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The design and synthetic studies of N Putland, Michael Stuart 1990

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THE DESIGN AND SYNTHETIC STUDIES OF N*-[1-(CARBOXY)-9,11,21-(TRIOXO)-HENEICOSYL]-L-ASPARTYL-L-LYSYL-(TERT-BUTYL)-L-ASPARAGINATE, A TRIPEPTIDE ANALOGUE OF IONOMYCIN BY MICHAEL STUART PUTLAND B . S c , The U n i v e r s i t y o f V i c t o r i a , 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1990 (c) Michael Stuart Putland In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Ionomycin i s a s t e r e o c h e m i c a l l y complex c a l c i u m ionophore. An analogue (34) o f ionomycin has been designed and i t s s y n t h e s i s i n i t i a t e d . Regions o f the analogue which p a r a l l e l p o r t i o n s of ionomycin do not i n c o r p o r a t e any of the c h i r a l c e n t e r s found i n the n a t u r a l product. The analogue 34 a l s o i n c o r p o r a t e s a h i g h l y conserved t r i p e p t i d e found i n the c a l c i u m b i n d i n g s i t e of t r o p o n i n C p r o t e i n s . i i i TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i i i LIST OF SCHEMES i v LIST OF FIGURES v LIST OF ABBREVIATIONS v i ACKNOWLEDGEMENTS v i i i DEDICATION ix INTRODUCTION GENERAL 1 HISTORY OF THE POLYETHER ANTIBIOTICS ' 1 CHARACTERISTICS OF THE POLYETHER ANTIBIOTICS 3 MODE OF ACTION 4 INTEREST IN IONOMYCIN 15 CALCIUM BINDING PROTEINS 18 OBJECTIVES OF THIS WORK. 21 RESULTS AND DISCUSSION DESIGNING THE ANALOGUE 23 RETROSYNTHETIC ANALYSIS OF THE ANALOGUE (34) 29 TOWARDS THE SYNTHESIS OF ANALOGUE 34 30 FRAGMENT A SYNTHESIS 30 FRAGMENT B SYNTHESIS 37 COUPLING OF THE FRAGMENTS 39 CONCLUSION 41 EXPERIMENTAL GENERAL 42 SYNTHESIS AND REACTIONS 44 REFERENCES 75 APPENDIX IR AND 1H NMR SPECTRA 78 LIST OF SCHEMES SCHEME TITLE PAGE 1 The enolization and io n i z a t i o n of a Ji-diketone..24 2 Retrosynthesis of the analogue 34 2 9 3 The synthesis of fragment A 31 4 The synthesis of fragment B 32 5 Fragment coupling and deprotection 33 6 Formation of the dianion of 6 35 7 The sel e c t i v e protection of the side chain amine of L-l y s i n e 39 V LIST OF FIGURES FIGURE TITLE PAGE 1 Crystal structures of the potassium s a l t of the monovalent a n t i b i o t i c monensin and the barium s a l t of the divalent a n t i b i o t i c l a s a l o c i d A 5 2 The U-tube used to measure ion transport by polyether a n t i b i o t i c s 10 3 Changes i n pH on the two sides of a U-tube as a r e s u l t of the counter current flow of metal ions with hydrogen atoms res u l t s i n a decreasing pH i n the high s a l t side of the tube 10 4 Transport mode for carboxylic acid ionophores..11 5 The o v e r a l l transport reactions and i n d i v i d u a l component reactions required to complete a single transport cycle for monovalent and divalent monocarboxylic ionophores 12 6 Changes i n pH of the donor phase of the U-tube using various cations at 1 M 13 7 Conformation of the ionomycin-Ca ion complex. A: The hydrophobic complex. B: I n t e r f a c i a l complex. . 14 8 Transition between the ionomycin-calcium i n t e r f a c i a l complex and the hydrophobic complex 15 9 Leukotriene synthesis from arachidonic acid....19 10 The EF Hand model for the calcium binding s i t e of various proteins 21 11 The X-ray structure of the calcium s a l t of ionomycin 24 12 Comparison of ionomycin (1) with the analogue (34) 28 LIST OF ABBREVIATIONS A angstrom ATP adenosine triphosphate br. dd. broad doublet of doublets br. s. broad singlet °C degrees c e l c i u s calcd calculated cAMP c y c l i c adenosine monophosphate Cbz carbobenzoxy CI chemical i o n i z a t i o n (with methane or ammonia) C-terminal carboxy terminal d doublet DCC 1,3-dicyclohexylcarbodiimide dd doublet of doublets DMAP 4-dimethylaminopyridine DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(IH)-pyrimidinone EI electron impact g gram •^H NMR proton nuclear magnetic resonance HRMS high resolution mass spectrum Hz Hertz IR in f r a r e d KBr potassium bromide p e l l e t LD50 l e t h a l dose to k i l l 50% of tes t subjects LDA lithium diisopropylamide LRMS low resolution mass spectrum m multiplet M molar M + parent ion MHz megahertz mL m i l l i l i t r e mmol millimole m/z mass to charge r a t i o N - t e r m i n a l amino-terminal ppm parts per m i l l i o n r b f round bottom flask s singlet SRS slow reacting substance t t r i p l e t TBDMS t e r t i a r y - b u t y l d i m e t h y l s i l y l THF . tetrahydrofuran TLC t h i n layer chromatography ACKNOWLEDGEMENTS I would l i k e to thank Professor Larry Weiler for his guidance and helpf u l suggestions throughout the duration of th i s research. His advice was greatly appreciated. Thanks also go to my friends and colleagues i n the Weiler group. The services of the NMR and Mass Spec. Departments were greatly appreciated. This thesis i s dedicated to my family and friends. INTRODUCTION GENERAL: Recently, the e f f o r t s of several research groups have been directed to the t o t a l synthesis of the polyether a n t i b i o t i c ionomycin (1) which i s produced by Streetomyces  conalobatus. Due to the complex stereochemical nature of th i s compound, i t s synthesis has not been a t r i v i a l task. 1 The goal of t h i s research i s to develop a simple ionomycin analogue which might mimic the a c t i v i t y of the a n t i b i o t i c . The analogue would incorporate a tr i p e p t i d e moxety found to be conserved i n the calcium ion (Ca ) binding s i t e of the troponin C family of proteins. HISTORY OF THE POLYETHER ANTIBIOTICS: In 1951, two groups reported the i s o l a t i o n of three novel a n t i b i o t i c s which are now known to be polyether 1 7 a n t i b i o t i c s . ' Berger and coworkers reported the i s o l a t i o n of a n t i b i o t i c s X-206, X-464 (later known as nigericin) and X-537A (later known as l a s a l o c i d A) . Harned and coworkers also reported the i s o l a t i o n of n i g e r i c i n . These three 2 a n t i b i o t i c s had common c h a r a c t e r i s t i c s which distinguished them from other known a n t i b i o t i c s and suggested they constituted a new c l a s s . For example, a l l were a c i d i c and very soluble i n organic solvents. An unusual c h a r a c t e r i s t i c was the fact that when organic solutions of the a n t i b i o t i c s were exposed to aqueous base, there was no extraction of the a n t i b i o t i c anion into the aqueous phase. In fact, when sodium carbonate was used as the base, sodium ions (Na+) were extracted into the organic phase. Evaporation of the solvent yielded a c r y s t a l l i n e sodium s a l t of the a n t i b i o t i c . The three new a n t i b i o t i c s showed antimicrobial a c t i v i t y against Gram p o s i t i v e bacteria and mycobacteria in vitro. Due to t h e i r r e l a t i v e l y high parenteral t o x i c i t y (X-206 has an L D 5 0 of 1.2 mg/kg of mouse), l i t t l e i n t e r e s t was shown i n them for the following sixteen years. In 1958, a fourth polyether a n t i b i o t i c c a l l e d dianemycin (A16183) was reported. 4 Lardy and coworkers used dianemycin, n i g e r i c i n , and a number of other a n t i b i o t i c s as tools for metabolic studies. They reported that dianemycin and n i g e r i c i n were powerful i n h i b i t o r s of the oxidation of most pyridine-nucleoside linked substrates. Nine years elapsed before the f i f t h polyether a n t i b i o t i c (monensin) was reported i n 1967.^ The structure of monensin was elucidated and the a n t i b i o t i c was stated to have potent a n t i c o c c i d i a l a c t i v i t y . In 1976, control of c o c c i d i o s i s i n the poultry industry was estimated to have cost $100 m i l l i o n worldwide. This a c t i v i t y , as well as an a b i l i t y to improve feed conversion i n ruminant animals sparked much of the recent interest i n the polyether a n t i b i o t i c s . In 1968, monensin's b i o l o g i c a l a c t i v i t y was attributed to i t s d i r e c t i n t e r a c t i o n with a l k a l i metal cations and the c a r r i e r mechanisms which regulate transport of potassium + 1 ions (K ) across mitochondrial membranes. In the ensuing years, many additional polyether a n t i b i o t i c s have been reported. The structure and absolute configuration have been f u l l y characterized for most compounds. The polyether a n t i b i o t i c s are produced primarily by members of the Streptomyces genus. However, some are produced by species of the Actinomadura and Dactylosporanaium genera. Many are harvested through large scale fermentations. CHARACTERISTICS OF THE POLYETHER ANTIBIOTICS: The polyether a n t i b i o t i c s are members of a large group D of compounds known as ionophores. A l l polyether a n t i b i o t i c s possess a carboxylic acid moiety introduced during the polyketide biosynthesis of the a n t i b i o t i c precursor. The term "polyether" refers to the tetrahydrofuran and tetrahydropyran rings found i n these compounds. Describing these compounds as ionophores refers to t h e i r a b i l i t y to form organic soluble complexes with a l k a l i earth cations, and mediate t h e i r transport across l i p i d b a r r i e r s . ^ The polyether a n t i b i o t i c s may be divided into two groups depending on t h e i r a b i l i t y to transport monovalent or o divalent cations. Most of the divalent cation transporters do so as a dimeric complex (2:1 r a t i o of a n t i b i o t i c : cation). Ionomycin i s unique because i t transports divalent calcium ions as a monomeric (1:1) complex. MODE OF A C T I O N : The polyether a n t i b i o t i c s act as catalysts to f a c i l i t a t e the transfer of cations through a non-aqueous b a r r i e r . In the a n t i b i o t i c , the energy required for t o t a l ion desolvation i s minimized by having oxygen or nitrogen atoms replace the solvent molecules around the ion. The ionophore-cation complex i s quite stable i n the non-aqueous 1 n phase while a dehydrated (naked) cation i s not. The formation of a s t r u c t u r a l l y d i s t i n c t complex between the cation and the ionophore, coupled with movement of the complex across a membrane, distinguishes the polyether a n t i b i o t i c s from other types of a n t i b i o t i c s and other agents which increase the permeability of the membranes to cations by d i f f e r e n t mechanisms. The polyethers possess multiple ether linkages usually i n the form of substituted tetrahydrofuran and tetrahydropyran rings. A l l ionophores of t h i s class have an in t e r n a l 5 l i g a t i n g s i t e with the outer surface of the complex being composed large l y of nonpolar hydrocarbon chains or heterocyclic and aromatic residues (Figure 1) . In each structure the polar l i g a t i n g portion of the complex i s shielded from the environment by the l i p o p h i l i c side chains of the a n t i b i o t i c molecule. The potassium complex i s i n a 1:1 r a t i o while the barium complex has a 2:1 stoichiometry of a n t i b i o t i c to cation. FIGURE 1: Crystal structures of the potassium s a l t of the monovalent a n t i b i o t i c monensin and the barium s a l t of the divalent a n t i b i o t i c l a s a l o c i d A. ' The a n t i b i o t i c ' s carbon backbone i s capable of assuming conformations that focus s t r a t e g i c a l l y placed electron donors about a cavity i n which a cation may f i t . Polyether ionophores possess f l e x i b l e carbon backbones and may open to permit the entry of a cation. The binding energy depends upon the conformational constraints on the f l e x i b i l i t y imparted by the s p e c i f i c s u b s t i t u t i o n a l and stereochemical arrays along the molecule. The nonligatmg substituents and t h e i r stereochemistry are important and exert t h e i r effect by preorganizing the structure to favour the binding c o n f o r m a t i o n . A n t i b i o t i c s with smaller carbon backbones w i l l usually have a very r i g i d conformation, while longer backbones w i l l permit more f l e x i b i l i t y and fo l d i n g i n the complexed state. The l i g a t i n g oxygen atoms are incorporated i n a v a r i e t y of functional groups such as ethers, alcohols, carboxylates, and amides. The neutral oxygen atoms chelate the cation v i a ion-dipole interactions analogous to those between the ion and h i g h - d i e l e c t r i c solvents. It i s important to note that the polyether a n t i b i o t i c s w i l l only form cation complexes when t h e i r carboxylic acid moiety i s deprotonated. Thus, the ionophores w i l l only carry ions as e l e c t r i c a l l y neutral, i o n i c complexes. However, the polyether may cross the membrane to pick up another cation i n i t s neutral state. The a n t i b i o t i c acts as an exchange d i f f u s i o n c a r r i e r by transporting hydrogen or a l k a l i metal ions down t h e i r concentration gradients across membranes. The polyether a n t i b i o t i c s w i l l usually form a 1:1 complex with an appropriate monovalent cation and a 2:1 complex of ionophore to divalent cation. One exception i s ionomycin which has an ionizable ii-diketone f u n c t i o n a l i t y , as well as the carboxylic acid moiety. This allows the a n t i b i o t i c to become doubly ionized so i t may transport divalent cations as a neutral 1:1 complex. The number of l i g a t i n g oxygens or nitrogens for a given cation often varies with the ionophore-cation complex. In some cases, water may take up some of the l i g a t i n g s i t e s on the ion. The l i p i d s o l u b i l i t y of the complex may be p a r t l y explained by e f f e c t i v e shielding of the polar i n t e r i o r which s t a b i l i z e s the cation charge. The compatibility of the exterior of the complex with l o w - d i e l e c t r i c solvents also improves l i p i d s o l u b i l i t y . Polyether a n t i b i o t i c s serve as the c a r r i e r i n a counter-current cation transport system. The l i g a t i n g cavity i s sim i l a r to an enzyme active s i t e , due to i t s a b i l i t y to discriminate between very s i m i l a r substrates (cations). Each a n t i b i o t i c has i t s own stable conformation for complexing with cations which may be maintained by hydrogen bonds. The bulky hydrophobic groups are arranged s t r a t e g i c a l l y to help maintain an active conformation. 1^ Conformational constraints i n the polyether a n t i b i o t i c s can l i m i t the strength with which cations of d i f f e r e n t sizes may be l i g a t e d . The dimensions of the l i g a t i n g cavity are reasonably sel e c t i v e for a cation of s p e c i f i c size and charge. The ion s e l e c t i v i t y of ionophores i s a function of the energy required for ion desolvation and the binding energy obtained on complexation. For polyether a n t i b i o t i c s with a r e l a t i v e l y unconstrained backbone, the free energy of ionophore complexation i s governed by the same considerations that determine the free energy of solvation. That i s , the charge density of the ion i s an important factor. Ionophores with highly constrained backbones w i l l select for a cation of c r i t i c a l i o n i c radius. Thus, form-f i t becomes very determinative i n the net free-energy i R difference between desolvation and complexation. ^ As a re s u l t of t h i s s e l e c t i v i t y , one cation i n a series (Table 1) i s usually accommodated more e f f i c i e n t l y than the others q which are smaller or larger than the optimal s i z e . i ft TABLE 1: Some metals and t h e i r i o n i c r a d i i . ° ELEMENT CHARGE ATOMIC NUMBER RADIUS (A) L i +1 3 0.68 Na +1 11 0.97 K +1 19 1.33 Rb +1 37 1.47 Cs +1 55 1.67 Mg +2 12 0.66 Ca +2 20 0.99 Ba +2 56 1.34 A balance between the energy required for desolvation (which need not be complete i n a l l cases), the energy of association with the ionophore, and the energy necessary to convert the ionophore from i t s uncomplexed conformation to that i n the complex a l l influence the complexation process. In general, movement of ionophores through membranes may be viewed as a problem of lipid-ionophore interactions while the complexation-decomplexation reactions are related 9 to i n t e r f a c i a l chemistry. E l e c t r o n e u t r a l i t y of t o t a l charge movements i s maintained i n the ov e r a l l transport cycle. The net charge of species which traverse the membrane are neutral. Consecutive reactions by which the transporting species form and dissociate (together with d i f f u s i o n reactions) constitute transport cycles. Each reaction could be described i n terms of an equilibrium constant and rate constants for forward and reverse d i r e c t i o n s . The d i r e c t i o n and extent of cation transport by polyether a n t i b i o t i c s are determined by metal and hydrogen ion concentration gradients. The transport of cations-across c e l l membranes may be e f f i c i e n t i f certain k i n e t i c c r i t e r i a are f u l f i l l e d . At the h i g h - d i e l e c t r i c region of the membrane interface, complexation and decomplexation reactions must be fast. The exchange of the ion's solvation s h e l l for the ionophore's l i g a t i n g system must be a concerted reaction i n order that the energy of act i v a t i o n for transport remain low. When the complex enters the low-d i e l e c t r i c region of the membrane i n t e r i o r , where i t i s immune to solvent attack, i t may then a t t a i n high s t a b i l i t y . 1 5 Ion transport by the polyethers may be measured by f i l l i n g a U-tube with antibiotic-doped chloroform to just above each bend (Figure 2). Each side arm of the apparatus i s then f i l l e d with an appropriate aqueous solution. The chloroform layer i s then gently s t i r r e d . I f one arm of the 10 U-tube i s f i l l e d with a s a l t solution and the other arm i s f i l l e d with a s a l t free solution, the pH of the s a l t free side r i s e s while that on the saline side decreases (Figure 3) . This re s u l t might be explained by the transport sequence that follows (Figure 4 ). The anionic ionophore on the high s a l t side of the "membrane" associates with an appropriate cation. The zwitte r i o n i c complex diffuses down the cation gradient across the membrane. The complexed cation i s released on the low s a l t side of the membrane. The. ionophore then picks up a proton from the solvent and diffuses back across the membrane to the high s a l t side. Release of the proton results i n a decrease i n pH on the high s a l t side while the pH on the low s a l t side increases. 10 Vrtfter-. borner (Ocn FIGURE 2 : polyether a n t i b i o t i c s . The U-tube used to measure ion transport by 10 S o h - f r e t \\it ( I t f f ) FIGURE 3: Changes i n pH on the two sides of a U-tube as a resul t of the counter current flow of metal ions with 11 hydrogen ions results i n a decreasing pH i n the high s a l t side of the tube 10 (H 20) n:M" nH 20 I I I h i g h s a l t d e c r e a s i n g pH HI H> M +I" HI I"M+ I — I I ««-I -IH M nH20 •H+ low s a l t i n c r e a s i n g pH FIGURE 4: Transport mode for carboxylic acid ionophores. I represents an ionophore, M represents an a l k a l i metal, and H represents hydrogen 15 This simple measurement i s a good method of i l l u s t r a t i n g the counter current transport catalyzed by polyether a n t i b i o t i c s . However, protons are not always the counter cation and the ionophore may be mono- or divalent. Counter transport of a mono- or divalent cation may provide the zwitterionic species that w i l l cross the membrane to complete the transport cycle ( F i g u r e 5). 12 o u r s i o e . o u r 6 « o e Pir^t + A i l FUMX FIGURE 5: The o v e r a l l t r a n s p o r t r e a c t i o n s ( l e f t ) and i n d i v i d u a l component r e a c t i o n s ( r i g h t ) r e q u i r e d t o complete a s i n g l e t r a n s p o r t c y c l e f o r monovalent (A) and d i v a l e n t (B) monocarboxylic ionophores. P a i r s o f v e r t i c a l l i n e s r e p r e s e n t the membrane acr o s s which t r a n s p o r t o c c u r s . S u b s c r i p t s i and o denote i n s i d e and o u t s i d e o f a membrane r e s p e c t i v e l y . Component r e a c t i o n s where d i f f u s i o n occurs a c r o s s the u n s t i r r e d aqueous l a y e r at the i n t e r f a c e are i n d i c a t e d w i t h an a s t e r i s k . Ion transport s p e c i f i c i t i e s of the ionophores can be determined i n the above system. By placing d i f f e r e n t cations i n the solution i n donor arm, the rate of appearance of cations i n the acceptor arm can be measured by atomic absorption spectrometry for example. On the other hand, the change i n pH of the saline solution can also be measured with time (Figure 6). Ions whose s p e c i f i c i t y i s high with a given ionophore would show a more rapid decrease i n pH i n the donor arm of the U-tube.. ( e ) (b) lc> FIGURE 6: Changes i n pH of the donor phase of the U-tube using various cations at 1.0 M. (a) Monensin. (b) 1 fi N i g e r i c i n . (c) Dianemycin. w It has been postulated that the ionophore conformation required for the complexation-decomplexation reactions may d i f f e r from that required for d i f f u s i o n of the complex across the membrane. Molecular modeling has been used to predict the l i k e l y conformations of ionomycin i n i t s 1 7 l i p o p h i l i c and lipid-water interface states. Molecular modeling i s a technique to calculate the most l i k e l y conformation of a molecule i n a solution of a given d i e l e c t r i c constant. The more stable conformations would 14 have minimal s t e r i c interactions between substituents. The d i e l e c t r i c constant discontinuity e x i s t i n g at the l i p i d -water interface mediates transformation of one conformation into another (Figure 7) . It was found that rotation about three of the bonds i n ionomycin can provide the change from one conformer to the other. Calcium ions leaving the cry p t i c cavity within the ionophore would be li b e r a t e d into the aqueous phase as the ionophore changed conformations due to changing solvent d i e l e c t r i c at the lipid-water interface. The degree of penetration of the ionophore into the membrane could provide the dr i v i n g force for the conformational change (Figure 8) . An important facet of these conformational changes i s that the molecule would only undergo a low energy t r a n s i t i o n (few bonds rotated) otherwise the b a r r i e r to ion complexation would be too large 1 7 for e f f i c i e n t transport across the membrane. FIGURE 7: Conformation of the ionomycin-Ca ion complex. A: The hydrophobic complex. B: I n t e r f a c i a l complex. The calcium ion i s shown as a closed c i r c l e . Open c i r c l e s r e f e r to carbon atoms and dotted c i r c l e s to oxygen atoms. The dotted l i n e delineates the hydrophobic (dielectric=3) and 1 7 the hydrophilic medium (dielectric=30). FIGURE 8: Transition between the ionomycin-calcium i n t e r f a c i a l complex and the hydrophobic complex. The dotted 1 7 l i n e denotes the surface of the membrane. INTEREST I N IONOMYCIN: C e l l s of m u l t i c e l l u l a r organisms communicate with each other v i a the passage of chemical messengers between c e l l s . These chemicals coordinate metabolic a c t i v i t y of various tissues, allow an organism to adapt to environmental changes, and prepare i t for reproduction. One major class of chemical messengers i s l i b e r a t e d by neurons i n response to stimulation. I f these compounds dif f u s e to neighboring c e l l s to activate s p e c i f i c responses, they are referred to as neurotransmitters. Another major class of chemical messengers i s known as hormones. A l l hormones act by binding to macromolecular receptors that are located either on the c e l l membrane or inside responsive c e l l s . Binding of the hormone induces a change in the conformation of the receptor; t h i s change i n conformation perturbs other molecules and e l i c i t s a chain of events that leads to a vast array of c e l l u l a r changes ranging from alt e r a t i o n s i n enzyme a c t i v i t y to changes i n gene expression. These ef f e c t s may lead to profound al t e r a t i o n s i n c e l l growth, morphology, and function. It i s important to note that d i f f e r e n t c e l l types may respond quite d i f f e r e n t l y to the same hormone. Two major signal transduction mechanisms are known to transmit signals across the c e l l membrane. One mechanism involves the a c t i v a t i o n of membrane-bound adenylate cyclase to convert adenosine triphosphate (ATP) to c y c l i c adenosine monophosphate (cAMP). C y c l i c AMP then serves to influence enzymes within the c e l l . The second mechanism used i n signal transduction u t i l i z e s several messengers such as calcium ions, i n o s i t o l triphosphate, and d i a c y l g l y c e r o l . It 7 + i s the influence of Ca on the c e l l which may be controlled by a transmembrane calcium transporter such as ionomycin. 7 + I n t r a c e l l u l a r Ca concentration has been found to influence several c e l l u l a r processes while bound to the calcium binding protein calmodulin. The calcium-calmodulin complex activates calcium stimulated ATPase, several kinases, adenylate cyclase, phospholipase A 2, and phosphodiesterase. ° Since these enzymes are activated by i n t r a c e l l u l a r calcium, c e l l u l a r processes such as protein phosphorylation, arachidonic acid oxygenation, glycogenolysis, muscle contraction, and cAMP level s are a l l influenced by calcium ion concentration. The role of calcium ions i n the oxygenation of arachidonic acid i s of p a r t i c u l a r i n t e r e s t i n the treatment of asthma. Calcium ions may come from the e x t r a c e l l u l a r f l u i d and t h e i r transport i s usually associated with potassium or hydrogen ion countertransport mediated by a membrane bound ion pump. I n t r a c e l l u l a r stores of calcium ions are usually associated with the cytoplasmic side of the plasma membrane or sequestered within the mitochondria or reticulum. A calcium gradient exists across the c e l l membrane where the e x t r a c e l l u l a r concentration i s approximately 100 times that found within the c e l l . C e l l u l a r processes are influenced by a change i n the i n t r a c e l l u l a r calcium concentration which can be perturbed quite e a s i l y as a resu l t of the transmembrane gradient. Chemicals released from mast c e l l s and immunological reactions can a l t e r the c a l i b r e of an airway. Mast c e l l s located i n the connective tissue underlying smooth muscle and i n airway walls release histamine and slow reacting substance (SRS). For many years, s c i e n t i s t s have been interested i n the biochemical processes involved with asthma. Possibly involved i n t h i s disease i s SRS, an extremely potent muscle contractant that can severely c o n s t r i c t small airways i n the lung. SRS, now known to be leukotriene C, i s a metabolite of arachidonic acid (Figure 9) whose synthesis i s stimulated by ionomycin mediated calcium transport. It has been shown that ionomycin can stimulate the acti v a t i o n of phospholipase A2 which i s involved i n the l i b e r a t i o n of arachidonic acid from d i a c y l g l y c e r o l . " However, prostaglandin synthesis from 0 1 arachidonic acid i s not stimulated by ionomycin. The prostaglandins are a group of compounds important to many physiological processes including c o n s t r i c t i o n and d i l a t i o n of the bronchioles of the lung. As a resu l t , the e f f e c t s of SRS may be assessed without the interference of potent chemicals such as the prostaglandins. It i s hoped that t h i s probe would give a better understanding of the biochemical basis of asthma and possibly lead to better treatments of the disease. C A L C I U M B I N D I N G P R O T E I N S : The i n t r a c e l l u l a r concentration of Ca^ must be kept at a low l e v e l due to the high i n t r a c e l l u l a r concentration of phosphate esters which form insoluble calcium s a l t s . As a resul t , a calcium gradient i s established across a c e l l membrane where the external concentration of calcium i s orders of magnitude higher than that found within the c e l l . This gradient has evolved into a signal mechanism where cy t o s o l i c C a 2 + concentration may be increased suddenly by 19 opening channels to calcium located i n the plasma or r e t i c u l a r membrane. / = V = V > V P 0 0 " L A = / W Arachldonlc add I Lipoxygenase oo-*-H]rt»rop«roxy-«,8,11,1 < alcotrtatiaonolc acid (5-HPETE) M.0 Dehydrase Gtulalhione-S-transterase COCT S - C H , I • CH—g—N—CH2COO" NH—C—CHjCH,—CH HjN* COO-i M k o t r t a T W C 1 Q FIGURE 9: Leukotriene biosynthesis from arachidonic acid. ^ Calcium ions are also capable of binding to proteins. The negatively charged oxygens found on the side chains of aspartate and glutamate residues as well as uncharged 7 + 7 + oxygens from mam-chain carbonyls bind to Ca . Since Ca^ can f a c i l i t a t e multiple coordination with six to eight oxygen atoms, i t may cross l i n k various regions of a protein and thus induce major conformational changes i n the protein. Also, the binding of Ca^ to a protein i s quite s e l e c t i v e due to the ion's high a f f i n i t y for uncharged oxygen atoms. 7+ Magnesium ions (Mg^ ) can serve as a competitor for protein 7 + bindmg s i t e s . However, i t turns out that Mg i s not very 7+ in-e f f e c t i v e as a competitor for Ca . Mg^ - has a low a f f i n i t y for uncharged oxygen atoms, and i t prefers to form small, 7+ symmetric coordination s h e l l s . Whereas Ca* forms asymmetric s h e l l s of larger radius. As a r e s u l t , Ca can bind to i r r e g u l a r l y shaped s i t e s i n proteins and can be selected over Mg* even i f the l a t t e r ion i s much more 7 7 abundant. X-ray crystallographic studies of calcium-binding proteins have suggested how calcium binding may occur. Parvalbumin, a carp muscle protein with two s i m i l a r calcium binding s i t e s , has been studied. The binding s i t e consists of a helix, a loop, and another h e l i x . Each calcium ion i s coordinated by eight oxygen atoms: three aspartate and three glutamate carboxylate oxygens, a main chain carbonyl oxygen, and one oxygen from a complexed water molecule. It has been suggested that the E and F helices of the binding s i t e resemble the forefinger and thumb of the right hand 7 + (Figure 10) . The Ca*1 binding s i t e i s formed by the loop found between these h e l i c e s . The calcium binding s i t e of parvalbumin and troponin C (involved i n muscle contraction) are believed to have evolved from the duplication of a primordial gene coding for the calcium-binding loop. The amino acid sequences of the binding s i t e s show some 21 s i m i l a r i t y and the EF hand has been found to recur i n the C a 2 + binding s i t e s of other p r o t e i n s . 2 2 Calmodulin i s a calcium binding protein found i n nearly a l l eukaryotic c e l l s . This protein has been constructed from repeating modules with the EF hand motif. Thus, certain amino acid sequences should be conserved throughout the evolution of the proteins i n order for the binding s i t e conformation to be conserved. This, i n fact, has been found to be the case 7 "3 within the troponin C family of proteins. FIGURE 10: The EF Hand model for the calcium binding s i t e 22 of various proteins. OBJECTIVES OF THIS WORK: It i s our objective to design a simple ionomycin analogue which would be capable of mimicking the a c t i v i t y of ionomycin without having the stereochemical complexity associated with the natural product. The analogue would incorporate part of the carbon skeleton of ionomycin, including the terminal carboxylate and the ii-diketone moieties, but without the a l k y l substituents which create the numerous stereochemical centers and complicate a synthesis. We wished to substitute a short peptide i n place of the bistetrahydrofuranyl portion of the natural product. A t r i p e p t i d e found i n the calcium binding s i t e of troponin C was chosen for t h i s purpose. It was deemed important to maintain the p o t e n t i a l membrane s o l u b i l i t y of the analogue and r e t a i n the charge balance which could be found i n an ionomycin-calcium complex. The aim was to design a simple calcium transporter which could not only be synthesized r e l a t i v e l y e a s i l y but might also provide information on the r e l a t i o n s h i p between C a 2 + a f f i n i t y and transport, as well as the t o x i c i t y of such compounds. RESULTS AND DISCUSSION DESIGNING THE ANALOGUE: The f i r s t stage of t h i s project involved the design of a novel molecule which would exhibit an a f f i n i t y for calcium ions and an a b i l i t y to transport those ions across a c e l l membrane. In short, we wanted to design a calcium ionophore based p a r t i a l l y on the information we had gathered about the mode of action of the polyether a n t i b i o t i c , .ionomycin. The analogue would be a molecule which would only possess those functional groups thought to be v i t a l to the function of the natural product. The target would also be s i m p l i f i e d by not incorporating the numerous stereochemical centers found along the carbon backbone of ionomycin. I n i t i a l l y , we studied the c r y s t a l structure of the p 4 calcium s a l t of ionomycin (Figure 11). In t h i s complex, i t was possible to determine which functional groups within ionomycin were important for the binding of a calcium ion. 9 + Ionomycin binds Ca as an e l e c t r i c a l l y neutral complex. Thus, i t was important to ensure that the analogue be O j . capable of binding Ca as an e l e c t r i c a l l y neutral complex. The natural product possesses two ionizable hydrogens from the carboxylic acid and the fi-diketone units. The 15-diketone exists primarily i n i t s keto-enol form with the enolic proton being l o s t upon the chelation of Ca*- (Scheme 1) . Since both of these functional groups were p i v o t a l i n Q -CARBON -OXYGEN -CALCIUM F I G U R E 11: The X-ray structure of the calcium s a l t of 24 ionomycin. c o n t r o l l i n g the charge balance of the calcium s a l t of ionomycin, i t was deemed important that those two functional groups be retained i n the design of an analogue. NOTE: R - an aJkyl chain no smaller than methyl Scheme 1: The enolization and ion i z a t i o n of a fi-diketone. o o BASE Location of the chelating functional groups along the carbon backbone of ionomycin allows the electron donating atoms to rotate into one of the octahedral chelation s i t e s on the calcium ion. In other words, the a b i l i t y of the molecule to wrap around the ion and bind snugly to i t would be an important consideration i n the design of the analogue. As a r e s u l t , our analogue would incorporate p o t e n t i a l l i g a t i n g functional groups at i n t e r v a l s along the carbon backbone approximating those found i n ionomycin. The X-ray structure of calcium s a l t of ionomycin reveals which atoms i n the molecule are important i n chelating the calcium ion. One of the oxygens found i n the carboxylic acid binds to the cation as expected, due to e l e c t r o s t a t i c a t t r a c t i o n s . Both oxygen atoms i n the J5-diketone moiety are involved i n the l i g a t i o n s i t e , l i k e l y due to e l e c t r o s t a t i c attractions as well. The a l c o h o l i c oxygens six and nine are also found to coordinate to the ce n t r a l l y complexed calcium ion. F i n a l l y , oxygen seven, found i n a tetrahydrofuran moiety, i s also involved i n cation chelation. Thus, there are six donor atoms located octahedrally around the central calcium ion. The analogue should attempt to incorporate six p o t e n t i a l l i g a t i n g atoms at distances approximating those found i n ionomycin. As mentioned previously, the analogue should also 7 + incorporate a t r i p e p t i d e found i n the Ca binding s i t e of the troponin C proteins. The sequence of amino acids i n a polypeptide confer a s p e c i f i c conformation upon that polypeptide, allowing i t to function i n a s p e c i f i c manner. Since the binding of calcium ions i s very important i n the regulation of many c e l l u l a r functions, i t i s possible that the amino acid sequence of a calcium binding s i t e i s u n l i k e l y to undergo r a d i c a l changes during evolution. This has been demonstrated for troponin C which i s an important protein for muscle contraction. The calcium binding loop found i n Troponin C has been i s o l a t e d from 149 binding s i t e s and the sequence of amino acids determined. J The binding s i t e consists of an °<-helix, a loop, and a second of-helix. The loop of amino acids runs from residues 13 to 24 i n a polypeptide that consists of 32 amino acids for the E and F helices and loop. It i s the loop of the binding s i t e which harbors the a b i l i t y to chelate calcium ions even a f t e r i t has been excised from the polypeptide. We a r b i t r a r i l y chose to l i m i t composition of our analogue to three amino acids i n order to maximize i t s calcium binding s i t e s i m i l a r i t y as well as minimizing synthetic demands. We chose three consecutive amino acids that are highly conserved i n the binding s i t e loop (residues 13 to 15) of troponin C. The N-terminal of the t r i p e p t i d e always consists of an L-aspartic acid residue (100% conservation). The higher the degree of conservation of a p a r t i c u l a r residue, the more c r u c i a l that amino acid i s thought to be in the structure and function of a protein. The second 27 amino acid of our, t r i p e p t i d e (derived from residue 14) was chosen to be L - l y s i n e which was the most highly conserved (29%) amino acid i n that p o s i t i o n . Another useful feature of using lysine as the second amino acid was the fact that i t has a basic side chain which may neutralize any charge on the a c i d i c side chain of the aspartic acid. The C-terminal amino acid of our tr i p e p t i d e would preferably be a neutral molecule to maintain charge n e u t r a l i t y required for calcium transport. Unfortunately, the most highly conserved amino acid i n that p o s i t i o n (residue 15) was L-aspartic acid (70%). The a c i d i c nature of t h i s molecule's side chain would make i t an undesirable choice for our analogue. The second most highly conserved amino acid was L-asparagine (24%), a molecule with a neutral side chain functional group. Thus, we planned to fuse a carbon chain analogous to the C-l to C-21 unit of ionomycin (Fragment A) to a tri p e p t i d e consisting of L-aspartic acid, L - l y s i n e , and L -asparagine. We were l e f t with one more consideration. The terminus of the t r i p e p t i d e fragment would be a free carboxylic acid. Leaving the acid i n i t s free form was thought to be undesirable since i t introduced a charge imbalance which we had been careful to avoid. Another possible problem with the free acid was the fact that i t might decrease l i p i d s o l u b i l i t y for the analogue by increasing the polar nature of the compound. Keeping these considerations i n mind, we decided to make a t e r t - b u t y l 28 ester at the C-terminus of the t r i p e p t i d e fragment. Not only would t h i s prevent formation of a charge imbalance, but i t could also improve the l i p i d s o l u b i l i t y of the analogue. The t r i p e p t i d e ester would constitute what w i l l be referred to as Fragment B. Thus, our complete analogue, N -(1-carboxy-9,11,21-trioxoheneicosyl) - L-aspartyl - L-lysyl-(tert-butyl) - L-asparaginate (34) could be divided into two main fragments (Scheme 2). The structure of ionomycin i s compared to the analogue 34 to i l l u s t r a t e that certain functional and s t r u c t u r a l features have been retained i n the proposed analogue (Figure 12). Ionomycin Os O: 34 The Analogue in-O H FIGURE 12: Comparison of ionomycin (1) with the analogue 34. RETROSYNTHETIC ANALYSIS OF THE ANALOGUE (34): Our synthetic plan was based on the retrosynthetic analysis of the desired compound 34 shown i n Scheme 2. The desired product 34 was divided into fragments A and B. Fragment A could be broken into three units: 1,7-heptanediol (7), acetylacetone (5), and 1,9-nonanediol (2). Fragment B could be broken into three units as well: L-aspartic acid (19), L-lysine (23), and L-asparagine (27). A great advantage to using these six simple s t a r t i n g materials was that they are commercially available and a l l were inexpensive. Using other simple s t a r t i n g materials would allow substitution of d i f f e r e n t amino acids and/or d i f f e r e n t d i o l s to make other analogues of ionomycin. 23 Scheme 2: Retrosynthesis of the analogue 34. TOWARDS THE SYNTHESIS OF ANALOGUE 34: The proposed synthesis of our t r i p e p t i d e analogue was broken into three phases: the synthesis of fragment A (Scheme 3), the synthesis of fragment B (Scheme 4), and the coupling of fragments A and B with t h e i r subsequent deprotection (Scheme 5) . A dotted l i n e across the schemes denotes the point up to which synthesis was performed i n the course of t h i s project. It i s important to note that i n the convergent synthesis of the analogue 34, the functional groups were protected i n a manner which would allow complete deprotection i n the f i n a l step of the synthesis. FRAGMENT A SYNTHESIS: Both 1,9-nonanediol (2) and 1,7-heptanediol (7) were monobrominated to give 9-bromo-l-nonanol (3) and 7-bromo-l-heptanol (8) respectively i n high y i e l d . Alcohol 3 was then protected using t e r t -b u t y l d i m e t h y l s i l y l chloride to give 9-bromo-l - r(tert-butyldimethylsilyl)oxy]-nonane (4) i n 99% y i e l d . 2 ^ The dianion of acetylacetone (5) was prepared using 2.1 equivalents of lithium diisopropylamide at 0°C i n THF and t h i s was monoalkylated on a primary carbon by slow addition 9 7 of 0.9 equivalents of the bromo compound 4. ' The a l k y l a t i o n reaction produced the desired 14-F (t e r t -butyldimethylsilyl)oxy]-2,4-tetradecadione (6) i n 61% y i e l d . A l k y l a t i o n can be shown to have occurred on a primary carbon Hm,B& CHjCI, 11 18 Fragment A Scheme 3: The synthesis of fragment A. 32 HjN 1 9 2 0 OH .OH PhCHjOH HjS04 OBz OH 2,2,2-triehloroethyl chloroformate 2 4 NaOH, water-dioxane benzyl chloroformats NaOH CljC O' 2 1 o A Synthesis completB above fine "OBz O DCC p-nitrophenol CH2Cl2 O JL. ci3cr o N H 2 2 f ^ O B z 6 Y D C C p-n i t rophenol o r ^ N H 2 A . ^OH 2 7 ° benzyl chkxDfonna t s NaOH O JL. BzO N H NHj .OH 2 8 isobutylene HjSO. NH2 O 2 9 ° H2/Pd-C MeOH NH, HjN .3.0.. o J^SJBZ O [^ TNOj C I 3 C ^ 0 ^ N ' ^ ^ H V ^ 0 ' ^ ^ 2 6 BzO. OBz O O o Y o Zn,THF-HjO Acetic acid Y o Fragment B O \ o\ C^^^N^Y^H'V0^ O 31 DBz O Y Scheme 4 : The synthesis of fragment B. 33 Fragment A Fragment B The Analogue Scheme 5: Fragment coupling and deprotection. by the presence of a methyl signal i n the 2.0 ppm region of the NMR spectrum integrating to only three protons. I f alk y l a t i o n had occurred at the central carbon, these methyl signals would integrate to six protons. The sin g l e t at 5.4 9 ppm would not be present i f the «(-alkylated product had been produced. Alcohol 8 was protected as i t s methoxymethyl ether to give 7-bromoheptyl-l-methoxymethyl ether (9) i n 67% y i e l d . ° The k i n e t i c a l l y deprotonated dianion of 6 (Scheme 6) was generated using 3.0 equivalents of lithium diisopropylamide in a 2:1 mixture of THF:DMPU.29 DMPU was necessary to get dianion formation. To t h i s dianion was added 1.5 equivalents of bromo compound 9 and the a l k y l a t i o n was allowed to take place over 12 hours at -78°C. The reaction produced 21-[ (tert-butyldimethylsilyl)oxy]-heneicosa-9,11-dioxo-l-methoxymethyl ether (10) i n good y i e l d (72%). Introduction of the methoxymethyl group was indicated by the two singlets at 4.61 ppm (2H) and 3.37 ppm (3H) i n the NMR spectrum of 10. Once again i t was important to v e r i f y that the a l k y l a t i o n had occurred at the primary rather than a secondary anionic center. A l k y l a t i o n at the methyl carbon derived from ketone 6 was supported by the presence of a singlet at 5.4 9 ppm and the disappearance of the methyl signal at 2.0 ppm i n the NMR spectrum. Mass spectral data also shows no s i g n i f i c a n t ion which would correspond to the loss of an ace t y l . The acetyl ion would be l o s t quite e a s i l y i f the i s o l a t e d product had been alkylated on the «<-carbon. o o ' T B D M S Kinetic Control 2.0 eq. LDA O O ( T B O M S 6 2.0 eq. L D A O O I T B D M S Thermodynamic Control Scheme 6: Formation of the dianion of 6. The methoxymethyl ether of compound 10 was removed -3(") using bromodimethyl borane i n methylene chloride. u Unfortunately, even using freshly d i s t i l l e d boron reagent, the reaction only gave s e l e c t i v e deprotection to y i e l d 50% of the expected 21-[(tert-butyldimethylsilyl)oxy]-9,11-dioxoheneicosanol (11) . The l i b e r a t i o n of the alcohol was v e r i f i e d by spectral data. The appearance of an alcohol stretch (3600 to 3200 cm - 1) i n the IR accompanied by disappearance of the methoxymethyl singlets i n the NMR suggest that deprotection did occur. It i s believed that the reagent was generating HBr which caused cleavage of the s i l y l ether as well. The alcohol 11 was subjected to Moffatt oxidation conditions to y i e l d the corresponding 36 aldehyde 12 i n greater than 90% y i e l d . The crude 21 -[(tert-butyldimethylsilyl)oxy] - 9 / 1 1-dioxoheneicosanal (12), which was d i f f i c u l t to purify, was oxidized using an excess of s i l v e r n i t r a t e and sodium hydroxide with C e l i t e to give 2 1 - [(tert-butyldimethylsilyl)oxy] - 9 , 1 1-dioxoheneicosanoic acid (13) i n 25% y i e l d . ^ 2 D i f f i c u l t i e s arose i n extracting the desired acid from the residues produced i n the reaction. The impure acid 13 was then e s t e r i f i e d using an excess of DCC and benzyl alcohol to give the desired ester, benzyl - 2 1 -[(tert-butyldimethylsilyl)oxy] - 9 , 1 1-dioxoheneicosanate (14) 3 3 i n low y i e l d . The appearance of aromatic protons at 7.35 ppm (5H) and methylene protons at 5.10 ppm i n the NMR spectrum of the product indicate that the e s t e r i f i c a t i o n was successful. The mass spectrum for t h i s compound has a signal at 338 corresponding to loss of s i l y l o x y and benzyloxy fragments. The s i l y l ether 14 was cleaved using tetrabutylammonium fl u o r i d e to give the corresponding alcohol 15 i n 75% y i e l d . ^ 4 Infrared v e r i f i e d the loss of the s i l y l protecting group by appearance of the alcohol stretch i n the 3600 cm region. Benzyl 21-hydroxy -9 ,11-dioxoheneicosanate (15) was the l a s t compound characterized i n the synthesis toward Fragment A. It i s proposed that 15 could be subjected to the Moffatt oxidation followed by another s i l v e r n i t r a t e oxidation to give aldehyde 16 and carboxylic acid 17 respectively."^ 1'^ 2 F i n a l l y , the preparation of Fragment A could be completed by forming the active ester 18 using an excess of DCC and p-nitrophenol. J The preparation of the activated ester, benzyl-20-(p-nitrophenyloxycarbonyl)-9,11-dioxoeicosanate (18) would leave Fragment A ready for coupling with the free amine of the t r i p e p t i d e . ^ As proposed, the completion of the synthesis of fragment A would involve r e p e t i t i o n of basic reaction conditions used e a r l i e r i n the preparation of the compound. FRAGMENT B SYNTHESIS: The synthesis of t h i s portion of the analogue primarily involved protecting various functional groups of the amino acids i n order to allow t h e i r l o g i c a l assembly into the desired t r i p e p t i d e . We chose to assemble our t r i p e p t i d e using activated ester methodology since t h i s technique suited our choice of protecting groups and f a c i l i t a t e d the introduction of the t e r t - b u t y l ester of L-asparagine. L-Aspartic acid (19) was e s t e r i f i e d with excess benzyl alcohol under a c i d i c conditions to s e l e c t i v e l y protect the side chain carboxylic acid and y i e l d B-benzyl-L-aspartic acid (20) i n 27% y i e l d . D e p r o t e c t i o n of t h i s functional group could l a t e r be achieved by hydrogenolysis. The next stage of preparing t h i s amino acid for peptide synthesis was proposed . to be the protection of the free amine by subjecting the compound to 2,2,2-trichloroethyl °.7 chloroformate i n pyridine. This should give r i s e to N-(2, 2, 2-trichloroethylformyl) - J J-benzyl - L-aspartic acid (21) . This amine protecting group was chosen because i t could be cleaved i n the presence of the benzyl protecting groups. The next step involved forming the active ester of the free acid 21 i n preparation for i t s coupling to the next amino acid chosen for the peptide. Compound 21 would be subjected to DCC and an excess of p-nitrophenol i n order to form the desired active p-nitrophenyl ester of aspartic acid (22). The second amino acid i n our t r i p e p t i d e was L - l y s i n e . This amino acid would require protection of i t s side chain amino group p r i o r to attempting a coupling with the aspartic acid derivative. L-Lysine monohydrochloride (23) was treated with a basic solution of copper (II) to form a dimeric complex (Scheme 7) involving the c<-amine as well as e l e c t r o s t a t i c complexation with the <?<-carboxyl group. Thus, the side chain amine could be s e l e c t i v e l y protected with benzyl chloroformate. The copper complex was subsequently destroyed, l i b e r a t i n g N -benzyloxycarbonyl - L-lysine (24) i n 57% y i e l d , by passing H2S through a solution of the dimer and r e s u l t i n g i n the formation of copper (II) s u l f i d e . It was proposed that mixing of the activated ester of aspartic acid 22 with the free amine of the protected lysine (24) would lead to the formation of a dipeptide 25 whose side chains were protected with groups that could be removed by hydrogenolysis. The free carboxyl of the l y s y l residue could then be e s t e r i f i e d with p-nitrophenol under previously described conditions to give the active ester 26. 39 Scheme 7: The s e l e c t i v e protection of the side chain amine of L - l y s i n e . Our task with L-asparagine (27) would involve formation of the t e r t - b u t y l ester without a f f e c t i n g the free amine of the molecule. As a re s u l t , the amine was protected by mixing with benzyl chloroformate under al k a l i n e conditions to give N^-benzyloxycarbonyl-L-asparagine (28) i n 50% y i e l d . 3 9 This derivative was then treated with isobutylene under a c i d i c conditions to form the desired t e r t - b u t y l ester (29) i n 97% y i e l d . 3 9 Subsequently, the benzyloxycarbonyl protecting group was s e l e c t i v e l y removed by hydrogenolysis to give tert-butyl-L-asparaginate (30) i n 30% y i e l d . F i n a l l y , i t has been proposed that the free amine of the asparagine derivative 30 would be coupled with the active ester of the dipeptide 26 to y i e l d the protected t r i p e p t i d e 31. The «K-amino protecting group found on the aspartic acid residue could f i n a l l y be removed by a zinc reduction to l i b e r a t e the amine i n preparation for coupling with Fragment A. 3 7 COUPLING THE FRAGMENTS AND COMPLETION OF THE SYNTHESIS: Mixing of the active ester of Fragment A 18 with the free amine of Fragment B 32 should r e s u l t i n the formation of an amide bond forming the protected analogue 33. F i n a l l y , subjecting 33 to hydrogenolysis conditions would result i n three simultaneous deprotections l i b e r a t i n g : the carboxyl group of Fragment A, the side chain carboxyl of the aspartyl residue, and the side chain amine of the l y s y l residue, to y i e l d the analogue 34. CONCLUSION Although completion of the synthesis was not achieved, a great deal has been accomplished i n t h i s early research. The l o g i c behind designing a peptide analogue of ionomycin has been explored. Further exploration of t h i s analogue, and others, may reveal some very i n t e r e s t i n g information on factors important to the binding and transport of calcium ions. 42 EXPERIMENTAL GENERAL: Unless otherwise stated, a l l reactions were performed under N 2 i n flame-dried glassware. The cold temperature baths used were: dry ice-acetone (-78°C), and ice-water (0°C). Anhydrous reagents and solvents were prepared according to the procedure given i n the l i t e r a t u r e . 4 ^ n-Butyllithium (Aldrich Chemical Co.) was standardized against diphenylacetic acid i n THF, a fa i n t yellow color being i n d i c a t i v e of the end point. S i l i c a gel 60, 230-400 mesh, supplied by E. Merck CO. was used for preparative f l a s h column chromatography. 4 1 Melting points were performed on a Fischer-Johns hot stage melting point apparatus and are corrected. Infrared (IR) spectra were recorded on a BOMEM FT-IR Michaelson-100 connected to an IBM compatible computer. IR spectra were taken neat using NaCl plates, i n a chloroform solution using NaCl c e l l s of 0.2 mm thickness, or i n the form of a KBr p e l l e t . IR spectra are uncalibrated. Proton nuclear magnetic resonance ( H NMR) spectra were recorded i n CDCI3 or DMSO-d^ solutions on a Bruker AC-200 (200 MHz) and Bruker WH-400 (400 MHz) instruments. Chemical s h i f t s are given in, parts per m i l l i o n (ppm) on the € scale versus tetramethyls i lane (8 0 ppm) or chloroform (S 7.27 ppm) as i n t e r n a l standards. S ignal m u l t i p l i c i t y , coupl ing constants, and in tegrat ion r a t i o s are given i n parentheses. Low reso lu t ion mass spectra (LRMS) were recorded on a Kratos-AEI model MS 50 or MS 9 spectrometer. Only peaks with greater than 20% r e l a t i v e i n t e n s i t y or those which were a n a l y t i c a l l y useful are reported. High re so lu t ion mass spectra (HRMS) were obtained from a Kratos-AEI model MS 50 spectrometer. An i o n i z a t i o n p o t e n t i a l of 70 eV was used i n a l l measurements. 44 9-Bromo-l-nonanol (3) A l i q u i d - l i q u i d extractor was charged with 100 mL of n-heptane followed by crushed 1,9-nonanediol (2) (10.00 g, 62.4 mmol) and 48% hydrobromic acid (16.0 mL, 141 mmol). A 500-mL rbf was f i l l e d with 300 mL n-heptane and attached to the side arm of the extractor. The aqueous phase i n the extractor was warmed to 80°C i n an o i l bath. The solvent reservoir was heated to reflux and n-heptane allowed to percolate through the aqueous layer for 24 hours. The organic and aqueous layers were separated and the pH of the aqueous layer was made alk a l i n e by the slow addition of concentrated ammonium hydroxide. The aqueous solution was extracted with d i e t h y l ether (3 x 20 mL) . The combined organic phases were washed with saturated sodium bicarbonate solution (2 x 20 mL) followed by brine (2 x 20 mL) . The organic extracts were dried over anhydrous magnesium sulfate, f i l t e r e d , then concentrated under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by fl a s h chromatography using petroleum ether/ethyl acetate (4:1) as eluent. The reaction produced 3 (11.84 g, 53.0 mmol, 85% yield) as a colourless o i l . 45 IR (CHCI3, cm" 1): 3624, 3535-3350, 2932, 2857, 1461, 1400, 1275, 1043. XH NMR (CDCI3, 400 MHz) 6: 3.65 (t, J=6.5 Hz, 2H) , 3.41 (t, J=6.8 Hz, 2H), 1.86 (m, 2H), 1.57 (m, 2H), 1.43 (m, 2H) , 1.38 (s, IH exchangeable i n D20) , 1.32 (br. s, 8H) . LRMS (m/z) from CI: 242 ( 8 1Br, M++18, 96), 240 ( 7 9Br, M++18, 100). LRMS (m/z) from EI i o n i z a t i o n : 206(0.1), 204(0.1), 150 (28), 148(30), 137 (45), 135 (46), 97 (64), 83(62), 82 (40), 81 (31), 70(33), 69 (87), 68 (46), 67 (41), 57 (39), 56 (45), 55 (100), 54 (35), 43(63), 42(51), 41 (91), 39 (45), 31(54), 29 (67), 28 (32) . HRMS: exact mass calcd for C9H 1 7 8 1Br (M+-H20): 206.0493/ found: 206.0474. Calcd for C9H 1 7 7 9Br (M+-H20): 204.0513; found: 204.0510. 9-Bromo-1- [ ( t e r t - b u t y l d i x n e t h y l s i l y l ) oxy] -nonane (4) : 46 ITBDMS A dry 500-mL rbf was charged with methylene ch lor ide (300 mL) and cooled to 0°C under an atmosphere of n i trogen . Compound 3 (11.84 g, 53.0 mmol) was d i s so lved i n the solvent p r i o r to adding t r i e t h y l amine (15.5 mL, 111 mmol) and- a c a t a l y t i c amount of 4-dimethylamino pyr id ine (100 mg) . A so lut ion of t e r t - b u t y l d i m e t h y l s i l y l ch lor ide (11.19 g, 74.3 mmol) i n methylene ch lor ide (50 mL) was slowly added to the react ion mixture which was slowly warmed to room temperature while s t i r r i n g for 24 hours. The mixture was poured into ice co ld 0.5 M hydrochlor ic ac id (150 mL) and the organic and aqueous layers were quickly separated. The aqueous phase was extracted with d i e t h y l ether (3 x 20 mL) and the combined organic phases were subsequently washed with saturated sodium bicarbonate so lut ion (2 x 20 mL) followed by brine (2 x 20 mL). The organic extracts were dr i ed over anhydrous magnesium su l fa te , f i l t e r e d , then concentrated under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by f l a sh chromatography using hexanes as e luent . The react ion produced 4 (17.72 g, 52.5 mmol, 99% yie ld) as a co lor l e s s o i l . 4 7 IR (neat, can"1) : 2905, 1465, 1387, 1361, 1252, 1 1 0 0 , 1006, 938, 840, 776, 721. XH NMR (CDCI3, 400 MHz) 8: 3.62 (t, J = 6 . 5 Hz, 2 H ) , 3.42 (t, J = 7 . 0 Hz, 2 H ) , 1 . 8 6 (m, 2 H ) , 1.51 (m, 2 H ) , 1 . 4 3 (m, 2 H ) , 1.31 (br. s, 8 H ) , 0.90 (s, 9 H ) , 0.05 (s, 6 H ) . LRMS (m/z) from CI: 3 3 9 ( 8 1Br, M ++l, 89), 3 3 7 ( 7 9Br, M ++l, 1 0 0 ) . LRMS (m/z) from EI i o n i z a t i o n : 2 8 1 ( 6 . 2 ) , 2 7 9 ( 6 . 2 ) , 1 6 9 ( 2 1 ) , 1 6 7 ( 2 1 ) , 125(30), 8 3 ( 4 5 ) , 7 5 ( 1 0 0 ) , 7 3 ( 4 3 ) , 70 (26), 69(24), 5 7 ( 7 5 ) , 5 5 ( 5 6 ) , 4 3 ( 2 2 ) , 41(41). HRMS: exact mass calcd for CnH240Si 8 1Br ( M + - C 4 H 9 ) : 281.0173; found: 281.0766. Calcd for C 1 : LH240Si 7 9Br (M+-C 4 H 9 ) : 279.0193; found: 279.0784. 48 1 4 - [ ( t e r t - B u t y l d i m e t h y l s i l y l ) o x y ] - 2 , 4 - t e t r a d e c a d i o n e (6): OTBDMS 6 A dry 500-mL, 2-neck rbf was equipped with an addition funnel and charged with anhydrous tetrahydrofuran (300 mL) which was subsequently cooled to 0°C under an atmosphere of nitrogen. Diisopropylamine (17.7 mL, 126 mmol) was added to the solvent p r i o r to the slow, dropwise addition of 1.6 M n-butylli t h i u m i n hexanes (75.5 mL, 120.77 mmol). The reaction was s t i r r e d for 45 minutes i n order to ensure formation of lithium diisopropylamine. Acetylacetone (5) (5 . 9 mL, 58 mmol) was slowly syringed into the LDA mixture over a period of one hour and the reaction was s t i r r e d for a further 45 minutes to ensure formation of the dianion. Compound 4 (17.72 g, 52.5 mmol) was dissolved i n THF (20 mL) and t h i s solution was added dropwise to the LDA solution over a period of 30 minutes by an addition funnel. The reaction was kept at 0°C and s t i r r e d overnight. The reaction was quenched by pouring i t into ice cold 0.5 M hydrochloric acid (50 mL) and quickly separating the aqueous and organic phases. The organic layer was washed with saturated sodium bicarbonate solution ( 2 x 2 0 mL) followed by brine (2 x 20 mL) . The organic phase was dried over anhydrous magnesium sulfate, f i l t e r e d , then concentrated 49 under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by f l a s h chromatography using petroleum ether/ethyl acetate (25:1) as eluent. The reaction produced 6 (11.42 g, 32.0 mmol, 61% yield) as a yellow o i l . IR (neat, cm" 1): 2906, 2853, 1742, 1713, 1613, 1463, 1390, 1361, 1250, 1100, 1006, 939, 840, 776. •^H NMR (CDCI3, 400 MHz) 6: 15.51 (br. s, 0. 9H exchangeable i n D 2 O ) , 5.49 (s, 0.9H), 3.61 (t, J=6.5 Hz), 2H) , 3.57 (s, 0.2H), 2.51 (t, J=7.5 Hz, 0.2H), 2.27 (t, J=7.5 Hz, 1.8H), 2.24 (s, 0.3H), 2.06 (s, 2. 7H) , 1.60 (m, 2H), 1.51 (m, 2H), 1.29 (br. s, 12H), 0.90 (s, 9H), 0.05 (s, 6H) . LRMS (m/z) from CI: 374 (M++18, 8), 357 (M++l, 69). LRMS (m/z) from EI i o n i z a t i o n : 356(0.1), 301(21), 300(48), 299(100), 85 (22), 75 (57), 73(25), 55 (23), 43 (41), 41 (21) . HRMS: exact mass calcd for C20 H40°3 s i < M + ) : 356.2747; found: 356.2753. 7-Bromo-l-heptanol (8): A l i q u i d - l i q u i d extractor was charged with 100 mL of n-heptane followed by 1,7-heptanediol, (7), (10.42 g, 78.8 mmol) and 48% hydrobromic acid (22.4 mL, 197 mmol). A 500-mL rbf was f i l l e d with 300 mL n-heptane and attached to the side arm of the extractor. The aqueous phase of the extractor was warmed to 80 °C i n an o i l bath. The solvent reservoir was brought to reflux and n-heptane allowed to percolate through the aqueous layer for 24 hours. The organic and aqueous layers were separated and the pH of the aqueous layer was made alk a l i n e by the slow addition of concentrated ammonium hydroxide and subsequently extracted with d i e t h y l ether (3 x 20 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (2 x 20 mL) followed by brine (2 x 20 mL). The organic extracts were dried over anhydrous magnesium sulfate, f i l t e r e d , then concentrated under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by f l a s h chromatography using petroleum ether/ethyl acetate (4:1) as eluent. The reaction produced 8 (13.99 g, 71.7 mmol, 91% yield) as a colourless 51 IR (neat, can"1) : 3650-3050, 2903, 2856, 1449, 1255, 1051, 725. XH NMR (CDCI3, 400 MHz) S: 3.64 (t, J=6.0 Hz, 2H) , 3.41 (t, J=6.8 Hz, 2H), 1.87 (m, 2H), 1.77 (s, IH exchangeable i n D20) , 1.58 (m, 2H) , 1.46 (m, 2H) , 1.37 (m, 4H) . LRMS (m/z) from CI: 214 ( 8 1Br, M++18, 100), 212 .( 7 9Br, M++18, 96). LRMS (m/z) from EI i o n i z a t i o n : 178(1.9), 176(1.9), 150(68), 148(68), 97(66), 81 (25), 70(26), 69 (91), 68 (46), 67(43), 57(23), 56(47), 55(100), 54(26), 43 (64), 42 (49), 41 (84), 39 (50), 31 (64) . HRMS: exact mass calcd for C 7 H 1 3 8 1 B r (M +-H 20): 178.0180; found: 178.0195. Calcd for C 7 H 1 3 7 9 B r (M +-H 20): 176.0200; found: 176.0207. 7-Bromoheptyl-l-methoxymethyl ether (9) : A 2-necked, 500-mL rbf was charged with methylene chloride (300 mL) under an atmosphere of nitrogen and equipped with a mechanical s t i r r e r . Dimethoxymethane (38.1 mL, 430 mmol) and 8 (13.99 g, 71.7 mmol) were slowly added with s t i r r i n g at room temperature. Phosphorus pentoxide (7.0 g, 4 9 mmol) was added to the reaction mixture i n one gram portions at 15 minute in t e r v a l s u n t i l the reaction was complete as judged from the TLC of the reaction mixture. The reaction mixture was poured into ice cold 1 M sodium carbonate solution (500 mL) and the gummy residue i n the rbf was washed with ice cold 1 M sodium carbonate solution (200 mL). The aqueous phase was separated from the organic phase without shaking. The methylene chloride was removed under reduced pressure and the remaining o i l was dissolved i n diet h y l ether (200 mL) . The aqueous phase was washed with di e t h y l ether (2 x 20 mL) and the combined organic phases were washed with brine (2 x 20 mL) then dried over anhydrous magnesium su l f a t e . The solution was f i l t e r e d and concentrated under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by fl a s h chromatography using petroleum ether/ethyl acetate (25:1) as eluent. The 53 reaction produced 9 (11.49 g, 48.1 mmol, 67% yield) as a yellow o i l . IR (neat, cm" 1): 2909, ,2853, 1738, 1453, 1385, 1236, 1200, 1146, l l l l , 1046, 919, 725. XH NMR ( C D C I 3 , 400 MHz) 6: 4.62 (s, 2H) , 3.53 (t, J=6.5 Hz, 2H) , 3.42 (t, J=7.0 Hz, 2H) , 3.37 (s, 3H) , 1.87 (m, 2H), 1.60 (m, 2H), 1.50 to 1.30 (m, 6H). LRMS (m/z) from CI: 258 ( 8 1Br, M++18, 84), 256 ( 7 9Br, M++18, 100). LRMS (m/z) from EI i o n i z a t i o n : 239(1.8), 237(1.9), 177(21), 97(100), 95(24), 75 (29), 69 (27), 57 (21), 55 (80), 45 (95), 43 (21) , 41(30) . HRMS: exact mass calcd for C9H 1 802 8 1Br (M +-H»): 239.0470/ found: 239.0470. Calcd for CgH 1 802 7 9Br (M +-H»): 237.0490; found: 237.0496. 21-[(tert-Butyldimethylsilyl)oxy]-heneicosa-9,11-dioxo-l-methoxymethyl ether (10): o o 'OTBDMS 10 A dry 250-mL, 2-neck rbf was equipped with an addition funnel and charged with anhydrous tetrahydrofuran (50 mL) which was subsequently cooled to 0°C under an atmosphere of nitrogen. Diisopropylamine (14.81 mL, 106 mmol) was dissolved i n the solvent p r i o r to the slow, dropwise addition of 1.6 M n-butyllithium i n hexanes (60.1 mL, 96.1 mmol) . The reaction was s t i r r e d for 10 minutes p r i o r to cooling to -78°C. A solution of 6 (11.42 g, 32.0 mmol) i n THF (30 mL) was added dropwise from the addition funnel over a period of 30 minutes. The addition funnel was rinsed with DMPU (55 mL) which was also added to the reaction vessel over a 10 minute period. The dianion was to form over an 8 hour period before 9 (11.48 g, 48.0 mmol) was added slowly by syringe. The reaction was s t i r r e d at -78°C for 12 hours before quenching with water and a saturated solution of ammonium chloride (100 mL). The organic and aqueous phases were separated and the aqueous phase was extracted with dieth y l ether (2 x 20 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (2 x 20 mL) followed by brine (2 x 20 mL) . The organic phase was dried over anhydrous magnesium sulfate, f i l t e r e d , then concentrated under reduced pressure to y i e l d a red o i l . The crude product was p u r i f i e d by f l a s h chromatography using petroleum ether/ethyl acetate (25:1) as eluent. The reaction produced 10 (11.87 g, 23.1 mmol, 72% yield) as a yellow o i l . IR (neat , c m - 1 ) : 2927, 2852, 1611, 1461, 1252, 1141, 1115, 1054, 920, 838, 776. X H NMR ( C D C 1 3 , 400 MHz) 6: 15.55 (br. s, 0.8H exchangeable with D 20), 5.49 (s, 0.8H), 4.61 (s, 2H), 3.60 (t, J=6.5 Hz, 2H) , 3.56 (s, 0.4H), 3.51 (t, J=6.5 Hz, 2H) , 3.37 (s, 3H) , 2.50 (t, J=7.5 Hz, 3.2H), 2.28 (t, J=7.5 Hz, 0.8H), 1.60 (m, 4H) , 1.50 (m, 4H) , 1.30 (m, 20H) , 0.90 (s, 9H), 0.05 (s, 6H). LRMS (m/z) from C I: 533 (M++18, 6). LRMS (m/z) from E I i o n i z a t i o n : 486(0.2), 458(32), 457(100), 425 (57), 395 (25), 75 (38), 69(27), 55(33), 45(35). HRMS: exact mass calcd for C25H4gSi05 (M+-OCH3): 457.3349; found 457.3348. 56 21 - [ ( t er t -Buty ld imethy l s i l y l )oxy ] -9 ,11 -d ioxohene icosan- l -o l (11) : A dry 500-mL rbf was charged with anhydrous methylene chloride (250 mL) i n which was dissolved 10 (11.87 g, 23.1 mmol). The solution was cooled to -78°C p r i o r to the slow addition of a freshly d i s t i l l e d 0.7 molar solution of bromodimethylborane i n methylene chloride (41.0 mL, 28.7 mmol) . The reaction was s t i r r e d for one hour at -78°C before i t was cannulated into a vigorously s t i r r e d mixture of tetrahydorfuran (70 mL) and saturated sodium bicarbonate solution (35 mL). The reaction vessel was kept at -78°C and i t was rinsed with methylene chloride (2 x 20 mL) . The washings were also cannulated into the aqueous mixture. After f i v e minutes, the mixture was di l u t e d with d i e t h y l ether (250 mL) , and the aqueous and organic layers were separated. The aqueous phase was extracted with d i e t h y l ether (2 x 20 mL) and then the combined organic phase was washed with water (2 x 20 mL), 10% sodium bisulphate (2 x 20 mL) , and f i n a l l y with brine (2 x 20 mL) . The organic phase was dried over anhydrous magnesium sulphate, f i l t e r e d , and concentrated under reduced pressure to y i e l d a white s o l i d . The crude product was immediately p u r i f i e d by f l a s h chromatography using petroleum ether/ethyl acetate (4:1) as 57 eluent. The reaction produced 11 (5.43 g, 11.5 mmol, 50% yield) as a yellow o i l . IR (neat, cm" 1): 3600-3200, 2930, 2856, 1738, 1609, 1464, 1368, 1249, 1098, 838, 777. XH NMR ( C D C I 3 , 400 MHz) 8: 15.65 (br. s, 0.8H exchangeable with D20) , 5.59 (s, 0.8H), 3.73 (t, J=6.5 Hz, 2H) , 3.70 (t, J=6.5 Hz, 2H), 3.64 (s, 0.4H), 2.60 (t, J=7.5 Hz, 0.8H), 2.38 (t, J=7.5 Hz, 3.2H), 1.68 (m, 5H with one being exchangeable with D20) , 1.40 (m, 24H), 0.90 (s, 9H) , 0 . 05 (s, 6H) . LRMS (m/z) from EI i o n i z a t i o n : 414(31), 413(100), 111 (21), 109(20), 97 (46), 95 (38), 85 (32), 83 (44), 81 (37), 75(75), 73(26), 71(22), 69 (72), 67 (27), 57 (21), 55 (64), 43 (24), 41 (21) . HRMS: exact mass calcd for C23H45SiC>4 (M+-C4Hg) : 413.3087; found 413.3068. 2 1 - [ ( t e r t - B u t y l d i m e t h y l s i l y l ) o x y ] - 9 , 1 1 - d i o x o h e n e i c o s a n a l (12) : A dry 250-mL rbf was charged with methylene chloride (75 mL) and DMSO (75 mL). To t h i s mixture was added alcohol 11 (5.43 g, 11.5 mmol), followed by DCC (14.27 g, 69.2 mmol). After the DCC had f u l l y dissolved, dichloroacetic acid (1.9 mL, 23 mmol) was added slowly by syringe and the reaction was s t i r r e d for four hours at 25°C. The mixture was d i l u t e d with ethyl acetate (100 mL) and oxalic acid (5.0 g) was added to convert the remaining DCC to the insoluble urea by-product. This mixture was decanted into brine (200 mL) and f i l t e r e d to remove any p r e c i p i t a t e which had formed. The organic and aqueous layers were separated and the aqueous was extracted with ethyl acetate (2 x 20 mL) . The combined organic phases were washed with brine (2 x 20 mL), dried over anhydrous magnesium sulphate, f i l t e r e d , and concentrated under reduced pressure- to y i e l d the crude aldehyde, 12, as • a yellow o i l (5.15 g, 11.0 mmol, 95% y i e l d ) . Due to d i f f i c u l t i e s i n p u r i f y i n g the product beyond t h i s point, spectra were c o l l e c t e d on the crude o i l only. IR (neat, c m - 1 ) : 2932, 2856, 1748, 1608, 1447, 1339, 1318, 1251, 1149, 1098, 960, 914, 837, 814, 776. LRMS (m/z) from EI i o n i z a t i o n : 468(0.1), 412(28), 411 (100), 241 (20), 83 (38), 81(29), 75 (34), 67 (29), 61 (31), 55 (79) , 43 (22) , 41.(56) . HRMS: exact mass calcd for C27H52Si04 (M+-H): 468.3635; found 468.3590. 21-[(tert-Butyldimethylsilyl)oxy]-9,11-dioxoheneicosanoic ac i d (13): ITBDMS 13 A 100-mL rbf was charged with tetrahydrofuran (30 mL) and water (30 mL) i n which was dissolved the aldehyde 12 (5.15 g, 11.0 mmol). To t h i s solution was added sodium hydroxide (8.76 g, 220 mmol), and C e l i t e (5.00 g) , followed by slow addition of s i l v e r n i t r a t e (18.62 g, 110 mmol) and the reaction was allowed to s t i r at 25°C for four hours. The black solution was then f i l t e r e d and the C e l i t e cake was washed with 1 M HCl (100 mL) and the f i l t r a t e was tested to be a c i d i c . The cake was then washed with ethyl acetate (100 mL) . The organic and aqueous phases were separated and the aqueous phase was extracted with d i e t h y l ether (2 x 20 mL). The combined organic phases were dried over anhydrous magnesium sulphate, f i l t e r e d , and concentrated under reduced pressure to y i e l d the crude acid, 13, as a yellow o i l (1.33 g, 2.74 mmol, 25% y i e l d ) . Due to d i f f i c u l t i e s i n p u r i f y i n g the product beyond t h i s point, spectra were c o l l e c t e d on the crude o i l only. IR (neat, cm - 1) : 2934, 2859, 2350, 1717, 1454, 1378, 1110, 1046, 905. 61 B e n z y l - 2 1 - [ ( t e r t - b u t y l d i m e t h y l s i l y l ) o x y ] - 9 , 1 1 -dioxoheneicosanate (14): A dry 250-mL rbf was charged with anhydrous methylene chloride (100 mL) i n which was dissolved the crude acid 13 (1.33 g, 2.74 mmol). To t h i s solution was added DCC (0.85 g, 4.1 mmol), benzyl alcohol (0.43 mL, 4.1 mmol), and DMAP (0.03 g, 0.3 mmol). The reaction was s t i r r e d over night at 25"C before i t was quenched by addition of oxalic acid (1.0 g). The solution was dried over anhydrous magnesium sulphate, f i l t e r e d and concentrated under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by fla s h chromatography using petroleum ether/ethyl acetate (25:1) as eluent. The reaction produced 14 (0.3945 g, 0.685 mmol, 25% yield) as a white s o l i d which melts at 45"C. IR (chloroform, cm" 1): 3418, 2936, 2858, 1691, 1519, 1452, 1346, 1061, 891. •^H NMR ( C D C I 3 , 200 MHz) 8: 15.55 (br. s, IH exchangeable with D 20), 7.35 (s, 5H), 5.45 (s, IH) , 5.10 (s, 2H) , 3.70 (t, J=7.5 Hz, 2H) , 2.30 (t, J=7.0 Hz, 2H) , 2.22 (t, J=7.0 Hz, 2H) , 2.21 (t, J=7.0 Hz, 2H) , 1.55 (m, 8H) , 1.25 (m, 20H), 0.90 (s, 9H), 0.05 (s, 6H). LRMS (m/z) from EI i o n i z a t i o n : 485(0.1), 445(0.1), 441(0.1), 334 (0.6), 322 (4), 310(0.8), 278 (1.3), 264 (8.7), 253 (12), 174(11), 147 (7), 128 (13), 91 (100), 83 (55), 82 (22), 67 (22), 55 (53), 41 (36) . Benzyl-21-hydroxy-9,11-dioxoheneicosanate (15): 63 o o o A dry 100-mL rbf was charged with anhydrous THF (30 mL) into which the s i l y l ether 14 (0.3945 g, 0.685 mmol) was dissolved. A I M solution of tetrabutylammonium flu o r i d e i n THF (1.37 mL, 1.37 mmol) was added dropwise by syringe to the s i l y l ether. The reaction was s t i r r e d at 25°C over night. The solvent was removed under reduced pressure and the residue was taken up i n d i e t h y l ether (50 mL) and washed with water (2 x 20 mL) then dried over anhydrous magnesium sulphate. The solution was f i l t e r e d and solvent removed under reduced pressure to y i e l d a yellow o i l . The crude product was p u r i f i e d by f l a s h chromatography using petroleum ether/ethyl acetate (3:1) as eluent. The reaction produced 15 (0.2367 g, 0.514 mmol, 75% yield) as a white s o l i d that melted at 37 to 38°C. IR (chloroform, c m - 1 ) : 3622, 2931, 2857, 1726, 1608, 1458, 1374, 1353, 1300, 1165, 1063, 997. XH NMR ( C D C I 3 , 400 MHz) 8: 15.55 (br. s, 0.8H exchangeable with D 2 O ) , 7.35 (m, 5H) , 5.48 (s, 0.4H), 5.12 (s, 2H), 3.63 (t, J=7.5 Hz, 2H), 3.60 (s, 0.8H), 3.55 (s, IH exchangeable with D20) , 2.51 (t, J=6.5 Hz, 0.4H), 2.50 (t, 64 J = 6 . 5 Hz, 0 . 4 H ) , 2 . 3 4 (t, J = 6 . 5 Hz, 2 H ) , 2.26 (t, J = 6 . 5 Hz, 1 . 6 H ) , 2.25 (t, J = 6 . 5 Hz, 1 . 6 H ) , 1.60 (m, 8 H ) , 1.25 (m, 18H) . LRMS (m/z) from EI i o n i z a t i o n : 4 6 0 ( 0 . 5 ) , 4 4 2 ( 0 . 5 ) , 427 ( 0 . 2 ) , 369 ( 1 ) , 351 ( 1 . 4 ) , 3 3 7 (15), 125 (19), 1 1 1 (24), 103 (69), 1 0 0 ( 3 2 ) , 98 ( 2 0 ) , 9 7 ( 4 0 ) , 9 5 ( 2 4 ) , 91 (65), 85 (67), 8 3 ( 4 3 ) , 81 (31), 7 5 ( 2 2 ) , 7 1 ( 2 2 ) , 6 9 ( 6 6 ) , 6 7 ( 3 5 ) , 5 7 ( 4 9 ) , 5 5 ( 1 0 0 ) , 4 3 ( 5 7 ) , 41 (46) . HRMS: exact mass calcd for C 2 8 H 4 4 O 5 (M+) : 460.3189; found 460.3195. B-Benzyl-L-aspartic a c i d (20) : Freshly d i s t i l l e d benzyl alcohol (100 mL, 966 mmol) was added to a mixture of di e t h y l ether (100 mL) and concentrated s u l f u r i c acid (10.0 mL) i n a 500-mL rbf. The die t h y l ether was removed under reduced pressure and f i n e l y ground L-aspartic acid (19) (13.31 g, 100 mmol) was added i n small portions with s t i r r i n g . The reaction was s t i r r e d at 25°C for 24 hours and was then d i l u t e d with 95% ethanol (200 mL) and neutralized by dropwise addition of pyridine (50 mL). The mixture was stored i n the r e f r i g e r a t o r over night and the c r y s t a l l i n e product c o l l e c t e d by f i l t r a t i o n . The white s o l i d was t r i t u r a t e d with d i e t h y l ether p r i o r to r e c r y s t a l l i z a t i o n from hot water containing a few drops of pyridine. The reaction produced 20 (6.03 g, 27 mmol, 27% yield) as a flaky white c r y s t a l that decomposed at 215 to 218 °C compared with a l i t e r a t u r e melting point of 218 to 220°C. 3 6 IR (KBr, cm" 1): 3018, 1736, 1620, 1404, 1361, 1309, 1225, 1166, 1022, 962, 862, 783, 738. 66 •••H NMR (DMSO-cr*, 400 MHz) 8: 8.30 (s, IH exchangeable with D20) , 7.40 (m, 5H) , 5.12 (s, 2H) , 3.54 (m, IH) , 2.95 (dd, J=5.0 Hz, J=17.0 Hz, IH) , 2.65 (dd, J=8.0 Hz, J=17.0 Hz, IH), 1.24 (s, 2H). LRMS (m/z) from CI: 224 (M++l, 100). LRMS (m/z) from EI i o n i z a t i o n : 223(0.4), 108(44), 107(32), 91(100), 79(62), 77(40), 65(42), 51 (21), 44 (25), 43 (21) , 39 (20) . HRMS: exact mass calcd for CnH 1 3N04 (M+) : 223.0844; found 223.0826. N -Benzyloxycarbonyl-L-lysine (24): 67 O In a 1000-mL rbf, L - l y s i n e monohydrochloride 23 (7.20 g, 39.4 mmol) was dissolved i n water (500 mL) and the solution was brought to a b o i l . Copper (II) carbonate (12.72 g, 53 mmol) was slowly added to the solution and then the solution was f i l t e r e d through a medium pore f r i t t e d glass f i l t e r . A 2 M sodium hydroxide solution (20.0 mL) was added and the dark blue solution was cooled i n an ice bath. A solution of benzyl chloroformate (8.0 mL, 53 mmol) i n 2 M sodium hydroxide (40.0 mL) was added i n ten portions over 30 minutes shaking and cooling the reaction over the duration. Care was taken to prevent the solution from becoming too alka l i n e at any given time. The copper complex which separates as a dark blue p e r c i p i t a t e was f i l t e r e d and washed with water (10 mL) and ethanol (10 mL) . The c r y s t a l l i n e compound was suspended i n water (500 mL) and a steady stream of hydrogen s u l f i d e gas was passed through the solution u n t i l the bubbles would no longer decrease i n size as they rose to the surface of the solution. The solution was brought to a b o i l and f i l t e r e d hot. Cooling of the f i l t r a t e 68 i n a r e f r i g e r a t o r resulted i n c r y s t a l l i z a t i o n of 24 (6.30 g, 22.5 mmol, 57% yield) as fine white flakes that decomposed from 240 to 242°C compared with 259°C (decomp) i n the l i t e r a t u r e . 3 8 IR (KBr, c m " 1 ): 3343, 2931, 2351, 1691, 1546, 1417, 1323, 1269, 1027, 743. X H NMR (DMSO-d 6 , 400 MHz) fi: 8.30 (s, IH exchangeable with D20) , 7.35 (m, 5H) , 7.20 (m, IH) , 5.01 (s, 2H) , 3.16 (m, IH) , 3.00 (m, 2H) , 1.71 (m, IH) , 1.56 (m, IH) , 1.35 (m, 4H), 1.23 (s, 2H). LRMS (m/z) from E I i o n i z a t i o n : 280(1.7), 235(37), 174(36), 128(73), 127 (68), 108 (93), 107 (65), 92 (33), 91(59), 84 (100), 82(27), 79(77), 77 (40) 74(36), 72 (21), 65 (30), 56(40) , 43 (21) , 30 (29) . HRMS: exact mass calcd for C 1 4H 2oN 204 (M +): 280.1423; found 280.1424. 69 N -Benzyloxycarbonyl-L-asparagine (28): o 2 8 BzO' X o N H 4 NH 2 .OH A 500-mL rbf was charged with water (100 mL) , 1 M sodium hydroxide (70.0 mL), and dioxane (100 mL) and the mixture was cooled to 0°C. Finely crushed L-asparagine 27 (8.00 g, 60.55 mmol) was slowly dissolved and to t h i s solution was added benzyl chloroformate (11.2 mL, 78.72 mmol) . Over the next 25 minutes, an additional equivalence of 1 M sodium hydroxide (70.0 mL) was added to neutralize any HCI that formed. The reaction was warmed to 25°C and s t i r r e d for one hour. After ensuring that the reaction mixture was alkaline, i t was extracted with ethyl acetate (2 x 100 mL) . The aqueous phase was a c i d i f i e d to pH 2 by adding concentrated HCI. The desired product 28 (8.06 g, 30.3 mmol, 50% yield) p r e c i p i t a t e d out of solution as white needles which melted at 159 to 160°C compared to 163 to 165*C i n the l i t e r a t u r e . 4 2 IR (KBr, cm" 1): 3412, 3336, 1699, 1644, 1584, 1538, 1427, 1319, 1269, 1230, 1200, 1063, 737. 70 X H NMR (DMSO-db, 400 MHz) 6: 1 2 . 5 5 (br. s, 1 H ) , 7 . 3 5 (m, 5 H ) , 7.30 (br. s, 2 H ) , 6.86 (br. s, 1 H ) , 5.03 (s, 2 H ) , 4 . 3 5 (m, IH), 2.50 (m, 2 H ) . LRMS (m/z) from EI i o n i z a t i o n : 2 6 6 ( 0 . 1 ) , 113(24), 1 1 2 ( 5 5 ) , 108 ( 1 0 0 ) , 107 (98), 91 (97), 90 ( 2 1 ) , 79(90), 77 (51). HRMS: exact mass calcd for C 1 2 H 1 4 N 2 O 5 (M +): 266.0902; found 266.0902. N -(Benzyloxycarbonyl)-tert-butyl-L-asparaginate (29): In a 500-mL pressure b o t t l e was mixed methylene chloride (30 mL) and dioxane (30 mL) . The carboxylic acid 28 (5.00 g, 18.8 mmol) was slowly dissolved and t h i s was followed by the addition of concentrated s u l f u r i c acid (3.0 mL) . Isobutylene was condensed (30 mL) i n a trap at -78°C and poured into the pressure b o t t l e which had been precooled in an ice bath. The pressure b o t t l e was wired closed with a rubber bung equipped with a valve and the valve was closed. The solution was s t i r r e d over night at 25°C. The reaction mixture was poured into ethyl acetate (200 mL) and the organic phase was washed with saturated sodium bicarbonate solution (2 x 20 mL) and brine (2 x 20 mL) . The organic solution was then dried over anhydrous magnesium sulphate, f i l t e r e d , and concentrated under reduced pressure. The ester 29 (5.87 g, 18.21 mmol, 97% yield) was r e c r y s t a l l i z e d from d i e t h y l ether as white flakes that melted from 85 to 86°C. IR (chloroform, cm - 1): 3526, 3414, 2968, 1711, 1594, 1499, 1455, 1396, 1356, 1156, 1059, 844. •"•H NMR ( C D C I 3 , 400 MHz) S: 7.35 (m, 5H) , 5.91 (br. d, J=6.5 Hz, IH), 5.61 (br. s, IH), 5.40 (br. s, IH), 5.12 (s, 2H) , 4.46 (m, IH) , 2.89 (br. dd, J=4.5 Hz, J=16.0 Hz, IH) , 2.73 (br. dd, J=4.0 Hz, J=16.0 Hz, IH), 1.45 (s, 9H). LRMS (m/z) from EI i o n i z a t i o n : 322(1.2), 305(0.2), 278 (2), 277 (2), 262 (2), 221 (4), 214(8), 199 (14), 177 (11), 159(13), 141(20), 113(23), 108(83), 107 (60), 91 (56), 79(66), 77(67), 70(34), 59(34), 58(100), 57(45), 56 (50), 55 (31), 51 (35), 44(49), 43(63), 42(38), 41 (62), 39 (60). HRMS: exact mass calcd for 0 3 ^ 2 2 ^ 0 5 (M+) : 322.1528; found: 322.1536. t e r t - B u t y l - L - a s p a r a g i n a t e (30): 73 A solution of the benzyloxycarbonyl compound 29 (5.00 g, 15.5 mmol) i n anhydrous methanol (40 mL) was prepared i n a dry 100-mL rbf under an atmosphere of nitrogen. A c a t a l y t i c amount of 10% palladium on activated carbon (0.18 g) was added to the solution and the atmosphere i n the reaction vessel was pruged with nitrogen for f i v e minutes p r i o r to being connected to a hydrogenation apparatus. The solution was s t i r r e d under a p o s i t i v e pressure of hydrogen (500 mL) over night. The reaction mixture was f i l t e r e d through a C e l i t e cake and the cake was rinsed with methanol (50 mL). The f i l t r a t e was concentrated under reduced pressure to give a yellow o i l . The crude compound formed pure amine 30 (0.88 g, 4.7 mmol, 30% yield) as white needles with a melting point of 132 to 135°C when r e c r y s t a l l i z e d from d i e t h y l ether. IR (chloroform, cm" 1): 3389, 2983, 2351, 1731, 1659, 1394, 1371, 1346, 1278, 1158, 839. AH NMR ( C D C I 3 , 400 MHz) S: 5.98 (br. s, 2H) , 3.85 (m, 1H) , 2.61 (dd, J=5.0 Hz, J=17.5 Hz, IH), 2.19 (dd, J=5.0 Hz, J=12.0 Hz), 1.79 (br. d, 2H), 1.46 (s, 9H). LRMS (m/z) from EI i o n i z a t i o n : 188(0.3), 129(54), 128 (41), 127 (94), 84 (100), 70 (33), 68 (22), 57 (35), 56 (24), 44 (26) , 42 (22) , 41 (32) . HRMS: exact mass calcd for C8H 1 6N 203 (M +): 188.1161; found: 188.1168. 75 REFERENCES 1. Berger, J., A.I. Rachlin, W.E. Scott, L.H. Sternbach, and M.W. Goldberg. J. Am. Chem. Soc. 1951, 73, 5295. 2. Harned, R.L., P.H. Hidy, C.J. Corum, and K.L. Jones. Antibiot. Chemother. 1951, I, 594. 3. Westley, J.W. ed. Polyether a n t i b i o t i c s : naturally occuring acid ionophores. Volume 1: Biology. Marcel Dekker Inc., New York. 1982. pp. v i i - x i and 1-184. 4. Lardy, H.A., D. Johnson, and W.C. McMurray. Archives of Biochemistry and Biophysics. 1958. 78, 587. 5. Agtarap, A., J.W. Chamberlin, M. Pinkerton, and L. Steinrauf. J. Am. Chem. Soc. 1967, 89, 5737. 6. Liu, W.C., D. Smith Slusarchyk, G. Astle, W.H. Trejo, W.E. Brown, and E. Meyers. J. Antibiot. 1978. 31(9), 815. 7. Estrada-O., S., B. Rightmire, and H.A. Lardy. Antimicrob. Agents Chemother. 1968. 1967, 279. 8. Westley, J.W. Advances in Applied Microbiology. 1977. 22, 111. 9. Reed, P.W. Methods in Enzymology. 1979. 55, 435. 10. Ashton, R., and L.K. Steinrauf. J. Mol. Biol. 1970. 49, 547. 11. Gale, E.F., E. Cundliffe, P.E. Reynolds, M.H. Richmond, and M.J. Waring. The Molecular Basis of A n t i b i o t i c Action. John Wiley and Sons, Toronto, 1972, pp. 154-164. 12. Johnson, S.M., J. Herrin, S.J. Liu, and I.C. Paul. J. Am. Chem. Soc. 1970. 92(14), 4428. 13. Smith, P.W., and W.C. S t i l l . J. Am. Chem. Soc. 1988. 110, 7917. 14. S t i l l , W.C., D. Cai, D. Lee, P. Hauck, A. Bernardi, and A. Romero. Lectures in Heterocyclic Chemistry. 1987. 9, S-33. 15. Pressman, B.C. Ann. Rev. Biochem. 1976. 45, 501. 76 16. Chemical Rubber Publishing Company. Handbook of Chemistry and Physics. Volume 61. 1980-1981. Cleveland, Ohio. 17. Brasseur, R., M. Notredame, and J.-M. Ruysschaert. Biochem. Biophys. Res. Comm. 1983. 114(2), 632. 18. Watterson, D.M. and F.F. Vincenzi. ed. Ann. N.Y. Acad. Sci. 1981. 356. 19. Zubay, G. Biochemistry. 1984. Addison Wesley Publishing Company, Don M i l l s , Ont. pp. 538-541. 20. Pollack, W.K., T.J. Rink, and R.F. Irvine. Biochem. J. 1986. 235, 869. 21. Rittenhouse, S.E., and W.C. Home. Biochem. Biophys. Res. Comm. 1984, 123, 393. 22. Stryer, L. Biochemistry (3 ed.). 1988. W.H. Freeman and Company, New York. pp. 988-989. 23. Boguta, G., and A. Bierzynski. Biophys. Chem. 1988, 31, 133. 24. Toeplitz, B.K., A.I. Cohen, P.T. Funke, W.L. Parker, and J.Z. Gougoutas. J. Am. Chem. Soc. 1978, 101, 3344. 25. Hendrich, C.A., Tetrahedron. 1977, 33, 1845. 26. Chaudhary, S.K., and 0. Hernandez. ret. Lett., 1979, 99. 27. Huckin, S.N., and L. Weiler. J. Am. Chem. Soc. 1974, 96, 1082. 28. F u j i , K., S. Nakano, and E. F u j i t a . Synthesis. 1975, 276. 29. Mukhopadhyay, T., and D. Seebach. Helv. Chim. Acta. 1982, 65(39), 385. 30. Guindon, Y., C. Yoakim, and H.E. Morton. J. Org. Chem. 1984, 49, 3912. 31. Moffatt, J.G. Orgr. Syn. 1967, 47, 25. 32. Corey, E.J., and J. Das. J. Am. Chem. Soc. 1982, 104, 5551. 33. Bodanszky, M., and V. du Vigneaud. Biochem. Prep. 1963, 9, 110. 77 34. Corey, E.J., and A. Venkateswarlu. J. Am. Chem. Soc. 1972, 94, 6190. 35. Khorana, H.G. Chem. Ind. (London). 1955, 1087. 36. Benoiton, L. Can. J. Chem. 1962, 40, 570. 37. Windholz, T.B., and D.B.R. Johnston. ret. Lett. 1967, 27, 2555. 38. Neuberger, A., and F. Sanger. Biochem. J. 1943, 37, 515. 39. Anderson, G.W., and F.M. Callahan. J. Am. Chem. Soc. I960, 82, 3359. 40. Perrin, D.D., and W.L.F. Armarego. Purification of Laboratory Chemicals, third ed. Permagon Press, Toronto, 1988. 41. S t i l l , W.C, M. Kahn, and A. Mitra. J. Org-. Chem. 1978, 43(14), 2923. 42. A l d r i c h Chemical Company, Inc. Aldrich Catalog Handbook of Fine Chemicals. 1990 to 1991. Milwaukee, Wisconsin, p. 260. a n d X H NMR S P E C T R A : APPENDIX 79 0.0057-p—i r— jjkk I. -O.O025-- « . 0 1 0 7 --e.oi9o--0.0272--0.0354_ ~i I I I I r-4000. 3400. -i 1 — | i 1 1 1— 2800. 2200. - 1 — I — J -1600. - :-0.015 ' I ' 1000. CH-1 81 'OTBDMS 6 16 I — c8 400 MHz 15 TMS 0 ppm .8350-EH M CO 2 W En Z M EH a - C 3 9 7 -0.0400. 1 ' „ 1000. CM-1 I RELATIVE INTENSITY •o -TJ -3 oo 83 9 ' i '. ••!•• . -j 1 . • • . , .. _. -. . , . 1_ 1 < \ J 400 MHz 0 ppm 11B.4-J I 1 1 r-H M CO Z w EH S3 5 94. H 71.1-47.4 23.7-0.0. —i 1 1 r 1 r- - i 1 1 1 1 1 r-4000. 3400. 1 r 8800. 2200. "T ' r— 1600. 1=57.367 1000. CH-1 84 102.22-1 i i — i r — i 1 1 1 1 r 11 TBDMS . , T . — ...... , i , , ,,. j ,,, ,. ^  t _ , U * " V ) > ' I ! 6 1 ?.( E B E . l *9 S I 2 t t'.t I B r r * 99.90" | » • 1 T"" 'I I I I "1 "T I T I * " ' T ' " I 1 I I »•-•"[' | r 4000. 3400. 2B00. 2200. 1600. 1000. CM-1 12 OTBDMS 87 88 o o o 'OTBDMS PPM 4000. 3400. 2800. 2200. 1600. 1000. CM-i 90 91 0 ppm 106.30 58 .650 O H 92 13 12 jf. ' L 400 MHz TMS ppm 92.39 V44.753 " 1 — 1 — 1 — 1 — r 2800. 2200. 1000. CM-1 93 , , , 1 , , , r ™ ~ i 1— r 1 1 r 1 1 i i 1 j r 1 4000. 3400. 2800. 2200. 1600. 1000. C M - i 94 

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