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Binding and transport of calcium by synthetic analogues of ionomycin Hu, Thomas Q. 1992

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BINDING AND TRANSPORT OF CALCIUM BYSYNTHETIC ANALOGUES OF IONOMYCINByThomas Qiuxiong HuB.Sc., South China Institute of Technology, 1985M.Sc., University of British Columbia, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of ChemistryWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1992© Thomas Qiuxiong Hu, 1992In 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.(SignatdreDepartment of C. F(ENtISTIZTThe University of British ColumbiaVancouver, CanadaDate  7.3A4‘)^117 3DE-6 (2/88)ABSTRACTThe syntheses of simple analogues of ionomycin (3), namely a series of f3-diketoco-carboxylic acids 4-6, 28-30 and 33-35, were achieved by consecutive alkylation of thedianion of 2,4-pentanedione (7) with appropriate bromides and subsequent oxidation of theP-diketo tau-alcohols.The transport of calcium across an organic barrier by these synthetic analogues ofionomycin was determined in a cylindrical glass cell using chloroform as the artificial membrane.The presence of the P-diketone and carboxyl groups within the same molecule and a sufficientlipid solubility of the compounds were shown to be necessary and sufficient conditions forcalcium transport. Small and no transport of calcium was found for analogue 4 and 6 respectivelydue to the low lipid solubility of these compounds. The relative calcium transport rates foranalogues 28 and 33-35 were 28 > 33 > 35 > 34, which demonstrated that optimum calciumtransport was achieved when the P-diketone group was separated from the carboxyl group byseven methylene units identical to the backbone found in ionomycin. Analogues 28-30 werecomparable to ionomycin and calcimycin in terms of calcium transport. The transport of calciumby synthetic analogues of ionomycin was found to be a saturable process which obeyedMichaelis-Menten kinetics. It was dependent of the pH in the aqueous source phase andindependent of the pH in the receiving phase. Both the carboxyl and the 13-diketone groups wereionized in the transport of calcium, indicating the stoichiometry of calcium complex in transportwas 1:1. Analogues 29 and 33 were found to cause transient calcium mobilization in culturedhuman leukemic cells.The pKa of the 0-diketone group of the analogues was determined to be in the range of10.90 to 11.16 in 80% Me0H-H20 by UV spectrophotometric titration. The pK a values wereii0 0 0^HO OHOH3HOdirectly related to the lipid solubility of the compounds and the hydrocarbon chain length betweenthe 13-diketone and the carboxyl groups.The binding constants of the analogues with calcium and magnesium in 80% Me0H-H20were determined to be in the order of 102 M-1 and 103 M-1 respectively using the pKa method.The binding constants of the analogues with magnesium were also determined by the mole ratiomethod which established the 1:1 stoichiometry in the magnesium complex. The selectivity inbinding was the same as the selectivity in transport, which was Mg2+ > Ca2+ >> Nat, K+.Dimethylation of the 13-diketone 42 proceeded with high diastereoselectivity. The majordiastereomer (43b) had the two methyl groups trans to each other. The characterization of thecalcium salt of analogue 28 showed that the stoichiometry of the calcium complex in the solidstate was 1:1.0^0 0^O^0 0HO•R^HO284, R = H5, R = OCH2CH20(CH2)50CH2Ph6, R = OCH2OCH329, R = (CH2)4CH330, R = (CH2)6CH34?..,./.0c0HO0HO0HO0 0330 0350 07^ 340 0^ 0 0TBDMSO^ TBDMSO42^ 4 3 biiiTABLE OF CONTENTSPageAbstract^ iiTable of Contents ^ ivList of Tables viiList of Figures ^ viiiList of Abbreviations xAcknowledgements ^ xiiDedication ^ xiiiCHAPTER ONE Introduction^ 11.1^Calcium in Biological Systems ^ 21.2^Calcium as a Cellular Messenger 41.3^Manipulation of Intracellular Calcium^ 81.4 Polyether Calcium Ionophores and Calcium Transport^91.5^Ionomycin and Its Unique Structure and Function 141.6 Calcium Ionophores Based on Ionomycin^ 18CHAPTER TWO Results and Discussion 192.1^Design and Synthesis of Ionomycin Analogues^ 192.2 Transport of Calcium across a Chloroform Liquid Membrane^292.3^Effect of Carrier Lipophilicity on Calcium Transport 332.4 Effect of Distance between the 13-Diketone and Carboxyl Groupson Calcium Transport^ 382.5 Comparison of Ionomycin Analogues to Calcimycin and Ionomycin^422.6^Cation Selectivity in Transport^ 442.7 Calcium Transport in Cultured Human Leukemic Cells ^46i vV2.8 Effect of Substrate Concentration on Calcium Transport ^502.9 Effect of pH on Calcium Transport^ 532.10 Characterization of Calcium Complex of Analogue 28^572.11 Binding of Ionomycin Analogues with Calcium Ions 622.12 Determination of pKa off3-Diketone by Ultraviolet Spectrophotometric Titration ^672.13 Binding of Calcium and Other Metal Ions by Ionomycin Analogues^712.14 Incorporation of Rigid Elements into Ionomycin Analogues^802.15 Conclusions and Future Consideration^ 83CHAPTER THREE Experimental^ 873.1^General^ 873.2^Synthesis of 9,11-Dioxopentadecanoic Acid (4)^ 943.3^Synthesis of 1542-(5-Benzyloxy)-pentyloxyl-ethoxy-9,11-dioxopentadecanoicAcid (5)^ 1003.4^Synthesis of 15-Methoxymethoxy-9,11-dioxopentadecanoic Acid (6)^ 1093.5^Synthesis of 9,11-Dioxododecanoic Acid (27)^ 1133.6^Synthesis of 9,11-Dioxooctadecanoic Acid (28) 1153.7^Synthesis of 9,11-Dioxoeicosanoic Acid (29)^ 1183.8^Synthesis of 9,11-Dioxodoeicosanoic Acid (30) 1213.9^Synthesis of 8,10-Octadecanedione (31)^ 1243.10 Synthesis of 7,9-Dioxooctadecanoic Acid (33) 1263.11 Synthesis of 11,13-Dioxooctadecanoic Acid (34) ^ 1323.12 Synthesis of 8,10-Dioxooctadecanoic Acid (35) 1383.13 Synthesis of Calcium Salt of 9,11-Dioxooctadecanoic Acid (36)^ 1443.14 Synthesis of 8,12-Dimethyl-9,11-dioxohexadecanoic Acid (41) ^ 1453.15 Transport of Calcium and Other Metal Ions by Synthetic Analogues of Ionomycin .1493.16 pKa of the 13-Diketone Groups of Synthetic Analogues of Ionomycin^ 1623.17 Binding of Calcium and Other Metal Ions by Synthetic Analogues of Ionomycin...167References^ 185Appendix 1^(Spectra)^ 191Appendix 2 (Preparation of Various Buffers) ^ 248viLIST OF TABLESvi iTable I.Table II.Table III.Table IV.Table V.Table VI.Table XVII.Table XVIII.PageAmount of Calcium in the Receiving Phase at Different Times^30Calcium Transport Rates (J) for Compounds 4, 5 and 6 32Calcium Transport Rates for Analogues 4 and 27-30^33Fragmental Constants for Various Fragments in Chloroform-Water System^36Calculated and Measured log P for Simple P-Diketones and Carboxylic Acids ^36Calcium Transport Rates (J) and Calculated log P Values forCompounds 4 and 2 7-3 0^ 37Number of Methylene Units (n) between the Two Functional Groups andCalcium Transport Rates (J) of Compounds 33, 35, 28 and 34^39Calcium Transport Rates for Calcimycin, Ionomycin and Compounds 28-30 ^42Calcium, Magnesium, Sodium and Potassium Transport Ratesfor Analogue 28^ 44Calcium Transport Rates (J) at Various Calcium Concentrationsin the Source Phase 51Calcium Transport Rates (J) at Various pH in the Receiving Phase^53Calcium Transport Rates (J) at Various pH in the Source Phase^54pKa of the p-Diketone Group of Ionomycin and of Analogues^69Binding Constants of Ionomycin and Analogues with Calcium and Magnesium ^72Binding Constants of Analogues with Potassium and Sodium^73Total Concentrations of Analogue 33 and Magnesium, Absorbance,Concentrations of the Complex, Analogue and Magnesium at EachTitration Point and Binding Constants Based on 1:1 Stoichiometry^76Binding Constants of Analogues with Magnesium Usingthe pKa and pKa. Method and the Mole Ratio Method^78Binding Constants of Various Stereoisomers of Lasalocid A with Barium Ions ^ 80Table VII.Table VIII.Table IXTable X.Table XI.Table XII.Table XIII.Table XIV.Table XV.Table XVI.LIST OF FIGURESPageFigure 1. Crystal structure of the barium salt of lasalocid A ^ 10Figure 2. Mechanism of calcium transport across a phospholipid membraneby calcium ionophores ^ 11Figure 3. Cross sections of the U-tube and the cylindrical glass cellfor the studies of calcium transport^ 13Figure 4. Crystal structure of the calcium salt of ionomycin^ 15Figure 5. The enolization and ionization of a 0-diketone 16Figure 6. Binding of the carboxylate and enolate of I3-diketone in thecalcium salt of ionomycin^ 20Figure 7. Construction of a f3-diketone by aldol condensation and oxidation^22Figure 8. Construction of a 0-diketone by dithiane alkylation, oxidation and hydrolysis^22Figure 9. Construction of a P-diketone by dianion alkylation of 2,4-pentanedione (7)^22Figure 10. Retrosynthetic analysis of analogues 4, 5 and 6^ 23Figure 11. Cross section and top view of the cylindrical glass cell for the studies ofcalcium transport across a chloroform liquid membrane^29Figure 12. Plot of the amount of calcium in the receiving phase versus time^31Figure 13. Graph of calcium transport rate versus the number of methylene unitsseparating the P-diketone and carboxyl groups ^ 40Figure 14. [CaIli in THP-1 cell stimulated by calcimycin bromide and analogue 6^46Figure 15. [Ca2-li in THP-1 cell stimulated by ionomycin and analogue 27^47Figure 16. [Ca2li in THP-1 cell stimulated by calcimycin bromide and analogue 28^47Figure 17. [Calli in THP-1 cell stimulated by ionomycin and analogue 29^48Figure 18. [Ca2i]i in THP-1 cell stimulated by calcimycin bromide and analogue 33^48Figure 19. The Michaelis-Menten expression for enzymatic catalysis^50viiiixFigure 20. The Michaelis-Menten expression for ionophore-catalyzed calcium transport^51Figure 21. Curve of the dependence of transport rates on calciumconcentration of the source phase^ 52Figure 22. Plot of log J as a function of pH in the source phase^55Figure 23. Plot of the dependence of transport rates on pH of the source phase^56Figure 24. Equilibrium between the calcium complex and the free ions in Me0H-d4^60Figure 25. Representative mole ratio plots of complexes of various stabilities^63Figure 26. Continuous variation curves plotted for hypothetical systems witha stoichiometry of 1:1 and binding constant^ 64Figure 27. UV spectrophotometric absorption spectra of analogue 33 as the pHof the solution increased from 5.80 to 13.00 67Figure 28. Plot of absorbance at 298 nm versus pH for analogue 33^68Figure 29. UV spectrophotometric absorption spectra of analogue 33 as the concentrationof MgC12 increased from 0.00 to 1.34 x 10 -3 M at pH = 9.10^75Figure 30. Plot of the number of moles of magnesium bound per mole ofanalogue 33 versus concentration of magnesium^ 77Figure 31. UV spectrophotometric absorption spectra of analogue 33 as the pH ofthe solution increased from 5.40 to 11.46 in the presence of CaC12^ 167Figure 32. Plot of the UV absorbance at 298 nm versus pH for analogue 33in the presence of CaC12^ 168Figure 33. UV spectrophotometric absorption spectra of analogue 33 as the pH ofthe solution increased from 5.61 to 9.51 in the presence of MgC12^ 171Figure 34. Plot of the UV absorbance at 298 nm versus pH for analogue 33in the presence of MgC12^ 172Figure 35. UV spectrophotometric absorption spectra of analogue 33 as the pH ofthe solution increased from 5.84 to 12.70 in the presence of NaCl^174Figure 36. Plot of the UV absorbance at 298 nm versus pH for analogue 33in the presence of NaC1^ 175Figure 37. UV spectrophotometric absorption spectra of analogue 33 as the pH ofthe solution increased from 5.84 to 12.70 in the presence of KC1^ 178Figure 38. Plot of the UV absorbance at 298 nm versus pH for analogue 33in the presence of KC1^ 179LIST OF ABBREVIATIONSA^ absorbanceA angstromATP^ adenosine triphosphatecAMP cyclic adenosine monophosphateCAPS^3-(cyclohexylamino)-propanesulfonic acidCHES 2-(cyclohexaylamino)-ethanesulfonic acidcalcd^ calculatedd doubletdd^ double doubletdt double tripletDCC^ 1,3-dicyclohexylcarbodiimideDMAP 4-dimethylaminopyridineDMSO^dirnethylsulfoxideElem. Anal. elemental analysisether^ diethyl ethereV electron voltsFAB-MS^fast atom bombardment mass spectrometryGC gas chromatographyh^ hour(s)HEPPS N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acidHOAc^acetic acidHPLC high performance liquid chromatographyHRMS^high resolution mass spectrometryIR infraredJ^ transport ratexxi1_,DA^ lithium diisopropylamideLRMS low resolution mass spectrometrym^ multipletM+ molecular ionMES^ 2-(N-morpholino)-ethanesulfonic acidmin minute(s)MOPS^3-(N-morpholino)-propanesulfonic acidmp melting pointnl/z^ mass to charge ratioNMR nuclear magnetic resonancePPTs^pyridinium p-toluenesulphonateRf the ratio of the distance a solute travels to the distancethe solvent front travelsr.p.m.^round per minuteRT retention timeS^ singlett tripletMA^ triethylamineTHE tetrahydrofuranTHP^ tetrahydropyranTLC thin layer chromatographyTBDMS^tert-butyldimethylsilylUV ultravioletACKNOWLEDGEMENTSI wish to express my sincere gratitude to my supervisor, Dr. Larry Weiler, for hisguidance and encouragement during the course of this work, and for his advice and assistanceduring the preparation of this thesis.I am indebted to Dr. Robert Young of Merck Frosst Canada for his valuable discussionsconcerning the progress of this research and to Dr. Edward Neeland of Okanagan College forproof reading this thesis. I am grateful to members of the Weiler Research Group, both past andpresent, for providing a pleasant work place.Thanks are also due to Dean Clyne, Ellay Harshenin, Nova Lee, Sulia Lo, RossLonergan, Margot Purdon, Annie Wong, Jackson Wu and Gerald Yeung whose friendships havemade the past four years a time to remember.In addition, financial assistance in the form of graduate fellowships from the University ofBritish Columbia and the efficient cooperation of the staff of the NMR, elemental analysis andmass spectrometry service are gratefully acknowledged.Finally and most importantly, I would like to thank my parents for their encouragement,patience and support throughout the course of my education. My gratitude to them is beyondmere words.xiiDEDICATIONThis thesis is dedicated to my parents.CHAPTER ONEINTRODUCTIONThe earth is replete with animals and plants in which calcium plays a vital part in theirskeletal structures and biochemistry. In the human body, calcium is the fifth most abundantelement and is mainly bound in bones and tooth enamel as calcium phosphate. Geologists haveused the calcareous remains of extinct organisms to study the fossil record and understand thecourse of evolution since the middle of the 19th century.Although the importance of calcium in the skeletal structures of living organisms has beenrecognized for more than a century, only recently have other biological roles for calcium,particularly its role as an intracellular messenger, drawn the attention of scientists from manydisciplines. A minute flow of calcium ions across cell membranes has been found to trigger suchdiverse processes as the regulation of muscle contraction, the secretion of hormones, the transportof salt and water across the intestinal lining and the control of glycogen metabolism in the liver. 1While biologists continue to elucidate calcium-induced cellular events, chemists have beencalled upon to develop natural and synthetic compounds that will specifically bind to calcium ionsand transport them across cell membranes. The development of such compounds would open thepossibility of regulating intracellular calcium levels and thus pharmacologically controlling a largenumber of physiological processes.12LI Calcium in Biological SystemsThe discovery of calcium dates back to the early 19th century when Humphry Davy at theRoyal Institution in London isolated the metal and named it after the Latin word 'calx' meaninglime 2 Following its discovery, biologists found that calcium carbonate and phosphate were themajor constituents of shell, bone and teeth in both living and extinct organisms.The first description of calcium as an important ion for the normal function and growth oforganisms was provided by the English physician and physiologist Sydney Ringer in 1883. 3Ringer was studying the effect of inorganic compounds on frog heart muscle contraction. Hefound that the muscle tissue failed to contract in a tissue culture medium made with distilled water,but it did contract in a medium made with tap water. The missing element was soon shown to becalcium. Further experiments by Ringer revealed that calcium was required for the developmentof fertilized eggs and for the adhesion of cells to each other. 4Subsequently Locke demonstrated that motor nerves exposed to a sodium chloridesolution lost their stimulant effect on muscle contractility. However, it could be restored byadding the proper proportions of calcium.5 At the turn of the century others, such as Loeb, Minesand Loewi, were able to show the involvement of calcium in the morphology and physiology ofcells, and in the action of cell stimuli by adrenaline and by drugs such as digitalis. 6Having established the universal requirement of calcium in living organisms, physiologistsand biologists started to investigate the sources of calcium and to define its biological role. In1937 Heilbrunn and Wilbur proposed that following various cell stimuli calcium ions could comefrom both external and internal pools of bound calcium. 7a Ten years later, Heilbrunn andWiercinski confirmed the regulatory role of intracellular calcium by an experiment in whichmuscle contraction was stimulated by an intracellular injection of calcium into cardiac cells.ThBy the middle of the 1960s four major roles of calcium ions in biological systems hadbeen demonstrated.8 In addition to providing rigidity to organisms as a major constituent of theskeletal structures, calcium carries charge across biological membranes of excitable cells andinfluences excitability by affecting the kinetics of sodium and potassium permeability. Calciumalso functions as a cofactor for extracellular enzymes and proteins. Finally, calcium serves as anintracellular messenger, conveying signals received at the cell surface to the inside of the cell andtriggering a range of cellular events such as muscle contraction and hormone secretion.Although the intracellular messenger role of calcium was recognized as early as the 1920sand was demonstrated by Heilbrunn and Wiercinski in 1947, the idea was slow to gain universalacceptance. There were three particular problems. Firstly, it was not generally realized that theconcentration of free calcium in the living cells was very low. Secondly, it was difficult toimagine how calcium ions, at low concentrations, could provide the energy for processes such asmuscle contraction. Thirdly, some so-called calcium-activated cells were found to work perfectlywell without external calcium. The role of calcium as an intracellular messenger was finallyaccepted universally by the middle of the 1960s with the development of techniques for the directmeasurement of intracellular calcium concentration, with the discovery and isolation of calciumbinding cellular proteins such as calmodulin and troponin C, and with the identification ofmechanisms responsible for the regulation of intracellular calcium.93Li Calcium as a Cellular MessengerIn a multicellular organism where each cell must perform a very specific function, it isessential that those cells performing the same function act in concert. This coordination isachieved by the use of chemical signals or messengers. One family of intercellular chemicalmessengers are the hormones.Hormones regulate the harmonious interplay of different tissues and organs in the body.They control metabolism and many other cellular functions such as cell growth, blood pressureand kidney function. Usually one organ will produce the hormone and secrete it into the bloodstream which will carry it to specific receptor sites on the cells of the target tissue.These intercellular messengers (primary messengers) arrive at a receptor site on the targetcell where they are detected and translated into internal signals. These signals are then carriedacross the cell membrane by a limited number of intracellular messengers (secondary messengers)to the specific cellular structure or enzyme that is the ultimate target of the hormone.Two pathways involved in the transmission of signals across the cell membranes byintracellular messengers have been identified. 1 One involves the activation of membrane-boundadenylate cyclase to convert adenosine triphosphate (ATP) to cyclic adenosine monophosphate(cAMP) which then serves to regulate enzymes within the ce11. 10 The other pathway utilizesseveral messengers such as inositol triphosphate, diacylglycerol and calcium ions. 1,10 Theseintracellular messengers bind directly to a cellular protein or activate an enzyme which thenphosphorylates a cellular protein. Both actions trigger a conformation change in the target proteinand lead to an appropriate physiological response.There are several other common ions besides calcium in the biological environment; theyinclude the doubly charged magnesium ion and singly charged sodium, potassium and chlorideions. The selection of calcium over these other common ions as an intracellular messenger is an4example of the remarkable ability of cells to differentiate and evolve in response to the organism'sneed. For a substance to act as an intracellular messenger, a target protein must be able to bind ittightly and with high specificity. It is also necessary that the messenger substance undergo a largeincrease in concentration. A tenfold increase in concentration may be needed to change an enzymefrom the "off' to the "on" state.Calcium is far better suited to tight and specific binding with proteins than other commonions are. The negatively charged oxygens found on the side chains of aspartate and glutamateresidues as well as uncharged oxygens from the carbonyls of the protein backbone bind to calciumwith high affinity. Potassium and chloride ions have relatively large radii (1.33 A and 1.81 Arespectively).9 Hence, they do not fit into the compact binding sites on proteins. The sodium ionhas a smaller radius (0.95 A), about the same as that of calcium ion (0.99 A), but because it bearsonly a single charge, sodium binds to proteins weakly. Although magnesium ion is capable ofstrong binding with proteins, its requirement for tetrahedral coordination and its smaller size (0.65A) make it difficult for a protein to adopt the binding conformation to accommodate it. 9To detect calcium signals, cells have evolved an elaborate system of proteins that bindcalcium with high selectivity at physiological concentrations (10 -7 to 10-5 M). Two examples ofsuch calcium-binding proteins are the regulatory proteins, calmodulin and troponin C of theskeletal muscle. These calcium-dependent regulatory molecules have binding sites that wraparound a calcium ion, thereby modifying their molecular conformations. This structural change isthen translated into an alteration in the activity of the regulated molecules such as myosin light-chain kinase or troponin I, to which the calcium-dependent regulatory molecules are bound. 11Not only does the cell have the ability to detect the calcium signal, but it is also capable ofremoving calcium and terminating the signal quickly. The regulation of intracellular calcium isusually achieved by intracellular organelles such as the mitochondria and endoplasmic reticulum.5The endoplasmic reticulum in various cell types (muscle, nerve, liver, etc.) has been found to beable to buffer and sequester free calcium. 12The resting intracellular concentration of free calcium is very low, on the order of 10 -7 Min virtually all animal cells. 13 Kretsinger has suggested that this low calcium concentration mayhave resulted from early cells avoiding the formation of insoluble calcium phosphate in thecytosol, so that they could utilize ATP as the energy carrier. 14 However, even if the secondarymessenger role of calcium was only an evolutionary afterthought, the low intracellular calciumconcentration has been utilized to an advantage in cells. A low intracellular calcium concentrationmakes the use of this ion as an intracellular messenger energetically inexpensive. The very lowcalcium concentration means that relatively few ions need to be moved, with a relatively smallexpenditure of energy, to raise the intracellular calcium concentration by a factor of 10 for theregulation of an enzyme. The intracellular concentrations of magnesium, potassium and sodiumare all too high to allow a large increase in concentration. Hence, these ions would be much lesseffective than calcium as intracellular messengers.In contrast to the low intracellular calcium concentration, free calcium concentration in theextracellular medium is high, on the order of 10 -3 M in vertebrates and higher in marineinvertebrates. This calcium concentration gradient, together with a membrane potential of -40 to-90 mV, allows for a rapid entry of calcium into the cell upon the activation of calcium-selectivechannels by appropriate electrical or chemical signals. 15 Alternatively, the sources of calcium maycome from intracellular storage sites where calcium is bound to the cytoplasmic surface of theplasma membrane, or sequestered in mitochondria or the endoplasmic reticulum. 16The intricacies of calcium regulation are only now being unraveled. Digitalis, a drugwhich has been used for thousands of years, exemplifies the clinical importance of controllingintracellular calcium concentration. The ability of digitalis to strengthen the heartbeat wasrecognized long ago. Today it is known that digitalis and other related drugs work by raising the6level of intracellular calcium in the heart by affecting the sodium-potassium pump in themembrane. For many heart patients the result of raising the level of intracellular calcium in theheart is a longer and more secure life.7La Manipulation of Intracellular CalciumBecause of the extensive role of calcium in the cell, it is essential to regulate intracellularcalcium level to be able to effectively control cellular processes. Two typical ways of alteringintracellular calcium concentrations are to inject calcium or a calcium chelating agent such asethylenecliaminetetraacetic acid (EDTA) into the cell or to add a substance to the extracellular fluidwhich will increase or decrease the concentration of intracellular calcium.The first approach was adopted by Heilbrunn and Wiercinski who showed that theinjection of calcium into frog muscle stimulated contraction. 7b Simple as this approach mightseem, it is fraught with several difficulties. Firstly, the micropipette can cause irreversible damageto the electrical and biochemical response of the cell. Secondly, the quantity of calcium that mustbe injected to significantly raise the concentration of calcium in the cytoplasm, and yet not to betoo high to cause damage, is often unknown. Finally, since calcium-buffering systems existinside cells, an elevated free calcium concentration in the cytoplasm for more than a few secondsis difficult to maintain by the injection method.An alternative approach is to introduce a substance which affects either the calciumpermeability of the cell membrane or intracellular calcium stores. Since the extracellular calciumconcentration is 10 3 to 106 higher than the intracellular calcium concentration, calcium may betransported across the cell membrane down its concentration gradient by lipophilic compoundsknown as calcium "ionophores" (phore from the Greek pherein to bear). The term ionophore isused in a biophysical context to mean a compound which facilitates the transport of an ion througha natural or artificial lipid membrane from one aqueous medium to another. 17 The search fornaturally-occurring and synthetic calcium ionophores, particularly the polyether calciumionophores, has attracted the attention of biologists and chemists since the early 1970s.8IA Polyether Calcium Ionophores and Calcium TransportIn 1951, the first polyether calcium ionophore, lasalocid A (1) (formerly known as X-537A) was extracted from Streptomyces lasaliensis. 18 More than twenty years elapsed before thesecond polyether calcium ionophore, calcimycin (2) (formerly known as A23187) was isolatedfrom Streptomyces chartreusensis. 19 The molecular structures of lasalocid A and calcimycin wereestablished in 1970 and in 1974 respectively by spectroscopic methods and a crystal structureanalysis of the free acids.20 The common structural characteristics of lasalocid A and calcimycinare the presence of a carboxyl group and a number of tetrahydrofuran and tetrahydropyran rings,the latter results in the term "polyether".9Both lasalocid A and calcimycin are very soluble in organic solvents. When their organicsolutions were exposed to aqueous alkali or alkaline base, there was no extraction of theionophore anion into the aqueous phase. In fact, cations of the base were extracted into theorganic phase! 18,19 This led to the investigation of the ability of these two compounds totransport divalent cations across an organic barrier and to the widespread use of these twocompounds, particularly calcimycin, in the elucidation of calcium-induced cellular events.The extraction of divalent cations from an aqueous phase into an organic phase bylasalocid A or calcimycin is due to the ability of these compounds to form a lipid solubleionophore-cation complex.21 The crystal structure of the 2:1 barium salt of lasalocid A wasdetermined by Johnson and co-workers (Figure 1). 220 = Carbon^• = Oxygen^O = BariumFigure 1. Crystal structure of the barium salt of lasalocid A. 22Oxygens from the carboxylate, the tetrahydrofuran and tetrahydropyran, the ketone andthe two hydroxyls of one ionophore molecule, together with the carboxylate oxygen and onehydroxyl oxygen from the second ionophore molecule, chelate the barium ion to form a 2:1ionophore-barium complex. The surface of the complex is composed largely of nonpolarhydrocarbon chains and the aromatic residues, which provide a lipophilic shield for the polarchelating portion of the complex, thereby rendering it soluble in organic solvents. The crystalstructure of the calcium salt of calcimycin was found to be almost identical to its solutionstructure.23 Calcimycin forms a lipid soluble 2:1 complex with calcium similar to the barium saltof lasalocid A.2310The nonchelating substituents of the ionophores and their stereochemistry are believed tobe important in preorganizing the conformations of the molecules for ion complexation. 24 Stilland co-workers proposed that particular arrays of the nonchelating substituents could destabilizeundesired rotomers and reduce the available conformations of the ionophores to those suitable formetal ion binding.24Both lasalocid A and calcimycin can act as mobile carriers to transport calcium across anorganic barrier such as a phospholipid membrane (Figure 2).25 At physiological pH, the carboxylgroup of the ionophore (HL) is likely to be ionized with the carboxylate group in the aqueousmedium, and the lipid soluble part of the molecule, inside the membrane. The extracellularcalcium ion approaches the ionophore and loses its water of solvation as successive chelatingoxygen and/or nitrogen atoms of the ionophore bind to it, forming the neutral, lipid soluble 2:1calcium complex (CaL2). Although this complex formation is undoubtedly a multistage reaction itcan be given a single composite rate constant, kf. Once the complex is formed it diffuses acrossthe membrane at a rate kdiff. At the inside surface, the reverse process occurs. This is also amultistep reaction which can be assigned the composite rate constant kd in which the calciumcomplex releases calcium into the cell interior with the regeneration of the neutral ionophore. Thereleased calcium ion is then available to stimulate the action of the cell concerned. The mechanism(Figure 2) involves the neutral ionophore diffusing back to the outside surface. In actual cells thisspecies may be another metal complex.In general, the direction and extent of such calcium transport are governed by the calciumconcentration gradients across the membrane. The transport of calcium across cell membraneswill be more efficient if certain kinetic criteria are fulfilled, such as a facile complexation reaction(kf >> kd) on the outside of the membrane, and a facile decomplexation reaction (kd >> kf) on theinside of the membrane.261 1Outside InsideCa2+ +kd2 H++ Ca2+ + 2 OFFIr kf+ 2 H2O12Figure 2. Mechanism of calcium transport across a phospholipid membraneby calcium ionophores.Efforts to develop synthetic calcium ionophores based on consideration of the structures oflasalocid A and calcimycin, and the desirable characteristics (e.g., ion selectivity, favorablebinding constant and calcium on/off kinetics) of an ideal calcium ionophore have been met withcertain success.27a ,b Two diglycolamic acids prepared by Umen and Scarpa were shown totransport calcium across an artificial membrane comparable to lasalocid A and calcimycin. 27a Theuse of lipophilic acyclic polyether dicarboxylic acids as mobile carriers to transport calcium,magnesium and barium across an artificial membrane was reported by Hiratani and co-workers.27bAn artificial membrane such as a bulk liquid membrane was used by the above authors andothers as a simple way to study ionophore-catalyzed calcium transport. 27 In this experiment twoaqueous compartments in a U-tube (Figure 3a)27g or a cylindrical glass cell (Figure 3b)27f areseparated by a gently stirred solution of the ionophore in chloroform. One aqueous compartmentis filled with solution containing calcium ions. The other compartment is filled with aqueoussolution without calcium ions. The appearance of calcium ions in this later compartment can befollowed by using a number of techniques including calcium atomic spectrophotometry or calciumspecific electrodes.3a^ 3bionophore in CHC13Figure 3. Cross sections of the U-tube and the cylindrical glass cell for the studies of calciumtransport.The U-tube or the cylindrical glass cell experiment can be used to determine the relativecalcium transport efficiency of different ionophores. It can also be used to determine the cationselectivity of an ionophore by placing competing cations in the source phase and following theirconcentrations in the receiving phase. Chloroform is usually employed in these transport studiesbecause its dielectric constant (e = 4.8) is similar to the dielectric constant of the interior of aphospholipid membrane (e = 2 - 4).28The U-tube or the cylindrical glass cell experiment is a very crude approximation ofcalcium transport across biological membranes in terms of the membrane size, membranecomposition and the transport time scale. However, it can serve as a model of transport behavior.Many factors which control the transport of calcium across biological membranes, such as thelipophilicity of an ionophore, can be studied in this simple system.13HO OHO^0 03HOLi Ionomycin and Its Unique Structure and FunctionIn 1975, a novel polyether calcium ionophore ionomycin (3) was isolated by Meyers, etal. as a fermentation product of Streptomyces conglobatus. 29 Its molecular structure wasdetermined by Toeplitz et al. using 1H NMR spectroscopy, mass spectrometry, and X-raycrystallographic analysis of three crystalline forms of its cadmium and calcium salts. 30Like other polyether calcium ionophores, ionomycin has been found to be a potentantimicrobial agent. It is active against Gram-positive bacteria with no demonstrable effect againstGram-negative bacteria. However, ionomycin was found to have very unusual physical,chemical, and biological properties. It possesses a 13-diketone group which, together with thecarboxyl group, confers the dibasic character to the ionophore. It has a very high affinity forcalcium and other divalent cations. In fact, the calcium salt of ionomycin was extracted from anaqueous solution of pH 12 which had been made alkaline with sodium hydroxide. 29Ionomycin is unique among polyether ionophores in that it forms a 1:1 calcium complex.As shown in the crystal structure of the calcium salt of ionomycin (Figure 4), the carboxylategroup, the enolate of the 13-diketone, two hydroxyl groups and a tetrahydrofuranyl oxygen forman octahedral complex around the calcium ion. The ligand wraps around the calcium ion with theoxygen atoms directed towards the inside of the sphere. The alkyl groups, on the other hand,protrude from the shell, providing the calcium complex with its lipophilic properties.14Q = Carbon^= Oxygen^0 = CalciumFigure 4. Crystal structure of the calcium salt of ionomycin. 30The structural characteristic which distinguishes ionomycin from other polyetherionophores is the presence of a 13-diketone group. The f3-diketone exists as an equilibriummixture of tautomeric keto and enol forms, the latter absorbs at 276 nm. 31 Ionization of the(3-diketone shifts the UV absorption to 300 nm (Figure 5). This permits the study of protonationand cation binding equilibria of ionomycin by UV spectrophotometric titration. The pKa of the(3-diketone group in ionomycin was determined to be 11.94 ± 0.02 in 80% Me0H-H20 using thistechnique.3215H0 0^ 0 ‘0,I)C)(1 1)/Lfkna, = 276 nmOH o + H2O16 Amax = 300 nmFigure 5. The enolization and ionization of a 13-diketone.Liu and Hermann studied the extraction of calcium ions from an aqueous phase into anorganic phase and the transport of calcium across an organic barrier by ionomycin.33 They foundthat the extraction of calcium by ionomycin was strongly dependent on the pH of the aqueousphase. Essentially no complexing of calcium occurred below pH 7.0 and complexation reached amaximum at about pH 9.5. In contrast, the extraction of calcium by calcimycin was observeddown to pH 5.0 and reached a maximum near pH 7.5. Under the same experimental conditions,ionomycin bound twice as much calcium as calcimycin and it had a greater selectivity for calciumover magnesium than calcimycin.33 The selectivity of ionomycin for divalent cations was shownto be Ca2+ > Mg2+ >>Sr2+ = Ba2+. No complexation with monovalent cations could bedetected.34In vivo studies also revealed that ionomycin was more effective than calcimycin as amobile calcium carrier. In studies of rat liver mitochondria, it was shown that ionomycinefficiently catalyzed the exchange of two protons for one calcium ion across the cell membrane. 34The turnover numbers for calcium transport by ionomycin were 3- to 5-fold greater than those bycalcimycin.35 Ionomycin has been found to stimulate the release of histamine from mast cells andcatecholamine from pheochromocytoma cells by transporting calcium across the cellmembranes.36 It has also been linked to the activation of human blood platelets by facilitatingtransport of calcium across the membrane and mobilizing calcium stored in organelles. 37Currently, ionomycin is being used to study the biochemical basis of asthma. Mast cellslocated in airway walls release histamine and leukotriene C. Leukotriene C is believed to be amediator in asthma as a potent muscle contractant that constricts small airways in the lung. It is ametabolite of arachidonic acid whose synthesis is shown to be stimulated by ionomycin mediatedcalcium transport. 38Since its discovery in 1976 ionomycin has been used in place of calcimycin and lasalocidA as a superior calcium ionophore. It has been shown to mimic, at least qualitatively, the effectsof many physiological cell stimuli. However, the scarcity of ionomycin limits its applications asan ionophore in biological studies, and its toxicity prevents its uses in the treatment of calciumrelated diseases.17J Calcium Ionophores Based on IonomycinAlthough the partial and total synthesis of ionomycin has been achieved, 39 the difficultiesassociated with the stereoselective introduction of the numerous asymmetric centers on themolecule have been significant. Provision of ionomycin through total synthesis is not aneconomical choice.Little is known about the structural features of ionomycin which control its calciumspecificity in transport. One objective of this project is to design and synthesize simple ionomycinanalogues to analyze the role of the functional groups of ionomycin in calcium binding andtransport and to study the effect of other structural features, such as lipophilicity and oxygencoordination, on calcium transport. It is also our objective to improve the calcium transportefficiency of these simple molecules based on structure-activity relationships and to finally providenew calcium ionophores based on ionomycin both as biological tools and potential therapeuticagents.18CHAFFER TWORESULTS AND DISCUSSION2J. Design and Synthesis of Ionomycin AnaloguesThe first stage of this project involved the design of ionomycin analogues based on theinformation gathered about the mode of action of ionomycin itself. The molecules would containthose functional groups thought to be vital to the function of ionomycin while maintaining thepotential membrane (lipid) solubility. The numerous stereochemical centers found along thecarbon backbone of ionomycin were omitted to simplify the preparation of the model compounds.The chemical and physical data on ionomycin suggest that the 13-diketone and carboxylgroups are crucial to its ionophoric properties. The crystal structure of the calcium salt ofionomycin (Figure 4) shows that both these groups are ionized and coordinated to the metal cationin the formation of the neutral 1:1 calcium complex. It was deemed important that these twofunctional groups be retained in the model compounds.In the natural product, there is a seven-carbon chain separating the 0-diketone and thecarboxyl groups. The presence of this seven-carbon chain could certainly contribute to the highlipid solubility of the ionophore-calcium complex. The distance between these two functionalgroups is such that a 12-membered chelate ring is formed on chelation of a calcium ion by thecarboxylate oxygen and the first oxygen of the f3-diketone enolate (Figure 6).19O^0 0HO20„0"'•Ws■Figure 6. Binding of the carboxylate and enolate of 0-diketone in the calcium salt of ionomycin.The formation of a 12-membered chelate ring would have less entropy to overcome thanthat of a larger chelate ring. On the other hand, the complex formed could be expected to havemore favorable enthalpy than a smaller chelate ring. The interplay of entropy and enthalpy effectsmay be optimized for efficient calcium transport when a seven-carbon chain separates the twofunctional groups. Thus, a seven methylene unit was chosen as the linkage of these twofunctional groups in the model compounds.To maintain the membrane solubility, high lipophilicity of the compounds may be needed.The simplest analogue fulfilling the above requirements would be a 9,11-dioxocarboxylic acidsuch as 9,11-dioxopentadecanoic acid (4) which was chosen as the first target molecule.0 0 0HOSince two hydroxyl and one tetrahydrofuranyl oxygen of the natural product are involvedin coordination to the calcium ion (Figure 4), we suspected it would be necessary to incorporate aside-chain which contained appropriately placed oxygen atoms to retain the six calcium bindingsites found in ionomycin. A side-chain that contains an ethylene glycol and an ether function fivemethylene units away was chosen to mimic the calcium coordination sites in ionomycin. An etherfunction, such as a benzyl ether, could compensate for the decrease of lipophilicity produced bythe introduction of three oxygen atoms in such a molecule. Therefore, abenzyloxypentyloxyethoxyl unit was incorporated into analogue 4 to give the second molecule1542-(5-benzyloxy)-pentyloxyl-ethoxy-9,11-dioxopentadecanoic acid (5).0^0 0HO 00W05The presence of six oxygen coordination sites may not be necessary for the binding andtransport of calcium. A molecule of water from the aqueous medium could occupy a calciumcoordination site as is often observed in the binding of calcium with proteins.40 The crystalstructure of the barium salt of lasalocid A shows one molecule of water in the ionophore-bariumcomplex (Figure 1). The water molecule not only occupies a barium coordination site, but it alsoholds the two ionophore molecules together through the formation of hydrogen bonds. Thus, itmay be sufficient to incorporate a side chain which contains only one or two oxygen atoms aspotential calcium coordination sites. With this in mind we incorporated a methoxymethoxyl unitin analogue 4 to give a further target molecule 15-methoxymethoxy-9,11-dioxopentadecanoic acid(6).0^0 0HO6The second stage of the project was to develop a synthetic route to each of the proposedanalogues. The carboxyl function of the compounds could be made by oxidation of a hydroxylgroup.41 The etheral units in analogues 5 and 6 could be prepared using known chemistry. 42The crucial part in the synthesis of the analogues appeared to be the introduction of the p-diketoneand the construction of the carbon backbone.21A 0-diketone could be prepared by oxidation of a (3-hydroxyl ketone which in turn couldbe generated by an aldol condensation between a methyl ketone and an aldehyde. This route wasused in the synthesis of ionomycin by Hanessian's group 39a and by Evan's group. 39b (Figure 7)220^0^HORAH + )(RI aldolR).. ^R'oxidation0 0A•)1R *R' Figure 7. Construction of a 0-diketone by aldol condensation and oxidation.Alternatively, the 0-diketone could be prepared by alkylation of the anion of a dithianewith an epoxide, followed by oxidation of the hydroxyl group and hydrolysis of the dithiane(Figure 8). This method was employed in the synthesis of an ionomycin fragment by Weiler andShelly.39cO S^HO r)^0 r )^0 0ii + ,)<S alkylation^1 S^oxidation A5<S hydrolysis )L)RN H R'^R•/` R'^R^R' R^R'Figure 8. Construction of a P-diketone by dithiane alkylation, oxidation and hydrolysis.These two methods could be applied to the synthesis of our proposed analogues ofionomycin. However, neither route appeared to be very efficient. The former method converted acarbonyl to an alcohol and later back to the carbonyl, and the latter had one more syntheticmanipulation than the former. A logical precursor of a 0-diketone appeared to be the simplest0-diketone, the commercially available 2,4-pentanedione (7). Consecutive alkylation of2,4-pentanedione at the methyl carbons could be achieved using the dianion chemistry (Figure 9).00 1. dianion^0 0^1. dianion)C)C  generation R ,.)LA, ^generationD■^ )2. R-Br 2. R'-Br70 0R^R'Figure 9. Construction of a p-diketone by dianion alkylation of 2,4-pentanedione (7).Using this method, the synthesis of analogues 4, 5 and 6 was reduced to the preparationof bromides 8-11 (Figure 10).0^0 0HO4,R = (CH2)2CH35,R = (CH2)30(CH2)20(CH2)5OCH2Ph6, R = (CH2)3OCH2OCH3,v 0 0TBDMSO L)L■RII.TBDMSOWBr +80 0)1■)L.R1i0 0+ Br —R9,R = (CH2)2CH310,R = (CH2)30(CH2)20(CH2)5OCH2Ph11, R = (CH2)3OCH2OCH3Figure 10. Retrosynthetic analysis of analogues 4, 5 and 6.Bromide 9 is commercially available. Our synthesis of bromide 8, a common intermediatein the preparation of the analogues, and bromide 11 is illustrated in Scheme I.23Scheme I.HO^OH12TBDMSOW Br8i HC)^,/■Br "1 3wBr •••'.%0H^Br^*V%*V14 11i. HBr, 72%; ii. TEA, DMAP, TBDMSC1, 82%; iii. P2O5, CH2(OCH3)2, 91%Monobromination of 1,7-heptanediol (12) was achieved by continuously extracting amixture of diol 12 and aqueous hydrobromic acid with heptane. 44 The hydroxyl group ofbromide 13 was protected as its tert-butyldimethylsilyl ether 45 to give bromide 8. Bromide 11was prepared by treatment of 3-bromo-l-propanol (14) with phosphorus pentoxide anddimethoxylmethane.46Our synthesis of bromide 10 is outlined in Scheme II. It was initiated with themomobenzylation of 1,5-pentanediol (15). 47 Treatment of diol 15 with two equivalents ofsodium hydride and one equivalent of benzyl bromide gave the monobenzylated alcohol 16 in54% yield, together with a small amount of dibenzylated product.The alcohol 16 was subjected to the modified Williamson ether synthesis procedure. 48This involved the vigorous mixing of a two-phase system containing the alcohol, 1-bromo-2-tetrahydropyranyloxylethane and aqueous sodium hydroxide in the presence of a phase transfercatalyst tetrabutylammonium hydrogen sulfate. The reaction gave the desired ether 17 in 83%yield. Only a small amount of alcohol 16 could be converted to ether 17 when the reaction wascarried out using sodium hydride as a base in polar aprotic solvents such as dimethylformamide.24Scheme II.HO^OH15i HO WO16i i THPOOWO17iii—o- HOOWO18TBDMS0/ 00WO19vHO/ 00W02 0Br ..• 0N)/WO1 0i. NaH, BrCH2Ph, 54%; ii. NaOH, Bu4NHSO4, THPO(CH2)2Br, 83%; iii. PPTs, 83%;iv. NaOH, Bu4NHSO4, TBDMSO(CH2)3Br, 83%; v. Bu4NF, 89%; vi. Ph3P, CBr4, 91%The tetrahydropyranyl ether protecting group in 17 was cleaved with pyridium p-toluenesulfonate to give alcohol 18 in 83% yield. 49 The alcohol 18 was subjected to the modifiedWilliamson ether synthesis procedure using 1-bromo-3-tert-butyldimethylsilyloxylpropane to givethe ether 19 in 83% yield. Deprotection of the TBDMS ether with tetrabutylammonium fluoride 50gave alcohol 20 which was converted to bromide 10 by treatment with triphenylphosphine andcarbon tetrabromide.51With bromides 8, 9, 10 and 11 in hand, we were ready to perform the dianion alkylationreactions on 2,4-pentanedione (7). The use of the dianion alkylation reactions in the synthesis ofanalogue 4 is shown in Scheme III. Treatment of 2,4-pentanedione (7) with one equivalent ofsodium hydride and one equivalent of n-butyllithium generated the dianion 21 which reacted withbromide 8 to give the monoalkylated 13-diketone 22 in 81% yield. 43a25Scheme III.0 0.^0 0A)C 1 A)L i7^210 0 iiiTBDMSOL)L220 0TBDMSO23iv 0 0TBDMSO2 4i. NaH, n -BuLi; ii.TBDMSO(CH2)7Br, 81%; iii. 2.0 LDA; iv. Br(CH2)2CH3, 81%The structure of the monoalkylated I3-diketone 22 was confirmed by 1H NMRspectroscopy. The spectrum of compound 22 exhibited a triplet at 5 2.51 which integrated to 0.4proton and a triplet at 8 2.27 which integrated to 1.6 protons ascribable to the methylene protonsat C-8 of the keto and enol forms of the 13-diketone respectively. A 0.6-proton singlet at 8 2.24and a 2.4-proton singlet at 8 2.06 were assigned to the methyl protons at C-12 of the keto andenol forms respectively. A 0.8-proton singlet at 8 15.50, a 0.8-proton singlet at 8 5.49 and a0.4-proton singlet at 8 3.58 were assigned to the enol hydroxyl proton, the vinyl proton and themethylene proton at C-10 respectively as shown below.8 3.580 0TBDMSO^ C: to 12^TBDMSO^ 1252.27t H52.51^52.24 ^82.065.49The second dianion alkylation reaction was achieved by treating the I3-diketone 22 withtwo equivalents of lithium diisopropylamide (LDA) and subsequently with one equivalent of268 15.500 H 'O1-bromopropane (9). The sterically hindered base LDA was used to regioselectively generatedianion 23.43b Reaction of dianion 23 with bromide 9 produced the 13-diketone 24 in 81% yield.The regioselective alkylation was also confirmed by 1H NMR spectroscopy. The spectrum ofcompound 24 showed two triplets at 8 2.53 - 2.24 integrating to a total of four protons ascribableto the methylene protons at C-8 and C-12. In addition, three singlets at 8 15.51, 5.48 and 3.54integrating to a total of two protons ascribable to the enol hydroxyl proton, the vinyl proton andthe methylene proton at C-10 were present in the spectrum.To complete the synthesis of analogue 4, the silyl ether protecting group of compound 24was cleaved with tetrabutylammonium fluoride to give alcohol 25 in 91% yield (Scheme IV). Thealcohol 25 was oxidized to aldehyde 26 with dimethylsulfoxide and dicyclohexylcarbodiimide inthe presence of dichloroacetic acid.52 Treatment of the aldehyde with silver nitrate and sodiumhydroxide53 produced the 13-diketone carboxylic acid 4 in 69% yield. Oxidation of alcohol 25using Jones oxidation54 gave a complex mixture of products which were difficult to purify. Nofurther effort was made to convert the alcohol 25 to the acid 4 in one step using Jones oxidation.Scheme IV.270 0TBDMSO240 0^O^0 0iiHO^ ----"" Hiii250HO 0 0264i. Bu4NF, 91%; ii. DMSO, DCC, C12CHCO2H; iii. AgNO3, NaOH, 69%iTo synthesize analogues 5 and 6, we used the 13-diketone 23 as the starting material.Regioselective dianion alkylation of 23 with bromide 10 and 11 respectively, subsequentdeprotection of the TBDMS ethers and oxidation of the alcohols furnished analogues 5 and 6 inyields similar to those of analogue 4 (Scheme V).Scheme V.0 0^.TBDMSOw.)L.A. 1230 0TBDMSO ,---\ w.0 0^0 iii0^0 0iv^v---0- --11.- HO5/—•0 0 WOTBDMSO^CIL)L0^ ili_a....230 0^iii^i v^vTBDMSO^ .00 -111.- .-----110► --ow.0^0 0HO6i. 2.0 LDA, Br(CH2)30(CH2)20(CH2)50CH2Ph;  ii. 2.0 LDA, Br(CH2)3OCH2OCH3;iii. Bu4NF; iv. DMSO, DCC, C12CHCO2H; v. AgNO3, NaOH2 8Top viewCross sectionglass dividerCa2+ + H2O5.3 cmH2O-L- - - CHC13 + ionophore • -EMII6.3 cm22 Transport of Calcium across a Chloroform Liquid MembraneWith ample quantities of analogues 4, 5 and 6 in hand, we proceeded to evaluate theability of these molecules to transport calcium across an organic barrier in a cylindrical glass cell(Figure 11).27d29Figure 11. Cross section and top view of the cylindrical glass cell for the studies of calciumtransport across a chloroform liquid membrane.A solution of analogue 5 (200 gM) in chloroform (40 mL) was placed at the bottom of thetransport vessel. The volume of the chloroform solution was such that two separatecompartments were created above the organic solution (Figure 11). An aqueous solution (10 mL)of calcium chloride (500 mM) and CHES/Me4NOH buffer (40 mM, pH = 9.5) was placed atop ofthe chloroform solution on one side of the transport vessel as the source phase. A solution ofdeionized distilled water (10 mL) containing MOPS/Me4NOH buffer (40 mM, pH = 7.0) wasplaced on the other side of the transport vessel as the receiving phase. The chloroform solutionwas stirred continuously and samples (0.2 mL) were withdrawn from the receiving phase atdifferent times. The organic phase remained clear during the experiment and subsequent studiesshowed that the concentration of ionophore was below the critical micelle concentration.0^0 030HO5Analogue 5 was chosen as the ionophore in our first transport experiment since thiscompound possessed more oxygen donor sites than analogues 4 and 6 and might mimic theionophoric properties of ionomycin better. The aqueous source phase was buffered at a higherpH and the receiving phase at a lower pH to possibly facilitate the transport process.To our satisfaction, calcium atomic absorption spectrophotometric analysis of the sampleswithdrawn from the receiving phase at different times showed a steady increase of calcium. Theresults are given in Table I. Each value in the Table is the average of three independentexperiments with a standard error of approximately ±10%.Table I. Amount of Calcium in the Receiving Phase at Different Times.Time (h) 0 12 16 20 24 36 42 48 60Ca2+ (=01) 0 5.8 7.8 10 12 17 19 22 25To determine if the above calcium transport was due to the leakage of calcium across thechloroform layer (blank transport of calcium), we carried out the transport experiment without theaddition of compound 5 in the chloroform layer. Analysis of the samples withdrawn from thereceiving phase using calcium atomic absorption spectrophotometry showed no detectablepresence of calcium. The ability of analogue 5 to function as a carrier for the transport of calciumacross an organic barrier was thus established.0 10^20^30Time (h)The data in Table I shows that the amount of calcium in the receiving phase increaseslinearly with time when the amount of calcium transported is small (less than 0.5% of calciumfrom the aqueous source phase). A plot of the amount of calcium transported versus time withinthis linear range is given in Figure 12. The slope of the straight line is 0.50 and the area betweenthe organic and the receiving phase is 6.8 cm -2, thus the initial transport rate, J, expressed as themoles of calcium transported per square centimeter of the chloroform per hour was calculated tobe (7.3 ± 0.7) x 10 -8 mole cm-2 h-1 .Figure 12. Plot of the amount of calcium in the receiving phase versus time. The points representthe average of three experimental values.This linear relationship between initial transport and time was observed in all oursubsequent transport experiments. These transport results will be given as the transport rate Jwith its upper and lower limit.Next, we carried out the transport experiments on compounds 4 and 6 to examine theeffect of minor structural changes on calcium transport. The results are summarized in Table II.0^0 031HOHO320^0 0Table II. Calcium Transport Rates (J) for Compounds 4, 5 and 6.Compound 4 5 6J (10-8 mole cm-2 h-1 ) 0.3 ± 0.1 7.3 ± 0.7 0Compound 5 was found to be the most efficient carrier for the transport of calcium.Presumably this was due to the effect of coordination of calcium by the three oxygen atoms on theside chain of the molecule. Surprisingly no detectable transport of calcium was observed forcompound 6 which had two additional oxygen donor atoms compared to compound 4. Thiscontradicted what was anticipated from the possible contribution of coordination of calcium byadditional oxygen atoms to the transport of calcium. In fact, it appeared that additional oxygenatoms on the side chain of compound 6 were detrimental to the transport of calcium.Besides providing potential coordination sites for calcium, additional oxygen atoms on theside chain of compounds 5 and 6 could change the lipophilicity of the molecules, which mayaffect the calcium transport ability of the compounds. Although the lipid solubility of thecompounds was not determined, it is very likely that they follow the order 5 > 4 > 6 based on theratio of the numbers of carbon and hydrogen atoms to oxygen atoms. This is the same order asthe calcium transport rate of the compounds.The hypothesis that the lipophilicity of the compounds may control calcium transport couldbe tested by varying the lipid solubility of the molecules in a predictable manner and study thetransport behavior of such molecules. To this end, compounds 27, 28, 29 and 30 which hadvarious alkyl side chains were prepared. The synthesis of these compounds was very similar tothat of analogue 4. Therefore, it will not be discussed here.0 0HO270 0HO40 0HO280 0HO29O 0HO0L.^000.3 ± 0.100015 ± 215 ± 213 ± 12 Effect of Carrier Lipophilicity on Calcium TransportThe transport experiments were carried out on compounds 27-30 to probe the effect oflipophilicity on calcium transport. The results are given in Table III.Table III. Calcium Transport Rates (J) for Compounds 4 and 27-30.Compound^ J (10-8 mole cm-2 h -1 )3330As seen from Table III, the lipophilicity of the compounds appears to play an importantrole in the transport of calcium. Compound 27 with no methylene units on the hydrocarbon sidechain did not transport calcium across the chloroform phase. Compound 4 had three methyleneunits on the side chain and was able to transport calcium. The addition of more methylene unitsresulted in a significant increase of calcium transport rate for compounds 28, 29 and 30. In fact,compounds 28-30 are more efficient calcium carriers than compound 5. It appears that additionaloxygen binding sites on the side chain of analogue 5 may not coordinate with calcium.Interestingly, the transport rate of compound 30 was comparable to compounds 28 and29 even though it had more methylene units on the side chain. This may be due to the lowsolubility of compound 30 in the high dielectric constant interface of the chloroform and aqueoussource phase where calcium transport is initiated. Compounds 28 and 29 appears to have optimallipid solubility for calcium transport.Additional evidence pointing to the importance of lipid solubility in calcium transport wasobtained when the concentration of the carrier in the chloroform phase was monitored during thetransport experiments. A small volume (1 mL) of the chloroform solution was withdrawn atdifferent times, the solvent was evaporated and the residue was dissolved in methanol. Themagnitude of the UV absorption at 276 nm of the methanol solution was used to determine thecarrier concentration. We found that the concentration of compounds 28, 29 and 30 in thechloroform phase remained constant throughout the transport experiments. In contrast, theconcentration of compound 27 in the chloroform phase steadily decreased. Using UVspectroscopy the lost compound was found to be in the aqueous source phase. Thus, compounds28, 29 and 30 were efficient carriers for the transport of calcium because these compounds andtheir calcium complexes had sufficient lipid solubility to remain in the chloroform phase.Compound 27 did not transport calcium across the chloroform layer because it was extracted intothe aqueous source phase due to its higher hydrophilicity.The results illustrate the importance of lipophilicity in calcium transport and suggest thatother compounds possessing the I3-diketone and carboxyl groups with different carbon backbonesmay also be efficient calcium carriers if their lipophilicity is similar to compounds 28-30. The34results with compound 27 illustrate the need to consider the partitioning of the carrier between theorganic and the aqueous phase.The partition coefficient (P) of a substance between an organic phase and an aqueousphase is defined as the ratio of equilibrium concentration of the substance in the organic phase tothat in the aqueous phase. 55 The partition coefficient, or its logarithm has been correlated to thevelocity with which a compound penetrates cells, and thus to the biological activity of thecompound.55 Much effort has been made to develop methods to estimate the partition coefficientof a substance based on its structure. Among them Rekker's hydrophobic fragmental constantmethod (f method) is considered to be the most reliable. 56 In this method, log P of a substance isan additive property of the substance and can be calculated using Equation 1, where fi representsthe hydrophobic fragmental constant of the various fragments in the molecule and ai is thefrequency of a given fragment in the molecule.35log P =^+ a2f2 + + anfn (1)Fragmental constants have been derived from the partition coefficients of a large numberof compounds for which reliable experimental data are available. Some fragments and their fvalues in CHC13-H20 calculated by Rekker56 are given in Table IV. The calculated values oflog P for a number of simple 13-diketones and carboxylic acids, together with their experimentalvalues56 , are given in Table V. These results demonstrate the utility of the f method.Table IV. Fragmental Constants for Various Fragments in the Chloroform-Water System. 56Fragment -CH3^-CH2-^-COOH -CH2COCH2COCH3 -CH2COCH2COCH2-fi^0.965^0.628^-2.485^0.90^0.56Table V. Calculated and Measured log P for Simple 13-Diketones and Carboxylic Acids. 56Compound log P (calculated) log P (found)560^01.24 1.40)0&,.)t,1.87 1.75)0L)OL3.00 2.970H0)1%• -0.26 -0.270HO) 0.36 0.34Using the f method, we calculated log P values for compounds 27-30 and 4 (Table VI).The calcium transport rates (J) by these compounds are also listed. Log P values for compounds5 and 6 were not calculated because the f values of their fragments are not available.36Table VI. Calcium Transport Rates (J) and Calculated log P Values for Compounds 4 and 27-30.Compound J (10-8 mole cm-2 h-1 ) log P0^0 0H0-1,-../N.../`..."...)1■....)1%, 0 2.18270^0 0HO 0.3 ± 0.1 4.0640^0 0HO 15 ± 2 5.95280^0 0HO 15 ± 2 7.2029O^0 0HO 13 ± 1 8.4530The data in Table VI suggests that the minimum value of log P for calcium transport isabout 4 and the optimal value of log P for calcium transport is in the range of 6-8. The futuredesign of simple analogues based on ionomycin should have a value of log P in this range.372.4 Effect of Distance between_the 0-Diketone and Carboxyl Groups on Calcium TransportThe studies of analogues with various alkyl side chains produced three simple compounds28, 29 and 30 as efficient calcium carriers. As a result, our efforts to further probe the structuralfeatures controlling the calcium transport of ionomycin and its simple analogues focussed on thesecompounds. The role of the P-diketone and carboxyl groups was investigated first.Compound 31 was prepared using the same synthetic procedures as before. Thiscompound and the commercially available octadecanoic acid (32) were subjected to the transportexperiments separately. No detectable transport of calcium was observed even though the lipidsolubility of these two compounds was sufficiently high compared to compounds 28-30. Thisindicates that both the 13-diketone and carboxyl groups are required for the transport of calcium.0 0310HO32To determine if the presence of the two functional groups within the same molecule isrequired, we performed a transport experiment with both compounds in the chloroform phase.Again no transport of calcium was detected. Therefore, the presence of the 13-diketone andcarboxyl groups within the same molecule is a necessary condition for the transport of calcium.In all the ionomycin analogues studied so far, the number of carbons between the13-diketone and carboxyl groups was the same as that in ionomycin. This was based on theassumption that a seven-carbon chain separating these two functional groups would lead tofavorable calcium transport. The requirement of the presence of the 13-diketone and carboxyl38groups within the same molecule indicates that the distance between the two functional groupsmay be important. We proceeded to study the effect of the distance between these groups oncalcium transport.Compounds 33 and 34 which had the same lipid solubility (log P) as compound 28 buthad the 13-diketone moved two carbons closer to and two carbons further away from the carboxylgroup respectively were synthesized. In addition, compound 35 which had the P-diketone in an'unnatural' position, i.e., the 13-diketone and carboxyl groups were in positions which would notbe expected from normal polyketide biosynthesis, 57 was prepared. The number of methyleneunits (n) between the two functional groups and the calcium transport rate (J) of these compoundsare given in Table VII. A graph of the calcium transport rate (J) versus the number of methyleneunits (n) is shown in Figure 13.Table VII. Number of Methylene Units (n) between the Two Functional Groups and CalciumTransport Rates (J) of Compounds 33, 35, 28 and 34.39Compound^ n^J (10-8 mole cm-2 h-1)O^0 0HO 5^3.5 ± 0.4330HOO 0^ 6^9.5 ± 0.9350^0 0HO 7^15+2280^0 0HO 9^0.9 ± 0.134Figure 13. Graph of calcium transport rate (J) versus number of methylene units (n) separatingthe P-diketone and carboxyl groups.Figure 13 shows that moving the 13-diketone closer to or further away from the carboxylgroup results in a significant decrease of calcium transport rate. The low calcium transport rate ofcompound 34 is likely due to the large unfavorable entropy change associated with the formationof its calcium complex. The first step in calcium transport is the binding of the ionophore withcalcium to form the calcium complex (Section 1.4). As the P-diketone is moved further awayfrom the carboxyl group, more entropy would need to be overcome to bring the two functionalgroups together for the binding and transport of calcium, resulting in a less effective calciumbinding and transport. This trend can be expected to continue until the 13-diketone is so far awayfrom the carboxyl group that the two functional groups act essentially as in separate molecules, inwhich case no transport of calcium would be observed. Our findings on the transport behavior ofcompounds 31 and 32 are illustrative of the entropy effect on calcium binding and transport.On the other hand, as the 13-diketone is moved closer to the carboxyl group, the enthalpyof the calcium complex would increase due to the increasing steric strains (bond angle strain,torsional angle strain and transannular hydrogen interactions) in the chelate rings. This400 07 ..^.7•c#2.*^O 0:.^ 0 071^ 1^.• cal'''^ Ca?'1unfavorable enthalpy may outweigh the more favorable entropy especially when the size of chelatering is reduced from 12-membered to 11-membered or 10--membered as in the calcium complexes36, 37 and 38 of compounds 28, 33 and 35. The enolate of the 13-diketone in the complexescould also increase the enthalpy in the order of 36 < 37 < 38. The seven atoms of the enolate areplanar (bonds between these atoms shown in bold), which, together with the fixed geometry ofthe carboxylate and enolate in the calcium complexes, restricts the number of bonds that canundergo rotations in the chelate rings to nine, eight and seven for complexes 36, 37 and 38respectively. This steric strain would increase in the order of 36 < 37 < 38 and may beresponsible for the transport rate of compounds 28 > 33 > 35.9 736^37^ 38It appears that the competing role of enthalpy and entropy may be minimized when sevenmethylene units separate the two functional groups. It would also appear that ionomycin wasbiosynthesized with the optimal distance between the two functional groups.41J (10-8 mole cm-2 10)Calcium ionophoreHO OH20 00-., N-H0HO12 ± 121 ± 221 Comparison of Ionomycin Analogues to Calcimycin and IonomycinTo compare compounds 28-30 with known calcium ionophores, we carried out thetransport experiments on ionomycin (3) and calcimycin (2). The results are given in Table VIII.Table VIII. Calcium Transport Rates (J) for Calcimycin, Ionomycin and Compounds 28-30.420 030HO280 0 0HO290 0 0HO3015 ± 215 ± 213± 1Calcium transport by ionomycin is about twice as fast as calcimycin at the same molarconcentration. This is in agreement with the transport results obtained for these two ionophores inother transport systems33 and is due to the difference in the mole ratio of the ionophore-calciumcomplex. Ionomycin transports calcium as a 1:1 complex while calcimycin transports calcium as a2:1 complex.Calcium transport by analogues 28, 29 and 30 is slower than ionomycin. The highercalcium transport rate of ionomycin compared to compounds 28-30 may be attributed to thesmaller entropy change associated with the formation of ionomycin-calcium complex. Althoughthe 0-diketone and carboxyl groups in both ionomycin and compounds 28-30 are separated by aseven-carbon chain, there are three methyl substituents on this chain in ionomycin. These threemethyl substituents, with their particular stereochemistry, may destabilize undesired rotomers ofthe acyclic bonds and reduce the number of conformations to those suitable for calciumcomplexation. This would reduce the entropy lost in the formation of calcium complex and thusincrease the calcium transport rate for ionomycin.Nevertheless, the comparative experiment demonstrates the high efficiency of compounds28-30 as carriers for calcium transport across artificial membranes such as a chloroform liquidmembrane.43The comparable calcium transport efficiency of compounds 28-30 to calcimycin andionomycin was very encouraging. However, a useful calcium ionophore should transport calciumboth efficiently and selectively. In particular, it should have selectivity for calcium over sodiumand potassium because of the ubiquitous presence of potassium and sodium ions in biologicalsystems. Calcimycin and ionomycin both have selectivity for calcium over sodium andpotassium. 19,34 Ionomycin is also selective for calcium over magnesium. 34To study the cation selectivity of our compounds, three transport experiments were carriedout on analogue 28. For each experiment, a solution of equimolar concentration of calciumchloride (250 mM) and chloride of the competing cation (magnesium chloride, sodium chlorideand potassium chloride respectively) was placed in the source phase. Samples were withdrawnfrom the receiving phase and analyzed for calcium and the competing cation by atomic absorptionspectroscopy. The results are given in Table IX.0^0 0HO28Table IX. Calcium, Magnesium, Sodium and Potassium Transport Rates for Analogue 28.Competing cations^Ca2+^Mg2+^Ca2+^Na+ (or K+)J (10-8 mole cm-2 h -1 )^2.2 ± 0.2^3.1 ± 0.3^5.5 ± 0.5^0As seen from Table IX, the requirement of cation selectivity for calcium over sodium andpotassium was met in analogue 28. No detectable transport of sodium or potassium wasobserved. Unlike ionomycin, analogue 28 transported magnesium faster than calcium.44The cation selectivity of compound 28 in transport is likely due to the selectivity inbinding which is expected to follow the order of ionic potentials of the cations Mg 2+ > Ca2+ >Na+ > K+. Magnesium ion and calcium ion are both doubly charged and will bind to the enolateof 13-diketone and the carboxylate more strongly than singly charged cations such as Na+ and K+.On the other hand, the smaller magnesium cation (0.65 A) will bind to the enolate of D-diketoneand the carboxylate more strongly than calcium ion (0.99 A).The cation selectivity of ionomycin for calcium over magnesium in transport may be due tothe size-match selectivity58 and/or the coordination of the cations by additional oxygen atomsalong the backbone of the ionophore. Ionomycin may have a limited number of low-energyconformations in which the size of the chelating portion of the molecule matches the size of acalcium ion but not the size of a magnesium ion. The coordination of cations by the additionaloxygen atoms of ionomycin could also be the source of its selectivity for calcium overmagnesium. The magnesium ion is small and its coordination with other oxygen atoms of theionophore would increase steric crowding of the molecule more than the coordination of a largercalcium ion. The less favorable enthalpy in the magnesium complex may lead to the reversedcation selectivity in transport by ionomycin.If one of the goals in the development of calcium ionophores based on ionomycin is tomaintain the cation selectivity for calcium over magnesium in transport, two approaches should beconsidered. One is to design molecules that have the carboxyl and the 13-diketone groups held inthe geometry found in the crystal structure of the calcium salt of ionomycin. 30 The other approachwould be to introduce additional oxygen atoms on appropriate positions into compounds such as28. The oxygen atoms should be on the hydrocarbon chain separating the carboxyl andI3-diketone groups in an effort to ensure that the coordination of magnesium ions is less favorable.450^44 86 128 170 212 254 296 3372,1 Calcium Transport in Cultured Human Leukemic Cells Analogues 6, 27, 28, 29 and 33 were submitted to Merck Frosst Canada for in vivotesting of calcium transport in cultured human leukemic cells (THP-1 cells). Intracellular calcium,[Ca2-1i, was determined using a calcium-specific fluorescent probe. 59 The calcium ionophorescalcimycin bromide and ionomycin were used as positive controls. Cumulative dose-responseswere obtained and the cell fluorescence was monitored continuously for 3-5 minutes. Data fromtwo separate experiments, one for positive control and the other for our analogues, are shown inFigure 14 - 18. Each curve represents the mean value of 2-3 determinations.Time (second)0^0 0Calcimycin bromideFigure 14. [Ca2li in THP-1 cell stimulated by calcimycin bromide and analogue 6.46[norm I1 10 MAcdIonomycinTime (second)0^0 0110)k■■■./■./%%A..#1C.Figure 15. [Ca2li in THP-1 cell stimulated by ionomycin and analogue 27.300250200-100 NA150100!TIM IIII7"" re.5" "'"^1 iö 212 254III -MITI 111Time (second)0^0 047Calcimycin bromide^"I" HOFigure 16. [Ca2-1 ]i in THP-1 cell stimulated by calcimycin bromide and analogue 28.Time (second)0^0 04- Ionomycin^+ HOFigure 17. [Ca24 ]i in THP-1 cell stimulated by ionomycin and analogue 29.300^250-86 128 1 0 212 254Time (second)0 0 04 Calcimycin bromide^+ HO^50 , 11111,1i^11111171^111111  ^-UTTTT11-1-1TTTIIT.I.FITId^44^'8'4 •128^170^21248Figure 18. [Ca2+]i in THP-1 cell stimulated by calcimycin bromide and analogue 33.As seen from Figure 14-18, the resting [Ca2÷Ji in THP-1 cells was in the range of 60-110nM, in line with observations in other cell types. 13 The THP-1 cells responded to eithercalcimycin bromide or ionomycin stimulation with large (up to 10-fold) and sustained increases in[Ca21-ii in a dose-dependent manner. Only analogues 29 and 33 (Figure 17 & 18) induced a clearresponse at or greater than 111M. These responses were transient and were significantly smallerthan those of calcimycin bromide or ionomycin. Analogues 6 and 27 did not transport calciumacross the membrane of THP-1 cell. This result is in agreement with those of the transportexperiments using chloroform as the artificial membrane and is likely due to the low lipophilicityof the compounds in the cell membrane.The transient and smaller responses induced by analogues 29 and 33 relative to the naturalionophores may be due to the lower chemical stability of the former compounds in the cellmembranes. The analogues might undergo relatively rapid transformation such astransesterification in the cell membranes, which would result in the need for more compound andthe transient calcium transport. It is also possible that the binding of the analogues with calcium istoo low to allow the complexation and the transport of calcium at the given extracellularconcentrations.Surprisingly, no calcium transport in THP-1 cells was observed for analogue 28 whichshowed efficient calcium transport across a chloroform phase. The chemical stability of theanalogues in biological membranes and its effect on calcium transport remained to be investigated.One correlation can be made between the cylindrical glass cell experiments and the in vivoexperiments. Compounds that show no calcium transport in the cylindrical glass cell would nottransport calcium across biological membranes. The cylindrical glass cell experiments can be usedto eliminate compounds from in vivo testing.49La Effect of Substrate Concentration on Calcium TransportTo gain insight into the transport behavior of the analogues, we next investigated the effectof substrate concentration and pH on calcium transport. The first step in the transport of calciumacross the chloroform phase is the binding of the ionophore to calcium at the interface of thechloroform and the aqueous source phase. This is followed by the diffusion of the calciumcomplex across the chloroform phase and the release of calcium into the aqueous receiving phase.If the binding of calcium is reversible, the ionophore-catalyzed calcium transport would showsaturation kinetics which could be treated in the same fashion as enzyme kinetics. 60In the case of an enzyme-catalyzed reaction, the substrate (S) binds reversibly to theenzyme (E) to form an enzyme-substrate complex (ES) which then reacts to give the product (P)with the regeneration of the free enzyme. This kinetic behavior was first described by Michaelisand Menten under equilibrium conditions 61 and later extended by Briggs and Haldane with thesteady-state approximation (Figure 19).625 0k 1E + S ---i". ESk2E + P k_ 1V =Vmax[S]KM + [5]Figure 19. The Michaelis-Menten expression for enzyme catalysis.This is formally the same as the ionophore (L) and calcium (Ca) forming an ionophore-calcium complex (CaL) which then diffuses across the membrane and releases calcium with theregeneration of the ionophore. The transport rate (J) can be related to the maximal rate (J max)through the Michealis-Menten equation (Equation 2) if the binding of calcium is reversible(Figure 20).L2- + Ca2+ k1k.. 1CaLk2 L2- + Ca2+51J=KM + [CallFigure 20. The Michaelis-Menten expression for ionophore-catalyzed calcium transport.The thermodynamic parameter KM represents (k2 + k_i)/ki. It is approximately equal tothe equilibrium dissociation constant of the enzyme-substrate complex or the ionophore-calciumcomplex if k2 << k_1 (the equilibrium assumption 61 ).The transport experiments were carried out on analogue 28 with various concentrations ofcalcium chloride in the source phase. The results are summarized in Table X.0^0 0HO28Table X. Calcium Transport Rates (J) at Various Calcium Concentrations in the Source Phase.[Ca2-1 (mM)^10^50^100^250^500^1000J (10-8 mole cm-2 10)^0.6 ± 0.1^4.4± 0.4^5.9± 0.6^13± 1^15± 2 16± 2When we fitted the transport rates (J) and substrate concentration ([Ca 2+]) into theMichaelis-Menten equation (Equation 2) by non-linear regression, we obtained a hyperbolic curve(Figure 21) with a standard error of less than 10%, which could be taken as confirmation of theadherence to the Michaelis-Menten kinetics 63Jmax [Ca2+]( 2 )0.20 ^0.40^0:60 ^0.6 1.00^1.20 10 310 11.801.601.401.201.004) 0.80000^0.60O0.400.200.000.00[Call (mM)Figure 21. Curve of the dependence of transport rates on calcium concentration of the sourcephase. The points represent the experimental values, the solid line is fitted to the data by non-linear regression analysis according to Equation 2.Therefore, calcium transport across the chloroform layer by analogue 28 is a saturableprocess which obeys the Michaelis-Menten kinetics. The values of the Michaelis-Menten constantKM and the maximal transport rate Jmax were found to be KM = 170 mM and J max = 20 x 10 -8mole cm-2 h-1 respectively by the above non-linear regression analysis. 63 The large KM indicatesthe binding of calcium by analogue 28 is weak (k..1 >> ki) and/or the diffusion of the analogue-calcium complex and the release of calcium is very fast compared to the binding of calcium(k2 >> k1).The rate limiting step in the transport of calcium by analogue 28 is likely the diffusion ofthe calcium complex across the chloroform layer. This could be verified by the studies of thedependence of calcium transport on the stirring speed.27e522,2 Effect of pH on Calcium TransportThe exchange of calcium ions from the ionophore-calcium complex (CaL) in thechloroform layer for protons from the aqueous receiving phase is the final step of the ionophore-catalyzed transport cycle. If this process is irreversible as suggested by the Michealis-Mentenkinetics, a small change in the concentration of hydrogen ions in the receiving phase should notsignificantly affect the transport rate. The previous transport experiments were carried out withthe aqueous receiving phase buffered at pH = 7.0.The transport experiments were carried out on analogue 28 with various pH in thereceiving phase. The pH of the aqueous source phase was buffered at pH = 9.5 as before and theresults are given in Table XI.0^0 0HO28Table XI. Calcium Transport Rates (J) at Various pH in the Receiving Phase.pH 8.0 7.0 6.0 5.0J (10-8 mole cm-2 h-1 ) 15 ± 2 16 ± 2 16 ± 2 15 ± 2As seen from Table XI, the calcium transport rate is essentially independent of the pH ofthe receiving phase in the pH range of 5.0-8.0. Thus, the exchange of calcium ions for protons,i.e., the release of calcium ions from the ionophore-calcium complex (a Michaelis complex), canbe treated as an irreversible process in this pH range, or at pH = 7.0 at least.53The presence of the carboxyl and 13-diketone groups in the same molecule has been shownto be necessary for calcium transport (section 2.4). However, it was not clear if ionization of theii-diketone is required for transport. The role of the enolate of the f3-diketone in the transport ofcalcium was investigated by varying the pH in the aqueous source phase above pH = 7.The transport experiments were carried out on analogue 5 with various pH's in the sourcephase. The concentration of the analogue in the chloroform layer was 300 i_LNI and the pH of thereceiving phase was buffered at pH = 6.5. The results are summarized in Table XII (no detectabletransport of calcium was found when the source phase was buffered at pH < 7.0).0^0 054HO r-N W.0 0^05Table XII. Calcium Transport Rates (J) at Various pH in the Source Phase.pH^7.5^8.0^8.5^9.0^9.5^10.0J(10-8 mole cm-2 h-1 )^1.3 ± 0.1 1.5 ± 0.1^2.5 ± 0.2^4.6 ± 0.4^9.0 ± 0.9 14+1The logarithm of calcium transport rate (log J) is linear with the pH in the source phase(Figure 22). Thus, transport of calcium by analogue 5 is directly dependent on the hydroxideconcentration in the source phase. The dependence of calcium transport on the hydroxideconcentration in the source phase suggests that the formation of the carboxylate and/or the enolateof the P-diketone of analogue 5 is required for the transport of calcium.1.6 41.4 11.21 .010.810.6)0.40.21007.0 8.0 9.0 10.0 11.055pHFigure 22. Plot of log J as a function of pH in the source phase. The points represent theexperimental values.Non-linear regression analysis of the transport rate data versus pH using Equation 3derived from the Henderson-Hasselbalch equationM gave a typical plot (Figure 23) for theionization of a monoprotic acid with a pKa of 9.6 ± 0.1.Jmin^imax X 10(PH - pKa)= ( 3 )10(1^pKa)^141 In their studies of competitive alkaline-earth cations extraction from aqueous solution intochloroform by polyether dicarboxylic acids, Kang et al. demonstrated that the carboxyl groups ofthe alkanoic acids were deprotonated completely at the interface of the aqueous solution andchloroform at pH (pH in the aqueous phase) lower than 7.0 and that the resulting dicarboxylatesextracted divalent cations as neutral complexes into the chloroform phase. 65a 5620.016.012.08.04.0ENOti0.0 ^, 7.0^8.0^9.0^10.0pH•11.0 12.0Figure 23. Plot of the dependence of transport rates on pH of the source phase. The pointsrepresent the experimental values and the solid line is determined by non-linear regression analysisaccording to Equation 3.Based on the results reported by Kang et al., the pK a of 9.6 was assigned to the pKa ofthe 0-diketone of analogue 5. Such a dependence of transport rate on pH of the source phaseindicated that the ionization of the D-diketone at the interface of the aqueous source phase andchloroform was required for calcium transport. The transport rate (section 2.4) and the pK a (seelater) depend on the distance between the 0-diketone group and the carboxyl group. We thereforeassociate this pKa with the ionization of the 13-diketone.The formation of the carboxylate and the enolate of the 13-diketone at the interface of theaqueous source phase and chloroform in the transport of calcium, the requirement of both thecarboxyl and 13-diketone groups within the same molecule for calcium transport (section 2.4) andthe requirement of electrical neutrality of the calcium complex in chloroform 65a indicates that thestoichiometry of the calcium complex involved in transport is 1:1.280HOU...0 0^ 02.10 Characterization of Calcium Complex of Analogue 28Further evidence for the formation and involvement of the neutral 1:1 calcium complex incalcium transport could be obtained by determining the stoichiometry of the calcium complex inthe solid state. To this end, the calcium salt of analogue 28 was isolated by reaction of theanalogue with calcium hydroxide.5736The UV spectrum of the calcium salt 36 in methanol showed an absorbance at 293 nm,indicating the presence of the enolate of the f3-diketone. When hydrochloric acid was added to thesolution of the calcium salt in methanol, the absorbance maximum was shifted from 293 nm to276 nm, indicating the generation of the enol form of the 13-diketone from the enolate. Ionizationof the (3-diketone group in the formation of calcium salt was thus confirmed.o o-.f)L•= 293 nm H0 sOf)L'LfXmax = 276 nmThe solid state IR spectrum of analogue 28 showed absorptions at 1719 cm -1 due to thefree acid, and at 1688 and 1642 cm -1 due to the ketone and the enol of the 13-diketone respectively.In contrast, the solid state IR spectrum of the calcium salt 36 had absorptions at 1577 and 1439cm-1 ascribable to the C=0 asymmetric and symmetric stretches of the carboxylate respectively,which indicated that both the carboxylate oxygens were coordinated to the calcium ions.65b Theabsorption at 1511 cm-1 could be assigned to the enolate of the P-diketone. The calcium salt also0H\0 0 00 0had weak absorptions at 3644 and 3527 cm-1 assigned to the O-H stretching absorptions of watermolecules perhaps bound to the calcium ions. Finally, it had a peak at 711 cm -1 assigned to Ca-0stretching. Thus, the formation of the enolate of the P-diketone group and the coordination ofcalcium with both the carboxylate oxygens were established from the IR. The presence of watermolecules in the complex was suggested from the IR.The 1H NMR spectrum of 28 in Me0H-d4 showed two sets of triplets at 2.52 and 2.51p.p.m., which were assigned to two sets of methylene protons Ha and Hb of the keto form of thep-diketone. Three sets of partially overlapped triplets at 2.30, 2.29 and 2.27 p.p.m. wereassigned to the methylene protons H e and Hd of the enol form of the 13-diketone and to themethylene protons He in both tautomers. The ratio of the enol tautomer to the keto tautomer was7:3 as indicated by the relative intensity of H e (or Hd) to Ha (or Hb).58HO HOThe 1 H NMR spectrum of the calcium salt 36 in Me0H-d4 showed a broad peak at 2.15p.p.m. and a less intense broad peak at 2.51 p.p.m. The peak at 2.15 p.p.m. could be assignedto the methylene protons He , alpha to the enolate anion of the P-diketone in the Z -geometry (78%by integration) and the methylene protons He , alpha to the carboxylate of the complex. Theupfield shifts of these methylene protons resulted from the formation of the enolate andcarboxylate. The less intense peak at 2.51 p.p.m. was assigned to the methylene protons (I -Ic")alpha to the enolate anion of the "free" ligand in the E -geometry (22%). The protons (H e-) of thetwo methylene groups had an averaged chemical shift at 2.51 p.p.m. presumbably due to the fastequilibrium between the two E -geometries as shown below.8 2.5159Z -geometry%He0^-**-- 8 2.51ft8 2.15^8 2.51E -geometry E -geometryThe 1H NMR spectra of alkali metal acetylacetonates in Me0H-d4 at -57 °C were studiedby Raban and coworkers.66a The methyl region of the 1 H NMR spectrum of sodiumacetylacetonate showed three singlets, two of which were of equal intensity. The singlet whichappeared at higher field (8 1.80) was assigned to the methyl protons of the complex in theZ -geometry (23%). The two equally intense singlets at lower field (8 1.89, 2.27) were assignedto the methyl protons of the free ligand in the E -geometry (77%).66a Based on this analogy, wesuggest that the species with the 8 2.51 1 H NMR signal is the "free" ligand. However, calciumions may be bound to the carboxylate (Figure 24) and/or the enolate of the 13-diketone in the •E -geometry.Na+0 0-^8 2.27+ Na+8 1.80 f^T 8 1.80^8 1.89 tZ -geometry E -geometry"free" enolate of the p-diketoneRaban and coworkers demonstrated that this dissociation was strongly dependent on thenature of the cation and represented a measurement of the ability of the acetylacetonate anion tocomplex with a cation.66a For example, the binding constant of acetylacetonate anion with Li+ is6.0 x 102 M-1 and lithium acetylacetonate was found to exist only as lithium complex insolution.66a,b The binding constant of acetylacetonate with Na+ is 4.0 x 10 M -1 and sodiumacetylacetonate existed predominantly as the dissociated enolate anion (77%). 66abThe presence of the "free" enolate anion in the solution NMR of the calcium complex 36indicated the existence of a similar equilibrium in Me0H-d4 (Figure 24). Additional evidence forsuch an equilibrium was obtained from temperature-dependent 1H NMR studies. The percentageof the "free" enolate anion changed from 15% to 22% and 24% when the temperature wasincreased from -25 °C to 25 °C and 45 °C respectively. Because the percentage of the "free"enolate anion from the calcium complex 36 was lower than that of sodium acetylacetonate andhigher than that of the lithium acetylacetonate, we predicted the binding constant of analogue 28with calcium to be in the range of 4.0 x 10 to 6.0 x 102 M-1 in Me0H, which was significantlylower than the binding constant of ionomycin with calcium (1.9 x 106 M-1 in 80% Me0H-H20).32calcium complex60Figure 24. Equilibrium between the calcium complex and the free ions in Me0H-d4.The mass spectrum of the calcium salt was obtained using fast atom bombardment (FAB)ionization technique. The prominent ion peaks at m/z = 388, 351 and 313 were assigned to theions [CaL•2H20 + H + H]+, [CaL + Hr and [H2L + H]+ where H2L represents analogue 28 andCaL represents the 1:1 calcium complex. The formation of 1:1 analogue-calcium complex wasthus confirmed. The presence of two molecules of water in the complex was likely but notconclusive .Assuming two molecules of water are indeed present in the 1:1 calcium complex and thecalcium ion has its usual six coordinations, we suggest structure 39 for the calcium salt ofanalogue 28. In this structure both oxygens of the carboxylate are involved in complexation.0::: .0: 02H0 -039Efforts to obtain a crystal of the calcium salt suitable for X-ray crystal structure analysiswere unsuccessful. However, the preparation and characterization of the calcium salt of analogue28 demonstrated that both the f3-diketone and the carboxyl groups were ionized in the formationof the calcium salt and the stoichiometry of the complex was 1:1. It also showed that the bindingconstant of analogue 28 with calcium was 4-5 orders of magnitude lower than that of ionomycinwith calcium.612.11 Binding of Ionornycin Analogues with Calcium IonsThe second phase of this project was to investigate the binding of ionomycin analogueswith calcium and other metal ions and determine if there was any correlation between calciumbinding and transport. The determination of binding constants of the analogues with calcium, thestoichiometry and cation selectivity in binding might also provide insight into the structuralfeatures controlling the calcium specificity of ionomycin.To systematically study the binding of the analogues with metal ions, we needed todevelop a general experimental procedure. There are many methods to determine the bindingconstant between an ionophore and a metal cation. 67 The most widely used experimentalapproach is by spectrophotometric titration. In this method, a solution of the ionophore is titratedwith the metal cation of interest while its absorbance is monitored as a function of the cationconcentration. The essential requirement for the use of this method is that a significant spectralchange occurs on the formation of the ionophore-cation complex. 67In many cases the stoichiometry of the ionophore complex and the binding constant maybe determined by the mole ratio method. 68 In this method the total ionophore concentration (Lt) isheld constant and the cation concentration (Mt) is varied. A plot of the maximal absorbancedifference at a chosen wavelength versus the ratio of ionophore and cation is examined fordiscontinuities or abrupt changes of slope corresponding to the stoichiometric ratio. Suppose thebinding constant 13 = 00 in such a system and for each m moles of the ionophore (L) in thesolution, addition of n moles of the cation (M) resulted in the formation of exactly one mole ofLmMn. The concentration of complex and thus the maximal absorbance difference will increaseuntil Lt/Mt = m/n; beyond this value no more complex can form because no more ionophore isavailable. Thus a break in the plot will be seen at this point (Figure 25, line A). 68b As thebinding constant decreases the extent of the complex dissociation increases, so the break becomesa gently rounded curve (Figure 25, lines B and C). 68b The discontinuity is then located by62extrapolation of the linear segments over considerable ranges. Obviously, the lower the bindingconstant is, the less accurate the determination of the stoichiometry will be.minMole ratio of ionophore and cation L t/M tFigure 25. Representative mole ratio plots of complexes of various stabilities. Line A is typicalof a relatively completely formed complex. Lines B and C are typical of successively weakercomplexes.68The other common method used in the determination of the stoichiometry in binding is thecontinuous variation method, often called Job's method. 69 In this method, a series of ionophoreand cation solutions having identical total molar concentrations but different mole fractions of thetwo components are prepared. If a single, strong complex is formed, a plot of the maximalabsorbance difference versus mole fraction of the solution gives a characteristic triangular plot(Figure 26, line A).67 The mole fraction of the maximum of this plot, the apex of the triangle,indicates the stoichiometry of the complex. However, if a weak complex is formed, a curved plotresults (Figure 26, line B and C).67 The stoichiometry of the complex can be obtained from thepoint of intersection of the tangents to the curves. Again, the lower the binding constant, the lessaccurate the determination of the stoichiometry will be.630.2^04^0.6^08Mole ratio of solutionFigure 26. Continuous variation curves plotted for hypothetical systems with a stoichiometry of1:1 and binding constant (from line A to line C) = 00, 103, 102.Since the extent of curvature in both the mole ratio and the continuous variation methodsdepends on the binding constant, it is possible to extract an estimate of the binding constant fromthe same data used to obtain the stoichiometric ratio. Many authors have described ways to dothis.67,69 For example, the extinction coefficients of the ionophore and the complex can bedetermined (the former from the absorbance in the absence of the metal cation and the latter fromthe absorbance at complete complexation). These extinction coefficients are then used, inconjunction with the known absorbance of the mixture, to calculate the concentration of thecomplex at each titration point, from which the concentration of the free ligand and free cation iscalculated. The stoichiometry determined from either the mole ratio or the continuous variationmethod is used to define the equation of the binding constant. The values of the concentrations ofthe complex, free ligand and cation are used in this equation to calculate the binding constant ateach titration point. The consistency of the calculated binding constants would indicate theaccuracy of the stoichiometry and the binding constant itself. 7064A more accurate method to deduce the binding constant is to fit the titration data, aftercertain manipulation, into the Scatchard equation by non-linear regression computer softwareprograms 63,71 Both the binding constant and the stoichiometry can be obtained from such ananalysis. Again, the accuracy of the calculation depends on the magnitude of the binding constantbecause the technique was developed to study complexes with high binding constants, such asthose between enzymes and substrates. 67In principle, the UV spectrophotometric titration method is applicable to the binding of ouranalogues because the UV absorption of the enol form of the 13-diketone group is shifted to longerwavelength upon complexation with cations. However, the mole ratio and the continuousvariation methods may not be suitable for the determination of the binding constants of theanalogues with calcium which, based on the transport results and the 1H NMR data of the calciumsalt, may be small. If the transport selectivity is due to the selectivity in binding, we would expectthe binding constant of the analogues with sodium and potassium to be even smaller than withcalcium. Examination of cation selectivity using the mole ratio or the continuous variationmethods would then be impossible.Clearly, a method for the determination of small binding constants is needed in ourstudies. The measurement of the binding constant of the enolate of a 13-diketone with a cation canbe carried out by measuring the difference of pKa of the 13-diketone group in the absence and thepresence of a large excess of metal ions. Let HL - represent a 13-diketo co-carboxylate, thedissociation of the enol form of a P-diketone group to the enolate of the 0-diketone (L 2-) andproton (H+) is given by Equation 4. The acid dissociation constant K a is given by Equation 5.6566HL^+^(4)[L2-] (5)Ka — [Hu]The binding of the enolate L2- with a divalent cation such as calcium ion is given byEquation 6 and the binding constant 13 by Equation 7, assuming a 1:1 stoichiometry in binding.L2- + Ca2+ ^ CaL^(6)[Cal.]=^ (7)[Ca2+][01In the presence of a large excess of calcium ions, the dissociation of the I3 -diketoco-carboxylate is given by Equation 8. The acid dissociation constant K a, can thus be defined byEquation 9 which is also applicable to the determination of the binding constant of the enolate L 2-with monovalent cations such as potassium and sodium ions under the assumption that thestoichiometry of the potassium (or sodium) complex is 1:2.+ Ca2+^H+ + CaL^(8)= ^a[CaL]^[CaL] ^[H+] [L21[HL-] [Ca21^[Ca2-1[L2-]^[1-11-1= Kalog 13 = PKa - pKa,^(9)^, ^ .268.8^288.8 388.8Wavelength (nm)328.8 348.82.12 Determination of pKa of 0-Diketones by Ultraviolet Spectrophotometric TitrationMeasurement of the pKa of the p-diketone group of the analogues by UVspectrophotometric titration is based on the different maximal absorptions of the enol form of theP-diketone and its conjugated base, the enolate of the 13-diketone. Figure 27 shows changes in theUV absorption spectra of analogue 33 in 80% Me0H-H20 as the pH of the solution is raisedfrom 5.80 to 13.0 by addition of tetramethylammonium hydroxide (Me4NOH). As the pH isincreased the concentration of the enolate rises. The absorption peak shifts to longer wavelengthand increases in magnitude. A clean isosbestic point is observed at 278 nm. The same generalbehavior was observed for all the analogues studied.0^0 0HO3367Figure 27. UV spectrophotometric absorption spectra of analogue 33 as the pH of the solutionincreased from 5.80 to 13.00.The absorbance at 298 nm (ascribable to the enolate of the 0-cliketone) versus pH wasfitted to a modified Henderson-Hasselbalch equation (3) by non-linear regression to calculate thepKa value.63 A pKa of 11.16 ± 0.02 was obtained. The curve calculated by such non-linearregression method is shown in Figure 28.Amin + Amax x 10(pH - pKa)A= ^pKa) + 168( 3 )1.601.501.401.301.201.10 •1.000.900.800.70 -0.60-0.50.0.40-0.30-■0.2C .^^r ^.5 0^6.0^7.0^8.0^9.0^10.0 ^11.0^12.0^13.0pHFigure 28. Plot of absorbance at 298 nm versus pH for analogue 33. The points represent theexperimental values and the solid line is determined by non-linear regression using Equation 3.Using this technique, we determined the pKa of the 13-diketone group of a number ofanalogues (Table XIII). The pKa values of the 13-diketone group of ionomycin and of4,6-nonanedione (40) were taken from the literature. 32Table XIII. pKa of the 0-Diketone Group of Ionomycin and of Analogues.Analogue^ pKa (± 0.02)Ionomycin^ 11.94690^0 0Ho)L.L.A./■/40 0 0HO290 0 0HO300 0 0HO330 0 0HO28O 0 0HO340 031JUL./..4010.9211.0411.0711.1611.0310.9010.8610.78As seen from Table XIII, analogues with higher lipid solubility have higher pK a values.Since binding of metal ions generally follows the same order as the binding of protons, a higherpKa would imply a stronger cation binding by the ligand. The solvent used in the pKadetermination (80% Me0H-H20) has been shown to mimic the interface of water andphospholipid. 32 The increasing lipid solubility of the analogue by the addition of methylene unitson the hydrocarbon side chain not only enhances the partition of cation complex into thechloroform phase but also result in stronger cation binding.Moving the 13-diketone group closer to the carboxylate results in an increase of its pK avalue. This is likely due to the formation of a less stable enolate anion with the increasing chargerepulsion between the enolate anion and the carboxylate as the P-diketone group is moved closerto the carboxylate.The pKa value of the 3-diketone group of ionomycin is significantly higher than that of theI3-diketone group of the analogues. We suspect that the higher pKa of ionomycin is due to thelower stability of its dianion in polar solvent such as 80% Me0H-H20. Ionomycin has severalalkyl groups which could destabilize its anion in a polar solvent. A similar hydrophobic effect canbe seen by comparing analogues 4, 29 and 30. Based on these pKa differences, the binding ofcalcium and other metal ions by synthetic analogues of ionomycin is expected to be weaker thanthat of ionomycin.702.13 Binding of Calcium and Other Metal Ions by lonomycin AnaloguesThe binding of calcium and other metal ions by ionomycin analogues was determined bymeasuring the pKa' values of the 13-diketone in the presence of a large excess of calcium or othermetal ion and applying Equation 9.log 0 = pKa - pKa .^ (9)For example, the UV spectrophotometric titration of analogue 33 was carried out in thepresence of a large excess of calcium chloride (molar ratio of calcium to analogue > 200).Determination of pKa' of analogue 33 was performed in the same way as in the determination ofpKa. A pKa' value of 8.69 ± 0.02 was obtained from which the binding constant of analogue 33with calcium was calculated to be [3 (Ca2+) = (3.0 ± 0.2) x 102 M-1 .0^0 0HO33In a same manner, the binding constants of analogue 33 with magnesium, potassium andsodium were determined as were the binding constants of other analogues with calcium,magnesium, potassium and sodium. The results are given in Table XIV and Table XV. Thebinding constants of ionomycin with calcium and magnesium ions were taken from theliterature.3271Table XIV. Binding Constants of Ionomycin 32 and Analogues with Calcium and Magnesium.Analogue^ 13 (Ca2+)^0 (vig2+)x102 M- 1^x103 M-1Ionomycin^ 1.9 x 104^8.9 x 1030 0 0HO 1.7 ± 0.1 4.4 ± 0.240 0 0HO 2.2 ± 0.2 5.6 ± 0.2290 0 0HO 2.0 ± 0.1 5.7 ± 0.3300 0 0HO 3.0 ± 0.2 7.6 ± 0.433O 0 0HO 1.8 ± 0.1 3.4 ± 0.228O 0 0HO 1.2±0.1 1.9±0.1347 2Table XV. Binding Constants of Analogues with Potassium and Sodium.Analogue^ (K+) N4-2^(Na+) A4-20HO0HO0HO0HOOHOOHOO 04O 029O 0300 033O 0280 0340.8 ± 0.1^1.0 ± 0.10.8 ± 0.1^1.2 ± 0.10.8 ± 0.1^1.1 ± 0.11.3 ± 0.1^1.8 ± 0.10.9 ± 0.1^1.2 ± 0.10.9 ± 0.1^0.8 ± 0.1As seen from Tables XIV and XV, binding of the metal ions follows the same trend,which is Mg2+ > Ca2+ >> Na+ > K+, for all the analogues studied. The ionomycin analogueswere selective for divalent cations over monovalent cation in binding and were selective formagnesium over calcium. Thus the cation transport selectivity results from the cation bindingselectivity.The binding constants of analogues with different cations correlates with the pKa values ofthe 13-diketone group. This is especially evident for analogues with same lipid solubility but with73the 13-diketone group at different positions. Moving the 13-diketone group closer to the carboxylgroup increases the pKa of the (3-diketone group and its binding with cations in the order, 33 >28 > 34.The binding constant of analogue 28 with calcium 13 (Ca2+) = (1.8 ± 0.1) x 102 M-1 isconsistent with the binding constant determined from the 1H NMR data of the calcium complex ofanalogue 28 r3 = 0.4 - 6.0 x 102 M-1 (Section 2.10). Ionomycin binds to calcium and magnesium3-4 orders of magnitude stronger than do the analogues. This is likely due to the fact thationomycin has a limited number of conformations available compared to the analogues andconsequently the loss of entropy in binding of ionomycin is significantly less than the loss ofentropy in binding of the analogues.In contrast to the mole ratio or the continuous variation method, the pK a and pKa, methodpermits the determination of small binding constants such as those of the analogues withpotassium and sodium. However, this method assumes a certain stoichiometry in binding and cannot be used to determine the stoichiometry of the complex. Because the binding constants of theanalogues with magnesium ions are significantly higher, it may be possible to determine thestoichiometry of magnesium binding using the mole ratio method. The results from the mole ratiomethod would test the accuracy of the pKa and pKa' method. To this end, a solution of analogue33 in 80% McOH-H20 was buffered at pH = 9.10 and titrated with a solution of magnesiumchloride. The pH of the solution was chosen so as not to induce significant ionization of the13-diketone group of the analogue but to force complete ionization of the (3-diketone group whensufficient magnesium ions were added, which was clearly demonstrated in the measurements ofpKa and pKa.(Mg2+) for the analogue.0 0 0HO7433: , 53 . 8280.HWavelength (nm)260.0 320.0 340.0Figure 29 shows changes in the UV absorption spectra of analogue 33 as theconcentration of magnesium chloride was increased from 0.00 to 1.34 x 10 -3 M. As theconcentration of magnesium chloride increased the concentration of the enolate rose. Theabsorption peak shifted to a longer wavelength and increased slightly in magnitude.Figure 29. UV spectrophotometric absorption spectra of analogue 33 as the concentration ofMgC12 increased from 0.00 to 1.34 x 10 -3 M at pH = 9.10.From the absorbance at 298 nm in the absence of magnesium ion (A0), the absorbance atcomplete complexation (A max) and the absorbance at each titration point (A), the concentration ofthe magnesium complex at equilibrium [Mg xLy] was calculated using Equation 10. From the totalconcentrations of the analogue [L]t and magnesium [Mg] t and the concentration of magnesiumcomplex at each titration point, the concentrations of free analogue [L] and free magnesium [Mg]were calculated respectively based on mass balances. Assuming the stoichiometry in binding wasn = x/y = 1/1, the binding constant (mg2+) could be calculated using Equation 11. TableXVI shows the results of these calculations.75(A - AO MtfMgxLy]Amax - Ao (10)13 1 : 1 (mg2+) _ [MgL][Mg2+] [1.21Table XVI. Total Concentrations of Analogue 33 and Magnesium, Absorbance, Concentrationsof the Complex, Analogue and Magnesium at Each Titration Point and Binding Constants Basedon 1:1 Stoichiometry.[Litx 10-4 M[Mg2-1-] tX 104 MA298 [Mgxli]x 10-4 M[Mg2+]x 10-4 M[L]x 1 0-4 m0 1 :1x 1030.80 0.00 0.384 0.080.79 0.50 0.608 0.17 0.33 0.62 8.30.79 0.98 0.741 0.27 0.71 0.52 7.30.78 1.47 0.853 0.35 1.12 0.43 7.20.78 2.68 1.032 0.48 2.20 0.30 7.20.78 3.88 1.132 0.56 3.32 0.22 7.70.77 6.28 1.246 0.63 5.65 0.14 8.00.77 8.65 1.302 0.68 7.97 0.09 9.4The calculated binding constants were consistent, which proved that the stoichiometry ofthe complex was 1:1. The average value of the binding constant of analogue 33 with magnesium76was (3 (Mg2+) = (7.9 ± 0.5) x 103 M -1 which was essentially the same as the value of 13 (Mg 2+) =(7.6 ± 0.4) x 103 M-1 obtained using the pKa and pKa' method.Alternatively, and more accurately, data of the number of moles of substrate (magnesium)bound per mole of ligand (analogue) v = [Mg xLy]/[L]t versus the concentration of substrate(magnesium) [S] = [Mg2+] were fitted to the Scatchard equation (11) by non-linearregression.63,71 The curve so obtained is shown in Figure 30. The binding constant of theanalogue with magnesium f3 (Mg2+) = 1/K.d was calculated to be (3 = (7.1 ± 0.1) x 10 3 M-1 andthe stoichiometry of binding was n = x/y = 1.0 by this non-linear regression method. 63v = n - Kd v/[S]771.000.900.800.700.600.500b1:1 .400.300.200.100.0010 10.00^0.20^0.40^0.60^0.80^1 .00^1.20[Mg21 x 10-4 (M)Figure 30. Plot of the number of moles of magnesium bound per mole of analogue 33 versusconcentration of magnesium. The points represent the experimental values and the solid line isdetermined by non-linear regression using Equation 11.Using the mole ratio method and the non-linear regression data treatments, we determinedthe binding constants of the other analogues with magnesium and the stoichiometry of binding.The stoichiometry in binding for all the analogues studied was 1:1. The binding constantsobtained from the pKa and pKa. method and the mole ratio method are given in Table XVII.Table XVII. Binding Constants of Analogues with Magnesium Using the pK a and pKa' Method(Method I) and the Mole Ratio Method (Method II).Analogue^ p, (Mg2+)^13 (Mg2+)X 103 M-1^x103 M- 1(method I)^(method II)0HO0HO0HO0HOOHOOHOO 040 029O 03 00 033O 0280 04.4 ± 0.2^5.5 ± 0.15.6 ± 0.2^6.2 ± 0.15.7 ± 0.3^6.2 ± 0.17.6 ± 0.4^7.1 ± 0.13.4 ± 0.2^5.6 ± 0.11.9 ± 0.1^4.5 ± 0.1347 8The binding constants of the analogues with magnesium determined by these two methodsare consistent. Therefore, the assumption of the stoichiometry of the calcium (or magnesium)complex and the sodium (or potassium) complex in the pK a and pKa' method is correct. Thebinding constants of the analogues with calcium ions determined by the pK a and pKa' could becompared with the binding constants of ionomycin and other calcium ionophores with calciumdetermined by the mole ratio method.All the experimental results, i.e., the stoichiometry of magnesium binding, the 1:1stoichiometry in the isolated calcium salt, the requirement of the presence of the carboxyl and f3-diketone groups within the same molecule and the formation of both the carboxylate and enolate ofthe 0-diketone in transport, suggested that the calcium complex had a 1:1 stoichiometry in bindingand transport.Because the binding of calcium by synthetic analogues of ionomycin is weaker than thatby ionomycin, the synthetic analogues could possibly be further modified to achieve a strongerbinding. This might be achieved by introducing some rigid structural elements into the moleculesto reduce the entropy losses on binding.792.14 Incorporation of Rigid Elements into lonomycin Analogues The polyether antibiotic calcium ionophores are unique in that they are stereochemicallycomplex acyclic chains of ligands. It has been proposed by Still and co-workers that particulararrays of chiral centers in these molecules could destabilize undesired rotomers of these acyclicchains and reduce the available conformations to those suitable for polydentate ion binding.23 Intheir elegant work towards the rational design of synthetic host molecules, Still and co-workersshowed that two chiral centers (shown by an asterisk) in lasalocid A (1) were partially responsiblefor the high affinity of lasalocid A for barium ions 23a The natural stereoisomer 1 binds barium 2-3 orders of magnitude more tightly than the three epimers la, lb and 1c (Table XVIII).Table XVIII. Binding Constants of Various Stereoisomers of Lasalocid A with Barium Ions. 23aStereoisomer^ 13 (Ba2+)2.1 x 1063.9 x 1048.8 x 1037.8 x 10380Although the substitutional and stereochemical arrays present in ionomycin might functionin the same way as those in lasalocid A to reduce the available conformations to those suitable forcalcium binding, there is no experimental data at this point as yet. The low binding constants ofthe analogues with calcium are suggestive, but not conclusive, that the stereochemical arrays inionomycin are, at least, partially responsible for its high affinity for calcium.To test the possibility of improving the ionophoric properties of the analogues byrestricting the available conformations for calcium complexation through the introduction of chiralcenters to the molecules, we set out to prepare analogues 41a and 41b. We envisaged thepreparation of these two compounds through dimethylation of an appropriate 13-diketo compoundand separation of the diastereomeric mixture. To this end, compound 42 was treated with twoequivalents of LDA and subsequently one equivalent of methyl iodide. Upon completion of thefirst methylation as shown by thin layer chromatography (TLC), one more equivalent of LDA andthen an additional equivalent of methyl iodide was added to the reaction mixture. Thedimethylated product 43, which was thought to be a mixture of diastereomers 43a and 43b andtheir enantiomers, showed a single spot on TLC.0^0 0^0^0 081HO HO41a^ 41b 0 0(1)2eq LDA(2)CH3I(3)LDA(4)CH3I0 0TBDMSO TBDMSO42 430 0^0 0TBDMSO TBDMSO43a^ 43 bInterestingly, subjection of product 43 to gas liquid chromatography (GC) revealed thepresence of two components in a ratio of 94:6. These two components had identical gas-liquidchromatography mass spectra (GCMS) in which there were two major peaks at m/z = 413 and355 corresponding to the molecular ion of [M + 11J+ and the fragment of [M - CH3]+. These twocomponents were found to be 43a and 43b by 1H NMR spectrometry. Thus, the dimethylationof compound 42 proceeded with high diastereoselectivity. The higher percentage of the enol formof the j3-diketone and the downfield chemical shifts of the methine protons alpha to the enol andketo form of the 13-diketone of the major isomer in the 1 H NMR spectrum of the mixturesuggested that the major isomer was 43b with the two methyl groups "trans " to each other.Unfortunately, efforts to separate 43a and 43b using column chromatography or highperformance liquid chromatography (HPLC) were unsuccessful. Attenipts to separate thealcohols 44a and 44b, or the acids 41a and 41b also failed.0 0^0 082HO HO44a 44bThe ratio of two diastereomeric alcohols and the acids prepared from the mixture of 43aand 43b were consistent with the ratio of 96:4 for 43a and 43b. The transport experiment onthe mixture of 41a and 41b gave a transport rate comparable to its structural isomer, analogue28. Unfortunately no conclusion of the effect of substitution and stereochemistry of analogues41a and 41b on calcium transport could be drawn at this stage because the configuration of thechiral centers of the major isomer 41b are opposite to those found in ionomycin.2,15. Conclusions and Future ConsiderationThe syntheses of simple analogues of ionomycin, namely a series of 13 -diketoco-carboxylic acids, were achieved by consecutive alkylation of the dianion of 2,4-pentanedionewith appropriate bromides and subsequent oxidation of the (3-diketo co-alcohols. This syntheticroute permitted us to vary the number of methylene units between the 124-diketone and the carboxylgroups and hence to probe the effect of the chain length between these two functional groups oncalcium binding and transport. It also allowed us to incorporate various alkyl side chains todetermine the effects of lipid solubility on calcium transport. Using this route gram quantities ofeach ionophore analogue was readily available.Transport of calcium across an organic barrier by these synthetic analogues of ionomycinwas determined in a cylindrical glass cell using chloroform as the artificial membrane. The basicstructural requirements for efficient calcium transport are the presence of the P-diketone and thecarboxyl groups within the same molecule, and a simple alkyl side chain with sufficient lipidsolubility. The lipid solubility of a p-diketo co-carboxylic acid could be represented by thelogarithm of its partition coefficient (log P) which was calculated by the hydrophobic fragmentalconstant method. The log P value of a good calcium ionophore is in the range of 6-8.The distance between the 0-diketone and the carboxyl groups is important in controllingthe efficiency of calcium transport. Optimum calcium transport is achieved when the 0-diketonegroup is separated from the carboxyl group by seven methylene units identical to the backbonefound in ionomycin. It appeared that additional oxygen binding sites on the side chain of theanalogues did not significantly contribute to the chelation and transport of calcium.Simple analogues with sufficient lipid solubility are comparable to calcimycin andionomycin in terms of calcium transport and selectivity. Two of the analogues were found tocause calcium flux in human leukemic cells. The calcium response induced by the analogues was83transient and small, presumably due to the low chemical stability of these molecules in the cellmembrane.Transport of calcium by synthetic analogues of ionomycin is a saturable process whichobeys Michaelis-Menten kinetics. Calcium transport is dependent of the pH in the aqueous sourcephase and independent of the pH in the receiving phase. Both the carboxyl and the 13-diketonegroups are ionized in the transport of calcium, indicating that the stoichiometry of calciumcomplex in transport is 1:1.The characterization of calcium salt of analogue 28 showed that the stoichiometry of thecalcium complex in the solid state was 1:1. It also revealed the weak binding of the analogue withcalcium.The pKa of the 13 -diketone group of the analogues was measured by UVspectrophotometric titration and found to be much lower than that of the f3-diketone group inionomycin. They were directly related to the lipid solubility of the molecules and the hydrocarbonchain length between the P-diketone and the carboxyl groups.The binding constants of the analogues with sodium, potassium, calcium and magnesiumwere determined by measuring the pK a of the 13-diketone group in the presence and absence of thecation. The selectivity in binding was the same as the selectivity in transport, which was Mg 2+ >Calf >> Na+, K+. Calcium and magnesium binding by the analogues were 3-4 orders ofmagnitude lower than that of ionomycin. The mole ratio method was used to determine thebinding constants of the analogues with magnesium. This established the 1:1 stoichiometry in themagnesium complex and verified the utility of the pKa method used for the measurements ofbinding constants of the analogues with sodium, potassium and calcium.8 4HODimethylation of the 0-diketone 42 in a one pot reaction proceeded with highdiastereoselectivity. The major diastereomers had the two methyl groups trans to each other.0 0TBDMSO42Future work on this project should address the two problems that have prevented the useof the analogues in biological systems: one is the proposed chemical instability of the analoguesin cells. It is very likely that the structurally simple analogues undergo rapid transformationwithin the cell which results in the transient nature of the effect of calcium mobilization in THP-1cells. These synthetic compounds may be transesterified and incorporated into the cell membrane.Introduction of bulky alkyl groups, such as a dimethyl group in 45, a to the carboxyl group, mayprevent the compound from being incorporated into the cell membrane and achieve a greater andmore sustained calcium transport into the cell.0^0 0HO45The second problem is the relative low binding constants of the analogues with calciumcompared to ionomycin. This problem is likely due to the presence of too many undesiredrotomers associated with the acyclic chain of the synthetic analogues. This may be partiallysolved by introducing two methyl groups cis to each other and adjacent to the f3-diketone groupas in analogue 41a.0^0 08541aHOAlternatively and likely more effectively, the entropy problem may be solved byconnecting the 13-carbons (shown by an asterisk) of the carboxyl and the 13-diketone groups toform a molecule such as analogue 47. Other derivatives such as analogues 48 and 49 are alsoworthy of testing. The cis and trans stereochemistry in analogues 47 and 48 respectively is tobring the 13-diketone and the carboxyl groups closer to each other to reduce the entropy losses onbinding.864748In conclusion, the study of calcium binding and transport of synthetic analogues based onionomycin has shed light on the structural features controlling calcium binding and transport byionomycin and by its simple analogues. It has also paved the way to the rational design of newcalcium ionophores both as potential biochemical tools and therapeutic agents.CHAPTER THREEEXPERIMENTAL.3.1. GeneralSolvents, reagents and equipment setup. Solvents were dried as follows: diethylether (ether) and tetrahydrofuran (THF) were distilled from sodium benzophenone ketyl radicalunder a dry nitrogen atmosphere. Methylene chloride was distilled from calcium hydride andmethanol from magnesium methoxide.Unless otherwise specified, all reagents were supplied by the Aldrich Chemical Companyand purified according to the procedure given in the literature. 72 n-Butyllithium (in hexanes) wasstandardized by titration against 2,2-diphenylacetic acid in THF at room temperature to the faintestappearance of a yellow color.Nitrogen was supplied by Union Carbide and prior to use was passed through twocolumns of indicating Drierite (CaSO4 impregnated with CoC12). Syringes and needles wereoven-dried at 120 °C for a minimum of 3-4 hours and stored in a desiccator. Unless statedotherwise, all reactions were carried out under a dry nitrogen atmosphere in an anhydrous solvent.The glassware (including the Teflon-coated magnetic stirring bar) was assembled and connected toa vacuum pump and flame-dried under vacuum. After the glassware had cooled, dry nitrogen wasintroduced to the system. Cold temperatures were maintained using either an ice/water bath (0 °C)or an acetone/dry ice bath (-78 °C). The concentration or evaporation of solvents under reducedpressure refers to the use of a Buchi rotary evaporator. Petroleum ether refers to the fractionwhich boils between 30-60 °C.87Reaction monitoring. All reactions were monitored by thin layer chromatography(TLC). Analytical TLC was performed on aluminum backed, precoated silica (SiO2) gel plates(E. Merck, type 5554). The plates were visualized by ultraviolet fluorescence or by heating theplates after spraying them with a mixture of methanol, acetic acid, sulfuric acid and anisaldehyde(90:10:5:1 by volume). Analytical gas liquid chromatography (GC) was performed on a HewlettPackard model 5880A gas chromatography using a 15 m x 0.2 mm capillary DB-210 column andwith the following temperature program: 100 °C for 2 min, then a temperature increase to 220 °Cat a rate of 20 °C per min, and a final time of 8 min at 220 °C.Product purification. Unless otherwise stated, all reaction products were purified byflash chromatography using 230-400 mesh ASTM silica gel supplied by E. Merck Co. In thosecases that a large amount of silica gel was used, the silica gel was recycled after columnchromatography. This involved discarding the upper 2-4 cm of silica gel in the column andflushing the remaining silica gel with methanol until clean. A hose connected to a water aspiratorwas attached to the column spigot and the silica gel was sucked to dryness (powder dry). Thesilica gel was subsequently regenerated by oven heating for 6-8 hours at 120 °C.Product characterization. Proton nuclear magnetic resonance ( 1 H NMR) spectrawere recorded at 400 MHz on a Bruker WH-400 spectrometer or at 300 MHz on a Varian XL-300spectrometer in deuterochloroform (CDC13) with signal positions given in parts per million (ppm)from the internal standard of tetramethylsilane (0.00 ppm) on the 8 scale. They are reported in theform: chemical shift (number of protons, signal multiplicities). The abbreviations used inreporting the data are: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet,dd = double doublet, dt = double triplet.Infrared (IR) spectra were recorded on a Bomem Michelson 100 FT-IR spectrophotometerusing internal calibration. Liquid samples were applied directly between two 3 mm NaCl plates.Solid samples were dissolved in chloroform and the spectra were taken and subsequently88.0 cmglass divider I6.3 cm115.3 cmsubtracted from a spectrum of chloroform. Solid state IR spectra were determined using presseddiscs of the samples in KBr. Absorption positions are given in cm -1 .Low and high resolution mass spectra were determined on a Kratos-AEI model MS 50spectrometer. The parent peak as well as major ion fragments are reported as percentages of thebase peak. Gas chromatography mass spectra were determined on a Kratos-MSAD massspectrometer. All instruments were operated at 70 eV. Melting points were determined on aMel-Temp II melting point apparatus and were not corrected.Microanalyses were carried out at the microanalytical laboratory of the University ofBritish Columbia using a Carlo Erba Elemental Analyzer 1106.Determination of metal ion transport. The transport of metal ions across achloroform liquid membrane by synthetic analogues of ionomycin was determined in a cylindricalglass cell of internal diameter of 5.0 cm (Figure 11). The height of the cell is 6.3 cm and the glassdivider is 0.4 cm wide and 5.3 cm from the top of the vessel.89Cross section Top ViewFigure 11. Cross section and top view of the cylindrical glass cell for the studies of calciumtransport across a chloroform liquid membrane.To determine the calcium transport efficiency of the various analogues, a solution of 200j.LM analogue in 40 mL of glass distilled chloroform (OmniSolv BDH Inc.) was placed at thebottom of the transport vessel. A solution of 500 mM of calcium chloride and 40 mM ofCHES/Me4NOH buffer in 10 mL of distilled deionized water at pH = 9.50 was placed on one sideof the transport vessel as the source phase. A solution of 40 mM MOPS/Me4NOH buffer in 10mL of distilled deionized water at pH = 7.00 was placed on the other side of the transport vesselas the receiving phase. The chloroform solution was stirred continuously with a 10 x 3 mmmicrostirring bar at a constant speed of 400 ± 20 r.p.m. as indicated by a chronometricspeedcounter.An aliquot of 200 pL of the receiving phase was withdrawn with a microsyringe at the endof 12, 16, 20 and 24 h, diluted with 4.80 mL of distilled deionized water and analyzed for thecalcium concentration by calcium atomic absorption spectrophotometry. The concentration ofcalcium in the receiving phase increased linearly with time when the amount of calciumtransported was small. The transport rate (J) is expressed as the moles of calcium transported persquare centimeter of the organic phase per hour. Results from separate runs were reproduciblewithin ± 10%. The reported J value is the average of two or three separate runs. For reason ofsimplicity, only one plot of the amount of calcium in the receiving phase versus time from arepresentative analogue is given.Cation selectivity in transport was determined using a source phase of 10 mL of distilleddeionized water containing equimolar concentration (250 mM) of calcium chloride and chloride ofthe competing cation (magnesium, sodium and potassium ions respectively), and 40 mM ofCHES/Me4NOH buffer at pH = 9.50. The samples taken from the receiving phase were analyzedfor the concentrations of both cations by atomic absorption spectrometry.90Calcium, magnesium, potassium and sodium atomic absorption spectrometry was carriedout on a Perkin-Elmer model 560 atomic absorption spectrometer. The setting of the instrumentwas: integration time = 3 seconds, lamp current = 6 mA, slit setting = 0.7 for calcium,magnesium and sodium, slit setting = 1.4 for potassium, wavelength = 422.7, 202.6, 589.0 and766.5 nm for calcium, magnesium, sodium and potassium lamp respectively.Calcium, magnesium or potassium solutions of 1.25, 2.50 and 5.00 ppm and sodiumsolutions of 0.25, 0.50 and 1.25 ppm prepared from 990 ppm atomic absorption standardsolution (Aldrich) were used to standardized the instrument. The absorbances were linear againstthe concentrations of the metal ions up to 5.00 ppm for calcium, magnesium and potassium, andup to 1.25 ppm for sodium. The linear equations were used to calculate the cation concentrationof the unknown samples whose absorbances were determined.Buffers which had negligible affinity for magnesium, calcium, sodium and potassium ionswere used to control the pH in the source phase and the receiving phase. They were prepared bymixing an aqueous solution of 10% tetramethylammonium hydroxide with an acid that has a pK aclose to the desired working pH and diluting the solution with distilled deionized water to a finalconcentration of 40 mM (Appendix 2).The effect of five selective analogues on intracellular calcium mobilization was examinedin cultured human leukemic cells known as THP-1 cells by Dr. C. Chan of Merck Frosst CentreFor Therapeutic Research. 73 THP-1 cells were suspended in L15 medium (Sigma, pH 7.4, with1 mM Hepes) at 107 cells/mL and were incubated with fura-2 acetocymethyl ester at 21.tg/mL for20 min at 37 °C. The cells were then washed three times with 20 mL of L15 to remove free dyeand fmally resuspended in L15 at 2 x 106 cells/mL. Cell fluorescence (2 mL cell suspension) wasmonitored using a Perkin-Elmer spectrofluorometer with excitation settings at 340 nm and 380nm. The ratio of fura-2 fluorescence with excitation at 340 nm to that at 380 nm was used as anindex to calculate intracellular calcium concentration according to the method of Grynkiewiez. 5991All compounds were dissolved in dimethylsulfoxide (DMSO) at 1000 times the final concentration(i.e. 2 'IL DMSO/compound in 2 mL cells). Calcimycin bromide and ionomycin were used aspositive controls. Cumulative dose-responses were obtained and the cell fluorescence weremonitored continuously for 3-4 minutes. The cells were constantly stirred and were kept at 37 °Cduring the course of the measurement.Determination of pKa of 13-diketones. The pKa of the 0-diketone group of thesynthetic analogues of ionomycin was determined in 80% Me0H-H20 by UV spectrophotometrictitration at 25 °C on a Perkin-Elmer UV-Visible Lambda 2 Spectrometer with a thermostatedsample cell. An aliquot of 1.0 mL 80% Me0H-H20 in a disposable cuvette (1.0 cm pathlength,InterScience) was placed in the sample cell and the baseline was recorded from 260 nm to 350 nmand stored. The solvent was then replaced by a solution of 80 j.t.M analogue and 50 mMEt4NC1O4 in 1.0 mL of 80% Me0H-H20. Small aliquots (4 to 20 pL) of 0.1%, 0.25%, 0.5%and 5.0% Me4NOH (the titrant) in 80% Me0H-H20 were added and the spectra were recorded.The addition of the titrant was terminated when the maximum absorption of the spectrum remainedconstant.The values of pH at each titration point were determined with the same solutions on a5-fold greater scale on a PHM82 STANDARD pH meter (Bach-Simpson Limited). The pH meterwas standardized with pH = 7.0 and pH = 10.0 aqueous buffer solutions. No correction wasmade for differences in the pH readings between pure aqueous solutions and 80% Me0H-H20solutions.The absorbance at 298 nm versus pH was computer fitted to a modified Henderson-Hasselbalch equation by non-linear regression using Enzfitter developed by Leatherbarrow tocalculate the pKa values.63Determination of metal ion binding constants. The binding constant of theanalogues with various metal ions was determined in 80% Me0H-1120 by UV spectrophotometric92titration at 25 °C. The UV spectrophotometric titration of the analogues was carried out in thepresence of 50 mM NaCl, 50 mM KBr, 16.7 mM of CaC12 and 16.7 mM MgC12 respectively (inplace of Et4NC1O4). The absorbance at 298 nm versus pH was computer fitted to a modifiedHenderson-Hasselbalch equation by non-linear regression to calculate the conditional pKa' of a 13-diketone in the presence of the metal ion. The difference between pK a' and pKa gave thelogarithm of the dissociation constant of the analogue-cation complex.The binding constant of the analogues with magnesium was also determined using themole ratio method. A solution of 80 g/vI analogue and 50 mM CHES/Me4NOH buffer in 80%Me0H-H20 was titrated with a solution of 5, 10, 25, 50, 100 and 250 mM MgC12 in 80%Me0H-H20. Calculation of the number of moles of magnesium bound per mole of the analogueversus the concentration of free magnesium ions at each titration point were computer fitted to theScatchard equation by non-linear regression to determine the binding constant.63 This methodalso allowed the determination of the stoichiometry of the complex.For reason of simplicity, only one set of the titration and the data fitting curves for thedetermination of pKa, pKat, the magnesium binding constant and stoichiometry of a representativeanalogue is presented. Results for other analogues are reported as absorbance at each pH or ateach magnesium ion concentration and the calculated binding constant and stoichiometry.9 3942,2^Synthesis of 9.11-Dioxopentadecanoic Acid (4)0^0 0HO3.2.1 7-Bromo-1-heptanol (13)HO^OH12 HOW' Br13A suspension of 1,7-heptanediol (12) (5.8 g, 44 mmol) in 7.5 mL of 48% HBr (66mmol) was prepared in a 1-L liquid-liquid continuous extractor. The suspension was heated to90 °C and was extracted with 300 mL of heptane at this temperature for 72 h. The extract wascooled, washed twice with saturated NaHCO3 and once with brine. The organic layer was driedover MgSO4 and concentrated under reduced pressure. The crude oil was purified by columnchromatography using petroleum ether : ethyl acetate (6:1) as eluent to give the monobrominatedalcohol (6.2 g, 72%) as a colorless liquid, which was one spot by TLC.Rt. (1:1 petroleum ether : ethyl acetate eluent) 0.54.1 H NMR (300 MHz, CDC13) 8 3.66 (2H, t), 3.42 (2H, t), 1.87 (2H, p), 1.57 (2H, p), 1.52-1.25 (7H, m).IR (neat, cm -1 ) 3347, 2930, 2858, 1452, 1254, 1054, 726.LRMS (m/z) 195 (Br81 : M+ - H, 0.2), 193 (Br79 : M+ - H, 0.2), 150 (55), 148 (56), 137 (2),136 (2), 135 (2), 134 (2), 109 (3), 107 (3), 98 (3), 97 (52), 96 (5), 95 (6), 83 (5), 81 (34), 70(34), 69 (90), 68 (51), 67 (47), 57 (34), 56 (55), 55 (100), 44 (21), 43 (66), 42 (57), 41 (87),40 (23), 39 (61), 31 (71).HRMS calcd for C7H15Br81O - H: 195.0204, found: 195.0213; calcd for C71115Br790 - H:193.0224, found: 193.0228.3.2.2 1-Bmmo-7-(tert-butyldimethylsilyloxy)-heptane (8)HO?.%•W Br13 TBDMSOW Br8Freshly distilled triethylamine (1.68 mL, 12.0 mmol) was injected into a solution of thealcohol 13 (1.17 g, 6.0 mmol) in 100 mL of CH2C12 at room temperature. A catalytic amount of4-dimethylaminopyridine (DMAP) and 1.36 g (9.0 mmol) of tert-butyldimethylsilyl chloride(TBDMS-Cl) were added. The reaction was stirred at room temperature for 24 h. The mixturewas then quenched with 1N HC1 and extracted with ether three times. The combined organiclayers were washed twice with saturated NaHCO3 and once with brine. The organic phase wasdried over MgSO4 and concentrated under reduced pressure. Purification of the crude product bycolumn chromatography using petroleum ether : ethyl acetate (15:1) as eluent gave the silyl ether 8(1.50 g, 82%) as a colorless oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.83.1H NMR (400 MHz, CDC13) 5 3.64 (2H, t), 3.44 (2H, t), 1.86 (2H, p), 1.53-1.31 (8H, m),0.89 (9H, s), 0.05 (6H, s).IR (neat, cm-1) 2937, 2858, 1466, 1388, 1361, 1254, 1101, 1006, 938, 838, 776.LRMS (m/z) 253 (Br81 : M+ - C4H9, 4.4), 251 (Br79 :M+ - C4H9, 4.6), 173 (2), 171 (7), 170(4), 169 (38), 167 (33), 140 (4), 139 (34), 138 (4), 137 (33), 101 (25), 99 (17), 98 (29), 97(75), 96 (12), 93 (9), 89 (25), 76 (12), 75 (53), 74 (12), 73 (44), 70 (9), 69 (50), 68 (8), 67(13), 61 (21), 60 (8), 59 (35), 58 (22), 57 (28), 56 (34), 55 (100), 47 (24), 45 (33), 43 (42), 41(43), 39 (22).HRMS calcd for C13H29SiBr 81 0 - C4H9: 253.0438, found: 253.0450; calcd forC13H29SiBr790 - C4H9: 251.0458, found: 251.0460.9 53.2.3 12-(Tert-butyldimethylsilyloxy)-2.4-dodecanedione (22)yoL^0 0^I.- TBDMSOL7 22Sodium hydride (610 mg, 50% in oil, 12.7 mmol) was added to a 100 mL, two-neckround bottom flask and washed twice with 20 mL of THF. Then 20 mL of THF was added andthe resulting suspension was cooled to 0 °C. A solution of 2,4-pentanedione (7) (1.36 mL,12.7 mmol) in 15 mL of THF was added through an addition funnel. The resulting white solutionwas stirred for 30 min and 7.84 mL of n-BuLi (1.62 M, 12.7 mmol) was added. The orangesolution was stirred for 20 min. A solution of the bromide 8 (3.26 g, 10.6 mmol) in 10 mL ofTHF was slowly added through the addition funnel and the mixture was stirred at 0 °C for lh andat room temperature for 20 min. The mixture was quenched with saturated NH4C1, acidified with1N HC1 and extracted with ether three times. The combined organic layers were washed twicewith saturated NaHCO3 and once with brine, dried over MgSO4 and concentrated under reducedpressure. Purification of the crude product by column chromatography using a mixture ofpetroleum ether : ethyl acetate (15:1) as eluent gave the 13-diketone 22 (2.20 g) and the bromide 8(700 mg). The reaction yield, based on the recovered bromide, was 81%.RI. (6:1 petroleum ether : ethyl acetate eluent) 0.61.1H NMR (400 MHz, CDC13) 8 15.50 (0.8 H, s), 5.49 (0.8 H, s), 3.61 (2H, t), 3.58 (0.4 H,s), 2.51 (0.4 H, t), 2.27 (1.6 H, t), 2.24 (0.6 H, s), 2.06 (2.4 H, s), 1.65-1.26 (12H, m), 0.89(9H, s), 0.04 (6H, s).IR (neat, cm -1 ) 2934, 2857, 1709, 1614, 1465, 1253, 1100, 1006, 939, 838, 776.LRMS (m/z) 313 (M+ - CH3, 6), 274 (3), 273 (14), 272 (44), 271 (100), 253 (2), 229 (1), 171131 (4), 117 (4), 115 (10), 101 (8), 100 (4), 99 (23), 8561 (6), 59 (9), 58 (3), 57 (4), 55 (24), 43 (39), 41 (18).HRMS calcd for C18H36SiO3 - CH3: 313.2190, found: 313.2208.96(6), 169 (8), 157 (5), 145 (4), 143 (6),(22), 75 (55), 73 (24), 69 (10), 67 (7),3.2.4 15-(Tert-butyldimethylsilyloxy)-5.7-pentadecanedione (24)0 0^ 0 0TBDMSOWC --- "TBDMSOW•22^ 24Lithium diisopropylamide (LDA, 2.6 mmol) was prepared at -78 °C by addition of n-BuLi(1.53 M, 1.70 mL, 2.60 mmol) to a solution of diisopropylamine (0.36 mL, 2.6 mmol) in 20 mLof THF and stirring of the mixture for 30 min. It was then cannulated to an additional funnel andadded to a solution of the p-diketone 22 (426 mg, 1.30 mmol) in 20 mL of THF at -78 °C over aperiod of 1 h. The mixture was stirred at -78 °C for 24 h and allowed to warm up to 0 °C.Freshly distilled 1-bromopropane (160 mg, 1.30 mmol) in 10 mL of THF was slowly added andthe mixture was stirred at 0 °C for 12 h. The mixture was then quenched with saturated NH4C1,acidified with IN HC1 and extracted with ether three times. The combined organic phases werewashed with saturated NaHCO3, brine, dried over MgSO4 and concentrated under reducedpressure. Purification of the crude product by column chromatography using petroleum ether :ethyl acetate (20:1) gave the desired P-diketone 24 (294 mg) and the starting 13-diketone 22 (294mg). The yield of the reaction, based on the recovered P-diketone 22, was 81 %.Rt. (6:1 petroleum ether : ethyl acetate eluent) 0.74.1 H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 3.61 (2H, t), 3.54 (0.4H, s),2.53-2.47 (0.8H, dt), 2.30-2.24 (3.2H, m), 1.66-1.25 (16H, m), 0.93 (3H, t), 0.90 (9H, s),0.06 (6H, s).IR (neat, cm -1 ) 2936, 2858, 1707, 1611, 1463, 1253, 1100, 949, 838, 776, 704.LRMS (m/z) 355 (M+ - CH3, 3), 315 (7), 314 (23), 313 (99), 271 (1), 229 (2), 213 (2), 211(3), 185 (2), 171 (3), 169 (3), 159 (2), 157 (3), 155 (3), 129 (3), 128 (4), 127 (4), 115 (5), 113(5), 107 (8), 105 (4), 101 (8), 99 (6), 97 (10), 95 (11), 93 (7), 89 (12), 86 (5), 85 (76), 81 (13),79 (8), 77 (14), 76 (8), 75 (100), 73 (44), 69 (29), 67 (14), 59 (16), 57 (62), 56 (13), 55 (51).HRMS calcd for C211-142SiO3 - CH3: 355.2658, found: 355.2662.971723, 1700, 1605, 1460, 1355, 1307, 1149, 1098,(1), 214 (1), 213 (1), 181 (1), 157 (2), 156 (2), 155IR (CHC13, cm-1 ) 3621, 3462, 2934, 2859,1005, 957, 916.LRMS (m/z) 256 (M+, 1), 239 (0.2), 2383.2.5 15-Hydroxy1-5.7-pentadecanedione (251 0 0TBDMSOL.)24 0 0HOCA./*■./25To a solution of the silyl ether 24 (444 mg, 1.20 mmol) in 40 mL of THF was injected asolution of tetrabutylammonium fluride in THF (1.0 M, 2.40 mL, 2.4 mmol). The solution wasstirred at room temperature for 12 h. The solvent was removed under reduced pressure and theresidue was taken up in ether. The organic layer was washed twice with brine, dried over MgSO4and concentrated under reduced pressure. Purification of the crude product by columnchromatography using petroleum ether : ethyl acetate (6:1) as eluent gave the alcohol 25 (277 mg,91%) as a colorless solid, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate eluent) 0.58.mp 41.5 °C1 H NMR (400 MHz, CDC13) 8 15.50 (0.7H, s), 5.48 (0.7H, s), 3.65 (2H, t), 3.53 (0.6H, s),2.52-2.51 (1.2H, dt), 2.29-2.27 (2.8H, dt), 1.66-1.25 (17H, m), 0.93 (3H, t).98(6), 143 (2), 142 (22), 141 (4), 128 (3), 127 (30), 114 (3), 113 (12), 101 (3), 100 (38), 99 (4), 98(3), 97 (14), 86 (6), 85 (100), 84 (9), 83 (8), 71 (14), 70 (5), 69 (33), 68 (4), 67 (13), 58 (19), 57(79), 56 (12), 55 (87), 44 (20), 43 (65), 42 (8), 41 (65), 40 (34).HRMS calcd for C15H2803: 256.2031, found: 256.2031.3.2.6 9.11-Dioxopentadecanoic Acid (4)0 0^ 0^0 0HO ^ HO0''-25 4To the alcohol 25 (384 mg, 1.34 mmol) in 15 mL of CH2C12 and 15 mL of DMSO wasadded 1,3-dicyclohexylcarbodiimide (DCC) (1.67 g, 7.95 mmol) and dichioroacetic acid (53 RL,0.69 mmol). The mixture was stirred for 2 h, diluted with ethyl acetate and treated with oxalicacid (1.02 g, 7.95 mmol). The mixture was poured into brine, filtered to remove the ureaprecipitate and extracted with ethyl acetate. The organic layer was concentrated under reducedpressure. The resulting oil was dissolved in 24 mL 50% THF- H2O. Silver nitrate (1.85 g, 10.8mmol) and NaOH (0.86 g, 22 mmol) were added and the mixture was stirred for 4 h. Theprecipitate was filtered and washed with ethyl acetate and water. The aqueous layer was acidifiedwith 1N HC1 and was then extracted with ethyl acetate. The organic extract was washed withbrine, dried over MgSO4 and concentrated under reduced pressure. The crude product waspurified by recrystallization from hexanes to give the acid 4 (270 mg, 69%) as a colorless crystal.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.54.mp 43.0 °C.1 H NMR (300 MHz, CDC13) 8 15.50 (0.7H, s), 5.48 (0.7H, s), 3.53 (0.6H, s), 2.52-2.51(1.2H, dt), 2.33 (2H, t), 2.28-2.26 (2.8H, dt), 1.70-1.27 (15H, m), 0.93 (3H, t).IR (CHC13, cm-1 ) 3346-2480, 2937, 2861, 1713, 1601, 1459, 1286, 1116, 968, 903.LRMS (m/z) 270 (Mt , 4.0), 252 (1), 229 (1), 228 (8), 227 (3), 210 (2), 196 (2), 195 (15), 182(3), 171 (25), 167 (5), 155 (10), 143 (4), 142 (33), 127 (51), 125 (16), 113 (18), 111 (8), 100(37), 98 (6), 97 (17), 85 (100), 84 (9), 83 (17), 69 (22), 57 (32), 55 (32), 43 (25), 41 (24).HRMS calcd for Ci5H2604: 270.1824, found: 270.1828.Elem. Anal, calcd for Ci5H2604: C, 66.67; H, 9.63. found: C, 66.60; H, 9.65.99100al^Synthesis of 1542-(5-Benzyloxyl-Rentyloxyl-ethoxy-9.11-dioxopentadecanoic Acid (5)0^0 0HO 0/---O WO3.3.1 5-Benzyloxy-l-pentanol (16)HO^OH ^PP' HO ''', 015 16Sodium hydride (4.58 g, 80% in oil, 153 mmol) was added to a 500-mL two-neck roundbottom flask and washed twice with 20 mL of THF. A solution of 1,5-pentanediol (15) (7.95 g,76.4 mmol) in 20 mL of THF and 200 mL of THF was then added. The mixture was stirred atroom temperature for 30 min. Benzyl bromide (9.1 mL, 77 mmol) was slowly added and themixture was refluxed overnight. The reaction mixture was quenched with H2O. The organiclayer was washed once with 1N HCl and twice with brine, dried over MgSO4 and concentratedunder reduced pressure. The crude product was chromatographed on a silica gel column usingpetroleum ether : ethyl acetate (3:1) as eluent to give the alcohol 16 (7.98 g, 54%) as a clear oil,which was one spot by TLC.Rf (1 : 1 petroleum ether : ethyl acetate eluent) 0.38.1 H NMR (400 MHz, CDC13) 8 7.40-7.24 (5H, m), 4.52 (2H, s), 3.65 (2H, t), 3.52 (2H, t),1.70-1.40 (7H, m).IR (neat, cm-1 ) 3372, 3090, 3063, 3030, 2897, 2850, 1496, 1454, 1363, 1206, 1077, 903.LRMS (m/z) 194 (M+, 7), 176 (0.1), 131 (1), 118 (1), 117 (2), 109 (2), 108 (30), 107 (75),(24), 79 (23), 78 (6), 77 (13), 69 (15), 6743 (16), 42 (6), 41 (32).HRMS calcd for Ci2H1802: 194.1302, found: 194.1304.106 (4), 105 (9), 92 (50), 91(100), 90 (5), 89 (6), 85(7), 65 (30), 63 (5), 57 (15), 55 (12), 51 (11), 44 (4),3.3.2 5-Benzyloxy-1-(2-tetrahydropyranyloxy)-ethoxy-pentane (17)101HOWsO16 17A 25-mL, three-neck round bottom flask was charged with 50% sodium hydroxide(4.0 mL, 50 mmol), 1-bromo-2-tetrahydropyranyloxy-ethane (4.18 g, 20.0 mmol) and thealcohol 16 (0.97 g, 5.0 mmol). Tetrabutylammonium hydrogen sulfate (0.15 g, 0.44 mmol)was added. The two-phase mixture was stirred vigorously and heated to 65 °C for 72 h. Thereaction mixture was cooled to room temperature and taken up into 100 mL of ether. The organiclayer was washed with H2O, brine, dried over MgSO4 and concentrated under reduced pressure.Purification of the crude product by column chromatography using petroleum ether : ethyl acetate(6:1) as eluent gave the desired compound 17 (1.34 g, 83%) as a light yellow oil, which was onespot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.16.1 H NMR (300 MHz, CDC13) 8 7.40-7.24 (5H, m), 4.64 (1H, t), 4.50 (2H, s), 3.94-3.46(10H, m), 1.90-1.40 (12H, m).IR (neat, cm-1 ) 3086, 3060, 3029, 2935, 2861, 1495, 1453, 1359, 1269, 1201, 1184, 1106,1032, 988, 929, 906, 872, 814, 739.LRMS (m/z) 322 (M+, 0.2), 321 (0.1), 238119 (0.4), 118 (2), 117 (1), 108 (2), 107 (8),84 (5), 79 (6), 78 (2), 77 (7), 67 (7), 66 (1),(23).(0.6), 237 (2), 177 (0.4), 176 (1), 132 (1), 131 (9),106 (2), 105 (4), 92 (13), 91 (100), 86 (4), 85 (67),65 (13), 57 (11), 56 (7), 55 (11), 45 (10), 43 (8), 41HRMS calcd for C19H3004: 322.2136, found: 322.2152.3.3.3 2-(5-Benzyloxy)-pentyloxy-l-ethanol (18)17^ 18Pyridinium p-toluenesulfonate (42 mg, 0.16 mmol) was added to a solution of the THPether 17 (550 mg, 1.60 mmol) in 15 mL of Me0H. The mixture was stirred at room temperaturefor 12 h. The solvent was removed under reduced pressure and the residue was taken up in ether,washed with saturated NaHCO3 and brine, dried over MgSO4 and concentrated under reducedpressure. The crude oil was chromatographed on a silica gel column using petroleum ether : ethylacetate (6:1) as eluent to give the alcohol 18 (368 mg, 91%) as a light yellow oil, which was onespot by TLC.Rf (1:1 petroleum ether : ethyl acetate eluent) 0.30.1 H NMR (300 MHz, CDC13) 8 7.40-7.24 (511, m), 4.52 (2H, s), 3.72 (211, m), 3.53 (211, t),3.49 (4H, t), 2.04 (1H, s), 1.69-1.40 (6H, m).IR (neat, cm-1 )^3414, 3082, 3063, 3030, 2932, 2862, 1495, 1454, 1362, 1207, 1086, 893,740.LRMS (m/z) 238 (M+, 0.6), 208 (0.2), 177 (0.1), 176 (1), 175 (1), 148 (0.2), 147 (1), 133(1), 132 (3), 131 (3), 108 (2), 107 (10), 106 (3), 105 (5), 92 (14), 91(100), 85 (20), 79 (7), 78(3), 77 (8), 65 (14), 63 (8), 57 (5), 56 (2), 55 (4), 45 (15), 44 (2), 43 (4), 42 (3), 41 (10).HRMS calcd for C14112203: 238.1563, found: 238.1559.1023.3.4103HO'- 0w0 TBDMS0'•/%0"ThwO18^19Following the procedure outlined on section 3.3.2, the alcohol 18 (300 mg, 1.26 mmol)was reacted with 1-bromo-3-tert-butyldimethylsilyloxy-propane (1.28 g, 5.04 mmol) to give thecorresponding ether. The crude product was purified by column chromatography using petroleumether : ethyl acetate (6:1) as eluent to give the TBDMS ether 19 (1.34 g, 83%) as a light yellowoil, which was one spot by TLC.Rt (6:1 petroleum ether : ethyl acetate eluent) 0.20.1 H NMR (300 MHz, CDC13) 8 7.36-7.24 (5H, m), 4.50 (2H, s), 3.70 (2H, s), 3.58-3.45(10H, m), 1.79 (2H, p), 1.69-1.40 (6H, m), 0.89 (9H, s), 0.04 (6H, s).IR (neat, cm-1) 3087, 3064, 3030, 2934, 2862, 1463, 1360, 1253, 1108, 1015, 841, 777, 737.LRMS (m/z) 410 (Mt, 0.3), 354 (0.2), 353 (1), 268 (0.2), 267 (1), 248 (0.3), 247 (1), 209(1), 205 (2), 178 (2), 177 (22), 176 (2), 175 (3), 173 (4), 161 (5), 159 (3), 133 (13), 132 (3),131 (12), 119 (8), 118 (3), 117 (24), 116 (2), 115 (11), 105 (12), 103 (6), 101 (9), 92 (42), 91(100), 89 (13), 87 (10), 86 (5), 85 (32), 79 (10), 77 (8), 75 (26), 73 (20), 69 (12), 57 (12), 56(10), 45 (14), 44 (5), 43 (9), 41 (27).HRMS calcd for C23H42SiO4: 410.2841, found: 410.2861.3.3.5 3-12-(5-Benzyloxy)-pentyloxyl-ethoxy-l-propanol (20) TBDMSOV"."-Vw0 HO 'B'O'-`0^'O19^ 20To a solution of the silyl ether 19 (430 mg, 1.05 mmol) in 15 mL of THF was injected asolution of tetrabutylammonium fluride in THF (1.0 M, 2.10 mL, 2.1 mmol). The solution wasstirred at room temperature for 12 h. The solvent was removed under reduced pressure and theresidue was taken up in ether. The organic layer was washed with brine, dried over MgSO4 andconcentrated under reduced pressure. Purification of the crude product by columnchromatography using petroleum ether : ethyl acetate (3:1) as eluent gave the alcohol 20 (275 mg,89%) as a colorless oil, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate eluent) 0.20.1H NMR (400 MHz, CDC13) 5 7.38-7.26 (5H, m), 4.50 (2H, s), 3.78 (2H, s), 3.69 (2H, t),3.62-3.54 (4H, m), 3.47 (2H, t), 3.45 (2H,t), 2.48 (1H, s), 1.83 (2H, p), 1.69-1.40 (6H, m).Ill (neat, cm -1 )^3426, 3085, 3063, 3030, 2932, 2863, 1495, 1454, 1361, 1296, 1204, 1091,740.LRMS (m/z) 236 (M+ - C31180, 0.1), 223 (0.1), 222 (1), 192 (1), 191 (3), 190 (2), 187 (1),176 (2), 175 (1), 149 (2), 148 (1), 147 (2), 131 (14), 124 (4), 123 (40), 122 (20), 121 (10), 108(6), 107 (23), 106 (25), 105 (100), 104 (18), 103 (12), 101 (13), 100 (10), 99 (20), 92 (27), 91(99), 90 (6), 89 (21), 87 (31), 86 (20), 85 (84), 79 (11), 78 (8), 77 (57), 75 (14), 73 (11), 71(13), 70 (13), 69 (74), 68 (27), 67 (12), 65 (10), 59 (38), 58 (16), 57 (18), 56 (14), 55 (17).HRMS calcd for CoH2804 - C3H80: 236.1407, found: 236.1400.10485 (81), 84 (22), 79 (20),57 (36), 56 (18), 55 (31).77 (31), 71 (23),3.3.6 5-Benzyloxy-1-12-(3-bromo)-propyloxy]-ethoxy-heptane (10)HO"•/'0'-‘O'O^ Br '.'01--%0W02 0^ 10A 25-mL round bottom flask was charged with the alcohol 20 (296 mg, 1.00 mmol) in12 mL of CH2C12. The solution was cooled to 0 °C. Freshly recrystallized triphenylphosphine(340 mg, 1.30 mmol) and carbon tetrabromide (415 mg, 1.25 mmol) were added. The mixturewas stirred at 0 °C for 30 min, allowed to warm up to room temperature and diluted with ether.The organic layer was washed with H2O, brine, dried over MgSO4 and concentrated underreduced pressure. The crude product was purified by column chromatography using petroleumether : ethyl acetate (9:1) as eluent to give the bromide 10 (315 mg, 88%) as a colorless oil, whichwas one spot by TLC.Rf (9:1 petroleum ether : ethyl acetate eluent) 0.14.1H NMR (400 MHz, CDC13) 8 7.38-7.25 (5H, m), 4.50 (2H, s), 3.66-3.44 (12H, m), 2.12(2H, p), 1.69-1.40 (6H, m).IR (neat, cm-1 ) 3080, 3063, 3030, 2930, 2862, 1495, 1454, 1360, 1256, 1209, 1110.LRMS (m/z) 360 (Br81 : M+ , 3.5), 358 (Br79 : M+, 3.9), 269 (2), 267 (2), 197 (2), 195 (2), 185167 (20), 166 (21), 165 (18), 153 (22), 151 (24),(42), 108 (16), 107 (42), 106 (25), 105 (52), 92(39),(18),HRMS calcd for C17H27Br81O3: 360.1116, found: 360.1109; calc. for C17H27Br 7903:358.1136, found: 358.1139.105(30), 183 (34), 177 (5), 176 (26), 168 (18),140 (15), 138 (21), 131 (25), 123 (53), 121(50), 91 (100), 90 (10), 89 (10), 87 (33), 8670 (31), 69 (57), 68 (28), 67 (23), 65 (32), 593.3.7 142-(5-Benzyloxy)pentyloxyl-ethoxy-15-(tert-butyldimethylsilyloxy)-5.7- pentadecanedione (50) 0 0TBDMSO L•220 0TBDMSONe-NOWO50Following the procedure outlined in section 3.2.4, the P-diketone 22 (426 mg, 1.30mmol) was alkylated with the bromide 10 (359 mg, 1.00 mmol) to give the P-diketone 50. Thecrude product was purified by column chromatography using silica gel and petroleum ether : ethylacetate (9:1) to give the 0-cliketone 50 (270 mg) as a clear oil and the bromide 10 (104 mg). Thereaction yield, based on the recovered bromide, was 85%.Rf (9:1 petroleum ether : ethyl acetate eluent) 0.12.1 H NMR (400 MHz, CDC13) 6 15.48 (0.8H, s), 7.38-7.26 (5H, m), 5.47 (0.811, s), 4.51(2H, s), 3.65-3.44 (12.4H, m), 2.59-2.49 (0.811, dt), 2.35-2.24 (3.2H, dt), 1.72-1.25 (22H,m), 0.90 (9H, s), 0.50 (6H, s).IR (neat, cm-1 ) 3080, 3060, 3026, 1710, 1703, 1609, 1460, 1360, 1252, 1110, 840.LRMS (m/z) 606 (Mt, 0.7), 591 (0.5), 551 (3), 550 (9), 549 (24), 458 (0.6), 457 (2), 444283 (4), 272 (2), 271 (10), 170(9), 99 (10),(4), 69 (24),106(0.6), 443 (2), 312 (3), 311 (12), 298 (0.3), 297 (2), 284 (1),(2), 169 (9), 132 (2), 131 (9), 107 (7), 106 (3), 105 (9), 101(12), 91 (100), 85 (26), 77 (14), 75 (45), 73 (15), 71 (8), 70(21), 43 (17), 41 (23).97 (9), 95 (12), 9257 (13), 56 96), 55HRMS calcd for C35H62S106: 606.4299, found: 606.4313.3.3,8 1-1-2-(5-13enz,yloxy)pentyloxyl-ethoxy-15-hydroxyl-5,7-pentadecanedione (Si')0 0TBDMSO.) 0P-ThwO50 0 0Ho^■^.^.^) *(rThwo51Following the procedure outlined in section 3.2.5, the silyl ether 50 (1.40 g, 2.58 mmol)was converted to the corresponding alcohol 51. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (6:1) as eluent to give the alcohol 51 (993mg, 87%) as a colorless oil, which was one spot by TLC.Rf ( 1 : 1 petroleum ether : ethyl acetate eluent) 0.30.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 7.38-7.24 (5H, m), 5.48 (0.8H, s), 4.50(2H, s), 3.64 (2H, t), 3.57 (4H, m), 3.53 (0.4H, s), 3.50-3.43 (61-1, m), 2.58-2.47 (0.8H, dt),2.35-2.24 (3.2H, dt), 1.72-1.25 (23H, m).107IR (neat, cm -1 )^3437, 3080, 3060, 3030, 2930, 2863, 1710,739.LRMS (m/z) 492 (Mt, 1.3), 474 (0.4), 386 (1), 317 (1), 257253 (13), 198 (1), 197 (7), 178 (1), 177 (4), 176 (11), 154 (3),(12), 131 (13), 125 (20), 108 (4), 107 (12), 101 (12), 100 (4),1702, 1606, 1453, 1361, 1096,(2), 256 (6), 255 (37), 254 (10),153 (13), 141 (9), 140 (22), 13999 (19), 98 (10), 97 (17), 95 (7),92 (13), 91 (100), 85 (31).HRMS calcd for C29114806: 492.3438, found: 492.3445.3.3.9 1542-(5-Benzyloxy)-pentyloxy)-ethoxy-9.11-dioxopentadecanoic Acid (5)0 0H0 /--‘0w0 510^0 0Ho ^0,0wo5Following the procedure outlined in section 3.2.6, the alcohol 51 (2.11 g, 4.29 mmol)was converted to the corresponding acid 5. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (6:1) which also contained 5% acetic acid aseluent to give the acid 5 (1.58 g, 74%) as a clear oil, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.30.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 7.38-7.24 (5H, m), 5.47 (0.8H, s), 4.50(2H, s), 3.60-3.55 (4H, m), 3.53 (0.4H, s), 3.50-3.42 (6H, m), 2.58-2.46 (0.8H, m), 2.36-2.22 (5.2H, m), 1.72-1.25 (21H, m).IR (neat, cm-1 ) 3120, 3080, 3060, 3028, 2933, 2859, 1726, 1604, 1455, 1360, 1246, 1105,928, 738.LRMS (m/z) 506 (M+, 2), 488 (0.3), 463 (0.3), 462 (0.8), 416 (0.7), 415 (1), 401 (0.3), 400(1), 332 (0.4), 331 (2), 314 (1), 313 (3), 270 (7), 269 (38), 268 (29), 267 (7), 252 94), 251 (19),250 (12), 249 (7), 172 (5), 171 (42), 153 (27), 141 (15), 140 (37), 125 (49), 124 (8), 123 (29),113 (8), 112 (15), 111 (12), 107 (11), 106 (9), 105 (81), 101 (26), 100 (10), 99 (22), 98 (22), 97(38), 92 (8), 91 (80), 87 (18), 86 (10), 85 (63), 84 (15), 83 (33), 77 (14), 71 (12), 70 (9), 69(100), 68 (19), 67 (12), 57 (13), 56 (10), 55 (48).HRMS calcd for C29H4607: 506.3231, found: 506.3237.108I4 Synthesis of 15-Methoxymethoxy-9.11-dioxopentadecanoic Acid (6) 0^0 0HO3.4.1 1-Bromo-3-methoxymethoxypropane (11)Br^'OH^Br^°O/CY14 11To a solution of 3-bromo- 1-propanol (14) (3.08 g, 22.1 mmol) in 35 mL of CH2C12 wasadded dimethoxymethane (30.3 g, 399 mmol). The solution was cooled to 0 °C and phosphoruspentoxide (approximately 1.0 g at a time) was added every 10 min until the reaction wascompleted as shown by TLC. The mixture was poured into 200 ml of an ice-cooled saturatedNaHCO3 and the gummy residue remaining in the reaction flask carefully quenched with saturatedNaHCO3. The aqueous layer from the combined work-up solutions was extracted with ether.The combined organic layers were washed with brine, dried over MgSO4 and concentrated underreduced pressure. Distillation of the crude product under reduced pressure (85 °C/32 torr) gavethe bromide 11 (3.67 g, 91%) as a colorless oil.1H NMR (400 MHz, CDC13) 8 4.63 (2H, s), 3.67 (2H, t), 3.54 (2H, t), 3.38 (3H, s), 2.14(2H, p).IR (neat, cm-1 ) 2931, 2886, 1476, 1449, 1383, 1284, 1258, 1216, 1144, 1045, 919, 880, 766.LRMS (m/z) 183 (Br81 : M+ - H, 16), 181 (Br79 : M+ - H, 16), 154 (10), 153 (7), 152 (12), 151(6), 123 (14), 122 (5), 121 (13), 95 (2), 93 (2), 75 (52), 61 (6), 45 (100), 41 (31).HRMS calcd for C5H11Br8102 - H: 182.9841, found: 182.9847.1093.4.2 1-Methoxymethoxy-15-(jert-butyldimethylsilyloxy)-5.7-pentadecanedione (52)0 0^ 0 0TBDMSOw^TBDMS0'0"0"22 52Following the procedure outlined in section 3.2.4, the 13-diketone 22 (2.16 g, 6.59 mmol)was alkylated with the bromide 11 (1.08 g, 5.93 mmol) to give 52. The crude product waspurified by column chromatography using petroleum ether : ethyl acetate (15:1) as eluent to givethe purified product 52 (1.56 g, 73% based on recovered starting material) as a clear oil, whichwas one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.24.1H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 4.62 (1.6H, s), 4.61 (0.4H,s), 3.61 (2H, t), 3.57 (0.4H, s), 3.56 (2H, t), 3.38 (3H, s), 2.59-2.49 (0.8H, dt), 2.35-2.24(3.2H, dt), 1.88-1.25 (16H, m), 0.90 (9H, s), 0.50 (6H, s).1R (neat, cm-1 ) 2936, 2858, 1708, 1701, 1611, 1463, 1387, 1253, 1148, 1103, 1044, 924.LRMS (m/z) 415 (M+ - CH3, 1.7), 400 (0.1), 375 (5), 374 (17), 373 (63), 343 (4), 342 (5),341 (18), 313 (4), 312 (6), 311 (21), 284 (1), 283 (6), 242 (3), 241 (17), 214 (3), 213 (18), 125(11), 171 (5), 169 (5), 115 (25), 111 (21), 109 (7), 107 (7), 105 (6), 101 (12), 97 (12), 95 (16),89 (15), 85 (12), 83 (12), 81 (15), 79 (7), 77 (10), 75 (67), 73 (30), 71 (9), 69 (30), 67 (14), 59(10), 57 (11), 56 (10), 55 (43), 45 (100), 44 (5), 43 (21), 42 (7), 41 (25).1 10HRMS calcd for C23146Si05 - CH3: 415.2868, found: 415.2875.3.4.3 1-Methoxymethoxy-15-hydroxyl-5.7-pentadecanedione (53) 0 0^ 0 0TBDMS00"0" ----'°- HO^0^0'52 53Following the procedure outlined in section 3.2.5, the silyl ether 52 (215 mg, 0.50 mmol)was converted to the alcohol 53. The crude product was purified by column chromatographyusing petroleum ether : ethyl acetate (6:1) as eluent to give the alcohol 53 (136 mg, 86%) as acolorless solid, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate eluent) 0.36.mp 42.0 °C.1 H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 4.62 (1.6H, s), 4.61 (0.4H,s), 3.65 (2H, t), 3.56 (0.4H, s), 3.55 (2H, t), 3.37 (3H, s), 2.59-2.49 (0.8H, dt), 2.35-2.25(3.2H, dt), 1.77-1.25 (17H, m).IR (CHC13, cm -1 ) 3635, 3458, 2932, 2860, 1704, 1610, 1452, 1217, 1148, 1112, 1039, 923.LRMS (m/z) 316 (M+, 2.5), 298 (0.3), 284 (10), 267 (10), 266 (15), 256 (8), 255 (33), 254(31), 253 (27), 239 (5), 238 (9), 237 (4), 236 (9), 202 (18), 182 (4), 181 (19), 171 (10), 170(43), 157 (20), 155 (37), 153, (41), 152 (24), 142 (26), 141 (21), 140 (62), 139 (47), 127 (33),126 (26), 125 (76), 124 (31), 121 (30), 115 (30), 113 (39), 112 (34), 111 (50), 110 (24), 109(37), 101 (54), 100 (40), 99 (47), 98 (60), 97 (75), 96 (22), 95 (41), 85 (63), 84 (51), 83 (54), 82(31), 81 (41), 79 (22), 71 (51), 70 (37), 69 (90), 68 (22), 67 (41), 57 (48), 56 (34), 55 (84), 45(100), 43 (76), 42 (43), 41 (60).111HRMS calcd for CrH3205: 316.2241, found: 316.2247.3.4.4 15-Methoxymethoxy-9.11-dioxopentadecanoic Acid (6)O o^o^o oHO^ 0"0'^11041L..^"'0"0-53 6Following the procedure outlined in section 3.2.6, the alcohol 53 (128 mg, 0.40 mmol)was converted to the corresponding acid 6. The crude product was purified by recrystallizationfrom hexanes to give the acid 6 (94 mg, 73%) as a clear solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.34.mp 43.0 °C.1 H NMR (300 MHz, CDC13) 5 15.50 (0.8H, s), 5.49 (0.8H, s), 4.63 (1.6H, s), 4.62 (0.4H,s), 3.57-3.52 (2.4H, m), 3.38 (311, s), 2.59-2.48 (0.8H, dt), 2.36 (211, t), 2.34-2.25 (3.2H,dt), 1.77-1.25 (15H, m).Ht (CHC13, cm-1 ) 3116, 2932, 2860, 1717, 1611, 1461, 1416, 1221, 1148, 1111, 1039, 923.LRMS (m/z) 330 (M+ , 0.5), 312 (0.2), 298 (6), 281 (6), 280 (4), 269 (14), 268 (24), 267(21), 251 (7), 250 (8), 249 (15), 239 (7), 196 (5), 195 (29), 172 (9), 171 (55), 170 (36), 155(24), 153 (38), 140 (55), 127 (18), 126 (19), 125 (79), 113 (25), 112 (19), 111 (34), 101 (44),100 (22), 99 (29), 98 (50), 97 (64), 85 (58), 84 (39), 83 (53), 81 (26), 71 (35), 70 (18), 69(77), 68 (13), 67 (22), 57 (25), 56 (19), 55 (81), 45 (100), 44 (10), 43 (72), 42 (25), 41 (53).HRMS calcd for C17H3006: 330.2034, found: 330.2036.112Elem. Anal. calcd for C17H3006: C, 61.78; H, 9.09. found: C, 61.72; H, 9.08.1133,1^Synthesis of 9.11-Dioxododecanoic Acid (27)O^0 0HOL)L•3.5.1 12-Hydroxyl-2.4-dodecanedione (5410 0^ 0 0TBDMSOW^ HOL)L%22 54Following the procedure outlined in section 3.2.5, the silyl ether 22 (2.01 g, 6.13 mmol)was converted to the alcohol 54. The crude product was purified by column chromatographyusing petroleum ether : ethyl acetate (6:1) as eluent to give the alcohol 54 (1.23 g, 94%) as acolorless solid, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate eluent) 0.50.mp 40.5 °C.1H NMR (400 MHz, CDC13) 8 15.48 (0.8H, s), 5.48 (0.8H, s), 3.65 (2H, t), 3.57 (0.4H, s),2.50, (0.4H, t), 2.27 (1.6H, t), 2.22 (0.6H, s), 2.05 (2.4H, s), 1.66-1.28 (13H, m).IR (CHC13, cm-1 ) 3621, 3460, 2932, 2858, 1720, 1702, 1610, 1440, 1362, 1304, 1157, 1074,1011, 956, 915.LRMS (m/z) 214 (Mt, 2.1), 196 (5), 178 (2), 155 (1), 153 (3), 142 (2), 139 (6), 138 (7), 114(6), 113 (43), 112 (3), 111 (9), 101 (25), 100 (79), 98 (7), 97 (26), 96 (5), 95 (14), 87 (8), 86(18), 85 (94), 84 (25), 83 (17), 82 (9), 81 (17), 72 (38), 71 (19), 70 (10), 69 (62), 68 (12), 67(30), 58 (38), 57 (25), 56 (15), 55 (67), 54 915), 53 (16), 43 (100), 42 (43), 41 (64), 39 (40).HRMS calcd for C12H2203: 214.1563, found: 214.1569.3.5.2 9.11-Dioxododecanoic Acid (27)0 0^ 0^0 0HOC HOL)L54^ 27Following the procedure outlined in section 3.2.6, the alcohol 54 (750 mg, 3.50 mmol)was converted to the corresponding acid 27. The crude product was purified by recrystallizationfrom hexanes to give the acid 27 (480 mg, 60%) as a colorless solid, which was one spot byTLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.48.mp 41.5 °C.1 H NMR (400 MHz, CDC13) 8 15.48 (0.8H, s), 5.48 (0.8H, s), 3.57 (0.4H, s), 2.48 (0.4H,t), 2.39 (2H, t), 2.23 (1.6H, t), 2.21 (0.6H, s), 2.03 (2.4H, s), 1.66-1.28 (11H, m).IR (CHC13, cm-1 ) 3340-2500, 2935, 2860, 1712, 1607, 1460, 1410, 1364, 1290, 1135, 1097,954, 915.LRMS (m/z) 228 (Mt, 0.4), 210 (0.4), 195 (0.3), 193 (0.4), 191 (0.3), 186 (0.6), 184 (0.6),182 (0.5), 171 (3), 169 (3), 168 (9), 141 (1), 140 (5), 113 (3), 112 (3), 111 (35), 101 (3), 100(9), 99 (3), 98 (18), 97 (15), 85 (20), 84 (14), 83 (39), 82 (13), 81 (11), 73 (17), 72 (4), 71 (29),70 (7), 69 (28), 68 (10), 67 (15), 60 (23), 59 (17), 58 (97), 57 (11), 56 (9), 55 (60), 45 (17), 44(14), 43 (100), 42 (12), 41 (27).114HRMS calcd for Ci2H2004: 228.1356, found: 228.1362.1151..6^Synthesis of 9.11-Dioxooctadecanoic Acid (28)0^0 0HO3.6.1 18-(Tent-butyldimethylsilyloxyl-8,10-octadecanedione (55)0 0TBDMSO -N.wi1/4.A.220 0TBDMSO'N'w55Following the procedure outlined in section 3.2.4, the 13-diketone 22 (4.00 g, 12.2 mmol)was alkylated with freshly distilled 1-bromohexane (1.81 g, 11.0 mmol) to give thecorresponding I3-diketone 55. The crude product was purified by column chromatography usingpetroleum ether : ethyl acetate (20:1) as eluent to give the 0-diketone 55 (3.50 g, 85 % based onthe recovered (3-diketone 22) as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.76.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s) 5.48 (0.8H, s), 3.61 (211, t), 3.53 (0.4H, s),2.50 (0.8H, t), 2.27 (3.2H, t), 1.66-1.25 (22H, m), 0.90 (9H, s), 0.88 (311, t), 0.06 (6H, s).IR (neat, cm-1) 2931, 2857, 1711, 1612, 1463, 1392, 1365, 1252, 1101, 837, 776.LRMS (m/z) 397 (M+ - CH3, 2.4), 357 (7), 356 (26), 355 (100), 314 (0.4), 313 (2), 272(0.2), 271 (1), 269 (0.4), 171 (2), 170 (1), 169 (4), 128 (2), 127 (7), 109 (2), 107 (3), 100 (1),99 (1), 97 (2), 95 (3), 93 (2), 89 (2), 85 (4), 83 (2), 82 (1), 81 (4), 77 (2), 76 (1), 75 (18), 73(8), 71 (2), 69 (6), 67 (3), 61 (1), 59 (2), 58 (1), 57 (16), 55 (10), 43 (7), 42 (1) 41 (6).HRMS calcd for C24H48SiO3 - CH3: 397.3126, found: 397.3137.16,2 18-Hydroxyl-8.10-octadecanedione (56) O oTBDmsoC)C.550 0licy■CA../w■56Following the procedure outlined in section 3.2.5, the silyl ether 55 (2.66 g, 6.46 mmol)was converted to the corresponding alcohol 56. The crude product was purified byrecrystallization from hexanes to give the alcohol 56 (1.68 g, 87%) as a colorless solid, whichwas one spot by TLC.Rf ( 1 : 1 petroleum ether : ethyl acetate eluent) 0.62.mp 56.0 °C.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.66 (2H, t), 3.53 (0.4H, s),2.51 (0.8H, t), 2.27 (3.2H, t), 1.66-1.20 (23H, m), 0.88 (3H, t).IR (CHC13, cm-1) 3623, 3460, 2932, 2857, 1724, 1708, 1604, 1460, 1372, 1294, 1146, 1112,917.LRMS (m/z) 298 (Mt, 3.1), 281 (1), 280 (4), 262 (1), 238 (0.6), 237 (1), 215 (1), 214 (6), 198(2), 197 (14), 185 (4), 184 (24), 170 (7), 169 (58), 167 (4), 140 (4), 139 (27), 138 (12), 137 (5),128 (14), 127 (100), 126 (9), 125 (6), 124 (7), 123 (7), 114 (7), 113 (32), 112 (6), 111 (14), 110(5), 109 (14), 101 (9), 100 (88), 99 (5), 98 (9), 97 (31), 96 (5), 95 (10), 85 (32), 84 (11), 83(12), 71 (7), 70 (3), 69 (22), 68 (2), 67 (5), 58 (3), 57 (16), 56 (2), 55 (11).116HRMS calcd for C18H3403: 298.2499, found: 298.2499.3.6.3 9. 11-Dioxooctadecanoic Acid (28)0 0^ 0^0 0Ho-.^H014.1../..5 6 28Following the procedure outlined in section 3.2.6, the alcohol 56 (850 mg, 2.85 mmol)was converted to the corresponding acid 28. The crude product was purified by recrystallizationfrom hexanes to give the acid 28 (500 mg, 56%) as a clear solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.55.mp 57.0 °C.1 H NMR (300 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 3.53 (0.4H, s),2.50 (0.8H, t), 2.36 (2H, t), 2.28 (3.2H, t), 1.70-1.24 (21H, m), 0.90 (3H, t).IR (CHC13, cm -1 ) 3310-2480, 2932, 2859, 1707, 1609, 1466, 1407, 1290, 1132, 951, 916.IR (KBr, cm-1 )^3543, 3483, 2931, 2849, 1719, 1688, 1642, 1461, 1440, 1421, 1310, 1237,1188, 1138, 903.LRMS (m/z) 312 (Mt, 5.9), 295 (1), 294 (3), 277 (0.5), 276 (2), 267 (0.6),248 (1), 229 (3), 228 (21), 211 (2), 210 (9), 198 (2), 197 (13), 196 (4), 195266 (2), 249 (0.2),(26), 185 (4), 184(22), 183 (3), 182 (13), 172 (7), 171 (77), 170 (9), 169 (71), 168 (4), 154 (2), 153 (11), 152(11), 151 (8), 128 (10), 127 (76), 126 (12), 125 (33), 124 (11), 114 (6), 113 (30), 112 (6), 111(16), 101 (10), 100 (100),99 (7), 98 (17), 97 (40), 96 (4), 95 (7), 86 (4), 85 (62), 84 (20), 83(32), 82 (6), 81 (14), 71 (15), 70 (5), 69 (37), 68 (5), 67 (12), 57 (54), 55 (49), 43 (40), 41 (21).HRMS calcd for C18H3204: 312.2292, found: 312.2293.11 7Elem. Anal. calcd for C18H3204: C, 69.23; H, 10.33. found: C, 69.22; H, 10.33.118^3,2 Synthraisgf2,11- ^ijactssraggstuskAciaa9a0^0 0HO3.9.1 1-(Tert-butyldimethylsilyloxy)-9.11-eicosanedione (57)0 0^ 0 0^TBDMSOw^TBDMS0""."."..A^2 2 57Following the procedure outlined in section 3.2.4, the I3-diketone 22 (1.97 g, 6.0 mmol)was alkylated with freshly distilled 1-bromooctane (1.04 g, 5.4 mmol) to give the 13-diketone 57.The crude product was purified by column chromatography using petroleum ether : ethyl acetate(20:1) as eluent to give the 13-diketone 57 (1.52 g, 73 % based on the recovered t3-diketone 22)as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.78.1 H NMR (400 MHz, CDC13) 5 15.50 (0.811, s), 5.48 (0.811, s), 3.61 (2H, t), 3.54 (0.411, s),2.50 (0.8H, t), 2.27 (3.2H, m), 1.66-1.25 (26H, m), 0.90 (9H, s), 0.88 (3H, t), 0.07 (6H, s).IR (neat, cm-1) 2930, 2856, 1707, 1611, 1463, 1366, 1253, 1100, 1044, 943, 837, 776.LRMS (m/z) 440 (Mt, 0.5), 386 (7), 385 (39), 384 (90), 383 (100), 366 (0.7), 365 (3), 326298 (0.5), 297 (2), 284 (1), 283 (7), 272 (2), 271 (11), 230 (3), 229 (15), 172 (3), 171 (16),170 (4), 169 (16), 157 (9), 156 (9), 155 (52), 132 (2), 131 (10), 129 (11), 128 (6), 116 (5), 115(27), 110 (2), 109 (12), 108 (4), 107 (32), 102 (3), 101 (22), 100 (12), 99 (14), 98 (6), 97 (27),95 (34), 90 (2), 89 (23), 88 (3), 87 (7), 86 (5), 85 (62), 84 (10), 83 (20), 82 (6), 81 (32), 76(11), 75 (86), 74 (7), 73 (64), 72 (4), 71 (49), 70 (6), 69 (51).HRMS calcd for C26H52SiO3: 440.3672, found: 440.3654.3.7.2 1-Hydroxyl-9.11-eicosanedione (58)O oTBDmso-•wA.)570 0HO58Following the procedure outlined in section 3.2.5, the silyl ether 57 (1.40 g, 3.2 mmol)was converted to the corresponding alcohol 58. The crude product was purified byrecrystallization from hexanes to give the alcohol 58 (940 g, 91%) as a colorless solid, which wasone spot by TLC.Rf (1: 1 petroleum ether : ethyl acetate eluent) 0.65.mp 64.5 °C.1H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.66 (2H, t), 3.54 (0.4H, s),2.50 (0.8H, t), 2.27 (3.2H, t), 1.64-1.22 (27H, m), 0.88 (3H, t).IR (cHc13, cm-1 ) 3622, 2930, 2857, 1724, 1700, 1608, 1460, 1364, 1308, 1047,LRMS (m/z) 326 (M+, 3.5), 309 (2), 308 (9), 291 (0.6), 290 (3), 279 (0.7), 278905.(2), 266 (0.6),265 (2), 254 (0.5), 253 (0.6), 252 (2), 227 (2), 226 (2), 225 (8), 215 (1), 214 (7), 213 (2), 212(8), 199 (6), 198 (5), 197 (37), 196 (12), 195 (3), 194 (16), 158 (1), 157 (7), 156 (10), 155 (42),142 (6), 141 (2), 140 (4), 139 (33), 138 (30), 137 (10), 136 (20), 135 (10), 125 (6), 124 (10),123 (5), 122 (3), 121 (22), 120 (8), 119 (2), 114 (8), 113 (23), 112 97), 111 (17), 109 (11), 108(5), 107 (4), 101 (9), 100 (100), 99 (6), 98 (11), 97 (38), 96 (8), 95 (27), 94 (11), 93 (9), 86 (4),85 (38), 84 (16), 83 (18), 82 (6), 81 (29), 71 (48), 70 (7), 69 (75), 67 (19), 57 (40), 55 (67).119HRMS calcd for C20113803: 326.2811, found: 326.2814.3.7.3 9,11-Dioxoeicosanoic Acid (29)0 0^ 0^0 0H0-......./......"...".................^Holk./../...,"..).CA...........58 29Following the procedure outlined in section 3.2.6, the alcohol 58 (630 mg, 2.11 mmol)was converted to the corresponding acid 29. The crude product was purified by recrystallizationfrom hexanes to give the acid 29 (520 mg, 79%) as a clear solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.56.mp 64.0 °C.1 H NMR (300 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.53 (0.4H, s),2.49 (0.8H, t), 2.36 (2H, t), 2.28 (3.2H, t), 1.70-1.22 (25H, m), 0.90 (3H, t).ER (CHC13, cm-1 ) 3355-2490, 2931, 2858, 1708, 1606, 1459, 1413, 1292. 1134, 1104, 953.LRMS (m/z) 340 (Mt, 2.8), 323 (1), 322 (3), 305 (0.7), 304 (1), 295 (0.5), 294 (2), 282 (0.7),281 (1), 229 (3), 228 (17), 226 (2), 225 (12), 198 (9), 197 (61), 196 (5), 195 (31), 183 (2), 182(14), 172 (10), 171 (100), 170 (3), 169 (8), 168 (4), 167 (11), 156 (10), 155 (70), 154 (7), 153(13), 144 (2), 143 (17), 141 (4), 140 (5), 127 (7), 126 (8), 125 (45), 124 (11), 115 (3), 114 (6),113 (26), 102 (1), 101 (11), 100 (94), 99 (6), 98 (20), 97 (53), 96 (5), 95 (14), 93 (5), 86 (4), 85(45), 84 (23), 83 (41), 82 (8), 81 (19), 80 (4), 71 (46), 70 (8), 69 (29), 68 (7), 67 (12), 58 (10),57 (34), 56 (8), 55 (65).HRMS calcd for C201-13604: 340.2604, found: 340.2620.120Elem. Anal. calcd for C20H3604: C, 70.59; H, 10.58. found: C, 70.32; H, 10.66.1216.^Synthesis of 9.11-Dioxodoeicosanoic Acid (30)0^0 0HO3.8.1 1-(Tert-butyldimethylsilyloxy)-9,11-doeicosanedione (59)0 0TBDMSOC220 0TBDMSO59Following the procedure outlined in section 3.2.4, the I3-diketone 22 (1.67 g, 5.1 mmol)was alkylated with freshly distilled 1-bromodecane (1.12 g, 5.1 mmol) to give the 13-diketone 59.The crude product was purified by column chromatography using petroleum ether : ethyl acetate(20:1) as eluent to give the (3-diketone 59 (0.95 g, 70% based on the recovered 13-diketone 22) asa clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.80.1H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 3.60 (2H, t), 3.54 (0.4H, s),2.50 (0.8H, t), 2.27 (3.2H, m), 1.66-1.22 (30H, m), 0.90 (9H, s), 0.89 (3H, t), 0.07 (6H, s).IR (neat, cm-1) 2928, 2856, 1711, 1611, 1463, 1390, 1365, 1252, 1101, 1011, 944, 837.LRMS (m/z) 467 (M+, 0.7), 453 (2), 439 (0.4), 412 (33), 413 (7), 412 (33), 411 (100), 384(8), 383 (36), 313 (2), 283 (2), 272 (0.5), 271 (4), 263 (0.4), 262 (2), 230 (0.6), 229 (2), 187(1), 185 (1), 183 (6), 181 (2), 171 (3), 170 (1), 169 (3), 157 (2), 156 (1), 155 (5), 110 (1), 109(5), 108 (2), 107 (10), 100 (3), 99 (5), 98 (2), 97 (8), 96 (1), 95 (10), 85 (14), 84 (2), 83 (10),76 (4), 75 (50), 74 (3), 73 (19), 59 (5), 58 (5), 57 (18), 56 (7), 55 (27).HRMS calcd for C281-156S103: 467.3906, found: 467.3960.3.8.2 1-Hydroxyl-9.11-doeicosanedione (60)0 0650 060Following the procedure outlined in section 3.2.5, the silyl ether 59 (870 mg, 1.86 mmol)was converted to the corresponding alcohol 60. The crude product was purified byrecrystallization from hexanes to give the alcohol 60 (560 mg, 85%) as a colorless solid, whichwas one spot by TLC.RR (1:1 petroleum ether : ethyl acetate eluent) 0.68.mp 70.5 °C.1H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.47 (0.8H, s), 3.64 (2H, t), 3.54 (0.4H, s),2.50 (0.8H, t), 2.24 (3.2H, t), 1.64 - 1.22 (31H, m), 0.89 (3H, t).IR (CHC13, cm-1 ) 3623, 3458, 2929, 2857, 1607, 1459, 1380, 1285, 1148, 1053, 947, 898.LRMS (m/z) 354 (M+, 0.9), 337 (1), 336 (4), 319 (0.2), 318 (1), 294 (0.4), 293 (1), 279 (0.5),278 (3), 255 (0.5), 254 (1), 253 (5), 252 (2), 227 (3), 226 (4), 225 (22), 223 (2), 222 (9), 198(1), 197 (3), 196 (8), 184 (6), 183 (23), 143 (1), 142 (7), 141 (5), 140 (6), 139 (29), 138 (29),137 (9), 136 (5), 125 (7), 124 (11), 123 (9), 122 (4), 121 (24), 120 (8), 115 (5), 114 (8), 113(23), 112 (5), 111 (18), 110 (8), 109 (31), 108 (7), 101 (9), 100 (100), 99 (9), 98 (13), 97 (44),96 (10), 95 (35), 86 (4), 85 (38), 84 (18), 83 (32), 82 (9), 81 (32), 72 (4),71 (48), 70 (9), 69(64), 67 (30), 58 (13), 57 (69), 56 (11), 55 (72), 43 (59), 42 (9), 41 (58).122TBDMSOHOHRMS calcd for C22H4203: 354.3123, found: 354.3132.3.8.3 9.11-Dioxodoeicosanoic Acid (30)1230 06 00 0HOOHO3 0Following the procedure outlined in section 3.2.6, the alcohol 60 (450 mg, 1.27 mmol)was converted to the acid 30. The crude product was purified by recrystallization from hexanesto give the acid 30 (140 mg, 34%) as a colorless solid, which was one spot by TLC.14 (5% HOAc in 3:1 petroleum ether : ethyl acetate eluent) 0.57.mp 69.5 °C.1 H NMR (300 MHz, CDC13) 5 15.50 (0.8H, s), 5.48 (0.8H, s), 3.54 (0.4H, s),2.50 (0.8H, t), 2.36 (2H, t), 2.26 (3.2H, t), 1.72-1.22 (29H, m), 0.89 (3H, t).IR (CHC13, cm-1 ) 3370-2510, 2929, 2857, 1718, 1607, 1460, 1408, 1292, 1135, 1090, 952.LRMS (m/z) 368 (Mt, 9.8), 351 (3), 350 (10), 333 (1), 332 (3), 323 (0.7), 322 (3), 305 (0.4),304 (2), 280 (0.6), 279 (1), 254 (2), 253 (11), 241 (4), 240 (10), 229 (5), 228 (36), 226 (8), 225(51), 211 (4), 210 (17), 196 (4), 195 (32), 184 (5), 183 (32), 182 (18), 172 (9), 171 (83), 151(5), 150 (24), 149 (5), 126 (10), 125 (35), 124 (12), 114 (5), 113 (22), 112 97), 111 (18), 110(6), 109 (12), 108 (6), 107 (11), 101 (10), 100 (100), 99 (8), 98 (18), 97 (48), 96 (5), 95 (17), 85(33), 84 (21), 83 (40), 71 (34), 70 (7), 69 (27), 58 (10), 57 (54), 56 (10), 55 (86).HRMS calcd for C22114004: 368.2916, found: 368.2935.Elem. Anal. calcd for C22H4004: C, 71.74; H, 10.87. found: C, 71.20; H, 10.94.1243,2^Synthesis of 8.10-Octadecanedione (31)0 03,11 2.4-Undecanedione (61)7 6 1Following the procedure outlined in section 3.2.3, 2,4-pentanedione (7) (4.10 mL, 40.0mmol) was alkylated with 1-bromohexane (4.50 mL, 32.0 mmol) to give the P-diketone 61.Distillation of the crude product under reduced pressure (155 °C/50 torr) gave the purified13-diketone 61 (3.70 g, 63 %) as a colorless oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.60.1 H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.49 (0.8H, s), 3.58 (0.4H, s), 2.51 (0.4H,t), 2.27 (1.6H, t), 2.26 (0.6H, s), 2.06 (2.4H, s), 1.65-1.20 (10H, m), 0.89 (3H, t).IR (neat, cm-1 ) 2930, 2858, 1711, 1613, 1446 ,1364, 1254, 947.LRMS (m/z) 184 (M+, 20), 170 (1), 169 (10), 167 (1),127 (24), 126 (8), 114 (9), 113 (70), 101 (28), 100 (33),(37), 84 (17), 83 (10), 82 (10), 81 (7), 73 (2), 72 (44), 71166 (10), 143 (1), 142 (15), 128 (8),99 (5), 98 (11), 97 (8), 86 (15), 85(2), 70 (4), 69 (17), 68 (3), 67 (13),59 (8), 58 (54), 57 (100), 56 (9), 55 (25), 44 (8), 43 (50), 42 (16), 41 (53).HRMS calcd for C111-12002: 184.1458, found: 184.1459.3.9.2 8.10-Octadecaneclione (31)1)(361 0 012531Following the procedure outlined in section 3.2.4, the 13-diketone 61 (756 mg, 4.11mmol) was alkylated with 1-bromoheptane (660 mg, 3.69 mmol) to give the 13-diketone 30.Purification of the crude product by column chromatography using petroleum ether : ethyl acetate(20:1) gave the desired f3-diketone 31 (803 mg, 77%) as a colorless oil, which was one spot byTLC.Rf (6:1 petroleum ether : ethyl acetate eluent) 0.78.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.53 (0.4H, s), 2.50 (0.8H,t), 226 (3.2H, t), 1.64 - 1.22 (22H, m), 0.89 (6H, t).IR (neat, cm-1 ) 2934, 2857, 1704, 1613, 1455, 1276, 1142, 1102, 942.LRMS (m/z) 282 (Mt, 4.6), 265 (0.7), 264 (3), 241 (0.3), 240 (2), 227 (0.2), 226 (2), 212(2), 211 (10), 199 (3), 198 (14), 197 (110, 185 (4), 184 (24), 183 (36), 170 (6), 169 (42), 142(20), 141 (80), 139 (3), 138 (6), 128 (13), 127 (100), 126 (9), 114 (6), 113 (38), 112 (4), 111(6), 101 (7), 100 (97), 99 (7), 98 (12), 97 (16), 86 (3), 85 (52), 84 (14), 71 (30), 70 (4), 69(14), 58 (5), 57 (36), 56 (4), 55 (14).HRMS calcd for C18H3402: 282.2550, found: 282.2565.Elem. Anal. calcd for C18113402: C, 76.52; H, 12.14. found: C, 76.80; H, 12.16.,1111 Synthesis of 7.9-Dioxooctadecanoic Acid (33)0^0 0HO3.10.1 5-Bromo-l-pentanol (62)HO^OH HO/W Br15 62Following the procedure outlined in section 3.2.1, 1,5-pentanediol (15) (15.0 g,140 mmol) was converted to the corresponding bromide 62. The crude product was purified bycolumn chromatography using petroleum ether : ethyl acetate (6:1) as eluent to give themonobrominated alcohol 62 (12.4 g, 52%) as a colorless liquid, which was one spot by TLC.Rf ( 1 :1 petroleum ether : ethyl acetate) 0.46.1 H NMR (400 MHz, CDC13) 5 3.70 (2H, t), 3.42 (2H, t), 2.72 (1H, s), 1.921.48 (4H, m).(2H, p), 1.64 -IR (neat, cm-1 ) 3346, 2936, 2865, 1456, 1434, 1274, 1238, 1137, 1061, 1014, 984, 950, 734.LRMS (m/z) 167 (Br81 : M+ - H, 0.1), 165 (Br79 : M+ - H, 0.1), 137 (3), 135 (3), 123 (0.3),121 (0.3), 109 (2), 107 (2), 97 (5), 87 (1), 86 (2), 85 (4), 84 (1), 79 (4), 71 (2), 70 (8), 69(100), 68 (18), 67 (10), 57 (15), 56 (16), 55 (40), 45 (8), 44 (7), 43 (17), 42 (17), 41 (73).HRMS calcd for C5H11Br810 - H: 166.9892, found: 166.9902; calcd for C5Hi1Br790 - H:164.9912, found: 164.9911.1263.10.2 1-Diamozatuautliglimrayisity_loLylpsatagffil( -HOW Br^ TBDMS OW Br62^ 63Following the procedure outlined in section 3.2.2, the alcohol 62 (2.90 g, 17.4 mmol)was converted to the corresponding ether 63. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (15:1) as eluent to give the silyl ether 63(3.81 g, 81%) as a colorless oil, which was one spot by TLC.RI, (6:1 petroleum ether : ethyl acetate) 0.80.1 H NMR (400 MHz, CDC13) 8 3.61 (2H, t), 3.41 (2H, t), 1.88 (2H, p), 1.58-1.45 (4H, m),0.90 (9H, s), 0.07 (6H, s).IR (neat, cm-1 ) 2940, 2858, 1467, 1388, 1361, 1254, 1102, 1006, 835, 775.LRMS (m/z) 281 (Br81 : M+ - H, 0.2), 227 (0.4), 225 (0.4), 201 (0.1), 181 (0.1), 171 (0.2),169 (2), 167 (2), 139 (3), 137 (3), 89 (2), 87 (1), 85 (2), 81 (2), 77 (1), 76 (1), 75 (16), 73(11), 71 (2), 70 (6), 69 (100), 68 (2), 67 (3), 59 (5), 58 (2), 57 (5), 56 (3), 55 (5), 53 (1), 43(5), 42 (3), 41 (25).HRMS calcd for C11H25SiBr810 - H: 281.0753, found: 281.0527; calcd for Ci11125SiBr 790 -H: 279.0771, found: 279.0772.1273.10.3 10-(Tert-butyldimethylsilyloxy)-2.4-decanedione (64)0 07 0 0TBDMSOL)L64Following the procedure outlined in section 3.2.3, 2,4-pentanedione (7) (1.72 g,17.2 mmol) was converted to the 13 -diketone 64. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (15:1) as eluent to give the I3-diketone 64(2.12 g, 60% based on the recovered bromide), which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.56.1H NMR (400 MHz, CDC13) 8 15.50 (0.8 H, s), 5.49 (0.8 H, s), 3.60 (2H, t), 3.59 (0.4 H,s), 2.52 (0.4 H, t), 2.26 (1.6 H, t), 2.24 (0.6 H, s), 2.07 (2.4 H, s), 1.68-1.25 (8H, m), 0.90(9H, s), 0.06 (6H, s).IR (neat, cm-1 ) 2936, 2858, 1716, 1614, 1465, 1361, 1252, 1100, 1005, 936, 837, 775.LRMS (m/z) 285 (M+ - CH3, 7.5), 246 (1), 245 (10), 244 (40), 243 (100), 227 (2), 225 (2),186 (3), 185 (20), 171 (4), 170 (2), 169 (13), 167 (3), 159 (2), 158 (2), 157 (7), 156 (3), 155(7), 145 (5), 144 (2), 143 (11), 142 (1), 141 (3), 118 (1), 117 (11), 116 (3), 115 (29), 102 (2),101 (14), 100 (5), 99 (9), 97 (4), 95 (6), 87 (11), 85 (53), 83 (14), 82 (2), 81 (14), 79 (10), 78(2), 77 (30), 76 (19), 75 (97), 74 (10), 73 (81), 69 (31), 61 (22), 60 (5), 59 (36), 58 (13), 57(17), 56 (9), 55 (58), 45 (22), 44 (6), 43 (90), 42 (7), 41 (59).HRMS calcd for C1a132SiO3 - CH3: 285.1879, found: 285.1884.1283.10.4 1-(Tert-butildimethylsilyloxy1-7.9-octadecanedione (65)0 0^ 0 0TBDMSOW'•.)C)L•^TBDMSO64^65Following the procedure outlined in section 3.2.4, the 13 -ciiketone 64 (1.2 g, 4.2 mmol)was alkylated with freshly distilled 1-bromooctane (0.77 g, 4.0 mmol) to give the 13-diketone 65.The crude product was purified by column chromatography using petroleum ether : ethyl acetate(20:1) as eluent to give the 13-dilcetone 65 (0.77 g, 75 % based on the recovered 0-diketone 64)as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.74.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.61 (2H, t), 3.53 (0.4H, s),2.50 (0.8H, dt), 2.27 (3.2H, dt), 1.66-1.22 (22H, m), 0.90 (9H, s), 0.89 (3H, t), 0.06 (6H, s).IR (neat, cm-1 ) 2930, 2856, 1722, 1707, 1611, 1463, 1387, 1360, 1252, 1101, 1006, 937,836, 812, 775.LRMS (m/z) 412 (Mt, 0.1), 398 (0.7), 397 (2), 357 (7), 356 (26), 355 (100), 297 (1), 283 (2),282 (1), 281 (4), 271 (1), 270 (3), 269 (14), 257 (2), 256 (1), 255 (7), 187 (1), 186 (2), 185(8), 171 (4), 170 (9), 169 (11), 167 (3), 157 (4), 156 (3), 155 (12), 114 (5), 113 (6), 111 (7),110 (5), 109 (10), 86 (3), 85 (25), 84 (4), 83 (15), 77 (14), 76 (6), 75 (73), 74 (3), 73 (20), 72(5), 71 (31), 70 (6), 69 (48), 57 (34), 56 (7), 55 (33), 43 (33), 42 (6), 41 (41).HRMS calcd for C24H48SiO3: 412.3360, found: 412.3367.1293.10.5 1-Hydroxyl-7.9-octadecanedione (66)0 0TBDmso••••.."■,.."....A....k.."...--s.."../■650 0Hoos../■/■..A.)66Following the procedure outlined in section 3.2.5, the silyl ether 65 (640 mg, 1.55 mmol)was converted to the alcohol 66. The crude product was purified by recrystallization fromhexanes to give the alcohol 66 (410 mg, 89%) as a colorless solid, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate) 0.56.mp 57.5 °C.1 H NMR (400 MHz, CDC13) 8^15.50 (0.8H, s) 5.48 (0.8H, s), 3.64 (2H, t), 3.55 (0.4H, s),2.51 (0.8H, dt), 2.27 (3.2H, dt), 1.70-1.18 (23H, m), 0.89 (3H, t).IR (CHC13, cm-1 ) 3623, 2930, 2858, 1607, 1459, 1329, 1141, 1052, 965, 904.LRMS (m/z) 298 (M+, 1.0), 281 (1), 280 (3), 226 (2), 225 (10), 213 (2), 212 (8), 198 (7), 197(56), 187 (1), 186 (12), 170 (3), 169 (5), 168 (11), 157 (2), 156 (15), 155 (86), 154 (7), 153(21), 130 (4), 129 (39), 128 (6), 127 (34), 114 (6), 113 (33), 112 (10), 111 (56), 101 (8), 100(100), 99 (10), 98 (15), 97 (27), 96 (5), 95 (18), 86 (5), 85 (54), 84 (22), 83 (57), 82 (10), 81(46), 72 (5), 71 (58), 70 (10), 69 (75), 58 (15), 57 (42), 56 (8), 55 (77), 43 (47), 42 (7), 41 (26).130HRMS calcd for CigH3403: 298.2499, found: 298.2505.3.10.6 7,9-Dioxooctadecanoic Acid (33)0 0^ 0^0 0HoW■. Hok■••■••■)66 33Following the procedure outlined in section 3.2.6, the alcohol 66 (298 mg, 1.00 mmol)was converted to the corresponding acid 33. The crude product was purified by recrystallizationfrom hexanes to give the acid 33 (210 mg, 67%) as a clear solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate) 0.52.mp 58.0 °C.1 H NMR (300 MHz, CDC13) 8 15.50 (0.8H, s), 5.49 (0.8H, s), 3.53 (0.4H, s),2.51 (0.8H, dt), 2.36 (2H, t), 2.28 (3.2H, dt), 1.76-1.22 (21H, m), 0.90 (3H, t).IR (CHC13, cm-1 ) 3340-2490, 2930, 2858, 1709, 1607, 1460, 1409, 1291, 1131, 950, 907.LRMS (m/z) 312 (Mt, 4.1), 295 (1), 294 (4), 277 (2), 276 (4), 252 (1), 251 (2), 226 (1), 225(8), 201 (4), 200 (30), 198 (9), 197 (53), 196 (2) 195 (9), 183 (4), 182 (27), 169 (4), 168 (6),167 (32), 156 (8), 155 (49), 154 (9), 153 (6), 145 (2), 144 (8), 143 (100), 141 (9), 140 (28), 126(9), 125 (88), 115 (9), 114 (13), 113 (20), 101 (10), 100 (51), 99 (14), 98 (32), 97 (61), 96 (12),95 (24), 86 (7), 85 (42), 84 (23), 83 (26), 82 (8), 81 (24), 73 (26), 72 (4), 71 (53), 70 (20), 69(85), 58 (14), 57 (38), 56 (11), 55 (78), 45 (7), 44 (33), 43 (46), 42 (12), 41 (27).HRMS calcd for C18H3204: 312.2292, found: 312.2291.131Elem. Anal. calcd for C18H3204: C, 69.23; H, 10.33. found: C, 69.24; H, 10.33.I. Synthesis of 11.13-Dioxooctadecanoic Acid (34)^0^0 0HO3.11.1 9-Bromo- l -nonanol (67) ^HO^OH^HO WW Br68^ 67Following the procedure outlined in section 3.2.1, 1,9-nonanediol (68) (10.0 g,62.5 mmol) was converted to the corresponding bromide 67. The crude product was purified bycolumn chromatography using petroleum ether : ethyl acetate (6:1) as eluent to give themonobrominated alcohol 67 (9.78 g, 70%) as a colorless solid, which was one spot by TLC.Rf ( 1 :1 petroleum ether : ethyl acetate) 0.58.mp 34.0 °C.1 H NMR (400 MHz, CDC13) 8 3.64 (2H, t), 3.42 (2H, t), 1.83 (211, p), 1.58 (211, p), 1.48 -1.30 (11H, m).IR (CHC13, cm-1): 3622, 3460, 2929, 2857, 1459, 1386, 1352, 1274, 1111, 1044, 889.LRMS (m/z) 206 (Br81 : M+ - H2O, 0.1), 204 (Br79 : M+ - H2O, 0.1), 178 (12), 176 (12), 164(21), 162 (22), 150 (35), 148 (36), 137 (50), 135 (50), 109 (10), 107 (10), 98 (11), 97 (68), 96(11), 95 (18), 84 (10), 83 (63), 82 (42), 81 (40), 71 (7), 70 (40), 69 (88), 68 (50), 67 (39), 57(43), 56 (48), 55 (100), 54 (38), 53 (23), 45 (9), 44 (13), 43 (62), 42 (51), 41 (86).HRMS calcd for C9H19Br810 - H2O: 206.0489, found: 206.0494; calcd for C9H19Br790 -H20: 204.0514, found: 204.0509.1323.11.2 1-Bromo-9-ftert-butyldimethylsilyloxy)-nonane (69)HO^Br^TBDMSOMBr67^ 69Following the procedure outlined in section 3.2.2, the alcohol 67 (2.66 g, 11.9 mmol)was converted to the corresponding ether 69. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (15:1) as eluent to give the silyl ether 69(3.60 g, 90%) as a colorless oil, which was one spot by TLC.It; (6:1 petroleum ether : ethyl acetate) 0.84.1H NMR (400 MHz, CDC13) 5 3.60 (2H, t), 3.41 (2H, t), 1.86 (2H, p), 1.54 - 1.26 (12H, m),0.89 (9H, s), 0.05 (6H, s).IR (neat, cm-1) 2933, 2856, 1466, 1387, 1360, 1252, 1101, 1006, 839, 775, 717.LRMS (m/z) 281 (Br81 : M+ - C4H9, 0.5), 279 (Br79 : M+ - C4H9, 0.5), 241 (0.3), 215 (0.1),200 (2), 199 (13), 169 (14), 167 (14), 139 (15), 137 (14), 125 (11), 115 (9), 101 (14), 99 (7),97 (1), 84 (10), 83 (97), 82 (3), 81 (14), 76 (9), 75 (92), 74 (4), 73 (36), 70 (13), 69 (100), 67(11), 59 (10), 57 (25), 55 (55).HRMS calcd for C15H33SiBr 81 0 - C4H9: 281.0173, found: 281.0748; calcd forCi5H33SiBr790 - C4H9: 279.0193, found: 279.0786.1333.11.3 14-(Tert-butyldimethylsilyloxy)-2.4-tetradecanedione (7o)0 0pp7 0 0TBDMSO )C7 0Following the procedure outlined in section 3.2.3, 2,4-pentanedione (7) (0.98 g,9.4 mmol)was converted to the f3-diketone 70. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (15:1) as eluent to give the 13-diketone 70(2.12 g,79% based on the recovered bromide), which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.66.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.49 (0.8H, s), 3.59 (211, t), 3.57 (0.4H, s),2.49 (0.4H, t), 2.27 (1.6H, t), 2.24 (0.6H, s), 2.06 (2.4H, s), 1.65-1.24 (16H, m), 0.89 (9H,s), 0.06 (6H, s).IR (neat, cm-1 ) 2932, 2856, 1709, 1616, 1465, 1387, 1361, 1251, 1100, 1006, 940, 838, 813,776.LRMS (m/z) 355 (M+ - H, 0.1), 301 (6), 300 (25), 299 (100), 298 (0.1), 281 (0.1), 169 (4),157 (2), 145 (1), 143 (3), 117 (0.7), 115 (4), 101 (3), 99 (2), 97 (1), 95 (4), 93 (2), 89 (3), 85(14), 83 (1), 81 (6), 77 (5), 76 (2), 75 (46), 73 (13), 69 (8), 67 (6), 59 (6), 55 (14), 43 (29), 41(15).HRMS calcd for C20F140SiO3 - H: 355.2658, found: 355.2676.1343.11.4 18-(Tert-butyldimethylsilyloxy)-6. 8-octadecanedione (7110 0TBDMSO ks)700 0TBDMSO%71Following the procedure outlined in section 3.2.4, the 13-diketone 70 (1.2 g, 3.2 mmol)was alkylated with freshly distilled 1-bromobutane (0.60 g, 3.2 mmol) to give the 13-diketone 71.The crude product was purified by column chromatography using petroleum ether : ethyl acetate(20:1) as eluent to give the 13-diketone 71 (0.65 g, 77% based on the recoveredfl-diketone 70) asa clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.77.1 H NMR (400 MHz, CDC13) 8 15.52 (0.8H, s), 5.49 (0.8H, s), 3.61 (2H, t), 3.54 (0.4H, s),2.49 (0.8H, t), 2.27 (3.2H, m), 1.66-1.22 (22H, m), 0.90IR (neat, cm-1 )^2934, 2856, 1730, 1707, 1612, 1463,838.(12H, s), 0.06 (6H, s).1386, 1361, 1252, 1100, 1006, 940,LRMS (m/z) 411 (M+ - H, 0.2), 399 (0.2), 398 (1), 397 (3), 357 (8), 356 (30), 355 (100), 341(2), 339 (2), 337 (2), 300 (0.4), 299 (2), 279 (0.6), 278 (1), 262 (2), 242 (0.5), 241 (3), 226(2), 225 (7), 170 (2), 169 (11), 168 (1), 167 (3), 159 (2), 157 (3), 156 (3), 155 (3), 149 (8),147 (8), 130 (2), 129 (28), 112 (8), 111 (12), 100 (7), 99 (25), 98 (2), 97 (12), 86 (2), 85 (16),84 (7), 83 (19), 76 (6), 75 (64), 74 (2), 73 (19), 72 (4), 71 (42), 58 (4), 57 (33), 56 (10), 55(36), 45 (10), 44 (3), 43 (36), 42 (7), 41 (33).135HRMS calcd for C24H48SiO3 - H: 411.3282, found: 411.3287.3.11.5 18-Hydroxyl-6.8-octadecanedione (72)0 0TBDA4soWL../■/■710 0HoW■/■/\)C,) ,../N,72Following the procedure outlined in section 3.2.5, the silyl ether 71 (360 mg, 0.87 mmol)was converted to the alcohol 72. The crude product was purified by recrystallization fromhexanes to give the alcohol 72 (250 mg, 96%) as a colorless solid, which was one spot by TLC.RR (1:1 petroleum ether : ethyl acetate) 0.66.mp 56.0 °C.1 H NMR (400 MHz, CDC13) 6 15.50 (0.8H, s), 5.48 (0.8H, s), 3.64 (2H, t), 3.53 (0.4H, s),2.51 (0.8H, t), 2.27 (3.2H, t), 1.64 - 1.22 (23H, m), 0.89 (3H, t).IR (CHC13, cm-1 ) 3621, 3456, 2974, 2931, 1700, 1608, 1455, 1390, 1047.LRMS (m/z) 298 (Mt, 5.4), 281 (3), 280 (12), 263 (1), 262 (3), 243 (0.7), 242 (4), 227 (5),225 (3), 224 (10), 210 (1), 209 (7), 195 (1), 194 (6), 170 (3), 169 (21), 168 (2), 167 (15), 166(8), 157 (9), 156 (49), 150 (3), 149 (23), 148 (9), 147 (3), 142 (9), 141 (77), 139 (7), 138 (26),101 (8), 100 (96), 99 (100), 98 (10), 97 (26), 87 (4), 85 (22), 84 (11), 83 (23), 72 (4), 71 (35),70 (6), 69 (28), 58 (7), 57 (8), 56 (5), 55 (38), 43 (45), 42 (5), 41 (29).136HRMS calcd for C18H3403: 298.2499, found: 298.2504.3.11.6 11. 13-Dioxooctadecanoic Acid (34)0 0^O^0 0HO^ Hco)L/*■/■./■)L.)C//'■7 2^ 34Following the procedure outlined in section 3.2.6, the alcohol 72 (200 mg, 0.67 mmol)was converted to the acid 34. The crude product was purified by recrystallization from hexanesto give the acid 34 (130 mg, 64%) as a colorless solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate) 0.56.mp 56.5 °C.1 H NMR (300 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.55 (0.4H, s),2.50 (0.8H, t), 2.35 (2H, t), 2.27 (3.2H, t), 1.70-1.22 (21H, m), 0.90 (3H, t).IR (CHC13, cm-1 ) 3350-2500, 2933, 2859, 1710, 1605, 1458, 1395, 1281, 1138, 1047, 951,879.LRMS (m/z) 312 (Mt, 6.1), 296 (0.8), 295 (5), 278 (0.4), 277 (2), 256 (8), 239 (6), 224 (4),223 (26), 200 (5), 199 (33), 157 (7), 156 (39), 142 (11), 141 (65), 139 (11), 138 (18), 137 (4),136 (4), 135 (21), 114 (13), 113 (27), 112 (13), 111 (23), 101 (10), 100 (68), 99 (72), 98 (38),97 (47), 96 (7), 95 (22), 93 (12), 85 (43), 84 (42), 83 (38), 82 (7), 81 (28), 73 (25), 72 (9), 71(75), 69 (85), 68 (12), 67 (36), 60 (37), 59 (12), 58 (24), 57 (51), 56 (23), 55 (88), 45 (36), 44(17), 43 (100), 42 (52), 41 (81).HRMS calcd for C18I-13204: 312.2292, found: 312.2294.Elem. Anal. calcd for C18H3204: C, 69.23; H, 10.33. found: C, 68.94; H, 10.25.137LIZ Synthesis of 8.10-Dioxooctadecanoic Acid (35)138 0HO0 03.12.1 6-Bromo-l-hexanol (73)II() OH ^7 4HO^Br73Following the procedure outlined in section 3.2.1, 1,6-hexanediol (74) (5.9 g, 50 mmol)was converted to the corresponding bromide 73. The crude product was purified by columnchromatography using petroleum ether : ethyl acetate (6:1) as eluent to give the monobrominatedalcohol 73 (4.2 g, 46%) as a colorless liquid, which was one spot by TLC.Rf (1:1 petroleum ether : ethyl acetate) 0.48.1H NMR (400 MHz, CDC13) 8 3.68 (2H, t), 3.43 (2H, t), 1.88 (2H, p), 1.62 (2H, p), 1.52 -1.34 (5H, m).IR (neat, cm-1): 3335, 2932, 2860, 1447, 1260, 1055.LRMS (m/z) 181 (Br81 : M+ - H, 1.0), 179 (Br79 : M+ - H, 1.2), 164 (3), 162 (3), 137 (3), 136(19), 135 (3), 134 (19), 117 (11), 109 (5), 107 (6), 101 (12), 100 (6), 99 (18), 85 (21), 84 (10),83 (100), 82 (38), 81 (19), 70 (6), 69 (28), 68 (6), 64 (27), 57 (17), 56 (11), 55 (86), 54 (10),53 (5), 43 (6), 42 (6), 41 (12).HRMS calcd for C61-113&810 - H: 181.0048, found: 181.0043; calcd for C61113Br 790 - H:179.0068, found: 179.0063.3.12.2 1-Bromo-6-(tert-butyldimethylsilyloxy)-hexane (75) Br^ BrHO^ TBDMSOW73 75Following the procedure outlined in section 3.2.2, the alcohol 73 (2.86 g, 15.8 mmol)was converted to the ether 75. The crude product was purified by column chromatography usingpetroleum ether : ethyl acetate (15:1) as eluent to give the silyl ether 75 (4.12 g, 88%) as acolorless oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.82.1 H NMR (400 MHz, CDC13) 8 3.61 (2H, t), 3.41 (2H, t), 1.87 (2H, p), 1.60-1.43 (6H, m),0.90 (9H, s), 0.06 (6H, s).IR (neat, cm-1 ) 2936, 2858, 1466, 1253, 1101, 837, 775.LRMS (m/z) 295 (Br81 : M+ - H, 0.1), 293 (Br79 : M+ - H, 0.1), 239 (2), 237 (2), 200 (0.2),199 (2), 170 (3), 169 (29), 168 (3), 167 (29), 159 (5), 158 (7), 157 (56), 139 (31), 138 (3), 137(28), 129 (7), 127 (15), 115 (13), 101 (27), 99 (18), 90 (30, 89 (27), 88 (4), 87 (5), 85 (8), 84(14), 83 (100), 82 (4), 77 (7), 76 (10), 75 (94), 74 (5), 73 (38), 61 (6), 59 (15), 58 (6), 57 (6),56 (7), 55 (68).HRMS calcd for C12H27SiBr81O - H: 295.2000, found: 295.0917; calcd for C12H27SiBr790 -H: 293.2020, found: 293.0928.1393.12.3 2.4-Dodecanedione (76)N)ry .^_ nr.,,,,0 0 0 07 76Following the procedure outlined in section 3.2.3, 2,4-pentanedione (7) (3.00 g, 30.0mmol) was alkylated with freshly distilled 1-bromoheptane (5.37 g, 30.0 mmol) to give thecorresponding 13-diketone 76. The crude product was purified by column chromatography usingpetroleum ether : ethyl acetate (15:1) as eluent to give the D-diketone 76 (2.70 g, 63% based onthe recovered bromide) as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.64.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.49 (0.8H, s), 3.58 (0.4H, s), 2.50 (0.4H,t), 2.27 (1.6H, t), 2.24 (0.6H, s), 2.07 (2.4H, s), 1.68-1.20 (12H, m), 0.89 (3H, t).ER (neat, cm-1 ) 2931, 2856, 1713, 1614, 1449, 1363, 1241, 946, 777.LRMS (m/z) 198 (M+, 3.3), 184 (0.4), 183 (4), 181 (0.4), 180 (2), 157 (0.2), 156 (2), 155(1), 142 (4), 141 (10), 140 (3), 114 (4), 113 (33), 101 (14), 100 (98), 96 (4), 95 (9), 86 (7), 85(100), 84 (9), 73 (2), 72 (21), 71 (24), 69 (12), 68 (6), 58 (14), 57 (20), 56 (4), 55 (18), 44 (4),43 (45).140HRMS calcd for Cl2H2202: 198.1614, found: 198.1626.3.12.4 1-(Tert-butyldimethylsilyloxy)-8.10-octadecanedione (77) Ny've../■..,""..,/s.,0 076 TBDMSOW`0 077Following the procedure outlined in section 3.2.4, the P-diketone 76 (2.50 g, 12.6 mmol)was alkylated with the bromide 75 (3.10 g, 10.5 mmol) to give the corresponding P-diketone 77.The crude product was purified by column chromatography using petroleum ether : ethyl acetate(20:1) as eluent to give the P-diketone 77 (2.30 g, 75 % based on the recovered f3-diketone 76)as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.76.1H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.61 (2H, t), 3.53 (0.4H, s),2.50 (0.8H, t), 2.27 (3.2H, t), 1.66-1.22 (2211, m), 0.90 (9H, s), 0.89 (3H, t), 0.06 (6H, s).IR (neat, cm-1 ) 2909, 2856, 1704, 1615, 1459, 1360, 1251, 1100, 945, 838, 775.LRMS (m/z) 412 (Mt, 0.1), 398 (0.9), 397 (3), 357 (7), 356 (26), 355 (100), 338 (0.5), 337(2), 270 (0.4), 269 (2), 257 (2), 241 (1), 215 (3), 213 (2), 199 (3), 171 (2), 170 (1), 169 (4),157 (3), 155 (1), 145 (2), 143 (4), 142 (2), 141 (12), 129 (3), 127 (2), 117 (2), 116 (1), 115(5), 86 (1), 85 (6), 84 (2), 83 (5), 82 (1), 81 (7), 77 (5), 76 (4), 75 (43), 73 (14), 71 (12), 57(13), 55 (13), 44 (7), 43 (6), 41(5).HRMS calcd for C24H48SiO3: 412.3360, found: 412.3377.1413.12.5 1-Hydroxyl-8.10-octadecanedione (78)TBDMSO0 077HO0 078Following the procedure outlined in section 3.2.5, the silyl ether 77 (2.00 g, 4.85 mmol)was converted to the alcohol 78. The crude product was purified by recrystallization fromhexanes to give the alcohol (1.26 g, 87%) as a colorless solid, which was one spot by TLC.Rt. (1:1 petroleum ether : ethyl acetate) 0.64.mp 57.0 °C.1 H NMR (400 MHz, CDC13) 8 15.50 (0.8H, s), 5.48 (0.8H, s), 3.64 (2H, dt), 3.54 (0.4H,s), 2.51 (0.8H, dt), 2.26 (3.2H, dt), 1.70-1.20 (23H, m), 0.89 (3H, t).IR (CHC13, cm-1 ) 3622, 3460, 2931, 2857, 1703, 1607, 1458, 1335, 1051, 902.LRMS (m/z) 298 (M+, 1.0), 281 (0.8), 280 (2), 263 (0.6), 262 (2), 252 (0.4), 251 (1), 239 (1),237 (2), 235 (1), 212 (3), 211 (14), 199 (2), 198 (10), 184 (8), 183 (58), 168 (2), 167 (13), 143(13), 142 (14), 141 (100), 126 (5), 125 (42), 124 (20), 123 (15), 122 (18), 121 (10), 114 (8), 113(32), 112 (10), 111 (16), 101 (9), 100 (83), 98 (19), 97 (63), 96 (16), 95 (41), 94 (8), 86 (4), 85(38), 84 (24), 83 (31), 82 (14), 81 (37), 80 (12), 79 (15), 77 (11), 73 (10), 72 (6), 71 (75), 69(73), 68 (11), 67 (24), 58 (15), 57 (68), 56 (12), 55 (99), 44 (7), 43 (34), 42 (6), 41 (21).142HRMS calcd for C18113403: 298.2499, found: 298.2514.3.12.6 8.10-Dioxooctadecanoic Acid (35)HO^.-^...'irr'•.^0 078 0Hoit...."...."......Thr■e...."....."...."..0 035Following the procedure outlined in section 3.2.6, the alcohol 78 (596 mg, 2.00 mmol)was converted to the corresponding acid 35. The crude product was purified by recrystallizationfrom hexanes to give the acid 35 (306 mg, 50%) as a clear solid, which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate) 0.53.mp 58.5 °C.1 H NMR (400 MHz, CDC13) 5 15.50 (0.8H, s), 5.49 (0.8H, s), 3.54 (0.4H, s),2.51 (0.8H, dt), 2.34 (2H, dt), 2.27 (3.2H, dt), 1.72-1.20 (21H, m), 0.90 (3H, t).IR (CHC13, cm -1 ) 3340-2480, 2931, 2858, 1708, 1608, 1459, 1300, 1127.LRMS (m/z) 312 (M+, 6.4), 295 (0.9), 294 (3), 277 (0.2), 276 (0.8), 266 (1), 253 (1), 227 (1),215 (4), 214 (32), 199 (7), 198 (10), 197 (3), 196 (17), 184 (7), 183 (63), 182 (3), 181 (27), 158(9), 157 (100), 142 (9), 141 (58), 140 (7), 139 (27), 138 (12), 137 (6), 136 (14), 135 (4), 114(4), 113 (24), 112 (10), 111 (38), 101 (5), 100 (79), 99 (5), 98 (9), 97 (26), 87 (5), 85 (23), 84(17), 83 (41), 71 (52), 70 (4), 69 (46), 58 (8), 57 (63), 56 (7), 55 (48), 43 (52), 42 (8), 41 (57).HRMS calcd for C18113204: 312.2292, found: 312.2307.Elem. Anal. calcd for C18H3204: C, 69.23; H, 10.33. found: C, 68.94; H, 10.38.1433.13 Synthesis of Calcium Salt of 9.11-Dioxooctadecanoic Acid (36)144HO39To a solution of 9,11-dioxooctadecanoic acid (28) (60 mg, 0.19 mmol) in 8 mL ofacetone was added calcium hydroxide (28 mg, 0.38 mmol) in 15 mL of distilled deionized water.The mixture was stirred at room temperature for 2 h. The solid precipitate was filtered, washedwith distilled deionized water and acetone, and dried under reduced pressure to give 67 mg solidcompound. The yield of the reaction, based on a 1:1 stoichiometry of the calcium complex 39and on the assumption that two molecules of H2O were bound to each calcium ion, was 86%.mp 200-220 °C.1 H NMR (400 MHz, CD3OD) 8 2.51 (0.9H, m), 2.15 (5.1H, m), 1.64-1.52 (6H, m),1.38-1.26 (14H, m), 0.95 - 0.87 (3H, m).IR (KBr, cm-1 ) 3644, 3527, 2928, 2852, 1577, 1511, 1439, 711.FAB -MS (m/z) 388 [CaC18H3004(H20)2 + H + Hr, 351 [CaC18H3004 + H]+, 313[C181-13204 + H]+, 235, 186, 169.Elem. Anal. calcd for CaC181-13004•(H20)2: C, 55.91; H, 8.87. found: C, 54.79; H, 8.06.HO(0.5), 313 (2), 272 (0.3), 271 (1), 230 (0.3), 229 (2), 199 (2), 173(3), 115 (4), 101 (3), 100 (3), 99 (22), 97 (3), 89 (4), 77 (4), 76 (2),69 (8), 67 (5), 57 (4), 55 (13), 43 (15), 41 (8).(2), 171 (3), 169 (3),75 (29), 73 (11), 71141(19),114 Synthesis of 8.12-Dimethy1-9.11-dioxohexadecanoic Acid (41)0^0 01453.14.1 16-(Tert-butyldimethylsilyloxy)-6.8-hexadecanedione (42)0 0^ 0 0TBDMSO ^TBDMSOC)22^ 42Following the procedure outlined in section 3.2.4, the 3-diketone 22 (4.26 g, 13.0 mmol)was alkylated with freshly distilled 1-bromobutane (1.64 g, 12.0 mmol) to give the p-diketone42. The crude product was purified by column chromatography using petroleum ether : ethylacetate (20:1) as eluent to give the 13-diketone 42 (2.33 g, 68% based on the recovered (3-diketone22) as a clear oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.60.1H NMR (400 MHz, CDC13) 8 15.50 (0.811, s), 5.48 (0.8H, s), 3.60 (2H, t), 3.53 (0.411, s),(611, s).1006, 949,(100), 3142.49 (0.811, t), 2.27 (3.2H, t), 1.66-1.22 (18H, m), 0.90 (9H, s), 0.89 (3H, t), 0.06IR (neat, cm-1 )^2954, 2931, 2857, 1706, 1613, 1463, 1386, 1253, 1200, 1100,838, 776.LRMS (m/z) 383 (M+ - H, 0.7), 371 (0.3), 370 (1), 369 (3), 329 (7), 328 (26), 327HRMS calcd for C22H44SiO3 - H: 383.2970, found: 383.2975.3.14.2 16-(Tert-buvldimethylsilyloxy)-5.9-dimethy1-6.8-hexadecanedione (43) 0 0^ 0 0TBDMSO^ TBDMSOCr*---.."•C)/w•42 43Lithium diisopropylamide (LDA, 8.4 mmol) was prepared at -78 °C by addition of n-BuLi(6.2 mL, 1.35 M, 8.4 mmol) to a solution of diisopropylamine (1.3 mL, 8.8 mmol) in 20 mL ofTHF and stirring of the mixture for 30 min. It was then added to a solution of the I3-diketone 42(0.16 g, 4.2 mmol) in 80 mL of THF at -78 °C through an addition funnel. The mixture wasstirred at -78 °C for 12 h. Iodomethane (0.59 g, 4.2 mmol) was injected and the mixture wasstirred at 0 °C for 4 h. The mixture was cooled to -78 °C and LDA (4.2 mmol) was added. Themixture was stirred overnight. Iodomethane (0.59 g, 4.2 mmol) was injected and the mixture wasstirred at 0 °C for 2 h. The mixture was quenched with saturated NH4C1, acidified with 1N HC1and extracted with ether three times. The organic layers were washed with saturated NaHCO3,brine, dried over MgSO4 and concentrated under reduced pressure. Purification of the crudeproduct by column chromatography using petroleum ether : ethyl acetate (20:1) gave thedimethylated13-diketone 43 (1.25 g, 72%) as a colorless oil, which was one spot by TLC.Rf (6:1 petroleum ether : ethyl acetate) 0.86.GC 94%, RT = 9.68 min, 6%, RT = 9.27 min.1 H NMR (400 MHz, CDC13) 8 15.70 (0.8H, s), 5.47 (0.8H, s), 3.62 (0.4H, s), 3.59 (2H, t),2.63 - 2.56 (0.38H, m), 2.50 - 2.42 (0.02H, m), 2.33 - 2.22 (1.52H, m), 2.14 - 2.08 (0.08H,m), 1.68-1.20 (18H, m), 1.16 - 1.06 (6H, dd), 0.92 - 0.86 (12H, m), 0.06 (6H, s).IR (neat, cm-1 ) 2937, 2857, 1608, 1463, 1381, 1360, 1253, 1101, 1006, 940, 838, 776.LRMS (m/z) 412 (M+, 0.1), 411 (0.3), 399 (0.4), 398 (2), 397 (5), 357 (7), 356 (26), 355(100), 195 (1), 184 (1), 155 (3), 135 (2), 115 (2), 113 (4), 101 (1), 99 (2), 97 (1), 95 (2), 89(2), 85 (12), 83 (2), 81 (2), 77 (1), 75 (11), 57 (3), 55 (6), 43 (8), 41 (4).HRMS calcd for C24H48SiO3: 412.3360, found: 412.3326.1463.14.3 16-Hydroxyl-5.9-dimethy1-6.8-hexadecanedione (44) 0 0^ 0 0147TBDMSO HO43 44Following the procedure outlined in section 3.2.5, the silyl ether 43 (1.0 g, 2.4 mmol)was converted to the corresponding alcohol 44. The crude product was purified by columnchromatography using hexanes : ethyl acetate (3:1) as eluent to give the alcohol 44 (0.68 g, 94%)as a colorless oil, which was one spot by TLC.Rf ( 1:1 petroleum ether : ethyl acetate) 0.74.GC 94%, RT = 7.85 min, 6%, RT = 7.68 min.1 H NMR (400 MHz, CDC13) 8 15.70 (0.8H, s), 5.48 (0.8H, s), 3.64 (2H, t), 3.62 (0.4H, s),2.66 - 2.44 (0.4H, m), 2.36 - 2.10 (1.6H, m), 1.70 -1.20 (19H, m), 1.18 - 1.06 (6H, dd), 0.90(3H, t).IR (neat, cm-1 ) 3385, 2929, 2857, 1603, 1459, 1352, 1055, 793.LRMS (tn/z) 298 (M+, 2.5), 281 (0.1), 280 (0.6), 255 (0.4), 253 (0.7), 214 (0.5) 213 (4), 212(2), 211 (2), 198 (1), 197 (5), 196 (1), 195 (7), 185 (5), 184 (39), 156 (11), 155 (100), 143 (7),142 (3), 141 (18), 129 (3), 128 (19), 127 (5), 126 (5), 125 (12), 124 (4), 123 (20), 114 (7), 113(69), 112 (15), 111 (19), 110 (4), 109 (11), 99(28), 98 (14), 97 (25), 95 (14), 86 (8), 85 (84), 84(6), 83 (39), 82 (5), 81 (20), 72 (9), 71 (21), 70 (8), 69 (77), 57 (24), 56 (5), 55 (33).HRMS calcd for CigH3403: 298.2499, found: 298.2509.3.14.4 8.12-Dimethy1-9.11-dioxohexadecanoic Acid (41)0 0^0^0 0148HO HO44 41Following the procedure outlined in section 3.2.6, the alcohol 44 (596 mg, 2 0 mmol)was converted to the acid 41. The crude product was purified by column chromotography usinghexane : ethyl acetate : acetic acid (6:1:0.7) to give the acid 41 (384 mg, 62%) as a clear oil,which was one spot by TLC.Rf (5% HOAc in 3:1 petroleum ether : ethyl acetate) 0.60.GC 95%, RT = 8.35 min, 5%, RT = 8.15 min.1 H NMR (400 MHz, CDC13) 8 15.70 (0.8H, s), 5.48 (0.8H, s), 3.62 (0.4H, s), 2.66-2.40(0.4H, m), 2.36 (2H, t), 2.34-2.08 (1.6H, m), 1.70-1.20 (17H, m), 1.14-1.06 (6H, dd), 0.88(3H, t).IR (neat, cm-1 ) 3320-2480, 2939, 2860, 1720, 1600, 1449, 1377, 1287, 1224, 1099, 931.LRMS (m/z) 312 (Mt, 4), 295 (0.3), 294 (0.8), 257 (1), 256 (11), 239 (0.8), 238 (3), 210 (4),209 (17), 185 (18), 184 (39), 182 (7), 181 (38), 157 (24), 156 (14), 155 (100), 141 (13), 140 (6),139 (32), 137 (6), 128 (14), 114 (5), 113 (58), 112 (12), 111 (18), 99 (25), 98 (16), 97 (28), 95(12), 86 (7), 85 (69), 71 (18), 70 (5), 69 (46), 57 (15), 56 (5), 55 (26).HRMS calcd for C18113204: 312.2292, found: 312.2299.Elem. Anal. calcd for C18H3204: C, 69.23; H, 10.33. found: C, 69.08; H, 10.43.1^ 110 20Time (h)• •0 302,11 Transport of Calcium and Other Metal Ions by Synthetic Analogues of Ionomycin3.15.1 Transport of Calcium by Synthetic Analogues of Ionomycin Source Phase: 500 mM CaC12, 40 mM CHES/Me4NOH buffer, pH = 9.5Receiving Phase: 40 mM MOPS/Me4NOH buffer, pH = 7.0Analogue: 1542-(5-Benzyloxy)-pentyloxy)kethoxy-9,11-dioxopentadecanoic Acid (5)0^0 0HO'`../N.) 0'--VW0Time (h) 12 16 20 24Ca24- Transported (Amol) 5.8 ± 0.6 7.8 ± 0.7 10 ± 1 12+1Transport Rate (10-8 mole cm-2 h-1 ) J= 7.3 ± 0.7149Figure 12. Plot of the amount of calcium in the receiving phase versus time.150Analogue:^9,11-Dioxopentadecanoic Acid (4)0^0 0HOTime (h)^12^16^20^24Cali- Transported (ilmol)^0.2± 0.1^0.3± 0.1^0.3± 0.1^0.4 ± 0.1Transport Rate (10-8 mole cm-2 h-1 )^J= 0.3 ± 0.1Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HOTime (h)^ 16^20^24Ca2+ Transported (i.unol)^16 ± 1^20 ± 2^23 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 15 ± 2Analogue:^9,11-Dioxoeicosanoic Acid (29)0^0 0HOTune (h)^ 12^16^20^24Ca2+ Transported (j.tmol)^13 ± 1^17 ± 1^20 ± 1^24 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 15 ± 2151Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^0 0HOTime (h)^ 12^16^20^24Calf Transported (gmol)^11 ± 1^15 ± 1^18 ± 1^21 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 13 ± 1Analogue:^7,9-Dioxooctadecanoic Acid (33)0^0 0HOTime (h)^ 16^20^24Ca24- Transported (1.unol)^3.9 ± 0.4^4.9 ± 0.4^5.8 ± 0.6Transport Rate (10-8 mole cm-2 h-1 )^J = 3.5 ± 0.4Analogue:^11,13-Dioxooctadecanoic Acid (34)0^ 0 0HOTime (h)^12^16^20^24Ca2+ Transported (gmol)^0.9 ± 0.1^1.1 ± 0.1^1.2 ± 0.1^1.3 ± 0.1Transport Rate (10-8 mole cm-2 h-1 )^J= 0.9 ± 0.1HOHO152Analogue:^8,10-Dioxooctadecanoic Acid (35)00 0Time (h)^ 16^20^24Ca2+ Transported (i.tmol)^9.3 ± 0.9^13 ± 1^16 ± 1Transport Rate (10-8 mole cm-2 h-1 )^J = 9.5 ± 0.9Analogue:^8 , 12-Dimethy1-9,11-dioxohexadecanoic Acid (41)0^0 0Time (h)^ 16^20^24Ca2+ Transported (i.tmol)^10 ± 1^14 ± 1^16 ± 1Transport Rate (10-8 mole cm-2 h -1 )^J = 10 ± 1HO OHO^0 0HOCalcimycin (2)Tune (h)^ 12^16^20^24Ca2+ Transported (j.unol)^14 ± 1^16 ± 1^18 ± 1^19 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 12 ± 1Ionomycin (3)Time (h)^ 16^20^24^40Ca24- Transported (gmol)^20 ± 2^30 ± 2^36 ± 2^56 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 21 ± 21533.15.2 Cation Selectivity in TransportAnalogue:^9,11-dioxooctadecanoic Acid (28)0^0 0HOReceiving Phase: 40 mM MOPS/Me4NOH buffer, pH = 7.5Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5250 mM CaC12, 250 mM in MgC12Time (h) 16 20 24Ca2+ Transported (gmol) 3.0 ± 0.2 3.2 ± 0.2 3.5 ± 0.3Mgt-1- Transported (gmol) 4.0 ± 0.3 4.4 ± 0.4 5.0 ± 0.4Ca2+ Transport Rate (10-8 mole cm-2 h-1 )^J = 2.2 ± 0.2Mg2+ Transport Rate (10 -8 mole cm-2 h-1 )^J = 3.1 ± 0.3154Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5250 mM CaC12, 250 mM in NaCITime (h)^ 16 24 40Ca2+ Transported (µmol)^6.7 ± 0.5 10 ± 1 15 ± 1Na+ Transported (gmol)^0.0 0.0 0.0Ca2+ Transport Rate (10-8 mole cm-2 h-1 ) J = 5.5 ± 0.5Na+ Transport Rate (10 -8 mole cm-2 h -1 ) J = 0.0Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5250 mM CaC12, 250 mM in KC1Time (h)^ 16 24 40Cat-1- Transported (i.tmol)^6.7 ± 0.5 10 ± 1 15 ± 1K+ Transported (=01)^0.0 0.0 0.0Ca2+ Transport Rate (10-8 mole cm -2 h-1 ) J = 5.5 ± 0.5K+ Transport Rate (10 -8 mole cm-2 h-1 ) J = 0.01553.15.3 effect of Substrate Concentration on Calcium TransportAnalogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HOReceiving Phase: 40 mM MOPS/Me4NOH buffer, pH = 7.5Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 10 mM CaC12Time (h)^ 16^20^24Ca2+ Transported (gmol)^0.6 ± 0.1^0.8 ± 0.1^0.9 ± 0.1Transport Rate (10-8 mole cm-2 h -1 )^J = 0.6 ± 0.1Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 50 mM CaC12Time (h)^ 16^20^24Ca2+ Transported (gmol)^4.6 ± 0.4^6.0 ± 0.4^7.1 ± 0.7Transport Rate (10-8 mole cm-2 h-1 )^J = 4.4 ± 0.4156Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 100 mM CaC12Time (h)^ 16^20^24Ca2+ Transported (1.tmol)^7.4 ± 0.6^8.4 ± 0.7^9.4 ± 0.9Transport Rate (10-8 mole cm-2 h-1 )^J = 5.9 ± 0.6Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 250 mM CaC12157Time (h)Ca2+ Transported (gmol)Transport Rate (10-8 mole cm-2 h-1 )16^20^2414+1^18 ± 1^21 ± 2J = 13 ± 1Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 500 mM CaC12Time (h)^ 16^20^24Ca2+ Transported (plop^16 ± 2^20 ± 2^23 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 15 ± 2Source Phase: 40 mM CHES/Me4NOH buffer, pH = 9.5, 1000 mM CaC12Time (h)^ 16^20^24Ca2+ Transported (i.tmol)^16 ± 1^21 ± 2^28 ± 2Transport Rate (10 -8 mole cm-2 h-1 )^J= 16± 23.15.4 effect of pH in Receiving Phase on Calcium TransportAnalogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HOSource Phase: 500 mM CaC12, 40 mM CHES/Me4NOH buffer, pH = 9.5Receiving Phase: 40 mM Succinic Acid/Me4NOH buffer, pH = 5.0Time (h)^ 16^20^24Ca2+ Transported (=01)^13 ± 1^16 ± 1^21 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 15 ± 2Receiving Phase: 40 mM MES/Me4NOH buffer, pH = 6.0Time (h)^ 16^20^24Ca2+ Transported (Juno°^13 ± 1^17 ± 1^20 ± 2Transport Rate (10 -8 mole cm-2 h-1 )^J= 16 ±2158Receiving Phase: 40 mM MOPS/Me4NOH buffer, pH = 7.0Time (h)^ 16^20^24Cali- Transported (gmol)^14 ± 1^17 ± 2^18 ± 2Transport Rate (10 -8 mole cm-2 h -1 )^J = 16 ±2Receiving Phase: 40 mM HEPPS/Me4NOH buffer, pH = 8.0Time (h)^ 16^20^24Ca21- Transported (gmol)^13 ± 1^16 ± 2^18 ± 2Transport Rate (10-8 mole cm-2 h -1 )^J = 15 ± 21593.15.5 Effect of pH in Source Phase on Calcium TransportAnalogue: 1542-(5-Benzyloxy)-pentyloxy)]-ethoxy-9,11-dioxopentadecanoic Acid (5)0^0 0HO ./ '-0W0Concentration: 30011MReceiving Phase: 40 mM MES/Me4NOH buffer, pH = 6.5Source Phase: 500 mM CaC12, 40 mM MOPS/Me4NOH buffer, pH = 7.5Time (h)^ 16^20^24Ca2+ Transported (=01)^1.2 ± 0.1^1.3 ± 0.1^1.6 ± 0.1Transport Rate (10-8 mole cm-2 h-1 )^J = 1.3 ± 0.1Source Phase: 500 mM CaC12, 40 mM HEPPS/Me4NOH buffer, pH = 8.0Time (h)^ 16^20^24Cali- Transported (gmol)^1.1 ± 0.1^1.2 ± 0.1^1.6 ± 0.1Transport Rate (10-8 mole cm-2 h-1 )^J = 1.5 ± 0.1160Source Phase: 500 mM CaC12, 40 mM HEPPS/Me4NOH buffer, pH = 8.5Time (h)^ 16^20^24Ca2+ Transported (gimp^2.2 ± 0.2^2.8 ± 0.2^3.8 ± 0.4Transport Rate (10-8 mole cm-2 h-1 )^J = 2.5 ± 0.2Source Phase: 500 mM CaC12, 40 mM MOPS/Me4NOH buffer, pH = 9.0Time (h)^ 16^20^24Ca2+ Transported (gmol)^6.2 ± 0.4^6.6 ± 0.5^7.8 ± 0.7Transport Rate (10-8 mole cm-2 h-1 )^J = 4.6 ± 0.4Source Phase: 500 mM CaC12, 40 mM CHES/Me4NOH buffer, pH = 9.5161Time (h)Ca2+ Transported (1.imol)Transport Rate (10-8 mole cm-2 h-1 )16^20^2411+1^13 ± 1^14 ± 1J = 9.0 ± 0.9Source Phase: 500 mM CaC12, 40 mM CHES/Me4NOH buffer, pH = 10.0Time (h)^ 16^20^24Ca2+ Transported (1.imol)^18 ± 1^20 ± 2^21 ± 2Transport Rate (10-8 mole cm-2 h-1 )^J = 14 ± 1pKa = 11.16 ± 0.021.68001.28088.96800.64808.32088.0888268.8 288.8^388.81uI<pH = 12.76340.8328.83,1k pKa of the D-Diketone Group of Synthetic Analogues of IonomycinSolvent: 80% Me0H-H20^Ionic Medium: 50 mM Et4NC104Concentration: 8011M Titrant: Me4NOHAnalogue:^7,9-Dioxooctadecanoic Acid (33)0^0 0HOpH^5.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.66A (298 nm) 0.248 0.257 0.276 0.283 0.288 0.310 0.336 0.439 0.581pH^11.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.76A (298 nm) 0.800 0.892 1.130 1.240 1.368 1.415 1.479 1.520 1.537Wavelength (run)Figure 27. UV spectrophotometric absorption spectra of analogue 33 as the pH of thesolution increased from 5.80 to 12.76.1621.60 p.1.50 •1 .40:1.301.20:1.101.000.90• 0.800.700.600.500.400.300.20 1.^50 163■6.0^7.0^h 9.0^10.0^11.0^12.0^13.0pHFigure 28. Plot of the UV absorbance at 298 nm versus pH for analogue 33.Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HOpHA (298 nm)pHA (298 nm)5.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.660.275 0.294 0.320 0.324 0.335 0.361 0.370 0.544 0.69811.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.760.900 1.033 1.224 1.322 1.410 1.437 1.500 1.531 1.539pKa = 11.03 ± 0.025.80 6.80 7.56 8.200.315 0.324 0.348 0.36611.00 11.19 11.44 11.601.138 1.278 1.480 1.572pHA (298 nm)pHA (298 nm)164Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 0HO8.90 9.54 9.86 10.42 10.660.375 0.403 0.451 0.724 0.90211.88 12.06 12.28 12.52 12.761.659 1.680 1.728 1.780 1.829pKa = 10.90 ± 0.02Analogue:^8,10-Octadecanedione (31)0 05.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.660.208 0.211 0.211 0.221 0.235 0.256 0.286 0.484 0.59511.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.760.719 0.817 0.920 0.985 1.032 1.050 1.084 1.109 1.120pHA (298 nm)pHA (298 tun)pKa = 10.86 ± 0.02165Analogue:^9,11-Dioxopentadecanoic Acid (4)0^0 0HOpHA (298 nm)pHA (298 nm)5.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.660.279 0.293 0.310 0.323 0.350 0.381 0.421 0.664 0.83411.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.761.033 1.137 1.348 1.448 1.520 1.553 1.613 1.634 1.665plCa = 10.92 ± 0.02Analogue:^9,11-Dioxoeicosanoic Acid (29)0^0 0HOpHA (298 nm)pHA (298 nm)5.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.660.278 0.287 0.299 0.310 0.315 0.320 0.341 0.515 0.69111.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.760.936 1.009 1.255 1.373 1.481 1.508 1.551 1.593 1.611pKa = 11.04 ± 0.02166Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^0 0HOpHA (298 nm)pHA (298 nm)5.80 6.80 7.56 8.20 8.90 9.54 9.86 10.42 10.660.282 0.288 0.306 0.315 0.318 0.318 0.329 0.561 0.65711.00 11.19 11.44 11.60 11.88 12.06 12.28 12.52 12.760.932 1.009 1.188 1.332 1.440 1.474 1.523 1.570 1.593pKa = 11.07 ± 0.02J,1 Binding of Calcium and Other Metal Ions by Synthetic Analogues of lonotrwcin3.17.1 Binding of CalciumSolvent: 80% Me0H-H20^Ionic Medium: 16,7 mM CaC12Concentration: 801.tM Titrant: Me4NOHAnalogue:^7,9-Dioxooctadecanoic Acid (33)1670HO0 05.40 6.36 7.11 7.60 8.02 8.32 8.48 9.19 9.880.272 0.283 0.334 0.427 0.543 0.662 0.758 1.181 1.44510.62 10.94 11.27 11.461.503 1.504 1.510 1.515pHA (298 nm)pHA (298 nm)pKa' = 8.69 ± 0.02^13 = (3.0 ± 0.2) x 102 M-1268.8^208.8^388,0^320.0^348.8Wavelength (nm)Figure 31. UV spectrophotometric absorption spectra of analogue 33 as the pH of thesolution increased from 5.40 to 11.46 in the presence of CaC12.1.401.1.501.401.301.20•§ooo■N1.10 •1.00•V 0.70•8 0.100.700.40•0.50:0.400.300.206.05.0 7.0 8.0 9.0^10.0^11.0^12.0^13.0pHFigure 32. Plot of the UV absorbance at 298 nm versus pH for analogue 33 in thepresence of CaC12.Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HO1685.40 6.36 7.11 7.60 8.02 8.32 8.48 9.19 9.880.323 0.334 0.382 0.472 0.584 0.724 0.840 1.470 1.74310.62 10.94 11.27 11.461.842 1.854 1.867 1.882pHA (298 nm)pHA (298 nm)plCie = 8.77 ± 0.02^13 = (1.8 ± 0.1) x 102 M-1169Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 0HOpHA (298 nm)pHA (298 nm)5.40 6.36 7.11 7.60 8.02 8.32 8.48 9.19 9.880.310 0.324 0.369 0.436 0.531 0.626 0.755 1.267 1.56010.62 10.94 11.27 11.461.668 1.677 1.686 1.692plC.a , = 8.84 ± 0.02^13 = (1.2 ± 0.2) x 102 M-1Analogue:^9,11-Dioxopentadecanoic Acid (4)0^0 0HOpHA (298 nm)pHA (298 nm)5.40 6.36 7.11 7.60 8.02 8.32 8.48 9.19 9.880.282 0.296 0.389 0.481 0.632 0.727 0.805 1.286 1.57610.62 10.94 11.27 11.461.627 1.632 1.636 1.637pKa, = 8.69 ± 0.02^13 = (1.7 ± 0.1) x 102 M-1170Analogue:^9,11-Dioxoeicosanoic Acid (29)0^0 0HOpHA (298 nm)pHA (298 nm)5.40 6.42 7.14 7.61 8.03 8.27 8.38 8.98 9.600.257 0.274 0.334 0.435 0.561 0.624 0.723 1.039 1.36710.46 10.81 11.21 11.411.498 1.508 1.516 1.519pKie = 8.68 ± 0.02^13 = (2.2 ± 0.2) x 102 M-1Analogue:^9,11-Dioxodoeicosanoic Acid (30)0HO0 05.46 6.42 7.14 7.61 8.03 8.27 8.38 8.98 9.600.251 0.276 0.322 0.405 0.488 0.543 0.590 1.012 1.26210.46 10.81 11.211.416 1.422 1.419pHA (298 nm)pHA (298 nm)pKa. = 8.77 ± 0.02^0 = (2.0 ± 0.1) x 102 M-1ILI binding of MunesiumSolvent: 80% Me0H-H20^Ionic Medium: 16,7 mM MgC12Concentration: 80 p.M Titrant: Me4NOHAnalogue:^7,9-Dioxooctadecanoic Acid (33)0^0 0HOpH 5.61 6.58 7.20 7.64 7.95 8.23 8.48 9.21 9.51A (298 nm) 0.303 0.533 0.856 1.095 1.251 1.335 1.397 1.473 1.478pKa' = 7.28 ± 0.02^13 = (7.6 ± 0.4) x 103 M- 1Wavelength (nm)171Figure 33. UV spectrophotometric absorption spectra of analogue 33 as the pH of thesolution increased from 5.61 to 9.51 in the presence of MgC12.1.601.301.401.2D:1.20:1.10 •1.000.30•■0.000.70 •0.30•0.40^0.30^■^0.20 ^-- ^5 0^6.0^7.0^8.0^9.0 10.0 11.0 12.0 13.0PHFigure 34. Plot of the UV absorbance at 298 nm versus pH for analogue 33 in thepresence of mgc12.Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HO172pH 5.50 6.49 7.10 7.56 7.95 8.22 8.53A (298 nm) 0.351 0.556 0.828 1.134 1.376 1.534 1.626pKa' =7.50 ± 0.02^13 = (3.4 ± 0.2) x 103 M -19.28 9.541.764 1.775Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 0HOpH 5.61 6.58 7.20 7.64 7.95 8.23 8.48A (298 nm) 0.341 0.493 0.769 1.045 1.280 1.436 1.549pKai = 7.61 ± 0.02^= (1.9 ± 0.1) x 103 M -19.21 9.511.678 1.699173Analogue:^9,11-Dioxopentadecanoic Acid (4)0^O 0HOpH 5.50 6.49 7.10 7.56 7.95 8.22 8.53 9.28 9.54A (298 nm) 0.307 0.586 0.764 1.198 1.356 1.479 1.535 1.596 1.599pKa' = 728 ± 0.02^E3 = (4.4 ± 0.2) x 103 M-1Analogue:^9,11-Dioxoeicosanoic Acid (29)0^O 0HOpH 5.50 6.49 7.10 7.56 7.95 8.22 8.53 9.28 9.54A (298 nm) 0.278 0.448 0.749 1.047 1.263 1.337 1.398 1.473 1.473pKa' = 7.29 ± 0.02^0 = (5.6 ± 0.2) x 103 M-1Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^O 0HOpH 5.61 6.58 7.20 7.64 7.95 8.23 8.48 9.21 9.51A (298 nm) 0.288 0.392 0.798 1.059 1.203 1.288 1.338 1.436 1.440pKa, = 7.31 ± 0.02^fi = (5.7 ± 0.3) x 103 M-13,2,4 binding of SodiumSolvent: 80% Me0H-H20^Ionic Medium: 50 mM NaC1Concentration: 801.1M Titrant: Me4NOHAnalogue:^7,9-Dioxooctadecanoic Acid (33)O^0 0HO1745.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.270 0.276 0.289 0.307 0.325 0.345 0.375 0.663 0.76811.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.700.976 1.070 1.214 1.304 1.378 1.402 1.462 1.513 1.537pHA (298 nm)pHA (298 nm)plCa, = 11.16 ± 0.02^13 = 1.8 ± 0.1 M-21268.8^2813.8^338.8^328.8^348.8Wavelength (nm)Figure 35. UV spectrophotometric absorption spectra of analogue 33 as the pH of thesolution increased from 5.84 to 12.70 in the presence of NaCl.1.50 •1.401.301.201.101.000.300.000.70 '0.600.500.40^0.30 ^0.20 ^5.01.W.i.•^■--, ,6.0^7.0^8.0^9.0^10.0pHFigure 36. Plot of the UV absorbance at 298 nm versus pH for analogue 33 in thepresence of NaCl.Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0HO17511.0^12.0^13.0pHA (298 nm)pHA (298 nm)5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.268 0.270 0.283 0.294 0.308 0.347 0.362 0.565 0.70611.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.700.945 1.081 1.274 1.373 1.466 1.489 1.539 1.565 1.570pKa' = 10.96 ± 0.02^ fl = 1.2 ± 0.1 M-25.84 6.82 7.52 8.12 8.740.280 0.281 0.290 0.299 0.31811.00 11.20 11.46 11.62 11.881.058 1.184 1.311 1.422 1.5249.34 9.70 10.34 10.600.332 0.342 0.608 0.80512.06 12.26 12.50 12.761.557 1.612 1.636 1.664± 0.1 114-2pHA (298 nm)pHA (298 nm)pKa' = 10.90 ± 0.02^ 13 = 1.0176Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 0HOpHA (298 nm)pHA (298 nm)5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.322 0.322 0.331 0.346 0.356 0.363 0.373 0.581 0.79311.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.701.107 1.245 1.489 1.613 1.725 1.755 1.815 1.843 1.850pKg = 11.00 ± 0.02^ 13 = 0.8 ± 0.1 M-2Analogue:^9,11-Dioxopentadecanoic Acid (4)0^0 0HO177Analogue:^9,11-Dioxoeicosanoic Acid (29)0^0 0HO5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.274 0.275 0.289 0.306 0.314 0.318 0.326 0.590 0.69611.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.760.957 1.102 1.296 1.394 1.470 1.495 1.544 1.570 1.587pHA (298 nm)pHA (298 nm)pKa' = 10.96 ± 0.02^ 13 = 1.2 ± 0.1 M-2Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^0 0HO5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.252 0.255 0.262 0.275 0.282 0.287 0.292 0.411 0.65511.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.760.866 0.981 1.163 1.273 1.330 1.395 1.447 1.476 1.483pHA (298 nm)pHA (298 nm)pKa' = 11.03 ± 0.02^ 13 = 1.1 ± 0.1 M-23.7.5 binding of PotassiumSolvent: 80% Me0H-H20^Ionic Medium: 50 mM KClConcentration: 80 01V1 Titrant: Me4NOHAnalogue:^7,9-Dioxooctadecanoic Acid (33)O^0 0HO1785.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.252 0.261 0.279 0.291 0.300 0.324 0.359 0.524 0.65211.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.700.854 0.978 1.164 1.248 1.349 1.380 1.436 1.468 1.482pHA (298 nm)pHA (298 nm)pKa' = 11.03 ± 0.02^fl = 1.3 ± 0.1 M-2268.8^288.8^388.8^320.8^340.0Wavelength (nm)Figure 37. UV spectrophotometric absorption spectra of analogue 33 as the pH of thesolution increased from 5.84 to 12.70 in the presence of KC1.1.401.501.40 1791.301.201.10001.00O.300.00 •0.700 .60 •0.500.400 30  0 .20 ^ •^5.0^6.0^7.0^8.0^9.0^10.0^11.0 12.0 13.0pHFigure 38. Plot of the UV absorbance at 298 nm versus pH for analogue 33 in thepresence of KC1.Analogue:^9,11-Dioxooctadecanoic Acid (28)pHA (298 nm)0HO0 05.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.260 0.278 0.299 0.312 0.330 0.332 0.352 0.500 0.61811.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.700.848 0.990 1.171 1.279 1.384 1.418 1.480 1.506 1.519pHA (298 nm)pKa, = 11.09 ± 0.02^ f3=0.9±0.1 M-2180Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 0HO5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.293 0.304 0.327 0.350 0.356 0.377 0.427 0.671 0.81411.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.701.073 1.220 1.406 1.507 1.610 1.633 1.682 1.726 1.751pHA (298 nm)pHA (298 nm)pKa, = 10.94 ± 0.02^ f3 = 0.9 ± 0.1 M-2Analogue:^9,11-Dioxopentadecanoic Acid (4)0^0 0HOpHA (298 nm)pHA (298 nm)5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.286 0.287 0.300 0.308 0.316 0.335 0.340 0.543 0.72511.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.760.991 1.060 1.308 1.441 1.543 1.576 1.618 1.662 1.680pKa, = 11.00 ± 0.02^ (3 = 0.8 ± 0.1 At2181Analogue:^9,11-Dioxoeicosanoic Acid (29)0^0 0HO5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.271 0.271 0.283 0.295 0.301 0.306 0.307 0.430 0.59811.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.760.822 0.962 1.221 1.314 1.449 1.488 1.548 1.576 1.596pHA (298 nm)pHA (298 nm)pKa, = 11.14 ± 0.02^0 = 0.8 ± 0.1 M-2Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^0 0HOpHA (298 nm)pHA (298 nm)5.84 6.82 7.52 8.12 8.74 9.34 9.70 10.34 10.600.251 0.255 0.270 0.278 0.277 0.278 0.280 0.409 0.55711.00 11.20 11.46 11.62 11.88 12.06 12.26 12.50 12.760.762 0.863 1.141 1.231 1.330 1.378 1.424 1.470 1.480pKa. = 11.14 ± 0.02^r3=0.8±0.1 M-23.17.6 Binding of MagnesiumSolvent: 80% Me0H-H20 Ionic Medium: 50 mM CHES/Me4NOH bufferpH = 9.10^Concentration: 801.1M^Titrant: MgC12 (10-4 M)Analogue:^7,9-Dioxooctadecanoic Acid (33)0^0 0HO[MgC12] 0.25 0.50 0.98 1.47 2.68 3.88 6.28 8.65 13.40A (298 nm) 0.511 0.608 0.741 0.853 1.032 1.132 1.246 1.302 1.359n=1.0±0.1^f3 = (7.1 ± 0.1) x 10 3 M -1182I1.60001.28000.93080.64880.32008.0008268.0^288.0^3E0.0^320.e^348.8Wavelength (nm)Figure 29. UV spectrophotometric absorption spectra of analogue 33 as the concentrationof MgC12 increased from 0.25 to 1.34 x 10-3 M at pH = 9.10.HOHO0.25 0.50 0.98 1.47 2.68 3.880.536 0.618 0.785 0.890 1.091 1.2396.28 8.65 13.401.409 1.496 1.5816.28 8.65 13.401.380 1.468 1.554[MgCl2]A (298 nm)0.25 0.50 0.98 1.47 2.68 3.880.526 0.630 0.804 0.913 1.128 1.259[MgC12]A (298 nm)n=1.0±0.1^13 = (5.6 ± 0.1) x 103 is,4-1Analogue:^11,13-Dioxooctadecanoic Acid (34)0^0 01Mg2+1 x 10-4 (M)Figure 30. Plot of the number of moles of magnesium bound per mole of analogue 33versus concentration of magnesium.Analogue:^9,11-Dioxooctadecanoic Acid (28)0^0 0183n=1.0±0.1^13 = (4.5 ± 0.1) x 103 M -1184Analogue:^9, 11-Dioxopentadecanoic Acid (4)0^O 0HO0.25 0.50 0.98 1.47 2.68 3.88 6.28 8.65 13.400.444 0.542 0.695 0.804 0.982 1.126 1.251 1.324 1.3900=(5.5±0.1)x 103M-1[MgC12]A (298 nm)n= 1.0±0.1Analogue:^9,11-Dioxoeicosanoic Acid (29)0^O 0HO[MgC12]A (298 nm)0.25 0.50 0.98 1.47 2.68 3.88 6.28 8.65 13.400.450 0.545 0.701 0.819 1.007 1.114 1.243 1.302 1.369n= 1.0 ± 0.1^13 = (6.2 ± 0.1) x 103 M-1Analogue:^9,11-Dioxodoeicosanoic Acid (30)0^O 0HO[MgC12]A (298 nm)0.25 0.50 0.98 1.47 2.68 3.88 6.28 8.65 13.400.442 0.536 0.672 0.786 0.964 1.071 1.199 1.258 1.325n=1.0±0.1^13 = (6.2 ± 0.1) x 10 3 M-1REFERENCES(1) (a) Berridge, M.J. Sci. Am. 1985, 253 (4), 142; (b) Carafoli, E. and Penniston, J.T.Sci. Am. 1985, 253 (5), 70; (c) Ochial, E. J. Chem. Educ. 1991, 68, 10.(2) Davy, H. Philos. Soc. Trans. R. Soc. 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CM-180 -) 1Zg 449 1200^4000.197i3 1^ 6 PPM1iMI)AYIHOW()16/.1.....-...-14MIv-- 1 12800.^2200.^1600.I^I^I1000.^CM-14000.0 '^I3400.19817I0 PPM3^ 2I^1.1,7..111W/1f 7,11711-1-1117,11IT-1117^ 6^ 5^ 410080-60-4o-120-JlI2200.^1600.0^4000.T--3400. 2800.I^I^I1000. CM-119918178T^11^.1■111.111.1■11-1-r-1^TT ^ 1,71^116^5 4^3 2 0 PPM'3400. 2800. CM- 12200. 1600.0 ^,4000.I^i^I1000.tA200TBDMS0"•.'%OnDwO191 - I-3400.^2600.-1-^T -1"2200. CM- I80-144• 4-I(13^1840 1• 120-f104000.T^11600.^1000.j11111.111111.1'7111,1/11,1111-■^11-T■11-71117,,I11-711111117-11-,^1111-17/11711,111.11117^6^5 4•^3^2 1^0 PPM201100^-r-t 1HC31-40Thwo200 PPM202Br"/'01ThwO10.A______203411 1 4114 1 11 /1111111111111,14^/111/11^y,^v,ive^■^•^1^■^■^r^■^1^Ile^[TT^■^Ir.^IF,^Tvill.)1■1•Iivi^v.^It7^6^ 5 4 3 2 1^0 PPMrI,01I0401■JaR014000.1^,3400.I^I^I2800.I^i^I2200. 1600.V^I^I1000. CM—i0 0TBDMSOCA''•./%0°ThWOS 02 0 PPM10080-z 6 -<I200^4000.i3400.^2800.^2200. 1600.^1000.^CM-I151642043 reH1o  14000. 3400.100^--7^-1-^—^ ---r--^r---^—t-------r-- r^-1z20 —1'r 7-^-T^r ---r— --T2800.^2200.^1600.^1000.^CM-10 0HOOr-ThwO512057 5188416 '5206O^0 0HON/%0'--bw0516^ 155^ 477 rpm1600. 1000. CM- 11V•20- I100so-6°1ao,0MOO.^2200.4000. 3400.Br o^o'1143 2 0 PPM2070 0TBDMS0'0''O'5 2__.----------"-----16^155 42082, ,,,,,,,if)/I11b0 PPM3100 I80—60—-140-, I20—MI0^4000.1^r-r ---7- -II-- --I -1--1^I 1 I^2600.^2200.^1600. 1000.T^ 1^'3400. CM-1O 053_P"13.11S.6^S'.13 T II^I'S^3'.1^3r35.1^I:1 h. 11 410020980 —60—40—20—01 e I —r4000.^3400. I^11000. 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IPPM .516.1 15.81000.^CM-15.6^5:. 4.4^4.2^4.0^3.8^3.6^Si3.2^3.^2.8^2.6^2.4^2.2^2.1^I C I^1.6^I. 4 T 1.2^1.0PPM04000. 2200.I1600.I^I^61000.I I I I Z -r^ -r - -80-6040-r-I3400. 2800.217100218100^r--2800. 1600.—r-2200.^1^ti1000.^CM—I0 ^, ,^t4000. 3400.0^0 0HO16.1^15.637.1113.6 - 3.43.2^3.1^1.8 j 2.6^2. 4^2.26 '.11^5'S29pGI's••I•-c) 60-80—•20—1.8^1. 6^1.4I^i3400.wMOO. 2200. 1600.6. 11 5.5 5.1 4.5 4.1^3.5PPM 3.1 2.5 2.1 1.50 0TBDMSO5916 1^15.140I^'1000. CM-14000.219S.6^6'2^3 '.1^4.11^4.6^4.1 2^4.11^3 ' 0^3 '.6^3.4^3 '.2^3.41^2.8^2.6^2.4^2^2.1^1 '.13^1.6^Lr^1.2 ' 1 '.11^.8PPM100 0 1^I^I --I-1r- t-t "1"----T.•••••••••■••\so —60-ti40-20-o  14000.11^'1000. CM-1I^r^I3400. MOO.I2200.11600.220100r2200.•1600.4000. CM-13400.^2800.11000.180I42210^0 0Hoi./'■■•■/■A,A■■■■././■3016.0^15.51,5.5^5.4^5.2^5.11^4.5^4.6^4.4^4.3^I I^3.11^3 6^3.5^3.2^3.6^2.51^2.4 ^32^2-.11^1.6^I I^1.2^1 '.8^.8^:2PPM0 0)L.)L./'■615:8^5:6^5.1^5-.2^5.11^1.8^4.6^4 r.1^4.2- 4,11 . 3.E1^3.6^3 ^3.2^3.11^2.8^2.6^2.-1^2:2^2.11^1.8 - 1.6^2^I^.4 10"00.180j60 -41- • ---r-----t - r- t^i -- r -^r -T^T-3400 .I^f^t2200. Ii600.^1000.^CM-1I^-12800..62220 031223I^•16.8^15.86.0^5.5^5.8^4.5^4.4^3.5^3.11^2.5^2.8^1.5^1.11^.5^0 8PPM100480-.1104000.^3400.^2800.I^I^I^I^I^I^I^I^I2200. i600. 1000.^CM-1224HO^Br62il---I-^--1--, -0 PPM5^, Li;^1^3 21'14000. 1000.^CM-1I^i3400.1^I^I2800.I^12200.1--1- 1^,1600.ee5.5^5.1^4.5^4.4^3.5^3.4^2.5^2.4^L 5^IPPMCM-1I^I1000.O 0TBDMSOL.)C6.6^5.5 5.6^4.5 4.4^5.5PPM6.5 6. 6226r- --t- Y 1- 1 62800.^2200.I I^g1600.4000.0 I3400.O 0TBDMSO6522716.6^15.6a 0 .5B. I 5.5^5.1 4. I^3. 5PPM3.11^2.5 2.6^1.5^1.1 8. 8100........6114.••••••••••••■•••■••••■•••■••■■\80-20-'^ -r4000.^3400.^2800. 2200.-r- ,1600.I^I^I1000. 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CM- 1..........^HO^Br73J236:^3^t^;^0 PPM237 5^4^3^2^ 0 PPM16^1520 0762381S 4^r 3^ 1^f1^ -'0 PPM100rd.....-•""•••"•-•Nr80-60-r04000. 3400.1^'^I2800. 2200.^I ^Ii6 . 1000.^CM-11,)...........)r^I^11600.r^I^12800.I^i^'2200.TBDMSOW0 077)239rJ 6^5 4^3^2 r1^0 PPM10080:,,----Th^li1I^I^11000. CM-10^,^1^.4000. 3400.240r-16^15HO0 078*.^43 2^1^6 0 PPM10080-40-04000.^I ^I^ I^I^I34 0. 2800.I^I^I^ I^ T2200. 1600.^1000.^CM-1OHull..."........„...0 035Lelo.aloolorolner.^is1^ ►^.^t^-t^,6 5 4t^i3^III!^2^1 0 PPMr^ r--t- n--r--r- -,----T-----T-2800.^2200.^1600.^1000.^CM-113400.rir- ii -r -r- ii2419995918783ZU7975716763592.8^2.7^2.6^2.3^2.4^2.3^2.2^2.1^2.0^1.9^1.8^1.7^116^1.5^1.4^1.3^1.2^1.1^I.0^0:9^al^0.7^00 PPM242* For IR spectrum of calcium complex 36 in the region of 1000-400 cm-1 , see page 247.04000. 3400..1T-"1 —1—T —1 -1—r— T-1 T—T2800.^2200.^1600.^1000.^CM-1O 0TBDMS0)42— J6 6 5.2 6 6 6 6 6 61 3:8 e r.6 632 Le Le 2.6 6 2.2 2.1^16 1:1^jf,P18243x 0.5x 0.516 I^15.1J-fly10080160g p -20_20-S1 2.7^111^17^I.2O 0TBDMSOC)Ci4324416 is6 • 0 PPM100 .1^I^I^ U^r-80 -z 60-az40-44V0 I^I2800.1^I2200.I^I^11000.^CM-14000.^3400. 1600.r41124510080— i120—;0 ^, '^1^'^I4000. - 3400. 2800.1^I^I2200.I^I^.^1^I1600. 1000. CM-1lePPM2I -36^ 3^ 42200.I^I1600.^1000.^CM-10 0HO41x 0.516 T 1304000. - 3400. 2800.I2460.7^186 PPM1.6^1.3^1.4^1.3^1.2^1.1^1.0^fig^0.02.1^1.7^2.6^2.3^2.4^2.3^2.2^2.1^2.0^1.0^1.11^1.7J,in KBr99.1798.7498.3197.8897.45 -97.02 -96.59 -96.16 -95.73 -95.30 -94.87 -zU0tf2900 •^800 700^600^500^CM-1247I^11APPENDIX 2248PREPARATION OF BUFFERS1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%)1.0 mL Me4NOH (10%) +1.0 mL Me4NOH (10%) +1.0 mL Me4NOH (10%) +1.0 mL Me4NOH (10%) +1.0 mL Me4NOH (10%) +1.0 mL Me4NOH (10%) +pH = 11.5pH = 11.0pH = 10.5pH = 10.0pH = 9.5pH = 9.0pH = 8.5pH = 8.0pH = 7.5pH = 7.0pH = 6.0pH = 5.0pH = 4.0pH = 3.0+ 264 mg CAPS (pKa = 10.40) + 28.8 mL H2O+ 305 mg CAPS (pKa = 10.40) + 33.5 mL H2O+ 438 mg CAPS (pKa = 10.40) + 48.5 mL H2O+ 304 mg CHES (pKa = 9.50) + 35.2 mL H2O+ 462 mg CHES (pKa = 9.50) + 54.2 mL H2O+ 954 mg CHES (pKa = 9.50) + 113 mL H2O+ 365 mg HEPPS (pKa = 8.00) + 35.2 mL H2O+ 556 mg HEPPS (pKa = 8.00) + 54.2 mL H2O347 mg MOPS (pK a = 7.20) + 40.4 mL H2O600 mg MOPS (pKa = 7.20) + 70.4 mL H2O520 mg MES (pKa = 6.15) + 65.6 mL H2O108 mg S.A. (pKa2 = 5.57) + 21.8 mL H2O334 mg S.A. (pKa i = 4.19) + 69.8 mL H2O456 mg C.A. (pKa i = 3.06) + 58.4 mL H2O249

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