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Antibody-catalyzed formation of a 14-membered ring lactone Pungente, Michael D. 1997

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ANTIBODY-CATALYZED FORMATION OF A 14-MEMBERED RING LACTONE by Michael D. Pungente  B.Sc.(Hons.), University of Victoria, 1989  A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Chemistry)  W e accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A October 1997 © Michael D. Pungente, 1997  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  The University of British Columbia Vancouver, Canada  I  I further  purposes  gain  the  shall  requirements  agree  that  agree  may  representatives.  financial  permission.  DE-6 (2/88)  study.  of  be  It not  that  the  be  an  by  understood allowed  advanced  Library shall  permission for  granted  is  for  the that  without  make  it  extensive  head  of  copying my  my or  written  ABSTRACT  A number of macrolide antibiotics have a 14-membered ring lactone skeleton. Besides the stereochemical challenges associated with the syntheses of these macrocyclic compounds, the difficulty in controlling the ring forming step has provided the basis for many synthetic organic methodology studies. Attempts to use enzymes in aqueous and organic solvents to catalyze medium to large ring lactonizations has met with limited success, particularly with secondary alcohols.  W e report monoclonal antibody F123, raised  against transition state analogue 50, which catalyzed the intramolecular transesterification of hydroxyester 57 to give the 14-membered ring lactone 19. The reaction of antibody F123 with substrate 57 displayed enzyme-like  o  50  57  19  Ill  Michaelis-Menten kinetics. The kinetic parameters of this antibody reaction were determined by two methods, namely a multiwell method and the spectrophotometric cuvette method. Analysis of the reaction of F123 with 57 via the multiwell method yielded a Km of 250 ± 10 uM, V x of 0.62 ± 0.01 ma  ixmol/min mg and a kcat of 1.1 min" . The spectrophotometric cuvette method 1  yielded a rCr, of 330 ± 50 uM, V  m a x  of 1.4 ± 0.1 umol/min mg and a kcat of 2.2  min' . The results obtained from these two methods were found to be in good 1  agreement once the delay times in determining initial rates, characteristic of the multiwell method, were accounted for.  In both methods, the observed  rates were  corrected for the background hydrolysis in buffer. competitive  inhibition by the hapten derivative 60  Substrate specificity and (K = 2.9 ± 0.4 u.M)  demonstrated that the catalytic activity was associated with binding in the antibody-combining site. Finally, the lactone product was isolated from pooled antibody-catalyzed reactions by ether extraction and identified using gas chromatography-mass spectroscopy by comparison with an authentic lactone sample.  iv TABLE OF CONTENTS ABSTRACT  ii  LIST O F T A B L E S  ix  LIST OF F I G U R E S  x  LIST O F S C H E M E S  xiii  ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS CHAPTER I  INTRODUCTION T O M A C R O L I D E S  xvii 1  1.1 M A C R O L I D E ANTIBIOTICS  1  1.2 LACTONIZATION M E T H O D S  4  1.2.1  Chemical Methods  4  1.2.2 Enzymatic Methods  8  1.2.3 Antibody Methods  9  C H A P T E R II  INTRODUCTION T O C A T A L Y T I C ANTIBODIES  10  2.1 HISTORICAL A S P E C T S  10  2.2 A N T I B O D Y S T R U C T U R E  13  2.3 T H E IMMUNE S Y S T E M / R E S P O N S E  16  2.4 A N T I B O D Y P R O D U C T I O N IN VIVO  20  2.5 DIVERSITY O F T H E IMMUNE R E S P O N S E  23  2.5.1 Recombination  ...23  2.5.2 Junctional Diversity  24  2.5.3 Mutation  26  2.5.4 Association Of Heavy And Light Chains  26  2.6 A N T I B O D Y G E N E R A T I O N  27  2.7 H Y B R I D O M A T E C H N O L O G Y  27  2.8 S C R E E N I N G O F H Y B R I D O M A S F O R H A P T E N BINDING  29  V  2.8.1 The ELISA Assay  29  2.8.2 The P C F I A Assay  31  2.9 A S C I T E S P R O D U C T I O N  33  2.10 E N Z Y M E KINETICS A N D R E L E V A N C E T O S Y N T H E S I S  34  2.11 E N Z Y M E S A N D C A T A L Y T I C ANTIBODIES IN O R G A N I C S O L V E N T S 4 2 2.12 D E S I G N O F TRANSITION S T A T E A N A L O G U E S  44  2.13 R E A C T I O N T Y P E S C A T A L Y Z E D B Y ANTIBODIES  45  2.13.1 Lactonization of Y,8-unsaturated acids  48  2.13.2 Enantioselective lactonization  49  2.13.3 Intramolecularacyl migration..  50  C H A P T E R III  RESULTS AND DISCUSSION  54  3.1 S Y N T H E S I S O F M A C R O C Y C L I C P H O S P H O N A T E 20  54  3.2 A N T I B O D Y P R O D U C T I O N  70  3.2.1 Preparation of Antigen KLH-50  70  3.2.2 Antibody Raised Against Antigen KLH-50  74  3.3 BINDING A S S A Y S  75  3.4 A N T I B O D Y F123 ISOTYPING  76  3.5 A S C I T E S P R O D U C T I O N  78  3.6 S Y N T H E S I S O F S U B S T R A T E S A N D H A P T E N D E R I V A T I V E S  79  3.6.1 Synthesis of Substrate 55  79  3.6.2 Synthesis of Substrate 57  83  3.6.3 Synthesis of Substrate 58  84  3.6.4 Synthesis of Hapten Derivatives 60a and 60b  84  3.6.5 Synthesis of Six-membered Ring PhenylPhosphonates 61a and 61b  85  3.7 INVESTIGATION INTO T H E RELATIVE S T E R E O C H E M I S T R Y O F C Y C L I C P H O S P H O N A T E S 38a A N D 38b  87  3.8 INITIAL INVESTIGATION F O R REACTIVITY O F M A B ' S WITH S U B S T R A T E 57  93  vi 3.9 ISOLATION O F F 1 2 3 - C Y C L I Z E D M A C R O L A C T O N E 19  96  3.10 SPECIFICITY O F M O N O C L O N A L F123  98  3.11 R E A C T I O N O F P - N I T R O P H E N Y L T E T R A D E C A N O A T E WITH F123...98 3.12 INHIBITION A S S A Y S 3.13 DETERMINATION O F Km, V WITH S U B S T R A T E 57  100 m a x  A N D kca, F O R A N T I B O D Y F123 103  3.13.1 Determination of the Solubility Limit of Substrate 57 and the pH of the Reaction Solution  104  3.13.2 Determination of Km, Vmax and kcat for Monoclonal F123 with Substrate 57  107  3.13.2.1 Determination Km, V x and kcat for Monoclonal F123 with 57 via the Multiwell Method  109  3.13.2.2 Determination of Km, V x and k^t for Monoclonal F123 with 57 via the Spectrophotometer Cuvette Method  110  ma  ma  3.14 S U M M A R Y A N D C O N C L U S I O N S  114  3.15 S U G G E S T I O N S F O R F U T U R E W O R K  117  C H A P T E R IV.  EXPERIMENTAL  4.1 BIOLOGICAL M E T H O D S  121 121  4.1.1 Monoclonal Antibody Purification  121  4.1.2 Preparation of Immuno- and Binding-conjugates of 50  122  4.1.2.1 KLH-50 Hapten Conjugate  122  4.1.2.2 BSA-49 Hapten Conjugate-Coated Carboxyl Polystyrene Particles  122  4.1.3 Kinetic Assays  123  4.1.3.1 Multiwell Method  123  4.1.3.2 Spectrophotometer Cuvette Method  124  4.1.4 General Immunological Techniques  125  4.1.4.1 Immunizations  125  4.1.4.2 Test Bleeds  125  4.1.5 Polyethylene Glycol (PEG) Fusion  126  vii 4.1.5.1 Cell Counting  127  4.1.5.2 Myelomas  128  4.1.5.3 P E G Solution for Cell Fusion  128  4.1.5.4 Harvesting Spleenocytes  129  4.1.5.5 Harvesting Myelomas  130  4.1.5.6 Fusion  130  4.1.5.7 Transferring Fused Cells to the Growth Plates  131  4.1.5.8 Selection and Expansion of Hybridomas  131  4.1.5.9 Cloning Cell Lines  132  4.1.5.10 Ascites Production  133  4.2 G E N E R A L C H E M I C A L M E T H O D S  134  4.3 C H E M I C A L M E T H O D S  138  11- (Tetrahydropyranyloxy)-1-dodecanal (33)  138  I -Diphenylphosphinyl-12-(tetrahydropyranyloxy)-1 -tridecene (36a and 36b)  139  12- Hydroxy-1-diphenylphosphinyl-tridecane (37)  141  I- Phenyl-1 oxo-1 -phospho-13-tetradecanolide (38a and 38b) and Dimer 39  142  I I - Dodecen-2-ol (40)  144  11-(Tetrahydropyranyloxy)-1-dodecene (41)  145  II -(Tetrahydropyranyloxy)-I -dodecanol (42)  147  1 -Diphenylphosphinyl-12-(tetrahydropyranyloxy)-tridecane (43)  148  Phenyl 12-Hydroxytridecane Phosphonic Acid (44)  149  N-Cbz-5-amino-1-pentanol (48)  150  1-0-Cbz-linker-1-oxo-1-phospho-13-tetradecanolide (49a)  152  1-0-Cbz-linker-1 -oxo-1 -phospho-13-tetradecanolide (49b)  153  1-0-linker-1 -oxo-1 -phospho-13-tetradecanolide (50)  154  4-Benzoxybutanoic Anhydride (52)  155  Indoyl 4-Benzoxybutanoate (53)  156  viii (Indoyl butanoate) 13-Hydroxytetradecanoate (55)  157  p-Nitrophenyl 13-Hydroxytetradecanoate (57)  158  p-Nitrophenyl Tetradecanoate (58)  160  1 -Oxo-1 -phosphonic acid-13-tetradecanolide (59)  161  1 -(p-Nitrophenyl)-l -oxo-1 -phospho-13-tetradecanolide (60a and 60b)  162  6-Methyl-2-oxo-2-phenoxy-1,2-oxaphosphorinane (61a and 61b)  164  1-Benzyloxy-3-butanol (62)  166  1-Benzyloxy-3-(Tetrahydropyranyloxy) butane (63)  167  3-(Tetrahydropyranyloxy) butan-1-ol (64)  168  3- (Tetrahydropyranyloxy) butanal (65)  169  1-Diphenylphosphinyl-4-(tetrahydropyranyloxy)-1-pentene (66)  170  4- Hydroxy-1-diphenyiphosphinyl-1-pentene (67)  171  1-Diphenylphosphinyl pentan-4-ol (68)  172  REFERENCES  174  SPECTRAL APPENDIX  184  ix LIST OF TABLES  Table 1.  Relative rate constants at 0 °C for the cyclization of various o-bromoalkyl-carboxylate ions  5  Table 2.  Characteristics of antibody classes  15  Table 3.  Reactions catalyzed by antibodies  47  Table 4.  Comparison of the H N M R chemical shift data for the methine and methyl signals of the open chain hydroxy acid 44 with those of themacrocyclic phosphonates 38a and 38b  70  Table 5.  1  Key H a n d P N M R chemical shifts (8 values) and T L C 1  3 1  Rr-values for the cyclic phosphonates  89  Table 6.  Results of initial antibody experiments  96  Table 7.  Solubility study of substrate 57 with Triton X-100  Table 8.  A comparison of the catalytic efficiency (kcat/Km) of antibody  Table 9.  F123 with that of the antibody reported by Napper, et al Media and components used in hybridoma production  104  59  113 126  x LIST OF FIGURES  Figure 1.  Examples of macrolides  Figure 2.  Free energy diagram of an antibody-catalyzed reaction compare to the corresponding uncatalyzed reaction  Figure 3.  2  11  The schematic structure of immunoglobins (antibodies), (taken from ref 64, p 288)  13  Figure 4.  Overview of the immune system  19  Figure 5.  Kappa (K) rearrangements in germ line and B-lymphocytes (taken from ref 65, p 13)  24  Figure 6.  Heavy chain V D J joining (taken from ref 65, p 15)  25  Figure 7.  Hybridoma technology for the production of monoclonal antibodies  28  Figure 8.  ELISA assay for screening mouse antibodies  30  Figure 9.  P C F I A assay for screening mouse antibodies  33  Figure 10.  The effect of substrate concentration on the rate of the enzyme-catalyzed reaction A double-reciprocal plot, or a Lineweaver-Burk plot, of enzyme kinetics: 1/v is plotted as a function of 1/[S] The putative transition state and the corresponding phosphonate transition state analogue for the displacement reaction of an ester  Figure 11. Figure 12.  Figure 13.  Figure 14.  39 40  46  Stereoview of an O R T E P representation of dimeric cyclic phosphonate, 39, showing 3 3 % probability ellipsoids  66  Relative stereochemistry about the phosphorus atom and the C-1 carbon atom of dimer 39 as interpreted from Figure 13  91  xi Figure 15.  Monoclonal antibodies F123 (0), F125 (•) and F150 (•), each at a concentration of 2 u,M, were assayed for activity with substrate 57 (200 uM) in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100 at 37 °C  94  Figure 16.  Initial experiments with antibody F123, substrate 57, and hapten derivative 60 using P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100 at 37 °C. Reactions were run; (i) (•) 88 uM in 57 and 0.88 uM in F123, (ii) (O) 88 uM in 57 and 0.44 u M in F123, (iii) (•) 88 u M in 57, 0.88 u M in F 1 2 3 and 0.22 u,M of 60 (1:1 ratio of diastereomers) 95  Figure 17.  G C chromatograms of: (a) an independently synthesized sample of lactone 19; (b) ether extracts from a large-scale F123 catalyzed cyclization reaction of substrate 57; (c) a spiked G C run of the ether extracts from the antibody experiment with an authentic sample of 19  97  The activity of monoclonal F123 (O) is compared to that of a control monoclonal antibody, HIL-20 (•), using substrate 57 in both of the experiments. Each experiment was run 200 uM in 57 and 2 u.M in antibody, carried out in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100  99  Comparison of antibody F123 activity with hydroxy ester substrate 57 (O); with ester 58 (•). In each case, antibody was 0.46 u,M; substrates were 100 uM; carried out in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100; total reaction volume was 225 uL  100  Antibody F123 activity with hydroxy ester substrate 57 (O); with hydroxy ester substrate 57 in the presence of an equimolar amount of lactone 19 (•). Experiments were carried out in P B S buffer (pH 7.4) with 10% D M S O ; 0.46 uM in antibody F123; 200 uM in substrate; total reaction volume was 250 uL  101  Figure 18.  Figure 19.  Figure 20.  xii Figure 21.  Figure 22.  Figure 23.  Figure 24.  Figure 25.  Competitive inhibition of the F123-catalyzed macrolactonization of substrate 57 by a one-to-one mixture of 4-nitrophenyl phosphonate diastereomers 60a and 60b. The reactions were performed in P B S buffer, pH 7.4, containing 10% D M S O and 0.5% Triton X-100. The concentrations of the one-to-one mixture of 60a and 60b were: (O) 0 uM; (•) 2 uM; and (•) 5 uM  102  Antibody F123 reaction with substrate 57 compared to the background hydrolysis of 57 in the reaction buffer alone  105  Comparison of the F123 reaction with substrate 57 and background hydrolysis of 57 using 10% D M S O and 1.0% Triton X-100 in 8 uM P B S buffer (pH 6.95); and F123 and 57 in a 10% D M S O with 1.0% Triton X-100 in 500 uM P B S buffer (pH 7.45)  107  A Lineweaver-Burk analysis of the F123-catalyzed macrolactonization of hydroxy ester substrate 57 using the multiwell method  110  A Lineweaver-Burk analysis of the F123-catalyzed macrolactonization of hydroxy ester substrate 57 using the spectrophotometer cuvette method  112  xiii LIST OF SCHEMES  Scheme 1.  Lactonization strategies which proceed through activated activated carboxyl intermediates; (a) Corey method (from ref 45); (b) Mukaiyama method (from ref 46); (c) Yamaguchi method (from ref 47)  6  Scheme 2.  Synthesis of macrocyclic phosphonate 22  55  Scheme 3.  Proposed synthetic sequence for compound 35  58  Scheme 4.  A proposed mechanism for the Mitsunobu reaction (from ref 138)  62  Scheme 5.  Possible pathways in the Mitsunobu reaction (from ref 138)  62  Scheme 6.  Synthesis of macrocyclic phosphonates 38a and 38b  65  Scheme 7.  Coupling of the BSA-50 conjugate with the carboxy polystyrene particles (PSP) Exocyclic (path a) versus endocyclic (path b) hydrolysis of immuno-conjugate KLH-50 in mouse serum  Scheme 8.  Scheme 9.  76 ,78  Chromogenic assay reported by Gong, et al. (taken from ref 88)  80  Scheme 10.  Synthesis of indoyl substrate 55  81  Scheme 11.  Synthesis of cyclic phosphonates 61 a and 61b  87  ABBREVIATIONS  Ac  acetyl  Anal  analysis  Bn  benzyl  bp  boiling point  br  broad  BSA  bovine serum albumin  Bu  butyl  cc  cubic centimeter  C region  constant region  Calcd  calculated  Cbz  (benzyloxy) carbonyl  CDR  complementarity determining regions  D region  diversity region  d  doublet  Da  Dalton  DCC  1,3-dicyclohexylcarbodiimide  DC I  desorption chemical ionization  DHP  3,4-dihydro-2H-pyran  DEAD  diethyl azodicarboxylate  DMAP  4,4-dimethylaminopyridine  DMF  N,N-dimethylformamide  DMSO  dimethyl sulphoxide  ee  enantiomeric excess  El  electron impact  ELISA  enzyme-linked immunosorbent assay  Et  ethyl  XV  FCS  fetal calf serum  GC  gas chromatography  H  proton  HPLC  high performance liquid chromatography  HRMS  high-resolution mass spectroscopy  /-Pr  isopropyl  lg  immunoglobulin  IR  infrared  J  coupling constant  J region  joining region  kcat  catalytic rate constant  KLH  keyhole limpet hemocyanin  Km  Michaelis constant  LAH  lithium aluminium hydride  lit  literature  LRMS  low-resolution mass spectroscopy  m  multiplet  M  molar  M+  molecular ion  m/z....  mass-to-charge ratio  MAb  monoclonal antibody  Me  methyl  Mes  4-morpholineethanesulfonic acid  MHz  megahertz  mp  melting point  n  normal  PNP  p-nitrophenol  p-TsOH  p-toluenesulfonic acid  xvi  s  singlet  t  triplet  Tris  tris(hydroxymethyl)aminomethane  XVII  ACKNOWLEDGEMENTS  I would like to thank Professor Larry Weiler for his guidance and encouragement throughout the course of this project, and in the preparation of this thesis.  For providing me with an introduction to immunology, and without whos help and expertise this project would not have been possible, I would like to thank Professor Hermann Ziltener and his technicians Helen Merkens and Michael Williams at the Biomedical Research Center.  I would also like to thank Professor Steve Withers and his research group for their generous guidance and support with the kinetic studies performed in this thesis.  I am indebted to the service personnel of the Chemistry Department including the Mass Spec, and the Microanalytical laboratories. I am especially grateful to the N M R staff for the numerous routine and non-routine experiments they performed on my behalf.  To my lab-mates, both past and present, I extend my thanks for  their  contributions and freindship throughout my stay in the Weiler lab.  Finally, I would like to thank my family and friends for their unending support and encouragement during the preparation of this thesis.  This thesis is dedicated to my parents.  1  CHAPTER I.  INTRODUCTION TO MACROLIDES  1.1 MACROLIDE ANTIBIOTICS  Macrolide  antibiotics,  a  class  of  natural  products  largely  isolated  from  microorganisms, have been characterized extensively with regard to  their  structure and function. Ever since the isolation and structure elucidation of this general class of naturally-occurring macrocyclic antibiotics starting in the 1950's, syntheses of these large ring compounds have posed a challenge to chemists. The first of these macrocyclic antibiotics to be isolated was pikromycin, in 1950.  1  O  Pikromycin Woodward originally suggested that the name "macrolide" apply only to those 2  compounds containing a macrocyclic lactone ring such as pikromycin  or  erythromycin A. Today, however, that definition has been expanded to include a wider range of macrocyclic compounds, for instance, polyene macrolides such  2  Figure 1. Examples of some macrolides.  as amphotericin B, lactams such as FK-506, and cyclic peptides such as cyclosporin A (Figure 1). The production, isolation, and biological activity of the macrolide class of compounds has been reviewed elsewhere. " 3  12  Although the macrolides have been studied for several years, they still remain of interest to researchers for a number of reasons. Some macrolides have been shown to function as insect pheromones, while many others are clinically important. disease,  13,14  Erythromycin, for example, used in the treatment of Legionnaires' is a commonly prescribed antibiotic. " 15  17  The macrolides FK-506  and cyclosporin A are immunosuppressants, while others have shown anticancer activity. " 18  21  And lastly, the complex structure and stereochemistry associated  with these compounds presents a significant challenge to synthetic chemists. Since the introduction of penicillin as a medicine half a century ago, more than 80 antibiotics have been developed as human medicines, agrochemicals and veterinary medicines.  22  The development of ampicillin in 1961 by chemical  modification of penicillin lead to the evolution of semisynthetic antibiotics through the considerable efforts of organic chemists.  Clearly, the  stereochemical  complexity of the macrocylic compounds attracts the interest  synthetic  of  chemists, however, their clinical importance necessitates a search for general, efficient methods for their preparation.  Some reviews have been published  which focus on the efforts towards the synthesis of these compounds. " 23  27  The  majority  of  synthetic  approaches  used to  build  these  macrocyclic  compounds rely on first constructing a complex acyclic precursor and then cyclizing this precursor late in the synthesis. An alternative approach that has been the focus of our research group for some time is the macrocyclic conformational approach. In this route, the macrocyclic ring is formed early in the synthesis and the conformation of the ring is used to introduce the various substituents with regiochemical and stereochemical control.  Efforts using this  conformational approach have been published by our research g r o u p ' 28  others. " 35  1.2  34  and  38  LACTONIZATION METHODS  Chemical Methods  1.2.1  Lactone  formation  becomes  significantly  slower  6-membered rings to large ring sizes (Table 1 ).  39  in  going  from  5-  and  The difficulty in carrying out  the ring-forming step has lead to the development of a number of methodologies to effect large ring lactonization.  40  The direct cyclization of ©-hydroxy acids in the presence of mineral or Lewis acids at high temperature and in dilute solution is not recommended because other functional groups present in the molecule often are destroyed under such conditions.  Research in this area began to focus on methods to activate the  reacting groups of the open-chain ©-hydroxy acids. Activation of the carboxylic acid, involving such derivatives as active esters, mixed anhydrides or thiol esters, is a common cyclization strategy.  Alternatively, one could enlist an  approach which activates the hydroxyl end of the ©-hydroxy acid, for example using the Mitsunobu method. " 41  43  The remainder of this section will detail  examples of both approaches.  Table 1.  Relative rate constants at 0°C for the following reaction (taken from ref 44); BrfCHs^CCV  •  (CH )H5—C=0 2  +  Br  -  ^ 0 Ring size 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 23  In 1974 Corey and Nicoloau  45  Relative rate 21.7 5.4 x 1 0 1.5 x 1 0 1.7 x 1 0 97.3 1.00 1.12 3.35 8.51 10.6 32.2 41.9 45.1 52.0 51.2 60.4  3  6  4  reported a lactonization method which involved  activation of the carboxyl moiety of ©-hydroxy acids using triphenylphosphine and 2,2'-dipyridyl disulfide in xylene at room temperature to form 2-pyridinethiol  6 esters (Scheme 1a).  Without isolation, the slow addition of these activated  intermediates to refluxing xylene to achieve high-dilution resulted in lactone formation. involved  Mukaiyama et a l . slow  addition  46  of  followed with an activation methodology which the  ©-hydroxy  acid  to  an  excess  of  2-chloro-1-methylpyridinium iodide and triethylamine in refluxing acetonitrile or dichloromethane to afford lactones. A third approach, reported by Yamaguchi  Scheme 1.  Lactonization strategies which proceed through activated carboxyl intermediates; (a) Corey method (ref 45); (b) Mukaiyama method (ref 46); (c) Yamaguchi method (ref 47).  7 and co-workers in 1979, involved the reaction of the ©-hydroxy acid in T H F with 47  one equivalent  of 2,4,6-trichlorobenzoyl  chloride.  After  removal  of  the  triethylammonium chloride, the filtrate was diluted with a large volume of toluene and slowly added to a solution of refluxing toluene containing 3-6 equivalents of 4-dimethylaminopyridine to effect lactonization.  Lactonization methods of ©-hydroxy acids via activation at the hydroxyl end are less common. The Mitsunobu method is a simple one-pot approach which uses a mixture of triphenylphosphine (Ph P) and diethyl azodicarboxylate (DEAD) as 3  dehydrating agent in the synthesis of lactones under mild, neutral conditions (Equation 1). " 41  43  It has been suggested that the key intermediate of this method  is the alkoxyphosphonium salt that undergoes nucleophilic displacement by the carboxylate anion with inversion of stereochemistry, releasing the very stable by-product triphenylphosphine oxide (Ph P=0). Smith et a l . 3  48  demonstrated the  use of the Mitsunobu protocol, in the total synthesis of the latrunculins (Equation 2), to effect a macrocyclization of compound 1 with inversion of configuration at the secondary hydroxyl position giving compound 2.  These chemical approaches require that at least an equimolar ratio of reagent to hydroxy acid be used. A catalytic reagent would be of value. A n added bonus would occur if the cyclizing reagent (or catalyst) was enantioselective.  8  (D EtOCONH-NHCOOEt  1  1.2.2  Ph P=0 3  2  Enzymatic Methods  Macrolactonization  strategies  involving  the  use  of  enzymes  stereoselective catalytic cyclizations have also been investigated. Gatfield  49  was  the  first  to  report  the  intramolecular  to  In 1984,  esterification  15-hydroxypentadecanoic acid catalyzed by Mucor miehei lipase to pentadecanolide.  Yamada and co-workers  50  effect  of obtain  reported that Pseudomonas sp.  and porcine pancreas lipases mediate macrolactonization of methyl esters of o-hydroxy acids C12-C16, producing mono- and dilactones. Subsequently others have also reported the use of lipases in macrolactonizations. '  51 53  For example,  Guo et al. found the direction of the reaction and the yield of mono-, di- and trilactones from 16-hydroxyhexadecanoic acid depended on the lipase u s e d .  53  Attempts to use enzymes in aqueous and organic solvents to catalyze medium to large ring lactonization of secondary alcohols have met with only success.  54  1.2.3  55  Antibody Methods  Since the first successful reports by Lerner antibodies  limited  to  catalyze  chemical  56  and Schultz  transformations,  a  57  on the use of  large  number  antibody-catalyzed reactions have been reported (Table 3, Chapter 2).  of Two  reports to date describe the use of catalytic antibodies to effect 5- and 6-membered ring lactone cyclizations.  58,59  These two examples illustrate the  successful use of antibodies to act as chiral catalysts in enantioselective lactonizations.  The difficulties associated with the macrolactonization reaction  mentioned earlier (section 1.2) pose a challenge in the syntheses of macrolide antibiotics. W e believe the use of catalytic antibodies may provide a key for the formation of 12-16 membered ring lactones (macrolides), and in addition, may function enantioselectively and catalytically as in the small ring examples. Chapter 2 of this thesis will examine the general use of antibodies as catalysts in a range of organic transformations.  10 CHAPTER II.  2.1  INTRODUCTION TO CATALYTIC ANTIBODIES  HISTORICAL ASPECTS  The vast repertoire of antibodies expressed by the immune system enables it to generate a response to virtually any foreign molecule. Scientists have been able to exploit this diversity, and use the immune response to study various chemical processes.  The first reports on the use of antibodies to catalyze chemical  transformations appeared in 1 9 8 6 ,  56,57  when two research groups independently  published their results on acyl transfer reactions.  However, the idea of  harnessing the adaptive ability of the immune system to produce chemically useful catalysts was first eluded to by Linus Pauling in 1946.  60  It was, in fact,  Pauling's recognition of the similarity of antibodies and enzymes with respect to complementarity and the nature of catalysis that is acknowledged as one of the most important contributions to the field of catalytic antibodies. Despite a lack of details about protein structure, Pauling concluded that enzymes were similar to antibodies in that they both are complementary to the species they bind.  The  fundamental difference between these two types of proteins is that an antibody binds a stable species (hapten), while an enzyme binds the activated complex of the substrate  in an enzymatic reaction.  Pauling also pointed  out that  non-covalent factors, such as strain and ground state destabilization, are important for enzymatic catalysis.  11  It was approximately 20 years later that Jencks wrote "if complementarity between the active site and the transition state contributes significantly to enzymatic catalysis, it should be possible to synthesize an enzyme by constructing such an active site. One way to do this is to prepare an antibody to a haptenic group which resembles the transition state of a given reaction. The combining site of such antibodies should be complementary to the transition state and should cause an acceleration by forcing bound substrates to resemble the transition state."  61  The stabilization gained in the binding associated with the  antibody combining site is illustrated in the free energy diagram below (Figure  AG  Ig + S Ig*S  f '' Ig + P Reaction Coordinate  Figure 2.  Free energy diagram of an antibody-catalyzed reaction compared to the corresponding uncatalyzed reaction.  12 The basis of this approach to the generation of catalytic antibodies requires a stable transition state analogue (a hapten) that is similar in size, shape, and charge distribution to the putative transition state of the reaction of interest. Furthermore, small molecules are generally not recognized by the immune system, and require coupling to a high molecular weight carrier protein before immunization,  typically  in  mice  or  rabbits.  Booster  injections  of  the  hapten-protein conjugate (antigen) are required at regular intervals, following the primary immune response to the initial immunization, until the antigen-antibody binding interaction has reached a plateau. That is to say, the primary immune response to the antigen is mounted by the animal's existing "naive" antibody repertoire, which upon repeated exposure to antigen, evolves through mutations to a more complimentary binding association between antigen and antibody. At this point, a blood sample is taken from the host animal and the antibodies are partially purified, and screened for binding and catalytic activity. Early attempts to  test  the  transition  state  hypothesis were  disappointing  because the  researchers used this polyclonal sera, i.e., a mixture of many antibodies. Later work using monoclonal antibodies, Milstein  63  62  the development for which Kohler and  were awarded the Nobel prize in Physiology and Medicine in 1984,  lead to the initial successful demonstrations of catalytic antibodies by the research groups of Lerner and Schultz. 56  57  13 2.2 ANTIBODY STRUCTURE  Antibodies are large Y-shaped molecules (molecular weight = 150 kDa) that belong to a group of proteins called immunoglobins.  The antibody molecule  consists of four polypeptide chains - two identical heavy chains (50 kDa each) and two identical light chains (25 kDa each) held together by covalent disulfide linkages between the two heavy chains and between the heavy and light chains (Figure 3).  The light chains are composed of two distinct structural domains  known as the variable (V ) and constant (C ) regions. The heavy chains contain L  ontigtn-binding » i t i  Figure 3.  L  ontigen-binding »it«  The schematic structure of immunoglobins (antibodies), from ref 64).  (taken  14 four domains: V  H l  Cm, C 2 , and C 3 , each of which is composed of approximately H  H  110 amino acids.  Enzymatic digestion of the antibody molecule with pepsin gives rise to three fragments; two of which are identical (Fab fragments) and contain the variable region where antigen binding occurs, the third fragment is readily crystal I izable (Fc) and is responsible for immune regulation (i.e. effector functions).  In the  uncleaved antibody, the Fab and F c fragments meet at a region called the hinge, which allows lateral and rotational movement of the molecule.  Antibodies are divided into five classes, IgG, IgM, IgA, IgE, and IgD.  Each  antibody class, or isotype, is distinguished by the type of heavy chain found in the molecule. The heavy chains of IgG molecules are known as y-chains, IgMs have u-chains, IgAs have a-chains, IgEs have e-chains, and IgDs have 8-chains. The differences in their heavy chains allows each isotype to function in different immune responses, and at different stages of the developing immune response. A s for the light chains, there are only two different types, K and X. Recall that in the Y-shape antibody molecule, the two light polypeptide chains each associate with one heavy chain (Figure 3).  Although there exist five classes of heavy  chains and two of light, each antibody molecule will have only one type of light chain and one type of heavy chain. Furthermore, different classes of antibodies may vary in the number of Y-like units that join to form the  complete  15 immunoglobin (Table 2). The IgG class along with the IgE and IgD antibodies are monomeric (only one Y unit), while the IgA class can form either monomeric, dimeric, or trimeric complexes, and finally, the IgM antibodies are pentameric (containing five Y units each). These Y units are joined at the base of the F c regions via short polypeptide chains in the oligomers.  IgM antibodies then,  being pentamers, have 10 identical antigen binding sites, while monomeric IgG antibodies have two identical binding sites.  In addition, the differences in the  heavy chains between the antibody classes accounts for the different molecular weights of the various monomers.  IgG monomers, for example, have a  molecular  150  weight  of  approximately  kDa, while  IgM  monomers  are  approximately 175 kDa.  Table 2.  Characteristics of the various Antibody classes (taken from ref 65).  Characteristics  IgG  IgM  IgA  IgE  Igp  Heavy Chain Light Chain Molecular Formula Y Structure  y K or A  n K or A  a K or A  e K or A  S K or A  Valency Concentration in Serum Function  2  10  2,4, or 6  2  2  8-16mg/ml Secondary response  0.5-2 rag/ml Primary response  1-4 mg/ml Protects mucous membranes  10-400 ng/mi Protects against parasites (?)  0-0.4 mg/ml ?  *n - 1, 2, or 3.  y K, or y Aj 2  2  (HJKJ),  or  (M,),  CO,K ), 2  * or  (o,Aj).  16 The variable or Fab region of an antibody consists of one V and one V chain, H  L  to form one antigen binding site. The diversity of the variable region provides the basis for the large repertoire of binding sites that constitutes an effective immune response. However, the diversity within the variable region is limited to only  those  regions where  hypervariable regions.  contact  with the  antigen  occurs, known  as  The light and heavy chains have three hypervariable  regions each, and these form the majority of contact residues for the binding of the antigen to the antibody. Because they are the actual contact points with the antigen, they are referred to as the complementarity determining regions or CDRs.  The nature of these structural details, including the secondary and  tertiary structure of antibodies and the features of the binding site, have come from X-ray crystallographic studies of variable fragments, some of which contain the complexed antigen.  66,67  In the case of small organic molecules, the binding  occurs in clefts with volumes on the order of 600 A . 3  6 8  The clefts exclude water  and antigen binding occurs via van der W a a l s forces, hydrophobic and electrostatic interactions, and hydrogen bonding, in the same manner as observed for substrate and inhibitor binding to enzymes.  2.3 THE IMMUNE SYSTEM / RESPONSE  The principal function of the immune system is to protect animals from foreign molecules and organisms. This highly efficient defense mechanism utilizes  17 proteins and cells which circulate throughout the body to perform this function. There are many different mechanisms which act in this system, all of which can be divided into two broad categories - nonadaptive and adaptive immunity.  Nonadaptive immunity is mediated by cells that respond nonspecifically to foreign molecules. This response does not improve with repeated exposure. Examples of nonadaptive immunity are phagocytosis by macrophages, secretion of lysozyme by lacrimal cells, and cell lysis by natural killer cells. Conversely, adaptive immunity is directed at specific molecules and is enhanced by repeated exposure.  Adaptive immunity is mediated by cells called lymphocytes, which  have two main functions, namely the synthesis of cell-surface receptors or secretion of proteins that bind specifically to foreign molecules. These secreted proteins are known as antibodies. The terms "antigen" and "immunogen" are used to describe different properties of molecules in the context of the immune system.  A n antigen is any molecule that will bind to an antibody, while an  immunogen is defined as a molecule that induces an immune response. Furthermore, a "hapten" is a small molecule which by itself cannot act as an immunogen, but when coupled to a high molecular weight carrier protein, the complex is capable of eliciting an immune response.  The immune system contains more than 10  9  lymphocyte cells which travel  throughout the blood and lymphatic system, enabling them to respond promptly  18 to foreign molecules. The lymphocytes are found in higher concentrations in the lymphoid organs, namely the lymph nodes and spleen. It is in these organs that the immunogens tend to accumulate, thus, becoming the focus of the immune response.  A number of different lymphocytes have been identified within the immune system. Three basic types of lymphocytes have been found to be associated with the cellular functions of the immune system, namely B cells, killer or cytotoxic T cells, and helper T cells (Figure 4). B lymphocytes, which originate in the bone marrow, secrete antibodies and carry a bound form of the same antibody on their cell surface, where it acts as a receptor for antigens.  T  lymphocytes, originate in the thymus, and carry out either a regulatory role (helper T cells) or an antigen attacking role (cytotoxic T cells) through their surface antigen receptors, known as T-cell receptors. While they share similar structural features, the surface antibodies on B cells and the T-cell receptors are encoded by separate gene families; and their expression is cell-type specific. In addition, the surface antibodies on B cells can bind to soluble antigens, while the T-cell receptors recognize antigens only when displayed on the surface of other cells. Once an antigen is bound to the surface antibody on a B cell, for instance, the helper T cells play an essential regulatory role in governing the subsequent response of B cells and cytotoxic T cells during an immune response.  The  molecular basis for the specificity of the immune response is controlled by a  19 simple mechanism - all the surface antigen receptors on a single lymphocyte cell, whether a B- or T-lymphocyte, are identical and recognize one and only one antigen.  Stem Cells  THYMUS  BONE MARROW  TrLymphocytes  B-Lymphocytes  Helper T-Cells  Figure 4.  Killer T-Cells  Plasma Cells  Memory Cells  Overview of the immune system.  While the immune system is challenged constantly by a large number of foreign molecules,  it has the  ability  to distinguish these  invaders from  natural  components of the body. By constantly eliminating lymphocytes that bind to and attack self molecules, an organism is said to have tolerance.  Should this  self-preserving mechanism, called clonal deletion, break down and the organism mounts a response against itself, a range of disorders known collectively as autoimmune diseases may result (e.g. rheumatoid arthritis, diabetes, Graves' disease).  Clonal deletion occurs in the thymus during the development of the  immune system by a poorly understood process. Another important feature of  20 the immune system is immunological memory. Upon first exposure to a foreign antigen, an animal's immune response is slow.  This slow response may be  insufficient to prevent disease if the antigen is pathogenic.  O n subsequent  exposures to the same antigen, the response is stronger and more rapid due to this immunological memory. The memory is specific for each antigen and can last as long as the animal lives.  2.4 A N T I B O D Y P R O D U C T I O N IN VIVO  Antibody molecules are produced by white blood cells called B cells (bone marrow derived cells committed to producing antibodies). With no prediction of the shapes of foreign molecules to be encountered during the lifetime of an organism, nature has evolved a "catch-all" defense mechanism which is strengthened by a positive feedback system. The production of naive antigen receptors occurs in the absence of antigen. A diverse repertoire of B cells each with a unique antigen receptor, more than  10  7  continuously and ready for interaction with antigen.  in number,  is produced  Each individual B cell or  colony of identical B cells ("clones") makes numerous antibody molecules. The naive repertoire is expressed on the surface of the B cells, with each individual cell expressing many molecules of a unique antibody. Initially, when an antigen or foreign material is encountered, it binds to a limited number of B cells which express an antibody with some affinity for that invader.  These cells are  21 stimulated and divide.  This involves a complicated series of events, and  ultimately is responsible for the differentiation of B cells into plasma cells and memory cells. Plasma cells are short-lived cells found primarily in the lymphoid organs that specialize in secreting large amounts of antibodies.  Memory cells,  however, are long-lived cells that remain in circulation and do not secrete antibodies. Rather, memory cells retain cell-surface antibodies and are primed to respond to any subsequent exposure to the antigen.  The differentiation of B cells has two important processes associated with it; affinity maturation, a process that involves point mutations in the immunoglobin (Ig) V genes followed by the selective proliferation  of B cells expressing  antibodies with increased affinity for antigen; and class switching, a process that involves rearrangement of the chromosomal DNA encoding for the heavy chain polypeptide.  Early in an immune response, division of B cells results in hypermutation of the antibody genes, leading to the production of many variants of the initial clones selected.  The antigen in turn binds to and stimulates most effectively those  clones having the highest affinity for the antigen.  Affinity maturation  is  effectively a Darwinian process, involving variation resulting from mutations, followed by selection of cells driven by the requirement for high antigen-affinity  22  for cell survival.  Rounds of mutation and selection thus act to single-out the  highest affinity clones; this is the basis of affinity maturation.  After the affinity maturation process, class switching follows which leads to the production of antibodies with different specific functions. This process involves rearrangement polypeptide.  of the  chromosomal  D N A encoding for  the  heavy  chain  The result of this heavy chain shuffling allows the same variable  region to be expressed with different constant regions. It is the heavy chain that dictates which class (isotype) the antibody belongs to, and these different classes have different functions.  During a typical immune response, class *  switching is responsible for the replacement of IgM antibodies, characteristic of the early immune response, by IgG antibodies.  A s well, because the affinity  maturation has taken place, IgG molecules typically have higher affinities for the antigen than the short-lived IgMs.  The level of antibodies in the system peaks about 7-10 days after first contact with the antigen and the secreted antibodies enhance the rate of clearance of the foreign molecule. If no further antigen is present after this time, the number of plasma cells will decrease but the helper T-cells and memory cells remain in circulation. Upon re-exposure to the antigen, the secondary response will occur. This secondary response involves a similar sequence of events as in the primary response; however, there are some differences.  Due to the high number of  23 helper T-cells and memory cells already in the system, the response is faster. Also, since the antibodies have already undergone affinity maturation, they have a  higher affinity for the  antigen  making the  response stronger.  This  immunological memory can remain with the animal for its entire lifetime, leaving the immune system primed for any further contact with the antigen.  2.5 DIVERSITY O F T H E IMMUNE R E S P O N S E  The naive immune repertoire consists of some 10 -10 7  9  different  molecules before any exposure to a foreign material or antigen.  antibody This large  repertoire is expected to be able to recognize, albeit with moderate affinity, virtually any foreign material.  The interpretation  of the diversity of this  extraordinary system has been the subject of several reviews. 69  72  This next  section will look at the four main sources of this diversity, namely recombination, junctional diversity, mutation, and association of heavy and light chains.  2.5.1 R e c o m b i n a t i o n  Recombination essentially involves the rearrangement of the germ-line D N A (the nonfunctional gene) of both the heavy and light chains to produce mature B-cell DNA (the functional gene). The coding sequences for the variable and constant regions of the germ-line D N A are separated by several hundred kilobase pairs, and as a result the gene is silent in these cells.  During B cell differentiation,  24 these intervening regions are excised and the variable and constant regions are joined, producing a functional B cell gene. The diversity in the recombination step arises from the number of different associations possible between the variable and constant regions of the germ-line DNA.  A n example which  illustrates this diversity involves cells synthesizing functional K chains.  In this  case, there are approximately 200 different K variable regions and only one constant region. During the recombination process, one of the variable regions is selected basically at random, and is joined to the constant region to produce a functional gene (Figure 5).  - 2 0 0 Variable Region* G E R M LINE D N A NONFUNCTIONAL OENE  4 J Regions  Constant Region  —ijHH.;rrX V-J JOINING  \ ConsianI Region MATURE B-CEll DNA J  -  FUNCTIONAL GENE  Figure 5.  Kappa (K) rearrangements in germ line and B-lymphocytes (taken from ref 65).  2.5.2 J u n c t i o n a l Diversity  It became clear however, after further studies of the fine structure of the recombination  process between the  variable and constant  regions, that  25 recombination alone does not account for all the variation detected in the immune repertoire.  A third segment, known as the J region for joining, was  discovered to exist between the variable and constant regions. Recombination, then, brings the randomly selected variable region together with one of the four functional J regions making a V - J fragment, which on association with the constant region produces a functional K gene.  In addition, the recombination  events between the variable and J regions do not always occur at exactly the same nucleotides, and this allows for further diversity in the V - J junction.  SO-lOO Variable Regions  12 D Regions  4 J Regions  Constant Region  lH•^ffl^a-  GERM UNE D N A  D-J JOINING Constant Region MATURE B-CBLL D N A  NONFUNCTIONAL GENE  MATURE B-CEU. D N A  FUNCTIONAL GENf  Figure 6.  Heavy chain V D J joining (taken from ref 65).  Heavy chains have, in addition to the variable , J , and constant regions, a fourth coding region referred to as the D, or diversity, region. Thus, two recombination  26 events are required for the production of a functional heavy chain gene, the first joins the D region to a J-constant region, and a second joins a variable region to the D region (Figure 6).  2.5.3  Mutation  Mutations arising from a complex process referred to as affinity maturation lead to fine-tuning of the binding site, thus creating a better fit for antigen-antibody interaction.  These mutations occur during an immune response to a foreign  molecule or antigen, and the mechanisms that produce these mutations are not known.  2.5.4 Association Of Heavy And Light Chains  The genetic events described above provide heterogeneity in both heavy and light chains. The complete antibody molecule then requires joining of a heavy and a light chain. Once recombination has lead to the generation of functional heavy and light chain genes, a poorly understood process, called allelic exclusion, prevents further recombination, fixing the antigen binding site for the life of the cell.  This explains why B lymphocytes secrete antibodies with only  one type of antigen binding site, and why antibodies have only one type of light chain. The type, or class, of heavy chain produced by a particular B lymphocyte is determined by the process of class switching described earlier. Because the  27 different classes and subclasses determine the functional roles of antibodies, class switching is an important mechanism for controlling where and how the antigen binding site will be used in the immune response.  2.6 A N T I B O D Y G E N E R A T I O N  The method used to produce catalytic monoclonal antibodies is the same as that used to produce monoclonal antibodies used for other purposes.  63  The  synthesis of the hapten is often the most difficult and time demanding part to the production of catalytic monoclonal antibodies. Once the appropriate hapten has been made, it is then coupled to a carrier protein such as keyhole limpet hemocyanin (KLH) for immunization.  The coupling strategies used must be  compatible with the hapten design, and produce a conjugate that is stable under physiological conditions.  The hapten is coupled to the carrier protein via a  spacer arm, which typically is on the order of 6 A , " 73  76  so as to avoid any steric  interference from the carrier. The spacer arm usually has a carboxy or an amine terminus to allow amide bond formation between the hapten and corresponding amino or carboxy groups on the surface of the carrier protein.  2.7 H Y B R I D O M A T E C H N O L O G Y  The basis of the hybridoma technique involves combining the nuclei of normal antibody-producing cells with those of their malignant counterparts with retention  28 of both types of function (Figure 7). In this way, an immortal hybridoma cell line that secretes an antigen-specific monoclonal antibody  is obtained.  This  monumental advancement in immunology made possible the isolation and  ANTIGEN  u—« "^C  a  ANTIGENIC DETERMINANT MYELOMA CELLS  SPLEEN  ©  o  o  HYBRIOMYELOMA CELLS  o  LYMPHOCYTES  /I  u—u ANT1SOOY  V.  ANTIGEN  <a  - J  CLCNE 1 2  §i »• \  x  3  4  I  ANTISERUM  MIXEO ANTIBODIES  Figure 7.  \2  MONOCLONAL ANTIBODIES  IMMUNE R E S P O N S E is initiated (a) when an antigen molecule carrying several antigeninc determinants enters the body of an animal. The immune system responds: lines of B-lymphocytes proliferate, each secreting an immunoglobulin molecule that fits a single antigenic determinant (or part of it). Conventional antiserum contains a mixture of these antibodies. Monoclonal antibodies are derived by fusing lymphocytes from the mouse spleen with malignant myeloma cells (b). Individual hybrid cells are cloned, and each of the clones secretes a monoclonal antibody that specifically binds a single antigenic determinant on the antibody molecule (from ref 77).  29 production of large quantities of monoclonal antibodies that would specifically bind virtually any molecule  6 3  2.8 S C R E E N I N G O F H Y B R I D O M A S F O R H A P T E N BINDING  Following the hybridization of the mouse spleen cells using the hybridoma technology, the stable clones are screened for binding activity against the same hapten to which they were raised. Currently, because there is no general assay available for directly screening the library of hybridomas for catalytic activity, clones must be selected for further studies based on their ability to bind to hapten.  The assumption is made that strong hapten binding translates to  catalytic activity.  The two binding assays that will be discussed in this section are, first, the enzyme-linked  immunosorbent  assay (ELISA), and  second, the  particle  concentration fluorescence immuno-assay (PCFIA).  2.8.1 The E L I S A A s s a y  The ELISA assay is widely used to study hapten binding of catalytic antibodies. With this assay, the hapten molecule is coupled to a carrier protein different from the protein used for the immunization protocol.  K L H is often chosen as the  immunization carrier protein, while bovine serum albumin (BSA) is frequently  30 chosen as the conjugating protein for binding assays.  This ensures that  antibodies are selected for binding specifically to the hapten, rather than the  HAPTEN-PROTEIN CONJUGATE - Incubate onto 96-well microtiter plates; - Wash off unbound conjugate and block rest of the plate with albumin. Hapten-protein conjugate ^  96-well microtiter plate  Step 1  • Add mouse antibodies,  nr  • Incubate, then wash away unbound antibodies. Step 2  \si/—v&—^LV Add enzyme-linked \ / anti-mouse antibody, Incubate, then wash away unbound antibody.  Step 3  ~^&/—v^y— • Add substrate; • Incubate, then detect absorbance of released chromophore ( *).  Step 4  Figure 8.  -^afr—\Jy— ELISA assay for screening mouse antibodies (figure provided by Gay Yuyitung).  31 carrier protein.  In the ELISA assay (Figure 8), the BSA-hapten conjugate is  affinity bound to the wells of a polystyrene microtiter plate via nonspecific interactions, and the excess conjugate is washed away (Step 1).  Any free  binding sites in the polystyrene wells are blocked with the protein albumin.  In  the next step, the antibody-containing serum or hybridoma supernatant of interest is incubated with the bound hapten-protein conjugate, and unbound portions are washed away (Step 2). A n enzyme-linked antibody (or secondary antibody) that binds to immunoglobulin bound to the plate (i.e. binding to antibody having specific affinity for the hapten) is added, and after  an  appropriate incubation time unbound secondary antibody is then washed away (Step 3). A substrate is added to each well and it is subsequently processed by the enzyme coupled to the secondary antibody, producing a chromogenic product (*) that is detected and quantified spectroscopically (Step 4).  2.8.2 The P C F I A A s s a y  The PCFIA assay, commonly referred to as the Pandex binding assay, can be used in place of the traditional hybridomas.  ELISA assay for the initial screening of  Because this technique uses fluorescence detection, it is more  sensitive than ELISA.  The PCFIA assay (see Figure 9) uses hapten-protein  conjugate-coated carboxyl polystyrene particles in 96-well filter assay plates, and a goat anti-mouse fluorescein isothiocyanate (FITC) secondary antibody for detection.  The filter assay plates allow loading and incubating of buffered  32 solutions in the wells, followed by suction filtration and washing of excess reagents  through the  bottoms of the wells.  Once the  hapten-protein  conjugate-coated carboxyl polystyrene particles (hapten-beads) are made, these are added to the 96-well filter assay plates (Step 1).  The antibody-containing  serum or hybridoma supernatant of interest is incubated with the hapten-beads, and unbound immunoglobulins are washed away (Step 2). Next, the fluorescent labelled secondary antibody is added and binds to antibodies recognizing the hapten. The excess FITC labelled secondary antibody is washed away prior to reading the wells for a fluorescent signal, indicating specific hapten binding (Step 3).  Once hapten-specific hybridomas are selected based on the binding assay results, large quantities of monoclonal antibodies can be obtained by expanding the cells in tissue culture and harvesting the antibody secreted into the supernatant, or more commonly, the cells can be propagated in vivo in other mice to produce tumors which secrete a high concentration of antibody (ascites fluid). The characterization of binding and reaction kinetics of the monoclonal antibody with various substrates requires large quantities of antibody.  33 HAPTEN-PROTEIN CONJUGATE Link to PANDEX beads  Hapten-protein conjugate PANDEX bead  Incubate onto 96-well microtiter plates.  J  Step 1 niif  - A d d mouse antibodies, |  Y  - Incubate, then wash away unbound antibody.  Step 2  .7  \iiin  • Add anti-mouse antibody labelled with FITC • Incubate, then wash away unbound antibody; • Read fluoresence of FITC.  Step 3  Figure 9.  P C F I A assay for screening mouse antibodies (figure provided by Gay Yuyitung).  2.9 A S C I T E S P R O D U C T I O N  Ascites fluid is an intraperitoneal fluid that forms as a result of an intraperitoneal tumor produced by injection of a hybridoma cell line into the mouse. Hybridoma cells are injected intraperitoneal^ into a mouse. The cells grow to a high density  34 and thus produce a high titer solution of the monoclonal antibody, typically between 0.5 and 10.0 mg/mL  2.10  6 5  ENZYMATIC KINETICS AND RELEVANCE TO SYNTHESIS  Enzymatic reactions can be broken down into two components: substrate binding and catalysis. Most enzymes are highly selective in binding their substrates, and particularly selective in binding the transition state of the reaction they catalyze, which explains the catalytic specificity of enzymes. In 1902, Brown  78  proposed that the substrate dependence of enzyme-catalyzed reaction rates could best be described qualitatively by Equation 3 below.  In the absence of  inhibition, the free enzyme (E) first complexes with the substrate (S) to form one distinct enzyme-substrate complex (ES), with a rate constant kv  Once formed,  the E S complex, according to Equation 3, has two possible fates.  It can  dissociate to E and S, with a rate constant k.^ or it can be converted to the free enzyme and product (P), with a rate constant k . 2  E + S  -  ES  E + P  (3)  This qualitative picture of enzyme reactions was subsequently put into a mathematical framework first by Henri Menten  80  79  in 1903 and later by Michaelis and  in 1913. The basic rate equation derived below is commonly referred  to as the Michaelis-Menten equation, although, some writers have recently taken  35 to referring to the equation as the Henri-Michaelis-Menten equation.  For the  situation where the initial step in formation of the E S complex is not rate-limiting, a general expression that relates the rate of reaction (v) to the concentrations of substrate and enzyme and the rates of the individual steps can be derived, starting with the general rate expression, v =  5£! dr  k [ES].  =  (4)  2  From Equation 3, the net change in concentration of the E S complex with time (d[ES]/df) can be expressed as follows:  2^1 dr  = *i[E][S]-MES]-fc[ES].  Equation 3 can represent steady-state conditions.  <> 5  In a steady state, the  concentrations of intermediates remain constant while the concentrations of starting materials and products are changing. Hence, the rate of formation of E S must equal its rate of consumption over most of the course of the reaction; that is,  ^  -  °  <> 6  at According to Equation 5, therefore,  *1[E][S] = ( * - i + * ) [ E S ] . 2  By rearranging Equation 7,  (7)  36  [ E S ]  .  '  E  "  S  '  .  (8)  (/f-1+/C2)/^1  Equation 8 can be simplified by combining the rate constants into a new constant, Km, referred to as the Michaelis constant:  (k.+k ) 2  Km =  [  0)  1  *1  and substituting it into Equation 8, which then becomes  [ES] =  [E][S] -i-ii-iKm  (10)  Therefore, Km is a ratio of the rate constants for the breakdown of the E S complex, k.1 + k , over the rate constant for the formation of E S , k i . The 2  magnitude of Km is routinely used as a measure of the inverse relative affinity an enzyme has for a particular substrate.  T h e significance of Km will later be  discussed in more detail.  Experimentally, substrate concentrations are typically very large compared to the enzyme concentration (e.g. mM in substrate and nM in enzyme).  Under these  conditions, the concentration of uncombined substrate, [S], is essentially equal to the total substrate concentration. The concentration of uncombined enzyme, [E], is equal to the total enzyme concentration, [E ], minus the concentration of T  the E S complex.  37 [E] =  (11)  [E ] - [ES] T  By substituting this expression for [E] in Equation 10, we get  [ES] =  ([E ] - [ES])[S] T  1  (12)  •  Km Solving Equation 12 for [ES] now gives  [ES] =  [S]/Km [ET]  —  —  (13)  —  1 + [S]/Km or [ET][S]  (14)  •  [ES] =  [S] + Km On substituting this expression for [ES] into Equation 4, the initial rate of the reaction can then be expressed in terms of the experimentally measurable quantities [E ] and [S], as shown below in the Michaelis-Menten equation: T  *cat[E ][S] T  (15)  [S] + Km  where a rate constant kcat is commonly written as k and often referred to as the 2  catalytic constant of the enzyme, kcat is also known as the turnover number of the enzyme because it is the number of reaction processes (turnovers) that each enzyme molecule catalyzes per unit time.  38 The effect of substrate concentration on the rate of the enzyme-catalyzed reaction is shown in Figure 10. At low substrate concentrations or high Km's, where [ S ] « Km, the initial rate of the reaction is proportional to the substrate concentration, and Equation 15 simplifies to:  ^cat [ET][S] v  =  6 3 1 1  J l  1  •  (16)  Km Conversely, at saturating substrate concentrations, where [ S ] » [S]/([S]  + Km)  approaches  1), the initial  rate  Km (so that  of the reaction  becomes  independent of the substrate concentration, and approaches a constant maximum rate, V x.' ma  v =/c t[E ]= V ca  T  m a x  .  (17)  Therefore, the parameter Km represents the concentration of substrate at which the initial rate of the reaction is equal to one-half of the maximal rate, i.e. v =V  max  / 2 , as shown in Figure 10. In addition, from Equation 3, when k.1 »  k, 2  Equation 9 simplifies to give Km « k-i /ki, and the value of Km gives an inverse measure of the stability of the enzyme-substrate complex (ES). A n enzyme with a low Km has a high affinity for a substrate.  The Michaelis constant, Km, and the maximal rate, Vmax, can be readily determined from rates of reaction measured at different substrate concentrations for enzyme systems that operate under steady state conditions a s in Equation 3.  39  VnuxIS] V  r  = k  m  'ma*  •—  is  ' I U X  ~T  Substrate concentration [S] Figure 10.  The effect of substrate concentration enzyme-catalyzed reaction.  on the rate  of the  Traditionally, the values of Km and V x were obtained by transforming the ma  Michaelis-Menten equation, Equation 15, into a straight line plot by substituting in the expression kcat[E ] = V x, then taking the reciprocal of both sides to give: T  ma  1 v  V  max  Km  1  V,  (18)  [S]  max  A plot of 1/v versus 1/[S], called a Lineweaver-Burk plot, yields a straight line with an intercept of 1 A/ x and a slope of KmA/ x as shown in Figure 11. More ma  ma  commonly today, these values are determined by fitting the rate data to a weighted nonlinear regression of the Michaelis-Menten equation using a computer program, such as Grafit™.  40  1_ _  1  +  *m  X-intercepc  (Substrate concentration)  [S]"  Figure 11.  A double-reciprocal plot, called a Lineweaver-Burk plot, of enzyme kinetics: 1/v is plotted as a function of 1/[S].  The quantity  KJKn,  referred  often referred to as the enzyme's catalytic efficiency, is also  to as the enzyme's specificity  constant.  W h e n the substrate  concentration is much greater than Km, the rate of catalysis is equal to k (or kcat) 2  which equals the turnover number, a s described above in Equation 16. Thus, when [S] « Km, the enzymatic rate depends on the value of k /Km, or kca,/Km, and 2  on [S]. It should be noted that this ratio depends on ki, k i , and k shown by substituting for Km:  2l  as can be  41  neat  =  Km  « *1 2  <  K  Y  (  1  9  )  (*-i+* ) 2  Therefore, the ultimate limit on the value of  KaJKn  is set by  the rate of  formation of the E S complex. This rate of formation of the E S complex cannot be faster than the diffusion-controlled encounter of an enzyme and its substrate. Hence, the upper limit on k^/Km is between 10 diffusion-controlled enzymes.  8  and 1 0  9  M"  1  s' , the 1  limit for small molecules with macromolecules such as  81  While knowledge of the kinetic constants for the enzymatic reaction used in a synthetic  organic  chemistry  application  is  not  required,  obtaining  and  understanding the kinetic constants enables maximum efficiency of the use of the enzyme. For instance, if the substrate concentration is too low, a certain fraction of enzyme active sites are vacant at equilibrium, whereas, in some cases, if substrate concentrations are too high, the enzyme may be deactivated (as the character of the solvent becomes markedly different from that of water). The Michaelis constant Km is therefore valuable in determining the minimum concentration of substrate which must be maintained in solution to achieve efficient use of the enzyme.  42 Without kinetic analyses of the enzyme system employed, often the catalytic activity of the enzyme observed under synthetic conditions is lower than the value obtainable under ideal (assay or Vmax) conditions.  This discrepancy in  rates may have several origins, a few being nonspecific deactivation of the enzyme by high or inappropriate concentrations of substrates, products, buffers, ions (pH), or co-solvents.  2.11  ENZYMES AND CATALYTIC ANTIBODIES IN ORGANIC SOLVENTS  Water is a poor solvent for the majority of reactions in organic chemistry because most organic compounds are insoluble in this medium. side-reactions  such  as  hydrolysis,  polymerization,  decomposition are common in the presence of water.  In addition,  racemization  and  These limitations were  avoided long ago by the use of organic solvents for nearly all organic chemical reactions. Conversely, biocatalysts have mainly been used in aqueous solutions since they generally display their highest catalytic power in water, and may be destroyed in organic solvents.  However, this commonly held perception is far  too general, and has likely impeded investigations into the utility of enzymes in nonaqueous media in the past. Review articles are becoming available dealing with the developing field of enzyme-catalyzed reactions in organic solvents.  82  While the optimal amount of exogenous water to be added to the organic solvent  43 is enzyme dependent, there is a general consensus that some water is required for the catalytic function of the enzyme.  83  Although activities are generally lower in an organic environment, many other advantages can be accrued by using biocatalysts in organic solvents.  Some  advantages of biocatalysts over chemical catalysts include: enzymes are very efficient catalysts, typically employed in a mole fraction of 10' -10" % whereas 3  4  most chemical catalysts are generally used in a mole fraction of 0.1-1%; enzymes are environmentally acceptable unlike heavy metals often employed in chemical catalysts; enzymes act under mild conditions; enzymes can catalyze a broad range of chemical transformations; and, enzymes often exhibit a high substrate tolerance by accepting a variety of unnatural substances and often they are not limited to a purely aqueous environment.  Both enzymes and catalytic antibodies have been successfully used in organic solvents under antibody  8691  a variety  of conditions,  including: (i) the  enzyme  84,85  or  dissolved in a monophasic aqueous-organic solution consisting  largely of water and a water-miscible organic co-solvent (such as dimethyl sulfoxide, dimethyl formamide, acetonitrile) or surfactant (such as Triton X-100) to facilitate substrate solubility; (ii) the e n z y m e  92,93  or antibody  94  dissolved in a  biphasic aqueous-organic solution consisting of two macroscopic phases; (iii) the enzyme  95,96  or antibody  97  suspended in a monophasic organic solution (with < 2%  44 water content); (iv) the e n z y m e " 98  103  or antibody " 104  106  immobilized onto solid  supports or in reverse micelles; and, (v) at least one example of a lipid-coated antibody in water-miscible organic solvents.  107  Such successful demonstrations of biocatalsyts in organic solvents continue to increase, providing organic chemists with alternate and often unique solutions to challenging synthetic transformations.  2.12 D E S I G N O F TRANSITION S T A T E A N A L O G U E S  The approaches used to generate catalytic antibodies have been based largely on principles of enzymatic catalysis such as transition state stabilization,  108  general acid-base catalysis, nucleophilic and electrophilic catalysis, as well as strain and proximity effects.  61,109  Clearly, the success of catalytic antibodies has  and will continue to depend on the clever design of haptens or transition state analogues.  This is the fundamental requirement when one sets out to study  catalytic antibodies.  A number of functional groups, some examples being  sulphones, phosphonates and phosphonamidates, quaternary ammonium ions, 1,3-diketones and secondary alcohols, have proved successful in mimicking the reactive centers in a transition state.  Phosphonates have been most widely  used, undoubtedly due to their proven utility as transition state inhibitors  108  for a  variety of enzymes including a number of proteases and esterases. The dipole  45 of the P=0 bond of phosphonate analogues reflects the developing negative charge on the carbonyl oxygen in the transition state of transesterification reactions (figure 12).  In addition, antibodies complementary to the tetrahedral  phosphonate haptens should have considerably lower affinity for the trigonal products, thereby reducing product inhibition. The efficiency of enzymes is the result of employing several of the above mentioned catalytic mechanisms simultaneously, to achieve large rate enhancements. In order to realize similar rate accelerations with antibodies, it may require the simultaneous application of more than one of these strategies for introducing catalytic activity into antibodies as well. There have been a few reports of hydrolytic antibodies that appear to utilize active site acid-base chemistry in addition to the expected transition state stabilization. ' ' ' 86  87  110  111  The great diversity offered by the immune system, coupled with well designed transition state analogues, has allowed chemists to exploit the immune system to study the antibody catalysis of a number of diverse reactions.  2.13 R E A C T I O N T Y P E S C A T A L Y Z E D B Y ANTIBODIES  The ability of enzymes to use binding energies to orient a substrate into a reactive conformation or to bring two molecules together in the proper orientation  46  Putative Transition State  R OH 2  0  Ri—P—OR  2  1  OR Transition State Analogue 3  Figure 12.  The putative transition state and the corresponding phosphonate transition state analogue for the displacement reaction of an ester.  for a reaction to occur represents an effective way to compensate for the entropy losses that may accompany such a chemical transformation.  Mechanistic  investigations of antibody-catalyzed reactions involving structural modifications to substrates and X-ray crystal structure studies have shown that antibodies utilize binding energies analogously to enzymes to effect chemical catalysis.  While all antibodies share a common structural motif, the shape of the binding site can vary extensively, from deep pockets, clefts and grooves to flatter surfaces, complimenting the antigen against which it was raised. This versatility, essential for the ability of these antibodies to maintain surveillance against foreign molecules in vivo, also accounts for the ability of these antibodies to distinguish subtle changes in substrate molecules, and is responsible for their  47 ability to catalyze different types of reactions. The field of catalytic antibodies has been extensively reviewed elsewhere, and since the first successful reports, has flourished  to  encompass an impressive array  of  antibody-catalyzed  reactions. Table 3 below outlines many of these antibody-catalyzed reactions.  Table 3.  Reactions Catalyzed by Antibodies. Reaction T y p e  Ester, carbonate hydrolysis  Reference 56,57  Lactonization of y,5-unsaturated acids  58  Enantioselective lactonization  59  Amide bond formation/hydrolysis  112-114  Redox  115,116  p-Elimination  117  Porphyrin-mediated metal chelation  118  Protecting group removal  119,120  Transesterification  86  Enantiofacial protonation  121  Cis-trans isomerization of alkenes  122  Glycosidic bond hydrolysis  123  Stereoelectronically disfavoured cyclization  88  Diels-Alder reactions  124-126  Regio- and enantioselective reduction  127  Michael addition  128  Aldol condensation  129-131  48 Two successful antibody-catalyzed reactions listed above, and a third attempt, not in the above list, are of particular relevance to this project and will be discussed in detail. They are: (1) Lactonization of y,8-unsaturated acids; (2) Enantioselective lactonization; (3) Intramolecular acyl migration.  2.13.1 Lactonization of v,S-unsaturated a c i d s  In 1995 Kitazume and Takeda described the use of antibodies to catalyze the cyclizatibn of unsaturated carboxylic acids to 5- and 6-membered ring lactones.  58  The authors employed hapten-conjugates 3 and 4, cyclic ethers bearing a sulfoxide group, to elicit antibodies to catalyze the  lactonization  of  the  y,8-unsaturated acid 5. Two diastereomers of the KLH-hapten conjugate 3 (the cis and trans  isomers; referring  to the  relative  stereochemistry  of  the  trifluoromethyl and sulfoxide containing side chains) were used to immunize BALB/c mice.  The antibodies induced from hapten 3, derived from the trans-y-lactone, acted to promote exclusively exocyclic ring closure of the y.S-unsaturated acid 5 to the trans^-lactone 6 with a kcat of 0.86 m i n , a Km of 190 LIM, and a selectivity of 1  > 97% de (58% yield). The antibodies raised against the cis isomer of hapten 3  49  o  catalyzed the formation of the cis^y-lactone isomer of 6, from reaction with the same substrate 5, with a keat of 0.64 min" , a Km of 170 u.M, and a selectivity of 1  > 96% de (61% yield).  In addition, antibodies were elicited to the trans  KLH-hapten conjugate 4, and were found to promote cyclization of 5 to form the 5-lactone 7 with a kcat of 0.81 min' , a Km of 180 LIM, and a selectivity of > 94% ee 1  (5-lactone 7 : y-lactone 6 = 93:7), and the antibody induced from the cis isomer of 4 acted to form the 8-lactone 7 (5-lactone : y-lactone = 94:6) with a comparable enantioselectivity.  2.13.2 Enantioselective lactonization  In 1987, Napper, et al. reported the enantioselective lactonization of the corresponding racemic 5-hydroxy ester 8 to lactone 9 by a catalytic monoclonal antibody.  59  Antibodies were raised against the cyclic phosphonate hapten 10.  50 This hapten is a transition state mimic for the six-membered ring cyclization. Lactonization of 8 was catalyzed until 50% of the initially added substrate was consumed. Upon addition of a second aliquot of substrate, the antibody again converted 50% of the freshly added substrate. substrate were  racemic, the  monoclonal  selectivity for the (R)-(-)-enantiomer of 8.  Although the hapten and the  antibody,  being  chiral,  showed  The monoclonal antibody (MAB)  displayed enzyme-like Michaelis-Menten kinetics with a k^t of 0.5 min , a Km of -1  76 uM and a kcaAuncat of 167, which afforded the 6-membered ring lactone 9 in an enantiomeric excess of 94 percent.  8  9  2.13.3 Intramolecular acyl migration  Antibodies were raised against the cyclic phosphonate 13 in an attempt to obtain antibodies capable of catalyzing an intramolecular acyl migration reaction.  132  51 Antibodies specific for phosphonate 13 were expected to bind substrate 11 with the hydroxyl group in close proximity to the carbonate, resulting in a rapid acyl migration to give 12. Monoclonal antibodies raised against hapten 13 did not catalyze the desired acyl migration of 11 to 12, but simply hydrolyzed the substrate, giving  14, C 0 , and R O H . 2  The author  speculated that the  hapten-protein conjugate 15 may have hydrolyzed during the course of the  13  immunization, yielding 16, a hapten now resembling the phosphonic acid transition  state analogues employed to obtain antibodies which catalyze  hydrolytic reactions.  52  If hydrolysis of the hapten did occur during the course of  immunization,  endocyclic cleavage would have had to occur (as illustrated in path a above) for the hapten to remain intact with the carrier protein, and lead to the production of  53 hydrolytic antibodies.  Exocyclic cleavage would have resulted in the hapten  molecule being cleaved from the carrier protein, and such small molecules alone usually do not induce an immune response.  The antibody-catalyzed lactonization by Napper, et  al.,  mentioned above,  provided the impetus for our investigation of catalytic antibodies in the synthesis of macrocyclic lactones. A s outlined in Chapter 1, investigators have searched for a general high-yielding method to prepare these macrocyclic compounds. A n antibody capable of catalyzing such a macrolactonization step, with high enantioselectivity, would be a welcome and interesting achievement in the field of macrolide chemistry.  54 C H A P T E R III.  3.1  R E S U L T S A N D DISCUSSION  S Y N T H E S I S O F M A C R O C Y C L I C P H O S P H O N A T E 20  Based on the success by Napper et a l . ,  59  our initial goal towards the generation  of a catalytic antibody for macrocyclic lactonization was the synthesis of the macrocyclic phosphonate hapten 20.  Phosphonate 20 is a stable analogue of  the putative transition state for the lactonization of hydroxy ester 18  to  macrolactone 19.  20  The synthesis of compound 20 was intended as an introductory study for the investigation  of the  phosphonates.  stability  and  spectral characteristics of  macrocyclic  Earlier attempts in our laboratory toward the synthesis of the  55 macrocyclic methyl phosphonate 22 from 21 via a direct displacement reaction (Scheme 2) carried out under high dilution conditions resulted in low and irreproducible yields of the desired product.  133  To our knowledge, this was the  first synthesis of such a macrocyclic phosphonate.  However, this is not a  practical method to prepare large quantities of these compounds, therefore we turned our attention to an alternate approach.  S c h e m e 2.  Synthesis of macrocylic phosphonate 22.  In this alternate approach, the final steps in the synthesis of phosphonate 20 were envisioned to proceed via an intramolecular Wittig reaction using the acyclic precursor 23, followed by hydrogenation of the vinyl phosphonate.  56  Carbon-carbon bond formation can be carried out under near-neutral conditions using a stabilized Wittig reagent. This methodology was successfully used by Wilson  134  to yield the 12-membered ring a,p-unsaturated ketone 25 from 24.  o  24  o  25 (20-30%)  Other r e p o r t s  135,136  demonstrate intramolecular cyclization reactions of aldehyde  phosphonates for the construction of macrocyclic a,p-unsaturated lactones of 13 and more carbon atoms with varying degrees of substitution. The 14-membered a,p-unsaturated lactone 27 was obtained by the intramolecular condensation of the phosphonate 26 in a 40-50% yield.  Such examples from the literature  suggested that our strategy for the synthesis of compound 20 was reasonable.  57  o  o  o  27  26  Before embarking on a synthesis of the cyclic phosphonate 20, however, we decided to carry out a model study using phosphonate 30.  Direct displacement  of the iodide of compound 28 with triphenylphosphine in an attempt to produce intermediate 29 was not successful. A report by Moffatt and coworkers  O  (Ph) P  II  .P(OEt)  137  3  2  28  detailed the synthesis of a diphenylphosphonate analogue of the Wittig reagent 30,  and in the same report, discussed the difficulties associated with the  displacement route from 28 to 29.  Using the Moffatt method, the stabilized  Wittig reagent 32 was synthesized in good yield.  Commercially available  chloromethylphosphonic dichloride was reacted with 2 equivalents of phenol to produce 31 in 90% yield.  Subsequently, quaternization of triphenylphosphine  with 31 at 175 °C and deprotonation lead to 32 in 77% yield.  58  2PhOH  0  Cl\  J>Ck  0  9  1.Ph P 3  —  C l ^ ^P(OPh)2  E t a N THF,O C 0  3 1  A  >•  P h 3 P ^ . P (OPh)  2.NaOH THF-H2O  3  2  2  Aldehyde 33 was also prepared (Scheme 6) for subsequent conversion to compound 34, which would be followed by coupling with 32 to produce the stabilized Wittig 35 as in Scheme 3 below. However, with 33 and 32 in hand, a Wittig coupling of these two compounds was carried out, and the  vinyl  phosphonates 36a and 36b were produced in a moderate yield of 5 2 % with a  35 S c h e m e 3.  Proposed synthetic sequence for compound 35.  4:1 ratio of trans to cis isomers, respectively, based on the integration of the 400 MHz H N M R of the crude mixture. 1  59  OTHP  O  36a + 36b  This synthesis of 36 inspired an alternate route towards the synthesis of macrocyclic phosphonate 20. With compound 36 in hand, all that remained was hydrogenation of the double bond followed by cleavage of the T H P ether giving rise to 37, and finally, a viable means of condensing the phosphonate moiety with the secondary hydroxyl of 37 to give 38.  36  37  38  60 Furthermore, we anticipated that the intended model compound 38 could be further modified at the phosphonate center with a linker molecule to enable  39  coupling to a carrier protein, and the resulting conjugate 39 might be used as an antigen for the production of a lactonizing catalytic antibody.  One concern we  had regarding the use of such a conjugate was the hydrolysis problem encounter by Jacobs with a similar 7-membered ring phosphonate mentioned in Chapter 1.  132  However, we felt our 14-membered ring hapten would not undergo  hydrolysis as readily as the 7-membered ring phosphonate employed by Jacobs, since 14-membered ring macrocycles in general possess less ring strain than their 7-membered ring counterparts.  To achieve cyclization of the hydroxy phosphonate ester 37 to give 38, we turned our attention to the Mitsunobu reaction. Over the past two decades, the Mitsunobu reaction has found extensive use in organic synthesis, particularly for the inversion of alcohols via an esterification/hydrolysis procedure.  42  The  61 Mitsunobu reaction has also been proven a mild coupling reaction for the preparation of mixed phosphonates from phosphonic acid monoesters and primary or secondary alcohols (Equation 3 ) .  O R-P-OH OR'  acids,  phenols,  P h P , DEAD 3  R"OH, THF  phosphates,  and  138  It has been used with carboxylic  ° R-P-OR" OR'  phosphonic  acids  (3)  as  138  the  nucleophilic  component. Thus we hoped the Mitsunobu reaction would prove successful in the transformation of the monophosphonic acid of 37 into the cyclic phosphonate 38. The precedent by Smith et al. in employing the Mitsunobu protocol in the key lactone forming step in the total synthesis of the latrunculins (14-membered ring lactone compounds; Equation 2 )  40  suggested that our strategy  was  reasonable. A proposed mechanism for the Mitsunobu reaction of benzoic acid and a secondary a l c o h o l  139  is shown in Scheme 4 below.  While a variety of acids have been employed in the Mitsunobu reaction, benzoic acid has been the most widely used. Subsequently, it has been found that the strength of the acid used has an effect on the relative reactivity of the phosphonium adduct. Thus, for a successful Mitsunobu esterification to occur, the alcohol must react faster (path A) than the carboxylate (path B) with the phosphonium adduct as shown in Scheme 5. If the conjugate acid of the  62  H  PhCOO"  I  R0 C—N=N-C0 R 2  2  +  P  P  n  2  PhCOOH  3  R0 C—N—N—C0 R P P h 3 Phosphonium adduct 2  +  OH R  0=PPh  if"*  'To  3  j^fat  PhCOO"  OCPh  S c h e m e 4.  •2 *  A proposed mechanism for the Mitsunobu reaction (from ref 139).  carboxylate is a relatively strong acid, then the carboxylate will be stable and a poor nucleophile, and path A is favoured.  If, however, the conjugate acid is a  weak acid, its conjugate base may be sufficiently nucleophilic to compete with the alcohol, resulting in path B being favoured and thus lowering the yield of the desired product. H  Path A  I  R0 C—N—N—C0 R 2  (OPPh  3  2  +PPh3  R /Vv'"H 1 kR  Phosphonium  OOCR  R  y OH  adduct R  1  R  2  + R  3  S c h e m e 5.  3  2  0=PPh  OOCR3 Possible pathways in the Mitsunobu reaction (from ref 139).  3  63 With the new synthetic strategy involving an intramolecular Mitsunobu reaction decided, the synthesis of phosphonate 38 (Scheme 6) was attempted, beginning with the Grignard reaction of commercially available undecylenic aldehyde and methylmagnesium bromide to give the hydroxy alkene 40 in 9 0 % yield.  The  hydroxyl group of 40 was protected as the T H P ether 41 in 84% yield.  This  addition of the T H P group created a second chiral centre in 41, and therefore, two diastereomers of 41 were isolated. The two diastereomers were obtained in a 1:1 ratio as seen in the H N M R spectrum of 41, which reveals two methyl 1  doublets of equal intensity and two methine multiplets, corresponding to the methine proton of the T H P group, also of equal intensity. The two diastereomers of 41 were not separated, but treated together in the following sequences. Hydroboration of 41 followed by oxidation gave 42 in 96% yield. The alcohol 42 was subsequently oxidized under the Swern conditions to give the aldehyde 33 in 87% yield. Aldehyde 33 was then reacted with the stabilized Wittig reagent 32 in refluxing toluene to give the vinyl phosphonate 36 in a moderate yield of 52% with a 4:1 ratio of trans to cis isomers based on the integration of the 400 MHz H N M R of the crude mixture. The alkene 36 was then reduced using H 1  2  and 10% P d / C in EtOAc to give the saturated diphenyl phosphonate 43 in 99% yield. Phosphonate 43 was subsequently reacted with p-toluenesulphonic acid to liberate the secondary hydroxyl of compound 37 in 86% yield. Removal of the T H P group from 43 eliminated one of the two chiral centres in the molecule, reflected in the H N M R spectrum of 37 which contains only one methyl doublet. 1  64  The hydrolysis of a single "OR" group of a symmetric phosphonate such as 37 is readily achieved under refluxing aqueous sodium or potassium hydroxide, while the hydrolysis of both "OR" groups can be achieved in refluxing hydrochloric acid.  140  Therefore, phosphonic acid monoester 44 was produced in 76% yield by  the base hydrolysis of 37.  The key step in the synthesis of the macrocyclic hapten phosphonate 38 was the cyclization of 44 to give the 14-membered ring phosphonate 38.  The hydroxy  phosphonic acid monoester 44 was reacted with D E A D and triphenylphosphine in benzene under high dilution conditions to produce the cyclic phosphonates 38a and 38b in an 8 2 % yield and a 5:1 ratio of diastereomers based on the H 1  NMR of the  crude.  For each pair of  14-membered  ring  phosphonate  diastereomers referred to in this thesis, the distinction between isomer 'a' and isomer 'b' of a pair of diastereomers was made based on their relative R v a l u e s r  from thin-layer chromatography (TLC) performed on Merck silica gel  F 54 2  pre-coated aluminium sheets. With each pair of 14-membered ring phosphonate diastereomers studied, the 'a' isomer has a higher R value (i.e. travelled faster f  on the T L C plate) than its corresponding 'b' diastereomer.  65 1)MeMgBr, THF Ooc XHO 2)DHP, CH Cfe 2  p-TsOH, rt  40; R = H (90%) 41; R = THP(84%) 1a) B H - T H F , - 7 8 ° C b) NaOH, H 0 , 3  2  2  2) DMSO, (COCI) , Et N, CHsCb, -78 oc 2  3  OTHP  O Ph3P*^(OPh)  2  32  42; R = CH OH(96%) 33; R = CHO (87%) 2  C6H5CH3, A  OTHP (PhO)  2  36a + 36b (52%; 4:1) 1) H , Pd/C, EtOH 2  2) p-TsOH, MeOH 3) 3N KOH - THF, A  43; R-, = (PhO) (0)P R = OTHP (99%) 37; R1 = (PhO)2(0)P R2 = OH (86%) 44; R, = (PhO)(HO)(0)P R = OH (76%) 2  2  2  PhsP, DEAD, PhH  —OPh  \  38a + 38b (82%; 5:1)  S c h e m e 6.  Synthesis of macrocyclic phosphonates 38a and 38b.  66 In addition to the two 14-membered ring diastereomers 38a and 38b, a cyclic dimer side product 39 was isolated as rod-shaped crystals on purification of 38a  Figure 13.  Stereoview of an O R T E P representation of dimeric cyclic phosphonate, 39, showing 3 3 % probability ellipsoids. Hydrogen atoms have been given arbitrary thermal parameters for clarity.  and 38b.  The structure of dimer 39 was determined by single-crystal X-ray  diffraction  (Figure  13).  Dimer 39, a centrosymmetric 28-membered  ring  67 compound with two parallel chains bridged at both ends by the phosphonate group, contains four stereogenic centres with R*R* stereochemistry on one bridge, and the mirror image S * S * stereochemistry at the opposite bridge. Dale has reported that cycloalkanes above twenty carbons have the collapsed shape of two parallel straight chains bridged at both e n d s  141  as in dimer 39.  This  collapsed shape is likely a result of a sufficiently large interior "hole" which allows for stabilizing hydrophobic interactions across the ring.  Groth reported  the X-ray structure of cyclohexaeicosane, which was also found to have a centre of symmetry.  142  Dimer 39 has a centre of symmetry as a result of the R*R* and  S * S * stereochemistry at the bridge ends.  In an attempt to explain why dimer 39 has the R*R* relative stereochemistry, rather than S*R*, molecular models of the transition states leading to the R*R* and S*R* products were examined.  The transition state leading to the R*R*  stereochemistry, illustrated by A below, proceeds with lower steric interactions than transition state B which would result in the S * R * stereochemistry.  In B  there is a steric interaction between the methyl group on the secondary alcohol and the phosphonate oxygen that is not found in model A.  If the A transition  state leading to the R*R* stereochemistry is extended to the monomers 38a and 38b, it is reasonable to suggest that the major monomer 38a might also exhibit the R*R* relative stereochemistry.  68  s-  ^o—- .~- . _ — Q P P h s r  PhO  M  sPhO R*R*  s-  .„o—PhO  --OPPha  M  PhO B  S*R*  M = cation or solvent molecule +  The bimolecular condensation of two molecules of 44  via the  Mitsunobu  methodology resulted in the R*R* stereochemistry, and it follows that closure of the opposite end of the acyclic dimer would be governed by the same steric control, resulting in either the S S or R R stereochemistry for the second phosphonate bridge.  Then the question to answer is why the only dimer  molecule isolated on cyclization of 44 was the R * R * S * S * dimer 39?  Molecular  models of the R * R * S * S * and R * R * R * R * diastereomers of dimer 39 constructed.  were  The R * R * S * S * model was constructed according to the X-ray  structure obtained for dimer 39.  The R * R * R * R * model was constructed from the  same conformation as 39, where the second R*R* bridge was created by  69 inverting the stereochemistry at the S * S * bridge. This R * R * R * R * model retained one R*R* bridge common to 39, where the aryloxy moiety attached to the phosphorus atom occupies an equatorial position, projecting away from the ring while the doubly-bonded oxygen atom projects into the ring (as in Figure 13). However, the R*R* stereochemistry at the opposite bridge of the R * R * R * R * model forces the methyl and aryloxy moieties to project in toward the ring, as illustrated  below.  MACROMODEL  calculations  R * R * R * R * dimers using the M M 2 force f i e l d  144  143  on the  R*R*S*S*  and  gave a lower energy conformation  for the R * R * S * S * dimer, 92.40 kJ/mol, compared to 109.23 kJ/mol for the R * R * R * R * dimer. These calculations suggest that the R * R * S * S * dimer is of lower energy, and may provide a rationale for why only this dimer was isolated on cyclization of compound 44. CH  3  H R*R*  R*R*  In the H N M R spectrum of the macrocyclic phosphonates, we observed a 1  downfield shift of the C-13 methine signal relative to that of the open-chain  70 precursor 44. In the synthesis of simple macrocyclic lactones, we also used the downfield shift of this methine proton in the cyclized product relative to the methine signal of the open chain hydroxy acid precursor to establish whether cyclization has taken place. Similarly, for the cyclic phosphonates, the electron withdrawing nature of the phosphonate moiety deshields the methine proton in the cyclic compound relative to the open chain precursor (Table 4), giving rise to the observed downfield shift.  In addition, the C-1 methyl signal of the cyclic  phosphonates 38a and 38b shifts downfield in the H N M R spectrum as is 1  observed for the 13-tetradecanolide lactones.  Table 4.  Comparison of the H N M R chemical shift data for the methine and methyl signals of the open chain hydroxy acid 44 with those of the macrocyclic phosphonates 38a and 38b. 1  Compound  Methine multiplet (ppm)  Methyl doublet (ppm)  44  3.78  1.17  38a  4.75  1.22  38b  4.65  1.43  3.2 A N T I B O D Y P R O D U C T I O N  3.2.1 Preparation of Antigen K L H - 5 0  A s stated earlier in Section 2.3, small hapten molecules must be coupled to a carrier protein prior to immunization, in order to elicit an immune response. This  71 coupling is achieved by means of a spacer molecule. A 5-carbon unit as linker has been shown to be the optimal length for a spacer, or linker, in a number of studies. " 73  76  For this purpose, the linker molecule 48 was synthesized in a 35%  yield by reaction of 5-amino-5-pentanol with benzyloxy chloride (Cbz-CI).  9  1) a q N a C 0 , 0 ° C 2  HO. ^ \ ^ x ^ N H  3  »•  2  HO^^-s^^v^NHCOCI-feCeHs  2) C6H5CH2OCOCI 48  Of the two cyclic phenyl phosphonate diastereomers, 38a and 38b, only the minor diastereomer 38b was coupled to a carrier protein (KLH) for immunization in mice for antibody production. Displacement of the phenoxy moiety of 38b by the linker molecule 48 produced 49a in a 59% yield. Finally, hydrogenolysis of 49a in ethanol with hydrogen and 10% Pd/C afforded 50 in 8 5 % yield.  38b  49a; R = Cbz(59%) 5 0 ; R = H(85%)  72 Amine 50 was coupled to carboxy residues of the carrier protein keyhole limpet hemocyanine  (KLH)  using  the  water  1-(3-dimethylaminopropyl)-3-ethylcarbodiimide  soluble  coupling  hydrochloride  (EDC).  reagent The  coupling takes place between the amino group of 50 and carboxyl residues on the carrier protein.  An attempt to generate the diastereomeric hapten of 49a, namely 49b, from phosphonate 38a using the same reaction scale and conditions that produced 49a from 38b was attempted.  However, isolation of the expected product by  flash chromatography was not successful due to a poor yield.  A subsequent  attempt to isolate and characterize 49b from the reaction of 38a with 48 after doubling the scale of the reaction resulted in a 14% yield of 49b.  Nucleophilic displacement reactions at phosphorus have been investigated.  145-147  Green and Hudson carried out a series of nucleophilic displacement reactions on phosphorus.  148  The stereochemical outcomes of these  displacement  73 reactions have been interpreted as proceeding with predominant inversion of the configuration at the phosphorus atom. Wadsworth reports mixed stereochemical results upon displacement at a phosphorus center, and the effect of added salt on the  stereochemistry  of  nucleophilic  phosphate esters and their analogues.  displacements  at  phosphorus  in  149  The nucleophilic reactions involving displacement of the phenoxy moieties of cyclic phosphonates 38a and 38b with the linker molecule 48 are believed to have undergone  inversion at the  phosphorus centers.  In both of  the  experiments, displacement of the phenoxy groups of compounds 38a and 38b with linker 48 produced clean diastereomeric products.  In addition, the faster  moving 'a' isomer of 38 (by T L C ) on reaction with 48 generated the slower moving isomer, 49b, while the reaction of 38b with 48 generated exclusively the faster moving isomer, 49a.  These results are suggestive of inversion at the  phosphorus centers of 38a and 38b on displacement with 48. Further evidence to support inversion at the phosphorus centers of 38a and 38b on displacement with 48 is found in the analysis of the N M R data of these compounds as outlined below in Section 3.7.  74 3.2.2 Antibody Raised Against Antigen KLH-50  Balb/c mice ( 6-8 week old females) were immunized with the KLH-50 conjugate. After the last injection, the mice were sacrificed and the spleens harvested. The mortal antibody-secreting spleen cells were fused with immortal myeloma cells to afford immortal antibody-producing hybrids (hybridomas), and these were plated into  96-well  culture  plates  in  HAT selection  hypoxanthine, aminopterine and thymidine). selected as follows:  media  (media  containing  Spleen-myeloma hybrids are  the fusion partners (myelomas; those used in this  experimental are P3-X63-Ag8-653, or clone 653) are deficient in the enzyme hypoxanthine quanine phosphoribosyl transferase ( H G P R T - or HPRT-) and cannot utilize exogeneous hypoxanthine in the synthesis of purine nucleotides. The presence of aminopterine, a dihydrofolate inhibitor of dihydrofolate reductase (K < 10" M ) , 9  of nucleotides.  62  analogue and  competitive  blocks the de novo synthesis  Thus in the presence of HAT selection media, unfused  myelomas do not survive. Since spleen cells do not grow well in tissue culture, they die in about a week. Hybridomas are H G P R T + (donated by the spleen cell) and can utilize exogeneous hypoxanthine and thymidine in their salvage pathway and survive. Cell lines obtained in this fashion can be maintained in continuous culture or frozen in liquid nitrogen indefinitely.  75 3.3 BINDING A S S A Y S  The particle concentration fluorescence immunoassay (PCFIA) technique  150  was  used in place of the traditional enzyme-linked immunosorbant assay (ELISA) for the initial screening of hybridomas. The P C F I A assays were carried out using hapten-protein conjugate-coated carboxyl polystyrene particles, 96 well filter assay plates, a goat antimouse fluorescein isothiocyanate (FITC) secondary antibody for detection at 385 nm wavelength, and a P C F I A buffer (2% fedal calf serum (FCS), 0.2% sodium azide, and 1x P B S , filter-sterilized through a 0.22 urn filter).  A typical P C F I A assay involved the screening of supernatants from the hybridoma cells for hapten affinity using BSA-50 conjugate-coated carboxyl polystyrene particles as a solid phase. The BSA-50 conjugate was produced under the same conditions as the KLH-50 conjugate, then the BSA-50 conjugate was coupled through amino residues on the B S A with the carboxylate of the carboxy polystyrene  particles using E D C as in Scheme 7.  Once  supernatants from the hybridoma cells have incubated with the  the  BSA-50  conjugate-coated carboxyl polystyrene particles, a FITC secondary antibody was used for detection (at 385 nm wavelength) of hapten-specific antibody binding. Positives from these screenings were subcloned by further limiting dilution onto 96-well microtiter plates to ensure we had stable monoclonal cell lines.  A  76 second P C F I A binding assay was then carried out to verify that the hybridomas were still secreting anti-hapten antibodies.  BSA-50  S c h e m e 7.  EDC PBS, pH 7.4  Coupling of the BSA-50 conjugate with the carboxy polystyrene particles (PSP).  3.4 A N T I B O D Y F123 ISOTYPING  Using a standard isotyping assay, our monoclonal antibody F123 was found to be an IgM class antibody. IgM class antibodies, as described in Chapter 1, are expressed in the primary immune response.  Typical immunization protocols, such as the one we followed, include a large initial dose (challenge) of antigen, followed by booster injections containing a lower dose of antigen. Our protocol called for 100 Lig of the immunoconjugate KLH-50 per mouse on immunization, and 50 |ig per mouse for each booster injection.  The time delay between the administration of the immunoconjugate  into the mouse and the triggering of the primary response is not clear, nor is it clear at which stage of B cell differentiation, from stem cell to mature B cell,  77 commitment to an ultimate antibody specificity occurs. Further refinement to the antibody specificity, by such mechanisms as affinity maturation and class switching (evolution, for example, from an IgM to an IgG class antibody), occurs under conditions in which the immune system is continuously challenged with the immuno-conjugate. A sufficiently stable hapten-protein conjugate is required to remain intact in vivo between booster injections to provide a constant challenge to the immune system of the mouse. One might speculate that if a sufficiently large immunizing dose of the KLH-50 conjugate was administered causing an initial immune response, while the amount of antigen in subsequent booster injections was not sufficiently large to allow sufficient lifetime of the conjugate for further refinement mechanisms to occur, fusion of these naive spleenocytes with immortal cells would result in IgM secreting hybridomas. Success in obtaining a lactone-forming catalytic antibody depended on the lifetime of the phosphonate immuno-conjugate, KLH-50, in the mouse. If KLH-50 was susceptible to hydrolysis in the mouse, exocyclic (path a) rather than endocyclic (path b) cleavage (Scheme 8) should prevail since the 14-membered ring does not suffer from the ring strain in the seven membered phosphonate.  In addition, endocyclic cleavage would result in the  ring intact  conjugate KLH-51, a hapten now resembling the phosphonic acid transition state analogues employed to obtain antibodies which catalyze hydrolytic reactions. However, as will be described later, we obtained a lactone-forming IgM antibody, one which was not found to have hydrolytic activity.  78  S c h e m e 8.  Exocyclic (path a) versus endocyclic (path immuno-conjugate KLH-50 in mouse serum.  b)  hydrolysis  of  3.5 A S C I T E S P R O D U C T I O N  Monoclonal antibodies selected for kinetic analysis, based on the binding assay results, were prepared by injecting a million hybridoma cells into pristane-primed Balb/c mice (6-8 week old females). The resulting ascites fluid was collected, and monoclonal antibodies were purified from ascites fluid by sheep-antimouse (SAM) affinity chromatography.  The antibody samples were dialyzed into P B S  buffer (8.2 mM N a H P 0 , 137 mM NaCI, 2.7 mM KCI, 1.5 mM H P 0 , pH 7.4) for 4  2  4  79 activity assays.  Antibody concentration was determined by the absorbance at  280 nm.  3.6 S Y N T H E S I S O F S U B S T R A T E S A N D H A P T E N D E R I V A T I V E S  3.6.1 S y n t h e s i s of Substrate 55  Gong,  et  al  89  reported  that  a  chromogenic  substrate  containing  a  3-hydroxyindole moiety (see Scheme 9) might be useful in a direct catalytic assay. The indoyl strategy was suggested to be a facile chromogenic assay that might lend itself to the screening of large antibody libraries. The insoluble indigo product obtained upon carbonate cleavage affords high detection sensitivity since the indigo accumulates at the site of reaction, rather than diffusing through the reaction medium as is the case with soluble chromogenic species such as the p-nitrophenolate anion.  The synthesis of 55, shown in Scheme 10, began with the hydrolysis of 3-indoyl acetate in a deoxygenated solution of refluxing 2N N a O H . The anhydride 52, in deoxygenated EtOH was then canulated into this reaction solution at 0 °C. Upon  80  O  H IgG  S c h e m e 9.  Chromogenic assay reported by Gong, et al. (taken from ref 89).  isolation of crude indole 53 by ether extraction, followed by hydrogenolysis of the crude with H gas and Pd/C in EtOAc, the resulting crude isolate was coupled to 2  the  hydroxy  acid  54*  with  1,3-dicyclohexylcarbodiimide  (DCC)  and  4-dimethylaminopyridine (DMAP) in C H C I yielding the target hydroxy ester 55. 2  * Prepared by 0 . Clyne.  2  81  1) NaOH-H 0, N 2  PhCH 0  2  2  2)  H  O (PhCHzO^^^Vfe 52  53  H  o 1) H , Pd/C, EtOAc 2  2)  OH  O  u (CH )TV"OH 2  54 DCC, DMAP CH2CI2  S c h e m e 10. Synthesis of indoyl substrate 55.  Anhydride 52 was prepared by the D C C coupling of carboxylic acid 56 .  PhCH 0  XOOH  2  56  *CH CI 2  2  (PhCH 0  yT2  2  52  Substrate 55 was found to be insoluble in the P B S buffer.  0  A variety of  co-solvents, including M e O H , DMF, C H C N and D M S O , were used in an attempt 3  to solublize 55. Of the co-solvents used, a P B S solution containing 10% D M S O was found to be the most effective, however, complete solubility of 55 was only achieved by employing 0.5% Triton X-100 with the 10% D M S O . Solutions were  * Prepared by J . P. Ounsworth.  82 determined to be homogeneous by examination under an inverted microscope (10x magnification).  Reaction of the indoyl substrate 55 with monoclonal antibody F123 did indeed lead to the expected insoluble byproduct, namely indigo, while none was observed in the blank experiment, however, the indigo could neither be easily nor conveniently quantified on the assay scale used.  Due to the low solubility of substrate 55 in the reaction buffer, along with the problem associated with quantifying the extent of the reaction (i.e. the amount of indigo formed), we elected to switch to the shorter, more soluble p-nitrophenyl ester substrate 57.  In addition, the soluble p-nitrophenolate anion, resulting  from reaction of 57 with the catalytic antibody, is a convenient chromophore for acquiring kinetic data spectrophotometrically.  Substrate 57, used in the catalytic assays for the monoclonal antibodies, differs from 55 in that 55 mimicks more closely the linker-arm portion of hapten molecule 50.  However, it should not be surprising that the antibodies are  tolerant to this substrate variability, since o t h e r s  89,151,152  suggest that antibody  83 binding specificity is relatively insensitive to elements of the hapten and substrate close to the conjugation site. Also, antibodies specific for the steroid androstenedione were s c r e e n e d for specificity using derivatives of this steroid. 75  This work showed that the antibodies were much less sensitive to changes in the ligand which were near the position of linker attachment on the steroid, and that the specificity was greatly affected by changes farthest from the position of attachment to the linker. This evidence supports the idea that the portion of the hapten which extends away from the carrier protein provides the most accessible epitopes for antibodies.  3.6.2 S y n t h e s i s of Substrate 57  The hydroxy p-nitrophenyl ester substrate 57 was obtained by reaction of 13-hydroxytetradecanoic acid (54) with p-nitrophenol in C H C I in the presence 2  2  of D C C and D M A P under a N atmosphere to produce the desired substrate in 2  87% yield. OH  o OH  54  OH  O  N0 57  (87%)  2  84  3.6.3 Synthesis of Substrate 58  Myristic acid, or tetradecanoic acid, was reacted with p-nitrophenol as below to produce p-nitrophenyl tetradecanoate (58) in 88% yield.  CH Cfc>, D C C . D M A P 2  t  o  3.6.4 Synthesis of Hapten Derivatives 60a and 60b  The synthesis of the p-nitrophenylphosphonate hapten derivatives 60a and 60b began with the hydrolysis of phenyl phosphonate 38b using refluxing 3N NaOH-THF produced the phosphonic acid derivative 59 in a 7 3 % yield. Conversion of 59 to the acid chloride with oxalyl chloride, followed by coupling of this phosphoryl chloride intermediate with p-nitrophenol (PNP) gave a 1.2:1 ratio of diastereomers 60a and 60b in 69% yield.  85  3.6.5 Synthesis of Six-membered Ring Phenyl Phosphonates, and 61b  61a  The six-membered ring phenyl phosphonates, 61a and 61b, were synthesized as an  offshoot  of  the  14-membered  ring  project,  and  as  such,  the  full  characterization of the intermediates that led to compounds 61a and 61b is not complete.  However, the targets, 61a and 61b, have been fully characterized,  and the stereochemistry of these two phenyl phosphonates has been firmly established, and this proved useful in elucidating the stereochemistry of the 14-membered ring analogues, 38a and 38b, discussed in Section 3.7.  The  synthesis of 61a and 61b (Scheme 11) began with the benzylation of the primary hydroxyl group of 1,3-butanediol to give 62 in 58% yield.  The secondary  hydroxyl of 62 was then protected as the T H P ether 63 in 76% yield.  This  addition of the T H P group created a second chiral center in 63, and therefore, two diastereomers of 63 were obtained. The two diastereomers were obtained in a 1:1 ratio as seen in the H N M R spectrum of 63, which reveals two methyl 1  doublets of equal intensity and two methine multiplets, corresponding to the  86 methine proton of the T H P group.  Hydrogenolysis of the benzyl moiety of  compound 63 gave alcohol 64 in an isolated yield of 85%.  The H N M R 1  spectrum of the flash chromatography-purified diastereomers of compound 64 reveals a diastereomeric ratio of approximately 1:4, rather than a 1:1 as would be expected, based on the relative intensities of the diagnostic methyl and methine signals. A portion of one of the diastereomers of 64 was accidently discarded during purification resulting in this 1:4 ratio.  This was of no  consequence, however, since the T H P group was subsequently removed, destroying one of the two stereogenic centres in the molecule. The 1:4 ratio of isomers of alcohol 64 were then oxidized under the Swern conditions to give the aldehyde 65 in 96% yield.  Aldehyde 65 was subsequently reacted with the  stabilized Wittig reagent 32 to give the trans vinyl phosphonate 66 in a moderate yield of 55%. Phosphonate 66 was then reacted with p-toluenesulphonic acid to liberate the secondary hydroxyl compound 67 in 70% yield. Removal of the T H P group from 66 eliminated one of the two stereogenic centres in the molecule giving a single diastereomer, reflected in the H N M R spectrum of 67 which 1  contains only one methyl doublet. The alkene 67 was then reduced to give the saturated diphenyl phosphonate 68 in 9 3 % yield. Finally, the hydroxy diphenyl phosphonate 68 was reacted with n-butyllithium in T H F at -78 °C to produce the cyclic phosphonates 61a  and 61b  in a 96% yield and a 6:1  diastereomers based on the H N M R spectrum of the crude product. 1  ratio of  87  ?  H  HO  OH  NaH, THF, 0°C BLUNI,  PhCH Br  PhCH 0'  2  CH CI  2  OTHP  DHP, p-TsOH 2  PhCH 0' 2  2  63 (76%)  62 (58%)  H , Pd-C 2  EtOAc O  OTHP  66 (55%)  OTHP  OTHP  32  DMSO, (COCI) ,  C6H5CH3, a  Et N, CH C(2 -78° C  2  3  65  (96%)  HO'  2  64 (85%)  p-TsOH, MeOH . O H , Pd-C  "BuLi, THF  2  67 (70%)  v  OPh  X -78 °C  EtOAc  ^O 61a + 61b (96%; 6:1)  68 (93%)  Scheme 11. Synthesis of cyclic phosphonates 61a and 61b.  3.7  INVESTIGATION INTO THE RELATIVE STEREOCHEMISTRY OF CYCLIC PHOSPHONATES 38a AND 38b  Table 5 below lists the H NMR chemical shifts of the methine proton and the 1  methyl protons for the 14-membered ring phosphonates 38a, 38b, 60a, 60b, 49a, 49b and 50, as well as those for the 28-membered ring dimer 39 and the six-membered ring phosphonates 61a and 61b.  In addition, Table 5 lists the  NMR signals and T L C R values for each compound. f  correlation between the macrocyclic  1  H and  phosphonate dimer  31  P  39  NMR  P  W e first looked for a  spectra of the  and those  31  of the  28-membered  cyclic monomeric  diastereomers, 38a and 38b. Since we only had a crystal structure for the dimer  88 39, we hoped that the H and 1  3 1  P N M R spectra of the 28-membered macrocyclic  phosphonate dimer 39, together with the structural information from the X-ray structure of 39 would allow us to infer structural information about monomers 38a and 38b, and ultimately the hapten molecule 50, to which antibody F123 was raised.  The chemical shifts of the C-1 methyl doublets for both isomer 38a and the dimer 39 are at higher field than the methylene envelope, while that of isomer 38b is at lower field than this envelope (see Spectral Appendix). In the case of compound 50, the methyl doublet lies just within the highfield region of the methylene envelope, that is, to the "isomer a side". This result is consistent with inversion at phosphorus, since 50 was produced on substitution of the phenol moiety of diastereomer  38b  with the  linker  48,  giving  rise  to  49a  hydrogenolysis of the C b z moiety of 49a yielded compound 50.  which  upon  Compound 49b  was synthesized by displacement of the phenol moiety of 38a with linker 48 giving rise to compound 49b exclusively.  Diastereomers 60a  and 60b were produced by conversion of the cyclic  phosphonic acid 59 into two phosphoryl chloride intermediates (as observed by  89  38a,38b 60a,60b 49a,49b 50  R = Ph R = PNP R = (CH^sNHCbz R = (CH )5NH  Table 5.  2  2  Key H and P N M R chemical shifts (8 values) and T L C R v a l u e s for the cyclic phosphonates. 1  3 1  r  TLC R, values  8 H for C H protons (ppm)  8 H for methine proton (ppm)  8 P (ppm)  38a  1.22  4.75  29.6  0.71  38b  1.43  4.65  27.8  0.57  60a  1.23  4.77  30.4  0.66  60b  1.43  4.65  28.5  0.56  49a  1.30  4.59  32.5  0.18  49b  1.35  4.43  31.3  0.12  50  1.32  4.95  33.1  0  39  1.15  4.66  29.0  0.62  61a  1.35  4.51  20.5  0.21  61b  1.36  4.68  23.4  0.10  Compound  1  3  1  Obtained using a 1:1 mixture of petroleum ether and ethyl acetate.  31  1  90 TLC) followed by substitution with p-nitrophenol. The H and 1  3 1  P N M R chemical  shifts (8 values) for compounds 49a and 49b as well as for diastereomers 60a and 60b are consistent with the pattern observed with 38a and 38b.  That is, within  each pair of 14-membered ring phosphonate diastereomers listed in Table 5, the 1  H N M R chemical shift of the C-1 methyl doublet is at higher field, while the shift  of the methine proton is at lower field for all the a isomers compared with those for the corresponding b isomers. The H N M R chemical shift of the C-1 methyl 1  doublet for dimer 39 is consistent with those of the a isomers of the 14-membered ring phosphonates. The stereoview of the O R T E P representation of the macrocyclic dimer 39 (Figure 13) reveals that the doubly-bonded oxygen on the phosphorus atom is 'syn' to the C-13 methine proton, and the C-1 methyl group is 'syn' to the oxygen of the phenol moiety as illustrated in Figure 14. W e are suggesting, therefore,  that the a isomers of the  14-membered  ring  phosphonates listed in Table 5 have the R*,R* relative stereochemistry between the C-1 methyl and the phosphorus atom consistent with that of compound 39. In addition, we believe that the C-1 methyl group and the O R group on phosphorus are mainly in a conformation in which both are equatorial in the a isomer as found in the X-ray structure of 39 (Figure 14).  Support for the  equatorial assignment of the O R groups on the phosphorus atoms of the 14-membered ring phosphonates listed in Table 5 comes from the chemical shift data.  It has been observed that the  3 1  3 1  P NMR  P N M R signal for the  equitorial P - O R isomer is approximately 2 ppm lower field than the  91  Figure 14.  Relative stereochemistry about the phosphorus atom and the C-1 carbon atom of dimer 39.  corresponding axial isomer in the cyclic phosphonates of hexopyranoses " 153  and in other six-membered ring phosphonates.  156,157  correlation between compounds 39 and 38a, since the  155  This trend supports our 3 1  P N M R shift for isomer  38a is 2 ppm downfield from that of isomer 38b.  A final piece of evidence to support the equatorial assignment for the O P h group in compound 38a  comes from a comparison of 38a  six-membered ring anaolgues, 61a and 61b.  and 38b  with the  The stereochemical assignments  (as shown below) for compounds 61a and 61b were determined on the basis of 1  H NMR N O E (nuclear overhausser effects) difference experiments.  Irradiation  of the methine signal at 4.51 ppm of phosphonate 61a resulted in enhancement of the  phenyl  signal  between  7.4-7.2 ppm, whereas,  irradiation  of  the  corresponding methine signal for 61b failed to produce an enhanced signal in the phenyl region.  This N O E data is consistent with the  assignments for compounds 61a and 61b.  stereochemical  92  CH OPh —  3 1  3  H 61b  61a  In addition, the  CH  3  P N M R shifts for compounds 61a and 61b, listed in Table 5, are  in agreement with the trends described above for similar six-membered ring phosphonates. That is, the  3 1  P signal for phosphonate 61b, with the O P h group  in the equitorial position, is at lower field than the corresponding axial isomer, 61a. With the stereochemistry of compounds 61a and 61b firmly established, a correlation was made between these compounds and the 14-membered ring analogues, 38a and 38b.  Considering the structures for compounds 61a and  61b along with H N M R data listed in Table 5, a 1,3-diaxial (or syn) relationship 1  between the methine proton and the doubly-bonded oxygen attached to the phosphorus atom (as in 61b) results in a shift in the methine signal to lower field compared to the higher field methine signal when these two groups are anti to one another (as in 61a).  The methine signal for compound 38a (at 4.75 ppm) is  at lower field than that of compound 38b (at 4.65 ppm), which supports our assignment of the O P h group being equatorial (and hence the doubly-bonded oxygen attached to the phosphorus atom in a 1,3-diaxial relationship with the methine proton) in phosphonate 38a.  93 3.8  INITIAL INVESTIGATION F O R REACTIVITY O F M A B ' S WITH S U B S T R A T E 57.  The concentrations of antibody stock solutions used in this study, obtained from immunoaffinity  purification of ascites preparations, were determined using  6 = 1.4 and a molecular weight of 175 kDa for IgM monomer.  Clones that showed the strongest binding to our hapten molecule were chosen for catalytic screening. Analyses were performed using 96-well microtiter plates and a microtiter plate reader (Bio-Tek Instruments) equipped with the Delta Soft II computer program (referred to later as the 'multiwell method'). Masamune and co-workers  90  A report by  also uses a microtiter plate reader for kinetic  analysis of their catalytic monoclonal antibodies. Furthermore, Copeland states that many workers now perform absorption measurements with 96-well microtiter plate readers.  158  All assays reported in this thesis by the microtiter method were  performed at least in duplicate.  Three monoclonal antibodies, F123, F125 and F150 were selected, based on binding assays, for a reactivity test against substrate 57 (Figure 15). clones  were  assayed  spectrophotometrically  (405  nm)  at  37  These °C  for  4-nitrophenolate anion release from substrate 57 on reaction with the antibody. Only monoclonal F123 showed significant reactivity with 57, and was selected for further study. A n initial test of the catalytic potential of monoclonal F123 was  94 carried out, involving three independent experiments on one microtiter plate. The first experiment was a reaction of substrate 57 (88 LIM) with antibody F123  0  10  20  30  40  50  60  Time (min) Figure 15.  Monoclonal antibodies F123 (O), F125 ( • ) and F150 (•), each at a concentration of 2 LIM, were assayed for activity with substrate 57 (200 uM) in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100 at 37 °C.  (0.88 LIM). The second experiment involved reaction of 57 (88 LIM) with antibody F123 (0.44 LIM). From the slopes (rates) of these two experiments (see Figure 16), we see that halving the antibody concentration resulted in a halving of the reaction rate (see Table 6). Finally, the third experiment involved reaction of 57 (88 LIM) in the presence of 0.25 equivalents (0.22 LIM) of hapten analogue 60 (a one-to-one mixture of both diastereomers) with antibody F123 (0.88 LIM).  Here  we expected to see inhibition of the p-nitrophenol release in the presence of the hapten derivative 60.  From the slopes in Table 6, it is clear that compound 60  95 inhibits the antibody activity, demonstrating that antibody activity is associated with binding in the antibody-combining site.  0.12  0  2  4  6  8  10  Time (min) Figure 16.  Initial experiments with antibody F123, substrate 57, and hapten derivative 60 using P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X - 1 0 0 at 37 ° C . Reactions were run; (i) (•) 88 LIM in 57 and 0.88 uM in F123, (ii) ( O ) 88 LIM in 57 and 0.44 L I M in F123, (iii) ( • ) 88 u.M in 57, 0.88 L I M in F123 and 0.22 LUVI of 60 (1:1 ratio of diastereomers).  The findings of these three experiments were encouraging, and suggested that monclonal F123 was behaving as a catalyst, and that the catalytic activity of F123 is associated with binding in the antibody-combining site. The next step was to prove that antibody F123 is indeed catalysing the cyclization of 57 to lactone 19, and to then carry out a kinetic analysis of the reaction between  96 antibody F123 and substrate 57 to determine the Michaelis-Menten parameters Km, Vmax and kcat-  Table 6. Results of initial F123 antibody experiments. Experiment  Slope (AOD/min)  Correlation Coefficient  (i)  0.0101  0.9995  (ii)  0.0056  0.9983  (iii)  0.0008  0.9935  3.9 ISOLATION OF F123-CYCLIZED MACROLACTONE 19  Confirmation that F123 catalyzed the cyclization of substrate 57 to lactone 19 was achieved by isolation of 19 through ether extractions from pooled catalytic experiments.  Several small-scale catalytic reactions of antibody F123 with  substrate 57 in P B S buffer, pH 7.4, containing 10% D M S O and 0.5% Triton X-100 at 37 °C (ranging from 100-400 LIM in 57, and each having a final volume of 250 |iL) were pooled for ether extractions.  Initial identification of the antibody generated lactone was achieved by G C analyses.  G C analysis of an independently synthesized sample of lactone 19  revealed a broad peak with a retention time of approximately 17 minutes, as seen in trace (a) of Figure 17 below. The G C trace of the ether extracts from the  pooled antibody experiments also contained a broad peak at approximately 17 minutes, Figure 17 trace (b). The G C run for the ether extracts from the antibody experiment spiked with lactone 19, G C trace (c), showed an increase in the peak  i 0  1  1 5  1  1 10  1  1 15  1  1 20  Retentiontint*(min)  Figure 17.  G C chromatograms of: (a) an independently synthesized sample of lactone 19; (b) ether extracts from the pooled F123 catalyzed cyclization reactions of substrate 57; (c) a spiked G C run of the ether extracts from the antibody experiment with an authentic sample of 19.  corresponding to lactone 19 having a retention time of approximately 17 minutes, confirming the presence of lactone 19 in the pooled antibody experiments.  Furthermore, the unambiguous identification of the antibody generated lactone was obtained by G C M S analysis.  The high resolution mass spectrum of the  98 G C M S lactone peak from the ether extracts was consistent with that of the independently synthesized lactone sample. A s expected, the molecular ion peak for lactone 19 at m/z 226 was observed in both mass spectra. In addition, the anticipated fragment at m/z 208 was present in both mass spectra.  Finally, a large scale background experiment with 57 (250 LIM) in the reaction buffer (containing no antibody) was conducted at 37 °C for five days.  GCMS  analysis of ether extracts from this background reaction revealed no detectable lactone.  3.10 SPECIFICITY OF MONOCLONAL F123  To illustrate that the activity of antibody F123 with 57 is not due to an esterolytic contaminant of the serum, an affinity purified control M A b raised against an unrelated hapten was run in parallel with F123. The control M A b showed no activity with substrate 57 (see Figure 18).  3.11 REACTION OF P-NITROPHENYL TETRADECANOATE WITH F123  Substrate 58, which lacks the secondary hydroxyl necessary for lactonization, was tested with catalytic antibody F123 and showed no activity.  This also  provided strong evidence that MAb F123 was not simply behaving as an  99 esterase by hydrolyzing the p-nitrophenol ester moiety of substrate 57, but rather, acted by carrying out the desired macrolactonization (Figure 19).  0  10  20  30  40  50  60  Time (min) Figure 18.  The activity of monoclonal F123 (0) is compared to that of a control monoclonal antibody, HIL-20 (•), using substrate 57 in both of the experiments. Each experiment was run 200 LIM in 57 and 2 lj.M in antibody, carried out in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100.  100  0.05  0  2  4  6  8  Time (min) Figure 19.  Comparison of antibody F123 activity with hydroxy ester substrate 57 (O); with ester 58 (•). In each case, antibody was 0.46 LIM; substrates were 100 u,M; carried out in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X - 1 0 0 ; total reaction volume was 225 \xL.  3.12 INHIBITION ASSAYS  The  F123 antibody catalyzed reaction of substrate 57 to lactone 19 was  analyzed for inhibition by an independently synthesized sample of lactone 19. Lactonization of 57 by F123 was not inhibited in the presence of one molar equivalent of 19 (relative to substrate 57) as shown in Figure 20 below.  101  Time (min) Figure 20.  Antibody F 1 2 3 activity with hydroxy ester substrate 57 (O); with hydroxy ester substrate 57 in the presence of an equimolar amount of lactone 19 (•). Experiments were carried out in P B S buffer (pH 7.4) with 1 0 % D M S O ; 0 . 4 6 \M in antibody F 1 2 3 ; 2 0 0 L I M in substrate; total reaction volume was 2 5 0 LIL.  Continuing from the initial reactivity studies of antibody F123 described in Section 3.8, inhibition assays with a one-to-one mixture of the 4-nitrophenyl phosphonate diastereomers, 60a and 60b, were performed in the presence of substrate 57 and MAb F123. A s Figure 21 shows, the mixture of 60a and 60b competitively inhibited the F123 antibody-catalyzed reaction of 57.  Each data  point in Figure 21 represents an average of two reaction runs, which agree within 10%.  102  4  h  2  h  c I ^  -0.01  0  0.01  0.02  1/[S] (10« M" ) 1  Figure 21.  Competitive inhibition of the F123-catalyzed macrolactonization of substrate 57 by a one-to-one mixture of 4-nitrophenyl phosphonate diastereomers, 60a and 60b. The reactions were performed in P B S buffer (pH 7.4) containing 10% D M S O and 0.5% Triton X-100. The concentrations of the mixture of 60a and 60b (1:1) were: (O) 0 uM; ( • ) 2 u M ; a n d ( D ) 5 u M .  By fitting the data from Figure 21 with the inhibitor concentrations to the computer program GraFit™, a value of Kj = 2.9 ± 0.4 uM was determined. Because of the chiral nature of enzymes and antibodies, we believe that this inhibition constant of 2.9 ± 0.4 LIM represents an upper limit (where a smaller Kj value is indicative of a more potent inhibitor), and the true K is expected to be one-quarter this value since only one of the four stereoisomers in the mixture of 60a and 60b diastereomers is believed to be responsible for the inhibition observed.  Further inhibition studies using the four stereoisomers of  60  103 separately are necessary to refine this inhibition constant.  Nonetheless, the  competitive inhibition of F123 with this mixture of 4-nitrophenyl phosphonate diastereomers,  60a and 60b, demonstrates that the  catalytic activity  is  associated with binding in the antibody-combining site, as expected.  3.13 D E T E R M I N A T I O N O F K , V M  M A X  AND  kc F O R A N T I B O D Y F 1 2 3 W I T H at  S U B S T R A T E 57  Approximate values of Km and V x may be determined for substrates by ma  measuring  initial reaction rates using three  concentrations of the substrate.  different  (and  wide-ranging)  Once approximate values are obtained,  accurate values of Km and V x are then determined by measuring initial reaction ma  rates using 5-8 different concentrations of the substrate, concentrations that typically range between 0.3-5 times the approximated Km value.  For substrate 5 7 , the experiment to determine approximate Km and V x values ma  was carried out with concentrations of 40, 100, 200, and 400 LIM of 5 7 in the reaction buffer consisting of 10% D M S O and 0.5% Triton X-100 in P B S buffer, pH 7.4. The approximate Km was found to be 250 LIM for substrate 5 7 . With a solubility limit of 400 LIM for 5 7 , and an approximate Km of 250 LIM, this meant that in order to achieve a more accurate value of Km, we would need to increase the solubility of 5 7 in the reaction buffer in order to bracket the approximate Km by 0.3-5 times. This lead to a solubility study of substrate 5 7 .  104 3.13.1  Determination of the Solubility Limit of Substrate 57 and the pH of the Reaction Solution  In an effort to attain a higher substrate solubility limit, the effect of varying the Triton X-100 concentration in the reaction buffer mixture was investigated. Solubility limits for substrate 57 in the reaction buffer containing 0%, 0.25%, 0.5% and 1.0% Triton X-100 were determined by examination of solutions under a microscope (10x magnification). A s shown in Table 7, the solubility limit of 57 increases with increasing concentration of Triton X-100 in the reaction buffer used in this study. Conversely, it was found that the pH of the reaction solution decreased on increasing the concentration of Triton X-100.  Table 7.  Solubility Study of Substrate 57 with Triton X-100.  Triton X-100 Content (%) 0  1  Solubility Limit of 57 (uM) 0  0  7  "  5  pH 5  0.25  200  7.58  0.50  400  7.35  1.00  800  6.95  However, it was found that a Triton X-100 content of 1 % or more in the reaction solution not only increased the solubility limit of 57 and decreased the pH of the solution, it appeared also to inhibit the antibody activity. To probe this apparent solubility/reactivity  problem, catalytic experiments were carried out  in the  presence of 0%, 0.25%, 0.5% and 1% Triton X-100 (with 10% D M S O , 200 u,M in  105 substrate 57, in P B S buffer, pH 7.4).  It was observed that antibody activity  appeared to decrease with increasing Triton X-100 content (see Figure 22 below). The pH of the four test buffers (containing 0%, 0.25%, 0.5% and 1.0%  Figure 22.  Antibody F123 reaction with substrate 57 compared to background hydrolysis of 57 in the reaction buffer alone.  the  Triton X-100) was determined, and found to be 7.55; 7.58; 7.35 and 6.95, respectively (Table 7).  At this point, we believed that the apparent loss in  antibody activity may be attributed to one of the following two factors, namely the increase in the surfactant concentration, Triton X-100, or the drop in pH of the reaction buffer as a result of increasing the surfactant concentration. decrease in pH of the reaction buffer  A  (on increasing the Triton X-100  concentration) would result in a shift in the equilibrium shown below toward the protonated  form,  p-nitrophenol,  which would  explain a decrease in  the  106 absorbance reading. Alternatively, the antibody may simple lose activity as the pH is reduced.  chromophore ( W = 405)  To determine if this pH depression was the cause of the apparent loss in antibody activity, the buffer strength of the P B S buffer used in the reaction solution was increased from 8 mM sodium phosphate to 500 mM sodium phosphate. The higher buffer capacity was sufficient to suppress the drop in pH, (pH was measured and found to be 7.45 for the 500 mM sodium phosphate solution, compared to pH 6.95 for the 8 mM sodium phosphate solution), however, catalysis (as measured by the rate of release of p-nitrophenolate anion above background) was not observed (see Figure 23).  This finding suggests  that the increased concentration of Triton X-100 does not affect the position of equilibrium between the p-nitrophenolate anion and the protonated form, but rather, in some way inhibits antibody activity when the concentration of Triton X-100 is 1% or higher.  Further experiments are required to investigate the  nature of this effect. Perhaps we have reached or exceeded the critical micelle concentration (cmc) of the surfactant for our buffer system, or perhaps substrate molecules are being partitioned within the micelle wall, and in effect, the Triton  107 X-100 is competing with the antibody for substrate binding.  If this is the case,  the loss of substrate molecules to micelles would make it impossible to know the actual substrate concentration in our reaction mixture.  Furthermore, at 1%  Triton X-100, perhaps the surfactant binds sufficient substrate such that none is available to the antibody, and we simply observe background hydrolysis, as in the background samples.  c 1 D O E  500 m M  8 mM  Substrate concentration  Figure 23.  Comparison of the F123 reaction with substrate 57 and background hydrolysis of 57 using 10% D M S O and 1.0% Triton X-100 in 8 mM P B S buffer (pH 6.95); and F123 and 57 in a 10% D M S O with 1.0% Triton X-100 in 500 mM P B S buffer (pH 7.45).  3.13.2 Determination of K , V Substrate 57 m  m a x  and k  cat  for Monoclonal F123 with  Km, Vmax and kcat determinations were performed on antibody F123 using two independent methods.  The first method will be referred to a s the 96-well  multiwell method, employing a microtiter plate reader (Bio-Tek Instruments)  108 equipped with the Delta Soft II computer program. The second method will be referred to as the spectrophotometer cuvette method.  The molar absorptivity (e) for the p-nitrophenolate anion in the reaction buffer was determined as follows. Measuring the absorbance (Xmax = 405 nm) of a 0.1 mM solution of p-nitrophenolate anion in the reaction buffer and applying Beer's Law, A = eel (where A is the absorbance, c is the solute concentration expressed in moles per liter , and I is the pathlength in centimeters of the solution being measured), e for the p-nitrophenolate anion was found to be 10,830 L/mol-cm.  While the pathlength of the 1 mL quartz cuvettes used with the Unicam UVA/IS spectrophotometer, employed in the spectrophotometer cuvette method, is known to be 1 cm, the pathlength of the 250 LIL volumes of reation buffer in the 96-well microtiter plates employed in the multiwell method had to be determined.  The absorbance of a 0.1 M solution of p-nitrophenolate in the reaction buffer was measured using the Unicam spectrometer (A™* = 405 nm) having a pathlength of 1 cm. This result was used to determine the pathlength of the 250 LIL reaction volume in the multiwell experiments by measuring the absorbance of a 250 LIL alliquot of this solution in a microtiter plate, and applying Beer's Law. The pathlength of the 250 u l volume of reaction buffer in the microtiter plate was found to be 0.64 cm.  109 3.13.2.1  Determination of K , V and kcat for Monoclonal F123 with 57 via the Multiwell Method m  m a x  Assays were performed in 96-well microtiter plates, and the reaction buffer consisted of P B S containing 10% D M S O and 0.5% Triton X-100 (for substrate solubility), at pH 7.4. Without Triton X-100, the solubility limit of substrate 57 is 200 uM, and with 0.5% Triton X-100, this limit is increased to 400 LIM.  Final  reaction volumes used were 250 u l , and the pathlength of the ELISA reader was determined above to be 0.64 cm.  Rates were determined by measuring  4-nitrophenolate release at 405 nm (e = 10,830 L/mol-cm) and defined as mOD/min.  The antibody concentration used was 0.23 L I M (based on IgM  monomer molecular weight of 175 kDa). Kinetic parameters were obtained using the method of initial rates and fitted to a Lineweaver-Burk analysis to obtain the Michaelis-Menten parameters Km, V  m a x  and kcat for substrate 57 (Figure 24).  It  should be noted that prior to reading the microtiter plates by the ELISA reader, mixing of the reaction buffer with a micro-pipette, until homogenous, caused the formation of bubbles at the liquid surface of the reaction solutions. Since the ELISA reader measures absorbance down through the top of each well, plate reading was delayed (approximately  10 minutes)  until the  bubbles had  collapsed, after which time, the initial rates were measured. Since the antibody rate is expected to decrease with time, one might expect the actual initial rates would be greater if the reactions were monitored immediately after mixing, without a 10 minute delay time.  110  -0.01  0  0.01  0.02  0.03  1/[S](10 M- ) 6  Figure 24.  1  A Lineweaver-Burk analysis of the F123-catalyzed macrolactonization of hydroxy ester substrate 57 using the multiwell method.  At an antibody concentration of 0.23 LIM, the multiwell method yielded a V  m a x  of  0.62 ± 0.01 iimol/min mg and a Km of 250 ± 1 0 LIM. The k^t for this experiment was calculated to be 1.1 min' . 1  3.13.2.2  Analyses for Km, V  Determination of K , V and kcat for Monoclonal F123 with 57 via the Spectrophotometer Cuvette Method m  m a x  m a x  and kca determinations were also performed using a t  Unicam UVA/IS spectrophotometer in 1 mL quartz cuvettes with 1 cm pathlength (600 LIL reaction volume) to compare techniques.  Rates were determined by  measuring 4-nitrophenolate release at 400 nm (e = 10,730 L/mol-cm), and the  111 same  reaction  measurements.  buffer  as  in the  multiwell  method  was  used  in  these  Kinetic parameters were obtained using the method of initial  rates and fitted to a Lineweaver-Burk analysis to obtain the Michaelis-Menten parameters Km, V x and kcat for substrate 57 (Figure 25). ma  The antibody  concentration used was 0.25 LIM (based on IgM monomer molecular weight of 175 kDa). With this approach, unlike the multiwell method, the absorption can be read immediately after mixing, to obtain more accurate initial rates. Once all reagents and buffers were added to the cuvettes, they were covered with Parafilm, and inverted several times to ensure thorough mixing, then inserted into the spectrophotometer. A delay time of 1.5 minutes was programed into the spectrophotometer prior to reading the cuvettes to obtain the initial rates. This delay time of 1.5 minutes was to ensure equilibration of the reaction solution temperature to that of the spectrophotometer, 37 °C, prior to reading the absorbances. The time required for mixing, together with the 1.5 minute delay, meant that the total delay time prior to recording the initial rates with this approach was approximately 2 minutes, compared to the 10 minute delay time for the multiwell method.  The Unicam UVA/IS spectrometer experiments gave a V x of 1.4 ± 0.1 ma  umol/min mg and a Km of 330 ± 50 LIM from Figure 25.  Since the antibody  concentration was 0.25 LIM for this experiment, the kcat was calculated to be 2.2 min" by this approach. The Km value obtained by this approach is in agreement 1  112 with that of the multiwell method, within experimental error. Of the two methods used, 0.1  c , o  E  0.05  .>  0 0  0.005  0.01  1/[S](10 M- ) 6  Figure 25.  1  A Lineweaver-Burk analysis of the F123-catalyzed macrolactonization of hydroxy ester substrate 57 using the spectrophotometer cuvette method.  however, the correlation proved better with the multiwell method. The kca values t  differ, however, by a factor of two.  A s we anticipated, the difference in delay  times for the two approaches resulted in a difference in kca, values. This can be accounted for by taking into consideration the delay time required, in the multiwell method, prior to beginning reading the multiwell experiments.  At 10  minutes, it is expected that the rates would have diminished somewhat from the true initial rates.  It is reasonable to assume, therefore, that this delay in the  measurement of initial rates for the multiwell experiments would result in a lower  113 V  m a x  , and ultimately a lower kcat for the multiwell method.  The Km values are,  however, in good experimental agreement, as one would expect, since Km determination is relatively insensitive to this time delay problem.  Finally, the catalytic efficiency, or kcat/Km, of antibody F123 is compared below in Table 8 with that of the antibody reported by Napper, et al. which catalyzes the 59  cyclization of a six-membered ring lactone (as described in Section 2.12.2).  Table 8.  A comparison of the catalytic efficiency (kcat/Km) of antibody F123 with that of the antibody reported by Napper, et al. 59  Ring Size  k^Cs' )  14  a  0.018  2.50X1C  4  72  14  b  0.037  3.30 X10"  4  112  0.008  7.6 x 1 0 "  5  105  6  K  1  C  g  m  (M)  U K n , (M"V ) 1  Data obtained by the multiwell method. Data obtained by the spectrophotometer cuvette method. Data obtained from ref 59. c  A s shown in Table 8, the antibody-catalyzed reaction for the formation of the six-membered ring lactone, 9, has a lower kcat value compared to the two values of  kcat for  the  14-membered  antibody-catalyzed  ring  lactone,  19.  reaction  leading to  formation  of  the  the  Km value  in  the  compared with those of  the  However,  six-membered ring data set of 7 . 6 x 1 0 * M 5  lower  14-membered ring data sets results in comparable values for the catalytic efficiencies for these two antibodies. kcat/Km values for other catalytic antibodies reported in the literature  159  range from 3.6 x l O "  4  M" s" for a decarboxylating 1  1  114 catalytic antibody to as high as 5.5 x 10 M ' s' for an antibody that catalyzes a 3  1  1  proton transfer.  3.14 S U M M A R Y A N D C O N C L U S I O N S  The macrocyclic phosphonate, 38, was synthesized in nine steps with a modest overall yield of 17%.  However, since only 20 mg of 38 was required for this  project, obtaining the target was of greater importance than the optimization of its yield, which can be accomplished at a later date.  The key step in the  synthesis of 38 was the use of the Mitsunobu reaction in the challenging ring-closing step, which proceeded in an 8 2 % yield giving a 5:1 ratio of the two possible diastereomers.  The successful coupling of the macrocyclic transition state analogue 38 to the carrier protein K L H , and subsequent production of monoclonal  antibodies  against the transition state analogue was evident by the positive results obtained in binding assays.  Eight monoclonal antibodies  showed binding  above  background to the BSA-transition state analogue conjugate in the P C F I A binding assay.  Ascites fluid was produced in Balb/c mice from the hybridomas of the three strongest binders, then affinity purified using a S A M column in preparation for  115 catalytic testing.  Of these three monoclonals, only F123 showed significant  catalytic  with  activity  substrate  57  as  measured  by  release  of  the  p-nitrophenolate anion.  Isotyping of the hybridoma supernatant revealed monoclonal F123 to be an IgM antibody. It is not clear why F123 is of the IgM class, however, this may suggest that the immuno-conjugate KLH-50 was stable in the mouse long enough to elicit a primary immune response, but not long enough to allow for class switching to occur.  Pooled catalytic experiments revealed that F123 was catalyzing the production of the macrocyclic lactone 19.  Ether extracts of the pooled reaction solutions  were compared with that of an independently synthesized sample of authentic lactone by G C / M S analysis. Conversely, a large scale background experiment with 57 carried out at 37 °C for five days, followed by extraction with ether and G C M S analysis of the ether extracts revealed no detectable lactone.  Comparison of the reaction of F123 with substrate 57 run in parallel with an affinity purified monoclonal antibody raised against an unrelated antigen with 57 confirmed, that the release of p-nitrophenolate anion was not the result of an esterase contaminant; that the release of p-nitrophenolate anion occurred only with F123; and that F123 functions as a specific catalyst. Further evidence of  116 the specific activity of F123 was established by the lack of activity of F123 with p-nitrophenyl tetradecanoate (59).  The reactivity of F123 with substrate 57 in the presence of one equivalent of lactone product 19 showed no product inhibition.  Hapten derivative 60 (in a one-to-one ratio of both diastereomers) proved to be a potent competitive inhibitor of the F123 catalysis with 57 (where the inhibition constant, Kj, for 60 was found to be 2.9 ± 0.4 LIM), demonstrating that the catalytic activity is associated with binding in the antibody-combining site.  The results of the solubility study of substrate 57 in the reaction buffer revealed that the maximum concentration of 57 in this buffer system to be 400 LIM. attempt was made to increase the concentration of 57 concentration of the Triton X-100.  An  by increasing the  However, it appeared that an increase in  Triton X-100 concentration resulted in inhibition of the antibody activity.  Hence,  400 LIM 57 was accepted as the upper solubility limit for this study.  Antibody F123 displayed saturation kinetics, and the Michaelis-Menten kinetic parameters of Km, V  m a x  and k^t for F123 with substrate 57 were obtained using  the method of initial rates and fitted to Lineweaver-Burk analyses using two independent methods. The multiwell method yielded a K„, of 250 ± 10 LIM, V  m a x  117 of 0.62 ± 0.01. Limol/min mg and kcat = 1.1 min' , while the spectrophotometer 1  cuvette method yielded a Km of 330 ± 50 LIM, V x of 1.4 ± 0.1 Limol/min mg and ma  kcat = 2.2 min" . These two methods were found to be in good agreement with 1  each other once we accounted for the time delay inherent in the multiwell method when determining initial rates.  In conclusion, while this work opens the door to the antibody-catalyzed formation of macrocyclic lactones, F123 is not yet viewed as a practical catalyst for preparative scale prodution of macrocyclic lactone 19. Much work remains in the full characterization of the activity of antibody F123 as a catalyst for this application.  In general, the chemical approaches towards the syntheses of macrocyclic lactones remain superior in terms of yields, however, biocatalytic methods offer promise as a result of the asymmetric control they often provide, an important factor to synthetic chemists.  3.15 S U G G E S T I O N S F O R F U T U R E W O R K  The work described in this thesis is the first step towards the characterization of a monoclonal antibody raised against a macrocyclic phosphonate, capable of catalyzing the formation of a 14-membered ring lactone.  118  Future directions for this project potentially include:  1)  Investigations into the use of organic solvents to raise the solubility limit  of substrate 57.  2)  Testing antibody F123 for stereospecific cyclization, as demonstrated in  the work by Napper, et  al.  involving the stereospecific cyclization of a  six-membered ring lactone by a catalytic antibody, by reacting F123 with enantiomerically pure isomers of substrate 57, namely 69 and 70.  69  3)  70  Varying the structure of the substrate molecule to further the study of the  specificity of F123.  Some suggestions for substrate modifications  include  compounds 71, 72 and 73. Compounds 71, 57 and 72 would allow a comparison between the rates of lactonization involving a primary, secondary and a tertiary hydroxyl group. Compound 73 would probe the effect of a double bond, placed at various positions along the open chain molecule, on the rate of lactonization.  119 Finally, the rates of lactonization for different ring sizes can be probed by varying the chain lengths of the substrate molecules.  4)  73  72  71  Production of a more efficient catalyst might be possible by modifying the  existing hapten molecule. Two new hapten designs are suggested below, each of which offers qualities not possessed by the hapten used in this study, namely 50.  Perhaps a hapten molecule such as 74 with a 14-membered ring P N P  phosphonate and a linker arm extending from a remote junction, rather than through the phosphonate center as in 50, would serve as a more appropriate transition  state  analogue for  eliciting  catalytic  macrolactonization reactions for two reasons.  antibodies  to  carry  out  First, the P N P phosphonate  moiety incorporates a nitroaryl ring, believed to be desirable for induction of a strong immune r e s p o n s e .  160  Secondly, linking the carrier protein through a more  remote position from the phosphonate center should allow greater exposure of the phosphonate center as the key antibody epitope.  The second proposed  hapten design, 75, may not resemble the presumed transition state for the reaction as well as 50 or 74, however, it may provide more stability than the  120 other potential haptens. This increased stability may lead to the production of a higher affinity IgG class antibody, rather than the IgM class obtained.  ,NO  5)  2  Enzymatic cleavage of the antibody molecule with pepsin, and isolation of  the resulting Fab fragments by affinity chromatography followed by a kinetic analysis of the Fab portion, which contains the antibody combining site, for the lactonization reaction to obtain the Michaelis-Menten constants, and compare them to those of the native antibody.  6)  The  Fab  fragment  might  allow  NMR  and  MS  studies  of  the  antibody-substrate, and antibody-hapten complexes.  7)  Crystallization of the Fab-hapten complex may be possible, and would  allow sequencing of the antibody combining site. sequence  of the  combining  site would  Knowing the amino acid  open the  door  to  mutagenesis  experiments for the purpose of probing the catalytic mechanism of the reaction.  121 CHAPTER IV  EXPERIMENTAL  4.1 BIOLOGICAL METHODS  Deionized water was used in the preparation of all buffers.  Phosphate buffered saline (PBS), available as a 10x solution, was diluted to give the 1x P B S (8.2 mM N a H P 0 , 137 mM NaCI, 2.7 mM KCI, 1.5 mM H P 0 , pH 4  2  4  7.4).  4.1.1  Monoclonal Antibody Purification  Monoclonal antibodies were purified from ascites fluid first by, filter-sterilization through a 0.22 urn filter, followed by immunoaffinity  purification using a  sheep-antimouse (SAM) affinity column.  The ascites fluid was diluted in half using 1x P B S before 0.80 Lim filtration, followed by 0.45 u,m, and finally 0.22 LUTI filtration. The ascites were then loaded onto a S A M affinity column and washed with four bed volumes of 1x P B S . Antibodies were eluted from the S A M column with five bed-volumes of 0.1 M glycine (pH 2.5) and collected in 1.5 mL centrifuge tubes.  Eluates were  neutralized by the addition of 50 LIL of saturated Tris buffer to each tube containing purified antibody.  The antibody samples were dialyzed against 1X  122 P B S buffer, pH 7.4, and the concentration was determined by measuring the absorbance at 280 nm (where the O D reading/1.4 gives the concentration in mg/mL) prior to kinetic assays.  4.1.2  Preparation of Immuno- and Binding-conjugates of 50  Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) were supplied by the Sigma Chemical Co.  4.1.2.1 KLH-50 Hapten Conjugate  To a solution of K L H (15 mg) and 50 (10.0 mg, 28.8 Limol) in 0.5 mL of 0.1 M  MES  buffer,  pH  4.5,  and  4.5  mL  of  water  was  added  1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) (25.0 mg, 0.131 mmol).  The mixture was stirred at room temperature overnight.  The  coupled KLH-50 conjugate was then dialyzed against P B S buffer, p H 7.4.  4.1.2.2 BSA-49 Hapten Conjugate-Coated Carboxyl Polystyrene Particles  Into a 30 mL siliconized Corex glass centifuge tube hapten 50 (1.0 mg, 2.9 Limol) and B S A (1 mg) dissolved in 8 mL of 0.1M M E S buffer, pH 4.5, were added, followed by 1 mL of a 5% (w/v) Fluoricon carboxyl polystyrene assay particle suspension and E D C (5.0 mg, 26 Limol).  The mixture was incubated at room  123 temperature for 24 hours.  The BSA-50 crosslinked particles were then  centrifuged (6000 rpm, 22 °C, 20 min, Sorval HB4 rotor) and the resulting pellet washed 2x in 20 mL of P C F I A buffer, pH 7.4 (1x P B S pH 7.4, 2 % F C S and 0.2% NaN ). 3  Finally, the particles were resuspended in a 40 mL volume of PCFIA  buffer, pH 7.4, to produce a final concentration of 0.125% (w/v) particles and 25 Lig/mL protein.  4.1.3  Kinetic A s s a y s  4.1.3.1 Multiwell Method  The catalytic reactions were performed in 96 well microtiter  plates, and  monitored in a microtiter plate reader (Bio-Tek Instruments) equipped with the Delta Soft II computer program. All reactions were performed in duplicates. Typical reactions were 250 LIL in total volume, and all contained final concentrations (v/v) of 10% D M S O , 0.5% Triton X-100 and 89.5% 1x P B S buffer, pH 7.4. Catalyzed reactions were performed at 37 °C in the presence of 0.23 LIM monoclonal antibody (based on IgM monomer molecular weight of 175 kDa) and were initiated by the addition of various amounts (50-400 jiM) of substrate from a D M S O stock solution. After addition of reagents, substrate, and antibody to the wells of the microtiter plate, a multichannel micropipette was used to mix contents until homogeneous. This mixing created bubbles at the surface of the reaction wells due to the Triton X-100, and a 10 minute delay was required for all  124 bubbles to collapse prior to reading the plates to obtain initial rate data.  The  rates were determined by measuring the initial change in absorbance, due to 4-nitrophenolate release, at 405 nm (e = 10,830 L/mol-cm).  The background  hydrolysis rate of the substrate (in the absence of antibody) was also measured each time. For antibody F123, the antibody-catalyzed reaction rates with four  different  substrate concentrations from 50 to 400 LIM were measured in the presence of 0.23 u.M of the antibody concentration. The Michaelis-Menten parameters were determined by standard methods.  4.1.3.2 Spectrophotometer Cuvette Method  Analysis for V  m a x  and Km determinations were also performed using a Unicam  UVA/IS spectrophotometer in 1 mL quartz cuvettes with 1 cm pathlength to compare this technique with the multiwell method.  Typical reactions were 600 LIL in total volume, and all contained final concentrations (v/v) of 10% D M S O , 0.5% Triton X-100 and 89.5% 1x P B S buffer, pH 7.4. Catalyzed reactions were performed at 37 °C in the presence of 0.25 LIM monoclonal antibody (based on IgM monomer molecular weight of 175 kDa) and were initiated by the addition of various amounts (50-400 LIM) of substrate from a D M S O stock solution. The top of the cuvette was covered with Parafilm and the cell shaken several times prior to placement in the cell holder, and observation  125 at the desired wavelength was obtained after a one-minute delay time.  The  rates were determined by measuring the initial change in absorbance, due to 4-nitrophenolate release, at 400 nm (e = 10,730 L/mol-cm).  The background  hydrolysis rate of the substrate (in the absence of antibody) was also measured each time.  The Michaelis-Menten parameters were determined by standard  methods.  4.1.4  General Immunological Techniques.  4.1.4.1 Immunizations.  Balb/c mice ( 6-8 week old females) were immunized with the K L H conjugate of hapten 50 (KLH-50) according to the following schedule:  day 1, 100 u g of  KLH-50 emulsified in complete Freund's adjuvant, subcutaneous injection; days 19, 50 and 117, administered booster immunizations of 50 Lig of KLH-50 emulsified in incomplete Freund's adjuvant, subcutaneous injection; day 127 the mice received an intraperotoneal booster immunization of 15 |ig of KLH-50 in saline (100 iiL).  Four days after this last booster injection, the mice were  sacrificed and the spleens harvested.  4.1.4.2 Test Bleeds.  The mouse was first warmed to dilate its blood vessels by shining a heat lamp directly into the open cage (from a distance of approximately 18 inches) until the  126 tail veins became visible (usually between 5-10 minutes).  The mouse was  removed and placed in a mouse restraint. The tail was wiped with 70% ethanol to enhance vasodilation of the tail veins. One of the tail veins was then pierced with a 26-gauge needle.  Blood from the puncture hole was collected with a  sterile heparinized capillary tube, and transferred to a microcentrifuge tube. The microcentrifuge tube was placed in a 37 °C water bath until the blood clotted (approximately 20 minutes). The blood was then centrifuged at 600 g to pellet the clotted cells and separate the serum. The serum was then drawn off and transferred to a new tube and stored at -20 °C until needed.  4.1.5  Polyethylene G l y c o l ( P E G ) F u s i o n  The procedures described here are derived from a protocol prepared by Ms. Helen Merkens in Prof. Hermann Ziltener's lab at The Biomedical Research  Centre entitled Fusion Protocol for Mouse Spleen Cell Hybrids for Monoclonal Antibody Production.  Table 9.  Media and components used in hybridoma production.  Media  Components  DMEM  1 M Hepes buffer 0.37% H C 0 " 2 mM L-Glutamine 3  HT Medium  1x10-4 M hypoxanthine 1.6x10" M thymidine 5  127 1x1 IT M hypoxanthine 4x10" M aminopterine 1.6x10" M thymidine 1 M NaOH  HAT Medium  4  7  5  Lysing Solution  0.17MNH CI 0.017 MTris-HCI, pH 7.2-7.4  Fusion Medium  D M E M High Glucose or R P M 1 1 6 4 0 20% F B S 5x10" M 23-mercaptoethanol 2% 10x 3T3 conditioned medium 2 mM 1-glutamine  4  4  Freeze Medium  90% F C S 10% D M S O  P C F I A Buffer  2% F C S 0.2% sodium azide 1xPBS  4.1.5.1 Cell Counting  To a 10 LIL aliquot of a thoroughly suspended cell sample in a microfusion tube is added 10 LIL of a 0.2% (w/v) solution of trypan blue, a cell staining dye, in aqueous 580 mM NaCI solution and the resulting suspension thoroughly mixed. A 10 LIL volume of the cell suspension is slowly pipetted into the sample groove of a clean hemacytometer. The hemacytometer is placed on a inverted phase microscope and the cells in the four counting grids are counted, divided by four, and multiplied by 1 x 10 . This number is then multiplied by two to account for 4  the trypan blue dilution to afford the number of cells/mL.  128 4.1.5.2 M y e l o m a s  A s the myeloma fusion partner, we used P3-X63-Ag8-653 (clone 653). The cells must be in the log phase (2 x 10 viable cells/mL) at the time of fusion. 5  Day 1.  The clone 653 is expanded into 4 x 3 0 mL of D M E M - 2 0 % heat  inactivated F C S at approximately 10 cells/mL. 3  Day 4. The cells are counted and each dish is split into four new dishes and brought up to a volume of 30 mL each with D M E M - 2 0 % heat inactivated F C S at approximately 10 cells/mL. 5  Day 5. The cell count is approximately 2 x 10 live cells per mL, and 2 x 10 live 5  7  cells with a 99% viability are required per fusion.  4.1.5.3 P E G Solution for C e l l F u s i o n  A 50% (w/w) solution of polyethylene glycol (Merck P E G 4000) was prepared by autoclaving 10 gram of polyethylene glycol at 121 °C for 10 minutes. The liquid was cooled to about 56 °C and 10 mL of warm D M E M was added. Finally, the mixture was neutralized with 7.5% H C C V and stored at room temperature.  129 4.1.5.4 Harvesting S p l e e n o c y t e s  All dissecting instruments are sterilized in 7 0 % ethanol prior to use.  The mouse was anesthetized with carbon dioxide and killed by cervical dislocation, then wetted with 70% ethanol. A small incision was made through the skin of the abdomen. The skin was then stretched and torn to expose the peritoneal cavity along the left side of the mouse. A n incision was made through the muscle of the peritoneal wall and the spleen carefully removed. Tweezers were used to extract the spleen and gently remove any connective tissue. The spleen was quickly transferred into a 10 mL sterile tube containing medium.  A spleen cell suspension was prepared by gently mashing the spleen through a fine mesh screen using the rubber end of a 10 cc syringe plunger into a Petri dish containing 2 % F C S - H a n k s * solution, pH 7.4. The cell suspension was then transferred into a 10 mL centrifuge tube, and the cells centrifuged at 300 g for 5 minutes. The supernatant was aspirated off and the cell pellet is resuspended in 5 mL of red blood cell lysing solution at 37 °C for 7 minutes. The lysing buffer was neutralized by adding 5 mL of 2 % F C S - H a n k s solution and the cell suspension then centrifuged at 300 g for 5 minutes.  The cell pellet was  resuspended in 10 mL of 2 % F C S - H a n k s solution and a 1 in 20 dilution was made from which the number of viable cells was counted. This procedure results * See ref 161 for a list of components contained in Hanks buffer.  130 in > 1 x 1 0 cells of approximately 9 5 % viability. 8  1 0 live spleen cells are used 8  per fusion.  4.1.5.5 Harvesting Myelomas  Healthy myelomas (in log phase, phase bright with smooth cell membrane, less than 2 % dead by trypan blue staining) were pelleted by centrifugation at 300 g for 5 minutes, decanted, resuspended in 30 mL of complete D M E M and then transferred to a 50 mL conical tube.  The cells were again pelleted by  centrifugation, washed twice with 50 mL of serum-free D M E M , and resuspended in 10 mL of D M E M .  A n aliquot was diluted 1:10 (v/v) and then the cells were  counted by the method described above.  4.1.5.6 Fusion  Myeloma and spleen cells are combined in a ratio of 1:5 in a 50 mL Falcon tube, centrifuged at 300 g for 5 minutes, and the supernatant was removed. The cell pellet was then resuspended in serum-free medium. repeated three times.  This wash step was  It is essential to remove all serum from the cell surface  since this inhibits the fusion process.  After the last wash the medium was  removed until there was only a meniscus of liquid over the cell pellet. The tube bottom was knocked on a hard surface to liquify the pellet. Prior to the fusion, the P E G and serum-free R P M I * solutions were warmed to 37 °C. Next, 0.8 mL * S e e ref 161 for a list of components contained in RPMI buffer.  131 of the P E G solution was added dropwise to the pellet over 60 seconds while gently tapping the tube. The mixture was allowed to stand for one minute before 1 mL of serum-free RPMI solution, pH 7.4, was added dropwise over 60 seconds, followed by an additional 20 mL over a 5 minute period.  The cells  were then centrifuged for 5 minutes at 300 g, and the supernatant was aspirated off. Finally, the pellet was resuspended in 40 mL of fusion medium.  4.1.5.7 Transferring Fused Cells to the Growth Plates  The cell suspension was plated into four 96-well flat bottomed plates, each well received 100 LIL or 5 x 1 0 cells, and the final volume of each well made up to 4  200 LIL with fusion medium. On days 1, 2, 3, 7, 10 and 14, approximately half of the medium in each well was removed using a sterile pasteur pipette and suction, and replaced with 100 LIL of fresh fusion medium. After approximately 7-8 days, colonies of hybridomas were visible.  4.1.5.8 Selection and Expansion of Hybridomas  The particle concentration fluorescence immunoassay (PCFIA) technique was used in place of the traditional enzyme-linked immunosorbant assay (ELISA) for the initial screening of hybridomas. T h e PCFIA assays were carried out using hapten-protein conjugate-coated carboxyl polystyrene particles, 96 well filter assay plates, a goat antimouse fluorescein isothiocyanate (FITC) secondary  132 antibody for detection at 535 nm wavelength (excitation at 485 nm), and a P C F I A buffer (filtered through a 0.22 urn filter), pH 7.4.  Supernatants from the hybridoma cells were screened for hapten affinity using the P C F I A immunoassay with BSA-50 conjugate-coated carboxyl polystyrene particles as the solid phase.  Cells from cultures displaying anti-hapten antibodies were subcloned by limited dilution in 96-well plates as described below.  4.1.5.9 C l o n i n g C e l l L i n e s  Any hybridoma cells from cultures displaying anti-hapten activity were cloned by limited dilution. The cells from a positive well (100 LIL of cell suspension) were transferred and titrated 1:2 dilution step down the first column of a fresh 96-well growth plate that contained 100 \iL of media per well. The column was then also titrated across the plate and each well made up to a final volume of 100 LIL with additional media. The cells were allowed to grow for approximately 10 days, supernatants  from  wells  with  hybridomas  were  assayed,  and  then  hapten-specific colonies were expanded as described above. Copies of these clones were expanded in 2 mL tissue culture dishes and frozen in liquid nitrogen. media.  To prepare the clones for freezing, they were expanded in growth Once a cell density of 1 - 2 x 1 0  5  cells/mL was reached the cells were  133 centrifuged at 300 g for 5 minutes and supernatant was removed. The cell pellet was resuspended in freeze medium at a concentration of 2 x 1 0 cells/mL and 6  transferred to a 1 mL freezing vial.  The cells were then cooled at a rate of  1 °C/minute until they were frozen at a temperature -195 °C. The frozen cells were then transferred into the liquid nitrogen storage tank. Once the hybridomas have been cloned and copies frozen in liquid nitrogen, they can be assayed for catalytic activity.  Large quantities of monoclonal antibodies were produced in  pristane-phmed Balb/c mice as described below.  4.1.5.10  Ascites Production  Prior to ascites production, the Balb/c mice (6-8 week old females) were primed by injection of 200 LIL of pristane (2,6,10,14-tetramethylpentadecane) into the interperitoneal cavity.  Pristane acts as an irritant causing macrophages to  secrete cytokines thus creating a good environment for growing hybridomas. Approximately 10-14 days after pristane priming, each mouse was injected with 1 x 10 hybridoma cells in 0.5 mL of P B S into the peritoneal cavity using a 6  22 gauge needle. After 10-14 days the liquid tumors (ascites fluid) were ready to harvest. The mice were anesthetized with carbon dioxide and killed by cervical dislocation. A small incision was made in the skin above the abdomen and the skin pulled back to expose the abdomen muscles. Using tweezers, the muscles were pinched and held upwards like a tent, and a small incision was made through the muscles just large enough to drain the ascites fluid using a  134 disposable Liquipette. The fluid was centrifuged to pellet any cells and the oily pristane layer on top was removed. The ascites fluid was decanted into a new tube and stored at -20 °C.  4.2 G E N E R A L C H E M I C A L M E T H O D S  Unless otherwise stated, all reactions were performed under a N atmosphere 2  using flame-dried glassware. Cold temperature baths were prepared as follows: -78 °C (dry ice-acetone), -20 °C (dry ice-CCI ), and 0 °C (ice-water). 4  Anhydrous reagents and solvents were purified and prepared according to literature procedures.  162  Chromatographic solvents and reagents were used as  received unless noted otherwise.  The low boiling fraction (35-60 °C) of  petroleum ether was used. All reagents were supplied by the Aldrich Chemical Co.  and unless otherwise stated were used without further  purification.  n-Butyllithium (n-BuLi) was standardized by titration against 2,2-diphenylacetic acid in T H F at room temperature to the appearance of a faint yellow colour.  All reactions were monitored by thin layer chromatography (TLC) and were judged to be completed when the starting material was consumed as determined by T L C . In the description of reaction work-ups, washing with brine refers to a saturated solution of NaCI, drying of the organic phase was accomplished with  135 M g S 0 and removal of solvent in vacuo or concentration of solvent refers to the 4  use of a rotary evaporator using a water aspirator and heating using a water bath.  Preparative flash chromatography  163  was performed using 230-400 mesh A S T M  silica gel supplied by E. Merck Co. A s an indicator of purity, all compounds were purified such that they showed a single spot by T L C .  Melting points (mp) were determined using a Mel-Temp III (Laboratory Devices) melting point apparatus and are uncorrected.  Infrared (IR)  spectra were recorded on a Bomem Michelson 100 FT-IR  spectrometer using internal calibration.  IR spectra were recorded on the neat  liquid or as a chloroform solution using NaCI solution cells.  Except where noted, proton nuclear magnetic resonance spectra ( H NMR) were 1  recorded in deuteriochloroform solutions on a Bruker A C - 2 0 0 (200 MHz) or Bruker WH-400 (400 MHz) spectrometer.  Chemical shifts are reported in parts  per million (ppm) on the 8 scale versus chloroform (8 7.24 ppm) as an internal standard. Signal multiplicity, spin-spin coupling constants (where possible) and integration ratios are indicated in parentheses. resonance spectra ( P 31  NMR) with proton  Phosphorus nuclear magnetic decoupling were  recorded  in  136 deuteriochloroform solutions on a Bruker A C - 2 0 0 (81.75 MHz) spectrometer. Chemical shifts are given in parts per million (ppm) on the 8 scale versus 85% phosphoric acid as an external standard.  Low- and high-resolution electron-impact (El) mass spectral analyses were performed on a Kratos-AEI model M S 50 mass spectrometer. energy of 70 e V was used in all measurements.  A n ionization  Low-resolution desorption  chemical ionization (DCI) mass spectral analyses, using C H or N H reagent gas 4  3  as indicated, were recorded on a Delsi Nermag R10-10C mass spectrometer. High-resolution DCI mass spectral analyses, using C H or N H reagent gas as 4  3  indicated, were recorded on a Kratos M S 80 R F A mass spectrometer.  Kinetic analyses using ultraviolet-visible (UV-Vis) spectroscopy were performed on a UV-4 Unicam spectrophotometer.  Thin-layer chromatography was performed on Merck silica gel 60 F  2 5 4  pre-coated  aluminium sheets. Visualization was achieved by irradiation with ultraviolet light at 254 nm and/or by spraying with anisaldehyde reagent (a solution of 1 mL anisaldehyde, 5 mL cone. H S 0 and 10 mL glacial acetic acid in 90 mL MeOH) 2  followed by heating.  4  137 Microanalyses were carried out at the microanalytical laboratory of the University of British Columbia Chemistry Department Analyzer  1106.  Samples for  using a Carlo Erba Elemental  microanalysis  were  purified  by  chromatography using the solvent system indicated for each compound.  column  138 4.3 C H E M I C A L M E T H O D S  11 -(Tetrahydropyranyloxy)-dodecanal (33). OTHP  To a stirred solution of oxalyl chloride (1.01 mL, 11.5 mmol) in 75 mL of dry C H C I under N at -78 °C was added DMSO (1.80 mL, 25.2 mmol) dissolved 2  2  2  in 5 mL C H C I via a stainless steel cannula. 2  2  After 10 min. of stirring,  compound 42 (3.00 g, 3.50 mmol) in 25 mL of C H C I was added to the 2  activated D M S O via a stainless steel cannula.  2  Finally, after stirring an  additional 10 min., EUN (7.31 mL, 52.5 mmol) was syringed in, and the reaction mixture was allowed to warm to room temperature while maintaining stirring. After 3 h, the solution was diluted with 100 mL of E t 0 followed by 2  100 mL of water. The aqueous layer was further extracted with 3 x 50 mL of E t 0 and the combined E t 0 extracts were dried over anhydrous M g S 0 , 2  2  4  filtered and concentrated under reduced pressure to a yellow oil. The crude product was then purified by flash chromatography using 4:1 petroleum ether and ethyl acetate to afford 2.59 g (87%) of 33 as a colourless oil.  IR(neat): 2927, 2854, 2728,1725,1458,1362,1322,1124,1075, 901, 867 cm" ; 1  1  H N M R (200 MHz, CDCI ) 8: 9.74 (t, J = 1.7 Hz, 1H), 4.68 (m, 0.5H), 4.62 3  139 (m, 0.5H), 3.88 (m, 1H), 3.75 (m, 0.5H), 3.68 (m, 0.5H), 3.46 (m, 1H), 2.38 (m, 2H), 1.22-1.90 (m, 22H), 1.18 (d, J = 6.2 Hz, 1.5H), 1.07 (d, J =6.2 Hz, 1.5H); LRMS (DCI, NH ) m/z (relative intensity): 302 (M + 18,12), 285 (M + 1, 3), +  +  3  218(30), 201 (15); HRMS (DCI, C H ) calcd. for C17H32O3: 284.2351, found 284.2409. 4  1 -Diphenylphosphinyl-12-(tetrahydropyranyloxy)-1 -tridecene (36a  and  36b). OTHP  (OPhb 36a + 36b The Wittig reagent 32 (2.15 g, 4.23 mmol) and aldehyde 33 (1.2 g, 4.2 mmol) were dissolved in 17 mL of toluene and refluxed under N for 3 days. The 2  solvent was removed under reduced pressure and the crude product was purified by flash chromatography using 5:1 petroleum ether and ethyl acetate to afford 1.12 g (52%) of a colourless oil which was a mixture of cis and trans isomers 36a and 36b. A small quantity of the crude product was repurified by flash chromatography using 5:1 petroleum ether and ethyl acetate and pure 36a, the cis isomer, and pure 36b, the trans isomer, were isolated separately for analysis.  140 Cis isomer 36a: IR(neat): 2929, 2855, 1623, 1593, 1490,1459,1377,1270, 1192, 1163, 1129,1074, 1025, 998, 931, 763 cm" ; 1  1  H NMR (400 MHz, CDCI ) 8: 7.15-7.38 (m, 10H), 6.59 (ddt, J = 7.8, 13.0, 3  57.4 Hz, 1H), 5.76 (dd, J = 13.0, 21.8 Hz, 1H), 4.74 (m, 0.5H), 4.67 (m, 0.5H), 3.94 (m, 1H), 3.81 (m, 0.5H), 3.74 (m, 0.5H), 3.52 (m, 1H), 2.27 (dt, J = 7.8, 7.8 Hz, 2H), 1.85 (m, 1H), 1.72 (m, 1H), 1.24-1.68 (m, 20H), 1.23 (d, J = 6.2 Hz, 1.5H), 1.13 (d, J = 6.2 Hz, 1.5H); 3 1  P NMR (81 MHz, CDCI ) 8: 9.7; 3  L R M S (El) m/z (relative intensity): 514 (0.2), 429 (6), 415 (20), 414 (54), 413 (100), 94(28), 85(31); H R M S calcd. for  C30H43O5P:  514.2848, found 514.2835;  Anal. Calcd. for C ^ O g P : C, 70.02; H, 8.42. Found: C, 69.93; H, 8.34.  Trans isomer 36b: IR (neat): 2929, 2854,1628, 1593, 1490, 1455,1374, 1273, 1198, 1164, 1128, 1075, 1025, 996, 932, 869, 826, 767 cm" ; 1  1  H N M R (400 MHz, CDCI ) 5: 7.10-7.38 (m, 10H), 7.20 (ddt, J = 6.6,17.0, 3  25.4 Hz, 1H), 5.88 (dd, J = 17.0, 23.4 Hz, 1H), 4.74 (m, 0.5H), 4.67 (m, 0.5H), 3.94 (m, 1H), 3.81 (m, 0.5H), 3.74 (m, 0.5H), 3.52 (m, 1H), 2.27 (dt, J = 6.6, 7.0 Hz, 2H), 1.85 (m, 1H), 1.72 (m, 1H), 1.24-1.68 (m, 20H), 1.23 (d, J = 6.2 Hz, 1.5H), 1.13 (d, J = 6.2 Hz, 1.5H);  141  3 1  P N M R (81 MHz, CDCI3) 8:11.9;  L R M S (El) m/z (relative intensity): 514 (0.2), 429 (6), 415 (20), 414 (54), 413 (100), 94 (28), 85 (31); H R M S calcd. for C30H43O5P: 514.2848, found 514.2852; Anal. Calcd. for C30H43O5P: C, 70.02; H, 8.42. Found: C, 69.91; H, 8.30.  12-Hydroxy-1-diphenylphosphinyl-tridecane (37).  37 p-TsOH (0.65 g, 3.4 mmol) was added to a stirred solution of compound 43 (8.75 g, 17.0 mmol) in 200 mL of M e O H at room temperature. After 18 h, the MeOH was removed under reduced pressure and the resulting oil was dissolved in E t 0 , washed with 2 x 50 mL of water followed by 2 x 50 mL of 2  brine, dried over anhydrous M g S 0 , filtered and concentrated under reduced 4  pressure.  The crude oil was purified by flash chromatography using 1:1  petroleum ether and ethyl acetate to afford 6.31 g (86%) of alcohol 37 as a colourless oil.  IR (neat): 3424, 3066, 2925, 2854,1592,1490,1460,1265,1214,1192, 1163,1071,1025,1007, 934, 802, 764 cm ; -1  1  H N M R (400 MHz, CDCI ) 8: 7.11-7.32 (m, 10H), 3.76 (m, 1H), 2.04 (m, 2H), 3  1.77 (m, 2H), 1.20-1.50 (m, 18H), 1.15 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 5: 25.9; 3  L R M S (El) m/z (relative intensity): 432 (1), 431 (4), 415 (7), 414 (19); H R M S calcd. for C25H37O4P: 432.2429, found 432.2398; Anal. Calcd. for C H370 P: C, 69.41; H, 8.63. Found: C, 69.62; H, 8.72. 25  4  1-Phenyl-1-oxo-1-phospho-13-tetradecanolide (38a and 38b) and Dimer 39. O  38a + 38b To a stirred solution of compound 44 (125 mg, 0.351 mmol) in 150 mL of dry PhH at room temperature under N was added P h P (368 mg, 1.40 mmol) 2  3  followed by DEAD (221 LII, 1.40 mmol). After 2 h, the solvent was removed under reduced pressure and the resulting crude mixture was purified by flash chromatography using 2:1 hexane and ethyl acetate to afford the separated diastereomers; 38a and 38b, in a 5:1 ratio based on the recovered yields, and a combined yield of 97.1 mg (82%). In addition, 6 mg of the dimeric side product, 39, was isolated as a colourless, crystalline solid. The R* values of  143 the three cyclic products, 38a, 38b and 39, were found to be 0.71, 0.57 and 0.62 respectively by thin-layer chromatography using a 1:1 mixture of petroleum ether and ethyl acetate.  Diastereomer 38a (faster moving by TLC): IR(neat): 2927, 2859, 1593, 1490, 1458, 1380, 1253, 1210, 1163, 1071, 991, 920, 766 cm ; -1  1  H NMR (400 MHz, CDCI ) 5: 7.05-7.38 (m, 5H), 4.75 (m, 1H), 1.24-1.88 (m, 3  22H), 1.22 (d, J = 6.3Hz, 3H); 3 1  P NMR (81 MHz, CDCI ) 5: 29.6; 3  L R M S (El) m/z (relative intensity): 338 (52), 323 (5), 227 (16), 199 (9), 185 (38), 172 (69), 94 (100), 93 (31), 77 (17), 43 (39); H R M S calcd. for C i H 0 P : 338.2011, found 338.2002; 9  31  3  Anal. Calcd. for C i H 0 P : C, 67.42; H, 9.24. Found: C, 67.20; H, 9.20. 9  31  3  Diastereomer 38b (slower moving by TLC): mp: 97-99 °C IR (CDCI ): 2932, 2860, 1593, 1492,1456, 1223, 1011 cm ; -1  3  1  H N M R (400 MHz, CDCI ) 6: 7.05-7.38 (m, 5H), 4.65 (m, 1H), 1.93 (m, 2H), 3  1.20-1.73 (m, 20H), 1.43 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 5: 27.8; 3  L R M S (El) m/z (relative intensity): 338 (26), 323 (2), 227 (11), 199 (7), 185  144 (30), 172 (59), 94 (100), 93 (3), 77 (20), 43 (75); H R M S calcd. for C i H i 0 P : 338.2011, found 338.2009; 9  3  3  Anal. Calcd. for C i H 0 P : C, 67.42; H, 9.24. Found: C, 67.15; H, 9.39. 9  3 1  3  Dimer 39: mp: 103-105°C IR (CDCI ): 2930, 2856, 1593, 1491, 1229, 1012 cm" ; 1  3  1  H NMR (400 MHz, CDCI ) 5: 7.10-7.33 (m, 10H), 4.66 (m, 2H), 1.84 (m, 4H), 3  1.68 (m, 4H), 1.56 (m, 4H), 1.20-1.45 (m, 40H), 1.15 (d, J = 6.2 Hz, 6H); 3 1  P NMR (81 MHz, CDCI ) 8: 29.0; 3  L R M S (El) m/z (relative intensity): 676 (1), 661 (1), 583 (100), 339 (19), 338 (10), 245 (7), 227 (12), 199 (6), 185 (15), 172 (21), 94 (27), 77 (5); H R M S calcd. for CasHezOePa: 676.4022,,found 676.4002; Anal. Calcd. for C M H K O S P * C, 67.42; H, 9.24. Found: C, 67.51; H, 9.28.  11-Dodecen-2-ol (40). OH 40  ^  To a stirred solution of commercially available 10-undecenal (8.1 g, 48 mmol) in 250 mL of T H F under N at 0 °C was slowly added 3 M methylmagnesium 2  bromide (18 mL, 53 mmol).  After 0.5 h the reaction was quenched with  145 saturated aqueous NH CI, diluted with E t 0 , and the resulting precipitate was 4  2  filtered and discarded. The filtrate was washed with 2 x 100 mL of brine, dried over anhydrous M g S 0 , filtered and the solvent removed under 4  reduced pressure to give 13.6 g of crude oil. The residue was purified by flash chromatography using 4:1 petroleum ether and ethyl acetate to afford 7.96 g (90%) of 40 as a colourless oil.  IR (neat): 3351, 3077, 2922, 2855, 1641, 1457, 1373, 1122, 994, 910, 842, 722 cm" ; 1  1  H NMR (400 MHz, CDCI ) 8: 5.78 (ddt, J = 6.7, 1.0.4, 17.3 Hz, 1H), 4.94 3  (d,d, J = 10.4,17.1 Hz, 2H), 3.75 (m, 1H), 2.02 (dt, J = 6.3, 6.7 Hz, 2H), 1.63 (br. s, 1H), 1.20-1.50 (m, 14H), 1.16 (d, J = 6.2 Hz, 3H); LRMS (El) m/z (relative intensity): 184 (0.4), 166 (2), 151 (1), 141 (2), 138 (3), 124 (10), 109 (10), 96 (29), 82 (51), 69 (40), 45 (100), 41 (62); HRMS calcd. for C i H 0 : 1 8 4 . 1 8 2 7 , found 184.1832; 2  24  Anal. Calcd. for C i H 0 : C, 78.20; H, 13.12. Found: C, 78.40; H, 12.99. 2  2 4  11 -(Tetrahydropyranyloxy)-I -dodecene (41). OTHP 41  To a stirred solution of 40 (10 g, 54 mmol) in 300 mL of C H C I was added 2  2  DHP (5.5 mL, 65 mmol) and p-TsOH (0.52 g, 2.7 mmol), and the mixture was stirred at room temperature under N for 2 h. The solvent was then removed 2  and the residue was diluted with E t 0 . The ether layer was washed with 2 x 2  25 mL of saturated N a H C 0 , 1 x 25 mL water and 1 x 50 mL of brine, and 3  dried over anhydrous M g S 0 and filtered. The solvent was removed under 4  reduced pressure and the crude material (15.7 g of oil) was purified by flash chromatography using 10:1 petroleum ether and ethyl acetate to afford 12.2 g (84%) of 41 as a colourless oil.  I R (neat): 3076, 2929, 2855, 1727, 1641, 1453, 1375, 1352, 1321, 1260, 1200, 1126, 1076, 1026, 996, 911, 869, 812, 734 cm" ; 1  1  H N M R (400 MHz, CDCI ) 5: 5.78 (ddt, J = 6.7, 10.4, 17.1 Hz, 1H), 4.93 3  (dd, J = 10.4, 17.1 Hz, 2H), 4.70 (m, 0.5H), 4.59 (m, 0.5H), 3.89 (m, 1H), 3.76 (m, 0.5H), 3.68 (m, 0.5H), 3.47 (m, 1H), 2.02 (dt, J = 6.3, 6.7 Hz, 2H), 1.22-1.90 (m, 20H), 1.07 (d, J = 6.2 Hz, 1.5H), 1.19 (d, J = 6.2 Hz, 1.5H); LRMS  (El) m/z (relative intensity): 268 (0.3), 182 (1), 167 (2), 129 (25), 101 (43), 97 (28), 85 (100), 83 (36), 69 (40), 55 (73), 41 (67);  HRMS  calcd. for C I T H K C V 268.2402, found 268.2404;  Anal. Calcd. for C i H 0 : C, 76.06; H, 12.02. Found: C, 76.23; H, 11.95. 7  3 2  2  11 -(Tetrahydropyranyloxy)-I -dodecanol (42).  OH  42  To compound 41 (1.00 g, 3.73 mmol) in 5 mL of dry T H F at 0 °C under N  2  was added 1 M BH .THF (3.0 mL, 3.0 mmol) dropwise via syringe. After 1 h, 3  the reaction was quenched with 1.0 mL of water, and oxidized by the addition of 1.2 mL of 3N N a O H and 1.2 mL of 30% H 0 . After 15 minutes of vigorous 2  2  stirring, the mixture was extracted with 3 x 50 mL of E t 0 . 2  The combined  ether extracts were dried over M g S 0 , filtered and concentrated under 4  reduced pressure. The crude material was purified by flash chromatography using 4:1 petroleum ether and ethyl acetate to afford 1.02 g (96%) of 42 as a colourless oil.  IR (neat): 3391, 2928, 2855, 1455,1375, 1339,1321,1260,1200, 1184, 1126, 1076, 1026, 995, 941, 904, 869, 811, 722 cm- ; 1  1  H NMR (400 MHz, CDCI ) 8: 4.70 (m, 0.5H), 4.59 (m, 0.5H), 3.89 (m, 1H), 3  3.76 (m, 0.5H), 3.68 (m, 0.5H), 3.62 (dt, J = 5.1, 6.4 Hz, 2H), 3.47 (m, 1H), 1.81 (m, 1H), 1.68 (m, 1H), 1.53 (m, 6H), 1.18-1.43 (m, 16H), 1.19 (d, J = 6.2 Hz, 1.5H), 1.08 (d, J = 6.2 Hz, 1.5H); L R M S (DCI, NH ) m/z (relative intensity): 304 (M + 18,100), 287 ( M + 1, +  3  53), 286 (M , 5), 262 (6); +  +  148 HRMS (DCI, C H ) calcd. for C17H34O3: 286.2508, found 286.2499; 4  Anal. Calcd. for C17H34O3: C, 71.27; H, 11.97. Found: C, 71.04; H, 11.91.  1 -Diphenylphosphinyl-12-(tetrahydropyranyloxy)-tridecane (43). OTHP (OPhb  A spatula tip of Pd-C (10%) in 250 mL of EtOAc was saturated with H for 15 2  h before compound 36 (9.00 g, 17.5 mmol) in 5 mL of EtOAc was added via a steel cannula. This mixture was allowed to stir for an additional 3 days under H gas at a pressure slightly greater than one atmosphere. 2  The reaction  mixture was filtered through Celite and concentrated under reduced pressure to afford 8.95 g (99%) of 43 as a colourless oil.  IR (neat): 2929, 2854, 1739,1593, 1491,1461, 1373,1272, 1200,1163, 1127, 1074, 1026, 931, 870, 808, 764 cm' ; 1  1  H NMR (400 MHz, CDCI ) 8: 7.11-7.32 (m, 10H), 4.68 (m, 0.5H), 4.62 (m, 3  0.5H), 3.88 (m, 1H), 3.72 (m, 1H), 3.47 (m, 1H), 2.04 (m, 2H), 1.22-1.90 (m, 26H), 1.08 (d, J = 6.2 Hz, 1.5H), 1.19 (d, J = 6.2 Hz, 1.5H); 3 1  P NMR (81 MHz, CDCI3) 6: 26.0;  LRMS (El) m/z (relative intensity): 516 (0.4), 472 (0.2), 416 (42), 415 (68),  149 388 (30), 359 (3), 345 (5), 303 (6), 289 (4), 261 (22), 248 (29), 172 (11), 141 (10), 94 (81), 85 (61), 78 (98), 77 (46), 55 (100); HRMS calcd. for Anal. Calcd. for  516.3005, found 516.3007;  C30H45O5P: C30H45O5P:  C, 69.73; H, 8.78. Found: C, 69.94; H, 8.90.  Phenyl 12-Hydroxytrideane Phosphonic acid (44).  OH OPh  Compound 37 (199 mg, 0.461 mmol) was dissolved in 5 mL of T H F and 20 mL of 3M KOH, and refluxed for 1 h. The T H F was removed under reduced pressure and the remaining aqueous solution was acidified with 4N HCI, then extracted with E t 0 . The combined E t 0 extracts were dried over anhydrous 2  2  M g S 0 , filtered and evaporated under reduced pressure to afford 125 mg 4  (76%) of 44 as a white solid.  mp: 72-73°C IR (CDCI3): 3614, 2929, 2856, 1593, 1492, 1456, 1213, 1010, 984 cm" ; 1  1  H N M R (400 MHz, CDCI ) 8: 7.08-7.30 (m, 5H), 6.82 (br s, 2H), 3.78 (m, 3  1H), 1.80 (m, 2H), 1.62 (m, 2H), 1.20-1.50 (m, 18H), 1.17( d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 6: 32.7. 3  L R M S (El) m/z (relative intensity): 356 (0.8), 355 (3), 341(8), 339 (2), 338 (5), 312(17), 227(12), 199 (12), 185 (49), 172 (79), 157(2), 110 (4), 94 (100), 77 (22); HRMS calcd. for  C19H33O4P:  356.2116, found 356.2108;  Anal. Calcd. for C19H33O4P: C, 64.01; H, 9.34. Found: C, 64.22; H, 9.45.  N-Cbz-5-amino-1-pentanol (48). O H  \ / \ ^ ^ ^  O  N  H  C  O  C  H  2  C  6  H  5  48  To a stirred solution of commercially available 5-amino-1-pentanol (1.00 g, 9.69 mmol) in 30 mL of aqueous N a C 0 2  3  at 0 °C was added benzyl  chloroformate (Cbz-CI) (0.96 mL, 11 mmol) drop wise. The reaction mixture was stirred and allowed to warm to room temperature. After one hour the solution was diluted with EtOAc (50 mL) and the organic layer was extracted. The aqueous layer was extracted twice more with EtOAc (75 mL). The combined organic extracts were washed with 1M HCI (50 mL), saturated aqueous N a H C 0 (50 mL), and finally with water (2 x 50 mL). The mixture 3  was dried over M g S 0 , filtered and the solvent was removed under reduced 4  pressure. The crude product was purified by flash chromatography using 2:1 petroleum ether and ethyl acetate to afforded 0.78 g (34%) of 48 as a white  151 solid.  In addition, 1.69 g of colourless oil, thought to be the di-protected  product, was isolated and subsequently hydrolyzed in 1M NaOH under reflux conditions. This reaction mixture was neutralized with 1M HCI, extracted with EtOAc, dried over anhydrous M g S 0 , filtered and the solvent was removed 4  under reduced pressure to yield an additional 1.08 g of compound 48. The combined yield of 48 was 81 %.  mp: 43-45 °C; IR (CDCI ): 3692, 3620, 3453, 2939, 2862,1718,1516,1232 cm" ; 1  3  1  H N M R (400 MHz, CDCI ) 6: 7.33 (m, 5H), 5.08 (s, 2H), 4.72 (br s, 1H), 3  3.62 (t, J = 6.4 Hz, 2H), 3.19 (q, J = 6.5 Hz, 2H), 1.65-1.30 (m, 6H); L R M S (DCI, NH ) m/z (relative intensity): 255 (M + 18,10), 238 ( M + 1, 75), +  +  3  237 (M , 3), 220 (1), 194 (99), 146 (2), 108 (62), 102 (15), 91 (100); +  H R M S calcd. for C i H N 0 : 237.1365, found 237.1373; 3  19  3  Anal. Calcd. for C i H N 0 : C, 65.78; H, 8.07; N, 5.91. Found: C, 65.86; 3  19  H, 8.23; N 5.93.  3  152 C b z linked 1-oxo-1-phospho-13-tetradecanolide (49a). 0  0  r^^P-0(CH )5NHCOCH C H5 2  2  6  49a The alcohol 48 (HO(CH ) NHCbz) (26.0 mg, 108 Limol) was dissolved in 4.0 2  5  mL of T H F at -78 °C under a N atmosphere and treated with 1.2 M nBuLi 2  (98.0 LIL, 118 Limol). The alkoxide was allowed to form for 10 min before this mixture was cannulated into a stirred solution of 38b (16.6 mg, 49.0 Limol) in 8.0 mL of T H F at -78 °C under a N atmosphere. This mixture was allowed to 2  warm slowly to room temperature, and was stirred overnight. The T H F was then removed under reduced pressure, and the crude oil purified by column chromatography (2% M e O H in CH CI ) to afford 13.8 mg (59%) of 49a, a 2  2  colourless oil.  IR(CDCI ): 3453, 2935, 2861,1715, 1516,1451,1233,1016 cm' ; 1  3  1  H NMR (400 MHz, CDCI ) 6: 7.34 (m, 5H), 5.07 (s, 2H), 4.86 (br s, 1H), 3  4.59 (ddq, J = 5.3, 6.2, 6.5 Hz, 1H), 3.95 (m, 2H), 3.17 (dt, J = 6.4, 6.5 Hz, 2H), 1.20-1.80 (m, 28H), 1.30 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 5: 32.5; 3  153 L R M S (DCI, NH ) m/z (relative intensity): 482 (M + 1,100), 374 (3), 284 (4), +  3  255 (14), 238 (30), 194 (31), 91 (10); H R M S calcd. for Anal. Calcd. for  C26H44NO5P: C26H44NO5P:  481.2957, found 482.3032 ( M + 1); +  C, 64.83; H, 9.21; N, 2.91. Found: C, 64.59; H,  9.10; N, 3.00.  C b z linked 1-oxo-1-phospho-13-tetradecanolide (49b). O O // II P-0(CH )5NHCOCH2C H5 2  6  49b Alcohol 48 (HO(CH ) NHCbz) (59.0 mg, 230 Limol) was dissolved in 15.0 mL 2  5  of THF at -78 °C under a N atmosphere and treated with 1.4 M nBuLi 2  (176 u.L, 250 Limol). The alkoxide was allowed to form for 10 min before this mixture was cannulated into a stirred solution of 38a (69.0 mg, 283 Limol) in 10.0 mL of T H F at -78 °C under a N atmosphere. This mixture was allowed 2  to warm slowly to room temperature, and was stirred overnight.  The T H F  was then removed under reduced pressure, and the crude oil purified by column chromatography (2% MeOH in CH CI ) to afford 18.5 mg (14%) of 2  49b, a colourless oil.  2  IR(neat): 3293, 3064, 3033, 2927, 2859,1711, 1533,1456,1242, 1013 cm' ; 1  1  H N M R (400 MHz, CDCI ) 5: 7.33 (m, 5H), 5.07 (s, 2H), 4.84 (br s, 1H), 3  4.43 (ddq, J = 2.7, 3.7, 6.3,1H), 4.01 (m, 2H), 3.18 (dt, J = 6.4, 6.6 Hz, 2H), 1.20-1.80 (m, 28H), 1.35 (d, J = 6.3 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 8: 31.3; 3  L R M S (El) m/z(relative intensity): 481 (15), 374 (55), 346 (20), 263 (100), 219(10), 193 (7), 91 (69); HRMS calcd. for  C26H44NO5P:  481.2957, found 481.2951.  1 -O-linker-1 -oxo-1 -phospho-13-tetradecanolide (50). 0 r^  N v  P-0(CH )5NH2 2  50 A spatula tip of Pd-C (10%) stirring in 15 mL of EtOH was saturated with H  2  for 12 h before a solution of compound 49a (13.8 mg, 28.6 Limol) in 5 mL of EtOH was added via a steel cannula. This mixture was allowed to stir for an additional five hours under H pressure of one atmosphere. 2  The reaction  155 mixture was filtered through Celite and concentrated under reduced pressure to afford 8.5 mg (85%) of 50 as a white solid.  IR (CDCI ): 3692, 3632, 3181, 2934, 2861, 1603, 1265 1209, 1008 cm ; -1  3  1  H N M R (400 MHz, CDCI ) 8: 8.37 (br s, 2 H), 4.61 (m, 1 H), 4.05 (m, 1H), 3  3.95 (m, 1H), 3.01 (brs, 2 H), 1.20-1.90 (m, 28 H), 1.32 (d, J = 6.2 Hz, 3H); 3 1  P NMR (81 MHz, CDCI3) 8: 33.1;  L R M S (DCI, NH ) m/z (relative intensity): 348 ( M + 1, 93), 345 (7), 344 (30), +  3  86(21);  H R M S (DCI, C H + NH ) calcd. for C^H^NC-aP: 347.2589, found 347.2593. 4  3  4-Benzoxybutanoic anhydride (52).  52  O  4-Benzoxybutanoic acid (56) (2.0 g, 10 mmoles) was dissolved in 50 mL of anhydrous E t 0 with D C C (1.1 g, 5.33 mmoles) at room temperature under 2  N . After 3.5 hours of stirring, the D C U (dicyclohexylurea) formed during the 2  reaction was filtered off by aspiration, and the ether was removed under reduced pressure to give a quantitative yield of anhydride 52 as a colourless  156 oil. The crude 52 obtained was carried on to the next step without further purification.  IR(CDCI ): 3031, 2932, 2857, 1814, 1719, 1643, 1496, 1452, 1274, 1101, 3  1067,741 cm" ; 1  1  H NMR (200 MHz, CDCI ) 8: 7.33 (m, 5H), 4.49 (s, 2H), 3.50 (t, J = 6.2 Hz, 3  2H), 2.57 (t, J = 6.2 Hz, 2H), 1.95 (quint, J = 6.2 Hz, 2 H); L R M S (DCI, NH ) m/z (relative intensity): 388 (M + 18, 70), 195 (9), 193 (3), +  3  178 (11), 177 (100), 108 (26), 91 (23); HRMS (DCI, CH ) calcd. for C12H22O5: (M + 1) 371.1856, found +  4  (M +1)371.1874. +  Indoyl 4-benzoxybutanoate (53).  H  To a N purged solution of 2N NaOH (5 mL) in a 3-neck round-bottom flask, 2  fitted with a condenser, was added 3-indolyl acetate (135 mg, 0.77 mmoles) via a stainless steel cannula. The mixture was heated to reflux until the 3-indolyl acetate had completely dissolved. The reaction mixture was then cooled to 0 °C, then anhydride 52 was added via cannula. After one hour of  157 stirring at 0 °C the mixture was transferred to a separatory funnel containing E t 0 and water. The layers were separated, and the ether layer was dried 2  over M g S 0 , filtered, and the solvent removed under reduced pressure to 4  yield 102 mg (43%) of 53 as a dark red-burgundy oil.  IR (CDCI ): 3478, 3065, 2959, 2931, 2868, 1747, 1223, 1156, 1128 cm' ; 1  3  1  H N M R (200 MHz, CDCI ) 8: 7.84 (br s, 1H), 7.60-7.08 (m, 10H), 4.54 (s, 3  2H), 3.62 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 6.2 Hz, 2H), 2.10 (m, J = 6.2 Hz, 2H); L R M S (El) m/z (relative intensity): 309 (0.4), 177 (4), 132 (40), 107 (36), 91 (100), 77 (7);  H R M S calcd. for C H N 0 : (M + 1) 310.1443, found (M + 1) 310.1445. +  1 9  1 9  +  3  (Indoyl butanoate) 13-hydroxytetradecanoate (55). OH O  Crude 53 (143 mg, 0.461 mmoles) was hydrogenolyzed overnight under an atmosphere of N (slightly greater than one atmosphere of pressure) with a 2  spatula tip of 10% Pd/C in 30 mL of EtOH. The mixture was filtered through  158 Celite, and the EtOH removed under reduced pressure, and the crude product was reacted with the hydroxy acid 54 (134 mg, 0.548 mmoles) using D C C (113 mg, 0.548 mmoles) and a spatula tip of DMAP (11 mg, 0.091 mmoles) in CH CI . After 2 hours of stirring, the reaction mixture was filtered 2  2  by aspiration to remove the DHU formed, and the C H C I was evaporated 2  2  under reduced pressure. The resulting crude red oil was purified on silica using a 2:1 mixture of petroleum ether and EtOAc to give 78 mg (38%) of the substrate 55.  IR (CDCI ): 3395, 2924, 2852, 1730, 1458, 1164, 740 cm" ; 1  3  1  H N M R (200 MHz, CDCI ) 5: 8.02 (br s, 1H), 7.58 (m, 5H), 4.21 (t, 3  J = 6.2 Hz, 2H), 3.78 (m, 1H), 2.73 (t, J = 6.2 Hz, 2H), 2.30 (t, J = 6.2 Hz, 2H), 2.14 (t, J = 6.2 Hz, 2H), 1.60 (t, J = 6.2 Hz, 2H), 1.40 (m, 2H), 1.25 (m, 16 H), 1.17 (d, J = 6.0Hz, 3H); L R M S (El) m/z (relative intensity): 445 (0.7), 226 (4), 184 (0.5), 141 (24), 133(100), 86(1); H R M S calcd. for C H39N0 : (M ) 445.2828, found (M*) 445.2829; +  26  5  p-Nitrophenyl 13-Hydroxytetradecanoate (57). OH  0  159  To a stirred solution of 54 (100 mg, 0.41 mmoles) in 20 mL of C H C I was 2  2  added p-nitrophenol (68 mg, 0.49 mmoles), D C C (102 mg, 0.38 mmoles) and DMAP (10 mg, 82 Limoles).  The mixture was allowed to stir at room  temperature under N overnight. 2  reduced  pressure,  and  the  The solvent was then removed under  crude  material  was  purified  by  flash  chromatography using 4:1 petroleum ether and ethyl acetate to afford 130 mg (87%) of 57 as a white solid. A small quantity of p-nitrophenol was found to be present in the purified sample of 57 as seen by the multiplets at 8.15 and 6.88 ppm in their H NMR spectra which correspond to the aromatic protons 1  of p-nitrophenol.  mp: 69-72°C; IR (CDCI ): 3613, 2930, 2856, 1762, 1616, 1594, 1492, 1348, 1208 cm ; -1  3  1  H N M R (400 MHz, CDCI ) 6: 8.25 (m, 2H), 7.25 (m, 2H), 3.78 (m, J = 6.2 Hz, 3  1H), 2.57 (t, J = 7.5 Hz, 2H), 1.74 (quint, J = 7.4 Hz, 2H), 1.21-1.63 (m, 19H), 1.18 (d, J = 6.2 Hz, 3H); L R M S (DCI, NH ) m/z (relative intensity): 383 (M* + 18, 85), 366 ( M + 1, 4), +  3  365 (3), 348 (77), 227 (10), 226 (22), 225 (100), 143 (15), 125 (10), 123 (35), 110(12), 109(16), 100(13), 99(25), 98(18); H R M S (DCI, NH ) calcd. for C20H32NO5: (M + 1) 366.2281, found +  3  (M +1)366.2271; +  160 Anal. Calcd. for C20H31NO5: C, 65.71; H, 8.55; N, 3.83. Found: C, 66.00; H, 8.49; N, 3.84.  p-Nitrophenyl Tetradecanoate (58). O  To a stirred solution of commercially available myristic acid (0.50 g, 2.2 mmoles) in 50 mL of C H C I was added p-nitrophenol (0.37 g, 2.6 mmoles), 2  2  D C C (0.70 g, 2.6 mmoles) and DMAP (54 mg, 0.44 mmoles). The mixture was allowed to stir at room temperature under N overnight. The solvent was 2  then removed under reduced pressure, and the crude material was purified by flash chromatography using 20:1 petroleum ether and ethyl acetate to afford 0.67 g (88%) of 58 as a white solid.  mp: 52-54°C; IR(CDCI ): 2931, 2856, 1762, 1616, 1593, 1492, 1457, 1348, 1207 cm' ; 1  3  1  H N M R (400 MHz, CDCI ) 8: 8.25 (m, 2H), 7.25 (m, 2H), 2.57 (t, J = 7.5 Hz, 3  2H), 1.73 (quint, J = 7.5 Hz, 2H), 1.45-1.15 (m, 23H); L R M S (El) m/z (relative intensity): 349 (0.1), 319 (0.6), 239 (4), 211 (100), 151 (0.8), 137 (3), 123 (4), 109 (27), 85 (16), 71 (28), 57 (45), 43 (38);  161 H R M S calcd. for Anal. Calcd. for  C20H31NO4:  C20H31NO4:  349.2253, found 349.2251;  C, 68.72; H, 8.95. Found: C, 68.89; H, 8.90.  1-Oxo-1-phosphonic acid-13-tetradecanolide (59). O  59 Compound 38b (62 mg, 0.18 mg) was dissolved in 20 mL of THF and 10 mL of 1.5M NaOH and the resulting solution was refluxed for 18 h. The T H F was removed and the residual solution was acidified with 4N HCI then extracted with E t 0 . The combined ether extracts were dried over anhydrous M g S 0 , 2  4  filtered and concentrated under reduced pressure to afford 35 mg (73%) of 59 as a white solid. This compound was used in subsequent steps without further purification.  mp: 65-68 °C; IR (CDCI3): 3160, 2932, 2861,1701,1452,1203, 1007 cm' ; 1  1  H N M R (400 MHz, CDCI ) 6: 4.54 (m, 1H), 3.75 (br s, 1H), 1.77 (m, 2H), 1.58 3  (m, 4H), 1.20-1.48 (m, 16H), 1.36 (d, J = 6.2 Hz, 3H);  162  31  P NMR (81 MHz, CDCI3) 8: 35.2;  LRMS (DCI, NH ) m/z (relative intensity): 280 ( M + 18, 4), 263 ( M + 1, 100) +  +  3  262 (9), 179 (25), 165 (35), 151 (48), 137 (25), 109 (14), 96 (16); HRMS (DCI, CH ) calcd. for Ci H 70 P: 262.1698, found 262.1627; 4  3  2  3  Anal. Calcd. for C i H 0 P : C, 59.50; H, 10.38. Found: C, 59.58; H, 10.47. 3  27  3  1-p-NitrophenyM -oxo-1 -phospho-13-tetradecanolide (60a and 60b).  60a + 60b Acid 59 (90.0 mg, 344 Limol) was dissolved in 40 mL of CH CI at 0 °C under 2  2  a N atmosphere. Then (COCI) (120 LIL, 1.37 mmol) was added, and the 2  2  mixture was allowed to stir for 20 hr. The solvent, as well as excess (COCI) , 2  were removed under reduced pressure.  The resulting acid chloride was  redissolved in 40 mL of CH CI , then p-nitrophenol (150 mg, 1.08 mmol) and 2  2  EUN (100 LIL, 717 Limol) were added, and the mixture was refluxed overnight. The reaction mixture was then washed with 3 x 25 mL of N a H C 0 , followed 3  by 3 x 25 mL of water. The CH CI layer was dried over M g S 0 , filtered and 2  2  4  the solvent removed under reduced pressure to afford 130 mg of crude  163 product. This crude material was purified by column chromatography using 3:1 petroleum ether and ethyl acetate to afford the separated diastereomers; 60a and 60b, in a 1.2:1 ratio based on the isolated yields and a combined yield of 91.0 mg (69%). A small quantity of p-nitrophenol was found to be present in both of the purified diastereomers as seen by the multiplets at 8.15 and 6.88 ppm in their H N M R spectra which correspond to the aromatic 1  protons of p-nitrophenol.  Diastereomer 60a: IR(neat): 3459, 3113, 3080, 2929, 2859, 1611, 1591, 1522, 1492, 1458, 1403, 1380, 1346, 1292, 1238, 1162, 1110, 995, 911, 860, 751 cm ; -1  1  H N M R (400 MHz, CDCI ) 5: 8.22 (m, 2H), 7.34 (m, 2H), 4.77 (m, 1H), 1.95 3  (m, 2H), 1.20-1.84 (m, 20H), 1.23 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 5: 30.4; 3  L R M S (El) m/z (relative intensity): 383 (43), 367 (20), 366 (77), 353 (21), 349 (35), 340 (17), 337 (4), 322 (17), 286 (18), 258 (25), 246 (8), 245 (56), 244 (16), 230 (44), 217 (100), 201 (10), 189 (16), 129 (28), 97 (11), 83 (21), 69 (43), 55(97); H R M S calcd. for  C19H30NO5P:  383.1862, found 383.1859.  Diastereomer 60b: IR(neat): 3450, 3113, 3080, 2929, 2859,1611,1591,1522, 1491,1459,  164 1345, 1235, 1163, 1110, 1004, 910, 859, 753 cm' ; 1  1  H NMR (400 MHz, CDCI ) 6: 8.22 (m, 2H), 7.36 (m, 2H), 4.65 (m, 1H), 2.20 3  (m, 2H), 1.20-1.84 (m, 20H),1.43 (d, J = 6.3 Hz, 3H); 31  P NMR (81 MHz, CDCI ) 6: 28.5; 3  LRMS (El) m/z (relative intensity): 383 (9), 367 (2), 366 (9), 353 (2), 349 (3), 340 (2), 337 (0.2), 322 (2), 286 (3), 258 (5), 246 (2), 245 (11), 244 (4), 230 (11), 217 (22), 201 (2), 189 (3), 129 (2), 109 (29), 97 (5), 83 (12), 81 (24), 69 (37), 55(100); HRMS calcd. for CisHaoNOgP: 383.1862, found 383.1861.  6-Methyl-2-oxo-2-phenoxy-1,2-oxaphosphorinane (61a and 61b). O  ,OPh  61a + 61b Compound 68 (495 mg, 1.55 mmol) was dissolved in 30 mL of dry T H F and stirred under N , then cooled to -78 °C with a dry-ice acetone bath. 2  1.2 M n-BuLi (94 LIL, 1.1 mmol) was then added via syringe, and after stirring at -78 °C for 2.5 h, the dry-ice acetone bath was removed.  The  solvent was removed under reduced pressure, and the crude product was purified by flash chromatography using 1:1 petroleum ether and ethyl  acetate to afford 0.286 mg (83%) of 61a and 0.57 mg (17%) of 61b colourless oils.  Isomer 61a: IR(neat): 2946, 1592, 1491, 1455, 1384, 1302, 1255, 1209, 1161, 1079, 1049, 1032, 980, 921, 839, 789, 765 cm' ; 1  1  H N M R (400 MHz, CDCI ) 6: 7.11-7.34 (m, 5H), 4.51 (m, 1H), 2.02-2.20 3  (m, 2H), 1.94 (m, 1H), 1.68-1.84 (m, 2H), 1.51 (m, 1H), 1.35 (dd, J = 2.3, 6.3 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 8: 20.5; 3  L R M S (El) m/z (relative intensity): 226 (29), 211 (6), 185 (23), 172 (25), 144 (24), 115 (9), 94 (100), 77 (18); H R M S calcd. for C n H 0 P : 226.0759, found 226.0756; 1 5  3  Isomer 61b: IR (neat): 2943, 1593, 1491,1383, 1291, 1250, 1210,1153,1026, 951,783 cm' ; 1  1  H NMR (400 MHz, CDCI ) 8: 7.11-7.34 (m, 5H), 4.68 (m, 1H), 1.96-2.22 3  (m, 3H), 1.71-1.86 (m, 2H), 1.45-1.54 (m, 1H), 1.36 (dd, J = 2.3, 6.3Hz,3H); 3 1  P N M R (81 MHz, CDCI ) 8: 23.4; 3  L R M S (El) m/z (relative intensity): 226 (41), 211 (7), 185 (27), 172 (30),  166 144 (26), 115 (9), 94(100), 77 (18); H R M S calcd. for C n H 0 P : 226.0759, found 226.0759; 1 5  3  Anal. Calcd. for C n H i 0 P : C, 58.39; H, 6.69. Found: C, 58.07; H, 6.71. 5  3  1-Benzyloxy-3-butanol (62). OH  62 1,3-Butanediol (5.0 g, 56 mmol) was dissolved in 125 mL of dry THF, and cooled to 0 °C under N prior to the addition of a 60% dispersion of NaH in 2  mineral oil (2.7 g, 67 mmol) over a 10 minute period. Upon addition of the NaH, the mixture was stirred for 10 minutes, then Bu NI (1.0 g, 2.8 mmol) was 4  added, followed by the dropwise addition of benzyl bromide (8.0 mL, 67 mmol).  The ice-water bath was removed, and the reaction mixture was  stirred at room temperature for 2.5 h before diluting the mixture with E t 0 , 2  and quenching with water, followed by 1 M HCI.  The mixture was then  extracted with E t 0 , and the combined E t 0 extracts were dried over 2  2  anhydrous MgSCM, filtered and solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography using 1:1 petroleum ether and ethyl acetate to afford 5.8 g (58%) of 62 as a colourless oil.  167 1  H N M R (200 MHz, CDCI ) 8: 7.32 (m, 5H), 4.52 (s, 2H), 4.00 (m, 1H), 3.65 3  (m, 2H), 2.63 (s, 1H), 1.73 (m, 2H), 1.18 (d, J = 6.2 Hz, 3H); L R M S (El) m/z (relative intensity): 180 (2), 161 (11), 120 (15), 108 (11), 107 (46), 92 (16), 91 (100), 79 (16), 65 (12).  1-Benzyloxy-3-(Tetrahydropyranyloxy) butane (63). OTHP  63 To a stirred solution of 62 (260 mg, 1.44 mmol) in 5 mL of C H C I was added 2  2  DHP (158 LIL, 1.73 mmol) and p-TsOH (14 mg, 0.072 mmol), and the mixture was stirred at room temperature under N for 2 h. The solvent was then 2  removed and the residue was diluted with E t 0 . The ether layer was washed 2  with 2 x 25 mL of saturated N a H C 0 , 1 x 25 mL water and 1 x 50 mL of brine, 3  dried over anhydrous M g S 0 and filtered. The solvent was removed under 4  reduced  pressure  and  the  crude  product  was  purified  by  flash  chromatography using 5:1 petroleum ether and ethyl acetate to afford 288 mg (76%) of 63 as a colourless oil.  1  H N M R (200 MHz, CDCI ) 5: 7.30 (m, 5H), 4.70 (m, 0.4H), 4.55 (m, 0.6H), 3  4.49 (m, 2H), 3.75-4.08 (m, 2H), 3.62 (m, 1H), 3.48 (m, 2H), 1.60-2.00 (m, 4H), 1.40-1.60 (m, 4H), 1.24 (d, J = 6.2 Hz, 1.5H), 1.11 (d,  168 J = 6.2 Hz, 1.5H); L R M S (DCI, NH ) m/z (relative intensity): 282 (M + 18, 4), 265 ( M + 1, 4), +  +  3  181 (66), 179 (16), 91 (88), 85 (100); H R M S (DCI, C H ) calcd. for C i H 4 0 : 264.1725, found 264.1648. 4  6  2  3  3-(Tetrahydropyranyloxy) butan-1-ol (64). OTHP HO 64  A spatula tip of Pd-C (10%) stirring in 20 mL of EtOAc was saturated with H  2  for 12 h before a solution of compound 63 (230 mg, 0.871 mmol) in 5 mL of EtOH was added via a steel cannula. This mixture was allowed to stir for an additional five hours under H pressure of one atmosphere. The reaction 2  mixture was filtered through Celite and concentrated under reduced pressure to give 146 mg of crude product, which was purified by flash chromatography using 1:1 petroleum ether and ethyl acetate to afford 129 mg (85%) of 64 as a colourless oil.  IR(neat): 3408, 2918,1447,1376,1322,1260,1201,1128, 1027,1001, 907,868,811 cm' ; 1  169 1  H N M R (200 MHz, CDCI ) 6: 4.68 (m, 0.2H), 4.55 (m, 0.8H), 3.40-4.13 3  (m, 6H), 2.35 (brs, 1H), 1.28 (d, J = 6.2 Hz, 0.6H), 1.15 (d, J = 6.2 Hz, 1.4H); L R M S (DCI, NH ) m/z (relative intensity): 192 (M + 18,1), 175 ( M + 1, +  +  3  4), 173 (2), 108 (6), 101 (13), 86 (10), 85 (100), 55 (18).  3-(Tetrahydropyranyloxy) butanal (65). OTHP  O  65 To a stirred solution of oxalyl chloride (75 LIL, 0.86 mmol) in 10 mL of dry CH2CI2 under N at -78 °C was added DMSO (102 u.L, 1.44 mmol) dissolved 2  in 2 mL CH2CI2 via a stainless steel cannula.  After 10 min. of stirring,  compound 64 (102 mg, 0.575 mmol) in 5 mL of CH2CI2 was added to the activated DMSO via a stainless steel cannula.  Finally, after stirring an  additional 10 min., Et^N (410 LIL, 2.95 mmol) was syringed in, and the reaction mixture was allowed to warm to room temperature while maintaining stirring. After 3 h, the solution was diluted with 50 mL of E t 0 followed by 50 2  mL of water. The aqueous layer was further extracted with 3 x 50 mL of E t 0 2  and the combined E t 0 extracts were dried over anhydrous M g S 0 , filtered 2  4  and concentrated under reduced pressure to a yellow oil. The crude product  170 was then purified by flash chromatography using 4:1 petroleum ether and ethyl acetate to afford 95 mg (96%) of 65 as a colourless oil.  1  H N M R (200 MHz, CDCI ) 5: 9.80 (m, 1H), 4.68 (m, 0.8H), 4.55 (m, 0.2H), 3  4.18-4.43 (m, 1H), 3.63-3.99 (m, 2H), 3.48 (m, 1H), 2.55 (m, 2H), 1.40-1.85 (m,8H), 1.26 (d, J = 6.2 Hz, 0.6H), 1.20 (d, J = 6.2 Hz, 2.4H).  1 -Diphenylphosphinyl-4-(tetrahydropyranyloxy)-1 -pentene (66). OTHP  O  66 The Wittig reagent 32 (1.9 g, 3.8 mmol) and aldehyde 65 (0.43 g, 2.5 mmol) were dissolved in 40 mL of toluene and refluxed under N for 3 days. The 2  solvent was removed under reduced pressure and the crude product was purified by flash chromatography using 5:1 petroleum ether and ethyl acetate to afford 0.55 g (55%) of 66 as a colourless oil.  IR(neat): 2941, 2870,1629,1592,1489,1455,1379,1341,1271, 1214, 1192,1163, 1127, 1074,1026, 994, 934, 767 cm' ; 1  1  H NMR (200 MHz, CDCI ) 8: 7.10-7.38 (m, 10H), 7.00 (m, 1H), 5.95 3  (m, 1H), 4.68 (m, 0.5H), 4.55 (m, 0.5H), 3.73-4.00 (m, 2H), 3.35-3.60  171 (m, 1H), 2.30-2.55 (m, 2H), 1.35-1.90 (m, 6H), 1.18 (d, J = 6.2 Hz, 1.5H), 1.08 (d, J = 6.2 Hz, 1.5H); 3 1  P N M R ( 8 1 MHz, C D C I ) 5 : 1 0 . 8 ; 3  L R M S (El) m/z (relative intensity): 402 (1), 318 (28), 301 (22), 275 (26), 274(100), 259(17), 181 (13), 162(10), 141 (13), 133(15), 118(18), 117 (96), 116 (65), 115 (47), 94 (85), 77 (87); H R M S calcd. for C ^ T O S P : 402.1596, found 402.1605.  4-Hydroxy-1 -diphenylphosphinyl-1 -pentene (67). OH  O  67 p-TsOH (26 mg, 0.14 mmol) was added to a stirred solution of compound 66 (0.55 g, 1.4 mmol) in 20 mL of MeOH at room temperature. After 5 h, the MeOH was removed under reduced pressure and the resulting oil was dissolved in E t 0 , washed with 2 x 20 mL of water followed by 2 x 20 mL of 2  brine, dried over anhydrous M g S 0 , filtered and concentrated under reduced 4  pressure.  The crude oil was purified by flash chromatography using 1:1  petroleum ether and ethyl acetate to afford 0.31 g (70%) of alcohol 67 as a colourless oil.  IR (neat): 3403, 2971,1629,1592, 1489,1456,1198,1164,1120, 1072,  172 1025,937,814,767 cm" ; 1  1  H NMR (400 MHz, CDCI ) 8: 7.12-7.35 (m, 10H), 6.94 (ddt, J = 7.2, 17.1, 3  23.1 Hz, 1H), 5.94 (ddt, J = 1.4,17.1, 23.1 Hz, 1H), 3.88 (m, 1H), 2.37 (dt, J = 1.4, 7.7 Hz, 2H), 1.61 (br s, 1H), 1.14 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 8: 10.8; 3  L R M S (El) m/z (relative intensity): 318 (16), 274 (48), 259 (8), 181 (6), 133 (7), 117 (830), 94 (82), 77 (100); H R M S calcd. for C H 0 P : 318.1021, found 318.1014; 1 7  1 9  4  Anal. Calcd. for C17H19O4P: C, 64.13; H, 6.02. Found: C, 64.03; H, 6.11.  1-Diphenylphosphinyl pentan-4-ol (68).  A spatula tip of Pd-C (10%) in 20 mL of EtOAc was saturated with H for 16 h 2  before compound 67 (0.31 g, 0.97 mmol) in 3 mL of EtOAc was added via a steel cannula. This mixture was allowed to stir for an additional 2 days under H gas at a pressure slightly greater than one atmosphere. 2  The reaction  mixture was filtered through Celite and concentrated under reduced pressure to afford 0.29 g (93%) of 68 as a colourless oil.  IR (neat): 3411, 2940,1592,1490, 1456,1374,1202,1164,1126, 1069,  1025, 1007, 935,763 cm' ; 1  1  H N M R (300 MHz, CDCI ) 5: 7.13-7.37 (m, 10H), 3.84 (m, 1H), 2.02-2.22 3  (m, 2H), 1.76-2.02 (m, 3H), 1.61 (dt, J = 6.4, 7.5 Hz, 2H), 1.21 (d, J = 6.2 Hz, 3H); 3 1  P N M R (81 MHz, CDCI ) 8: 25.6; 3  L R M S (El) m/z (relative intensity): 320 (1), 305 (10), 227 (100), 185 (15), 172 (25), 140 (25), 95 (57), 94 (96), 91 (75), 77 (86).  174 REFERENCES  1)  Brockmann, H.; Henkel, W . 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Chem. 1978, 43, 2923.  184  S P E C T R A L APPENDIX  T—'—'—'—'—I—•—•—•—'—1—••—'•I • • •—•I • " ' '—I ' • • • I • • ^ 3.0  9 . 0  1  8 . 0  7 . 0  6 . 0  5 . 0 PPM  4 . 0  I ' ' ••I ' ' ^ I  3 . 0  2 . 0  1.0  0  oo H  o 1  1  1  r  1  3200  1  1  1  2400 Wave  number  1  1  1  1 600 (cm—1)  1  i  P  i 800  185  186  187  188  !  _j  (  !  2400 Wove  number  ,  !  ,  1 600 (cm—1)  !  ,  ,  ,  800  189  I  ,  ,  3200  |  1  1  2400 Wave  number  1  I  1 1600  (cm—1)  1  1  1  1  800  191  193  194  196  1  o°  ,  ,  ,  1  1  3200  1  1  1  2400 Wave  number  1  1  '  1600 (cm—1)  <  '  1  1  800  197  198  199  200  Wove n u m b e r  (cm—1)  201  203  205  207  208  1 oo H  3200  "^2"400^ Wove  number  1  1600 ^ (cm —  1)  SOO  209  i i |—I—i—r—i—j—i—i—i—I—|—r—i—II | i—i—i—i—|—i—i—i i |—iM—i i | — i i i—r—|—r—i—i 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0  I |—I  2.0  ' i i i | I—R-R—t—F 1.0 0.  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