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Macrolides : chemistry and progress towards a catalytic antibody Spracklin, Douglas K. 1994

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MACROLIDES: CHEMISTRY AND PROGRESS TOWARDS ACATALYTIC ANTIBODYbyDouglas K. SpracklinB.Sc.(Hons.), University of Toronto, 1986M.Sc., University of British Columbia, 1989A THESIS SUBMuTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYInTHE FACULTY OF GRADUATE STUDIES(Department of Chemistry)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIANovember 1994© Douglas K. Spracklin, 1994in presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of --.The University of British ColumbiaVancouver, CanadaDate____________DE-6 (2/88)IIABSTRACTAs part of a continuing study examining the chemistry and conformationalbehaviour of keto 1 3-tetradecanolides, ii -oxo- 1 3-tetradecanolide (24) was synthesized.In the preparation of the acyclic precursor, optimal yields were obtained when the C-liketone was protected until the macrocyclic ring had been formed. Otherwise, a protected-hydroxy group was eliminated under both acidic and basic conditions. The cyclizationwas accomplished via a trichlorobenzoyl chloride intermediate.Reduction of 24 using lithium tri-sec-butylborohydride (L-Selectride) gave the(1 JR , 13S*)isomer of 49 in >99.8% diastereoselectivity. The relative stereochemistryof 49 was determined by reduction and conversion of the 1,3-diol to an acetonide for 13CNMR analysis.Molecular mechanics calculations were used to determine the possible low energyconformations for compounds 24 and 49. Reduction of 24 from the preferred twist[3434] conformations would be expected to give rise to product 49 as is indeed observed.The calculations also predicted 1H NMR coupling constants in good agreement with theobserved values for both 24 and 49.L-Selectride24 49ifiAs part of an effort to develop a catalytic antibody for the synthesis of macrocycliclactones, polyclonal antibodies against the phosphonate hapten 130 were raised. Theseantibodies demonstrated hapten-specific binding, but they did not catalyze the lactonizationof a corresponding hydroxy ester substrate under the conditions examined.00130ivTABLE OF CONTENTSABSTRACT iiLIST OFTABLES ixLIST OF FIGURES xLIST OF SCHEMES xiABBREVIATIONS xiiACKNOWLEDGMENTS xvDEDICATION xviCHAPTER I INTRODUCTION TO MACROLIDES 11.1 HISTORICAL PERSPECTIVE 11.2 SYNTHESIS OF MACROLIDES 41.3 AIM 10CHAPTER II RESULTS AND DISCUSSION: SYNTHESIS, CHEMISTRYAND CONFORMATIONAL ANALYSIS OF MACROLIDES 122.1 SYNTHESIS OF MACROLIDE 24 122.2 REDUCTION OF MACROLIDE 24 212.3 NMR ANALYSIS OF MACROCYCLIC COMPOUNDS 262.4 CONFORMATIONAL ANALYSIS OF MACROLIDES 24 AND 49....342.5 CONCLUSION 372.6 SUGGESTIONS FOR FUTURE WORK 38CHAPTER III INTRODUCTION TO CATALYTIC ANTIBODIES 393.1 THE IMMUNE SYSTEM 393.2 ANTIBODY PRODUCTION in VIVO 403.3 ANTIBODY STRUCTURE 423.4 DIVERSITY OF THE IMMUNE RESPONSE 443.4.1 Recombination 44V3.4.2 Junctional Diversity .453.4.3 Mutation 463.4.4 Association of Heavy and Light Chains 463.5 HISTORICAL BACKGROUND TO CATALYTIC ANTIBODIES 473.6 REACTIONS CATALYZED BY ANTIBODIES 513.6.1 Ester Hydrolysis 523.6.2 Transesterification 543.6.3 Stereospecific Lactonization 55CHAPTER IV RESULTS AND DISCUSSION: PROGRESS TOWARDS ALACTONIZING CATALYTIC ANTIBODY 574.1 SYNTHESIS OF MACROCYCLIC PHOSPHONATE 77 574.2 SYNTHESIS OF HAPTEN 130 634.3 POLYCLONAL ANTIBODIES 894.3.1 Screening of Serum for Hapten-Specific Binding 924.3.1.1 Carrier Protein Control Experiment 924.3.1.2 Rabbit Serum Control Experiment 954.3.1.3 Protein-Hapten Coupling Control Experiment 974.3.2 Purification of Polyclonal Antibodies 994.3.3 Synthesis of Substrate 148 1004.3.4 Assay to Determine Catalytic Activity 1044.4 CONCLUSION 1064.5 SUGGESTIONS FOR FUTURE WORK 106CHAPTER V EXPERIMENTAL 1085.1 GENERAL CHEMICAL METHODS 1085.2 CHEMICAL METHODS 1105.2.1 1 1-(1,3-Dithian-2-yl)-13-tetradecanolide (35) 110vi5.2.2 2-Benzyloxy-4-( 1 ,3-dithian-2-yl)- 14-(2-tetrahydropyranyloxy)-tetradecane (36) 1125.2.3 2-Benzyloxy-4-oxo-14-(2-tetrahydropyranyloxy)-tetradecane (37) 1135.2.4 13-Benzyloxy-1 1-oxo-tetradecan-1-ol (38) 1145.2.5 1 3-Benzyloxy- 11 -oxo-tetradecanoic acid (39) 1155.2.6 2-Acetoxy-4-oxo- 14-(2-tetrahydropyranyloxy)-tetradecane (40) 1165.2.7 13-Acetoxy-1 1-oxo-tetradecan-1-ol (41) 1175.2.8 1 3-Acetoxy- 1 1-oxo-tetradecanoic acid (42) 1185.2.9 1 1-Oxo-12-tetradecenoic acid (44) 1195.2.10 13-Hydroxy-1 1-oxo-tetradecanoic acid (43) 1205.2.11 1 3-Methoxy- 11 -oxo-tetradecanoic acid (45) 1205.2.12 1 3-Acetoxy- 11 -oxo-tetradecanal (46) 1225.2.13 11 -Hydroxy- 1 3-tetradecanolide (49) 1235.2.14 11 -Bromoacetyl- 1 3-tetradecanolide (50) 1245.2.15 1,1 1,13-Tetradecanetriol (51) 1265.2.16 4-( 1 0-hydroxydecane)-2,2,6-trimethy1- 1 ,3-dioxane (52) 1275.2.17 10-Undecen-1-ol (67) 1285.2.18 10-Undecen-1-p-toluenesulphonate (68) 1295.2.19 1 1-Undecen-1-nitrile (69) 1305.2.20 11-Dodecenal(70) 1315.2.21 11 -Dodecenoic acid (71) 1325.2.22 11-Dodecen-1-ol(72) 1335.2.23 1 1-Dodecen-1-p-toluenesulphonate (73) 1345.2.24 12-Iodo-1-dodecene (74) 1355.2.25 Dimethyl 12-tridecenyiphosphonate (75) 1365.2.26 Dimethyl 12-hydroxytridecanyiphosphonate (76) 137vii5.2.27 1 -Oxo- 1 -methoxy- 1 -phospho- 1 3-tetradecanolide (77) 1385.2.28 Methyl benzyl-( 1 2-hydroxytridecanyl)phosphinate (78) 1395.2.29 Dimethyl 4-pentenylphosphonate (112a) 1405.2.30 Diethyl 4-pentenyiphosphonate (112b) 1415.2.31 5-Bromo-1,2-epoxypentane (116) 1425.2.3 2 Dimethyl 4,5-epoxypentanyiphosphonate (113a) 1445.2.33 Diethyl 4,5-epoxypentanylphosphonate (113b) 1455.2.34 4-Bromo- 1 ,2-epoxybutane (123) 1465.2.35 Dimethyl 5-halo-4-hydroxypentanylphosphonate(124, 125) 1475.2.36 6-(Hydroxymethyl)-2-methoxy-2-oxo- 1,2-oxaphosphorinane (115a) 1485.2.37 Dimethyl 5-halo-4-acetoxypentanylphosphonate(126, 127) 1495.2.3 8 Phenyl phosphorodichloridite (88) 1505.2.39 Diisopropyl phenyl phosphite (89) 1515.2.40 Phenyl isopropyl 4-pentenylphosphonate (90) 1535.2.41 6-(Iodomethyl)-2-oxo-2-phenoxy- 1,2-oxaphosphorinane (91) 1545.2.42 6-(Azidomethyl)-2-oxo-2-phenoxy- 1,2-oxaphosphorinane (92) 1555.2.43 6-(Azidomethyl)-2-methoxy-2-oxo- 1,2-oxaphosphorinane (128) 1575.2.44 6-(Aminomethyl)-2-methoxy--2-oxo-1 ,2-oxaphosphorinane (129) 1595.2.45 Mono N-hydroxysuccinimide glutarate 1605.2.46 Mono N-hydroxysuccinimide glutaryl chloride (94) 1615.2.47 Mono N-hydroxysuccinimide glutaryl 6-(amidomethyl)-2-methoxy-2-oxo- 1 ,2-oxaphosphorinane (130) 1625.2.48 5-Hexenoic acid (146) 164vifi5.2.49 6-Hydroxymethyl 6-valerolactone (149) . 1655.2.50 4-Nitrophenyl 5-hexenoate (150) 1665.2.51 4-Nitrophenyl 5,6-epoxyhexanoate (151) 1675.2.52 Acetamido 5,6-epoxyhexanamide (153) 1685.2.5 3 Methyl glutaryl chloride (157) 1695.2.54 Methyl glutaryl cyanide (158) 1705.2.55 Methyl 6-acetamido-5-oxo-hexanoate (159) 1715.2.56 Methyl 6-acetamido-5-hydroxy-hexanoate (148) 1725.2.57 6-Acetamidomethyl 6-valerolactone (160) 1735.3 GENERAL BIOLOGICAL METHODS 1745.4 BIOLOGICAL METHODS 1755.4.1 Antigen Preparation— Protein (KLH)-HaptenConjugate (143) 1755.4.2 Rabbit Immunizations 1755.4.3 Test Bleeds 1765.4.4 ELISA Assays 1765.4.5 Affinity Column Purification 1785.4.6 Catalytic Assay 180REFERENCES 181SPECTRAL APPENDIX 192ixLIST OF TABLESTable I. Summary of the NMR Data for Macrolide 49 28Table II. Selected Dihedral Angles in Calculated Conformations of 24 34Table ifi. Selected Calculated and Observed Coupling Constants in Compound 24 36Table IV. Reactions Catalyzed by Antibodies 51Table V. Experimental Conditions for Cyclization of Hydroxy Phosphonate 76 59Table VI. Comparison of 31P NMR Shifts in Diastereomers 91, 92, and 128 84Table VII. Conditions Used for the Hapten Model Coupling Reaction 88xLIST OF FIGURESFigure 1. Examples of Macrolides.3Figure 2. 50 MHz 13C NMR APT Spectrum of Compound 52 25Figure 3a. 200 MHz 1H NMR of Compound 49 in CDC13 30Figure 3b. 200 MHz 1H NMR of Compound 49 in C6D 31Figure 3c. 200 MHz 1H NMR of Compound 49 in Acetone-d6 32Figure 3d. 200 MHz 1H NMR of Compound 49 in MeOH-d4 33Figure 4. Overview of the Immune System 39Figure 5. Antigen Processing by B-Cells 41Figure 6. Interactions Between Helper T-Cells and B-Cells 41Figure 7. The Schematic Structure of Immunoglobulins (Antibodies) 43Figure 8. Kappa (ic) Rearrangements in Germ Line and B-Lymphocytes 45Figure 9. Heavy Chain VDJ Joining 46Figure 10. Free Energy Diagram of an Antibody-Catalyzed Reaction Compared tothe Corresponding Uncatalyzed Reaction 49Figure 11. Hybridoma Technology for the Production of Monoclonal Antibodies 50Figure 12. Crystal Structure of Compound 92 from X-ray 78Figure 13. ELISA Assay for Screening of Rabbit Serum 91Figure 14. ELISA Comparison of Carrier Protein Controls with (a) H23and (b) H24 94Figure 15. ELISA Comparison of Serum Controls H14 and H22 with (a) H23and (b) H24 96Figure 16. ELISA Comparison of Coupling Reaction Controls with (a) H23and (b) H24 98Figure 17. Preparation of Hapten 144-Coupled Sepharose Affinity Column for thePurification of Polyclonal Antibodies 99)LIST OF SCHEMESScheme 1. Conformational Control of a Medium-Sized Ring in a Synthesis ofErythronolide A (13) 5Scheme 2. Conformational Control of Reactions of a Large Ring in a Synthesisof (±)-3-Deoxyrosaranolide (16) 6Scheme 3. Conformational Control in a Synthesis of a Derivative ofErythromycin A 7Scheme 4. Conformational Control of a Cyclization in a Synthesis ofBryostatin 1 (23) 9Scheme 5. Biosynthesis of Erythromycin A (6) 11Scheme 6. Original Synthesis of Macrolide 24 12Scheme 7. Modified Synthesis of Macrolide 24 13Scheme 8. Acetate Modified Synthesis of Macrolide 24 16Scheme 9. Yamaguchi Cyclization Procedure 20Scheme 10. Mechanism for Base-Catalyzed Ester Hydrolysis 52Scheme 11. Synthesis of Macrocyclic Phosphonate 77 58Scheme 12. Alternative Synthesis of Phosphonate 76 62Scheme 13. Synthesis of Compound 95 64Scheme 14. Mechanism for Formation of Phosphorinane 104 68Scheme 15. Synthesis of Phosphonate 115 70Scheme 16. Modified Benkovic Synthesis of 130 75Scheme 17. Mechanism for Methanolysis of 92 85Scheme 18. Initial Synthetic Plan for Substrate 148 100Scheme 19. Modified Synthetic Plan for Substrate 148 102Scheme 20. Synthesis of Substrate 148 104xiiABBREVIATIONS1D . one-dimensional2D.two-dimensionalAc acetylAnal analysisAPT attached proton testBn benzylbp boiling pointhr broadBSA bovine serum albuminBu butylC region constant regionCalcd calculatedCbz (benzyloxy) carbonylCDR complementarity determining regionsCOSY correlation spectroscopyD region diversity regiond doubletDa DaltonDCC 1,3-dicyclohexylcarbodiimideDCI desorption chemical ionizationdec decompositionD HP 3 ,4-dihydro-2H-pyranDIBAL diisobutylaluminium hydrideDMAP 4,4-dimethylaminopyridineDMF N,N-dimethylformamideXIIIDMSO.dimethyl suiphoxideee enantiomeric excessEl electron impactELISA enzyme-linked immunosorbent assayEt ethylFT Fourier transformGC gas chromatographyHETCOR heteronuclear correlation spectroscopyHPLC high performance liquid chromatographyHRMS high-resolution mass spectrometryi-Pr isopropylIg immunoglobulinJR infraredJ coupling constantJ region joining region‘cat catalytic rate constantKLH keyhole limpet hemocyaninKm Michaelis constantLAH lithium aluminium hydrideLDA lithium diisopropylaniidelit literatureLRMS low-resolution mass spectrometrym multipletM + molecular ionm-CPBA meta-chloroperbenzoic acidm/z mass-to-charge ratioMe methylxivMEM . 2-methoxyethoxymethylMHC major histocompatability complexmp melting pointn normalNB S N-bromosuccinimideNHS N-hydroxysuccinimideNMR nuclear magnetic resonancePBS phosphate buffered salinePE petroleum etherPh phenylPIVIB p-methoxybenzylppm parts per millionpy pyridineq quartetRf retardation factorS singletsept septett tripletTHF tetrahydrofuranTHP tetrahydropyranTLC thin-layer chromatographyTMSC1 trimethylsilyl chlorideTMSI trimethylsilyl iodideTris tris[hydroxymethyl]aminomethaneT s toluenesulphonylV region variable regionxvACKNOWLEDGMENTSForemost, I would like to thank Professor Larry Weiler for his guidance throughoutthe duration of my tenure in his laboratory. His patience and steady manner steered theproject through some difficult times.A project that crosses traditional disciplinary barriers as this one has done requiresmuch learning in the new field. For providing me with an introduction to immunology, Iwould like to thank Professor Hermann Ziltener and his technicians Helen Merkens andMike Williams at the Biomedical Research Center.I am indebted to the service personnel of the Chemistry Department including themass spec lab and the microanalytical laboratory. I am especially grateful to the NMR stafffor the numerous routine and non-routine experiments they performed.To the people who shared the day-to-day highs and lows, my labmates, I extendmy thanks and best wishes for your success in the future. They are too numerous tomention by name, but assuredly each has made a positive contribution to my stay here.Finally, I would like to thank Debbie. She was here for the most hectic phase ofmy time here and her encouragement and understanding helped make it possible.xviThis thesis is dedicated to my parents.1CHAPTER IINTRODUCTION TO MACROLIDES1.1 HISTORICAL PERSPECTIVEInterest in the chemistry of macrocyclic compounds dates back as far as 1926 whenRuzicka carried out degradative studies which led to the structure of civetone (1) andmuscone (2), components of musk oil,1 and 1927 when Kerschbaum isolated the lactonesambrettolide (3) and exaltolide (4),2 also musk oil components. The first macrolideantibiotic to be isolated was pikromycin (5), in 195O, and since then, many more of thesecompounds have been isolated and characterized.41Civetone4Ambrettolide Exaltolide2MusconeINTRODUCTION: MACROLIDES 2‘IIINMe2PikromycinOriginally, Woodward5proposed that the name “macrolide” apply only to thosecompounds containing a macrocyclic lactone ring such as erythromycin A (6). However,today that definition has been expanded to include lactams such as the immunosuppressantFK-506 (7), polyene macrolides such as amphotericin B (8), and cyclic peptides such ascyclosporin A (9), another immunosuppressant (Figure 1).Although the macrolides have been studied for several years, they remain of interestto scientists today for several reasons. First, their complex structure presents a significantchallenge to synthetic chemists. For example, the successful synthesis of a macrolideantibiotic has been used by Hanessian and co-workers (chiron approach),6-8Woodwardand co-workers (ring cleavage approach),9Masamune et al. (diastereoselective aldolapproach),’° and Still and Novak (conformational approach)” to illustrate the applicabilityof a variety of new synthetic concepts. Scientists also remain interested in the macrolidesbecause they are clinically relevant. For example, erythromycin is a commonly prescribedantibiotic.1246 Macrolides are used in the treatment of Legionnaires’ disease,178 andthey have shown anticancer activity. 19-22 The production, isolation, and biological activityof the macrolides has been reviewed in detail elsewhere.2332‘IIIEt’05INTRODUCTION: MACROLIDES 30 HO1HO)1OH MeOEt”Lo ii - 0OMeoH0HO “Q06 - ‘OMe FK-506Erythromycin A OMeZSOHH: I NH2HO 0 OH OH OH OH 0ICO2H0 - OH- OHOH8Amphotericin BMe 0 Me 0NNMeHO ‘ o /N-MeO 9N — Me\\NCyclosporin A0 MeFigure 1. Examples of Macrolides.INTRODUCTION: MACROLIDES 41.2 SYNTHESIS OF MACROLIDESClearly, the stereochemical complexity of these compounds attracts the interest ofsynthetic chemists, but their clinical importance necessitates a search for general, efficientmethods for their preparation. The efforts towards the synthesis of these compounds havebeen previously reviewed.4’334One strategy mentioned above- the conformational approach - has been the focus ofresearch in this group for some time.3541 In this strategy, the macrocyclic ring is formedearly in the synthesis and the conformation of the ring is used to introduce the varioussubstituents with regiochemical and stereochemical control. This contrasts with previousapproaches where a complex acyclic precursor is constructed and then cyclized late in thesynthesis. Some examples of the diverse reactions that have been successfully used in thisconformational approach are given below.INTRODUCTION: MACROLIDES 5In 1983, Vedejs and co-workers42 (Scheme 1) exploited the conformationalpreferences of a 9—membered ring in a synthesis of erythronolide A (13). The ring of 10assumes a crown-like conformation such that the alkyl substituent alpha to the olefinoccupies a pseudo-equatorial position. Osmylation of the double bond from the leasthindered face gave the diol 11 with 30:1 diastereoselectivity, which was further elaboratedto erythronolide A (13).OBnCO2EtFH0s04C5HN10 11‘IIIOH“bR12 13Scheme 1. Conformational Control of a Medium-Sized Ring in a Synthesis ofErythronolide A (13).42INTRODUCTION: MACROLIDES 6In 1984, Still and co-workers illustrated their seminal findings relating chemicalreactivity and conformation43 in the synthesis of (±)-3-deoxyrosaranolide (16)11(Scheme 2), whose parent compound, rosaramicin, has shown potent antibioticactivity.44’5 In this work, no less than eleven reactions were used to establish thestereochemical centers about the ring. Eight of these reactions gave stereoselectivity >15:1,while the rest were in the range of 5-10:1 selectivity. Shown in Scheme 2 is one alkylationthat illustrates this approach. The diastereoselectivity of the reaction giving 15 is 20:1(8a:8j3).0141) KN(SiMe322) CH3IEt”•II’O1516Scheme 2. Conformational Control of Reactions of a Large Ring in a Synthesis of(±)-3-Deoxyrosaranolide (16).1 10Et’INTRODUCTION: MACROLIDES 7More recently, Baker et al.46 reported a stereoselective intramolecular Michaelreaction used in the synthesis of erythromycin A derivative 19 (Scheme 3). Thestereochemistry at C-il of the cyclic carbamate 19 arises from the stereochemistry of C- 12and the conformation of the c-unsaturated ketone 17 shown below. Protonation of theresulting enolate 18 from the outside face of the ring results in a single stereoisomer atC-b.OH019R = carbohydrate= carbohydratet-BuO.Me o18III’Et”OMe‘‘I’’“ORScheme 3. Conformational Control in a Synthesis of a Derivative of Erythromycin A.46INTRODUCTION: MACROLIDES 8Finally, in 1990, Evans and Carreira47 reported a conformationally controlledreaction in studies directed toward a synthesis of bryostatin 1 (23) (Scheme 4). Thereaction of interest is a macrocyclic olefination which controls the geometry of the alkene atc-i 3. Molecular mechanics calculations predicted that the Z-olefin 21 is more stable thanthe E-olefin by approximately 10 kcallmol. This large energy difference arises because the14-membered dilactone ring in compound 21 (Z-olefin) can adopt a conformation thatminimizes unfavourable transannular interactions and satisfies the stereoelectronicrequirement that both esters have the s-trans geometry. conversely, in the E-olefin, theconjugated ester exists in the higher energy s-cis arrangement. Because the reaction iskinetically controlled, these effects are reflected in the transition state and the Z-olefin isformed exclusively, as determined by 1H NMR.INTRODUCTION: MACROL1DES 9P(O)(OMe)2J o MeO OMEM13CH3NoO LiC1, Et3N1mMOMe HMeO20Li2CO3 I21OMeHOOAcMe2Cfyy”MeOfM2CHOHMe(CH2)(CH)4CO” OHOMeH CO2Me22 23Scheme 4. Conformational Control of a Cyclization in a Synthesis ofBryostatin 1 (23).INTRODUCTION: MACROLIDES 101.3 AIMAlthough the aforementioned results are impressive, there are relatively fewexamples of this conformational control in the macrolide literature. Part of the reason forthis is that the conformational behaviour of these large rings can be quite complex. Our aimis to study simpler systems with the goal to generate a general conformational model of thebehaviour of these large ring compounds which might be useful in predicting their chemicalproperties.In earlier work, our research group has studied the chemistry of the 3-, 5-, 7-, and9-oxo-13-tetradecanolides. To complete the series, we wanted to study the 1 1-oxo-1 3-tetradecanolide (24).Note that this would represent a study of the five 14-membered keto lactoneshaving the ketone at the ‘oxygenated” position expected for an acetate biogenetic pathwayfor such compounds. This biosynthetic pathway for erythromycin A (6) is illustrated inScheme 548-5324INTRODUCTION: MACROLIDES 11[EnzymeI2)H(optional)i1L[EnzymejRepeat 1) and 2)for 6 cycles‘OHCyclization, releasefrom enzyme andfurther elaborations[Enymej 1) Acyl TransferI’ll“OH0Et”06Erythromycin AScheme 5. Biosynthesis of Erythromycin A (6).485312CHAPTER IIRESULTS AND DISCUSSION: SYNTHESIS, CHEMISTRY ANDCONFORMATIONAL ANALYSIS OF MACROLIDES2.1 SYNTHESIS OF MACROLIDE 24The first task was to prepare 1 1-oxo- 13-tetradecanolide (24). The synthesis of thiscompound is outlined in Scheme 6 and the experimental details can be found in a previousreport.541,3-Dithiane [‘1) HBr (aq) X(CH2)100R nBuLiHO(CH2)100 S S2) p-TsOH THF H><2)925 DHP 26;X=Br,R=H(75%) -20.CTHF 27; X = Br, R = fliP (80%) 28; R THP (78%)la) n-BuLiTHF-20 °CRO 1) DMSO, py, CF3H, DCC, PhHb) Propylene OxideJ<CH29R 2) AgNO3,NaOH, H20, THF2) Et3N, DMAPAc20, CH2C12 3) NaOH, MeOH:H20 (3:1), reflux29; R’=H,R=THP(85%)3) p-TsOH, MeOH30; R’=Ac,R=THP(91%)31; R=Ac,R=H(84%)1) tBuNC:1-Phenyl-1H-tetrazole-5-thiolROPhCH3reflux2) NBS, Acetone:H20 (9:1)32; R =Ac,X=H33; R’ = Ac, X = OH (50% for two steps)35; X = S(CH2)3(20%)34; R=H,X=OH(53%)24; X=O(42%)Scheme 6. Original Synthesis of MacrolideRESULTS & DISCUSSION: MACROLIDES 13In the course of preparing more compound, we tried to optimize the yield of 24 byaltering the synthetic route. Specifically, the problems we wanted to address were:(1) The poor yield of the two-step oxidation of alcohol 31 to acid 33;(2) The poor yield for the removal of the acetate protecting group from compound 33;(3) The poor yield of the cyclization step to give macrolide 35.In the oxidation step, we had previously found that the dithiane protecting groupwas not compatible with a one-step Jones oxidation55 as the dithiane was destroyed.56’7We thought we could utilize the Jones oxidation if the ketone at C-il were simplyunprotected. Another concern was that in chromium-based oxidations, we have found thathighly oxygenated molecules can be difficult to remove from the chromium salts,58presumably due to complexation. Therefore, judicious choice of the protecting group forthe C-i 3 alcohol could facilitate the work-up of the oxidation step, while at the same timeaddress the problem of the poor yield in the C-13 deprotection. For this reason, we chosethe benzyl group for the C-13 protection and embarked on the modified synthesis shown inScheme 7.1) NaH, BnBr, THFHO 2) HgO-HgC1 RO x(CH2)90R Acetone:H20(4:1) IJi(CH2)9R3) p-TsOH, MeOH36; R’ = Bn, X = S(CH2)3,R = THP (83%)2937;R’=Bn,X=O,R= THP (70%)38; R’ = Bn, X =0, R = H (80%)Jones R 1) H2/catalystoxidation (CH)8cOH 2) Cychze39; R’ = Bn (32%)24Scheme 7. Modified Synthesis of Macrolide 24.RESULTS & DISCUSSION: MACROLIDES 14The first four steps are the same as those in our previous route (Scheme 6) to givealcohol 29. Benzylation of this alcohol proceeded slowly, giving the benzyl protectedalcohol 36 in only 36% yield after 24 hours at room temperature. However, the yield wasimproved to 83% by refluxing the solution. It has been previously observed54’9 thatf3-hydroxy esters cannot be efficiently benzylated under basic conditions. This is due to thecomplexation of the metal cation as shown below. A similar effect could occur in thedithiane 29, which would slow the rate of the reaction.NateROO100R29Evidence for formation of the benzylated alcohol 36 is the appearance of aromaticsignals in the 1H NMR at 6 7.3 ppm in the product . It is interesting that the chemicalshift of the methine at C-13 in compound 36 shifts upfield to 6 3.9 ppm from 64.2 ppmin the alcohol 29. We use this signal as a diagnostic for our cyclization reactions where itshifts downfield from 6 4.2 ppm in the open chain hydroxy acid to 6 5.2 ppm in thecyclized product. The electron withdrawing nature of the acylated product (lactone) isresponsible for this latter shift.The next step was the removal of the dithiane protecting group. Initially we usedN-bromosuccinimide (NBS) to hydrolyze the dithiane to ketone 37. However, under theseconditions, the THP group was partially hydrolyzed as well and the overall yield for thetwo steps was low (40%). We investigated the removal of the dithiane using HgOHgC126°which gave the desired product 37 in good yield (70%). In the 1H NMRspectrum of 37, we see a loss of the 6 2.8 ppm signal assigned to the protons adjacent toRESULTS & DISCUSSION: MACROLIDES 15sulphur in the dithiane and the appearance of the four-proton signals at 2.3-2.8 ppm,arising from C-b and C-12 protons, alpha to the carbonyl at C-li. The geminal protonsof C-12 are well separated in their chemical shift: 2.79 ppm (J = 16, 7 Hz, 1 H) andö 2.37 ppm (J 16, 7 Hz, 1 H), reflecting their proximity to the chiral center at C-13,while the protons at C-10 appear as a triplet 2.32 ppm (J = 8 Hz, 2 H). In the IR, wesee the appearance of a carbonyl stretch (1713 cm-1) as further evidence for the formationof 37.Next, the THP of 37 was removed in acidic methanol to give free alcohol 38 ingood yield (80%).Unfortunately, the key oxidation step to convert alcohol 38 to acid 39 using theJones reagent was inefficient. The desired acid was isolated in only 32% yield. This maybe attributable to the polar nature of the compound. As we have observed before, it can bedifficult to remove highly oxygenated compounds from the chromium salts.58 In the 1HNMR spectrum of the reaction product, we observe the loss of the triplet at ö 3.6 ppmassigned to the methylene protons adjacent to the alcohol and the appearance of a newtriplet at ö 2.3 ppm, consistent with formation of the acid 39. We also observed otherproducts from the reaction. One of these was a compound in which the benzyloxy grouphad been eliminated to give the cz,3-unsaturated ketone. Although there were only smallamounts of this compound, its appearance was somewhat surprising because the benzylgroup is known to be stable under acidic conditions.61Finally, removal of the benzyl group of 39 via catalytic hydrogenation was notsuccessful. After 24 hours under a hydrogen atmosphere at room temperature, the benzylgroup of 39 remained intact and the recovery of the product from the catalyst proved to bedifficult.RESULTS & DISCUSSION: MACROLIDES 16Next, we chose to return to the use of an acyl group to protect the alcohol.Although its removal previously went in only 53% yield (Scheme 6), if the Jonesoxidation were efficient, this low yield might be offset by a shorter route. The proposednew route is shown below (Scheme 8).AcOCH2)9R29; R = CH2OT P1) HgO-HgC12Acetone:H20(4:1)2) p-TsOH, MeOH3) Jones Oxidation(CH29R40; R = CH2OT P (87%)41; R = CH2O (80%)42; R CO2H (57%)NaOHMeOH:H 02).(3:1)(CH2)9C0H4366%CycizeScheme 8. Acetate Modified Synthesis of Macrolide 24.Initially, we used NBS to liberate the masked ketone of 29. As observed before,the THP group was partially removed as well. For instance, in one experiment, weobtained a 50% yield of the THP protected product 40 and 25% of the THP cleavedproduct 41. Fortunately, the dithiane could be removed cleanly using HgO-HgC126°togive the desired product 40 in good yield (87%). In addition to the higher yield, theadvantage of the HgO-HgCl2 reagent compared to NBS was that the product from thisreaction was easier to purify by column chromatography and it was cleaner by TLC.24RESULTS & DISCUSSION: MACROLIDES 17Evidence in the 1H NMR spectrum for the removal of the dithiane included loss of the2.8 ppm signal (from protons adjacent to the sulphur) and the appearance of a methylenesignal at ö 2.4 ppm (t, J = 7.5 Hz, 2 H) assigned to the protons at C-b. The geminalproton signals at C-l2 were clearly separated: 2.79 (dd, J = 16, 7 Hz, 1 H) andö 2.52 ppm (dd, J = 16, 6 Hz, 1 H) and a 13C APT NMR experiment confirmed thepresence of the ketone functionality (ö 208 ppm). The THP protected alcohol 40 wassubjected to acidic methanol conditions and this gave alcohol 41 in 80% yield.We were now ready to try the Jones oxidation of alcohol 41. In the 1H NMRspectrum of the product 42, we observed loss of the alcohol methylene signal atö 3.6 ppm and the presence of two triplets at ö 2.40 ppm (J = 8 Hz, 2 H) and2.35 ppm (J = 8 Hz, 2 H), all consistent with the formation of acid 42. The yield forthe reaction was 57%. Again, other products could be detected, including unreactedstarting material and an alcohol where the acetate group had been eliminated. Mostprominent of these by products was the elimination product 44. Consistent with theproposed structure for this compound is the appearance of vinyl signals in the 1H NMR atö 6.85 ppm (dq, J = 16, 8 Hz, 1 H) and ö 6.15 ppm (dq, J = 16, 2 Hz, 1 H) and theC-14 methyl signal at 1.9 ppm (dd, J= 8, 2 Hz). In the IR, we see the differentcarbonyl stretches for the acid (1710 cm1) and for the conjugated ketone (1663 cm1),and we also observe an olefinic band at 1628 cm1. The low resolution mass spectrumshowed a molecular ion peak at m/z = 240 which was further evidence for compound 44.(CH2)8C0H44The next step was the hydrolysis of the acetate group. Conditions used previously(NaOH in MeOH:H20,reflux) were harsh; hence we examined milder conditions, i.e., theRESULTS & DISCUSSION: MACROLIDES 18same conditions but at room temperature. Although the reaction progressed slowly, after2.5 days we observed complete hydrolysis of the acetate group. The isolated yield ofcompound 43 was 66%, an improvement over the yield of 53% using the refluxconditions. However, this result was not reproducible. In some experiments the majorproduct isolated was the methyl ether 45. In the 1H NMR of this product, we observe theloss of the methyl signal of the acetate group at 6 2.0 ppm, the upfield shift of the methinesignal from 6 5.3 to 6 3.7 ppm, the methyl ether signal at 6 3.3 ppm (s, 3 H), and alsoan upfield shift of the C-14 doublet. In addition, we observe an M peak at m/z = 272 inthe low resolution mass spectrum. This ether could form after the initial elimination of theacetate of 42 to give 44. Subsequent Michael addition of methoxide to 44 and protonationwould give the observed compound 45.MeO 0(CH2)8COH45The observation of even traces of elimination of the acetate group was a concern.When the ketone at C-il was liberated, we saw some elimination in both the Jonesoxidation and the hydrolysis of the acetate group. We examined the oxidation further,hoping that different conditions would be more compatible with our functional groups.Since there seemed to be elimination of the acetate under the acidic conditions of the Jonesoxidation, we reasoned that switching to basic conditions might be successful. Althoughwe observed elimination under the strongly basic hydrolysis conditions, the more mildlybasic conditions of the Swern oxidation62might be more suitable. Oxidation of the alcohol41 under the Swern conditions (Equation 1) gave the desired aldehyde as evidenced by theappearance of the characteristic aldehyde signal at 6 9.7 ppm in the 1H NMR , along withthe loss of the alcohol methylene triplet at 6 3.6 ppm and the appearance of a triplet at6 2.4 ppm, assigned to the protons at C-2, adjacent to the aldehyde. Unfortunately, theRESULTS & DISCUSSION: MACROLIDES 19product could only be isolated in 55% yield. Since the original two-step oxidation ofalcohol 31 went in 50% overall and the Jones oxidation of 41 went in 57% yield, theSwern oxidation was not a significantly better alternative.Et3NAcO 0 DMSO AcO 0(CH)9O (COd)2 (CH28CHO (1)-78 °C41 4655%At this point, it seemed that our attempts to optimize the yield of 24 were notsuccessful and our original route was determined to be the best overall method.Finally, the cyclization of 34 was examined. There has been much effort to devisea cyclization procedure that is general and high yielding for macrocyclic lactones. Many ofthe cyclization methods employed work for simple, unsubstituted macrocycles but do notwork well in more complex cases. In our previous work, we used a double activationmethod63 but the low yield (20%) was a detriment to this synthesis. We decided toinvestigate the Yamaguchi reagent64 which has recently proven to be effective forlactonizations (Scheme 9).RESULTS & DISCUSSION: MACROLIDES 20(CH2)8COHEt3N, THF2,4,6-tnchloro- Ibenzoyl chloride -Cl Cl34 47HO 0N(CH2)7JILL+MeMeMeMe 48Scheme 9. Yamaguchi Cycization Procedure.MThe first step in this reaction is the formation of the mixed anhydride 47•65 Thetrichlorobenzoyl group was chosen over other activators because it is a good leaving groupfor the lactonization step and there is some steric impediment about its carbonyl centerforcing the nucleophilic secondary alcohol to attack the desired carbonyl in the lactonizationstep. A comparison to other activators has shown the trichlorobenzoyl group to be optimalin terms of both product yield and rate of reaction and a survey of solvents found thataromatic hydrocarbons were most suitable.64The remarkable catalytic effect of DMAP in acyl transfer reactions has been knownfor some time6669 and it accelerates the acylation in even the most sterically demandingcases. Its role in our intramolecular acylation is suggested in intermediate 48 (Scheme 9).Yamaguchi and co-workers found that it was best to use greater than one equivalent ofDMAP.643547%RESULTS & DISCUSSION: MACROLIDES 21Thus, the acid 34 was activated as its trichlorobenzoyl anhydride 47 and cyclizedin the presence of DMAP under high dilution conditions. The product 35 was isolated in47% yield, which is only moderate, but represents a significant improvement on ourprevious results.54 In the 1H NMR of compound 35, we see the shift of the C-13 methinesignal downfield from 6 4.2 ppm to 6 5.2 ppm as a clear indicator that the cyclization hastaken place.Hydrolysis of the dithiane of compound 35 gave ketone 24, which was the startingpoint for our studies.2.2 REDUCTION OF MACROLIDE 24With compound 24 in hand, we were ready to study its reduction. We chose to usethe reducing agent L-Selectride, feeling that its steric bulk would give us the best chance ofobtaining high diastereoselectivity in the reaction. Treatment of the ketone 24 with1.1 equivalents of L-Selectride at -78 °C (Equation 2) afforded the alcohol 49 in 62%yield after purification by column chromatography.Li(sec-Bu)3BH(2)THF-78 °C62%In the 1H NMR spectrum of compound 49, we observed the appearance of a newmethine signal at 6 3.9 ppm (m, 1 H), which we assigned to the proton at C-il, alongwith a simplification of the region 6 2.0-2.4 ppm, indicating loss of the C-li carbonyl.24 49RESULTS & DISCUSSION: MACROLIDES 22The TLC of the crude reaction mixture of the reduction reaction showed a singlespot, the 1H NMR of the isolated product showed no evidence of another diastereomer andthe GC of the isolated product showed a single peak. From these results, we believe thediastereoselectivity of the reaction is in excess of 99.8%. To prove this further and todetermine the stereochemistry of the product obtained, we attempted to prepare andcrystallize the bromoacetate derivative of the alcohol product 49 as we have previouslydone successfully in the case of the 3-hydroxy- 1 3-tetradecanolides.70 Conversion of thealcohol 49 to the bromoacetate 50 (Equation 3) proceeded smoothly (6 1%).py, DMAPEt20, 0°C3)BrBr50; R = BrCH2C(O)61%The downfield shift of the C-il methine signal in the 1H NMR spectrum of 50from ö 3.9 ppm to ö 5.2 ppm (a proton-proton COSY experiment was used todistinguish between the C-il and C- 13 methine signals) and the appearance of a singlet atö 3.8 ppm (2 H), assigned to the methylene protons alpha to the bromine atom,confirmed that the desired product was obtained. Unfortunately, we were unable to obtaina suitable crystal of 50 for x-ray analysis.We then turned our attention to other methods to determine the stereochemistry of49. Recent reports correlating the synlanti stereochemistry of 1,3-diols with the 13C NMRspectra of their acetonide derivatives offered the promise that we might be able to assign therelative stereochemistry of C-il and C- 13 in compound 49 via conversion to the49RESULTS & DISCUSSION: MACROLIDES 23corresponding acetonide.7173 The basis of this correlation between the 13C NMR spectraand the stereochemistry of 1 ,3-diols is that there are conformational differences between theacetonides of syn and anti 1 ,3-diols and this manifests itself in significantly different 13Cshifts for the ketal carbon and the attached methyl groups as shown below.CACB13C SHIFT ()30.0 ± 0.15 ppm19.4 ± 0.21 ppm98.1 ± 0.83 ppm13C SHIFT (ö)CA 24.6 ± 0.76 ppmCBC* 100.6 ± 0.25 ppmIn the syn arrangement, the acetonide adopts the chair conformation A in which thetwo alkyl groups from the original diol are equatorial. The methyl groups of the acetonideare in different chemical environments and therefore, have significantly differentresonances in the 13C NMR spectra. Conversely, in the anti-acetonide, a twist boatconformation B avoids 1 ,3-diaxial interactions of the alkyl substituents and the methylgroups of the acetonide are in much more similar chemical environments, resulting in asingle resonance in the 13C NMR spectra. In addition, the resonance of the quaternarycarbon of the syn ketal is above 100 ppm, while for the anti ketal it is below100 ppm.7173HA BRESULTS & DISCUSSION: MACROLIDES 24LiAIH4 AcetoneTHF CuSO4 (4)0°C p-TsOH5248% for two stepsReduction of macrolide 49 to triol 51, followed by formation of the acetonide gavecompound 52 in 48% yield for two steps (Equation 4). In the 1H NMR spectrum of 52,there are two methine signals at 3.95 and ö 3.80 ppm and a triplet at ö 3.62 ppm (J =7 Hz), assigned to the protons adjacent to the alcohol. In the 13C APT NMR experiment(Figure 2), we clearly see resonances for the axial and equatorial methyl carbons of thegem dimethyl group at ö 19 and ö 30 ppm respectively, as well as a signal for thequaternary ketal carbon at 98 ppm. The methyl signal from C-l4 appears at ö 22 ppm,the signals from C-il and C- 13 appear at 6 64 and 6 68 ppm, which were not uniquelyassigned, and the signal from C-12 appears at 6 62 ppm. This is convincing evidence thatacetomde 52 has the syn arrangement, and exists in the chair conformation A. Hence the1 ,3-diol in 51 must also be syn. Therefore, the L-Selectride reduction of macrolide 24proceeds to give the (11R*, ]3S*)isomer of compound 49.49 5110080PPM.1I1r,,-.-uiLJIJ,1LLLu..hf1hlLuIdllLlihiitLLdiJjULL&44-,.—III,..jriI[1L1rrj..1..t4II•II180160140120I‘---—IIII6040200—20Figure2.50MHz13CNMRAPTSpectrumofCompound52.RESULTS & DISCUSSION: MACROLIDES 262.3 NMR ANALYSIS OF MACROCYCLIC COMPOUNDSWe sought information on the conformation of macrolide 49 through the use ofNMR experiments. NMR spectroscopy has previously been used to assign the relativestereochemistry and the solution conformation of macrolide compounds. Shown below arefour recent examples of complex macrolide compounds where the stereochemistry andconformation have been ascertained via NMR experiments.7477CH3O’53RoxithromycinR, R’ = Carbohydrate- OHII54Flavofungin55(+)-HitachimycinFor this approach to be successful, it requires complete assignment of the 1H and13C NMR spectra. The one key that all these examples have in common that makes thisIIIIIIIIOHO\ —H56LL-F28249RESULTS & DISCUSSION: MACROLIDES 27possible is the chemical shift dispersion, especially in the proton signals. Illustrative of thisapproach is the analysis of roxithromycin (53), a 14-membered erythromycin A analogue.Using homonuclear and heteronuclear COSY experiments, it was possible to assign all ofthe proton and carbon signals in 53. In this compound there are 36 protons to accountfor, comparable to the 26 protons we must assign in compound 49. However, there ismuch greater dispersion in both the 1H and 13C NMR of roxithromycin (53) than in 49.First, the proton-proton three bond (3J) connectivities of 53 were established by 2Dproton-proton COSY experiments in a variety of solvents. Some of the signals thatoverlapped in one solvent were well separated in another and in this way, all the proton-proton connectivities were determined.Second, the 13C NMR signals of 53 were assigned using proton decouplingexperiments and proton-carbon HETCOR experiments, which also confirmed the protonassignments made previously. Again, changing solvents resulted in changes in thechemical shift of overlapping signals which made it possible to assign the 13C NMRspectrum of 53 unambiguously.Third, many of the coupling constants were determined from a series of 2DJ-resolved experiments. For more complex systems, the coupling constants weredetermined by an NMR simulation and iteration program. The coupling constants gavevaluable stereochemical and conformational information.Finally, a series of 1D and 2D nuclear Overhauser experiments allowed theseworkers to construct a spatial proximity map for the protons. Analysis of these results ledto the conclusion that 53 exists predominantly in a single conformation in solution which issimilar to the solid state conformation.Clearly, the task for compound 49 will be much more difficult since there are16 protons in the methylene region from 6 1.2-1.4 ppm. The results of several NMRexperiments on compound 49 are summarized in Table I.RESULTS & DISCUSSION: MACROLIDES 28Table I. Summary of the NMR Data for Macrolide 49.Position oH (ppm) JHH (Hz) 0C (ppm)1 167.02 2.41 14, 10, 4 34.452.24 14, 10, 43 1.64, 1.50 23.824 m 25.93a5 m 25.48a6 m 25.28a7 m 24.27a8 m 24.lOa9 m 22.8410 1.57, 1.21 32.7111 3.90 8,4 66.0012 1.95 14, 10, 4 43.631.65 14,8,213 4.94 10, 6, 2 68.4414 1.29 6 20.75a Could not be uniquely assignedm Signals with overlapping multiplets that could not be distinguishedRESULTS & DISCUSSION: MACROLIDES 29The 1H assignments were made mainly on the basis of chemical shift and proton-proton COSY experiments. The 13C assignments were made on the basis of chemicalshift, HETCOR and attached proton (APT) experiments. Proton-proton couplinginformation was determined primarily from decoupling and nuclear Overhauser differenceexperiments. Unfortunately, changing solvents did not result in separation of overlappingsignals and the task of complete assignment proved too difficult as there was simply notenough dispersion in the 1H NMR spectrum to be able to make definitive assignments.It is possible though to extract some interesting conformational information fromthe above. The C- 13 methine shows a sharp, well resolved 11-line pattern. This in itself isnot indicative of a conformationally rigid system.78 However, molecular mechanicscalculations (see section 2.4 Conformational Analysis of Macrolides 24 and 49) indicatethat compound 49 does exist in predominantly one conformation in solution. In light ofthis additional information, we believe that in the case of 49, the 11-line pattern for C- 13 isconsistent with a conformationally rigid system. Other indicators of a conformationallyrigid system include:79’80(1) Large chemical shift differences for diastereotopic geminal protons;(2) Vicinal coupling constants varying significantly from the average of 6-8 Hz;(3) Little or no dependence of coupling constants on solvent or temperature.For example, in Table I we see the geminal C-2 protons show this large separation(zS.ö = 0.17 ppm) and the vicinal coupling constants of the protons attached to C-il, C- 12and C- 13 vary significantly from 6-8 Hz. In addition, the spectra in CDC13,benzene-d6,acetone-d6 and MeOH-d4show similar well resolved patterns for the protons at C-2, C-il,C-12 and C-13 (Figure 3). Deuterochloroform was then chosen as the solvent for higherfield studies. The NMR data suggest that compound 49 exists in predominantly oneconformation in solution.rn ‘I) CII,II•IIIIIIII•IIIIIIIIIIIIIIIIIII‘ 0.0Figure3a.200MHz1HNMRofCompound49inCDC13.I••,III•IlIII4. z C rn Cl)I1I1—i.•IIF8.‘HNMRof Compound49inC6D6.— C’, C z C tnIIIIIIIT5.04.0PPM—-—-I•’’I10.09.0IIIII8.,4.02:52.01.5PPMFigure3d.200MHz1HNMRofCompound49inMeOH-d4.RESULTS & DISCUSSION: MACROLIDES 342.4 CONFORMATIONAL ANALYSIS OF MACROLIDES 24 AND 49To rationalize these chemical and spectroscopic results, we also undertook aconformational analysis of the starting ketone 24. A MACROMODEL calculation81on thismolecule using the MM2 force field82 gave several conformations all within 2 kcal/mol ofthe global minimum. Four of the five lowest energy conformations follow the SchweitzerDunitz rule,83 which says that the dihedral angle, C-0-C-H, of esters of secondaryalcohols is in the range of 0-60°. This is illustrated below.0600o/-Newman Projection Through C(O)-O-C-H BondIn addition, these five lowest energy conformations have the same dihedral angles(endocyclic to the ring) about C- 10 to 0-14, i.e., they share a similar local conformation.The dihedral angles of this region of the ring are summarized in Table II.Table II. Selected Dihedral Angles in Calculated Conformations of 24.Dihedral Angle (endocycic to the ring)Conformation 10 11 12 13 1424a 171 -162 67 -142 -17924b 164 -156 66 -154 -17524c 165 -150 68 -162 -17324e 63 -162 65 -152 -178RESULTS & DISCUSSION: MACROLIDES 35Furthermore, in these conformations, the two carbonyl groups have similarorientations and the methyl group occupies a pseudo-equatorial position. The two carbonylgroups are within 4.4 A of each other, providing the opportunity for the lithium cation ofthe L-Selectride to coordinate to the carbonyl groups in the reduction.41 Attack of thehydride from the more open face43 of any of these conformations will give rise to the(1JR*, ]3S*).isomer of 49 as was observed (Equation 5).H‘dH(5)24 49These conformations of ketone 24 (Table II) are not regular [3434] conformations,but are much closer to the twist [3434] conformations (shown below) that we havepreviously observed.37 We carried out an NMR analysis of the ketone 24. However,there was insufficient dispersion in the 1H NMR even at 400 MHz to assign all of theproton signals. In addition, the fact that the C-2 methylene proton signals were not wellseparated suggested that the conformational behaviour of this compound might be complexand we did not study its behaviour in detail.The calculatedJ12,3 coupling constants for each conformation of 24 are given inTable III. The observed coupling constants for J12,3 are 2 and 10 Hz respectively,which is consistent with any one or a mixture of these conformations in solution.RESULTS & DISCUSSION: MACROLIDES 36//0Table III. Selected Calculated and Observed Coupling Constants in Compound 24.Conformation J12,3 (Calculated)24a l.6&llHz24b 1.6&llHz24c l.5&llHz24e 1.7&llHzobserved 2 & 10 HzThe conformation of the product alcohol 49 was also interesting. The above NMRdata suggest that 49 exists in a single conformation at room temperature. MACROMODELcalculations with the MM2 force field show that the calculated global energy minimumconformation violates the Schweitzer-Dunitz rule. There have not been any observedexceptions to this rule in the x-ray structures of macrocyclic lactones. Therefore, we didnot consider it further. The next lowest energy conformation is 0.52 kcal/mol above theglobal minimum and is a [3344] conformation. The next one above that was 1.00 kcallmolabove the global minimum. It is unusual to have such a large energy separation betweenconformers in these systems and therefore, we believe that 49 exists mainly in the [3344]conformation shown below which is consistent with our NMR analysis.24RESULTS & DISCUSSION: MACROLIDES 37From 1H NMR experiments, we have determined that theJ11,2 values were 4 and8 Hz and theJ12,3 values were 2 and 10 Hz. The calculated values for the conformationof 49 shown above were Jii,12 = 2.0 and 11.6 Hz andJ12,3 = 1.0 and 9.3 Hz which isin reasonable agreement with the observed values.Another interesting feature in this conformation of 49 is the possibility ofintramolecular hydrogen bonding between the alcohol and the carbonyl of the lactone.Indeed, the JR showed a carbonyl peak at 1720 cm1 which is slightly lower than wenormally observe in these lactones. We attribute the shift to lower frequency to theformation of the intramolecular hydrogen bond. The MACROMODEL calculations alsosuggest the formation of such a hydrogen bond.In summary, the MACROMODEL conformational analysis is consistent with ourexperimental findings. Reduction of the ketone 24 from its lowest energy calculatedconformation gives rise to the observed product 49. The observed coupling constants inboth the ketone and the reduction product are consistent with the conformations suggestedby the NMR data and these calculations.2.5 CONCLUSIONThe synthesis of 11 -oxo- 1 3-tetradecanolide (24) was examined. In the preparationof the acyclic precursor to this compound, it was observed that when the ketone at C-ilwas unprotected, the C-13 protected alcohol was eliminated under basic and acidicconditions. The C-il carbonyl should remain protected until the cyclization to the49RESULTS & DISCUSSION: MACROLIDES 38macrolide has been completed to prevent this elimination. Using the Yamaguchi reagent,cyclization of hydroxy acid 34 gave macrolide 35 in much higher yield than the previouslyexamined cyclization procedure.Reduction of keto lactone 24 to hydroxy lactone 49 was accomplished with highdiastereoselectivity using L-Selectride as the reducing agent. The relative stereochemistryat C-li and C-13 in compound 49 was determined by NMR experiments on the derivedacetonide.Molecular mechanics calculations suggested low energy conformations forcompounds 24 and 49 which were in agreement with the data from NMR experiments.2.6 SUGGESTIONS FOR FUTURE WORKAs mentioned in the introduction, this work completes the study of the keto lactoneshaving a single carbonyl at the “oxygenated” position expected for an acetate biogeneticpathway for these compounds. Much information has been gathered regarding theirconformational behaviour and the stereoselectivity obtained in reduction by a variety ofreducing agents. It would be interesting to study the regiochemistry of such reductionswhen two or more carbonyls are present. Theoretically, further substitution on themolecule should simplify the conformational behaviour and by prudent choice of reducingagents, it should be possible to obtain high diastereo- and stereoselectivities in a predictablemanner. Based on the work described here, NMR experiments can provide valuableinformation when the signals are well dispersed. A logical starting compound would be3,1 i-dioxo-13-tetradecanolide. The product obtained by reduction of either of these twocarbonyls would offer a good opportunity for the necessary dispersion in the resultingNMR spectrum.39CHAPTER IIIINTRODUCTION TO CATALYTIC ANTIBODIES3.1 THE IMMUNE SYSTEMThe immune system is nature’s highly efficient defence mechanism used to protectanimals from foreign molecules and organisms. There are many ways by which thisoccurs, but they can all be classified as either adaptive or non-adaptive forms of immunity.Adaptive immunity is mediated by cells called lymphocytes (Figure 4).” One function ofthe lymphocytes is to secrete proteins called antibodies that bind specifically to foreignmolecules or antigens. There is a distinction between an antigen, which is any moleculethat will bind to an antibody, and an immunogen, a molecule that induces an immuneresponse. A hapten is a small molecule that by itself cannot act as an immunogen, butwhen coupled to a high molecular weight carrier, the complex is capable of eliciting animmune response.Stem CellsTHYMUS BONE MARROW* *T-Lymphocytes B-LymphocytesHelper Killer Plasma MemoryT-Cells T-Cells Cells CellsFigure 4. Overview of the Immune System.84INTRODUCTION: CATALYTIC ANTIBODIES 40The immune system contains approximately 1012 of these lymphocyte cells whichcirculate throughout the system. There are two types of lymphocytes, both of whichoriginate from the stem cells (Figure 4). The lymphocytes can be categorized as eitherB-lymphocytes, which originate in the bone marrow and are responsible for antibodysecretion, or T-lymphocytes, which originate in the thymus and may have either aregulatory or an antigen attacking role. An interesting feature of the immune system is thespecificity of the response it can mount to virtually any foreign molecule. The basis for thisspecificity is that a single lymphocyte cell recognizes one and only one antigen because allthe glycoprotein receptors found on the surface of the cell, whether a B- or T-lymphocyte,are identical.Another feature of the immune system is that it can usually distinguish foreignmolecules from natural components of the body. By constantly eliminating lymphocytesthat bind to self molecules, an organism is said to have tolerance. Should this mechanismbreak down, and the organism mounts a response against itself, an autoimmune diseasemay result (e.g. rheumatoid arthritis, diabetes, Graves’ disease). Another property of theimmune system is immunological memory. Upon first exposure to an immunogen, theresponse is slow. Upon subsequent exposure to the same immunogen, the response isstronger and more rapid due to this immunological memory. The memory is specific foreach antigen and lasts for the entire life of the animal.3.2 ANTIBODY PRODUCTION in VIVOAn effective immune response is a complicated event. It involves a number ofregulatory molecules, contact between various lymphocytes and macrophages, and a largenumber of receptors and signalling molecules which mediate this cellular communication.Virgin B-cells have a modified antibody on their surface and when they encounteran antigen, this antibody binds the antigen and the whole antibody-antigen complex isinternalized and degraded (Figure 5). Fragments of the antigen then appear on the surfaceINTRODUCTION: CATALYTIC ANTIBODIES 41of the B-cell where they are complexed to a cell surface glycoprotein called the majorhistocompatability complex (MHC) class II protein.85I, AntigenAntigenBinding,Processing C_ AntigenPresentationFigure 5. Antigen Processing by B-Cells.85Helper T-cells recognize this antigen-MHC complex and bind to the B-cell(Figure 6). This binding event is a major regulatory step in the production of antibodies asit causes the T-cells to produce a growth factor, interleukin-4 (IL-4), and a celldifferentiation factor, interleukin-5 (IL-5), both of which bind to the B-cell. IL-4 causesthe B-cells to divide exponentially while IL-5 causes the B-cell to differentiate into either aplasma or a memory cell.Figure 6. Interactions Between Helper T-Cells and B-Cells.85Plasma cells are responsible for secreting antibodies, they have only a short lifetime(3-4 days), and they are found primarily in the lymphoid organs. Conversely, memoryB-cellB-cellIIB-cell Proliferation,DifferentiationHelper Plasma MemoryT-cell Cell CelliNTRODUCTION: CATALYTIC ANTIBODIES 42cells do not secrete antibodies, they are long-lived, and they circulate throughout the systemto respond to any subsequent exposure to the antigen.Differentiation of the B-cells is a critical event as there are two important processesassociated with it. First, a process called affinity maturation occurs, i.e., somaticmutations take place which lead to antibodies that have a higher affinity for the antigen thanthe original B-cell. Second, class shifting takes place. Antibodies are classed according tothe type of heavy chain they possess (see section 3.3 Antibody Structure) and thesedifferent classes have different functions. It is after B-cell differentiation that antibodieswith different specific functions are selected by the system. In a typical immune response,this means there is a class switch from short-lived 1gM to longer lasting IgG antibodies.The level of antibodies in the system peaks about 7-10 days after first contact withthe antigen and the secreted antibodies enhance the rate of clearance of the foreignmolecule. If no further antigen is present after this time, the number of plasma cells willdecrease but the helper T-cells and memory cells remain in circulation. Upon re-exposureto the antigen, the secondary response will take place. This involves a similar sequence ofevents as in the primary response; however, there are some differences. Because of thehigh number of helper T-cells and memory cells already in the system, the response isfaster. Also, because the antibodies have undergone affinity maturation, they have a higheraffinity for the antigen so the response is stronger. This immunological memory remainswith the animal for its lifetime, leaving the immune system primed for any further contactwith the antigen.3.3 ANTIBODY STRUCTUREAntibodies belong to a group of proteins called immunoglobulins. The antibodymolecule is a large Y-shaped protein (molecular weight 150 kDa) consisting of fourpolypeptide chains - two identical heavy chains (50 kDa) and two identical light chains(25 kDa) joined by disulphide bridges (Figure 7)•8689 The light chain is comprised ofINTRODUCTION: CATALYTIC ANTIBODIES 43two domains called the variable (VL) and constant (CL) regions, each made up ofapproximately 110 amino acids. The heavy chains consist of a variable region (VH) andthree constant regions (CHI, C, CH3), each of which is also composed of approximately110 amino acids.antigen—bindin9 siteFigure 7. The Schematic Structure of Immunoglobulins (Antibodies).90Enzymatic cleavage of the antibody molecule gives rise to three fragments. Two ofthese are identical fragments that contain the variable region where antigen binding occurs(Fab). The other is a fragment that is readily crystallizable (Fe) and is involved in immuneregulation. The Fab and F regions meet at a region called the hinge, which allows lateraland rotational movement of the molecule.The antigen binding site is found in the variable region of both the heavy and lightchains in the Fab domain. Amino acid analysis has shown that this variability iscntigenbinding site$4)heavy choinFoblight choin.Fobco?iNTRODUCTION: CATALYTIC ANTIBODIES 44concentrated in short regions of the chain where most of the contact with antigens takesplace. There are three of these hypervariable regions on each of the heavy and light chains.Because they are the actual contact points with the antigen, they are called thecomplementarity determining regions (CDR). Many of these structural details, includingthe secondary and tertiary structure of the molecule and the nature of the binding site, havecome from x-ray crystallographic studies of Fab fragments, some of which contain boundantigen.89’9 In the case of small organic molecules, the binding occurs in clefts withvolumes on the order of 600 A3.92 The cleft excludes water and binding occurs via vander Waals forces, hydrophobic and electrostatic interactions, and hydrogen bondinginteractions, in the same manner as observed for substrate and inhibitor binding inenzymes.3.4 DIVERSITY OF THE IMMUNE RESPONSEIt has been estimated that the immune systems of higher organisms have the abilityto produce approximately 1012 different antibodies - a response to virtually any foreignmolecule. The explanation of the diversity of this remarkable system has been the subjectof several reviews.9395 There are four main sources of this diversity:(1) Recombination;(2) Junctional Diversity;(3) Mutation;(4) Association of heavy and light chains.3.4.1 RecombinationIn the germ-line DNA (prior to recombination) for both the light and heavy chaingenes, the coding sequences for the variable and constant regions are not adjacent to eachother but are separated by several hundred kilobase pairs and the DNA must be rearrangedto produce a functional gene (Figure 8).85 These intervening regions are excised and the VINTRODUCTION: CATALYTIC ANTIBODIES 45and C regions are joined to produce the functional gene. For example, in the mouse, thereare two kinds of light chains. One of these, the K chain, contains approximately 200 Vregions and one C region. One of these variable regions is selected randomly and joined tothe constant region to produce a functional gene.GERM LINE DNAFigure 8. Kappa (K) Rearrangements in Germ Line and B-Lymphocytes.853.4.2 Junctional DiversityBut recombination alone does not account for the diversity observed in the immunesystem. Further studies on the joining of the V and C regions revealed a third segmentknown as the joining region J was also present in the functional gene (Figure 8).85Recombination brings the randomly selected V region together with one of four J regions togive a V-J fragment, which together with the constant region gives the complete K gene.Furthermore, these recombination events do not always occur at exactly the samenucleotide in the joining region, so this gives further diversity to the V-J junction.Similar events occur in the formation of a heavy chain, with even greater diversitypossible (Figure 9).85 In addition to the V, J and C regions, heavy chains contain a fourth—200 VorIabl RegionsNONFUNCTIONAL GENEMATURE B•CELL DNAv1J2FUNCTIONAL GENEINTRODUCTION: CATALYTIC ANTIBODIES 46coding region known as the diversity region D. Thus two DNA rearrangements arerequired to form a complete heavy chain gene consisting of a V. D, J and C region.50-100 VarIable R.gions 12 D RegionsGERM LINE DNA_____ _____H-]Ill!]1[ffl]—-!i—€NONFUNCTIONAL GENEConstantRegionMATURE B’CELL DNA—i__j-E]41J- F—V2DYJFUNCTIONAL GENEFigure 9. Heavy Chain VDJ Joining.853.4.3 MutationIn addition to these recombination events, a high rate of somatic mutation,especially in the variable region, contributes to the diversity of the immune system.3.4.4 Association of Heavy and Light ChainsThese previously described genetic events generate variety in both the heavy andlight chains. A complete antibody molecule requires joining of a heavy and light chain.Since all combinations are allowed, this further increases the diversity of the immuneresponse.Constant4.1 Regions RegionMATURE BCELL DNANONFUNCTIONALGENEiNTRODUCTION: CATALYTIC ANTIBODIES 473.5 HISTORICAL BACKGROUND TO CATALYTIC ANTIBODIESBecause the immune system can mount a response to virtually any foreignmolecule, scientists have been interested in using this system as a tool to study variousphysiological phenomena and chemical processes. The first reports on the use ofantibodies to catalyze chemical reactions appeared in 1986,96,97 when the groups ofSchultz and Lerner independently published their results on acyl transfer reactions. Theunderlying concepts to these catalytic antibodies predated this by about 40 years.In 1946, Linus Pauling cited two subjects of personal interest.98 The firstconcerned the detailed atomic structure of molecules, crystals and cells, and the second wasthe basis of the physiological activity of substances. His conclusion that the two wereintimately related led him to believe that the answers to many of the biological problemse.g. growth processes, the replication of viruses, genes, and cells, the mechanism of actionof enzymes, and the physiological activity of drugs, hormones and vitamins lay in atomicstructural determination of the molecules of interest. He cited the example that only whenthe crystal structure of penicillin was determined99 could the structural basis for itsmechanism of bacteriostatic activity be considered.Despite a lack of details about protein structure, Pauling said there was compellingevidence that the specificity of the physiological activity of these substances wasdetermined by their molecular size and shape. Size and shape determined the specificity ofthe interaction between a protein and another molecule - the two have a “lock and key”complementarity.’°°The notion of complementarity of shape between the surface structure of antigensand antibodies was first suggested by Ehrlich101 and later by others.102104 Pauling andco-workers published evidence of antibody-antigen complementarity to explain serologicalspecificity.’°5’106 However, it was Pauling’s recognition of the similarity of antibodiesand enzymes with respect to complementarity and the nature of catalysis that isacknowledged as one of the most important contributions to the field of catalytic antibodies.INTRODUCTION: CATALYTIC ANTIBODIES 48Pauling concluded that enzymes were similar to antibodies in that they both arecomplementary to the species they bind. The difference is that antibodies bind the groundstate of the antigen, while enzymes bind the activated complex of the substrate. Paulingstated that “the only reasonable picture of the catalytic activity of enzymes is that whichinvolves an active region of the surface of the enzyme which is closely complementary instructure not to the substrate molecule itself, in its normal configuration, but rather to thesubstrate molecule in a strained configuration, corresponding to the ‘activated complex’ forthe reaction catalyzed by the enzyme.”98 This insight into the importance of shape is quiteremarkable in light of the fact that at that time, the structural details of only a few aminoacids were known, and the details of protein composition, sequence and structure werequite vague.Complementarity is essential to understanding the catalytic activity of enzymaticreactions. As Pauling postulated in 1948, “the enzyme would show a small power ofattraction for the substrate molecule or molecules, which would become attached to it in itsactive surface region. This substrate molecule, or these molecules, would then be strainedby the forces of attraction to the enzyme, which would tend to deform it into theconfiguration of the activated complex, for which the power of attraction by the enzyme isgreatest. The activated complex would then, under the influence of ordinary thermalagitation, either reassume the configuration corresponding to the reactants, or assume theconfiguration corresponding to the products. The assumption made above that the enzymehas a configuration complementary to the activated complex, and accordingly has thestrongest power of attraction for the activated complex, means that the activation energy forthe reaction is less in the presence of the enzyme than in its absence, and accordingly thatthe reaction would be speeded up by the enzyme.”07It was approximately 20 years later that Jencks suggested that “if complementaritybetween the active site and the transition state contributes significantly to enzymaticcatalysis, it should be possible to synthesize an enzyme by constructing such an active site.INTRODUCTION: CATALYTIC ANTIBODIES 49One way to do this is to prepare an antibody to a haptenic group which resembles thetransition state of a given reaction. The combining site of such antibodies should becomplementary to the transition state and should cause an acceleration by forcing boundsubstrates to resemble the transition state.”108 This is illustrated in Figure 10.StAGIg + SIg + PIg*SReaction CoordinateFigure 10. Free Energy Diagram of an Antibody-Catalyzed Reaction Compared to theCorresponding Uncatalyzed Reaction.109To test this transition state analogue hypothesis, a molecule (hapten) is prepared thatis similar to the transition state in size, shape, charge distribution, etc. Generally, thesesmall molecules are not recognized by the immune system so they are coupled to a highmolecular weight carrier protein. HO The protein-hapten conjugate (antigen) is introducedinto the animal of choice, often mice or rabbits. After the immune system has mounted itsprimary response to the antigen (7-10 days), a subsequent injection of the antigen is made.These boosts are given at regular intervals until the levels of antigen-specific antibody aresatisfactory. Then a sample of blood is collected and the antibodies are separated from theother components of the serum and tested for catalytic activity. The first attempts to test thetransition state hypothesis failed because the researchers used this polyclonal sera, i.e., aINTRODUCTION: CATALYTIC ANTIBODIES 50mixture of many antibodies.1113 However, hybridoma technology, which involves theisolation of a single antibody-producing cell and its fusion to a myeloma cell andsubsequent cloning (Figure 11), ensures immortality of the cell line and plentiful quantitiesof monoclonal antibodies.114 This technique, for which Köhler and Milstein won a Nobelprize, paved the way for the successful use of catalytic antibodies by Schultz andLerner.96’7bLYMPHOCYTES MYELOMA CELLS[FUSEjHYBRIDMYELOMACELLS_/JANTIBODY CLONE 1 2ANTIGENFigure 11. IMMUNE RESPONSE is initiated (a) when an antigen molecule carryingseveral antigenic determinants enters the body of an animal. The immune system responds:lines of B-lymphocytes proliferate, each secreting an immunoglobulin molecule that fits asingle antigenic determinant (or part of it). A conventional antiserum contains a mixture ofthese antibodies. Monoclonal antibodies are derived by fusing lymphocytes from themouse spleen with malignant myeloma cells (b). Individual hybrid cells are cloned, andeach of the clones secretes a monoclonal antibody that specifically binds a single antigenicdeterminant on the antibody molecule.”5a ANTIGEN— ANTIGENICDETERMINANT‘I,SP7J\\0000LYMPHOCYTES14.01ANTISERUMMIXED ANTIBODIESLJLELJMONOCL.ONAL ANTIBODIESINTRODUCTION: CATALYTIC ANTIBODIES 513.6 REACTIONS CATALYZED BY ANTIBODIESThe field of catalytic antibodies has been extensively reviewed90’116121 and sincethe first successful reports of Schultz and Lerner,96’7 an impressive array of antibody-catalyzed reactions has been reported (Table IV). This suggests that antibodies have greatpotential for use in synthesis, in much the same way that enzymes have come to berecognized as valuable synthetic tools.’22’123Table IV. Reactions Catalyzed by Antibodies.Reaction Type ReferenceEster, carbonate hydrolysis 96,97Stereospecific lactonization 124Amide bond formation/hydrolysis 125-127Redox 128,129f3-Elimination 130Porphyrin-mediated oxidations 131Trityl Protecting Group Removal 132Transesterification 133Enantiofacial Protonation 134Cis-trans Isomerization 135Glycosidic bond hydrolysis 136Stereoelectronically disfavoured 137cyclization (anti Baldwin’s rule)Diels-Alder reactions 138-140Regio- and stereoselective reduction 141INTRODUCTION: CATALYTIC ANTIBODIES 52Three of these reactions, which are relevant to this project, will be discussed indetail. They are:(1) Ester hydrolysis;97(2) Transesterification;133(3) Stereospecific lactonization.’243.6.1 Ester HydrolysisLerner and co-workers97reported the first example of a catalytic antibody in thehydrolysis of an ester. The mechanism of this reaction has been well studied. The base-catalyzed reaction is believed to proceed via the rate-determining attack of water orhydroxide ion at the carbonyl carbon to afford a negatively charged, sp3-hybridizedintermediate which rapidly breaks down to products (Scheme 10). 142 The transition stateto this tetrahedral intermediate is shown in Scheme 10. Phosphonates andphosphonamidates resemble this transition state and have been shown to be potenttransition state inhibitors of proteolytic enzymes such as pepsin and thermolysin whichreact via this mechanism.’43 During ester hydrolysis, significant electronic and geometricchanges occur - the substrate goes from ansp2-hybridized, planar, uncharged species to acharged pyramidal transition state.oO OH *HO____+ 0R’R OR’ R OHR OR’Scheme 10. Mechanism for Base-Catalyzed Ester Hydrolysis.’42Lerner et al.97 prepared the hapten 57, against which monoclonal antibodies wereraised. In addition to possessing the phosphonate moiety as a transition state analogue, theINTRODUCTION: CATALYTIC ANTIBODIES 53hapten incorporated a dipicolinic acid moiety as a potential metal ion chelator. Inmetalloenzymes such as carboxypeptidase A and thermolysin, metal ions play a key role inthe catalytic mechanism by polarizing the amide carbonyl through coordination or bydelivering a coordinated hydroxide ion to the amide carbonyl.144146 Thus, Lerner andco-workers reasoned that incorporating a metal chelating site into the hapten would generatea better transition state analogue.0/=\F3CC(O)NH—Kj---—-R=z(X02C N CONHCH2One antibody raised against 57 was in fact found to catalyze the hydrolysis of ester58. Kinetic analysis revealed a rate enhancement of 960 for the antibody-catalyzed reactionrelative to the uncatalyzed reaction. Substrates that did not incorporate the trifluoromethyland acetamido groups were not hydrolyzed by the antibody.F3CC(O)NHONHC(O)CHR5758INTRODUCTION: CATALYTIC ANTIBODIES 54Further experiments using this system’47 resulted in the isolation of anotherantibody that had remarkable catalytic activity. This particular antibody catalyzed thehydrolysis of a substrate similar to 58, with keat = 20 sec-’, corresponding to a rateenhancement of 6.25 x 106, which is comparable to that found for hydrolytic enzymes.3.6.2 TransesterificationThe success of the phosphonate transition state analogues in eliciting antibodies thatcatalyze the analogous hydrolytic reaction led investigators to study more sophisticatedprocesses, such as bimolecular transesterifications. In 1991, Lerner, Benkovic andco-workers reported the first antibody-catalyzed transesterification reaction.133 This is adifficult transformation to carry out in water because water itself is a competing reactant andis present in large excess. Therefore, any catalyst for a transesterification in water musthave a high specificity for the alcohol and the ester.These workers obtained monoclonal antibodies against the racemic phosphonate59. These antibodies, previously shown to act as esterases,148 were examined for theirability to catalyze the ester synthesis shown in Equation 6.HCH3 N0RO +R (6)60 61 62R CH3(O)NHCH4259H HINTRODUCTION: CATALYTIC ANTIBODIES 55The vinyl ester was chosen because it is a good leaving group and upon release andsubsequent protonation, it tautomerizes to acetaldehyde, which can be spectroscopicallydetermined. One antibody was found to catalyze the reaction of ester 60 and alcohol 61with high enantioselectivity - only the (S)-(-)-isomer of alcohol 61 was a substrate. Adetailed kinetic analysis was carried out and kcat was determined to be 21 minute-. Themechanism of the antibody-catalyzed reaction and the uncatalyzed reaction are different;however the effective molarity of the alcohol was estimated to be iO- 106 M.3.6.3 Stereospecific LactonizationIn 1987, Benkovic et al.124 reported the antibody-catalyzed stereospecificlactonization of compound 63 (Equation 7). The antibodies obtained in this study wereraised against hapten 65.0AcNH (7)O OPhNHc(:cH2)4c0—NThe lactonization of 63 was catalyzed until 50% of the initially added substrate wasconsumed. Upon addition of a second aliquot of substrate, the antibody again consumedapproximately 50% of the freshly added substrate. Although the hapten and the substratewere racemic, this antibody was able to distinguish between the enantiomers of alcohol 63,INTRODUCTION: CATALYTIC ANTIBODIES 56showing selectivity for the (R)-(-)-enantiomer (94% ee). Kinetic analysis yielded a kcat of0.50 minute-1,corresponding to a rate enhancement of 167. The stereochemistry at C-5was determined from NMR experiments and an independent synthesis of compound 64.This last example provided the impetus for our study of catalytic antibodies in thegeneration of macrocyclic lactones. As outlined in Chapter I, investigators have searchedfor a general high-yielding method to prepare these compounds. An antibody that couldcatalyze the cyclization to a macrocyclic lactone, with high stereoselectivity, would be awelcome and interesting achievement.57CHAPTER IVRESULTS AND DISCUSSION: PROGRESS TOWARDS ALACTONIZING CATALYTIC ANTIBODY4.1 SYNTHESIS OF MACROCYCLIC PHOSPHONATE 77Based on the work of Benkovic et al.,124 our initial goal was the synthesis ofmacrocyclic phosphonate 77 (Scheme 11). Reduction of undecylenic acid (66) withlithium aluminium hydride gave the alcohol 67 in good yield. This alcohol was convertedto the corresponding tosylate 68 and then displacement of the tosylate by the cyanide aniongave compound 69 in moderate yield (66%).’We examined two methods to convert the nitrile 69 to the alcohol 72. In the first,reduction of the cyanide group by diisobutyl aluminium hydride, followed by hydrolysis ofthe resulting imine gave the corresponding aldehyde 70 (not shown).149 Reduction of thiscompound with lithium aluminium hydride gave the alcohol 72 in 58% yield for the twosteps. In the alternative procedure, hydrolysis of the cyanide group under basic conditionsto the corresponding carboxyl compound 71 (not shown), followed by lithium aluminiumhydride reduction afforded compound 72. The overall yield for these two reactions was61%. The difference in the overall yield for the two procedures was not significant enoughto favour one route over the other; however, the hydrolysis reaction was simplerexperimentally, since the DIBAL reduction required the use of inert atmosphere techniques.In addition, the aldehyde was moderately unstable whereas the acid showed no suchinstability.Alcohol 72 was converted to the tosylate 73 in good yield. But all attempts todisplace the tosylate of 73 with the anion of dimethyl methylphosphonate’5°wereunsuccessful and the starting material was recovered in high yield (80-90%). We wereconfident that the anion of the phosphonate was indeed being formed under theseRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 58conditions54 so we speculated that the phosphonate anion was deprotonating the methylgroup of the tosylate.LiA1H466THF0°C‘NCH2)9OH6792%1) p-TsC1, Et3NDMAP, CH212) KCN,DMS0,NCH2)9X68; X = OTs (89%)69; X = CN (66%)la) DIBAL, CH2I-78 °Cb) H3O (69%)/i,TH\0°C (84%)la) NaOH, EtOH,tb) H3O (72%)/2) LiA1H4,THF0°C (85%)NCH210011721) p-TsC1, Et3NDMAP, CH2I2) Nal, Acetone,73; X = OTs (88%)74;X=I(73%)n-BuLiCH3P(O)(OCH)2THF-78 °C(CH2)11OCH3)27552%la) Hg(OAc)2THF:H0(1:1)b) NaOH, NaBH4OH0(CH2)11(OCH3)27660%NaH, PhH,t77Scheme 11. Synthesis of Macrocyclic Phosphonate 77.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 59There is precedence for such a deprotonation - in the Shapiro reaction for example.Sometimes 2,4,6-triisopropylbenzenesulphonyl hydrazides are used to prevent suchdeprotonation.’5’Our solution was to prepare iodide 74 and then displace the iodide by the anion ofdimethyl methylphosphonate to give compound 75, in moderate yield (52%). The finalstep before we could attempt the cyclization reaction was a Markovnikov hydration of thedouble bond of compound 75. Oxidation of the alkene by mercuric acetate, followed by insitu reduction with sodium borohydride gave the alcohol 76.152We were now ready to try the cyclization to the phosphonate 77. The results of thevarious conditions that we examined are given in Table V.Table V. Experimental Conditions for Cyclization of Hydroxy Phosphonate 76.Entry Conditions Yield of 771 NaH, PhCH3A 5%2 NaH, PhH, A 30%3 Et3N, PhCH3A -4 NaN(SiMe3)2,PhCH3,A <5%5 NaN(SiMe3)2,PhH, A <5%6 n-BuLi, THF, -78 °C to reflux <5%7 LDA, THF, -78 °C to reflux <5%8 n-BuLi, PhCH3-78 °C to reflux <5%9 n-BuLi, PhH, 0 °C to reflux <5%Our first strategy was to deprotonate the alcohol of compound 76 with NaH andcarry out the cyclization under high dilution conditions (Entry l).153 Using toluene as thesolvent, we were able to isolate a very small amount of the desired cyclic material (5%). InRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 60the 1H NMR spectrum of the product, we observed a downfield shift of the C-13 methinesignal from 6 3.5 to 6 4.5 ppm. In the macrocyclic lactones, we use the downfield shiftof this methine proton in the cyclized product relative to the methine of the open chainhydroxy acid precursor to help prove that the cycization has taken place. Similarly, in thephosphonate 77, the electron withdrawing nature of the phosphonate deshields the methineproton in the cyclized compound relative to the open chain precursor, giving rise to theobserved downfield shift. The electron withdrawing effect of the cyclic phosphonate is notas strong as that of the lactone and therefore the methine signal in the phosphonate is not asfar downfield as in the lactone (approximately 64.5 vs. 5.5 ppm). In addition, the C-14methyl signal of the cyclic phosphonate shifts downfield in the 1H NMR as was observedfor the lactones.Other products can be observed in the reaction as well. One of these by-products78 results from deprotonation of the methyl group of toluene, and the resulting aniondisplaces one of the methoxy groups from the phosphonate. As evidence for thiscompound, we see the appearance of aromatic signals and a signal at 6 5.1 ppm (d, J =9.5 Hz, 2 H) due to the benzylic protons which are coupled to the phosphorus in the 1HNMR spectrum. In addition, the integration of the phosphonate methyl signal at6 3.65 ppm shows the loss of one methoxy group. The mass spectrum (El) of thisproduct has a peak due to (M- 1) at m/z = 367, as well as a peak at m/z = 91, characteristicof a benzyl group. This compound was also isolated in very small amounts (5%).OH02 11 \OCH3CH2Ph78RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 61We also observed small amounts of starting material (5%), another compound inwhich the hydroxyl group has been eliminated, and other products which were notidentified.To try to overcome this deprotonation of the toluene solvent, we tried benzene asthe solvent and the yield of the reaction improved to 30% (25% recovered starting material)(Entry 2).We also examined the effect of the base upon the reaction. First, we tried theexperiment without deprotonation of the alcohol to determine if the compound wouldcyclize by simply heating it under high dilution conditions (Entry 3). However, werecovered only starting material and concluded that deprotonation prior to cyclization wasnecessary. We tried sodium 1,1,1,3,3 ,3-hexamethyldisilazane as the base, but were onlyable to isolate small amounts of the desired compound (< 5%) using either toluene orbenzene as solvent (Entries 4 and 5).We next examined the effect of temperature on the reaction. The alcohol wasdeprotonated using n-BuLi in THF, and the solution was gradually heated to reflux.Again, this resulted in only small amounts of product with the majority of materialrecovered being starting material (Entry 6). We also carried out this experiment usingLDA in TFIF (Entry 7), as well as n-BuLi in both toluene and benzene (Entries 8 and 9),all with similarly disappointing results.To our knowledge, this is the first synthesis of a macrocyclic phosphonate, but thisis not a practical method to prepare large quantities of these compounds. In fact, this studyshows that the problems encountered in synthesizing macrocyclic lactones, namely the pooryielding lactonization step can also be a problem in forming large-ring phosphonates.While we needed only small amounts of the phosphonate compound for the antibodyproduction, the poor yield of this route still makes it impractical for such a study.We did examine another route to the open-chain phosphonate 76, i.e., altering theorder of reactions to go from compound 69 to 76 as outlined in Scheme 12.154 However,RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 62this alternative is two steps longer than Scheme 11 and did not result in a higher yield Hg(OAc)2 RO la) NaOH, ROTI:HO(1:1) EtOH,(CH2)9COHNCH2)9CN b) NaOH, NaBH4 CNb) H3O69 2) p-TsOH, DHPCH2C12 79;R=H(31%) 8180; R = THP (54%) 71%1) LiA1H4,THF,O°Cn-BuLi2) p-TsC1, Et3N, RODMAP, CH21-NCH2)9CHCH3P(O)(OCH)2X THF (CH2)11P(OCH3)23) Nal, Acetone,-78 °C85; R=THP82;R=THP,X=OH(90%)66%83; R = THP, X = OTs (60%)84;R=THP,X=I(78%) : -THPV76Scheme 12. Alternative Synthesis of Phosphonate 76.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 634.2 SYNTHESIS OF HAPTEN 130We next turned our attention to preparing the small ring phosphonate compound87, analogous to the compound 95 synthesized by Benkovic et al.124 The synthesis ofhapten 95 as described by Benkovic is shown in Scheme 13.124,156//,L}00NH2At the same time, other studies in our laboratory led to an efficient synthesis of thephosphonate 86155 and this compound was selected for antibody production.86NO287RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 64PhOH ÷ PCi3______2x i-PrOH)‘ PhOPC1 (i-PrO)2POPhEt3N88 Et20 89BrNal185°CPhOi-PrO901)12, CHC132) NaN3, (Bu)4NBr,PhH:DMF(1:1), 60°C3) H2, Pd/C, 40 psiO OPh\\/91; X=I92; X= N393; X=NH2Et3NCH21a94 0Scheme 13. Synthesis of Compound 95124156We chose to prepare a similar compound as a control experiment - it was to serve asa test of our experimental efficiency in producing and isolating antibodies, techniques ourlaboratory would be using for the first time. In addition, it would be interesting to comparethe binding and catalytic activity of antibodies raised to 86 to those raised to 87. We095RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 65intended to modify the Benkovic hapten 95 by replacing the phenoxy group with the4-nitrophenol analogue. The rationale for this replacement was that in the assay forcatalytic activity in the reaction shown in equation 8, it would be more convenient tospectroscopically measure the release of the 4-nitrophenol anion rather than the phenolate.In addition, the nitrophenol group is known to be highly immunogenic.86Antibody+ArOH (8)IThe first strategy in the synthesis of 87 was simply to use 4-nitrophenol in place ofphenol in the formation of phosphorodichloridite 88 (Scheme 13). Attempted purificationof the reaction mixture by distillation resulted in an explosion. We believe that the desired4-nitrophenol analogue of phosphorodichloridite 88 was initially formed. The thermalinstability of this compound was surprising in light of the fact that the followingcompounds have all been successfully prepared and purified by distillation:157OArHRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 66ROPC1296;R =X3CCH297; =PhCH298; =CH399; = NCCH2CH100; =Cl—K101; = PhCH2CH102; =ON__Q__CHCHThere have been reports of explosions during the preparation of benzylphosphorodichloridite 97• 158,159 These workers attributed this to the benzyl alcohol actingas a “tertiary and secondary alcohol rather than a primary” resulting in the formation of“benzyl chloride, P and gases.” Indeed, PCi3 is a commonly used chlorinating agent foralcohols.160 However, Ogilvie et al. were able to successfully prepare this compoundwithout incident, so there was no reason to foresee any problem with the 4-nitrophenolanalogue.’57We attribute the explosion to the formation of the 2,4,6-tris(aryloxy)-l,3,5,2,4,6-trioxytriphosphorinane 103. In 1986, Chasar and co-workers reported the preparation,characterization and chemistry of 104161,162 The stability of these trivalent phosphoruscompounds is due to the steric hindrance of the ligands, which prevents “alternativeRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 67reactions from occurring.’63 The proposed mechanism of formation of 104, based on31P NMR observations, is shown in Scheme 14.OAr104Ar =R = alkyl, 0-alkyl, C02-alkylThese workers report that these compounds are generally air and moisture stable,especially when free of phosphorous acid 105. However, they report these compounds tobe thermally unstable and observe decomposition during melting. Calorimetric expenmentsconfirmed that “more than just melting” was occurring.’6’There is literature precedent for the formation of 106.164 Stepwise reaction of 106with itself could give the product 104.’ Alternatively, 13-elimination in 106 could yield107, which is very reactive and undergoes reaction with itself to give 104 and 108. Analkyl phosphenite analogous to 107 has been detected as a reaction intermediate165 andsimilar species have been proposed as intermediates in other reactions. 166In analogy with this mechanism, we suggest the formation of 103 because in ourexperiment, during the course of the reaction, the 4-nitrophenol was not as miscible withthe PCi3 as phenol, so we added Et3N and observed the formation of a white, flocculentprecipitate, presumablyEt3NHC1.103t-BuRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 680II Et3N ClROH ROPC12 + H20 RO—POH OH881051061+ Et3NHC1[RO—P=O1107OAr___P_AiO O OArp —O/RO104 108Scheme 14. Mechanism for Formation of Phosphorinane 104.Finally, 107 may undergo a cycloaddition reaction167 with 02 to give 109,analogous to an ozonide, a class of compounds which is also known to be thermallyunstable.OAr9_I0—01091.ORRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 69We explored the possibility of introducing the 4-nitrophenol at a later stage.Attempts to displace the phenoxide of compound 93 with the anion of 4-nitrophenol byheating 93 and 4-nitrophenol to reflux in CH21 were unsuccessful (Equation 9). Theresonance stabilization of the 4-nitrophenolate anion makes it a poor nucleophile. Otherworkers have reported the conversion of an acyl chloride to the corresponding4-nitrophenolate ester only after heating the reagents to 150 °C in the absence of solvent forseveral hours.168NO2O OPh\\/ Et3N /0 4-nitrophenol o(9)NH2 CH21 NH293 87We decided to alter our strategy and prepare 115. It would not be as convenient tomonitor the lactonization of the corresponding methyl or ethyl ester spectroscopically;however, we thought that we could devise an assay that would circumvent this difficulty bymeasuring product formation, rather than substrate depletion. Our proposed route to 115is shown in Scheme 15.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 70Br (RO)3P (OR)2 m-CPBA111 112a;R=Me(14%) OC 113112b; R = Et(63%) 25%HydrolysisR\ /OROHCycizaon [o ]115a;R=Me 114115b;RzEtScheme 15. Synthesis of Phosphonate 115.We first tried the Arbuzov reaction shown in Scheme 15 using trimethyiphosphite.However, the yields of the desired product 112a were low (10-15%) and there weremultiple spots in the TLC analysis of the reaction mixture. At this point, we did notidentify these by-products. Upon changing to triethyiphosphite, the yield of the product112b could be improved significantly (65%). Compound 112a was epoxidized to yield113 (25%) using m-CPBA. The poor yield led us to consider other alternatives for thepreparation of 113.We explored the reverse order of reactions to see if we could prepare 113 moreefficiently (Equation 10).RESULTS & DISCUSSION: CATALYTIC ANTII3ODIES 71Br m-CPBA Br O)3P (10)CH21 170 °C0°C111 116 113a;R=Me(50%)83% 113b;R=Et(68%)The first step was the epoxidation of 5-bromopentene (111) to give 116.169 Anumber of factors were critical to optimizing the yield of this reaction. It was important tomaintain an excess of oxidizing agent throughout the reaction (monitored by Kllstarchpaper). The initial 1.3 equivalents of m-CPBA seemed to be consumed before the reactionreached completion. The product 116 was relatively volatile (lit.’69bp 75-83 °C125 torr), so it was necessary to remove the solvent from the reaction mixturecarefully by concentrating on the rotary evaporator.The second step in this sequence was the preparation of phosphonate 113 by anArbuzov reaction. It was difficult to completely separate the unreacted trialkylphosphitefrom 113 by column chromatography. It could be removed by an aqueous workup, butthe yields were lowered since the product was partially soluble in the aqueous phase.Initially, trimethylphosphite was used in this reaction; however, the yields of 113a werelow (2O-5O%) as we saw in the previous Arbuzov reaction using (MeO)3P. We tried toform the more reactive iodo analogue of 116 in situ by the addition of Nal but this did notimprove the yield. Upon changing to triethyiphosphite, the yields of 113b improved aswe observed before (65-75%).In the case of the (MeO)3P, a by-product with a TLC Rf similar to that of thedesired product was determined to be dimethyl methyiphosphonate (121). The 1H NMRof this compound had signals at 6 3.6 (d, J = 12 Hz, 6 H) and 6 1.3 ppm (d, J =18 Hz, 3 H). The formation of compound 121 is explained in the mechanism shownbelow. Attack of (MeO)3Pon compound 119 ultimately leads to the by-product 121. Analkyl methyl group is approximately 30 times more susceptible to nucleophilic attack thanRESULTS & DISCUSSION: CATALYTIC ANTIBODiES 72an alkyl ethyl group,’70”1 explaining why we did not observe the correspondingby-product in the reaction using (EtO)3P.RBr(CH3O)P:+ Br RLOCH + CH3BrOCH3118 119(MeO)P1?OCH3121Br +120We examined one more possible route to obtain the dimethyl phosphonate 113shown in equation 11.CH3P(O)(OMe)2113n-BuLiThe epoxidation of 122 proceeded smoothly (89%) to give compound 123. Wethought we could displace the bromide with the anion of dimethyl methyiphosphonatebased on a literature precedent’72that stated that in aprotic solvents, and using delocalizedanions, nucleophiles reacted exclusively to displace the bromide of epoxyalkyl bromidessuch as 123. In aprotic solvents, there is no hydrogen bonding to the oxirane which117[ IBr122m-CPBABrCH210°C 12389%THF-78 °C(11)RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 73would weaken the C-O bond making it more susceptible to nucleophilic attack. Therefore,the C-Br bond is the preferred site of nucleophilic attack. In addition, the phosphonateanion is a soft nucleophile, and since the carbon of the C-Br bond is soft electrophile, thisalso is expected to favour attack at this position. However, under these reactionconditions, we observed complete loss of the epoxide functionality and we did not pursuethis further.With epoxy-phosphonates 113a and 113b in hand, we were ready to try the keyreactions to form the cyclic phosphonate 115 (Scheme 15). The first step involvedhydrolysis of the phosphonate ester. The first conditions we tried were piperidinelDMF(20% v/v)’73 at room temperature overnight but we recovered only unreacted startingmaterial for both 113a and 113b. Next we tried refluxing with t-BuNH2 overnight,’74but again, we observed no reaction and recovered our starting materials. On extending thereaction time to six days we obtained a mixture of products. Some hydrolysis wasobserved but in addition, we found incorporation of the t-butylamine moiety into someproducts.Next we examined the use of trimethylsilyl iodide which is commonly used tohydrolyze methyl and phosphonate esters.176 Using one equivalent of TMSI,generated in situ from TMSC1 and NaT, we observed loss of our epoxide functionality from113a; however, we isolated only trace amounts of the desired cyclized product liSa, andanother product which we could not immediately identify. We observed the same resultsfor 113b. The IR of the unknown product showed an OH stretch (3378 cm1) and wesuspected that the epoxide had been opened by the chloride and/or the iodide of the TMSC1-NaT reagent to give 124 and 125. A Beilstein test for the presence of an organic halidewas positive’77 and there was evidence in the low resolution mass spectrum (El) for boththe chloro compound (m/z = 231 (35C1, M+l), 233(37C1, M+1) in a 3:1 ratio) and theiodo compound (m/z = 323, M+1). The 1H NMR also suggested there were twocompounds present. There appeared to be a multiplet signal overlapping the P(O)(OMe)2RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 74signal at 6 3.85-3.70 ppm. In addition, there were four distinct geminal signals at 6 3.6(dd, J = 10, 3.5 Hz, 1 H), 3.5 (dd, J = 10, 7 Hz, 1 H), 3.35 (dd, J = 10, 4 Hz,1 H), and 3.25 ppm (dd, J = 10, 7 Hz, 1 H), and one complex signal overlapping thegeminal signal at 6 3.6 ppm. We assigned the complex signals at 6 3.8 and 6 3.6 ppmto be the methine protons of the compounds 124 and 125. A decoupling experiment at6 3.5 ppm simplified the signals at 6 3.35 and 6 3.25 to doublets, both with J = 10 Hz.Therefore, the methine at 6 3.5 ppm is coupled with the methylenes at 6 3.3 and3.2 ppm, and the methine at 6 3.8 ppm with the methylenes at 6 3.6 and 3.5 ppm.OH 0 OAc 0124;X=C1 126;X=Cl125;X=I 127;X=ITo confirm this, the suspected alcohols were converted to the acetates 126 and127. We observed two methine signals at 6 5.0 (m, 1 H) and 4.7 ppm (m, 1 H). Themethylene signals of the acetate compounds were not as well separated as they were in thealcohols. They appear as multiplets at 6 3.5 and 3.2 ppm. We could not completelyseparate the mixture chromatographically, but we could enhance the composition of126:127 (3:1).The final possibility we investigated to hydrolyze the phosphonate in 113 wasbasic hydrolysis. However, using 3 M NaOH, we were not able to hydrolyze thephosphonate of 113 in the presence of the epoxide. Clearly, this tandem hydrolysiscyclization approach (Scheme 15) would not be a viable route to the cyclic phosphonate115 since the phosphonate could not be hydrolyzed in the presence of the epoxide underany of the conditions tried.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 75We then turned our attention to preparing compound 130 by a route similar to theroute reported by Benkovic (Scheme 16).PhOH + PCi3_____2x i-PrOHPhOPCl . (i-PrO)2POPhEt3N88 Et20 8981% 85%BrNal185°CPhO—i-PrO9093%1)12, CHC132) NaN3, (Bu)4NBr,PhH:DMF (1:1), 60°C3) n-BuLi, MeOHTHF, -78 °C4) H2, PdJC, 40 psiO OR\\/91;R=Ph,X=I (95%)92;R=Ph,X=N3(53%)128; R = Me, X = N3 (74%)129; R = Me, X = NH2 (100% crude)Et3NCH210 0 0\\Ci94 00.013058%Scheme 16. Modified Benkovic Synthesis of 130.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 76We examined two methods to prepare diisopropyl phenyl phosphite (89)directly.’78 In the first, two moles of i-PrOH are allowed to react with PC13, then PhOH isadded to form the phosphite 89 (Equation 12). In our hands, we could only isolate lowyields of the desired product (3O%). Purification of the reaction mixture by distillationyielded multiple fractions, each of which contained multiple peaks by 31P NMR, andshowed little incorporation of PhOH as determined by 1H NMR.PC13PhOH+‘ [(i-PrO)2PC1] (i-PrO)2POPh (12)2x i-PrOH 89The other method to prepare 89 directly involves addition of i-PrOH to (PhO)3(Equation 13) but again, we were only able to isolate small quantities of the desiredproduct (15%).(PhO)3 Na (i-PrO)2POPh (13)2x i-PrOH89In both of these cases, it seems there is incomplete addition of the ROH to thephosphorus species. The resulting mixed-phosphite intermediate (RO)PCl (fromEquation 12) and trialkyl phosphite (RO)3P(from Equation 13) species are known to bereadily hydrolyzed to give compound 131 which is in equilibrium with 132. Indeed, inthe 1H NMR, we see a signal that integrates for one proton and has a very large couplingconstant (J 690 Hz), consistent with structure 132.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 77RO\_____,P—OH ROHRO RO131 132We then resorted to the two-step route to 89 (Scheme 16).179 Addition of PhOHto PCi3 gave phenyl phosphorodichioridite (88) in good yield (8 1%). This must be storedunder N2 and used immediately as it reacts with moisture in the air. Displacement of thechlorides of 88 by i-PrOH gave diisopropyl phenyl phosphite (89) in good yield (85%).The overall yield for the preparation of 89 via these two steps was 68%, which wassignificantly better than either of the one-step procedures outlined previously.The next step is an Arbuzov reaction of 89 with 5-bromopentene which yieldedphenyl isopropyl 4-pentenylphosphonate (90) in 93% yield. Cyclization of compound 90by an ‘2 mediated 80 gave 2-phenoxy-2-oxo-6-iodomethyl- 1,2-oxaphosphorinane (91) in 95% yield as a mixture of diastereomers.Displacement of the iodide in 91 by azide gave compound 92 (53%) as a mixtureof diastereomers. These diastereomers were separated by column chromatography and oneof them, 92a, crystallized upon purification. X-ray analysis of 92a showed the structureto be as shown in Figure 12, with the CH2N3 and the OPh in a 1,3-trans orientation.Benkovic et al. reported a crystal structure for one of the iodo isomers of 91181 whichshowed it was the 1,3-trans orientation.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 78H12Hil cliCl 2NiC8H7HIH2Figure 12. Crystal Structure of Compound 92a from X-ray.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 79For the isomers of compounds 91 and 92 that crystallized, i.e., 91a and 92a, the1 ,2-oxaphosphorinane moiety adopts a chair conformation. Interestingly, there has been areport in which the 1,2-oxaphosphorinane adopts a boat conformation.’82 In compound133, the severe 1 ,3-diaxial steric interactions present in the chair conformation force the1,2-oxaphosphorinane to adopt the boat conformation shown below.0 0°HOMeOTs133The trans relationship of the CH2I and the OPh substituents of compound 91aoriginates in the 12 mediated cyclization. The suggested mechanism is shownbelow.183’4i-PrO2[I jOPh OPh OPhI90 134 91aThe cyclization is stereoselective and gives the 1,3-trans product 91a with aminimum stereoselectivity of 75% for a number of experiments. The stereochemistry ofthe iodomethyl group at C-6 is determined in the intermediate 134. The equatorialorientation of the iodomethyl group avoids 1 ,3-diaxial interactions in the transition state.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 80The reason for the axial orientation of the OPh group may be due to electronicconsiderations. If the reaction has a late transition state, then this transition state will beproduct-like.’85 If the OPh is in the axial orientation, the molecule can be stabilized by theanomeric effect. Therefore, the axial orientation of the OPh group is more stable.A series of decoupling experiments indicated that compound 92a - the 1,3-transdiastereomer - exists predominantly in the chair conformation A as shown below and foundin the solid state. In a 1H NMR decoupling experiment, when the methylene protonsadjacent to the azide were decoupled, the methine proton at C-6 simplified to a doublet ofdoublets (J = 11.5, 1.6 Hz). The large coupling results from the H6axH5ax interaction,while the smaller is due to the H6axH5eq interaction. A Karplus-type relationship doeshold for 3J H-P coupling,178”86’87and a Newman projection along the C5-O bond showsthat H6 has a gauche relationship to the phosphorus in A and therefore, the coupling will besmall, or non-existent. If conformer B were significantly populated in solution, then wewould expect to observe a 3J coupling on the order of 22Hz178’867 between H6 andthe phosphorus due to their anti orientation. This is seen in a Newman projection along theC5-O bond in conformation B. Because we do not observe this large coupling and inaddition, the coupling we do observe is much closer to the ideal value for conformation A,rather than a time weighted average of the two, we conclude that 92a is predominantly inconformation A.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 81N H6OPhA0PhO92aOPh N3OPhA92aBOne might ask what the contribution of the anomeric effect is to the conformationalequilibrium shown above. The term “anomeric effect” was first introduced in 1958188 andrefers to the tendency of an alkoxy group at C-i of a pyranose ring to assume the axialrather than the equatorial orientation despite unfavourable steric interactions. There havebeen three influential monographs published on this effect’899’and recent studies on thissubject have been reviewed.192’3 There has been much debate about the origin of theanomeric effect. One explanation is that unfavourable dipole-dipole interactions in Bdestabilize the pyranose ring and favour conformation A as illustrated below. 194BRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 82A BAnother explanation is that there is a stabilizing interaction of the ring-heteroatomlone pairs with an antibonding a*orbital of the carbon-ligand bond which also favoursconformation A as shown below.195In fact, the accumulated evidence suggests that both factors contribute to theanomeric effect.Investigators have previously examined the anomeric effect in phosphorinanesystems; however, most of these efforts have focused on the 2-oxo-l,3,2-dioxaphosphorinanes in light of their importance in biological systems.178186196199 Forexample, it has been observed that compounds such as 135 can adopt a twist-boatconformation to take advantage of the anomeric effect.178 However, in 2-oxo-1,2-phosphorinane systems such as 92, the magnitude of the anomeric effect should bereduced because of the absence of the second oxygen atom and the presence of 1 ,3-stericinteractions due to the methylene group.0R,A BRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 83OR135In the solid state of 92a (Figure 12), the P-O bond exocyclic to the ring is longerthan the P-O bond endocyclic to the ring (1.598 A vs. 1.574 A). This is consistent withthe orbital overlap argument used to explain the anomeric effect. However, the 1 ,3-stericinteractions also favour conformation A with an equatorial C6 substituent, so this does notgive an indication of the magnitude of the anomeric effect. The anomeric effect can becompared to the 1,3-diaxial interaction in 92b - the 1,3-cis diastereomer which wasseparated by chromatography and is a liquid. Its conformational equilibrium is illustratedbelow.PhO92bA series of decoupling experiments suggested that in 92b, A is the predominantconformer in solution. In this case, decoupling of the methylene protons adjacent to theazide simplified the H6 signal to a doublet of triplets (J = 11, 2.9 Hz). It is possible thatthere is accidental overlap of the 3J coupling of H6 to phosphorus (gauche relationship) andthe H6H5eq coupling so that we observe a triplet. Significantly, we do not observe a largeBRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 843J coupling of H6 to the phosphorus (22 Hz) which would be observed in conformationB. It appears that the stabilization gained from the anomeric effect in conformation B isnot enough to offset the destabilization of the 1 ,3-diaxial steric interaction.The next step was to introduce the OMe by displacement of the OPh of 92a to give128 in 74% yield as a mixture of diastereomers (Scheme 16). This resulted in inversionof stereochemistry about the phosphorus to give mainly 128a - the 1,3-cis diastereomer -in the chair conformation shown below. A comparison of the shift of the 31P NMR signalsfor the major and minor diastereomers of these compounds is consistent with this analysis(Table VI). The 31P NMR signal of oxaphosphorinane diastereomers in which the P-ORsubstituent is axial, as in compounds 91a and 92a, has been shown to be upfield of thecorresponding compounds in which the P-OR substituent is equatorial as in128a.78’200203 A possible mechanism of this reaction is shown in Scheme 17.0MeO /PII H0128aTable VI. Comparison of 31P NMR Shifts in Diastereomers of 91, 92, and 128.O OPh 0 OPh 0 OMecx—91a 91b 92a 92b 128b 128a(major) (minor) (major) (minor) (minor) (major)trans cis trans cis trans cis31P 21.0 24.5 20.0 23.6 25.9 28.3(8 ppm)RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 85OPhH I ?CH3 pseudorotationCH3O-4’P—OPh(I0N3CH2 0N392a 136HOCH3N3CH2.I0IOCH3PhON3137 128bRetention Product(minor)OPh 0H OPh H II_P0 CH3O- i /_7OCH3O1CH 0N3CH2N3 N392a 138 128aInversion Product(major)Scheme 17. Mechanism for Methanolysis of 92a.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 86Attack of the methoxide from the “axial” direction relative to the P=0 bond in 92agives intermediate 136. Because the leaving group must depart from an axial position,intermediate 136 must undergo pseudorotation to 137 prior to elimination of the phenoxygroup.204 However, placing a carbon atom of the ring at the axial position of the trigonalbipyramid has a high energy barrier204207 because the bond angle of the 0-P-C within thering is approximately 900 compared to 1200 in 136. This makes the retention product128b kinetically disfavoured.Conversely, if the methoxide attacks from the “equatorial” direction relative to theP=0 bond in 92a, the resulting intermediate 138 has the phenoxy group in an axialposition and direct breakdown of 138 gives the inversion product 128a. It has beenshown that the amount of inversion product formed by this direct displacement mechanismis enhanced when a good leaving group such as phenoxy is used.208Reduction of the azide 128a to give the amine 129 (Scheme 16) proved to beproblematic. We first examined reduction by 1 ,3-propanedithiol in the presence ofEt3N.209 There was evidence in the crude reaction mixture for the desired compound(76%) (two peaks in the 31P NMR, broad peak in the 1H NMR due to the protons of theamine, and an (M+1) peak in both the LRJHRMS). The product was quite polar and hadto be extracted with n-BuOH from the aqueous phase. However, the yield using theseconditions was not reproducible. In addition, we observed (by 1H NMR) that thecompound decomposed over time. We next examined hydrogenation of the azide. Again,the product was observed in the crude reaction mixture (100%). However, attempts topurify the compound by chromatography resulted in decomposition (multiple spots byTLC). To determine if the difficulties were associated with this particular compound, wecarried out the reduction on the Benkovic azide 92 (Scheme 13) under both these reactionconditions. Reduction using 1,3-propanedithiollEt3N showed the amine product 93 in thecrude reaction mixture; however, the yield again was low. Hydrogenation also gave theRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 87product 93 in the crude mixture, but we could not isolate reasonable yields of the desiredproduct after chromatography.A search of the literature revealed some examples of amino phosphonates whichwere used immediately after formation of the amino group, without further characterization,although no specific details about any inherent instability were described (see for exampleEquations 142b0,2i and 15212). It is possible that the amino group reacts with thephosphonate esters to form the corresponding phosphonamidates.OR OR 0I 1) Ph3, THF:H20 IM—P2)MeO —P (14)139;R=steroid 14001) N2H4,MeOHN—CH2-P(OMe) Cbz-NH—CH2--P(OMe) (15)2) Cbz-C1, Et3N,CHCI3142141We did examine the possibility of converting the amine to the correspondingpyridinium bisulphate salt for characterization and storage. We obtained some evidence forthis salt in the 1H NMR; however, the next step consisting of regeneration of the free base,followed by coupling to the N-hydroxysuccinimide glutaryl chloride 94, was notsuccessful. Thus we were forced to reduce the azide and use it immediately in the nextreaction without further characterization.The amine was to be coupled to a small molecule which would serve as a linkbetween the hapten and the carrier protein. The C-5 unit has been shown to be the optimalRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 88length for such a spacer in a number of studies.2 3216 Because of the problems with theinstability of the amine, we carried out a model study to optimize the conditions for thiscoupling. We used benzyl amine as the model compound and examined three commonlyused methods to form amide bonds. The results are summarized in Table VII and led us touse the acid chloride method with amine 129 which gave 130 in 58% yield as shownpreviously in Scheme 16.BnNH2 +Table VII. Conditions Used for the Hapten Model Coupling Reaction.x= Yield0—C6H11—N=<NHC6H1130%0-JNOClNo reaction70%Finally, compound 130 was coupled to the carrier protein keyhole limpethemocyanin (KLH).”0 It has been observed that KLH conjugates are more immunogenicthan bovine serum albumin (BSA) conjugates although the reasons for this are unclear.2170RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 89The coupling takes place between amino groups on the protein and theN-hydroxysuccinimide activated ester218 130 as shown below.+ H3NDMF4.3 POLYCLONAL ANTIBODIESThe failure to obtain catalytic antibodies in earlier mice and rabbit studies wasascribed to the fact that polyclonal antibodies were used.111-113 However, Gallacher andco-workers219’20recently reported carbonate and p-nitroanilide hydrolysis catalyzed bypolyclonal antibodies isolated from the serum of sheep immunized with an arylphosphonate derivative. There has also been a report on the catalytic activity of antibodiesfrom the serum of patients, although interestingly, they had not been intentionallyimmunized with a transition state analogue.221’2 In 1993, Stephens and Iverson223reported the details of the catalytic activity of polyclonal antibodies intentionally elicited inrabbits. They obtained antibodies against a phosphonium hapten that catalyzed removal ofthe corresponding trityl group.130 0n143RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 90The advantages of polyclonal antibodies over monoclonal antibodies are that theycan be isolated more quickly and cost effectively. Also, because they represent the entireimmune response, they offer a more general assessment of an antigen’s ability to induce acatalytic antibody. For these reasons, we chose to prepare polyclonal antibodies against thehapten-protein conjugate (antigen) 143.Two New Zealand White rabbits (female, nonpregnant) were initially immunizedwith a 150 jig aliquot of antigen 143 and aluminium hydroxide in PBS buffer. The initialimmunization was intradermal at multiple sites along the back, encircling an injection ofFreund’s complete adjuvant. Four weeks after the initial immunization, a boostimmunization containing 100 jig of antigen 143 and aluminium hydroxide in PBS bufferwas given intradermally in the thigh. Two weeks after the first boost, a blood sample wasdrawn. The blood sample was stored on ice (0 °C) and after 20 minutes, the resulting clotwas removed from the walls of the test tube. The sample was centrifuged and the strawcoloured serum was separated from the red blood cells, then heat inactivated at 56 °C for35 minutes. It was stored at -20 °C and thawed prior to use in the assay described below.The presence of antiligand antibodies, i.e., antibodies specific to antigen 143, wasdetermined by an enzyme-linked immunosorbent assay (ELISA) shown in Figure 13. Analiquot of the antigen was bound to a polystyrene well via nonspecific interactions and theexcess antigen was washed away (step 1). A series of dilutions from the antibody-containing serum of interest was incubated with the bound antigen, then unbound portionswere washed away (step 2). An enzyme-linked anti-antibody was added which binds toany rabbit immunoglobulins attached to the plate (step 3). Unbound anti-antibody wasthoroughly washed away and then a substrate was added. The substrate was processed bythe enzyme to yield a chromogenic product (*) which was detected and quantifiedspectroscopically.A protein, different from the protein used for the immunization, was chosen to bindthe hapten to the ELISA plate. This should eliminate any response from antibodies anti toLARESULTS & DISCUSSION: CATALYTIC ANTIBODIES 91the original carrier protein. The protein used for conjugation in the ELISA experimentswas BSA.I(1)(2)(3)A AFE FEI * 1*— —* *Figure 13. ELISA Assay for Screening of Rabbit Serum.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 924.3.1 Screening of Serum for Hapten-Specific BindingThere are three controls that must be run before we can ascertain that the antibodiesare binding specifically to the cyclic phosphonate moiety of hapten 143. These are:(1) The carrier protein of the conjugate;(2) Other rabbit serum;(3) Reagents of the protein-hapten coupling reaction. Carrier Protein Control ExperimentAs mentioned, a protein other than the carrier protein (KLH) used for theimmunization is used in the screening assay to eliminate any response from antibodieswhich bind to the carrier protein. Shown in Figure 14 are the ELISA results for thescreening of the serum obtained from the two rabbits (H23 and H24) in our study againstthe hapten-BSA conjugate 144 and against BSA alone. The positive response of theconjugate as compared to the BSA alone shows that the binding is not due to associationwith the carrier protein (BSA).The concentration range shown in Figure 14 was chosen arbitrarily using a startingdilution of serum:buffer = 1:10. The approximately constant fluorescence (Figure 14a) orslightly increasing fluorescence (Figure 14b) observed over this range is due to aconcentration phenomena known as the prozone effect.”224 At low concentrations ofn144RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 93antibody, each antibody molecule binds a hapten molecule at both of the Fab sites (seesection 3.3 Antibody Structure) as illustrated in Figure 13. At high concentrations, theantibodies aggregate and they can bind the hapten at only one of the antigen binding sites.These antibodies are weakly bound to the well wall and they are removed in subsequentwashing steps so that although the concentration of antibodies in the serum is high, theamount of antibody that remains bound to the hapten on the assay plate is low and adecrease in fluorescence is observed as the concentration of antibody increases(Figure 14b). As the antibody concentration decreases, the aggregation decreases;however, there is still an excess of antibody compared to hapten. The binding of antibodyis limited by the amount of hapten bound to the ELISA plate and therefore, a nearly steadyfluorescence is observed (Figure 14a). Beyond this range, the hapten is in excess ofantibody so that the fluorescence should decrease with the decreasing amount antibodypresent. This is observed in Figure 15.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 94(a)H23: Carrier Protein Control Experiment1.0-, 0.9- H23vs.1440.8- • H23 vs. BSA0.70.5-o 0.4-0.3-B ... a—.0.2-0.1-0.0-. 4. 4,-0.1- Ilol io2Reciprocal Dilution of Serum(b)H24: Carrier Protein Control Experiment1.00.9 0 H24vs.1440.8 • H24 vs. BSA0.70.60.5;!101 io2Reciprocal Dilution of SerumFigure 14. ELISA Comparison of Carrier Protein Controls with (a) H23 and (b) H24.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 954.3.1.2 Rabbit Serum Control ExperimentNext, we examined the effect of other rabbit serum. It is possible that theantibodies from any rabbit serum may have a non-specific affinity for our hapten. Wecompared the binding of the serum from the rabbits in our study to the serum of two otherrabbits (H14 and H22) from unrelated studies (Figure 15). A positive response of theserum from rabbits H23 and H24 to the hapten-BSA conjugate 144 compared to theserum of the other two rabbits eliminates the possibility of non-specific binding of rabbitantibodies to 144.Note that over this range of antibody concentration, the amount of hapten is inexcess of the antibody present and the fluorescence decreases with decreasing antibodyconcentration. This is in accord with the prozone effect described previously.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 96(a)H23: Serum Control Experiment1.0-0.9‘ H23vs.1440.8-. Hl4vs.1440.7- H22vs.1440.6-0.50.4-0.3-0.2• 0.1-—0.0--0.1-io2 io io io5 io6Reciprocal Dilution of Serum(b)H24: Serum Control Experiment1.00.9 0 H24vs.144.—0.8 • H14 vs. 1440.7 H22vs.1440.6C.?•—0.0•-0.1•io2 io io 1o5 1o6Reciprocal Dilution of SerumFigure 15. ELISA Comparison of Serum Controls H14 and H22 with (a) H23and (b) H24.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 974.3.1.3 Protein-Hapten Coupling Control ExperimentFinally, we examined the possibility that the antibodies may be binding to the linkerin 143 or to the reagents from the coupling reaction and hence interfering with the ELISAresults. We compared the hapten-BSA conjugate 144 to a mixture ofN-hydroxysuccinimide and BSA, and also to a mixture of compound 145 and BSA in anELISA assay (Figure 16). Again, a positive response of the serum from rabbits H23 andH24 to the hapten conjugate 144 as compared to the controls eliminates the possibility thatthe linker or any of the reagents from the protein coupling reaction had elicited an immuneresponse in the animals which was being measured in the ELISA assay.Over this concentration range of antibody, the nearly steady fluorescence readingcan again be ascribed to the prozone effect.145RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 98(a)H23: Coupling Control Experiment1.0•0.9 H23vs.1440.8 • H23vs.NHS0.7 -• H23 vs. 1450.6-0.5-o 0.4-—:0.0.-0.1- Iio2 io ioReciprocal Dilution of Serum(b)H24: Coupling Control Experiment1.00.9 H24vs.1440.8 • H24 vs. NHS0.7 H24vs. 1450.60.50 0.4—0.2_______ri0.0 I I I-0.1 Iio2 io ioReciprocal Dilution of SerumFigure 16. ELISA Comparison of Coupling Reaction Controls with (a) 1123and (b) 1124.ÉMRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 994.3.2 Purification of Polyclonal AntibodiesWe examined the use of affinity chromatography to purify the hapten-specificpolyclonal antibodies from the serum obtained from rabbits H23 and H24. Generally, thehapten of interest is coupled to a cyanogen bromide-activated Sepharose support via aurethane bond as illustrated in Figure 17. Because hapten 130 (see Scheme 16) does notpossess a nucleophilic group to react with the cyanogen bromide intermediate, we used theBSA-conjugate 144 (see section, reasoning that the amino groups of the proteinwould be suitable nucleophiles for this purpose.0NR’1 HI BrCN WNH2_____R RH20[—OH R RSepharose SupportFigure 17. Preparation of Hapten 144-Coupled Sepharose Affinity Colunm for thePurification of Polyclonal Antibodies.The serum is then passed over the functionalized support and the hapten-specificantibodies are retained on the column while any non-specific antibodies pass through. Thedesired antibodies are eluted from the column using an acidic glycine solution. Todetermine the relative concentration of antibodies in solution, the optical density at 2 =280 nm is measured.The amount of antibody retained on the column is governed by the affinity of theantibody for the hapten. If the affinity is low, then the antibodies of interest will simplyRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 100pass through the column without being retained. In fact, this was observed for the serumwe obtained from the rabbits in our study.We next examined a method that is similar to the affinity column protocol. In thiscase, instead of using the hapten of interest coupled to a Sepharose support, the column iscomposed of protein A. Protein A has an affinity for all mammalian IgG antibodies andthey are retained on the column by an interaction of their FC regions with protein A.225This method was successful in purifying antibodies from the serum of the rabbits of ourstudy.4.3.3 Synthesis of Substrate 148The initial route for the synthesis of substrate 148 is shown in Scheme 18. Theadvantage of this plan was that it would allow for the preparation of a variety of esterswhich would enable us to examine the effect of altering the substrate on the specificity ofany catalytic antibodies obtained. In addition, it was anticipated that the acetamide could beintroduced in a single step via the opening of the epoxide of compound 147.2261) Mg m-CPBAEt20 CH212)CO111 2 14683%°CO2H------ - -CO2H32) NaHCH3(O)NH 0147 DIVIF 148Scheme 18. Initial Synthetic Plan for Substrate 148.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 101Addition of carbon dioxide to the Grignard reagent formed from 111 gave the acid146 in good yield; however, the epoxidation of 146 to give 147 was unsuccessful. Themajor product isolated after chromatography was the lactone 149. Although evidence forthe desired epoxide 147 was observed in the 1H NMR of the crude reaction mixture, itwas not possible to isolate a sample of compound 147 that was not contaminated by thelactone 149, although the two compounds had significantly different Rf values by TLCanalysis. The cyclization of the epoxy acid 147 to the lactone 149 occurs under acidicconditions, as in the m-CPBA reaction, so it is possible that the cyclization may also takeplace under the mildly acidic conditions of the silica gel chromatography. This is asignificant result because it is analogous to the type of cyclization we had envisioned usingto obtain phosphonate 115 (see Scheme 15). It suggests that a successful route to 115might involve first hydrolyzing the phosphonate of 112, then carrying out the epoxidationon the corresponding alkenyl phosphonic acid. This is the reverse order in which wecarried out these transformations.OH149The route to compound 148 was modified as shown in Scheme 19. Afterpreparing the ester 150, the alkene was successfully epoxidized using m-CPBA.However, the anion of acetaniide did not open the epoxide of 151 as anticipated but reactedat the ester moiety of 151 to give the imide 153. The 4-nitrophenyl ester is verysusceptible to nucleophilic attack due to the good leaving group ability of the4-nitrophenolate anion. The 4-nitrophenyl ester had been prepared as a matter ofRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 102convenience because the corresponding methyl ester was more volatile and difficult to workwith.0_________m-CPBA1) SOC12ONOCH212) pyridine4-nitrophenolCH21 15014646%0 OHNaH2 CH3(O)NHC264NOONO - -DMF o151 15266%OH1) Hydrolyze2) EsteriC230148Scheme 19. Modified Synthetic Plan for Substrate 148.0 0ockH153RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 103To determine what conditions were necessary to open the epoxide with the anion ofacetamide, the THP-protected alcohol 154 was subjected to the reaction conditions shownin Equation 16. After heating to 110 °C for two hours, the starting material remained intactas indicated by TLC analysis. Addition of BF3Et2O, an effective Lewis acid which isknown to enhance epoxide opening by nucleophiles, resulted in loss of the starting materialbut none of the desired material could be isolated. In light of this, it is reasonable toassume that the methyl ester would not survive the harsh conditions necessary to open theepoxide.OT - - - - (16)CH3(O)NH2 IIDMF154 155Finally we examined the route to substrate 148 as shown in Scheme 20 andreported by Benkovic.124 Formation of the acid chloride 157, followed by displacementof the chloride by cyanide anion gave the acyl cyanide 158 (63% for two steps). Thecyanide was reduced via hydrogenation in the presence of acetic anhydride to yield theacetamide 159 (64%). The acidic conditions for the reduction of 159 to 148 are essential.We examined the reduction using NaBH4 without the addition of acid and found that thealkoxide intermediate in the reaction cydlized readily to the corresponding lactone.RESULTS & DISCUSSION: CATALYTIC ANTIBODIES 104HOOCH3sod2cOCH3 CH3N156 15792%NCOCH3 Pd/C OCH3H2 (40 psi) 0158 15969% 64%NaBH3CN‘OCHMeOHH+ 0148Scheme 20. Synthesis of Substrate 148.1244.3.4 Assay to Determine Catalytic ActivityAn HPLC assay was used for the direct detection of the product 160 of thecyclization reaction (Equation 17). Known amounts of 160 were injected onto a C-18reverse phase column using anCH3N:H20:CFOHsolvent system to establish a limitRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 105of detection for this compound under these conditions. The solvent ratios were chosensuch that compound 160 was separated from the substrate 148 by approximately3 minutes and both gave symmetric peak shapes.0CH3ONyAthod (17)0 pH=7.40148 160In the experiments to test for catalytic activity, the concentration of substrate 148(100 mM) was chosen such that it was well above the Km for typical polyclonal catalyticantibody systems.219’203 Similarly, the antibody concentration (5 ElM) was chosenbased on previously reported catalytic antibody experiments.124 Another factor inchoosing the concentration of antibody was the potential turnover rate of the antibody.Assuming a modest kcat = 0.5 minute-1,24 we can calculate the turnover of substrate toproduct at a given time for the reaction. The concentration of antibody was chosen suchthat the amount of product formed was estimated to be above the limit of detection for thesystem, ensuring that catalytic activity could be measured.Aliquots from the reaction mixture, taken at 30 minute intervals over the firsttwo hours and finally after 22 hours, showed no evidence for catalysis. While there wasno evidence for product formation, hydrolysis of the substrate was observed; however, thiswas not above the background levels of the control experiment.The lack of observed catalytic activity is possibly due to several factors. First, inpolyclonal serum, there are antibodies to many epitopes of the complete antigenicconjugate. The portion of the antigen represented by the haptenic molecule is quite smalland correspondingly, the number of antibodies in the serum that will recognize theRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 106substrate is quite small. Second, not every antibody that binds the hapten is catalytic, so ofthe small portion of antibodies in the polyclonal serum that bind the haptenic portion of theantigen, only a portion of these will be catalytic.4.4 CONCLUSIONThe cyclic phosphonate hapten 130 was prepared as a transition state analogue forthe lactonization of a hydroxy ester. This compound, when coupled to a carrier protein,was used to elicit an immune response in two rabbits. The resulting serum was shown tocontain antibodies which specifically bound to the haptenic portion of the antigen. AnHPLC assay used to examine these antibodies for their ability to catalyze the lactonizationreaction detected no catalysis under the conditions of the experiment.4.5 SUGGESTIONS FOR FUTURE WORKThis screening for catalytic activity of these polyclonal antibodies represents apreliminary survey of the conditions that might be necessary to observe catalysis. Forexample, most enzymes function over a pH range rather than at a single pH. It is possiblethat the pH of the experiment was outside the optimal working range for these antibodies sothe experiments should be repeated in solutions of different pH values.It is more likely that if these antibodies are catalytic, then the amount of catalysiswill be very small and the task will be to measure and quantify this. In that regard, a moresensitive assay system will be required. It might be possible to use the HPLC system if amore suitable substrate could be prepared. The ester/lactone chromophore we used fordetection does not have a large molar absorptivity. A suitable substrate would contain achromophore with as large a molar absorptivity as possible. However, the obvious choiceof a 4-nitrophenyl ester is inappropriate for two reasons. First, it would be dissimilar tothe hapten used for immunization (a methyl phosphonate ester) and it has been shown thateven subtle changes in the structure of the substrate can drastically reduce catalyticRESULTS & DISCUSSION: CATALYTIC ANTIBODIES 107activity.227 Second, the background rate of hydrolysis of a 4-nitrophenyl ester is greaterthan that for a methyl ester. To detect what is probably a small catalytic rate, it is desirablethat the difference in the catalyzed and uncatalyzed rates of reaction be as large as possible.Another possible substrate might contain a diene system, located on the carbonyl side of theamide. This would have a large molar absorptivity and would not significantly alter thereacting portion of the molecule.Alternatively, it might be necessary to use an entirely different assay system withhigher sensitivity. For example, detection of a radioactively labelled product wouldprovide a much more sensitive assay system. This method would involve incorporation ofa radioisotope (e.g. tritium) into the substrate, separation of substrate and product in thecatalytic reaction mixture, and scintillation counting of the resulting fractions to determinethe relative amounts of each. Fersht reports that depending on the specific activity of theisotope used, radioactivity-based assays can detect amounts as small as 10-17 moles ofcompound.228 This is several orders of magnitude below the limit of detection ofspectrophotometric methods.108CHAPTER VEXPERIMENTAL5.1 GENERAL CHEMICAL METHODSUnless otherwise stated, all reactions were performed under a nitrogenatmosphere using flame-dried glassware. Cold temperature baths were prepared asfollows: -78 °C (dry ice - acetone), -20 °C (dry ice - carbon tetrachloride), and 0 °C (ice -water).Anhydrous reagents and solvents were purified and prepared according toliterature procedures.229 The low boiling fraction (35-60 °C) of petroleum ether wasused. n-BuLi, purchased from Aldrich Chemical Co., was standardized by titrationagainst diphenylacetic acid230 or 2,5-dimethoxybenzyl alcohol in THF.23’All reactions were monitored by thin-layer chromatography (TLC) and werejudged to be completed when the starting material was consumed as determined by TLC.In the description of reaction work-ups, washing with brine refers to a saturated solutionof NaC1, drying of the organic phase was accomplished with magnesium sulphate andremoval of solvent in vacuo or concentration of solvent refers to the use of a rotaryevaporator using a water aspirator and heating using a water bath.Preparative flash column chromatography232was performed using silica gel, 230-400 mesh, supplied by E. Merck Co. A solvent system was chosen such that the desiredcompound had an Rf of approximately 0.35 on TLC. As an indicator of purity, allcompounds were purified such that they showed a single spot by TLC.Infrared (IR) spectra were recorded on a Bomem Michelson 100 FT-IRspectrometer using internal calibration. IR spectra were recorded on the neat liquid or asa chloroform solution between two NaC1 plates.Melting points were recorded on a Mel-Temp III (Laboratory Devices) meltingpoint apparatus and are uncorrected.EXPERIMENTAL 109Except where noted, proton nuclear magnetic resonance spectra (1H NMR) wererecorded in deuterochloroform solutions on a Bruker AC-200 (200 MHz), BrukerWH-400 (400 MHz), or a Bruker AMX (500 MHz) spectrometer. Chemical shifts aregiven in parts per million (ppm) on the 6 scale versus tetramethylsilane (3 0 ppm) orchloroform (3 7.24 ppm) as internal standards. Signal multiplicity, spin-spin couplingconstants (where possible), and integration ratios are indicated in parentheses.Phosphorus nuclear magnetic resonance spectra (31 NMR) were recorded indeuterochloroform solutions (except where noted) on a Bruker AC-200 (81 MHz).Chemical shifts are given in parts per million (ppm) on the 6 scale versus 85%phosphoric acid as an external standard.Low- and high-resolution electron-impact mass spectral analyses were performedon a Kratos-AEI model MS 50 mass spectrometer. An ionization potential of 70 eV wasused in all measurements. Low-resolution chemical-ionization mass spectral analyseswere recorded on a Delsi Nermag Rb-bC mass spectrometer. High-resolutionchemical-ionization mass spectral analyses were recorded on a Kratos MS 80 RFA massspectrometer. Only peaks with greater than 20% relative intensity or those which wereanalytically useful are reported.Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254pre-coated aluminium sheets. Visualization was achieved by irradiation with ultravioletlight at 254 nm and/or by spraying with anisaldehyde reagent (a solution of 1 mLanisaldehyde, 5 mL conc. H2S04and 10 mL glacial acetic acid in 90 mL methanol)followed by heating.Microanalyses were carried out at the microanalytical laboratory of the Universityof British Columbia Chemistry Department using a Carlo Erba Elemental Analyzer 1106.Samples for microanalyses were purified by column chromatography using the solventsystem indicated for each compound.EXPERIMENTAL 1105.2 CHEMICAL METHODS5.2.1 11-(1,3-Dithian-2-yl)-13-tetradecanolide (35)35; X = S(CH2)3A solution of 1 1-(1,3-dithian-2-yl)-13-hydroxy-tetradecanoic acid (34)54 (0.20 g,0.57 mmol) and Et3N (89 jiL, 0.63 mmol) in THF (6 mL) was stirred for 10 minutes atroom temperature, and then trichlorobenzoyl chloride (89 IlL, 0.57 mmol) was added andthe resulting solution was stirred for a further 3.5 hours at room temperature. Theresulting white precipitate was removed by filtration, washed with Et20, and the filtratewas concentrated by rotary evaporation and dried in vacuo for 20 minutes to yield themixed trichlorobenzoyl anhydride 47 as a yellow oil. This was used in the next stepwithout further purification.A solution of DMAP (0.42 g, 3.4 mrnol) in toluene (60 mL) was prepared andheated to reflux. The previously formed anhydride 47, diluted in 250 mL of toluene wasadded dropwise to the refluxing DMAP solution and the refluxing was continuedovernight. The volume of solvent was reduced to 100 mL by rotary evaporation, theresulting solution was diluted with Et20 and then washed successively with 0.5 M HC1,H20, NaHCO3and H20. The solution was dried and the solvent removed. Columnchromatography using EtOAc:PE (1:10) as eluant gave 35 as a yellow oil (0.090 g, 47%).IR (CHC13)v: 2931, 2860, 1721, 1488, 1460, 1371, 1258, 1109, 1048, 1018 cm1;EXPERIMENTAL 1111H NMR (CDCI3,400 MHz) ö: 5.18 (m, 1 H), 2.95-2.68 (m, 4 H), 2.40-2.20 (m,4 H), 2.05 (m, 1 H), 1.95-1.83 (m, 2 H), 1.80-1.65 (m, 2 H), 1.45-1.32 (m, 13 H),1.30 (d, 1= 6 Hz, 3 H);LRMS (El) mjz: 330 (M, 73), 256 (38), 223 (25), 159 (100), 149 (22), 106 (28), 100(46), 85 (22), 81(23), 69 (26), 55 (46);HRMS (El) Calcd forC17H3002S:330.1687; found: 330.1685.EXPERIMENTAL 1125.2.2 2-Benzyloxy-4-(1,3-dithian-2-yl)-14-(2-tetrahydropyranyloxy)-tetradecane (36)BnO 11(CH)9OT P362-Hydroxy-4-( 1 ,3-dithian-2-yl)- 14-(2-tetrahydropyranyloxy)-tetradecane (29)(1.0 g, 2.4 mmol) was dissolved in THF (15 mL), NaH (50% dispersion in oil, 0.086 g,3.6 mmol) was added and the mixture was stirred for 10 minutes. Benzyl bromide(0.61 g, 0.42 mL, 3.6 mmol) was added and the mixture was stirred at room temperaturefor 20 hours, then heated to reflux for 20 hours. The reaction was quenched with waterand the mixture was extracted with EtOAc. The combined extracts were washed withbrine and dried. Concentration of the solution and column chromatography usingEtOAc:PE (1:11) as eluant yielded 36 as a yellow oil (1.0 g, 83%).IR (neat) v: 3062, 3029, 2902, 1495, 1448, 1359, 1274, 1201, 1082, 906, 869, 813,738 cm*1H N1\’IR (CDC13,400 MHz) 6: 7.3 (m, 5 H), 4.58 (m, 1 H), 4.52 (d, J = 12 Hz, 1 H),4.48 (d, J = 12 Hz, 1 H), 3.90 (m, 2 H), 3.75 (m, 1 H), 3.50 (m, 1 H), 3.40 (m,1 H), 2.80 (m, 5 H), 2.30 (dd, J = 15, 7 Hz, 1 H), 2.05 (dd, J = 15, 3 Hz, 1 H),1.90-1.78 (m, 6 H), 1.70 (m, 1 H), 1.60-1.45 (m, 9 H), 1.50-1.20 (m, 12 H);LR1’VIS (El) m/z: 509 (M+l, 4), 508 (M, 11), 424 (59), 423 (28), 400 (18), 275 (21),161 (23), 106 (28), 105 (28), 91(100), 85 (64), 55 (27);HRMS (El) Calcd forC29H4803S:508.3045; found: 508.3035.EXPERIMENTAL 1135.2.3 2-Benzyloxy-4-oxo-14-(2-tetrahydropyranyloxy)-tetradecane (37)(CH)9OT P372-Benzyloxy-4-( 1,3 -dithian-2-yl)- 1 4-(2-tetrahydropyranyloxy)-tetradecane (36)(0.50 g, 0.98 mmol) was dissolved in acetone:water (4:1), then HgO (0.85 g, 3.9 mmol)and HgC12 (1.1 g, 3.9 mmol) were added and the mixture was stirred at room temperaturefor 15 minutes. The resulting precipitate was filtered through Celite, washed withacetone, and the filtrate was poured into a solution of 10% KI. The acetone was removedby rotary evaporation and the aqueous phase was extracted with CHC13. The organicphase was washed with 10% KI, brine and dried. Concentration and columnchromatography using EtOAc :PE (1:5) as eluant yielded 37 as a clear, colourless liquid(0.29 g, 70%).IR (neat) v: 2929, 2855, 1713, 1456, 1365, 1201, 1128, 1072, 1031, 905, 813,737 cm1;1H NMR (CDC13,400 MHz) ö: 7.3 (m, 5 H), 4.58 (m, 1 H), 4.52 (d, J = 12 Hz, 1 H),4.45 (d, J = 12 Hz, 1 H), 4.05 (m, 1 H), 3.87 (m, 1 H), 3.62 (m, 1 H), 3.50 (m,1 H), 3.38 (m, 1 H), 2.79 (dd, J = 16, 7 Hz, 1 H), 2.50 (t, J = 7 Hz, 2 H), 2.45-2.35(m, 4 H), 1.85 (m, 1 H), 1.75-1.45 (m, 7 H), 1.40-1.20 (m, 14 H);LRMS (El) m/z: 418 (M, 14), 333 (26), 228 (23), 185 (21), 107 (20), 91(92), 87(24), 86 (34), 85 (100), 84 (42), 71(21), 69 (43), 55 (39), 41(22);HRMS (El) Calcd forC26H420:418.3083; found: 418.3088;Anal. Calcd forC26H420:C, 74.60; H, 10.11; found: C, 74.40; H, 10.20.EXPERIMENTAL 1145.2.4 13-Benzyloxy-11-oxo-tetradecan-1-ol (38)(CH2)90382-Benzyloxy-4-oxo- 1 4-(2-tetrahydropyranyloxy)-tetradecane (37) (0.25 g,0.60 mmol) was dissolved in MeOH (5 mL) and p-TsOH (0.01 g, 0.06 mmol) was addedand the solution was stirred at room temperature for 45 minutes. It was diluted withEt20, washed with water, brine and dried. Concentration of the solution and columnchromatography using EtOAc:PE (1:3) as eluant yielded 38 as a clear, colourless oil(0.16 g, 80%).IR(neat)v: 3411, 2927, 2855, 1710, 1496, 1456, 1373, 1076, 738 cm-1;1H NMR (CDC13,400 MHz) ö: 7.3 (m, 5 H), 4.52 (d, J = 11 Hz, 1 H), 4.45 (d, J =11Hz, 1 H), 4.05 (m, 1 H), 3.6 (t, J= 7Hz, 2 H), 2.75 (dd, J= 14, 8Hz, 1 H),2.5-2.35 (m, 3 H), 1.68 (br s, 1 H), 1.55 (m, 5 H), 1.40-1.30 (m, 11 H), 1.27 (d,J=6Hz,3H);LRMS (El) m/z: 335 (M+1, 35), 228 (52), 181 (56), 167 (61), 149 (51), 107 (80), 91(100), 86 (88), 79 (71), 69 (92), 65 (72), 55 (92);HRMS (El) Calcd forC21H350(M+1): 335.2586; found: 335.2557;Anal. Calcd forC21H340:C, 75.39; H, 10.25; found: C, 75.10; H, 10.15.EXPERIMENTAL 1155.2.5 13-Benzyloxy-11-oxo-tetradecanoic acid (39)(CH)8COH3913-Benzyloxy-1 1-oxo-tetradecan-1-ol (38) (0.15 g, 0.45 mmol) was dissolved inacetone (3 mL) and the solution was cooled to 0 °C. Jones reagent was added until thecolour of the solution remained a persistent dark brown, then isopropanol wasimmediately added until the solution turned colourless with the formation of a greenprecipitate. The acetone was removed by rotary evaporation and the precipitate wasdissolved in 1 M HC1 and extracted with EtOAc. The combined extracts were dried andconcentrated. Column chromatography using EtOAc:PE (1:1) as eluant yielded 39 as ayellow oil (0.050 g, 32%).IR (neat) v: 3600-2500 (br), 2928, 2855, 1715, 1495, 1455, 1409, 1374, 1097, 1027,930, 739 cm1;1H NMR (CDC13,400 MHz) : 7.3 (m, 5 H), 4.52 (d, J = 12 Hz, 1 H), 4.43 (d, J =12 Hz, 1 H), 4.05 (m, 1 H), 2.75 (dd, J = 16, 8 Hz, 1 H), 2.45-2.35 (m, 3 H), 2.30(t, J = 8 Hz, 2 H), 1.65-1.50 (m, 5 H), 1.35-1.22 (m, 9 H), 1.20 (d, J = 6 Hz, 3 H);LRMS (El) m/z: 349 (M+1, 3), 330 (2), 302 (2), 242 (20), 105 (25), 91(100), 84(45), 69 (52), 55 (27), 43 (22), 41(26);LR1’IS (DCI) m/z: 366 (M+18, 100), 349 (M-i-1, 18);HRMS (DCI) Calcd forC21H3304(M+1): 349.2379; found: 349.2378.EXPERIMENTAL 1165.2.6 2-Acetoxy-4-oxo-14-(2-tetrahydropyranyloxy)-tetradecane (40)(CH)9OT P402-Acetoxy-4-( 1, 3-dithian-2-yl)- 1 4-(2-tetrahydropyranyloxy)-tetradecane (30)(0.50 g, 1.1 mmol) was dissolved in acetone:water (4:1), then HgO (0.94 g, 4.3 mmol)and HgC12 (1.2 g, 4.3 mmol) were added and the mixture was stirred at room temperaturefor 10 minutes. The resulting precipitate was filtered through Celite, washed withacetone, and the filtrate was poured into a solution of 10% KI. The acetone was removedby rotary evaporation and the aqueous phase was extracted with CHC13. The organicphase was washed with 10% KI, brine and dried. Concentration and columnchromatography using EtOAc:PE (1:5) as eluant yielded 40 as a clear, colourless liquid(0.35 g, 87%).IR (neat) v: 2930, 2855, 1740, 1717, 1454, 1409, 1372, 1243, 1200, 1130, 1076,1030, 958, 904, 868, 813 cm1;1H NMR (CDC13,400 MHz) & 5.35 (m, 1 H), 4.58 (m, 1 H), 3.88 (m, 1 H), 3.72 (m,1 H), 3.50 (m, 1 H), 3.38 (m, 1 H), 2.79 (dd, J = 16, 7 Hz, 1 H), 2.52 (dd, J = 16,6 Hz, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.0 (s, 3 H), 1.85 (m, 1 H), 1.70 (m, 1 H),1.65-1.45 (m, 8 H), 1.40-1.20 (m, 15 H);LRMS (El) m/z: 370 (M, 9), 255 (22), 85 (100), 84 (64), 69 (57), 55 (28), 43 (30);HRMS (El) Calcd forC21H3805:370.2719; found: 370.2713;Anal. Calcd forC21H3805:C, 68.07; H, 10.34; found: C, 67.79; H, 10.44.EXPERIMENTAL 1175.2.7 13-Acetoxy-11-oxo-tetradecan-1-ol (41)(CH2)90412-Acetoxy-4-oxo- 14-(2-tetrahydropyranyloxy)-tetradecane (40) (0.29 g,0.78 mmol) was dissolved in MeOH (5 mL) and p-TsOH (0.010 g, 0.058 mmol) wasadded and the solution was stirred at room temperature for 45 minutes. It was dilutedwith Et20, washed with water, brine and dried. Concentration and columnchromatography using EtOAc:PE (1:3) as eluant yielded 41 as a clear, colourless oil(0.18 g, 80%).IR (neat) v: 3429, 2927, 2855, 1739, 1716, 1459, 1409, 1373, 1247, 1138, 1101,1043, 957, 721 cm1;1H NMR (CDC13,400 MHz) ö: 5.25 (m, 1 H), 3.60 (t, J 8 Hz, 2 H), 2.74 (dd, J16, 8 Hz, 1 H), 2.50 (dd, J = 16, 6 Hz, 1 H), 2.35 (t, J 7.5 Hz, 2 H), 2.0 (s, 3 H),1.85 (br s, 1 H), 1.55 (m, 4 H), 1.35-1.20 (m, 15 H);LRMS (El) m/z: 287 (M-i-1, 61), 227 (98), 209 (59), 167 (72), 149 (72), 125 (73), 97(88), 84 (98), 69 (100), 55 (94);HRMS (El) Calcd forC16H3104(M+1): 287.2222; found: 287.2233;Anal. Calcd forC16H3004:C, 67.08; H, 10.56; found: C, 66.86; H, 10.76.EXPERIMENTAL 1185.2.8 13-Acetoxy-li-oxo-tetradecanoic acid (42)(CH2)8C0H4213-Acetoxy-1 l-oxo-tetradecan-l-ol (41) (1.4 g, 4.9 mmol) was dissolved inacetone (25 mL) and the solution was cooled to 0 °C. Jones reagent was added until thecolour of the solution remained a persistent dark brown, then isopropanol wasimmediately added until the solution turned colourless with the formation of a greenprecipitate. The acetone was removed by rotary evaporation and the precipitate wasdissolved in 1 M HC1 and extracted with EtOAc. The combined extracts were dried andconcentrated. Colunm chromatography using EtOAc : PE (1:1) as eluant yielded 42 as awhite solid (0.84 g, 57%).mp: 44.5-46.0 °C;IR (CDC13)v: 3515, 2930, 2857, 1731, 1712, 1458, 1373, 1265, 1135, 1049,957 cm;1H NMR (CDC13,400 MHz) ö: 5.30 (m, 1 H), 2.80 (dd, J = 16, 8 Hz, 1 H), 2.54 (dd,J = 16, 6 Hz, 1 H), 2.40 (t, J = 8 Hz, 2 H), 2.35 (t, J = 8 Hz, 2 H), 2.01 (s, 3 H),1.70-1.50 (m, 5 H), 1.40-1.20 (m, 12 H);LRMS (El) m/z: 301 (M+1, 28), 300 (M, 10), 97 (29), 85 (67), 84 (78), 83 (32), 69(100), 57 (38), 55 (42);HRMS (El) Calcd forC16H2905(M-i-1): 301.2015; found: 301.2011;Anal. Calcd forC16H2805:C, 63.97; H, 9.40; found: C, 64.06; H, 9.33.EXPERIMENTAL 119The following compound was also isolated from this reaction and purified bycolumn chromatography using EtOAc:PE (1:1) as eluant (1%):5.2.9 11-Oxo-12-tetradecenoic acid (44)(CH)8COH44mp: 70.0-74.0 °C;IR (CDC13)v: 3400-2400 (br), 2930, 2856, 1710, 1663, 1628, 1440, 1406, 1291,1245, 1220, 1174, 974 cm-1;1H NMR (CDC13,400 MHz) ö: 6.85 (dq, I = 16, 8 Hz, 1 H), 6.15 (dq, J = 16, 2 Hz,1 H), 2.52 (t, J = 7.5 Hz, 2 H), 2.35 (t, J = 7.5 Hz, 2 H), 1.90 (dd, J 8, 2Hz,3H), 1.60 (m,5H), 1.40-l.20(m, 1OH);LRMS (El) m/z: 240 (M, 5), 222 (16), 207 (23), 194 (38), 153 (20), 97 (38), 84 (85),69 (100), 55 (27);HRMS (El) Calcd forC14H203:240.1725; found: 240.1730.EXPERIMENTAL 1205.2.10 13-Hydroxy-li-oxo-tetradecanoic acid (43)(CH2)8C0H4313-Acetoxy-1 1-oxo-tetradecanoic acid (42) (0.99 g, 3.3 mmol) was dissolved inMeOH:H20(3:1) (15 mL), NaOH was added until the solution was basic (pH >12) andthe mixture was stirred at room temperature for 60 hours. The resulting yellow solutionwas acidified with concentrated HC1, then the MeOH was removed by rotary evaporation.The remaining aqueous portion was extracted with EtOAc, and the combined extractswere dried. Concentration and column chromatography using EtOAc:PE (1:2) as eluantyielded 43 as a yellow oil (0.57 g, 66%).1H NMR (CDC13,200 MHz) ö: 7.6 (br s, 2 H), 4.2 (m, 1 H), 2.5 (m, 2 H), 2.30 (m,5 H), 2.1 (m, 3 H), 1.8-1.5 (m, 6 H), 1.4-1.2 (m, 4 H), 1.05 (t, J = 6Hz, 3 H).Subsequent attempts to form compound 43 under the same conditions wereunsuccessful. Instead, compound 45, a white solid, was isolated and purified by columnchromatography using EtOAc:PE (1:1) in all experiments (80%).5.2.11 13-Methoxy-11-oxo-tetradecanoic acid (45)MeO 0(CH2)8C0H45mp: 32.5-34.0 °C;EXPERIMENTAL 121IR (CHC13)v: 3400-2400 (br), 1713, 1526, 1418, 1351, 1287, 1205, 1135, 1077,830 cm-1;1H NMR (CDC13,400 MHz) 6: 3.80 (m, 1 H), 3.30 (s, 3 H), 2.70 (dd, J = 16, 7 Hz,1 H), 2.42 (m, 3 H), 2.35 (m, 3 H), 1.70-1.50 (m, 5 H), 1.35-1.20 (m, 8 H), 1.18(d,J=7Hz,3H);LRMS (El) m/z: 272 (M, 17), 240 (23), 239 (34), 222 (21), 116 (32), 101 (33), 84(60), 69 (60), 59 (100), 41(20);HRMS (El) Calcd forC15H2804:272.1987; found: 272.1984;Anal. Calcd forC15H2804:C, 66.14; H, 10.36; found: C, 66.51; H, 10.46.•(Hii‘w)Oii-‘(Hc‘m)OVI-0L1‘(H‘s)0‘(H9‘w)E1-91‘(Hj‘zH8‘ci=f‘pp)081‘(HI‘ui)c‘(HI‘s)L6:(zpjoo‘EJ3Q)UPINHjnopu.woousiMuoizuirtqIduJoDi‘iqiinj(upiioqipious1?tpunodwoDsiqiwiprnjqiprnkiiqusuionu(%cc‘oco)piosojpis9f,pppiAuonuuopuppuiuuqqipqsuqi‘QZqiMpinIJpsiainixuqjsnurtuJ1AOainiijdujiwoosoiuJ.woipojsiarnixiwqiprnppps(lounu91‘Tn11‘91)N1JSIflUW01qinJ‘°JD08L-ItpaiJBSS1MainIXHUqi)U1pppiS1M(‘uii)1TDH3in(jouuu1E‘160)(It’)o--uj-xoi-1JOUOTflOSVD0SL-VSflUTW01qiou1JOJpaUTISSMUOTIflJOSqiU11’PSM(10mw9L“iI7co‘6co)pixoqdnsJAqiT-u•8L-oipi°°SMuoTlnjosqipui(‘1w01)1DH3UTPA10SSTPSUM(10mwç•“lulOO‘17V0)pTJ0)1l(TtXO91’OHD 8(HD)(91’)innpJJ-oxo-JJ-xoJv-Jzurs111‘TVIN3JAJIc1XEXPERIMENTAL 1235.2.13 11-Hydroxy-13-tetradecanolide (49)To a cooled solution of 1 1-oxo-13-tetradecanolide (24) (0.032 g, 0.13 mmol) inTHF (5 mL) at -78 °C, a solution of L-Selectride (0.15 mL, 0.15 mmol, 1 M in THF) wasadded and the mixture was stirred at -78 °C for 2 hours. The solution was quenched with30% H20(0.5 mL) and 1 M NaOH (0.5 mL), acidified with 1 M HC1 and then extractedwith Et20. The combined extracts were washed with brine and dried. Concentration andcolumn chromatography using EtOAc:PE (1:5) as eluant gave the alcohol 49 as a clear,colourless oil (0.020 g, 62%).IR (CHC13)v: 2931, 2858, 1720, 1459, 1362, 1119, 998 cm1;1H NMR (CDC13,400 MHz) 8: 4.94 (m, J = 10, 6, 2 Hz, 1 H), 3.90 (m, J = 8, 4 Hz,1 H), 2.41 (ddd, J = 14, 10, 4 Hz, 1 H), 2.24 (ddd, J = 14, 10, 4 Hz, 1 H), 1.95(ddd, J = 14, 10, 4 Hz, 1 H), 1.75-1.50 (m, 5 H), 1.45-1.20 (m, 16 H);LRMS (El) m/z: 242 (M, 0.1), 224 (M-18, 7), 183 (44), 112 (25), 111 (28), 98(73),97 (32), 96 (25), 95 (42), 84 (42), 83 (30), 82 (37), 81(62), 73 (30), 71(62), 69(59), 68 (53), 67 (52), 55 (100), 45 (31), 44 (31), 43 (88), 41(81);HRMS (El) Calcd forC14H2603:242.1882; found: 242.1877.EXPERIMENTAL 1245.2.14 1 1-Bromoacetyl-13-tetradecanolide (50)50; R = BrCH2C(O)1 1-Hydroxy-13-tetradecanolide (49) (0.023 g, 0.095 mmol) was dissolved in Et20(1 mL) and the mixture was cooled to 0 °C. DMAP (1.0 mg, 9.5 pmol), pyridine(0.045 g, 0.046 mL, 0.57 mmol), and bromoacetyl bromide (0.077 g, 0.033 mL,0.38 mmol) were added and the mixture was stirred for 2 hours. The reaction wasquenched with water and then diluted with Et20. The layers were separated and theaqueous phase was extracted with Et20. The combined extracts were washed with 1 MHC1, saturated NaHCO3,brine and dried. Concentration of the solution followed bycolumn chromatography using EtOAc:PE (1:7) as eluant yielded 50 as a yellow oil(0.021 g, 61%).1H NMR (CDC13,400 MHz) ö: 5.18 (m, 1 H), 4.95 (m, 1 H), 3.80 (s, 2 H), 2.49(ddd, J = 14.2, 6, 3.4 Hz, 1 H), 2.25 (ddd, J = 14.2, 8.5, 3.4 Hz, 1 H), 2.00 (m,1 H), 1.80-1.65 (m, 3 H), 1.60-1.45 (m, 4 H), 1.40-1.20 (m, 13 H);LRMS (DCI) m/z: 365(81Br, M+1, 27), 363 (79Br, M-i-1, 27), 225 (92), 224 (96),223 (31), 207 (55), 206 (42), 205 (25), 183 (28), 181 (26), 180 (41), 137 (22), 135(24), 123 (45), 122 (22), 121 (30), 112 (39), 111 (31), 110 (26), 109 (54), 108(21), 98 (75), 97 (38), 96 (46), 95 (91), 94 (24), 93 (20), 91(72), 85 (22), 84 (27),83 (46), 82 (62), 81(100), 71(28), 69 (70), 68 (80), 67 (60), 57 (23), 55 (83), 54(24), 43 (69), 41(38);EXPERIMENTAL 125fIRMS (DCI) Calcd forC16H28O481Br(Mfll): 365.1150; found: 365.1156;Caled forC16H28O479Br(M+1): 363.1170; found: 363.1166.EXPERIMENTAL 1265.2.15 1,11,13-Tetradecanetriol (51)LiA1H4 (7.0 mg, 0.18 mmol) was added to a solution of il-hydroxy13-tetradecanolide (49) (0.020 g, 0.083 mmol) in THF (5 mL) and the reaction mixturewas stirred for 20 minutes at room temperature. The reaction was quenched with 1 MNaOH (1 mL) and the resulting precipitate was filtered and washed with Et20. Thelayers of the filtrate were separated and the organic phase was washed with brine anddried. Concentration gave 51 as a yellow oil which was used in the next step withoutfurther purification.51EXPERIMENTAL 1275.2.16 4-(1O’-hydroxydecane)-2,2,6-trimethyl-1,3-dioxane (52)Crude 1,1 1,13-tetradecanetriol (51) was dissolved in acetone (5 mL), a catalyticamount of p-TsOH and CuSO4were added, and the reaction was stirred at roomtemperature for 10 minutes. The mixture was then diluted with Et20, washed withNaHCO3, brine, and dried. Concentration of solvent followed by columnchromatography using EtOAc:PE (1:3) as eluant gave 52 as a yellow oil (0.012 g, 49%for two steps).1H NMR (CDC13,400 MHz) ö: 3.95 (m, 1 H), 3.80 (m, 1 H), 3.62 (t, J 7 Hz, 2 H),1.60-1.30 (m, 27 H), 1.16 (d,J=6Hz,3H);l3 NMR (CDC13,50 MHz) ö: 98 (C), 68 (CH), 64 (CH), 62 (CH), 39 (CR2), 37(CR2), 33 (CR2), 30 (CR3), 29 (CH2), 28 (CR2), 26 (CH2), 25 (CR2), 22 (CH3),19 (CH3), 9 (CH2);LRMS (DCI) m/z: 287 (M+1, 8), 272 (25), 271 (100), 229 (32), 211 (31), 137 (26),129 (32), 123 (44), 111 (25), 109 (69), 97(41), 95(91), 83 (40), 81(80), 71(30),69 (60), 68 (21), 67 (40), 59 (81), 56 (28), 55 (53), 43 (47), 41(21);HRMS (DCI) Calcd forC17H350(M+1): 287.2586; found: 287.2580.EXPERIMENTAL 1285.2.17 1O-Undecen-1-ol (67)NCH2)9O67Lithium aluminium hydride (LAH) (4.63 g, 122 mmol) was added to THF(300 mL) and the resulting gray suspension was cooled to 0 °C. A THF solution of10-undecenoic acid (66) (15.0 g, 81.4 mmol) was added to the cooled LAH suspensionand the resulting mixture was allowed to warm to room temperature and stirredovernight. After cooling to 0 °C, the reaction was quenched with 1 M NaOH to give aclear solution containing a white precipitate. This precipitate was removed by suctionfiltration and washed with Et20. The layers of the filtrate were separated, and the organiclayer was washed with brine and dried. Concentration of solvent, followed by columnchromatography using EtOAc:PE (1:3) as eluant yielded 67 as a clear, colourless liquid(12.8 g, 92%).IR (CDC13)v: 3622, 3076, 2934, 2861, 1639, 1426, 1639, 1426, 1274, 1045 cnr1;1H NMR (CDC13,400 MHz) & 5.82 (m, J = 17, 10 Hz, 1 H), 4.98 (m, 3 = 17, 2 Hz,1 H), 4.94 (m, J = 10, 2 Hz, 1 H), 3.64 (t, J = 6 Hz, 2 H), 2.05 (m, 2 H), 1.57 (m,2 H), 1.42-1.22 (m, 13 H);LRMS (DCI) m/z: 188 (M+18), 169 (M-1), 152 (M-18);LRMS (El) m/z: 96 (27), 95 (33), 82 (62), 81(60), 69 (47), 68 (70), 67 (82), 55 (100),54 (47), 43 (24), 41(81), 39 (21), 31(12);HRMS (El) Calcd forC11H20 (M-18): 152.1565; found: 152.1556;Anal. Calcd forC11H220:C, 77.57; H, 13.03; found: C, 77.62; H, 13.00.EXPERIMENTAL 1295.2.18 1O-Undecen-1-p-toluenesulphonate (68)NCH2)9OTs6810-Undecen-1-ol (67) (4.37 g, 25.6 mmol) was dissolved in CH2C12 (100 mL)and the following were added successively: Et3N (5.20 g, 7.15 mL, 51.3 mmol), DMAP(0.31 g, 2.6 mmol), and p-toluenesulphonyl chloride (6.36 g, 33.3 mmol) and theresulting mixture was stirred overnight at room temperature. It was then diluted withEt20, washed with NaHCO3, brine and dried. Concentration of solvent and columnchromatography using EtOAc:PE (1:3) as eluant yielded 68 as a clear, colourless oil(7.45 g, 89%).IR (CHCT3)v: 2977, 2930, 2858, 1598, 1445, 1356, 1189, 1177, 1114 cm;1H NMR (CDC13,400 MHz) 6: 7.78 (d, J = 7.5 Hz, 2 H), 7.35 (d, J = 7.5 Hz, 2 H),5.81 (m, J = 17, 10Hz, 1 H), 4.99 (m, J = 17, 2Hz, 1 H), 4.92 (m, J = 10, 2Hz,1 H), 4.03 (t, J = 6 Hz, 2 H), 2.46 (s, 3 H), 2.04 (m, 2 H), 1.63 (m, 2 H), 1.40-1.20(m, 12 H);LR1VIS (DCI) m/z: 342 (M+18) (100), 152 (2), 108 (2), 81(1), 54 (1);HRMS (El) Calcd forC11H20 (M-171): 152.1565; found 152.1571;Anal. Calcd forC18H203S:C, 66.63; H, 8.70; found: C, 66.90; H, 8.65.EXPERIMENTAL 1305.2.19 11-Undecen-1-nitrile (69)NCH2)9CN69A mixture of 10-undecen-1-p-toluenesulphonate (68) (3.01 g, 9.28 mmol) andpotassium cyanide (1.21 g, 18.5 mmol) in DMSO (20 mL) was heated to 70 °C for30 minutes. The cooled reaction mixture was diluted with Et20, washed with water,brine, and dried. Concentration of solvent and column chromatography using EtOAc:PE(1:15) as eluant yielded 69 as a clear, colourless oil (1.10 g, 66%).IR (CDC13)v: 3076, 2930, 2857, 2243, 1639, 1440, 1287, 998 cm1;1-H NMR (CDC13,400 MHz) ö: 5.82 (m, J = 17, 10 Hz, 1 H), 5.00 (m, J 17, 2 Hz,1 H), 4.93 (m, J = 10, 2 Hz, 1 H), 2.34 (t, J = 7.5 Hz, 2 H), 2.05 (m, 2 H), 1.66 (m,2 H), 1.50-1.22 (m, 12 H);LRMS (El) m/z: 179 (M, 2), 178 (3), 150 (25), 136 (60), 122 (71), 108 (24), 96 (22),94 (26), 83 (22), 82 (24), 69 (46), 56 (28), 55 (100), 54 (24), 41(92);HRMS (El) Calcd forC12H21N: 179.1674; found: 179.1672;Anal. Calcd forC12H21N: C, 80.37; H, 11.80; N, 7.81; found: C, 80.25; H, 11.90; N,8.00.EXPERIMENTAL 1315.2.20 11-Dodecenal (70)‘NCH2)9CHO701 1-Undecen-1-nitrile (69) (3.52 g, 19.6 mmol) was dissolved in CH21 (100 mL)and the solution was cooled to -78 °C. Diisobutylaluminium hydride (DIBAL) (54.9 mL,1 M in hexanes, 54.9 mmol) was added dropwise and the resulting mixture was stirred at-78 °C for 4 hours. The reaction was quenched with 1 M HC1, and then diluted withEt20. The layers were separated and the aqueous layer was extracted with Et20. Thecombined organic extracts were washed with 1 M HC1, brine and dried. Concentration ofthe solvent yielded 70 as a yellow liquid (2.94 g (85% pure by GC), 70%) that was usedwithout further purification.IR (CHC13)v: 3077, 2929, 2856, 2727, 1722, 1639, 1447, 1350, 1128, 1035 cm1;1H NMR (CDC13,400 MHz) 6: 9.78 (s, 1 H), 5.85 (m, 1 H), 4.95 (m, 2 H), 2.45 (td,J = 8, 2 Hz, 2 H), 2.05 (m, 2 H), 1.65 (m, 2 H), 1.58-1.20 (m, 12 H);LRMS (El) m/z: 181 (M-1, 0.4), 96 (20), 95 (20), 84 (25), 83 (31), 82 (41), 81(30),73 (20), 69 (53), 68 (36), 67 (34), 56 (21), 55 (100), 54 (25), 43 (21), 41(76), 39(26);HRMS (El) Calcd forC12H210(M-1): 181.1592; found: 181.1580.EXPERIMENTAL 1325.2.21 11-Dodecenoic acid (71)‘NCH2)9COH711 1-Undecen-1-nitrile (69) (14.49 g, 80.81 mmol) was dissolved in EtOH(300 mL) and NaOH was added until the solution was saturated, and then the solutionwas heated to reflux. After 24 hours, the solution was light brown and contained a whiteprecipitate. Water was added and the aqueous phase was acidified with concentratedHC1, and extracted with EtOAc. The combined organic extracts were washed with brine,dried, and concentrated in vacuo to give 71 as a yellow oil (11.56 g, 72%) which wasused without further purification.IR (CDC13) v: 3500-2500 (s, br), 3190, 3056, 2930, 2856, 1711, 1639, 1421, 1275,1017, 843 cm1;1H NMR (CDC13, 200 MHz) 6: 11.3 (br s, 1 H), 5.9-5.7 (m, 1 H), 5.0-4.8 (m, 2 H),2.3 (t, J = 7.5 Hz, 2 H), 2.0 (m, 2 H), 1.6 (m, 2 H), 1.4-1.0 (m, 12 H);LRMS (El) m/z: 198 (M, 1), 180 (3), 97 (23), 96 (37), 95 (27), 86 (44), 84 (69), 83(34), 82 (49), 81(37), 69 (56), 68 (41), 67 (44), 60 (77), 59 (53), 56 (22), 55(100), 54 (28), 45 (69), 43 (93), 41(74), 32 (34), 31(36);HRMS (El) Calcd forC12H20:198.1620; found: 198.1615.EXPERIMENTAL 1335.2.22 11-Dodecen-1-ol (72)72(i) Lithium aluminium hydride (4.43 g, 117 mmol) was suspended in THF(300 mL) and the solution cooled toO °C. A solution of 1 1-dodecenoic acid (71) (11.6 g,58.3 mmol) in THF (75 mL) was added to the LAH suspension and the mixture wasallowed to warm to room temperature overnight. After cooling to 0 °C, the reaction wasquenched with 1 M NaOH and the resulting precipitate was removed by filtration andwashed with Et20. The layers of the filtrate were separated and the organic layer waswashed with brine, dried and concentrated to give 72 as a yellow oil (9.10 g, 85%).(ii) An identical procedure using lithium aluminium hydride (0.78 g, 18 mrnol)and 1 1-dodecenal (70) (2.5 g, 14 mmol) yielded 72 as a yellow oil (2.1 g, 84%).IR (CHC13)v: 3612, 3075, 3003, 2939, 2860, 1677, 1638, 1426, 1318, 1193, 1138,1042 cm1;1H NMR (CDC13,200 MHz) ö: 5.9 (m, 1 H), 4.9 (m, 2 H), 3.6 (t, I 6.5 Hz, 2 H),2.0 (m, 3 H), 1.5 (m, 2 H), 1.4-1.0 (m, 14 H);LRMS (El) m/z: 185 (M+1, 0.5), 184 (M, 0.1), 166 (1), 96 (31), 95 (36), 83 (26),82 (64), 81(47), 69 (48), 68 (59), 67 (55), 55 (100), 54 (43), 43 (25), 41(61), 31(11);HRMS (El) Calcd forC12H240: 184.1827; found: 184.183 1.EXPERIMENTAL 1345.2.23 11-Dodecen-1-p-toluenesulphonate (73)‘NCH2)1oOTs731 1-Dodecen-1-ol (72) (9.10 g, 49.4 mmol) was dissolved in CH21 (250 mL) andthe following were added sequentially: Et3N (10.9 g, 15.0 mL, 108 mmol),4-pyrryolidinopyridine (0.73 g, 4.94 mmol) and p-toluenesulphonyl chloride (13.2 g,69.2 mmol). The mixture was stirred overnight at room temperature, after which time itwas diluted with Et20, washed successively with 1 M HC1, saturated NaHCO3,brine, anddried. Concentration of the solvent and colunm chromatography using EtOAc:PE (1:20)as eluant yielded 73 as a white solid (14.8 g, 88%).mp: 40.5-43.0 °C;IR (CHC13)v: 2928, 2856, 1638, 1599, 1460, 1358, 1174, 1098, 959, 910 cm1;1H NMR (CDC13,200 MHz) 6: 7.8 (d, J = 8 Hz, 2 H) 7.3 (d, J = 8 Hz, 2 H), 5.8 (m,1 H), 5.0 (m, 2 H), 4.0 (t, J = 6.5 Hz, 2 H), 2.4 (s, 3 H), 2.0 (m, 2 H), 1.6 (m, 2 H),1.4-1.1 (m, 14 H);LR1’1S (DCI) m/z: 356 (M+18, 95), 339 (M+1, 2), 338 (M, 1), 167 (100), 166(98), 155 (60), 138 (47), 137 (40), 125 (37), 124 (86), 123 (61), 111 (92), 96(92),81(95), 68 (88), 55 (59);HRMS (DCI) Calcd. forC19H310S(M+1): 339.1994; found: 339.1990;Anal. Calcd forC19H300S:C, 67.42; H, 8.94; found: C, 67.49; H, 8.98.EXPERIMENTAL 1355.2.24 12-Iodo-1-dodecene (74)NCH2)10’741 1-Dodecen-1-p-toluenesulphonate (73) (14.22 g, 42.01 mmol) was dissolved inacetone (200 mL), NaT (18.90 g, 126.1 mmol) was added and the mixture heated at refluxfor 1 hour, resulting in a brown solution with a white precipitate. The volume of solventwas reduced in vacuo to one half the original volume and the remainder of the mixturewas partitioned between EtOAc and H20. The organic layer was washed with saturatedNa2SO3,brine, dried and concentrated. Column chromatography using EtOAc:PE(1:20) as eluant yielded 74 as a light orange oil (9.03 g, 73%).IR (CHC13)v: 2928, 2855, 1639, 1445 cm1;1H NMR (CDC13,400 MHz) 6: 5.8 (m, 1 H), 5.0 (m, 2 H), 3.2 (t, J = 7 Hz, 2 H), 2.05(m, 2 H), 1.85 (m, 2 H), 1.5-1.2 (m, 14 H);LRMS (El) m/z: 294 (M, 16), 252 (13), 111(33), 97(64), 83 (81), 69(89), 57(28),55 (100), 43 (35), 41(56);HR1VIS (El) Calcd forC12H23: 294.0844; found: 294.0840.EXPERIMENTAL 1365.2.25 Dimethyl 12-tridecenyiphosphonate (75)(CH2)11(OCH3)275Dimethyl methyiphosphonate (63 j.tL, 0.58 mmol) was dissolved in THF (2 mL)and the solution was cooled to -78 °C. n-BuLi (0.34 mL, 1.7 M, 0.57 mmol) was addedand the mixture was stirred at -78 °C for 1 hour. A solution of 12-iodo-1-dodecene (74)(69 mg, 0.23 mmol) in THF (1 mL) was added to the anion of dimethylmethyiphosphonate, and the mixture was stirred at -78 °C for 2 hours. The reaction wasallowed to warm to room temperature, after which the reaction was quenched with 1120and the mixture diluted with Et20. The layers were separated, the aqueous phase wasacidified with 1 M HC1 and then extracted with Et20. The combined organic extractswere washed with brine, dried, and concentrated. Column chromatography usingEtOAc:PE (8:1) as eluant yielded 75 as a yellow oil (35 mg, 52%).IR (CHC13)v: 2980, 2927, 2854, 1639, 1459, 1282, 1063, 1048 cm1;1H NMR (CDC13,200 MHz) ö: 5.8 (m, 1 H), 4.9 (m, 2 H), 3.7 (d, J = 10 Hz, 6 H),2.0 (m, 2 H), 1.8-1.45 (m, 4 H), 1.40-1.1 (m, 16 H);3lp NMR (CDC13,81 MHz) ö: 35.1;ERMS (El) m/z: 291 (M+1, 3), 290 (M, 14), 179 (20), 138 (20), 137 (51), 124(100), 110 (28), 41(23);HRMS (El) Calcd forC15H310P:290.2011; found: 290.2010.EXPERIMENTAL 1375.2.26 Dimethyl 12-hydroxytridecanylphosphonate (76)( 2)11 ( 3)276Mercuric acetate (0.95 g, 3.0 mmol) was dissolved in H20 (3 mL) and a solutionof dimethyl 12-tridecenylphosphonate (75) (0.87 g, 3.0 mmol) in THF (3 mL) was added.After 25 minutes, a solution of NaOH (10 mL, 3 M) was added, followed by a solution ofNaBH4 (10 mL, 0.5 M NaBH4in 3 M NaOH), and the mixture was stirred for 5 minutes.Sodium chloride was added to saturate the aqueous layer, the layers were separated andthe aqueous layer was extracted with Et20. The combined organic extracts were washedwith brine, dried and concentrated. Column chromatography using EtOAc:PE (5:1) aseluant yielded 76 as a white solid (0.61 g, 60%).mp: 43-45 °C;IR (CHC13)v: 3604, 3406, 2991, 2927, 2853, 1460, 1374, 1285, 1083, 1009,842 cm-1;1H NMR (CDC13,200 MHz) ö: 3.5 (d, J = 11 Hz, 6 H), 3.5 (m, 1 H), 2.7 (s, 1 H),1.7-1.1 (m, 22 H), 1.05 (d, 3 = 6 Hz, 3 H);3lp NMR (CDC13,81 MHz) 8: 34.9;LRMS (DCI) m/z: 309 (M+1, 100);LR1’IS (El) m/z: 307 (M-1, 3), 264 (20), 151 (20), 138 (22), 137 (40), 124 (100),110 (21);HRMS (DCI) Calcd forC15H3404P(M+1): 309.2195; found: 309.2192;Anal. Calcd forC15H3304P:C, 58.42; H, 10.79; found: C, 58.18; H, 10.79.EXPERIMENTAL 1385.2.27 1-Methoxy-1-oxo-1-phospho-13-tetradecanolide (77)ECH3Dimethyl 12-hydroxytridecanyiphosphonate (76) (0.270 g, 0.875 mmol) wasdissolved in toluene (500 mL), NaH (0.0440 g, 50% dispersion in oil, 0.916 mmol) wasadded and the mixture was heated to reflux. After 24 hours, the toluene was removed invacuo and the residue was chromatographed using EtOAc:PE (8:1) as eluant to yield 77as a clear, colourless oil (0.0109 g, 5%).IR (CHC13)v: 2991, 2931, 2857, 1718, 1602, 1459, 1380, 1271, 1059, 966 cm;1H NMR (CDC13,400 MHz) 6: 4.5 (m, 1 H), 3.75 (d, J = 12 Hz, 6 H), 1.85-1.75 (dt,J 16, 8 Hz, 2 H), 1.65-1.20 (m, 20 H);31p NMR (CDC13,81 MHz) 6: 32.5;LRMS (El) m/z: 276 (M, 24), 165 (25), 151 (25), 137 (20), 124 (23), 123 (43), 110(100), 55 (36), 41(47);HRMS (El) Calcd forC14H2903P:276.1854; found: 276.1859.The following compound was also isolated from this procedure ( 5%) and purified bycolumn chromatography using EtOAc:PE (8:1) as eluant.EXPERIMENTAL 1395.2.28 Methyl benzyl-(12-hydroxytridecanyl)phosphinate (78)CH( 2)11 \OCH3CH2Ph781H NMR (CDC13,400 MHz) & 7.4 (m, 5 H), 5.1 (d, J = 10 Hz, 2 H), 3.8 (m, 1 H),3.65 (d, J = 12 Hz, 3 H), 1.8-1.20 (m, 23 H), 1.15 (d, J = 6.5 Hz, 3 H);31P NMR (CDCI3,81 MHz) ö: 34.4;LR1’IS (El) nilz: 367 (M, 5), 275 (20), 249 (24), 110 (43), 94(25), 91(100), 79 (46),69 (36), 65 (34), 55 (68), 45 (75), 43 (42), 41(48);HRMS (El) Calcd forC21H370P:368.2480; found: 368.2449.EXPERIMENTAL 1405.2.29 Dimethyl 4-pentenyiphosphonate (112a)0P(OMe)2112a5-Bromo-1-pentene (111) (6.00 g, 40.3 mmol) and trimethyl phosphite (14.98 g,14.24 mL, 121 mmol) were combined and heated to 180 °C for 2 hours. Columnchromatography of the crude reaction mixture using EtOAc:PE (7:1) as eluant yielded112a as a yellow oil (0.98 g, 14%) which could not be completely purified.1H NMR (CDC13,200 MHz) 6: 5.75 (m, 1 H), 5.0 (m, 2 H), 3.7 (d, J = 11 Hz, 6 H),2.1 (m, 2 H), 1.8-1.6 (m, 4 H);31p NMR (CDC13,81 MHz) 6: 34.9;LRMS (El) m/z: 178 (M, 5), 124 (42), 111(100), 94 (29), 93 (60), 79 (24), 68 (44),67 (41), 41(20), 32 (34);HRMS (El) Calcd forC7H1503P: 178.0759; found: 178.0758.EXPERIMENTAL 1415.2.30 Diethyl 4-pentenyiphosphonate (112b)112b5-Bromo-1-pentene (111) (5.00 g, 33.5 mmol) and triethyl phosphite (29.0 g,29.9 mL, 174 mmol) were combined and heated to 180 °C for 2 hours. Columnchromatography of the crude reaction mixture using EtOAc:PE (1:3) as eluant yielded112b as a clear, colourless oil (4.39 g, 63%).1H NMR (CDC13,200 MHz) ö: 5.7 (m, 1 H), 4.9 (m, 2 H), 4.0 (m, 4 H), 2.05 (m,2 H), 1.70 (m, 4 H), 1.2 (td, J = 7, 2 Hz, 6 H);31P NMR (CDC13,81 MHz) & 33.2;LRMS (El) m/z: 206 (M, 18), 152 (73), 138 (23), 133 (21), 125 (100), 124 (21), 111(29), 109 (47), 108 (31), 97 (67), 96 (21), 81(26), 67 (26), 41(35);HRMS (El) Calcd forC9H1903P:206.1072; found: 206.1065.EXPERIMENTAL 1425.2.31 5-Bromo-1,2-epoxypentane (116)Br1165-Bromo-1-pentene (111) (2.00 g, 13.4 mmol) was dissolved in CH21 (50 mL)and the solution cooled to 0 °C. m-CPBA (2.54 g, 80 mol%, 14.7 mmol) was added tothis solution in one portion and the mixture was stirred at 0 °C for 1 hour, and thenallowed to warm to room temperature. After 15 hours at room temperature, the solutiongave a negative test to Kllstarch paper so further m-CPBA (0.50 g, 2.89 mmol) wasadded. After 6 more hours at room temperature, the solution gave a negative test toKI/starch paper so further m-CPBA (0.50 g, 2.89 mrnol) was added. After 2 more hoursat room temperature, the solution gave a negative test to Kllstarch paper so furtherm-CPBA (1.00 g, 5.78 mmol) was added and the mixture stirred at room temperature fora further 16 hours (40 hours total). At this time, a white precipitate was filtered and thefiltrate was washed with NaHCO3, brine, dried and concentrated. Columnchromatography using EtOAc:PE (1:10) as eluant yielded 116 as a clear, colourless liquid(1.83 g, 83%).IR (neat) v: 2980, 2920, 1481, 1438, 1410, 1254, 1208, 1133, 915, 852, 787 cm1;1H NMR (CDC13,200 MHz) ö: 3.3 (t, J = 7 Hz, 2 H), 2.8 (m, 1 H), 2.6 (t, J = 4.5 Hz,1 H), 2.35 (dd, J = 5, 3 Hz, 1 H), 2.0-1.8 (m, 2 H), 1.75-1.60 (m, 1 H), 1.55-1.30(m, 1 H);LRIvlS (DCI) m/z: 184(81Br, M+18, 15), 182(79Br, M+18, 13), 167(81Br, M+1,90), 165(79Br, M+1, 100);LRMS (El) m/z: 166(81Br, M, 0.1), 164(79Br, M, 0.3), 108 (18), 106 (18), 86(44), 85 (100), 84 (57), 71(37), 58 (19), 57 (25), 55 (30);EXPERIMENTAL 143HRMS (El) Calcd for C5H9O81Br: 165.98 17; found: 165.9809;Calcd forC5H9O79Br: 163.9837; found: 163.9836;Anal. Calcd forC5H9OBr: C, 36.39; H, 5.50; found: C, 37.01; H, 5.44.EXPERiMENTAL 1445.2.32 Dimethyl 4,5-epoxypentanyiphosphonate (113a)113a5-Bromo-1,2-epoxypentane (116) (1.70 g, 10.3 mmol) was combined withtrimethyl phosphite (7.67 g, 7.30 mL, 61.8 mmol) and the mixture was heated to 170 °Cfor 6 hours, after which time the reaction mixture was cooled and chromatographed usingEtOAc (100%) as eluant to yield 113a as a clear, colourless oil (1.00 g, 50%).1H NMR (CDC13,200 MHz) ö: 3.65 (d, J 11 Hz, 6 H), 2.85 (m, 1 H), 2.70 (t, J =4.5 Hz, 1 H), 2.40 (dd, J = 5,3 Hz, 1 H), 1.90-1.30 (m, 6 H);31p NMR (CDC13,81 MHz) 3: 34.0;LR1’1S (DCI) m/z: 212 (M+18), 195 (M-i-1);LRMS (El) m/z: 193 (M-1, 4.3), 165 (35), 151 (41), 137 (52), 124 (85), 111 (23),110 (100), 109 (57), 94 (42), 93 (24), 82 (20), 79 (46);HR1’IS (El) Calcd forC7H1404P(M-1): 193.0630; found: 193.0626;Anal. Calcd forC7H1504P:C, 43.30; H, 7.79; found: C, 43.61; H, 7.96.EXPERIMENTAL 1455.2.33 Diethyl 4,5-epoxypentanyiphosphonate (113b)113b5-Bromo-1,2-epoxypentane (116) (0.20 g, 1.2 mmol) was combined with tnethylphosphite (1.21 g, 1.25 mL, 7.3 mmol) and the mixture was heated to 170 °C for 6 hours,after which time the reaction mixture was cooled and chromatographed using EtOAc(100%) as eluant to yield 113b as a clear, colourless oil (0.20 g, 74%).1H NMR (CDC13, 200 MHz) ö: 3.80 (m, 4 H), 2.65 (m, 1 H), 2.45 (t, J = 4.5 Hz,1 H), 2.20 (dd, J = 5, 2.6 Hz, 1 H), 1.60-1.20 (m, 6 H), 1.0 (t, J = 7.5 Hz, 6 H);3lp NMR (CDC13,81 MHz) & 32.2;LRMS (El) m/z: 223 (M+1, 1.8), 222 (M, 0.9), 193 (43), 179 (67), 166 (29), 165(85), 152 (89), 151 (22), 149 (94), 137 (100), 125 (97), 123 (49), 111 (76), 110(32), 109 (83);HRMS (El) Calcd forC9H1904P:222.1021; found: 222.1025;Anal. Calcd forC9H1904P:C, 48.64; H, 8.62; found: C, 48.36; H, 8.80.EXPERIMENTAL 1465.2.34 4-Bromo-1,2-epoxybutane (123)Br1234-Bromo-1-butene (122) (5.00 g, 33.1 mmol) was dissolved in CH21 (150 mL)and the mixture was cooled to 0 °C. m-CPBA (16.6 g, 50 mol%, 48.1 mmol) was addedand the mixture was allowed to warm to room temperature and stirred for 60 hours. Atthis time, the mixture was washed with 4 M NaOH and the aqueous phase was extractedwith CHCJ3. The combined organic extracts were washed with brine, dried andconcentrated. Column chromatography using EtOAc:PE (1:15) as eluant yielded 123 as aclear, colourless oil (4.98 g, 89%).IR (neat) v: 2952, 1481, 1438, 1410, 1253, 1207, 1133, 915, 851, 787 cm1;1H NMR (CDC13,200 MHz) 6: 3.45 (dd, 3 = 7, 7 Hz, 2 H), 3.10 (m, 1 H), 2.80 (t, I =4 Hz, 1 H), 2.55 (dd, J = 5, 4Hz, 1 H), 2.20-1.80 (m, 2 H);LRMS (DCI) m/z: 170(81Br, M+18, 55), 168(79Br, M+18, 56), 153 (81Br, M,52), 151 (79Br, M, 54), 134 (53), 132 (50), 125 (47), 123 (80), 121 (52), 111(22), 109 (41), 107 (31), 97 (69), 95 (69), 85 (62), 83 (80), 73 (41), 72 (52), 71(100).EXPERIMENTAL 1475.2.35 Dimethyl 5-halo-4-hydroxypentanylphosphonate (124, 125)124; X = Cl125; X=IDimethyl 4,5-epoxypentanyiphosphonate (113a) (0.20 g, 1.0 mmol) wasdissolved in CH3N (1 mL). Chlorotrimethylsilane (0.14 mL, 1.1 mmol) and NaT(0.16 g, 1.1 mmol) were added and the solution immediately turned yellow with theformation of a white precipitate. The mixture was stirred at room temperature for 1 hour,then H20 was added and the aqueous phase was separated and extracted with Et20. Theorganic extracts were dried and concentrated. Column chromatography usingCHC13:MeOH (20:1) as eluant yielded a yellow oil (0.096 g) which was a mixture of 124and 125.IR (CHC13)v: 3683, 3378, 3078, 2942, 1448, 1071, 842 cm1;1H NMR (CDC13,400 MHz) & 3.85-3.75 (m, 1 H), 3.75 (d, J = 10 Hz, 12 H),3.65-3.55 (m, 1 H), 3.60 (dd, J = 10, 3.5 Hz, 1 H), 3.50 (dd, J = 10, 7 Hz, 1 H),3.35 (dd, J = 10, 4 Hz, 1 H), 3.25 (dd, J = 10, 7 Hz, 1 H), 2.50 (s, 2 H), 1.90- 1.60(m, 12 H);3lp NMR (CDC13,81 MHz) : 34.5;LRMS (El) m/z: 323 (1271, M+1, 0.6), 233 (37Cl, M+1, 2.8), 231 (35Cl, M+1,7.1), 230(35Cl, M, 0.1), 181 (82), 177 (81), 163 (20), 152 (29), 151 (52), 149(54), 137 (24), 124 (100), 111 (32), 110 (61), 109 (77), 94 (82), 93 (28), 85 (25),83 (42), 80 (26), 79 (97), 71(33), 67 (58), 55 (38), 47 (25), 43 (34), 41(34);EXPERIMENTAL 148HRMS (El) Calcd forC7H1604P1(M+1): 322.9909; found: 322.9914;Calcd for1635C1: 230.0475; found: 230.0440.The following compound was also isolated as a yellow oil (in trace amounts) from thisprocedure. It was purified by column chromatography using CHC13:MeOH (20:1) aseluant.5.2.36 6-(Hydroxymethyl)-2-methoxy-2-oxo-1,2-oxaphosphorinane (115a)o OMeOH115aIR (CHC13)v: 3678, 3589, 3366, 2944, 2853, 1718, 1602, 1433, 1408, 1238, 1200,1046, 827 cm1;111 NMR (CDC13,400 MHz) ö: 3.80 (m, 1 H), 3.75 (d, J = 11 Hz, 3 H), 3.55 (dd, J =12, 4 Hz, 1 H), 3.45 (dd, J = 12, 6 Hz, 1 H), 1.90-1.55 (m, 7 H);31p NMR (CDC13,81 MHz) 6: 34.6;LRMS (DCI) m/z: 198 (M-i-18, 60), 181 (M+l, 60), 177 (70), 151 (40), 128 (45);HRMS (DCI) Calcd forC6H14O4P(M-i-1): 181.0630; found: 181.0629.EXPERIMENTAL 1495.2.37 Dimethyl 5-halo-4-acetoxypentanylphosphonate (126, 127)126; X = Cl127; X = IThe mixture (0.096 g) of alcohols 124 and 125 was dissolved in CH21 (5 mL),and then Et3N (1 mL), Ac20 (1 mL), and DMAP (catalytic) were added and the mixturewas stirred at room temperature overnight. The solution was diluted with Et20, thenwashed with 5% HC1, brine, dried and concentrated. Column chromatography usingCHC13:MeOH (20:1) as eluant yielded a mixture of acetates 126 and 127 (0.04 g).IR (CHC13)v: 3408, 2993, 2953, 2853, 1736, 1457, 1373, 1269, 1066, 1020, 901,838 cm1;1H NMR (CDC13,200 MHz) ö: 5.0 (m, 1 H), 4.7 (m, 1 H), 3.7 (d, J = 11 Hz, 12 H),3.5 (m, 2 H), 3.2 (m, 2 H), 2.05 (s, 6 H), 1.9-1.5 (m, 12 H);31p NMR (CDC13,81 MHz) ö: 33.8;LRMS (DCI) m/z: 382 (1271, M+18, 15), 365 (1271, M+1, 100), 292(37C1, M+18,2.5), 290(35C1, M+18, 6.2), 275(37C1, M+1, 25), 273(35Cl, M+1, 75);HRMS (El) Calcd forC9H1805P1: 363.9937; found: 363.9933;Calcd for1835C1: 272.0581; found: 272.0576.EXPERIMENTAL 1505.2.38 Phenyl phosphorodichloridite (88)PhOPC1288A flask, fitted with an outlet for HC1, was charged with PCi3 (263 g, 167 mL,1.91 mmol) and PhOH (30.0 g, 319 mmol) was added portion-wise over 3 hours. Thesolution was stirred at room temperature for a further 5 hours, then the gas outlet wasremoved and the mixture was heated to reflux overnight. Excess PCi3 was removed bydistillation at atmospheric pressure. The product was distilled (bp 88-94 °CI 10 mm Hg;lit. 179 bp 90 °CI 10 mm Hg) to yield 88 as a clear, colourless oil (50.2 g, 8 1%).IR (CHC13)v: 3070, 3042, 1942, 1872, 1725, 1590, 1489, 1454, 1286, 1236, 1188,1155, 1070, 1024, 1005, 956, 867, 825 cnr1;1H NMR (CDC13,200 MHz) 6: 7.4 (m, 2 H), 7.3 (m, 1 H), 7.2 (m, 2 H);31p NMR (CDC13,81 MHz) 8: 7.3.EXPERIMENTAL 1515.2.39 Dilsopropyl phenyl phosphite (89)(i-PrO)2POPh89(i) Et3N (60.7 g, 83.3 mL, 600 mL) and PCi3 (27.4 g, 17.4 mL, 200 mmol) wereadded to Et20 (500 mL) that had been previously cooled to 0 °C. A solution of i-PrOH(24.0 g, 30.5 mL, 400 mmol) in Et20 (60 mL) was added dropwise over 2 hours, andafter 5 h, a solution of PhOH (18.8 g, 200 mmol) in Et20 (50 mL) was added dropwise.The mixture was allowed to warm to room temperature and stirred for a further 62 hours.The resulting precipitate was filtered and washed with Et20 and the filtrate wasconcentrated to yield a yellow oil. Distillation of this crude material (bp 91-95 °CI0.5 mm Hg; lit.178 bp 65-68 °CI 0.2 mm Hg) yielded 89 as a clear, colourless liquid(15.2 g, 31%).(ii) Triphenyl phosphite (31.0 g, 26.2 mL, 100 mmol) and sodium (4.60 g,200 mmol) were added to THF (150 mL), the mixture was cooled to 0 °C, and a solutionof i PrOH (12.0 g, 15.3 mL, 200 mmol) in THF (30 mL) was added dropwise. Theresulting mixture was heated to reflux for 5 hours, cooled, and the THF was removed byrotary evaporation. The resulting white precipitate was filtered and washed with Et20.The filtrate was washed with 1 M NaOH, brine, dried and concentrated. Distillation(bp 115-118 °C/ 8 mm Hg; lit.178 bp 65-68 °CI 0.2 mm Hg) yielded 89 as a clear,colourless liquid (3.79 g, 16%).(iii) A flask, fitted with an outlet for HC1, was charged with Et20 (700 mL),phenyl phosphorodichloridite (88) (47.1 g, 242 mmol) and Et3N (48.9 g, 67.2 mL,483 mmol). This mixture was cooled to 15 °C and i-PrOH (29.0 g, 37.0 mL, 483 mmol)was added to it dropwise over 3 hours. The mixture was allowed to warm to roomtemperature and was stirred overnight. The resulting precipitate was filtered and washedEXPERIMENTAL 152with Et20. The filtrate was concentrated to give a yellow oil. Distillation (bp 93-98 °C/0.8 mm Hg; lit.’78 bp 65-68 °CI 0.2 mm Hg) of this material yielded 89 as a clear,colorless liquid (49.7 g, 85%).IR (CHC13)v: 3067, 3099, 2972, 2933, 2878, 2240, 1936, 1936, 1706, 1590, 1496,1450, 1348, 1224, 1178, 1138, 1071, 1013, 837 cm1;1H NMR (CDC13,200 MHz) & 7.23 (m, 2 H), 7.05 (m, 3 H), 4.6 (d, sept, J = 9, 6 Hz,2H), 1.3 (d,J=6Hz, 12H);3lp NMR (CDC13,81 MHz) & 135.3;LRMS (El) m/z: 242 (M, 2.6), 141 (20), 107 (24), 94 (100), 43 (21);HRMS (El) Calcd forC12H903P:242.1072; found: 242.1072.EXPERIMENTAL 1535.2.40 Phenyl isopropyl 4-pentenyiphosphonate (90)0PhOi-PrO905-Bromo-1-pentene (111) (10.2 g, 68.4 mmol), diisopropyl phenyl phosphite(49.7 g, 205 mmol), and NaT (1.02 g, 6.81 mmol) were combined and heated to 185 °Cfor 4 hours. The mixture was filtered, the precipitate washed with Et20, and the filtratewas concentrated. Column chromatography using EtOAc:PE (1:3) as eluant yielded 90as a clear, colourless liquid (17.1 g, 93%).IR (neat) v: 3071, 2979, 2935, 1641, 1593, 1490, 1454, 1381, 1251, 1210, 1164,1106, 1071, 999, 919, 765 cm1;1H NMR (CDC13,400 MHz) & 7.35 (t, J = 7.5 Hz, 2 H), 7.23 (dd, J = 8 Hz, 2 H),7.15 (t, J= 8Hz, 1 H), 5.65 (m, 1= 17, 11Hz, 1 H), 5.01 (m, J= 17, 2Hz, 1 H),5.0 (m, J = 11, 2Hz, 1 H), 4.90 (m, J = 6Hz, 1 H), 2.1 (m, 2 H), 1.95-1.65 (m,4H),1.30(d,J=6Hz,3H),1.15(d,J=6Hz,3H);3lp NMR (CDC13,81 MHz) 8: 28.2;LRMS (El) m/z: 268 (M, 7.3), 226 (58), 172 (64), 94 (100), 91(52), 41(26);HRMS (El) Calcd forC14H2103P:268.1228; found: 268.1236;Anal. Calcd forC14H2103P:C, 62.68; H, 7.89; found: C, 62.40; H, 7.92.EXPERIMENTAL 1545.2.41 6-(Iodomethyl)-2-oxo-2-phenoxy-1,2-oxaphosphorinane (91a,b)0 OPh\\/Cu91a,bPhenyl isopropyl 4-pentenyiphosphonate (90) (2.00 g, 7.45 mmol) and iodine(4.16 g, 18.1 mmol) were added to CHC13 (40 mL) and the mixture was stirred at roomtemperature for 113 hours. The mixture was washed with saturated NaHSO3,brine, driedand concentrated to yield a dark brown solid. Column chromatography using CHC13(100%) as eluant yielded a mixture of 91a,b as a brown solid (2.50 g, 95%). The ratio of91a:91b was 3:1 as judged by 1H 103-120 °C (dec.);IR (CHC13)v: 2956, 2876, 1593, 1490, 1456, 1375, 1304, 1157, 1105, 1070, 1043,997, 967, 900, 879 cm1;1H NMR (CDC13,400 MHz) & 7.50-7.10 (m, 5 H), 4.50-4.40 (m, 1 H), 3.45-3.20 (m,2 H), 2.30-1.50 (m, 6 H);31P NMR (CDC13,81 MHz) & 23.2, 20.2;LRMS (El) m/z: 352 (M, 8), 225 (78), 183 (22), 94 (61), 91(28), 77 (24), 67 (100),65 (26), 41(49), 39 (32);HR1’1S (El) Calcd forC11H403P : 351.9725; found: 351.9720.EXPERIMENTAL 1555.2.42 6-(Azidomethyl)-2-oxo-2-phenoxy-1,2-oxaphosphorinane (92a,b)PhO92a 92b6-(Iodomethyl)-2-oxo-2-phenoxy- 1 ,2-oxaphosphorinane (91a,b) (12.6 g,35.8 mmol), sodium azide (4.65 g, 71.5 mmol) and tetrabutylammonium bromide (1.15 g,3.56 mmol) were combined in benzene-DMF (1:1) (180 mL) and the mixture heated to80 °C for 24 h. The mixture was partitioned between Et20 and H20, solid NaHSO3wasadded and the layers were separated. The aqueous phase was extracted with Et20 and thecombined organic extracts were washed with H20, saturated NaHSO3,brine, then driedand concentrated to give a mixture of 92a and 92b (5:1 by 1H NMR) as a yellow oilcontaining white crystals (5.12 g, 53%). Repeated column chromatography usingCHC13:MeOH (35:1) as eluant yielded a sample of 92a as a white solid and 92b as ayellow oil.Diastereomer 92amp: 74-77 °C;IR (CHC13)v : 2943, 2877, 2107, 1594, 1490, 1456, 1304, 1164, 1144, 1071, 979,944, 899 cm1;1H NMR (CDC13,400 MHz) ö: 7.40-7. 10 (m, 5 H), 4.50 (m, J = 11.5, 1.6 Hz, 1 H),3.50 (ddd, J = 13, 7, 1 Hz, 1 H), 3.35 (ddd, J = 13, 4, 2.5 Hz, 1 H), 2.30-2.12 (m,2 H), 2.10-1.92 (m, 1 H), 1.90-1.75 (m, 2 H), 1.70-1.55 (m, J = 14, 3 Hz, 1 H);31P NMR (CDC13,81 MHz) ö: 20.2.EXPERIMENTAL 156Diastereomer 92bIR (CHC13)v: 2993, 2939, 2876, 2109, 1594, 1490, 1456, 1294, 1157, 1071, 1007,965, 904, 839 cm-1;1H NMR (CDC13,400 MHz) ö: 7.40-7.10 (m, 5 H), 4.70 (m, J = 11, 2.9 Hz, 1 H),3.40 (dd, J = 4.8, 1.9 Hz, 2 H), 2.30-2.05 (m, 3 H), 1.90-1.72 (m, 2 H), 1.70-1.60(m, 1 H);31p NMR (CDCJ3,81 MHz) ö: 23.6.MixtureLRMS (El) m/z: 267 (M, 19), 212 (19), 211 (100), 174 (33), 130 (20), 94 (59), 91(11), 82(22), 77 (75), 65 (32), 55 (47), 51(24), 41(21);HRMS (El) Calcd forC11H4N30P:267.0773; found: 267.0774.EXPERIMENTAL 1575.2.43 6-(Azidomethyl)-2-methoxy-2-oxo-1,2-oxaphosphorinane (128a,b)MeOJ0 OMe128a 128bMethanol (0.26 g, 0.33 mL, 8.1 mmol) was dissolved in THF (45 mL), thesolution cooled to -78 °C, n-BuLi (5.6 mL, 1.3 M, 7.4 mmol) was added dropwise, andthe mixture was stirred at -78 °C for 40 minutes. 6-(Azidomethyl)-2-oxo-2-phenoxy-1,2-oxaphosphorinane (92a) (2.0 g, 7.5 mmol) was added as a THF solution (10 mL). Thesolution was stirred at -78 °C for 3 hours, and allowed to warm to room temperatureovernight. The mixture was acidified with 1 M HC1, and extracted with EtOAc. Theorganic layer was dried and concentrated to give a yellow oil. Column chromatographyusing CHC13:MeOH (35:1) as eluant yielded 128a,b (3:1 by 1H NMR) as a yellow oil(1.1 g, 73%). Repeated column chromatography using this solvent system as eluantyielded a partially separated mixture of diastereomers 128a and 128b.Diastereomer 128a1H NMR (CDC13,400 MHz) & 4.60 (m, J = 11 Hz, 1 H), 3.82 (d, J = 11 Hz, 3 H),3.40 (d, J = 4.4 Hz, 2 H), 2.30-1.90 (m, 2 H), 1.80-1.60 (m, 4 H);31p NMR (CDC13,81 MHz) 6: 28.1.Conformation 128b (As determined from mixture)H NMR (CDC13,400 MHz) 6: 4.30 (m, 1 H), 3.79 (d, J = 11 Hz, 3 H), 3.49 (ddd, J =14, 7, 1 Hz, 1 H), 3.40-3.30 (ddd, J = 14, 4, 2 Hz, 1 H), 2.30-1.90 (m, 3 H),1.80-1.50 (m, 3 H);31p NMR (CDC13,81 MHz) 6: 24.9.EXPERIMENTAL 158MixtureIR (CHC13)v: 2932, 2855, 2106, 1724, 1457, 1377, 1290, 1061, 1046, 969 cm1;LRMS (DCI) m/z: 223 (M+18, 2.4), 206 (M+1, 100), 149 (10);HRMS (DCI) Calcd forC6H13N30P(M+1): 206.0694; found: 206.0697.EXPERIMENTAL 1595.2.44 6-(Aminomethyl)-2-methoxy-2-oxo-1,2-oxaphosphorinane (129)0 OMer—PYNH2129A mixture of 6-(azidomethyl)-2-methoxy-2-oxo- 1 ,2-oxaphosphorinane (128a,b)(0.54 g, 2.6 mmol) and Pd/C (10%) (0.21 g, 40% w/w) in EtOH (30 mL) was stirredunder a H2 atmosphere (40 psi) at room temperature overnight. Celite was added, themixture was filtered and the filtrate concentrated to give 129 as a yellow, foam-likematerial (0.47 g, 100%) which was used without further purification.1H NMR (CDC13,200 MHz) ö: 5.60 (hr s), 4.80 and 4.60 (m, 2 H), 3.90 (d, J =11Hz) and 3.85 (d, J = 11Hz) (6 H), 3.15 (m, 4 H), 2.20-1.40 (m, 12 H);31p NMR (CDC13,81 MHz) 6: 28.6, 26.4;LRMS (DCI) m/z: 194 (M-i-18, 80), 180 (M+1, 70), 166 (100), 74 (21);fIRMS (DCI) Calcd forC6H15N03P(M+1): 180.0789; found: 180.0783.EXPERIMENTAL 1605.2.45 Mono N-hydroxysuccinimide glutarateHO)AONJGlutaric anhydride (2.00 g, 17.5 mmol) and N-hydroxysuccinimide (2.00 g,17.4 mmol) were added to CH21 (85 mL). Et3N (2.13 g, 2.92 mL, 21.0 mmol) wasadded and the heterogeneous mixture became homogeneous. The solution was stirred for90 minutes, acidified with 1 M HC1, and extracted exhaustively with EtOAc. Thecombined organic extracts were dried and concentrated to give a white precipitate.Column chromatography using EtOAc:EtOH:H2(10:1:1) as eluant yielded monoN-hydroxysuccinimide glutarate as a white solid (2.97 g, 74%).mp: 43-52 °C;IR (CHC13)v: 3400-2600 (br), 1816, 1787, 1742, 1714, 1367, 1291, 1077, 991,886 cm1;111 NMR (CDC13,200 MHz) 3: 9.0 (br s, 1 H), 2.85 (s, 4 H), 2.70 (t, J = 7.5 Hz, 2 H),2.5 (t, J = 7.5 Hz, 2 H), 2.05 (m, 2 H);LRMS (DCI) m/z: 247 (M+18, 100), 230 (M+1, 56), 202 (12), 188 (18), 150 (18),133 (18), 132 (26), 102 (24), 83 (30);HRMS (DCI) Calcd forC9H12N06(M+1): 230.0665; found: 230.0659.EXPERIMENTAL 1615.2.46 Mono N-hydroxysuccinimide glutaryl chloride (94)ClO-NJMono N-hydroxysuccinimide glutarate (0.75 g, 3.27 mmol) was combined withoxalyl chloride (2.07 g, 1.43 mL, 16.3 mmol) and the mixture was stirred at roomtemperature for 3 hours. Excess oxalyl chloride and other volatiles were removed bypumping for 1.5 hours to yield 94 as a yellow-green solid which was used immediately inthe next reaction.IR (CHC13)v: 3524, 2946, 1809, 1780, 1748, 1430, 1410, 1361, 1207, 1062, 993,826 cm1;1H NMR (CDC13,200 MHz) & 3.1 (t, J = 7 Hz, 2 H), 2.8 (s, 4 H), 2.7 (t, J = 7 Hz,2 H), 2.1 (m, 2 H);LRMS (DCI) ,m/z: 267(37C1, M+18, 6), 265(35C1, M+18, 18);LRMS (El) m/z: 212 (M-35, 19), 135 (31), 133 (100), 107 (8), 105 (23), 97 (57), 77(20), 55 (83).EXPERIMENTAL 1625.2.47 Mono N-hydroxysuccinimide glutaryl 6-(amidomethyl)-2-methoxy-2-oxo-1,2-oxaphosphorinane (130)0 OMe130A solution of 6-(aminomethyl)-2-methoxy-2-oxo- 1 ,2-oxaphosphorinane (129)(0.55 g, 3.1 mmol) in CH21 (12 mL) was prepared and cooled to 0 °C. MonoN-hydroxysuccinimide glutaryl chloride (94) (0.50 g, 2.0 mmol) and Et3N (0.31 g,0.43 mL, 3.1 mmol) were added to this solution and the resulting mixture was stirred at0 °C. After 40 minutes, further mono N-hydroxysuccinimide glutaryl chloride (0.25 g,1.0 mmol) was added and after another 1 hour, further Et3N (0.04 g, 50 j.iL, 0.36 mmol)was added and the solution was allowed to warm to room temperature over 1 hour. Atthis time, the solvent was removed by rotary evaporation and column chromatography ofthe residue using acetone (100%) as eluant yielded 130 as a yellow foamy material(0.46 g, 58%). Repeated column chromatography using this solvent system as eluantyielded a sample of each of the two diastereomers of 130.Diastereomer 130aIR (CHC13)v: 2937, 1810, 1784, 1738, 1712, 1669, 1528, 1430, 1361, 1230, 1220,1066, 972, 830 cnr1;1H NMR (Acetone-d6, 400 MHz) & 6.75 (t, J = 4 Hz, 1 H), 4.45 (m, 1 H), 3.79 (d,J = 11 Hz, 3 H), 3.57 (dq, J = 14, 4 Hz, 1 H), 3.35 (ddd, J = 14, 8, 4 Hz, 1 H), 2.85(br s, 4 H), 2.68 (t, J = 7 Hz, 2 H), 2.65 (t, J = 7 Hz, 2 H), 2.20-1.40 (m, 8 H);3lp NMR (Acetone-d6, 81 MHz) ö: 28.4;EXPERIMENTAL 163LRMS (DCI) m/z: 408 (M+18, 4), 391 (M+1, 100), 276 (34), 161 (27), 150 (32);fiRMS (DCI) Calcd forC15H24N08P(M+1): 391.1270; found: 391.1264.Diastereomer 130bIR (CHC13)v: 2944, 1815, 1786, 1738, 1669, 1287, 1074, 982 cm1;1H NMR (Acetone-d6, 400 MHz) 6: 7.10 (t, J = 4 Hz, 1 H), 4.30 (m, 1 H), 3.70 (d,J = 10 Hz, 3 H), 3.55 (dq, J = 15, 3.4 Hz, 1 H), 3.40 (m, J = 2 Hz, 1 H), 2.85 (m,4 H), 2.70 (m, 4 H), 2.20-1.40 (m, 8 H);3lp NMR (Acetone-d6, 81 MHz) 6: 25.6;LRMS (DCI) m/z: 408 (M+18, 2), 391 (M+1, 100), 276 (70), 163 (42), 162 (29),150 (43), 149 (70), 55 (24);HRMS (DCI) Calcd forC15H24N08P(M+1): 391.1270; found: 391.1267.EXPERIMENTAL 1645.2.48 5-Hexenoic acid (146)CO2H146Magnesium (1.44 g, 59.2 mmol) was flame dried and then suspended in Et20(250 mL). A few crystals of iodine were added and the mixture turned deep red.5-Bromopentene (111) (8.40 g, 56.4 mmol) was added as a solution in Et20 (25 mL) andthe red colour of the iodine gradually faded to yield a clear mixture. This was heated toreflux for 30 minutes, then cooled to room temperature, and CO2 was bubbled into thesolution for 1 hour. The solution was acidified with 1 M HC1, extracted with Et20, andthen the combined extracts were dried. Concentration yielded 146 as a yellow oil (5.33 g,83%) which was used without further purification in the next step.IR (neat) v: 3480-2400 (br), 1712, 1641, 1424, 1269, 993, 914 cmt;1H NMR (CDC13,200 MHz) ö: 11.4 (br s, 1 H), 5.8 (m, 1 H), 5.0 (m, 2 H), 2.35 (t,J = 7.6 Hz, 2 H), 2.10 (m, 2 H), 1.70 (m, 2 H).EXPERIMENTAL 1655.2.49 6-Hydroxymethyl ö-valerolactone (149)OH1495-Hexenoic acid (146) (0.50 g, 4.4 mmol) was dissolved in CH21 (25 mL),m-CPBA (1.2 g, 80 mol%, 5.7 mmol) was added and the mixture was stirred at roomtemperature overnight. The resulting precipitate was removed by filtration and the filtratewas concentrated to give a white solid and yellow oil. Column chromatography usingCHC13:MeOH (9:2) as eluant yielded 149 as a clear, colourless oil (0.34 g, 60%).IR (CHC13)v: 3675, 3600, 3444, 2944, 2881, 1728, 1604, 1443, 1342, 1245, 1186,1182, 1053, 982, 909 cm1;1H NMR (CDC13,200 MHz) ö: 4.45 (m, 1 H), 3.80 (dd, J = 12, 3 Hz, 1 H), 3.65 (dd,J = 12, 5 Hz, 1 H), 2.65 (m, 1 H), 2.48 (m, 1 H), 2.10 (br s, 1 H), 1.95-1.60 (m,4H);LRMS (El) m/z: 131 (M+1, 4), 113 (4), 100 (22), 99(100), 71(96), 55 (58), 43 (31).EXPERIMENTAL 1665.2.50 4-Nitrophenyl 5-hexenoate (150)1505-Hexenoic acid (146) (0.50 g, 4.4 mmol) and thionyl chloride (1.6 g, 0.95 mL,13 nmol) were combined and heated to 80 °C for 30 minutes. The solution was cooledand the excess thionyl chloride was removed by distillation to yield the correspondingacid chloride which was used immediately in the next step.The acid chloride was dissolved in CH21 (20 mL) and 4-nitrophenol (1.8 g,13 mmol) and pyridine (0.38 g, 0.39 mL, 4.8 mmol) were added. The mixture was heatedto reflux overnight and upon cooling, a precipitate resulted. The mixture was washedwith 1 M HC1, saturated NaHCO3,brine and then dried. Concentration and columnchromatography using Et20:PE (1:10) as eluant yielded 150 as a yellow oil (0.47 g,46%).IR (neat) v: 3116, 3080, 2937, 1765, 1641, 1615, 1593, 1525, 1490, 1415, 1348,1208, 1117, 1012, 915, 862 cm-1;1H NMR (CDC13,400 MHz) ö: 8.29 (d, J = 9 Hz, 2 H), 7.30 (d, J = 9 Hz, 2 H), 5.85(m, 1 H), 5.05 (m, 2 H), 2.60 (t, J = 7 Hz, 2 H), 2.20 (m, 2 H), 1.85 (m, 2 H);LRMS (El) m/z: 235 (M, 5), 109 (28), 97 (100), 69 (75), 55 (41), 41(55);HRMS (El) Calcd forC12H3N04:235.0845; found: 235.0850.EXPERIMENTAL 1675.2.51 4-Nitrophenyl 5,6-epoxyhexanoate (151)1514-Nitrophenyl 5-hexenoate (150) (0.45 g, 1.9 mmol) was dissolved in CH21(10 mL), m-CPBA (0.43 g, 100 mol%, 2.5 mmol) was added and the mixture was stirredat room temperature overnight. The resulting precipitate was removed by filtration andthe filtrate was washed with 1 M HC1, dried and concentrated. Column chromatographyusing EtOAc:PE (1:2) as eluant yielded 151 as a yellow oil (0.32 g, 66%).IR (neat) v: 3115, 3081, 2944, 2864, 1763, 1614, 1592, 1523, 1490, 1454, 1413,1345, 1155, 1012, 918, 856, 750cm-1;1H NMR (CDC13,400 MHz) ö: 8.29 (d, J = 9 Hz, 2 H), 7.3 (d, J = 9 Hz, 2 H), 2.95(m, 1 H), 2.80 (t, J = 4 Hz, 1 H), 2.70 (t, J = 7.5 Hz, 2 H), 2.51 (dd, J = 5, 2.5 Hz,1 H), 2.0-1.90 (m, 2 H), 1.85-1.75 (m, 1 H), 1.65-1.50 (m, 1 H);LRMS (El) mlz: 251 (M, 6), 221 (48), 113 (70), 109 (75), 83 (35), 71(31), 69 (80),67 (56), 57 (22), 55 (100), 43 (31), 41(99), 39 (27);HRMS (El) Calcd forC12H3N05:251.0794; found: 251.0801.EXPERIMENTAL 1685.2.52 Acetamido 5,6-epoxyhexanamide (153)153Acetamide (0.020 g, 0.34 mmol) was dissolved in DMF (2 mL), NaH (0.020 g,50% dispersion in oil, 0.42 mmol) was added and the mixture was stirred for 10 minutes.4-Nitrophenyl 5,6-epoxyhexanoate (151) (0.10 g, 0.40 mmol) was added as a solution inDMF (1 mL) and the mixture immediately turned yellow. After 10 minutes, the solutionwas extracted with EtOAc and the combined extracts were dried and concentrated.Column chromatography using EtOAc:PE (3:1) as eluant yielded 153 as a clear,colourless oil (0.010 g, 15%).IR (CHC13)v: 3396, 3271, 3213, 2933, 2858, 1736, 1704, 1464, 1414, 1377, 1271,1208, 1142,1028,907cm-;1H NMR (CDC13,400 MHz) & 8.7 (br s, 1 H), 2.95 (m, 1 H), 2.78 (t, J = 4.4 Hz,1 H), 2.62 (t, J = 7 Hz, 2 H), 2.50 (dd, J = 5, 2.5 Hz, 1 H), 2.35 (s, 3 H), 1.90-1.80(m, 2 H), 1.78-1.67 (m, 1 H), 1.55-1.45 (m, 1 H);LRMS (El) m/z: 171 (M, 0.7), 101 (24), 98 (21), 94 (35), 86 (25), 60 (53), 55 (29),43 (100);HRMS (El) Calcd forC8H13N03:17 1.0895; found: 17 1.0890.EXPERIMENTAL 1695.2.53 Methyl glutaryl chloride (157)ClOCH3157Monomethyl glutarate (156) (5.00 g, 34.2 mmol) was combined with thionylchloride (6.10 g, 3.75 mL, 51.3 mmol) and stirred at room temperature for 1.5 hours. Theexcess thionyl chloride was removed by distillation and the remaining yellow oil wasdistilled (bp 89 °CI 8 mm Hg) to yield 157 as a clear, colourless oil (5.19 g, 92%) whichwas used immediately in the next step.IR (neat) v: 2954, 1799, 1737, 1441, 1373, 1205, 1057, 976, 873, 802, 734 cnr1;1H NMR (CDC13,200 MHz) : 3.65 (s, 3 H), 2.95 (t, 3 = 7 Hz, 2 H), 2.35 (t, J =7 Hz, 2 H), 1.95 (m, 2 H);LRMS (El) m/z: 129 (M-35, 18), 115 (83), 101 (15), 100 (100), 87 (91), 86 (83), 84(37), 74 (30), 59 (58), 55 (67), 43 (23).EXPERiMENTAL 1705.2.54 Methyl glutaryl cyanide (158)NCOCH3158Methyl glutaryl chloride (157) (5.19 g, 31.5 mmol) was dissolved in CH3N(250 mL), CuCN (3.39 g, 37.8 mmol) was added and the mixture was heated to reflux for1 hour. The solution was cooled and the solvent was removed by rotary evaporation togive a greenish solid. The solid was filtered and washed with Et20 until it remainedgray. The filtrate was concentrated to give a dark green oil which was distilled(bp 102-110 °C/ 8 mm Hg) to give 158 as a clear, colourless oil (3.38 g, 69%) that wasused immediately in the next step.IR (CDC13)v: 3433, 3004, 2953, 2891, 2850, 2221, 1727, 1424, 1317, 1247, 1196,1151, 1044, 825 cm1;1H NMR (CDCI3,200 MHz) 6: 3.65 (s, 3 H), 2.85 (t, J = 7 Hz, 2 H), 2.35 (t, J =7 Hz, 2 H), 2.0 (m, 2 H);LRMS (El) m/z: 155 (M, 0.3), 129 (14), 124 (52), 115 (32), 100 (100), 96 (54), 95(34), 87 (48), 86 (31), 74 (28), 69 (39), 68 (22), 59 (87), 55 (87).EXPERIMENTAL 1715.2.55 Methyl 6-acetamido-5-oxo-hexanoate (159)159A mixture of methyl glutaryl cyanide (158) (4.00 g, 25.8 mmol), acetic anhydride(5.39 g, 4.99 mL, 52.8 mmol) and Pd/C (10%) (0.91 g, 20% w/w) in acetic acid (45 mL)was stirred under a H2 atmosphere (40 psi) at room temperature for 48 hours. Themixture was filtered through Celite and the catalyst was washed with EtOH. The filtratewas concentrated to yield a yellow oil. Column chromatography using EtOAc (1QO%) aseluant yielded 159 as a pale yellow solid (3.30 g, 64%).mp: 52.5-54.0 °C;IR (CDC13)v: 3425, 2948, 1730, 1673, 1514, 1430, 1337, 1227, 1152, 1037 cm1;1H NMR (CDC13,400 MHz) ö: 6.35 (br s, 1 H), 4.15 (d, J = 5 Hz, 2 H), 3.65 (s, 3 H),2.55 (t, I = 7 Hz, 2 H), 2.35 (t, J = 7 Hz, 2 H), 2.05 (s, 3 H), 1.95 (m, 2 H);LRMS (El) m/z: 201 (M, 1.5), 129 (100), 128 (40), 101 (42), 73 (23), 72 (25), 59(38), 55 (26), 43 (29);HR1’1S (El) Calcd forC9H15N04:201.1001; found: 201.1002;Anal. Calcd forC9H15N04:C, 53.72; H, 7.51; N, 6.96; found: C, 53.59; H, 7.67;N, 6.73.EXPERIMENTAL 1725.2.56 Methyl 6-acetamido-5-hydroxy-hexanoate (148)148Methyl 6-acetamido-5-oxo-hexanoate (159) (.1079 g, 0.5362 mmol) andbromocresol green (a few crystals) were dissolved in MeOH (2.5 mL) to give a yellowsolution. NaBH3CN (0.1079 g, 1.717 mmol) was added and the solution immediatelyturned dark blue. A solution of 2 M HC1 in MeOH was added as required to return thesolution to a yellow colour. The mixture was stirred at room temperature for 2 hours,then brine and EtOAc were added. The layers were separated, the organic phase wasdried and concentrated to yield a white solid (0.1971 g, >100%). A sample was purifiedby column chromatography using EtOAc:MeOH (10:0.5) as eluant to yield 148 as a gum-like residue.IR(nujol)v: 3446 (br), 2915, 1722, 1643, 1554, 1453, 1375, 1115 cm-1;1H NMR (CDC13,400 MHz) 6: 3.72 (m, 1 H), 3.68 (s, 3 H), 3.28 (dd, J = 14, 4.2 Hz,1 H), 3.15 (dd, J = 14, 7.3 Hz, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.0 (s, 3 H), 1.8-1.4(m, 4 H);LRMS (El) rn/v 203 (M, 6), 143 (20), 112 (22), 100 (26), 99 (42), 84 (20), 73 (100),72 (31), 71(39), 55 (30), 43 (22);fiRMS (EI)CalcdforC9H17NO4:203.1158; found: 203.1160.EXPERIMENTAL 1735.2.57 6-Acetamidomethyl ö-valerolactone (160)00NT160Methyl 6-acetamido-5-oxo-hexanoate (159) (0.25 g, 1.2 mmol) was dissolved inEtOH (6 mL) and NaBH4 (0.056 g, 1.5 mmol) was added in one portion. After30 minutes, the mixture was acidified with 1 M HC1, then concentrated to yield a clear,colourless oil. Column chromatography using CHC13:MeOH (7:3) as eluant yielded 160as a clear, colourless oil (0.19 g, 75%).IR (CHC13)v: 3618, 3421 (EtOH), 2936, 1724, 1667, 1525, 1427, 1258, 1198, 1120,1049 cm1;1H NMR (CDC13,400 MHz) ö: 6.0 (s, 1 H), 3.7 (m, 1 H), 3.62 (t, J 6 Hz, 2 H),3.45 (ddd, J = 14, 6.4, 2.9 Hz, 1 H), 3.12 (ddd, J = 14, 7.5, 5.5 Hz, 1 H), 2.0 (s,3 H), 1.6-1.4 (m, 4 H);LRMS (El) rn/v 171 (M, 0.1), 102 (29), 98 (22), 85 (28), 74 (34), 73 (20), 72 (21),60 (100), 58 (26), 56 (22), 55 (20), 45 (68), 43 (38), 41(36), 31(93), 30 (35);HRMS (El) Calcd forC8H13N03:17 1.0895; found: 17 1.0892.EXPERIMENTAL 1745.3 GENERAL BIOLOGICAL METHODSKeyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) weresupplied by the Sigma Chemical Co.ELISA assays were performed using Falcon 3911 Microtest III Flexible AssayPlates supplied by Becton Dickinson Labware. The fluorescences were measured usingan EL 309 Microplate Autoreader, recording at 2. = 405 and 490 nm, supplied by Bio-TekInstruments.Water was distilled and de-ionized before use.Phosphate buffered saline (PBS), available as a lOx solution, was diluted to givethe lx PBS. The lOx solution consisted of NaCl (80.0 g), KC1 (2.0 g), Na2HPO4 (11.5 g)and KH2PO4(1.0 g) dissolved in 800 mL H20. The pH was adjusted to 7.4 and thevolume was brought up to 1000 mL.The following stock solutions were prepared at the concentrations indicated andwere stored at -70 °C.Solution A: Hapten 130 in DMF: [Hapten] = 5.9 mg/mLSolution B: KLH in lx PBS buffer: [KLH] = 10 mg/mLSolution C: BSA in lx PBS buffer: [BSA] = 10.8 mg/mLTwo rabbits, designated as H23 and H24, were acquired for this study and housedat the Animal Care Facility at the University of B.C. All immunizations were done bythe workers at the facility.EXPERIMENTAL 1755.4 BIOLOGICAL METHODS5.4.1 Antigen Preparation— Protein (KLH)-Hapten Conjugate (143)[KLH1Keyhole limpet hemocyanin (250 jiL of solution B, 2.5 mg, 5 nmol), lx PBSbuffer (500 jiL) and hapten 130 (250 jiL of solution A, 1.5 mg, 3.8 jimol) were combinedand left standing at room temperature for 2 hours, then stored at -70 °C.This was referred to as solution D: [KLH] = 2.5 mg/mL; [130] = 1.5 mg/mL5.4.2 Rabbit ImmunizationsThe immunization schedule was as follows:Week 0: The hapten-protein conjugate 143 (300 jiL of solution D, 0.45 mg hapten)was diluted in lx PBS buffer (3 mL) and then aluminium hydroxide was addeduntil the solution was slightly cloudy. Each rabbit was immunized with anemulsified mixture of this solution (1 mL, 150 jig hapten), and Freund’s completeadjuvant (1 mL).Week 4: The hapten-protein conjugate (200 jiL of solution D, 0.30 mg hapten) wasdiluted in lx PBS buffer (3 mL) and then aluminium hydroxide was added untilthe solution was slightly cloudy. Each rabbit was immunized with 1 mL (100 jighapten) of this booster solution.Weeks 8, 12, 16, 20, 26: A boost of hapten as described for week 4 was administeredto each animal.n143EXPERIMENTAL 1765.4.3 Test BleedsWeek 6: The blood, obtained from an ear vein of the animal, was received from theAnimal Care Facility. It was stored on ice until it clotted and the clot wasremoved by a toothpick. The blood sample was centrifuged at 3000 rpm for5 minutes and then the supernatant was decanted, leaving the red blood cellsbehind. The supernatant was centrifuged again at 3000 rpm for 5 minutes. Thestraw coloured supernatant serum was removed by pipette and stored at -20 °C.Weeks 10, 14, 18, 22: A sample of blood was collected and treated as described forweek 6.Week 28: The animals were sacrificed and a whole-body bleed was performed. Thesample was treated as described for week ELISA AssaysConjugate Preparation — Protein (BSA)-Hapten Conjugate (144)Bovine serum albumin (250 ilL of solution C, 2.5 mg, 31 nmol), lx PBS buffer(500 jiL) and hapten 130 (250 .tL of solution A, 1.5 mg, 3.8 l.tmol) were combined andleft standing at room temperature for 2 hours, then stored at -70 °C.This was referred to as solution E: [BSA] = 2.5 mglmL; [130] = 1.5 mg/mLOMe/n144EXPERIMENTAL 177The following stock solutions were prepared as follows:Solution F: An aliquot of stock solution E was dissolved in lx PBS buffer such thatthe [144] 5 pg/mLSolution G: Carnation Skim Milk was dissolved in lx PBS buffer such that the[skim milk] = 0.5%Solution H: Citrate buffer: citric acid (1.29 g) and Na2HPO4O(1.37 g) (or1.1 gNa2HPO4)were combined and made up to 100 mL with H20.Solution I: Enzyme-linked secondary antibody: Goat cL-rabbit peroxidase wasdissolved in skim milk (solution G) at a dilution of 1:2000.Solution J: 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) wasdissolved in citrate buffer (solution H) such that the [ABTS] = 1 mglmL. Asolution of 3% H20was added at a dilution of 1:500.ELISA Protocol(1) Coat Plates with Antigen.(a) Add 50 ji.L of BSA-conjugate (144) (solution F) to each well.(b) Incubate 2-24 hours.(2) Remove Excess Antigen.Thoroughly empty the wells by slapping the inverted plate against a papertowel. Fill the wells with skim milk (solution G) and empty. Repeat twicemore.(3) Titration of Antibodies in Serum(a) Prepare desired dilution of serum in lx PBS buffer, i.e., a 1:200 dilutionrequires 5 j.iL serum in 1 mL of skim milk (solution G).(b) Fill the top row of wells of the plate with 100 iL of solution from (3 a).EXPERIMENTAL 178(c) Fill wells of rows 2 through 12 with 50 j.iL of skim milk (solution G).(d) Prepare a serial dilution in rows 2 through 11. Leave row 12 blank.(e) Incubate for 1 hour.(4) Remove Excess Antibody.Empty the wells and wash 3x with skim milk (solution G).(5) Addition of Secondary Antibody(a) Add 50 p.L of secondary antibody (solution I) to each well.(b) Incubate 1 hour.(6) Remove Excess Antibody.Empty the wells and wash once with skim milk (solution G), followed by threewashes with H20. Repeat the washes with three changes ofH20.(7) Add Substrate.(a) Add 50 jiL ABTS (solution J) to each well.(b) Incubate at 37 °C for 0.5 hours.(8) Read Fluorescences using the Microplate Autoreader.5.4.5 Affinity Column PurificationAffinity Column PreparationSolution K: Approximately 1 mL of glycine powder in a graduated tube was made upto 10 mL with H20 such that the [Glycine] 0.1 M.(1) Weigh out 0.36 g of freeze-dried cyanogen bromide activated Sepharose-4Bbeads into a 50 mL tube.(2) Rehydrate Beads.(a) Wash beads with 1 mM HC1 (50 mL) for 10 minutes.(b) Centrifuge at 1000 rpm for 5 minutes.(c) Remove the supernatant by aspiration.(d) Repeat wash twice more.EXPERIMENTAL 179(3) Suspend beads in lx PBS buffer (5 mL).(4) Add solution of BSA-conjugate 144.(5) Rotate mixture of beads and BSA-conjugate 144 on a mechanical rotator for1 hour.(6) Centrifuge at 1000 rpm for 5 minutes.(7) Remove supernatant and record its OD at 2 = 280 nm to determine if couplingtook place.(8) Add glycine (solution K) and rotate beads and glycine on a mechanical rotor for1 hour.(9) Centrifuge at 1000 rpm for 5 minutes.(10) Remove supernatant by aspiration.(11) Resuspend beads in lx PBS buffer (10 mL) and quantitatively transfer beads toa column apparatus.(12) Store column at 4 °C in lx PBS buffer containing 0.05% NaN3.Affinity Column/Protein A Protocol(1) Prefilter serum (10 mL) to be applied to column successively throughSyrfil-MF filters of pore size 0.8 jim, 0.45, 0.22 urn.(2) Drain storage solution from column. Wash to remove any contaminants using:(a) lx PBS buffer (5x void volume);(b) 0.1 M glycine (5x void volume);(c) lx PBS (5x void volume).(3) Apply sample to column. Collect eluant and reapply to column. Keep depletedflow-through for OD reading. Add lx PBS buffer to push remaining sampleinto flow-through.(4) Wash excess antibody from column with lx PBS buffer (lOx void volume).(5) Add elution buffer of 0.1 M glycine. Collect 0.5x void volume with wash, thencollect 1.5 mL fractions.EXPERIMENTAL 180(6) Neutralize each fraction with saturated Tris base and record its OD at 2.. =280 nm.(7) Clean column with elution buffer (lOx void volume).(8) Equilibrate column with lx PBS buffer (lOx void volume).(9) Store column at 4 °C with lx PBS buffer containing 0.05% NaN3.5.4.6 Catalytic AssayAn aliquot (250 !.tL) of a stock solution of the antibody (1.4 mg/mL) was dilutedwith 200 jiL of lx PBS buffer. An aliquot of a 1 M stock solution of 148 (50 I.iL) wasthen added to the antibody solution. 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CM—I0HNOWL6Twddkj’HODHD)01£00tc9L6XII?3JdV1YLL3dS9L0t’nuo 6(tHD)0o’v—WD001uzd4001£PSZHIA{OGP861XIUN3JdV‘1VLJ.3JS;-H30wdd___•viHO6(tH3)fZHW0O.00t661XILNJdV‘1VaLJ3JSSPECTRAL 200400 MHz(CH2)8C0H42_ppm100, . . • . • , ,4000. 3400. 2800. 2200. 1600. 1000. CM—ISPECTRAL 201400 MHz(CH2)8C0H44i*ii.Uppm1003600. 3040. 2480. i920. 1360. 800. C.1:-SPECTRAL APPENDiX 202200 MHzJ_JL_CH28COH43aII I II•II•1 III.- I I I•I I I 1 I I I I 18 4 3 2 i 0ppm—lHto3R(ZHD)0073zC,,20CT’00Tuidd9f¶.4spLOtXIUNKJJVTflLLD3dSHPSPECTRAL 205400 MHz—______iJ5 4 3 2 1 0ppm100 fl1I0_-- r4000. 3400. 2800. 2200. 1600. 1000. CM—ISPECTRAL APPENDiX 206Ill50; R = BrCH2C(O)_ppmJJu4SPECTRAL APPENDIX 207400 MHz523 2ppm1 001z-wD0wdd001ES9L1rm-“.--L9HO6(tHD).Zfl00V8oXLc1ddV1VTJ3cISSPECTRAL APPENDDC 209400 MHzNCH29OTS68•______I hppm000 • • 1 I I’ I I • • • .1 •4000. 3400. 2800. 2200. 2600. 1000. CM—ISPECTRAL 210400 MHzNCH2)9CN69ppmI I I II IJ0‘ I ‘I • I I I I • I I I I I I j I4000. 3400. 2800. 2200. 1600. 00O. Ci—10ZH.4OOViucldc90tOLOHD 6(HD)01001£.--IIXIGNJdY‘1VLI3dS.Qoo.0ItucidOUtIIcotILH6ZTT\XIG?JdV‘iVLD3dSSPECTRAL APPENDIX 213200 MHzNCH2)1o01172I I I I I I I I I I I I I I I I I7 6 5 4 3 2 1 0ppm100 I 11111111J•1I0 I I I I I I I I T I [4000. 3400. 2800. 2200. i600. 1000. C—1—piaooo;oog;•ooOO9OOE‘OOO’09>zflm00twdd5COtSb0’(tFJD)7J3GM3JJV1VTJ.33dSSPECTRAL 215400 )Uz74itppm100I0— I I I I I’ J I I I I4000. 3400. 2800. 2200. 1600. 1000. CM—ISPECTRAL APPENDIX 216200 MHz‘(CH2)11’(OCH3)275I 1 1 I I I I 1 I I I I I I 1 j I I I .1 1 1 I7 6 5 4 3 2 1 0ppm‘°JLV.Z,’H&zzIAJSI/i;rj0 —j—----——-—-- , 1 1 I I 1 1 I I I I I J I4000. 3400. 2600. 2200. 1600. 1000. Ct4—i0zC’,00t—H3wddLZHN0OZ9LuDo)g T‘(HD)Lu3aUtZJdY1VLL)dS0IriZHNOOt811XtddV1VUD3dSzC-,>-4’3000T009TOOOO900’EOOO’ .,I.Iit_Lr1Fr[F.0001uiddpLLSPECTRAL 219400MHzCH2Ph78/IL._L6 4 3ppmSPECTRAL APD1X 2200II112a200 MHz7 6 5 4 ppmU’wa’>C.I’)SPECTRAL 222200 MHz116_______________________U1 1 1 -T I I 1 1 1 1 F1 1 11 I 1T I J I 1 1 I II I I I 1 I 1 I I 1 17 6 5 4 3 2 1 0ppmF‘‘•••1’0I I I4000. 3400. 2800. 2200. 1600. 1000. CM—iSPECTRAL 223200MHz°l>P(OMe)2113a r1H.ppmSPECTRAL APPENDIX 224200 MHzOo113bI I‘‘III I3 24 3 2ppm1 0-2Dot0wddc.€ZHWOOZLUXIUJdV‘IVLI3aJSSPECTRAL 226400MHz/124;X=C1 /125;X=I— I I • • . . .‘0 II.,Ir,II1I4000. 3400. 2800. 2200. 1600. 1000. CM—ISPECTRAL APPENDIX227400 MHzO OMe\\/P%,.o115aOH6 5ppm1007. L.0_-,__-__—-—-—1 • • • • 1 I 1 ‘ ‘4000. 3400. 2800. 2200. i600. 1000. CM—iSPECTRAL APPENDIX 228I 200MHZ126;X=C1127; X=I•_—‘ I I I I I I I T I I I • 1 I 1111 I I I7 6 5 4 3 2 1 0ppm1000 II1pII J T I I I •1 I I4000. 3400. 2800. 2200. 1600. 1000. CM—I /SPECTRAL 2200 MHzPhOPCJ288ppm100 —- T - 1 1 ---_-—_.---jI j]II,-.—-—- •‘i4000 3400. 2800. 2200. 1600. 1000. CM—iSPECTRAL APPENDIX 230200 MHz(i-PrO)2POPh89Till), Ill I I I I I I III 1111111 I I I — I I I100 -- -------i---—-— -r-—-—T-——I-----r--—1———r—-—,0 ----r r-” ‘4000. 3400. 2800. 2200. 1600. 1000. CM—ISPECTRAL APPEND]X 2310II— 400MHzi-PrO90L_:._________J U__________________l \________— 7 6 5 4 — 3 2 1 0ppm100 .......... i , r I I0 — • • • 1 I • • I4000. 3400. 2800. 2200. 1600. i000. CM—iSPECTRAL APPENDD 232400 MHzo OPh/16J3ppm100 ——-—-—i—-----—,-—---,-———-—,—-——. — •I f/1 1vjl 1’I]0 ‘111[ • r i ‘ —4000. 3400. 2800. 2200. 1600. 1000.0•iA•7’•i•.r6/ZHXIG?JJV1VLLDdSwddp00c9LSPECTRAL APPEND]X 234O—..... /Z-1 400 MHzPhO /N’II H092b7 6ico— • • • • • . .0 I I I I •4000. 3400. 2800. 2200. i600. i000. CM—i01tudd£eRZTZfflAQOXIG!.Z3ddVWT.L)aJSSPECTRAL 236Io-. 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CM—I009T0I?wdcl£--i.t00ILPc.• LffLj6IIXIcNJdV1VLJ.3JSooo;009TooF.F1.i Li)iJlXIUIZKJdV1VLLJS?08IiI•1.1I,’.ooo01Zwdd£c9L8IZIDCoyoISPECTRAL APPENDIX 245LQNO2400 fflzI H—I—— Ill JIII/_________‘—— ,__________—- 8 7 6 5 4 3 2 1 0ppm100— —tf\j :11 ;1 II i I If! hR I II )—I I I J I I I I I I I I I I I I I3400. 2800. 2200. i600. ±000.0IZEpcs’r9L860uidd00t97XIcEddV1V’N1D3JSC C0 nCTRANSMITTANCE-J4 .i0Bka 0,C,.[E-:XIG?2ddV1VTJ.JacIS—;D3D,Y009OO8OO’E0-9znen001wddcot£HDODNSPECTRAL APPENDIX 2490 0 400MHz159___i7 6 5 4 3 2 1 0ppmI cc0 —. . ••.•—-••••3400. 2800. 2200. 1600. 0OO. C’-0HDO0HOO09aoQO8CO’E ;—a.900;-O001puidd£81’IZHO0VocXIGddV1VLL33dSS•VL7_V1091HIj::“so;oog;oo1---.Vr_rrIIIIOO8OOE_•I.Irr-oott•V_I••wddKooi3acnIaddV1VLJS


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