0 (2 x 50 mL). The organic phase was washed with brine, dried with MgSCL, and the resulting solution was used without further purification in the following step. 2-(3-Bromophenyl)-2-methylpropionitrile (11) A 1000-mL round-bottom flask equipped with a magnetic stirring bar was charged with potassium ferf-butoxide (45 g, 0.40 mol) and Et2 0 (200 mL) and fitted with an addition funnel. Methyl iodide (25 mL, 0.40 mol) was added to the solution of 10 from the previous step and mixed thoroughly. The mixture was transferred into the addition funnel, nitrogen was introduced, and the suspension was cooled to 0 °C on ice. The resulting solution was added to ether suspension drop wise with continuous stirring. After the addition was complete the thick suspension was warmed to room temperature and the course of the reaction was monitored via GC. Upon completion H20 (100 mL) was added and the phases were separated. The organic phase was washed with 1 M aqueous HC1, saturated solution of NaHC(>3, brine, and dried over MgSCL. Et20 was removed in vacuo and the resulting dark red mixture was vacuum distilled to give the title product as a clear liquid (36 g, 80% over two steps). !H NMR (250 MHz, CDC13): 8 7.71 (t, 1H, J H -h = 1.9 Hz), 7.60-7.50 (m, 2H), 7.38 (t, 1 H, Jh-h = 8.0 Hz), 1.81 (s, 6H). 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. °C NMR (90 MHz, CDC13 ): 8 146.6, 130.9, 130.4, 128.2, 123.83, 123.77, 122.9, 38.9, 28.9. 2-(3-Bromophenyl)-2-methylpropionic acid (12) A 1000-mL round-bottom flask equipped with a magnetic stirring bar was charged with 11 (36 g, 0.16 mol), EtOH (92 mL), 30% aqueous H2G2, and the resulting mixture was cooled to 0 °C on an ice bath. 40% aqueous NaOH (92 mL) was carefully added to the mixture in portions. The resulting mixture was allowed to warm to room temperature, as appreciable effervescence was clearly noticeable. The flask was fitted with a reflux condenser and the mixture was slowly heated to a gentle reflux. The mixture was heated for 36 hours as the reaction was monitored via GC. When judged complete the reaction was allowed to cool and acidified with 6 M HC1 (160 mL). The resulting solution was extracted with EtaO (2 x 100 mL, 3 x 50 mL) and Et20 was removed in vacuo. The resulting oil was treated with NaHCCh (35 g) and dissolved in H2O (350 mL) with the help of heating. The resulting mixture was treated with decolorizing charcoal and filtered. Acidification of the resulting mixture precipitated the title compound as a white solid (35 g, 90%). NMR (360 MHz, CDCI3): 6 7.54 (s, 1H,), 7.39 (d, 1H, Jh-h = 8.2 Hz), 7.32 (d, 1H, J h- h = 8.2 Hz), 7.20 (t, 1H, J H .H = 8.2 Hz), 1.58 (s, 6H). 1 3 C NMR (90 MHz, CDCI3): 5 182.8, 146.0, 130.1, 129.9, 129.1, 124.7, 122.6, 46.2, 26.1. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tetrakis(triphenyIphospIiie)paUacliiiin A 100-mL round-bottom flask equipped with a magnetic stirring bar was charged with palladium chloride (0.50 g, 2.8 mmol) and DMSO (30 mL). To the dark mixture was added triphenylphosphine (3.7 g, 14 mmol) instantaneously forming a yellow suspension. Raising the temperature slowly the mixture was heated until all solids completely dissolved (sand bath at -170 °C) to form a red solution. Hydrazine hydrate (0.50 mL, 1.6 mmol) was added causing the mixture to turn dark yet homogenous. The solution was quickly cooled in a water bath forming a fine precipitate. The mixture was let to attain room temperature by standing in air, and the resulting bright yellow solids were collected by vacuum filtration under nitrogen. The crystalline solid was washed with EtOH (2 x 20 mL) and Et20 (3 x 10 mL), dried in vacuo, and transferred into a glove box. The title compound was obtained as a bright yellow crystalline solid (3.0 g, 92%) 2,7-Di-p-tolyl-9H-fluorene (13) A dry Schlenk flask equipped with a magnetic stirring bar was charged with 4-bromotoluene (3.9 g, 23 mmol) and dry THF (40 mL) under nitrogen. The solution was cooled to -78 °C in dry ice/acetone bath and 5.2 M w-hexyllithium in hexanes (4.8 mL, 25 mmol) was added drop wise. The cloudy solution was stirred for additional 30 minutes. A separate Schlenk flask equipped with a magnetic stirring bar was charged with ZnBri (5.6 g, 25 mmol) and THF (20 mL). The 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. resulting clear solution was cannulated into the aryl-lithium reaction mixture at -78 °C and the mixture was allowed to warm up to room temperature. The relative percent conversion and formation of the zinc reagent was determined indirectly. An aliquot (50 jil) was withdrawn from the reaction mixture and injected into a THF solution of iodine (1 mL), thus converting the zinc reagent to the corresponding aryl iodide derivative. After 5 minutes the aliquot was treated with saturated solution of NaiSaCb, and the organic layer was analyzed via GC. The analysis showed complete reaction with 97% conversion. The major impurity was toluene, the hydrolyzed product. The overall purity of the mixture was estimated at greater than 95%. The aryl-zinc mixture was treated under a strong stream of nitrogen with 2 (3.3 g, 10 mmol) and Pd(Ph.3P) 4 (260 mg, 23 jimol). The Schlenk flask was fitted with a reflux condenser and the yellow/orange solution was stirred at 60 °C for 3 days. A white solid formed during the course of the reaction. Saturated solution of NH4CI (50 mL) was added and the reaction mixture was filtered. The white solid was washed with EtOH (20 mL) and dried to yield 3.3 g (92%) of the title compound. ‘H NMR (500 MHz, CDCI3 - CD3COCD3): 5 7.82 (d, 2H, J H -h = 7.8 Hz), 7.75 (s, 2H), 7.59 (d, 2H, Jh-h = 7.8 Hz), 7.55 (d, 4H, J H .H = 7.8 Hz), 7.25 (d, 4H, J H - H = 7.8 Hz), 3.99 (s, 2H), 1.54 (s, 6 H). 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-(3-Bromophenyl)-2-methylpropionyI chloride (14) A 50-mL round-bottom flask equipped with a magnetic stirring bar was charged with SOCI2 (5 mL. 70 mmol) and 12 (5.0 g, 21 mmol). The solution was stirred at room temperature while monitoring the progress via GC, about 6 hours. At this point the excess solvent was removed in vacuo and the resulting liquid was dissolved in CH2CI2 (5 mL) and the solvent was removed. This process was repeated four more times to remove as much of SOCI2 as possible providing 14 as a pale yellow liquid. This compound was dissolved in CH2CI2 (50 mL) and used directly in the next step without further purification. 2-[l-(3-Bromophenyl)-l-methylet!iyl]-4,4-dimethyl-4,5-dihydrooxazole (15) A 250-mL round-bottom flask equipped with a magnetic stirring bar was charged with 2,2-dimethylpropanolamine (3.8 g, 43 mmol) and dry CH2CI2 (50 mL). The flask was fitted with an addition funnel, flushed with nitrogen, and cooled to 0 °C on ice. Solution of 14 from the previous step was transferred to the addition funnel via cannula and added at a moderate rate. After the addition was complete the mixture was allowed to warm to room temperature and the addition funnel was charged with a solution of SOCI2 (1.5 mL, 21 mmol) dissolved in CH2CI2 (8 mL). This mixture was added drop wise to the reaction mixture. After 30 minutes the reaction was quenched with H2O (120 mL) and the organic phase was washed sequentially with H2O (2 x 50 mL), neutralized with saturated solution of NaHCOg, 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. washed with brine, and dried with MgS0 4 . CH2CI2 was removed in vacuo to yield 15 (5.0 g, 83%) as a pale yellow liquid. ' l R NMR (250 MHz, CDCI3): 8 7.46 (s, 1H), 7.30 (d, 1H, J H -H = 8.0 Hz), 7.24 (d, 1H, Jh-h = 8.0 Hz), 7.12 (t, 1H, J H - h = 8.0 Hz), 3.80 (s, 2H), 1.52 (s, 6H), 1.25 (s, 6H). Compound 16 A dry Schlenk flask equipped with a magnetic stirring bar was charged with 15 (3.1 g, 10 mmol) and dry THF (30 mL) under nitrogen. The solution was cooled to -100 °C in EtaO/liquid N2 bath and 5.2 M n-hexyllithium in hexanes (2.2 mL, 11 mmol) was added drop wise. The solution was stirred for 15 minutes. A separate Schlenk flask equipped with a magnetic stirring bar was charged with ZnBr2 (2.6 g, 12 mmol) and THF (15 mL). The resulting clear solution was cannulated into the aryl-lithium reaction mixture at -100 °C and the mixture was allowed to warm up to room temperature. The relative percent conversion and formation of the zinc reagent was determined following the procedure described for compound 13. The mixture showed 95% conversion with less then 5% of the hydrolyzed derivative being detected. The aryl-zinc mixture was treated under a strong stream of nitrogen with 2 (1.3 g, 4.0 mmol) and Pd(Ph3P) 4 (120 mg, 10 jimol). The Schlenk flask was fitted with a reflux condenser and the rusty/orange solution was stirred at 60 °C overnight. The reaction was quenched with saturated solution of NH4CI (10 mL), washed with 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HaO (2x30 mL), washed with brine, and dried with MgSCL. THF was removed in vacuo leaving a brown oily residue, which was stirred in boiling hexane (50 mL) and filtered. 16 crystallized from this mixture as a white solid (0.77 g, 32%). ' lM NMR (250 MHz, CDCI3): 5 7.84 (d, 2H, J H -h = 7.7 Hz), 7.75 (s, 2H), 7.64 (s, 2H), 7.60 (d, 2H, J H-h = 7.7 Hz), 7.52 (d, 2H, J H -H = 7.6 Hz), 7.41 (t, 2H, J H -H = 7.6 Hz), 7.34 (d, 2H, J h- h = 7.6 Hz), 4.00 (s, 2H), 3.89 (s, 4H), 1.65 (s, 12H), 1.32 (s, 12H). Compound 17 A 50-mL round-bottom flask equipped with a magnetic stirring bar was charged with 16 (0.77 g, 1.3 mmol) and 3 M aqueous HC1 (20 mL). The flask was fitted with a reflux condenser and the solution was heated at reflux for 2 hours. A white precipitate formed which was filtered, washed with H2O, and air-dried to yield 17 (0.79 g, 87%) as a white powder. *H NMR (250 MHz, DMSO-£&): 8 8.18 (s, 6 H), 7.81 (d, 2H, JH -h = 8.0 Hz), 7.73 (s, 2H), 7.59-7.51 (m, 4H), 7.46 (d, 2H, Jh-h = 7.4 Hz), 7.35 (t, 2H, J H .H = 7.4 Hz), 7.28 (d, 2H, Jh-h = 7.4 Hz), 4.04-3.96 (m, 6H), 1.62 (s, 12H), 1.18 (s, 12H). 2-(3-Bromophenyl)-2-methylpropionic acid tert-butyl ester (18) A 250-mL round-bottom flask equipped with a magnetic stirring bar was charged with potassium fert-butoxide (20 g, 0.17 mol) and EtaO (200 mL). The flask was fitted with an addition funnel, flushed with nitrogen, and cooled to -78 °C 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in dry ice/acetone bath. Compound 14 prepared from 13 (35 g, 0.14 mol) was dissolved in EtaO (200 mL) and transferred to the addition funnel via cannula. The solution was added to the suspension at a moderate rate forming a syrupy mixture. The progress of the reaction was monitored via GC. After the addition was complete the mixture was allowed to warm up to room temperature and saturated solution of NH4CI (20 mL) was added followed by H2O (200 mL). The organic layer was separated and the aqueous layer was further extracted with pentane (4 x 50 mL). The combined fractions were dried over MgSCL, treated with decolorizing charcoal, and Et20 was removed in vacuo. The resulting light yellow liquid was vacuum distilled providing 18 (32 g, 74%) as a clear liquid. 1 H NMR (500 MHz, CDC13 ): 6 7.46 (t, 1H, JH -h = 2.0 Hz), 7.32 (ddd, 1H, J H . H = 8.1 Hz, 2.0 Hz, 1.0 Hz), 7.24 (ddd, 1H, JH -h = 8.0 Hz, 2.0 Hz, 1.0 Hz), 7.14 (t, 1H, Jh-h = 8.0 Hz), 1.48 (s, 6H), 1.36 (s, 9H). 1 3 C NMR (126 MHz, CDC13 ): 5 175.0, 147.5, 129.6, 129.4, 128.8, 124.3, 122.3, 80.4, 46.8,' 27.7, 26.3 Mass spectroscopy (FAB, MH+ ): m/e calc. 299.0647, found 299.0636. 2-(3-{7-[3-(l-#erf-Butoxycarbonyl-l-methylethyI)phenyl]-9H-fluoreii-2- yl} phenyl)-2-methylpropionic acid ferf-buty! ester (19) A dry Schlenk flask equipped with a magnetic stirring bar was charged with 18 (2.6 g, 8.7 mmol) and dry THF (30 mL) under nitrogen. The solution was cooled to -100 °C in Et2 0 /iiquid N2 bath and 5.2 M K-hexyllithium in hexanes (1.8 mL, 9.9 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mmol) was added drop wise. The solution was stirred for 15 minutes. A separate Schlenk flask equipped with a magnetic stirring bar was charged with ZnBra (2.2 g, 9.8 mmol) and THF (10 mL). The resulting clear solution was cannulated into the aryl-lithium reaction mixture at -100 °€ and the mixture was allowed to warm up to room temperature. The relative percent conversion and formation of the zinc reagent was determined following the procedure described for compound 13. The mixture analysis showed 93% conversion and the overall purity was estimated at greater than 85%. The aryl-zinc mixture was treated under a strong stream of nitrogen with 2 (1.1 g, 3.4 mmol) and Pd(Pfi3P) 4 (0.10 g, 8.6 jimol). The Schlenk flask was fitted with a reflux condenser and the solution was stirred at 60 °C overnight. The reaction was quenched with saturated solution of NH4CI (10 mL), and H2O (50 mL). The aqueous layer was extracted with CH2CI2 (2 x 50 mL) and the combined organic fractions were washed with brine, and dried with MgSCL. Removal of the solvents left behind yellow oil, which was dissolved in toluene (15 mL) and flashed through silica gel column with toluene (200 mL). The combined fractions were evaporated in vacuo and stirred in hexanes causing solidification, thus obtaining after filtration the title product as and off-white solid (1.4 g, 70%). ' X H NMR (360 MHz, CDCI3): 6 7.85 (d, 2H, J H - h = 7.9 Hz), 7.76 (s, 2H), 7.65-7.58 (m, 4H), 7.52 (d, 2H, J H - h = 7.9 Hz), 7.40 (t, 2H, J H .H = 7.9 Hz), 7.33 (d, 2H, Jh-h = 7.9 Hz), 4.02 (s, 2H), 1.59 (s, 12H), 1.40 (s, 18H). 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2,7 -Bis[3-(l-tert-butoxycarbonyl-l-methylethyl)phenyl]-9-metliyl-9H-fluorene- 9-carboxylic acid ethyl ester (20). A dry Schlenk flask equipped with a magnetic stirring bar was charged with 18 (10 g, 34 mmol) and dry THF (75 mL) under nitrogen. The solution was cooled to -100 °C in Et20/liquid N2 bath and 5.2 M w-hexyllithium in hexanes (6.7 mL, 37 mmol) was added drop wise. The solution was stirred for 15 minutes. A separate Schlenk flask equipped with a magnetic stirring bar was charged with ZnBt2 (8.9 g, 39 mmol) and THF (45 mL). The resulting clear solution was cannulated into the aryl-lithium reaction mixture at -100 °C and the mixture was allowed to warm up to room temperature. The relative percent conversion and formation of the zinc reagent was determined following the procedure described for compound 13. The mixture analysis showed 96% conversion and the overall purity was estimated at 94%. The aryl-zinc mixture was treated under a strong stream of nitrogen with 4 (6.3 g, 15 mmol) and Pd(Ph3 ? ) 4 (0.20 g, 0.17 mmol). The Schlenk flask was fitted with a reflux condenser and the solution was stirred at 70 °C overnight. The reaction mixture was transferred into a round-bottom flask and THF was removed in vacuo. The resulting oil was treated with saturated solution of NH4CI (50 mL), and H2 0 (50 mL), and pentane (100 mL). The mixture was filtered and the aqueous layer was further extracted with pentane (3 x 50 mL). The combined pentane extracts were dried over MgSCL and removed in vacuo to yield a yellow oil, a mixture of the product and hydrolyzed zinc reagent. The latter was removed by vacuum distillation and the resulting crude 20 was used in the next step without further purification. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2,7-Bis[3-(l-carboxyi-l-methylethyl)phenyI]-9-metliyl-9H-fluorene-9-carboxylic ad d ethyl ester (21) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with crude 20 from previous step and dry CH2CI2 (75 mL). The yellow solution was treated with triethylsilane (10 mL, 63 mmol) and trifluoroacetic acid (30 mL, 0.39 mol), and the solution was stirred at room temperature for 4 hours. The solvents were removed in vacuo and the resulting dark residue was treated with benzene (100 mL) and H2O (200 mL). Solid NaHCOs was added to the mixture in portions until the aqueous layer was basic to a litmus paper. The mixture was brought to a reflux and stirring was continued until all of the acid dissolved and two clear layers were visible. The aqueous layer was separated, treated with decolorizing charcoal and filtered. Acidification with 6 M aqueous HC1 precipitated 21, which was extracted with CH2CI2 (3 x 50 mL), washed with brine, and dried with MgSCL. CH2CI2 was removed in vacuo to yield 21 as a yellowish fluffy solid (6.7 g, 75% over two steps). *H NMR (360 MHz, CDCI3): 8 7.82 (d, 2H, J H .H = 7.6 Hz), 7.76 (s, 2H), 7.67-7.54 (m, 6H), 7.48 (t, 2H, / H-h = 7.2 Hz), 7.33 (d, 2H, J H -h = 7.2 Hz), 4.14 (q, 2H, / h - h = 7.0 Hz), 1.86 (s, 3H), 1.76 (s, 12H), 1.18 ( t , 3H, / H .H = 7.0 Hz). 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-(3-Chlorophenyl)-2-methylpropionitrile (23) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 22 (100 g, 0.62 mol), benzene (100 mL), NaCN (33 g, 0.66 mmol), HaO (40 mL), and tetrabutylammonium hydrogen sulfate (10 g, 28 mmol). An exothermic reaction took place and the biphasic mixture was stirred overnight. The solution turned dark. The reaction was monitored GC, and upon completion H2O (100 mL) was added to the yellowish reaction mixture. The phases were separated and the aqueous phase was further extracted with benzene (2 x 50 mL). The organic phase was washed with brine, dried with MgSO^ and benzene was removed in vacuo to give the crude 3-chlorophenyl acetonitrile as a dark liquid. A 1000-mL round-bottom flask equipped with a magnetic stirring bar was charged with potassium feri-butoxide (160 g, 1.4 mol) and THF (400 mL), and fitted with an addition funnel. The flask was cooled to 0 °C on an ice bath. The crude 3- chlorophenyl acetonitrile dissolved in THF (200 mL) was transferred to the addition funnel and added drop wise to the potassium feri-butoxide suspension. Methyl iodide (87 mL, 1.4 mol) was added drop wise to the dark, blood red solution. After the addition was complete the thick suspension was warmed to room temperature and the reaction was monitored via GC. Upon completion THF was removed in vacuo and HaO (200 mL), and EtaO was added and the phases separated. The aqueous phase was further extracted with Et20 (4 x 50 mL). The combined EtaO fractions were washed with brine, dried with MgSCA, and EtaO was removed in 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vacuo. The resulting dark mixture was vacuum distilled to give the title product as a clear liquid (60 g, 54% over two steps). 2-(3-Chlorophenyl)-2-metIiylpropiomc acid (24) This compound was prepared following the protocol for 12. The amounts used were 23 (60 g, 0.40 mol), EtOH (190 mL), 30% aqueous H2O2 (190 mL), and 40% aqueous NaOH (190 mL). The title compound was isolated as a white solid (57 g, 86%). 1 HNM R(250M Hz, CDCI3): 6 11.87 (s, 1H,), 7.40-7.35 (m, 1Hz), 7.30-7.20 (m, 3H), 1.57 (s, 6H). 1 3 C NMR (90 MHz, CDCI3): 5 182.9, 145.7, 134.4, 129.7, 127.2, 126.3, 124.2, 46.2, 26.1. 2-(3-Chlorophenyl)-2-methylpropionic acid ferf-butyl ester (25) A 250-mL round-bottom flask was charged with 24 (3.2 g, 16 mmol) and SOCI2 (3 mL, 41 mmol), and the solution was stirred at room temperature. The conversion to the acid chloride was monitored via GC. Once complete the solution was worked up following the protocol for 14. The acid chloride was isolated as a light yellow liquid, which was dissolved in dry THF (20 mL) and the flask was fitted with an addition funnel. Potassium fert-butoxide (2.0 g, 18 mmol) dissolved in THF (30 mL) was added to the addition funnel and the flask was cooled to 0 °C on ice. The solution was added drop wise to the acid chloride monitoring the progress of the 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reaction via GC. After completion THF was removed in vacuo and H2O (50 mL), saturated solution ofNHUCl (10 mL), and pentane (50 mL) were added. The aqueous layer was further extracted with pentane (3 x 25 mL) and the organic extracts were washed with brine, and dried with MgSO.*. Pentane was removed in vacuo to yield the title product as a pale yellow liquid (3.3 g, 80%), which was pure enough to use in the next step without further purification. 1 H NMR (250 MHz, CDCI3): 8 7.32-7.29 (m, 1H), 7.23-7.13 (m, 3H), 1.48 (s, 6 H), 1.35 (s, 9H). 1 3 C NMR (90 MHz, CDCI3): 8 175.2,147.3, 134.0, 129.4, 126.5, 125.9, 123.9, 80.6, 46.9, 27.7, 26.3. 1-Histidine methyl ester dihydrochloride (27) A 1000 mL round-bottom flask was charged with /-histidine (26) (50 g, 0.32 mol) and MeOH (500 mL). The flask was fitted with an addition funnel and cooled to 0 °C on ice. The addition funnel was charged with SOCI2 (100 mL, 1.4 mol), which was added drop wise to the suspension. After the addition was complete the addition funnel was replaced with a reflux condenser and the mixture was refluxed until all solids dissolved. The reflux was then continued further for 2 hours after which the heating was stopped. The yellow solution was slowly layered with EtiO (500 mL) and set aside to crystallize overnight. The precipitate was collected, washed with Et2 0 , and air-dried to yield the title compound as a white crystalline solid (74 g, 95%). 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lm NMR (250 MHz, D20): 8 8.72 (s, 1H), 7.47 (s, 1H), 4.53 (i, 1H, 7H -h = 6 . 8 Hz), 3.86 (s, 3H), 3.59-3.39 (m, 2H). 25 7-Bis[3-(2-imidazol-l-yl-l,l-dimethyl-2-oxoethyI)phenyl]-9-methyl-9H- fluorene-9-carboxylic acid ethyl ester (28) A 50-mL round-bottom flask was charged with 21 (0.70 g, 1.2 mmol) and CHCI3 (14 mL). 1,1 ’-Carbonyldiimidazole (0.48 g, 3.0 mmol) was added and the solution was stirred for 1 hour at room temperature. C 02 evolution was noticeable. The progress of the reaction was monitored via HPLC. The resulting mixture was washed with saturated solution ofNaHCCb (5 x 10 mL), dried over MgSCL, and CHCI3 was removed in vacuo to yield the title product as a light yellow solid. 2,7-Bis[3-(l-chlorocarbonylmethylethyl)phenyl]-9-methyl-9H-fluorene-9- carboxylic acid ethyl ester (30) A 20-mL round-bottom flask equipped with a magnetic stirring bar was charged with 21 (0.12 g, 0.21 mmol) and SOCI2 (1 mL, 14 mmol). The solution was stirred at room temperature overnight. The excess solvent was removed in vacuo and the resulting solid was dissolved in dry C H C I3 (1 mL) and the solvent was removed. This process was repeated four more times to remove as much of SOCI2 as possible providing 31 as an off-white solid. This compound was dissolved in CHCI3 (10 mL) and used directly in the next step without further purification. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2,7-Bis(3-{l-.[2-(lH-imidazoI-4-yI)-l-methoxycarbonylethylcarbamoyl]-l - methylethyl}phenyl)-9-methyl-9H-iiiorene-9-carboxyIlc acid ethyl ester (31) A 50-mL round-bottom flask equipped with a magnetic stirring bar was charged with 27 (0.28 g, 1.2 mmol), triethylamine (0.33 mL, 2.4 mmol), and CHCI3 (10 mL). The flask was fitted with an addition funnel and the solution was cooled to 0 °C on ice. Solution of 30 from the previous step was transferred to the addition funnel via cannula and added drop wise. The resulting light yellow mixture was allowed to warm to room temperature and was stirred for additional 1 hour. The progress of the reaction was followed via HPLC. The solvent was removed in vacuo and the resulting oil was treated with HaO (10 mL), saturated solution of NaHCCh (10 mL) and ethyl acetate (20 mL). The aqueous layer was further extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were dried over MgSCL and ethyl acetate was removed in vacuo to yield 31 as a yellow solid (0.13 mg, 71%). l-a Ns W,-Ditritylhistidine methyl ester (32) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 27 (10 g, 41 mmol), trityl chloride (23 g, 82 mmol), and CHCI3 (180 mL). The flask was fitted with an addition funnel filled with triethylamine (24 mL, 172 mmol). The setup was flushed with nitrogen and cooled to 0 °C on ice. Triethylamine was added drop wise to the cool suspension. After the addition was complete, the solution was allowed to warm up to room temperature and was stirred further for 1 hour. The solvent was removed in vacuo and the oily residue was ■ 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dissolved in ethyl acetate (100 mL) and BbO (100 mL). The organic layer was collected and the aqueous layer was further extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were washed sequentially with saturated solution of NaHCOs (50 mL), BbO (50 mL), brine (50 mL), and dried over MgSCL. Ethyl acetate was removed in vacuo producing the title compound as a fluffy white solid (27 g, 99%). *H NMR (500 MHz, CDCb): 5 7.41 (d, 6H, Jh-h = 8.0 Hz), 7.34 (s, 1H), 7.26 (d, 9H, J h-h = 7.0 Hz), 7.17 (t, 6H, Jh -h = 7.5 Hz), 7.11 (t, 9H, 6.5 Hz), 6.61 (s, 1H), 3.64 (ddd, 1H, J H .H = 6.0 Hz, 6.5 Hz, 10.5 Hz), 3.02 (s, 3H), 2.94 (dd, 1H, J h-h = 6.0 Hz, 14.0 Hz), 2.77 (dd, 1H, Jh -h = 6.5 Hz, 14 Hz), 2.70 (d, 1H, J H -h = 10.5 Hz). I- W-Tritylhistidine methyl ester trifluoroacetate (33) A 200-mL round-bottom flask fitted with a magnetic stirring bar was charged with 32 (5.0g, 7.6 mmol), triethylsilane (3.2 mL, 20 mmol), and CTbCb (25 mL). Trifluoroacetic acid (1.2 mL, 16 mmol) was added drop wise to the vigorously stirred solution. After 10 minutes the solvent was removed in vacuo and the oily yellow residue was dissolved in Et20 (10 mL). The solution was cooled on ice and pentane (40 mL) was added drop wise to the stirred solution. White fine solid separated. The suspension was treated further with Et20 (10 mL) and pentane (40 mL). The mixture was set-aside in a freezer overnight. The resulting suspension was filtered and washed with pentane. After drying the title product was obtained as a white solid (3.4 g, 85%). 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. *H NMR (500 MHz, CDCI3): 8 9.87 (s, 3H), 8.08 (s, 1H), 7.43-7.32 (m, 9H), 7.17-7.08 (m, 6H), 6.90 (s, 1H), 4.48 (t, 1H, J H .H = 5.5 Hz), 3.66 (s, 3H), 3.46 (m, 2H). 2,7-Bis(3-{l-[2-(l-methoxycarbonyl-2-(l-trityI-lH-imidazol-4- yl)ethylcarbainoyS]-l-methyletliyl}plieiiyl)-9-iiiethyl-9H-iiiorene-9-carboxySic acid ethyl ester (34) A 100-mL round-bottom flask was charged with 33 (6.3g, 12 mmol), and CHCI3 (15 mL). Triethylamine (3.5 mL, 25 mmol) was added in one portion and the flask was fitted with an addition funnel, flushed with nitrogen, and cooled on ice to 0 °C. The addition funnel was charged with 30 dissolved in CHCI3 (15 mL), which was prepared from 21 (3.3 g, 5.7 mmol). The light yellow solution was added drop wise and progress of the reaction was monitored via HPLC. Workup was performed as described for compound 31. Isolated 34 as a light yellow powder (6.5 g, 83%). *H NMR (500 MHz, CDCI3): 5 7.75 (dd, 2H, Jh-h = 6.5 Hz, 1.0 Hz), 7.66 (s, 2H), 7.64 (d, 2H, JH -h = 8.0 Hz), 7.65 (dt, 2H, J H .H = 7.5 Hz, 2.0 Hz), 7.40-7.22 (m, 26H), 7.10 (d, 2H, J h-h = 7.0 Hz), 6.98 (d, 12H, J H -h = 7.0 Hz), 6.43 (s, 2H), 4.71(m, 2H), 4.06 (q, 2H, J H .H = 7.0 Hz), 3.58 (d, 6H, J H -h = 8.5 Hz), 3.04-2.86 (m, 4H), 1.79 (s, 3H), 1.62 (d, 12H, J H .H = 6.0 Hz), 1.10 (t, 3H, JH -H = 7.0 Hz). 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.7-Bis(3-{l-[2-(lH-imidazol-4-yl)-l-methoxycarbonylethylcarbamoyI]-1- methyletbyl}phenyl)-9"inethyI-9H-flttorene-9-carboxylic acid ethyl ester (35) A 50-mL round-bottom flask equipped with a magnetic stirring bar was charged with 34 (3.8 g, 2.8 mmol), triethylsilane (1 mL, 6.3 mmol), and CH2CI2 (8 mL). Trifluoroacetic acid (4.3 mL, 56 mmol) was added drop wise to the solution. The orange solution was stirred for 3 hours at room temperature. The reaction was quenched with H2O (20 mL) and the pH was adjusted with NaHCOs until the aqueous layer was basic to a litmus paper. The layers were separated and the aqueous layer was further extracted with CH2CI2 (2x10 mL). The combined organic fractions were dried with MgSC>4 and the solvent was removed in vacuo. The resulting off-white solid was suspended in EtaO (20 mL) and washed thoroughly with the help of a sonication bath. The solids were collected to give upon drying the title compound as an off-white solid (1.9 g, 78%). 2.7-Bis(3-{l-[l-carboxy-2-(lH-imidazol-4-yl)ethylcarbamoyI]-l- methyletfayl}pfaenyI)-9“metfayl-9H-fIiioreiie-9-carboxyIate trisodiiim salt (36) A 10-mL round-bottom flask equipped with a magnetic stirring bar was charged with 35 (0.30 g, 0.34 mmol) and EtOH (1.2 mL). The yellow solution was treated with 2.5 M aqueous NaOH (0.60 mL, 1.5 mmol) and the resulting inhomogeneous mixture was stirred overnight. The mixture was evaporated to near dryness and acetonitrile (5 mL) was added. The suspension was stirred for 5 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. minutes, filtered, and dried to yield the target compound as an off-white solid (0.28 g, 90%). Fe(OTf)2 (CH3CN)2 A 100 mL Schlenk flask equipped with a magnetic stirring bar was charged with iron powder (2.0g, 36 mmol) and dry acetonitrile (36 mL) under nitrogen. The flask was fitted with an addition funnel filled with triflic acid (6.8 mL, 77 mmol) and cooled to 0 °C on ice. The triflic acid was added drop wise to the iron suspension. After the addition completed, the addition funnel was replaced with a cold finger and the mixture was stirred first at room temperature for one hour followed by an additional hour at 60 °C. The resulting light brown reaction mixture was filtered through celite to yield a light green solution from which about 25% of solvent was removed in vacuo. The solution was allowed to stand at -20 °C overnight. The supernatant was cannulated off and the white solid was washed with Et20 (3x7 mL). The white solid was dried in vacuo and transferred to a glove box. The collected washings were layered with Et20 (17 mL) and were allowed to stand at -20 °C overnight to obtain a second crop of crystals, which were washed with Et2 0 (3 x 5 mL) and dried in vacuo. By combining the first crop (9.3 g) and the second (5.7 g) the title compound was obtained as a white powder (15.0 g, 96%). 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2,7-Bis(3-{l-[l-carboxy-2-(l-trityl-lH-imidazol-4-yl)ethylcarbamoyl]-l- methylethyl}phenyl)-9-methyl-9H-fluorene-9-carboxylic add dihydrochloride (37) A 10-mL round-bottom flask equipped with a magnetic stirring bar was charged with 34 (0.50 g, 0.37 mmol) and THF (0.56 mL). 2.5M aqueous NaOH (0.52 mL, 1.3 mmol) was added and the suspension was stirred overnight. THF was removed in vacuo and H2O (10 mL) was added. CH2CI2 (10 mL) was added and 2.2M aqueous HC1 (2 mL, 4.4 mmol) was used to acidify the system. The resulting mixture was stirred for several minutes and the phases were allowed to separate. The organic layer was collected and the aqueous layer was further extracted with CH2CI2 (2 x 10 mL). The combined organic fractions were washed with brine, dried with MgSCL, and the solvent was removed in vacuo to yield the title compound as a white powder (0.45 g, 89%). Methyl-2,7-dibromofluorene-9-carboxylate (38) A 200-mL round-bottom flask equipped with a magnetic stirring bar was charged with 3 (4.0 g, 10 mmol) and MeOH (40 mL). Concentrated H2SO4 (1 mL) was added to the solution and the mixture was refluxed overnight. The progress of the reaction was monitored by HPLC. The mixture was allowed to cool and H2O (100 mL) was added. The stirring was continued for additional 5 minutes and the fine solid was filtered and dried. The title product was obtained as a white powder (3.5 g, 91%). 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Methyl-2,7-dibromofluorene-9-methyl-9-carboxylate (39) The title compound was prepared following the synthetic procedure for compound 4. X H NMR (500 MHz, Acetone-^): 8 7.82-7.76 (m, 4H), 7.60 (dd, 2H, J H -h = 8.5 Hz, 1.5 Hz), 3.60 (s, 3H), 1.77 (s, 3H). 1 3 C NMR (126 MHz, CDC13 ): 8 173.1, 150.1, 139.2, 132.2, 128.4, 122.9, 122.1, 58.1, 53.1, 24.0. 2,7 -Bis[3-(l-ferf-butoxycarbonyI-l-methylethyl)phenyl]-9-nithyl-9H-fliioreiie- 9-carboxylic acid methyl ester (40) The title compound was prepared following the synthetic procedure for compound 2 0 . 2.7-Bis[3-(l-carboxyl-l-methylethyl)phenyl]-9-methyl-9H-fluorene-9-carboxylic acid methyl ester (41) The title product was prepared following the synthetic procedure for compound 2 1 . 2.7-Bis[3-(l-chIorocarbonylmethylethyl)phenyl]-9-methyl-9H-fluorene-9- carboxylic acid methyl ester (42) A 20-mL round-bottom flask equipped with a magnetic stirring bar was charged with 41 (2 . 0 g, 3.5 mmol) and SOCI2 (4.0 mL, 56 mmol). The solution was 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stirred at room temperature overnight. The excess solvent was removed In vacuo and the resulting solid was dissolved in dry CHCI3 (1 mL) and the solvent was removed. The white solid was suspended in pentane (10 mL) and stirred vigorously. The solids were filtered, washed with pentane and quickly dissolved in CHCI3 (20 mL). The solution was used directly in the next step without further purification. 2,7-Bis(3-{l-[2-(l-methoxycarbonyI-2-(l-trityl-lH-imidazol-4- yl)ethylcarbamoyl]-l-methylethyI}phenyl)-9-methyI-9H-fluorene-9-carboxylic acid ethyl ester (43) The title product was prepared following the synthetic procedure for compound 34 (3.1g, 65%). 5-Oxo-5,6,7,8-tetrahydroimidazo[l,5-c]pyrimidine-7-carboxylic acid methyl ester (44) A 500-mL round-bottom flask was charged with 1,1 ’-carbonyldiimidazole (11.6 g, 71.5 mmol), and CH3CN (200 mL). Triethylamine (10.0 mL, 71.6 mmol) was added and nitrogen was introduced. The reaction mixture was cooled to 0° C on ice for 5 minutes. Finely powdered 27 (17.3 g, 71.5 mmol) was added in one sum and the reaction mixture was stirred at 0° C for 40 minutes followed by additional 3 hours at room temperature. The solvent was removed in vacuo and the resulting viscous solid was treated with NaHCCb ( 1 0 g), H2O (100 mL), and CH2CI2 ( 1 0 0 mL). The mixture was mixed thoroughly and the phases were separated. The 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aqueous phase was further extracted with CH2CI2 (8 x 50 mL). The combined extracts were dried over MgS(>4, and the solvent was removed in vacuo. The crude product was stirred in ice-cold acetomtrile (20 mL) and filtered to give the title compound as a white solid (9.1 g, 65%). *H NMR (250 MHz, DMSO-4?): 6 8.57 (d, 1H, J h-h = 3.8 Hz), 8.10 (s, 1H), 6.80 (s, 1H), 4.44 (q, 1H, J H -h = 4.5 Hz), 3.61 (s, 1H), 3.21 (d, 2H, J H .H = 4.5 Hz). 7-Methoxycarboiiyl-2-methyl-5-oxo-5,6,7,8-tetrahydroiiiiidazo[l,5-c]pyriinidin- 2-ium iodide (45) A 100-mL round-bottom flask was charged with 44 (2.4 g, 12 mmol), methyl iodide (1.5 mL, 24 mmol), and CH3CN (25 mL). The mixture was brought to gentle reflux and was stirred at this temperature for 3 hours. Product started separate from the mixture at this point. If not, the inner surface of the flask was scratched. The mixture was allowed to cool on ice and Et2 0 (75 mL) was added in portions. The solid was filtered, washed with ether, and allowed to air dry. The title compound was obtained as a white solid, 3.9 g (94%). 7-Methoxycarb0nyI-2-methyl-5-0xo-5? 6,7,8-tetra!iydroimidazo[l,5-c]pyriiitidin- 2-ium methylsulfonate (46) A 250-mL round-bottom flask was charged with 44 (8.0 g, 40 mmol), dimethyl sulfate (8.0 mL, 84 mmol), and CH3CN (80 mL). The flask was fitted with a reflux condenser. The mixture was brought to 60 °C and was maintained at this 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. temperature for 3 hours. Product started to separate from the mixture at this point. If not, the inner surface of the flask was scratched. The mixture was allowed to cool on ice and Et2 0 (40 mL) was added in portions. The solid was collected, washed with ether, and allowed to air dry. The title compound was obtained as a white solid, 12.6 g (95%). /-*jV-Methylhistidine (47) A 250-mL round-bottom flask equipped with a stirring bar was charged with 46 (12.6 g, 39 mmol), H2O (70 mL), and conc. HC1 (13 mL). The flask was fitted with a reflux condenser and the solution was gently refluxed overnight. The reaction mixture was allowed to cool and the pH was adjusted to 7.5 by a careful addition of solid NaHC(>3. Water was removed in vacuo and MeOH (130 mL) was added. The resulting mixture was warmed to 60 °C in a water bath with vigorous stirring for 10 minutes. All solids were filtered and MeOH was removed in vacuo. After thorough drying the title compound was obtained as a white powder, and was used in the following step without further purification. *H NMR (250 MHz, D20): 8 9.16 (s, 1H), 7.23 (s, 1H), 4.47 (t, 1H, J H-h = 5.6 Hz), 3.77 (s, 3H), 3.28 (d, 2H, JH .H = 5.6 Hz). /-*jV-Methylhistidine methyl ester dihydrochloride (48) A 250-mL round-bottom flask equipped with a stirring bar was charged with 47 from the previous step, and MeOH (50 mL). The flask was fitted with an addition 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. funnel and the mixture was cooled to 0 °C on ice. The addition funnel was charged with SOCI2 (10 mL, 0.14 mol), which was added drop wise to the suspension. After the addition was complete the addition funnel was replaced with a reflux condenser and the mixture was refluxed overnight. The heating was stopped and the resulting light yellow solution was carefully layered with EtiO (70 mL), and the product was allowed to crystallize. The title compound was obtained as a white crystalline solid, 8.0 g (80% from 46). 1 H NMR (250 MHz, D2 0): 8 8.47 (s, 1H), 7.24 (s, 1H), 4.32 (t, 1H, J h- h = 6.8 Hz), 3.71 (s, 3H), 3.68 (s, 3H), 3.35-3.15 (m, 2H). 2,7-Dibromo-9-ethoxycarbonylmethyl-9H-fluorene-9-caboxyIic acid ethyl ester (49) A 250-mL round-bottom flask equipped with a magnetic stirring bar was charged with EtOH (70 mL) in which sodium (1.70 g, 73.9 mmol) was carefully dissolved forming an alcoholic solution of sodium ethoxide. The flask was flushed with nitrogen and solution of 3 (23.7 g, 59.8 mmol) in THF (90 mL) was added via cannula. The resulting yellow solution was charged with ethyl bromoacetate and the progress of the reaction was monitored via HPLC. The sodium bromide was filtered off and the solvents were removed in vacuo. The resulting yellow solid was stirred in pentane (30 mL) on ice for 10 minutes. The solids were filtered, washed with pentane (2 x 10 mL), and air-dried. The titled product was obtained as a white solid Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and was used in the next step without further purification. An analytical sample was obtained by recrystallization from MeOH. JH NMR (500 MHz, CDC13 ): 8 7.81 (s, 2H), 7.51 (s, 4H), 4.19-4.06 (m, 4H), 3.06 (s, 2H), 1.22-1.12 (m, 6H). 1 3 C NMR (126 MHz, CDC13 ): 5 171.0, 170.2, 146.4, 138.6, 131.8, 128.6, 121.7, 121.3, 62.0, 61.0, 58.3, 42.6, 14.0, 13.9. 2.7-Dibromo-9H-fluoren-9-yI-acetic acid (50) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 49 from previous step, aqueous 1.5 M NaOH (200 mL), and EtOH (50 mL). The mixture was refluxed until all solids dissolved. The absence of starting material was conformed via HPLC, and the solution was cooled on ice. Cone. HC1 was added in portions till acidic to litmus paper. The mixture was stirred for 5 minutes and the solids were collected by filtration. After air-drying the title compound was obtained as an off-white solid, 21.6 g (94% from 3). 2.7-Dibromo-9H-fluoren-9-yl-acetic acid ethyl ester (51) A 250-mL round-bottom flask equipped with a magnetic stirring bar was charged with 50 (21.6 g, 56.5 mmol), EtOH (100 mL), and conc. H2SO4 (ImL). The flask was fitted with a reflux condenser, and the mixture was refluxed for 3 hours forming a yellow solution. HPLC showed 95% conversion after 2 hours. The heating was stopped and crystallization commenced. Quick agitation in a sonicator 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or continuous stirring decreased the ievei of color entrapment. The solids were collected and air-dried to give the title compound as an off-white solid, 18.5 g (80%). X H NMR (500 MHz, Acetone-*): 8 7.79(s, 2H), 7.77 (d, 2H, J H -h = 8.2 Hz), 7.55 (d, 2H, J h-h = 8.2 Hz), 4.40 (t, 1H, J H -h = 6.7 Hz), 4.17 (q, 2H, JH .H = 7.1 Hz), 2.91 (d, 2H, J h-h = 6.7 Hz), 1.22 (t, 3H, JH .H = 7.1 Hz). 1 3 C NMR (126 MHz, CDC13 ): 8 172.1, 149.4, 139.8, 131.5, 128.7, 122.6, 121.7, 61.2, 44.4, 38.1, 14.5. Mass spectroscopy (EI+, M*): m/e calc. 407.9360, found 407.9354. 2-(3-{7-[3-(l-tert-B«toxycarbonyI-l-iuethyIethyl)-phenyI]-9- ethoxycarbonylmethyl-9H-fluoren-2-yl}phenyl)-2-methylpropionic acid tert- butyl ester (52) A dry Schlenk flask equipped with a magnetic stirring bar was charged with 18 (5.8 g, 19 mmol) and dry THF (60 mL) under nitrogen. The solution was cooled to -100 °C in EtaO/liquid N2 bath and 6.3 M n-hexyllithium in hexanes (3.2 mL, 21 mmol) was added drop wise. The resulting orange solution was stirred at -100 °C for 30 minutes. A separate Schlenk flask equipped with a magnetic stirring bar was charged with dry ZnBra (4.5 g, 20 mmol) and THF (25 mL). The resulting clear solution was cannulated into the aryl-lithium reaction mixture at -100 °C. The solution turned nearly water white and the mixture was allowed to warm up to room temperature. The relative percent conversion and formation of the zinc reagent was 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determined following the procedure described for compound 13. The mixture analysis showed 95% conversion and the overall purity was estimated at 90%. The aryl-zinc mixture was treated under a strong stream of nitrogen with 51 (3.2 g, 7.8 mmol) and Pd(Ph3P)4 (0.110 g, 0.095 mmol). The Schlenk flask was fitted with a reflux condenser and the solution was stirred at 70 °C overnight while being monitored via HPLC. The reaction was quenched with saturated solution of NH4CI (30 mL), and H2O (30 mL), and allowed to warm to room temperature. The organic layer was separated and the solvent was removed in vacuo. The resulting orange oil was treated with pentane (100 mL) upon which a yellow solid separated. The mixture was stirred for 5 minutes and filtered. Two additional pentane washes (2 x 50 mL) were conducted, and the combined fractions were washed with brine, and dried with MgSCL. Pentane was removed in vacuo, and the crude product was subjected to column chromatography (silica gel, 90/10 hexane/ethyl acetate). The title compound was obtained as yellow oil. 2-(3-{7-[3-(l-Carboxy-l-methylethyl)phenyl]-9-ethoxycarbonyImethyl-9H- fluoren-2-yl}phenyI)-2-methylpropionic acid (53) A 250-mL round-bottom flask equipped with a magnetic stirring bar was charged with 52 and dry CH2CI2 (50 mL). The yellow solution was treated with triethylsilane (4.0 mL, 25 mmol) and trifluoroacetic acid (13 mL, 0.17 mol), and the solution was stirred at room temperature for 4 hours. The solvents were removed in vacuo and the resulting residue was refluxed with CH3CN (50 mL). The 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. heterogenious mixture was allowed to cool to room temperature. The product was collected and allowed to air dry. The title compound was obtained as a white powder, 2.9 g (65% over two steps). !H NMR (500 MHz, CDCI3/TFA): 8 7.84 (d, 2H, J H-h = 8-0 Hz), 7.68 (s, 2H), 7.66-7.61 (m, 4H), 7.55 (d, 2H, JH -h = 8.0 Hz), 7.46 (t, 2H, J H .H = 8.0 Hz), 7.40 (d, 2H, J h-h = 8.0 Hz), 4.50 (t, 1H, JH -h = 6.3 Hz), 4.25 (q, 2H, JH -h = 7.2 Hz), 3.03 (d, 2H, J h-h = 6.3 Hz), 1.72 (s, 12H), 1.22 (t, 3H, J H-h = 7.2 Hz). 1 3 C NMR (126 MHz, CDCI3/TFA): 5 185.0, 176.0, 146.5, 143.5, 141.8, 140.5, 139.9, 129.2, 127.3, 126.4, 124.9, 124.7, 123.1, 120.6, 62.68, 46.67, 43.72, 38.91,26.08, 13.72. Mass spectroscopy (MALDI, M+ ): m/e calc. 576.2506, found 576.2534. {2,7-Bis[3-(l-chIorocarbonyl-l-methyIethyl)phenyl]-9H-fluoren-9-yl}actic acid ethyl ester (54) A 50-mL round-bottom flask equipped with a magnetic stirring bar was charge with 53 (l.Og, 1.7 mmol) and SOCI2 (3 mL, 41 mmol). The flask was fitted with a reflux condenser and stirred overnight at 45 °C. The reflux condenser was emptied of water, fitted with a distillation head, and the excess SOCI2 was removed in vacuo. The resulting light yellow solid was treated with CHCI3 ( 1 0 mL), which was subsequently removed in vacuo. The process was repeated one more time. The title compound was obtained as a solution in CHCI3 (10 mL), which was used directly in the next step without further purification. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-(2-{3-[9-Ethoxycarbonylmethyl-7-(3-{l-[l-raetlioxycarbonyl-2-(l-methyl-lH- imidazol-4-yl)ethylearbamoyl]-l-methylethyl}phenyl)-9H-fluoren-2-yl]phenyl}- 2-methylpopiony!amino)-3-3-(l-methyl-lH-imidazoI-4-yl)propioiiic acid methyl ester (55) A 100-mL round-bottom flask equipped with a magnetic stirring bar was charged with 48 (0.93 g, 3.6 mmol), and CHCI3 (10 mL). The flask was fitted with a rubber septum and cooled to 0 °C on ice. Triethylamine (1.5 mL, 11 mmol) was added to the suspension. The rubber stopper was replaced with an addition funnel and the system was flushed with nitrogen. The solution of 54 from the previous step was transferred to the addition funnel under a nitrogen stream, and was drop wise added to the reaction mixture. After the addition was complete, the reaction was allowed to warm to room temperature and stirred overnight. Saturated solution of NaHCCb (25 mL) and H2O (25 mL) were added and the reaction mixture was transferred into a separatory funnel. The organic layer was separated, and the aqueous layer was further extracted with CHCI3 (2x10 mL). The combined organic fractions were dried over MgSCL, and CHCI3 was removed in vacuo. The title compound was obtained as a yellowish solid, 1.4 g (89%). 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-(2-{3-[9-Carboxymethyl-7-{4-{l-hydroxymethyl-2-(l-methyl-lH-imidazol-4- yI)ethylcarbamoyl]-l-methylenenbut-2-enyl}-9H-fluoren-2-yl)phenyl]-2- methylpropionylamino}-3-(l-methyl-lH-imidazol-4-yl)propioiiic acid trisodium salt (56) A 25-mL round-bottom flask equipped with a magnetic stirring bar was charged with 55 (0.50 g, 0.55 mmol), THF (5.0 mL) and 2.5M aqueous NaOH (0.90 mL, 2.25 mmol). The mixture was stirred at room temperature while the progress of the reaction was monitored via HPLC. The reaction was worked up based on the desired counter ion and desired form of the ligand. 1,8-Octane-dimesylate (58) A 250 mL flask equipped with a magnetic stirring bar was charged with 1,8- octane-diol (57) (5.0g, 34 mmol), triethylamine (10 mL, 71 mmol), and THF (70 mL). The mixture was cooled to 0 °C on ice. The flask was fitted with an addition funnel charged with methanesulfonyl chloride (5.5 mL, 71 mmol). The addition was done drop wise, regenerating the ice bath as necessary. The formation of a white precipitate was immediately noticeable. After the addition was complete, the reaction mixture was stirred for 30 minutes at 0 °C followed by additional hour at room temperature. THF was removed in vacuo followed by the addition of 10% aqueous HC1 (50 mL) and CH2CI2 (50 mL). The phases were separated and the aqueous phase was further extracted with CH2CI2 (4 x 50 mL). The resulting yellow solution was dried with MgS04, and the bulk solvent was removed in vacuo. 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recrystallization from EtOH gave the title compound as a light, white solid, 9.1 g (88%). 1,8-Octane-ditriflate (59) A 250-mL round-bottom flask equipped with a magnetic stirring bar and a rubber septum was flushed with nitrogen, and charged with dry CH2CI2 (20 mL). The flask was cooled to 0 °C on ice and triflic anhydride (4.0 mL, 24 mmol) was added in one portion. A separate 250-mL round-bottom flask was charged with 1,8- octyldiol (57) (1.8 g, 12 mmol), pyridine (2.0 mL, 25 mmol) and CH2CI2 (100 mL). All solids were dissolved with gentle heating if necessary, and the entire contents were transferred to the addition funnel via a cannula. The solution was added drop wise while maintaining the ice bath. After the addition was complete the reaction was stirred for additional 10 minutes. H2O (20 mL) was added and the reaction mixture was transferred into a separatory funnel. The organic phase was collected, washed with one more portion of H 2O (20 mL), dried over MgSCL, and filtered into a 500-mL round-bottom flask. The title compound was used directly in the next step without further purification. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7-Methoxycarbonyl-2-[8-(7-methoxycarb<myl-5-oxo-5,6,7,8- tetrahydroimidazo[l,5-c]pyrimidin-2-yl)octyl]-5-oxo-5,6,7,8- tetrahydrolmidazo [1,5-c] pyrimidin-2-ium ditriflate (60) To the solution of 59 from the previous step was added 44 (5.0 g, 26 mmol) and a magnetic stirring bar. The mixture was corked and vigorously mixed overnight. The mixing was stopped causing clear oil to separate. The solvent was carefully decanted and CH2CI2 (50 mL) was added. The mixture was vigorously stirred for 15 minutes and the oil was allowed to settle. The solvent was once again carefully decanted and the process was repeated two more times. Finally, excess CH2C S 2 was removed in vacuo and the title compound was obtained as a white, fluffy, and highly hygroscopic solid, 9.0 g (92%). X H NMR (250 MHz, DMSCMs): 8 9.82 (s, 2H), 9.52 (d, 2H, Jh-h = 4.0 Hz), 7.68 (s, 2H), 4.65 (dd, 2H, JH -h = 4.8 Hz, 4.0 Hz), 4.18 (t, 4H, JH -h = 6.9 Hz), 3.66 (s, 6H), 3.35 (d, 4H, J H .H = 4.8 Hz), 1.94-1.63 (m, 4H), 1.40-1.06 (m, 8H). 2-Amino-3-(l-{8-[4-(2-ainio-2-caboxyethyl)iinidazoH-yl]o€tyl}-lH-iiiiidazol-4- yl)propionic acid dihydrochloride-ditriflate salt (61) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 60 (9.0 g, 11 mmol), H2O (50 mL), and conc. HC1 (10 mL). The flask was fitted with a reflux condenser and the solution was gently refluxed overnight. Decolorizing charcoal was added and the mixture was filtered. The clear solution was evaporated in vacuo and any residual water was removed under high vacuum in 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a water bath maintained around 50 °C. The title compound was obtained as a white, highly hygroscopic solid, 7.8 g (87%). NMR (500 MHz, DMSO-4s): § 9.09 (s, 2H), 7.59 (s, 2H), 4.28 (t, 2H, J H-h = 6.8 Hz), 4.13 (t, 4H, J H-h = 6.5 Hz), 3.34-3.18 (m, 4H), 1.85-1.70 (m, 4H), 1.37- 1.18 (m, 8H). 1 3 C NMR (126 MHz, DMSO-J6 ): 8 169.2, 134.9, 127.7, 120.5, 99.74, 51.00, 29.24, 28.02, 25.31, 25.21. 2-Ami no-3-( 1 - {8-[4-(2-amio-2-methoxy carbonylethy l)imidazol-1-yl] octyl} - 1H- imidazol-4-yl)propionic acid methyl ester dihydrochloride-ditriflate salt (62) A 500-mL round-bottom flask equipped with a magnetic stirring bar was charged with 61 (7.8 g, 9.8 mmol) and 1M HC1 in MeOH (40 mL). The flask was fitted with a reflux condenser and the mixture was refluxed overnight. Next day the solvent was removed in vacuo and the solid was dried under high vacuum to insure that all of residual HC1 has been removed. The title compound was obtained as a white, hygroscopic solid, 7.9 g (97%). lH NMR (500 MHz, DMSO-ufe): 8 9.10 (s, 2H), 7.59 (s, 2H), 4.41 (t, 2H, J H -h - 6.5 Hz), 4.13 (t, 4H, Jh-h = 6.2 Hz), 3.73 (s, 6H), 3.28 (d, 4H, J H - H = 6.5 Hz), 1.82- 1.70 (m, 4H), 1.33-1.15 (m, 8H). 1 3 C NMR (126 MHz, DMSO-ufe): 5 168.2, 134.9, 127.2, 120.6, 102.7, 52.87, 50.97, 29.19, 27.99, 25.27, 25.11. Mass spectroscopy (FAR+ [M4+-3H~r]: m/e calc. 449.2876, found 449.2863. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Compound (63) A 1000-mL three neck round-bottom flask equipped with a magnetic stirring bar and two addition tunnels was flushed with nitrogen and charged with CHCI3 (400 mL). One addition funnel was charged under a stream of nitrogen with a solution of 62 in CHCI3 prepared as follows: a 100-mL round-bottom flask equipped with a magnetic stirring bar was charged with 62 (1.2 g, 1.5 mmol), triethylamine (1.9 mL, 14 mmol), and CHCI3 (50 mL). The second addition funnel was charged under a stream of nitrogen with a solution of 54 prepared as follows: a 50-mL round-bottom flask equipped with a magnetic stirring bar was charge with 53 (0.80 g, 1.4 mmol) and SOCI2 (3 mL, 41 mmol). The flask was fitted with a reflux condenser and stirred overnight at 45 °C. The reflux condenser was emptied of water, fitted with a distillation head, and the excess SOCI2 was removed in vacuo. The resulting light yellow solid was treated with CHCI3 (10 mL), which was subsequently removed in vacuo. The process was repeated one more time and the solid was dissolved in CHCI3 (50 mL). The system was maintained under nitrogen and the two solutions were simultaneously added drop wise to the reaction vessel. After the addition was complete the mixture was stirred for 5 days. The solvent was reduced in vacuo to ca. 50 mL, saturated solution of NaHCOs (25 mL) and H2O (25 mL) were added, and the reaction mixture was transferred into a separatory funnel. The organic layer was separated, and the aqueous layer was further extracted with CHCI3 (2 x 25 mL). The 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. combined organic fractions were dried over MgSCL, and CHCI3 was removed in vacuo to produce the title compound as a light yellow fluffy solid. Mass spectroscopy (MALDI, MH+ ) m/e calc. 989.5171, found 989.5120. Compound (64) A 10-mL round-bottom flask equipped with a magnetic stirring bar was charged with 63 (0.20 g, 0.20 mmol), MeOH (1.0 mL), and 0.88M aqueous NaOH (0.92 mL, 0.81 mmol). The mixture was stirred at room temperature while the progress of the reaction was monitored via HPLC. The reaction was worked up based on the desired counter ion and desired form of the ligand. Mass spectroscopy (MALDI, MH+ ): m/e calc. 933.4545, found 933.4606. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Alphabetized Bibliography Armstrong, W. H.; Lippard, S. I. Inrog. Chem. 1985, 24, 981. Baldwin, T. R.; Burch, R.; Squire, G. D.; Tsang, S. C. Appl. Catal. 1991, 74, 137. Barrios, A. M ; Lippard, S. J. Inorg. Chem, 2001, 40, 1060. Claridge, I. B.; Green, M. L. H.; Tsang, S. York, A. P. E. Appl. Catal. 1992, 89, 103. Claridge, J. B.; York, A, P. E.; Brungs, A. J.; Marquez-Alvarez, C.; Sloan, J.;Tsang, S. C.; Green, M. L. H. J. Catalysis 1998,180, 85. Comot-Gandolphe, S. Energy Exploration & Exploitation 1995, 13, 3. Costas, M.; Rohde, J. U.; Stubna, A.; Ho, R. Y. N.; Quaroni, L.; Miinck, E.; Que, L., Ir. J. Am. Chem. Soc. 2001,123, 12931. Crowther, G. P.; Kaiser, E. M.; Woodruff, R. A.; Hauser, C. R. Org. Synth., Collect. 1988, 6, 259. Green, M. L. H.; Tsang, S. C.; Vemon, P. D. F.; York, A. P. E. Catal. Lett. 1992, 13, 341. Hagen, K. S.; Inorg. Chem. 2000, 39, 5867. He, C.; Lippard, J. S. J. Am. Chem. Soc. 2000,122, 184. Kelley, I. L.; Miller, C. A.; McLean, E. W. J. Med. Chem. 1977, 20, 721. Kopp, D. A.; Gassner, T .; Blazyk, J. L.; Lippard, S. I. Biochemistry 2001, 40, 14932. Lee, D.; Krebs, C.; Huynh, B. H.; Hendrick, M. P.; Lippard, S.J. J. Am. Chem. Soc. 2000,122, 5000. Leprince, P.; Valais, M. Energy Sources 1993, 15, 95. Liu, K. E.; Valentine, A. M.; Wang, d.; Huynh, B. H.; Edmondson, D. E.; Salifoglou, A.; Lippard, S. I. J. Am. Chem. Soc. 1995,117, 10174. Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Tetrahedron Lett. 1992, 33, 5441. Miyaura, I. N. Chem. Commun. 1996, 2073. Nguyen, H. H. T.; Elliott, S. I.; Yip, J. H. K.; Chan, S. I. J. Biol Chem. 1998, 273, 7957. Noordham, A.; Maat, L.; Beyerman, H. C. Reel. Trav. Chim. Pays-Bas 1978, 97, 293. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Papaioannou, D.; Athanassopoulos, C.; Magafa, V.; Karigiannis, G.; Karamanos, N.; Stavropoulos, G.; Napoli, A.; Sindona, G.; Aksnes, D. Acta Chem. Scand. 1995, 49, 103. PCMODEL, version 7.0; Serena Software: Bloomington, IN, 1998. Periana, R. A.; Taube, D. J.; Evitt, E. R.; Loftier, D. G.; Wentrcek, P. R.; Voss, G.; Masada, T. Science 1993, 259, 340. Ranger, M.; Rondeau, D.; Leclerc, M. Macromolecules 1997, 30, 7686. Rostrup-Nielsen, J. R. in “Catalysis Science and Technology” (J. R. Anderson and M. Boudart, Eds.), Vol. 5, p. 1. Springer-Verlag, Berlin, 1984. Schow, S. R.; Bloom, J. D.; Thomposn, A. S.; Winzenberg, K. N.; Smith III, A. B. J. Am. Chem. Soc. 1986,108, 2662. Seigel, E L ; McCormick, D. B.;Acc. Chem. Res. 1970, 3, 201. Shu, L.; Nesheim, J. C.; Kauffmann, K.; Munck, E.; Lipscomb, J. D.; Que, L., Jr. Science 1997, 275, 515. Solomon, M. F.; Solomon, R. G. J. Am. Chem. Soc. 1979, 15, 4290. Valentine, A. M.; Stahl, s. S.; Lippard, S. J. J. Am. Chem. Soc. 1999, 121, 3876. White, M. C.; Doyle, A. G.; Jacobsen, E. N. J. Am. Chem. Soc. 2001,123, 7194. Whittington, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2001,123, 827. Ill Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Syntheses and photophysics of organic light -harvesting lanthanide complexes

PDF

Design and synthesis of novel eicosanoids

PDF

Synthesis of novel compounds from boronic acids, amines, and carbonyl derivatives

PDF

Design and synthesis of novel lipid mediators

PDF

Ruthenium catalyzed synthesis of novel aromatic quinones /siloxane copolymers and photochemistry of the polymers and model systems

PDF

Synthesis and biological evaluation of novel cidofovir prodrugs targeting HPepT1

PDF

Design, synthesis and characterization of novel nonlinear optical chromophores for electro-optical applications

PDF

Living anionic polymerization of 2-vinylnaphthalene. Synthesis and characterization of macrocyclic poly(2 -vinylnaphthalene)

PDF

Stereoselective synthesis of trifluoromethylated amines, allylic amines, diamines and amino alcohols using (trifluoromethyl)trimethylsilane

PDF

Synthesis and fluorescence of macrocyclic vinyl polymers

PDF

Novel materials and techniques of fabrication for organic light emitting diodes

PDF

Synthesis and characterization of cyclometalated iridium (III) complexes and their use in organic electroluminescent devices

PDF

Validation and optimization of microscopic molecular simulation methods for biological systems

PDF

Synthesis and characterization of functional oligomers and polymers for organic light emitting diodes

PDF

Synthesis of polyfunctional molecules using organoboron compounds

PDF

Synthesis, polymerization and modifications of cyclotetra- and cyclotrisiloxanes

PDF

Synthesis and characterization of poly(silyl ether)s and modified poly(siloxane)s

PDF

Synthesis and characterization of macrocyclic vinyl aromatic homopolymers and block copolymers

PDF

The influence of drug copay change on drug utilization: The case of small-firm employees in California

PDF

Novel troika acid derivatives: Photochemistry and metal chelation

Asset Metadata

Creator Vagner, Patrick (author)

Core Title The design and synthesis of novel ligands as possible mimics of methane monooxygenase

Degree Doctor of Philosophy

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, general,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-497684

Unique identifier UC11335780

Identifier 3133347.pdf (filename),usctheses-c16-497684 (legacy record id)

Legacy Identifier 3133347.pdf

Dmrecord 497684

Document Type Dissertation

Rights Vagner, Patrick

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA