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Isolation and structure determination of a novel glycolipid pachymoside A from marine sponge Pachymatisma… Shen, Jingkai 2003

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ISOLATION AND STRUCTURE DETERMINATION OF A N O V E L GLYCOLIPID PACHYMOSJDE A FROM MARINE SPONGE PACHYMATISMA JOHNSTONIA by JINGKAI SHEN M. Sc., Wuhan University, 2000 B. Sc., Wuhan University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCES in THE F A C U L T Y OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 2003 Jingkai Shen, 2003 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c opying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n permission. Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada Date Oct. to. JL»»$ 11 ABSTRACT The search for new pharmacologically active agents by screening natural sources has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases. The goal of my thesis was to discover structurally novel type III secretion inhibitors. Active extracts from marine sponge P. johnstonia were identified via high throughput bioassays for the EPEC type III secretion inhibitors. Guided by this novel bioassay model, a new glycolipid family, the pachymosides, was fractionated and identified. To determine the structure of the pachymosides, chemical degradation was performed and the glycolipids were divided into aglycon and monosaccharide parts. Three aglycons were isolated and identified as 6, 8 and 9. 26 o Ill Two monosaccharide residues were identified by NMR as glucose and galactose. Further chiral GC analysis determined their absolute configuration as D-glucose and D-galactose. Acetylation was performed on the pachymosides followed by 1 3 C labelling. The structure of pachymoside A (16), a member of a novel bioactive glycolipids family, was figured out. This is the second identified structure for the type III secretion inhibitor bioassay. 16 iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVATIONS x ACKNOWLEDGEMENTS xiii 1. Introduction 1 1.1 Marine natural products 1 1.2 Concept of pathogenicity island and TTSS 5 1.3 Type III secretion system 7 1.3.1 Pathways of protein secretion in Gram-negative 7 bacteria 1.3.2 TTSS in bacterial pathogenesis 9 1.3.3 Virulence proteins secreted via type III secretion 13 pathways 1.3.4 Process of TTSS 15 1.3.5 Regulation of type III secretion by contact with 17 eukaryotic cells 1.4 Potential antimicrobial inhibitor targets 19 1.5 Screening model of type III secretion inhibitors 20 1.6 Aim of this study 22 1.7 References 23 2. Isolation and structure determination of aglycons 26 and monosaccharides 2.1 Isolation of an active glycolipid from P. johnstonia 27 2.2 Biological activity of fraction F 30 2.3 Chemical degradation of the glycolipids 31 2.4 Aglycons 33 2.4.1 Isolations of the aglycons 33 2.4.2 Structure determination ofFYOP2 by NMR and MS 34 2.4.3 Structure confirmed by oxime formation 43 2.4.4 Structures of the other two aglycons 45 2.5 Structure elucidation of the sugars in the pachymosides 45 2.5.1 Determination of glucose 45 2.5.2 Determination of galactose 53 2.6 Absolute stereochemistry of glucose and galactose 2.7 References 57 61 3. The isolation and structure determination of pachymoside A 62 3.1 Isolation of the acetylated pachymoside A methyl ester 63 3.2 Structure elucidation of acetylated pachymoside A methyl ester 63 3.3 1 3 C Labeling for determination of acetylated positions 69 3.4 Significance of the result and future research 72 3.5 References 74 4. Experimental 75 4.1 General procedures 75 4.2 Isolation 77 4.3 Synthesis 79 LIST OF TABLES vi Table 1.1 Proteins via the Type III Secretion Pathway 14 Table 2.1 NMR data for aglycon FYOP2 recorded in CDC1 3 at 400 MHz 40 Table 2.2 Analysis of high resolution EIMS fragments of FYOP2 42 Table 2.3 Analysis of the MS fragments of the oxime 44 Table 2.4 Assignment of the coupling constant of the FYA1 major component 51 Table 2.5 Assignment of the coupling constant of the FYA2 major component 53 Table 2.6 ! H NMR spectrum assignment of the minor component 56 Table 3.1 lH NMR spectrum assignment of the saccharide protons 68 Vll LIST OF FIGURES Fig. 1.1 Marine natural products, 1965 onwards 2 Fig. 1.2 Distribution of aarine natural products by Phylum, 2001 3 Fig. 1.3 Type I, Type II and Type III secretion 8 Fig. 1.4 Type III secretion infection of Yersinia spp. 9 Fig. 1.5 Shigella induced Apoptosis 10 Fig. 1.6 Salmonella invasion 11 Fig. 1.7 EPEC attaching and effacing 12 Fig. 1.8 Erwinia hypersensitive response induction 13 Fig. 1.9 Delivery of virulence proteins into host cell 15 Fig.1.10 Schematic diagram of Esc forming a syringe and needle 16 through membranes Fig.1.11 Regulation of the Type III secretion 18 Fig.1.12 Homologies between different Type III secretion proteins 20 Fig. 2.1 Maine sponge Pachymatisma johnstonia 26 Fig. 2.2 Isolation scheme of active glycolipid from P. johnstonia 28 Fig. 2.3 lH and 1 3 C NMR spectrum of the glycolipids recorded in 29 CDC1 3 at 500 MHz Fig. 2.4 Bioassay result of fractions 16-35 and 53-62 from LH-20 30 Fig. 2.5 Bioassay result of F at different concentrations 31 Fig. 2.6 HPLC analysis of the three aglycons of FYOPl ,FYOP2 and FYOP3 33 Fig. 2.7 Comformers of the aglycons 35 Fig. 2.8 *H and 1 3 C NMR spectrum of aglycon FYOP2 recorded in CDC1 3 at 36 400 MHz Vlll Fig. 2.9 2D COSY spectrum of aglycon FYOP2 recorded in CDC1 3 at 37 400 MHz Fig. 2.10 2D H M B C spectrum of aglycon FYOP2 recorded in CDC1 3 at 38 400 MHz Fig. 2.11 Low resolution EI MS spectrum of the aglycon FYOP2 41 Fig. 2.12 ! H NMR spectrum of FYOP1 and FYOP3 recorded in 46 CDC13 at 400 MHz Fig. 2.13 'H NMR spectrum o f F Y A recorded in CD3OD at 400 MHz 47 shows the anomeric proton couplings Fig. 2.14 HP L C spectrum of the separation of FYA1 and F Y A2 49 Fig. 2.15 *H NMR spectrum of FYA1 (top) and FYA2 (bottom) 50 Fig. 2.16 Methyl-tetra-O-acetyl-a-glucopyranoside (10) has three 51 a-a couplings Fig. 2.17 COSY spectrum of FYA1 recorded in CDC1 3 at 400 MHz 52 Fig. 2.18 Methyl 2,3,4,6-tetra-O-acetyl-P-glucopyranoside has four 53 a-a couplings Fig. 2.19 lH NMR spectrum of the standard methyl 2,3,4,6-tetra-O- 55 acetyl-L-glucopyranoside (top) and methyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside (bottom), recorded in CDC1S at 400 MHz Fig. 2.20 Methyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside (11) 57 has only one a-a coupling Fig. 2. 21 Reduction and acetylation of the monosaccharides D- and 58 L - glucose Fig. 2.22 GC spectrum of alditol peracetates. The retention times are 60 28.3 min. for L-glucitol peracetate (top, 12), 28.7 min. for D-glucitol peracetate (bottom left, 13) and 30.7 min. for D-galacitol peracetate (bottom right, 14), respectively Fig. 3.1 HPLC spectrum of the acetylated glycolipid, fraction A, B, C, D, E, F and G were collected in sequence (top) and HPLC spectrum of purification of fraction A (bottom) 64 Fig. 3.2 'H NMR spectra of pachymoside A methyl ester (15) recorded 65 in C 6 D 6 at 800 MHz Fig. 3.3 COSY NMR spectra of pachymoside A methyl ester (15) recorded 66 in C 6 D 6 at 800 MHz Fig. 3. 4 Enhanced H M B C spectrum of 1 3 C labeled pachymoside A methyl 71 Ester in C6D 6 at 500 MHz LIST OF ABBREVIATIONS ATPase adenosine triphosphatase br broad CeD6 hexadeutereobenzene-^6 CDCI3 deuterated chloroform-d} CD3OD deuterated methanol-^ CH2CI2 methylene chloride COSY correlation spectroscopy 13 C NMR carbon nuclear magnetic resonance 5 chemical shift in parts per million from tetrathylsilane d doublet dd doublet of doublets DMAP dimethyl amino pyridin DMSO dimethyl sulfoxide 2D NMR 2 dimensional nuclear magnetic resonance EHEC Enterohemorrhagic Escherichia coli EIMS electron ionization mass spectrometry ELISA enzyme-linked immunosorbent EPEC Enteropathogenic Escherichia coli Esc E. coli secreted complex ESIMS electrospray ionization mass spectrometry Esp E. coli secreted proteins EtOAc ethyl acetate XI FABMS G G L GSL H M B C H M Q C ' H N M R HPLC Hz J kDa L C B M MeOH H-g pX mL mmol MS NMR PAI ppm s Tir fast atom bombardment mass spectrometry glycoglycerolipid glycosphingolipid heteronuclear multiple-bond correlation heteronuclear multiple-quantum correlation proton nuclear magnetic resonance high performance liquid chromatography hertz coupling constant in hertz kilo Daltons long-chain base molar methanol micrograms microliters milliliters millimolar mass spectrometry nuclear magnetic resonance pathogenicity island parts per million singlet translocted intimin receptor thin layer chromatography type III secretion system xiii A C K N O W L E D G E M E N T S I want to gratefully acknowledge all the support and encouragement from my supervisor Dr. Raymond Andersen. I would also like to thank him for the opportunity to work in his marine natural products laboratory. , I want to thank Dr. Brett Finlay and his microbiology group for all their enthusiasm and expertise in bioassays. I would like also to thank Dr. William Zimmerman and Dr. Kaoru Warabi for their help in the structure elucidation of Pachymoside A. We found some exciting and prospective results by our cooperation. I would especially like to thank Dr. David Williams, Xinghui Huang for passing on their expertise in separation, structural elucidation and synthesis. I also thank all the other members in our group. I was really fortunate to have the experience of working with them in the lab. N ' I would finally like to acknowledge my family and friends for all their support outside of the lab. 1 Chapter 1 Introduction The intricate mechanisms of biochemical processes continue to be a topic of much interest in the understanding of the type III secretion system (TTSS) and in the development of therapeutics. The pursuit of these interests incorporates a wide range of disciplines including microbiology, chemistry, and genetics. The research described in this thesis is the product of a close collaboration between Ray Andersen's marine natural product chemistry group and Brett Finlay's microbiology group at the University of British Columbia. 1.1 Marine natural products The isolation and structure determination of the secondary metabolites from living organisms remains a key focus of natural products chemistry. The extensive array of structurally diverse metabolites is a rich resource of potential therapeutics—actually, nearly half of all pharmaceuticals available for treating human diseases are developed from natural products. For example, paclitaxel (1) is a well-known anticancer agent isolated from the yew tree. Ph 1 2 Since the 1960's, there has been a remarkable and stable increase in the number of marine natural products reported in the literature. 4000 3500 3000 2500 2000 1500 1000 500 0 i l Total • N-containing I J III 1965-70 1971-75 1976-80 1981-85 1986-90 1991-95 1996-00 2001 «5 Fig. 1.1 Marine natural products, 1965 onwards 1 Since 2000, the number of papers published has declined slightly, which means a decreasing rate of discovery of new compounds from the marine environment.2'3 Also depicted in Fig. 1.1 is the rate of discovery of N-containing compounds over the same period.2 The steady increase in reports of nitrogenous compounds corresponds to the greater emphasis on bioactivities. The marine natural product resource distribution breakdown by phylum is shown in Fig. 1.2. Sponges are still the most productive of the marine organisms, closely followed by coelenterates, as shown. 3 • Molluscs • Tunicates • Microorg. & Phytoplankton • Echinodems 3% 15% 2% 1% Fig. 1.2 Distribution of marine natural products by Phylum, 20011 Among all the chemistry structures reported from marine organisms, glycolipids, as ubiquitous membrane constituents of animals and plants, have attracted broad interest in natural products research in the last few decades. One reason is that they are thought to play a major role as 'cell-surface-associated antigens and recognition factors'.4 What's more, in marine sponges, the saccharide heads of glycolipid molecules are involved in the mediation of sponges' asexual reproduction. Glycolipids from natural sources can be divided into three main classes. 1. Glycoglycerolipids (GGLs)— glycolipid containing a glycerol backbone. Structure (2)5 is a typical G G L with only one sugar. 4 2 2. Glycosphingolipids (GSLs)— glycolipid built around a long-chain aminoalcohol known as a sphingoid base or long-chain base (LCB). The most commonly found sphingoid bases are sphingosine in higher animals and phytosphigosine in plants. When a fatty acid residue is attached to the amino group of the sphingoid base the molecule is called a ceramide, as shown in structure (3).6 In this structure, the saccharide head is linked to the ceramide through the primary alcohol group of the sphingoid base. Carbohydrate chain Q ^ ^ ^ ^ v \ Fatty acid C21H43 NH 3 S > * ^ ^ 1 h 2 3 O H L C B Ceramide 3 3. Polyisoprenoid glycolipid— those with isoprenoid unit side chains as shown in structure (4).7 Most of them exist in invertebrates belonging to the phylum Echinodermata, while some others are found from sponges. 5 H 4 1.2 Concept of pathogenicity islands and TTSS Investigation of the mechanism of bacterial pathogenesis in humans, animals, and plants has been a major focus of microbiology. It is used to refine diagnostics, develop new antibiotics and improve vaccines. In addition, with the development of genetic and biochemical analyses of bacteria and tissue culture models of bacterial infection, the research of bacterial pathogenesis also offers insights into the interactions between pathogens and their hosts at the cellular and molecular levels. Initially it appeared that each disease might be created by a distinct molecular mechanism. However, the spectrum of methods is not as broad as first expected.8 Actually, there are some specific pathogenicity genes which are responsible for most common pathogenic mechanisms. These genes are often organized in so-called pathogenicity islands (PAIs). PAIs are clusters of genes which provide necessary components for the survival of the pathogen in the hostile environment of the infected host.9'10 It can be easily understood that the difference between pathogens and their nonpathogenic relatives is the presence of extra genes in the PAIs. 1 1 PAIs, which encode virulence traits in bacterial pathogens of animals 6 and plants normally appear to have foreign origins.12'13 And their homology suggests that many divergent bacterial pathogens may acquire a similar system from a common source during their evolution.8 Thus, pathogens which are distantly related have turned out to share closely related virulence genes. This point is particularly apparent for a set of approximately 20 genes which encode a pathogenicity mechanism named type III secretion system (TTSS). TTSS was first identified in the late 1980's. In this system, animal and plant pathogens deliver virulence factors into host cells by secreting and translocating specific proteins from their cytoplasm. The translocation pathway crosses several barriers, such as the bacterial inner membrane, peptidoglycan layer and outer membranes as well as host plasma membrane.14 These translocated proteins can resemble eukaryotic factors with signal transduction and interfere with eukaryotic signaling pathways, which may depress host immune responses or cytoskeletal re-organization, and establish subcellular niches for bacterial colonization.15 To thoroughly understand the TTSS, knowledge of the interaction of bacterial pathogens with host cells and pathways of protein secretion is necessary. 1.3 Type III secretion system 1. 3.1 Pathways of protein secretion in Gram-negative bacteria Although the proteins secreted by bacteria are diverse and characterized by various functions including proteolysis, haemolysis, cytotoxicity, and protein phosphorylation and 7 dephosphorylation, the research of the interaction of bacterial pathogens with host cells shows only a few pathways. The proteins involved in these pathways are transported from the bacterial cytoplasm to the extracellular space. Among these pathways, four of them have been described in gram-negative bacteria and are named type I to type IV secretion pathways separately. These four pathways can be divided into two catalogs — sec-dependent and sec-independent pathways.16'17 1. .Sec-dependent pathways Type II and type IV protein secretion pathways are sec-dependent, meaning that the proteins are exported from the cytoplasm into the periplasm via a sec system. Thus, there exists a separate step of transport across the inner membrane prior to transport across the cell envelope. A short amino-terminal signal sequence (about 30 amino acids) is added to the exported proteins (the broken line in Fig. 1.3). This signal sequence helps protein export and itself is cut down by a periplasmic signal peptidase when the exported protein gets to the periplasm. The difference between these two systems is the way in which the proteins are transported across the outer membrane. In type II secretion pathways, an additional set of inner and outer membrane proteins are required. However, in type IV secretion pathways, the proteins contain residues which are necessary for the transport across the outer membrane. Thus these so-called 'auto-transporters' form a pore in the outer membrane. After passing through the pore, these proteins are auto-proteolytically cleaved and released into the supernatant.18 8 Type I Type II Type III Fig. 1.3 Type I, Type II and Type III secretion 2.5V?c-independent pathways In contrast, type I and type III systems are both sec-independent pathways, which means no amino-terminal processing of the secreted proteins are involved. Type I secretion requires three secretory proteins: an inner membrane ATPase (energy provider), an outer membrane protein, and a membrane fusion protein. Thus, the proteins in this system are not subject to proteolytic cleavage and their secretion signal is located in the carboxy-terminal 60 amino acids.19 The TTSS has a specific property. The secretion occurs in a continuous process without the distinct presence of periplasmic intermediates. 9 1.3. 2 TTSS in bacterial pathogenesis Since the 1980's, a number of diverse gram-negative pathogens have been found to use the type III secretion pathway as their virulence mechanism. Although a variety of diseases are caused by these pathogens in different hosts, most of the secreted proteins interact directly with the host eukaryotic cells in which they are translocated. Yersinia Species Three Yersinia species, Y. pestis, Y.entercolitica and Y.pseudotuberculosis, are pathogenic to human beings and rodents.20 They can cause bubonic plague, gastrointestinal syndromes, or human self-limiting gastroenteritis. All these three pathogenic Yersinia spp. share a common ability to resist the post primary immune defense by the inhibition of phagocytosis. Fig. 1.4 Type III secretion infection of Yersinia spp. 10 Fig. 1.4 (left part) shows YopH (light part in panel B), a type III secreted protein by Y. pseudotuberculosis, injected into the cytosols of HeLa cells. The YopH in the macrophages results in inhibition of phagocytosis. While panel A exhibits this protein is only detected in association with the bacteria. Fig. 1.4 (right part) shows the cytotoxic effect of Y. pseudotuberculosis for epithelial cells by another type III secreted protein, YopE. Panel A is uninfected HeLa cells and panel B shows that the injection of YopE leads to a collapse of the cytoskeleton. Shigella flexneri Shigella spp. can cause diarrhea with a variety of clinical symptoms such as bacillary dysentery, a bloody diarrhea from the colon. Shigellosis occurs mostly in developing countries and causes half a million deaths to children younger than 5 years annually.2 2'2 3 Fig. 1.5 Shigella induced apoptosis Fig. 1.5 is an in vitro tissue culture infection experiment.24 In this case, S. flexneri invades into nonpolarized epithelial cells by inducing cytoskeletal rearrangements and forms protrusions at the contacting sites between bacterial membrane and the host cell. Panel A is an uninfected macrophage. Panel B is an apoptotic macrophage infected with wild-type S. 11 flexneri. Panel C shows a macrophage infected with a S. flexneri type III secretion mutant. It is found that, different from pathogenic Yersinia spp., Shigella spp. prefer intracellular locations. Salmonella typhimurium Salmonella spp. infect vertebrate hosts and result in diseases such as gastroenteritis, bacteremia, and enteric fever. S. typhi is responsible for systemic typhoid fever in humans, which results in half a million deaths per year all over the world, mostly in developing countries. Even in developed countries, S. typhi and S. enteritidis may still cause food poisoning.25'26 In systemic infection in mice, Salmonella can penetrate mucosa of the small intestine and destroy the host cells and the adjacent epithelium. Fig. 1.6 shows an invaded Hep-2 epithelial cell via the induction of membrane ruffling by S. typhimurium.21 Fig. 1.6 Salmonella invasion 12 Enteropathogenic Escherichia colL (EPEC) EPEC is a leading cause of diarrhea in infants in developing countries. It is one of the several categories of diarrheagenic E. coli. strains. The difference with other E coli. (such as Enterohemorrhagic E. Coli 0157: H - EHEC) is the ability of EPEC to inflict characteristic lesions in small intestine enterocytes and adhere to epithelial cells with a cluster pattern. After adherence, EPEC strains attach to the host epithelial cell surface, and efface microvilli beneath the bacteria.28 At the site of contact between the bacteria and the epithelial cell surface, cup-like pseudopods can be found, as in Fig. 1.7. These pseudopodia can form elongating pedestals carrying individual bacteria on their tops.29 Fig. 1.7 EPEC attaching and effacing Other bacteria with TTSS include Pseudomonas aeruginosa, Chlamydia Species, and even some plant-pathogenic bacteria. The plant diseases caused by type III secretion pathogens include fire blight of rosaceous plants and soft rot (by Erwinia spp.), bacterial spot disease of pepper and tomato (by Xanthomonas capestris), and bacterial wilt of solanaceous plants 13 (by Ralstonia solanacearum).30'31 Fig. 1.8 shows a macroscopically visible picture in laboratory conditions when the plant tissue is infiltrated with a large amount of phytopathogenic bacteria. We can see the induction of localized tissue necrosis in a tobacco leaf at the sites of infiltration with Erwinia spp in area 1, buffer in area 4, type III secretion mutants in area 2 and 5, and complemented mutants in area 3 and 6. Fig. 1.8 Erwinia hypersensitive response induction 1.3.3 Virulence proteins secreted via type III secretion pathways The type III secreted proteins by different pathogenic bacteria vary in size, structure and function. Some of them are virulent to host cells and others play an accessory role of secreting and translocating the actual virulence factors. What's more, some proteins have homologies in different TTSSs. For example, a secreted Salmonella protein contains two domains and each of them is similar to a different Yersinia antihost factor, respectively. Some secreted factors are even shared by Salmonella, Yersinia and the plant pathogen X. campestris.14 Thus, investigations of these proteins and their functions in TTSSs are 14 essential to understand the pathogenic mechanism. Below is a summary of some well-understood proteins as well as their bioactivities in the interaction with host or other proteins. Table 1.1 Proteins via the Type III Secretion Pathway Organisms Secreted proteins Biochemical activity Yersinia spp YopE, YopH, YopA, PTPase dephosphorylates YopM. YopM, LcrV, paxillin, Binds to thrombin LcrQ, LcrG P. aeruginosa ExoS,, ExoU, PcrV, Cytotoxic, F-actin PopB disruption S. Flexneri IpaB, IpaC, Ipad, IpaA, Modulation of cell VirA invasion S. Typhimurium SPI-1 AvrA, Sip/SspB, SopE, Induction of apoptosis SptP... EPEC EspA, EspB, EspC, Tir A / E lesion formation, receptor for EPEC attachment P. Syringae AvrPto, HrpA, HrpZ Elicitation of HR E. Amylovora HrpN Eli citation of HR R. solanacearum PopAl Elicitation of HR 1.3.4 Process of TTSS Among all of these pathogenic bacteria, the TTSS of EPEC is a well-studied model. As mentioned above, there are several different proteins among them Tir is the actual virulence factor. EPEC pathogens use more than twenty E. coli secreted complex (Esc) and proteins (Esp) to help deliver the 'translocated intimin receptor' (Tir) into the host cell. Tir 15 will then combine with its intimin ligand in the bacterial membrane to cause disease. Fig. 1.9 is the schematic representation of this mechanism.33 Fig. 1.9 Delivery of virulence proteins into the host cell This mechanism has been viewed as a three-stage process. The first stage involves the initial adherence of the bacteria to the host's intestinal epithelium. EPEC forms dense micro colonies on the surface of tissue culture cells in a pattern known as localized adherence. EPEC is thought to initially attach to the host cell through a plasmid-encoded bundle forming pilus. This step also contains a regulatory function for type III secretion, which will be mentioned in the next section. The second stage involves the production of bacterial proteins and a translocation channel. EscN is located in the bacterial cytoplasm along the inner membrane. Due to its ATPase 16 activity, EscN can convert biochemicals into mechanical energy within the secretion machinery. EscV, with eight transmembrane domains and a large cytoplasmic carboxyl - terminal domain, is associated with the bacteria inner membrane. EscJ, with a lipid attachment site motif, is thought to span the periplasmic region and connect type III components between the inner and outer membranes. EscC, another protein called secretin, forms a channel in the outer membrane and is responsible for transporting large molecules across the outer membrane. All these membrane components act as a syringe or basal body for the TTSS. Meanwhile, EspA and EscF form a filamentous organelle on the bacterial surface. It looks like a needle next to the syringe with the width of about 15 nm. EscF forms the translocation tube and EspA acts as a sheath or coat around the needle. IS rim: Fig. 1.10 Schematic diagram of Esc forming a syringe and needle through membranes 17 Two other proteins secreted by TTSS, EspB and EspD, are then translocated via this apparatus into the host plasma membrane, where they form a translocation pore. Each of these pores consists of six to eight subunits with the width of 3-5 nm. Thus, a whole channel is constructed between the bacteria and the host cell. The third stage of EPEC infection is characterized by enterocyte effacement and intimate bacteria attachment to the host cell. With the help of a chaperone called CestT, a 78kDa protein -Tir travels through this channel and is inserted into the host membrane, where it undergoes a series of phosphorylations on tyrosine, serine and probably threonine. After these modifications, Tir has two predicted transmembrane domains with a hairpin model proposed for Tir conformation in the host membrane. And its N- and C- terminal regions are located within the cell whereas the intervening region forms an extracellular loop (as shown in Fig. 1.9). Thus, the ligand intimin, a 94 kDa outer membrane bacterial protein, can binds to this extracellular loop via its C- terminal region. This Tir- intimin binding is essential for pedestal formation and actin condensation, which will result in continued signal transductions and symptoms. The TTSS mechanisms in other pathogens are thought to be similar to this process. Protein homologues are also found in these pathogens with strikingly similar functions. 1.3.5 Regulation of type III secretion by contact with eukaryotic cells Normally, the TTSSs are activated and regulated by contact of the bacteria with the surface of eukaryotic target cells. Some type III secreted proteins are not directly involved in the 18 core secretion mechanism but function in a sophisticated manner to regulate this secretion system. These proteins are thus called regulatory proteins. The TTSS of Yersinia species is employed to elucidate the regulation mechanism in this sector. Yersinia Fig. 1.11 Regulation of the type III secretion As shown in Fig. 1.11, a 293-aa type III secreted protein, YopN, is exposed on the bacterial surface.34 In contrast to other Yops such as YopE/H, YopN is never translocated into eukaryotic cells but functions as a 'surface-exposed sensor or a regulatory factor' to control the polarized secretion and translocation of other Yop proteins.35 Prior to contact with a host cell, the type III secretion channel (Ysc) is shut by YopN. And the cytoplasmic accumulation of LcrQ (a negative regulatory factor) leads to transcriptional repression of yop genes. After cell contact, YopN interacts with an unknown component on the eukaryotic cell surface which may change the conformation of YopN. Thus, YopN is released, and a localized opening of the secretion channel at the site the contact is facilitated. 19 Meanwhile, secretion of LcrQ is also activated after YopN release, which in turn relieves the block from yop gene expression. Anti-host proteins are thus rapidly secreted and translocated into the host cell across the type III machinery and YopB/D. 2 1 ' 3 6 Actually, some other factors besides YopN are also involved in the regulation of TTSS in this model. They may do that by interacting with YopN or with the core secretion apparatus, while the details are not clear yet.14 1.4 Potential antimicrobial inhibitor targets Recently, experiments proved that mutating or deleting part of TTSS decreases the virulence of the pathogens. Thus, people assume that compounds inhibiting the TTSS may prevent diseases by impairing the essential virulence properties, i.e., TTSS may be a potential therapeutic target for inhibitors. TTSS has many advantages as a good target for potential therapeutics. These include: 1. Because the TTSS is specific to pathogenic bacteria, inhibitors to specific components of this system would not harm the ordinary micro flora that do not possess type III secretion virulence mechanism. Thus, these compounds can avoid killing a broad range of bacteria without discrimination, as most current antibiotics do. 2. The genes for the TTSS are clustered as PAIs, and these extra genes are specific only in pathogenic bacteria. Thus, there is little selective pressure for viability. The 20 development of antibiotic resistance to the potential therapeutics will be significantly decreased. 3. Current inhibitors based on other systems are not specific to virulence mechanisms, but also involved in normal cellular functions, which may cause side effects. However, inhibitors of the TTSS would specifically target a virulence mechanism, decreasing the chance for potential side effects. 4. As we know, different type III secretion factors may share the same or similar sequences. For example, the protein tyrosine phosphatase SptP of S.typhimurium, the cytotoxin YopE and the tyrosine phosphatase YopH of Yersinia spp., and the ExoS ADP-ribosyltransferase of P. aeruginosa are close homologs. The identically shaded boxes in Fig. 1.12 indicate the regions of sequence similarities. C479 SptP YopE ExoS G403 YopH 100 aa Fig. 1.12 Homologies between different type III secretion proteins 21 1.5 Screening model of type III secretion inhibitors A high-throughput bioassay model has been developed in Dr. Finlay's lab in UBC. In this model, compounds are tested in vitro using mammalian cell cultures infected with EPEC to ensure that there is no toxicity to animal cells. These potential inhibitors may inhibit type III secreted proteins of EPEC without affecting general secretion of the bacteria. There are several choices for appropriate target proteins. At the beginning of screening, EspC, which is not secreted by the Type III pathway, was chosen to test the ability of compounds to inhibit type III secretion. Finlay's group found that it inhibits general bacterial secretion, not just type III secretion.33 The ATPase of the EscN family is the only type III secrection system protein with known enzymatic activity. It is located inside the bacterial membrane and also involved in ordinary nonpathogenic processes. So, it is not an attractive target for further screening either. Better targets are focused in those components exposed on the bacterial surface, although little is known about their molecular architecture or function in TTSS. Among them, EspB, which forms the pores in host cell membranes, is considered as a potential target. In Finlay's lab, the percentage of EspB secretion as determined by ELISA is used as an index of inhibition ability. 22 A number of extracts from marine organisms and plants have been screened by this model. Marine organisms are bathed in pathogenic bacteria environment and some plants lack active immune protection but are still in direct contact with microorganisms. In the screen of these extracts, 86% showed no effect on the bacteria, and 11% inhibited the general growth of EPEC (not specific to type III secretion). Further screening is still in progress and the first potential inhibitor was isolated from the sponge Caminus sphaeroconia Sollas. It is a complex family of novel glycolipids named caminosides.38 The major component of mixture, caminoside A, is shown below (5). PH 1.6 A i m of this study The goal of this study is to isolate the biologically active metabolites from Pachymatisma johnstonia, a sponge collected from shallow waters in the eastern Atlantic, using the type III secretion inhibitor bioassay to guide fractionations, and to elucidate their structures with physical and chemical methods. 23 1.7 References 1. J. W. Blunt, B. R. Copp, M . H.G. Munro, P. T. Northcote and M . R. Prinsep, Nat. Prod. Rep., 2003, 20, 1 2. S. Urban, S. J. H. Hickford, J. W. Blunt, M . H. G. Munro and M . Kelly, Curr. Org. Chem., 2000, 7, 765 3. D. J. Faulkner, Nat. Prod. Rep., 2002, 19, 1 4. V. Costantino, E. Fattorusso and A. Mangoni, Bioactive Compounds from Natural Sources, 2001, Chapter 14, 556 5. D. Colombo; F. Compostella; F. Ronchetti; A. Scala; L. Toma; M . Kuchide; H. Tokuda; H. Nishino, Cancer letters, 2000, 6, 201 6. F. Cafieri; E. Fattoorusso; A. Mangoni, Gazzetta Chimica Italian, 1996, 126, 711 7. V. Costantino; E. Fattorusso; C. Imperatore and A. Mangoni, Eur. J. Org. Chem., 2001,4457 8. J. Mecsas and E. J. Strauss, Emerging Infectious Diseases, 1996, 4, 271 9. C. A. Lee, Infect. Agents Dis., 1996, 5,1 10. J. Hacker G. Blum-Oehler, I. Muhldorfer and H. Tschape, Mol. Microbiol. 1997, 23, 1089 11. D. Macel and J. Davies, CMLS cellular and Molecular Life Sciences, 1999, 56, 742 12. S. Knapp, J. Hacker, T. Jarchau, and W. L. Goebel, Journal of Bacterial, 1986, 168, 22 13. J. Hacker, L. Bender, M . Ott, J. Wingender, B. Lund, R. Marre, et al., Microbial Pathogens, 1990, 8, 2213 14. C. J. Hueck, Microbiol. Mol. Biol. Rev., 1998, 62, 379 15. R. DeVinney; A. Gauthier, A. Abe and B. B. Finlay, Cell. Mol Life Sci,. 1999, 55, 961 16. A. P. Pugsley, Microbiol Rev., 1993, 57, 50 17. G. P. C. Salmond and'P. J. Reeves, Trends Biochem. Sci., 1993, 18, 7 18. F. C. Neidhardt; R. Curtiss III; J. L. Ingraham; E. C. C. Lin; K. B. Low; B. Magasanik; M . Riley; W. S. Reznikoff; M . Schaechter and H. E. Umbarger, Escherichia coli and 24 Salmonella, cellular and molecular biology, 2nd ed. A S M Press, Washington, D.C., 1996 19. C. Chervaux and I. B. Holland, J. Bacteriol, 1996, 178, 1232 20. T. Burrows and G. A. Bacon, Br. J. Exp. Pathol, 1956, 37, 481 21. K. Andersson; N. Carballeira; K. -E. Magnusson; C. Persson; O. Stendahl; H. Wolf-Watz and M . Fallmann, Mol. Microbiol, 1996, 1057 22. A. A. Lindberg and T. Pal., Vaccine, 1996, 11, 168 23. A. T. Maurelli and P. J. Sansonetti, Anna. Rev. Microbiol, 1988, 42, 127 24. A. Zychlinsky; M.-C. Prevos and P. J., Nature, 1992, 358, 167 25. T. Pang; Z. A. Bhutta; B. B. Finlay and M . Altwegg, Trends Microbiol 1995, 3, 253 26. S. I. Miller; E. L. Hohmann and D. A. Pegues, Salmonella (including Salmonella typhi), p. 2013-2033. In G. L. Mandell, J. E. Bennett, and D. Raphael (ed.), Principles and practice of infectious diseases, vol. 2. Churchill Livingstone, Inc., New York, N.Y., 1995 27. B. B.Finlay and S. Ruschkowski, J. Cell Sci, 1991, 99, 283 28.1. C. A. Scaletzky; M . L. M . Silva and L. R. Trabulsi, Infect. Immun., 1984, 45, 534 29.1., S. Rosenshine; Ruschkowski, M . Stein; D. J. Reinscheid; S. D. Mills and B. B. Finlay, EMBOJ., 1996, 15, 2613 30. A. Collmer and D. W. Bauer, Curr. Top. Microbiol Immunol, 1994, 192, 43 31. U. Bonas, Curr. Top. Microbiol Immunol, 1994, 192, 79 32. D. W. Bauer; Z. M . Wei; S. V. Beer and A. Collmer, Mol. Plant-Microbe Interact. ,1995, 8, 484 33. A. Gauthier and B. B. Finlay, ASM News, 2002, 68, 383 34. A. Forsberg; A . - M . Viitanen; M . Skurnik and H. Wolf-Watz, Mol. Microbiol 1991, 5, 977 35. A. Boland; M . P. Sory; M . Iriarte; C. Kerbourch; P. Wattiau and G. R. Cornells, EMBO 7.1996,15,5191 25 36. A . - M . Viitanen; P. Toivanen and M. Skurnik, J. Bacterioi, 1990, 172, 3152 37. S. M . Kulich; T. L. Yahr; L. M . Mende-Mueller; J. T. Barbieri and D. W. Frank, J. Biol. Chem. 1994, 269, 10431 38. R. Linlington; M . Robertson; A. Gauthier; B. B. Finlay; R. Soest and R. J. Andersen, Organic Letters, 2002, 4, 23 26 Chapter 2 Isolation and Structure Determination of Aglycons and Monosaccharides Pachymatisma johnstonia (Phylum Demospongiae, order Astrophorida, and family Geodiidae) is a large sponge (up to 15 cm in length) found in the eastern Atlantic. The surface of the sponge is smooth, with a 1 mm distinct skin as shown in Fig. 2.1. A crude MeOH extract of P. johnstonia gave a positive response in the type III secretion inhibitor bioassay during high throughput screening of thousands of marine and terrestrial extracts in Finlay's lab. The biological activity of the crude coupled with no previous literature reports on the chemical constituents of P. johnstonia made it an ideal candidate for further studies. Fig. 2.1 Pachymatisma johnstonia 27 2.1 Isolation of an active glycolipid from P. johnstonia Methanol (3 X 1200 mL) was used to extract the secondary metabolites from 245 g of frozen P. johnstonia. After evaporating of the combined MeOH extracts, 1.36 g of a greenish solid material was obtained. The solid was partitioned between EtOAc and H 2 0 to divide the extract into an aqueous soluble fraction and an organic soluble fraction. The organic soluble material (857 mg) was dissolved in MeOH and loaded directly onto a Sephadex LH-20 column and eluted in 100% MeOH. The fractions obtained from this size-exclusion separation were collected and submitted for type III secretion inhibitor bioassay. Activity was observed in fractions 27-33. Combined into one fraction, this active part, weighing 435 mg, was named F. Normal phase silica gel thin layer chromatography (TLC) of F was run in 15% MeOH : 85% CH 2 C1 2 and visualized with a vanillin spray. The result revealed a series of compounds with a long streak on the plate, forecasting the difficulty in the following separation. Fraction F was loaded onto a Sephadex LH-20 column and eluted with EtOAc : MeOH : H 2 0 = 20 : 5 : 2. F was also preloaded onto silica gel and separated using normal phase column chromatography with different elution systems. Neither of the procedures showed much improvement in the purification, indicating that this series of compounds may be a family of closely related compounds and therefore hard to separate. 28 Sponge P. johnstonia MeOH extraction Greenish material Partition between EtOAc : H2O Aqueous fraction Normal phase chromatography column Organic fraction 100% MeOH LH20 27-33 Active combined into F 53-62 Inactive (terpenoid) in type III secretion inhibitor bioassay NMR data indicate glycolipid structure FAB Mass Spectrum Sephadex LH20 column in different elution system Glycolipid family Fig. 2.2 Isolation scheme of active glycolipids from P. johnstonia The lH and 1 3 C NMR data recorded for F in C D 3 O D are shown in Fig. 2.3. The peaks around 5 100 ppm in 1 3 C spectrum were assigned to anomeric carbons,1 indicating polysaccharide structures. The patch of peaks around 2.0 ppm was assigned to acetyl methyl protons indicating extensive acetylation on the sugars. Several sharp singlets at 5 3.78 ppm, 8 2.98 ppm and 2.87 may come from nonpolar aglycon parts. A huge methylene envelope at ~ 5 1.3 ppm was also assigned to the aglycon. ^ 29 \ (ppm) Fig. 2.3 'Hand13 C NMR spectrum of the glycolipids recorded in CDCl3 at 500 MHz 30 Fast atom bombardment mass spectrum (FAB MS) was performed by the UBC chemistry MS lab. A series of peaks differing by 42 and 14 Daltons come from different numbers of acetyl and methylene groups, which suggests the compounds differ by the number of CH3CO- residues on the sugars and - C H 2 - residues in the aglycon. 2.2 Biological activity of fraction F Bioassays were performed on the LH-20 fractions 16-35 and 53-62 using ELISA to detect inhibitors of EPEC's TTSS. Antimicrobial activity was indicated when the extract negatively affects growth of EPEC, which is assessed by the size of the bacterial pellet.2 A percentage of EspB secretion was employed to measure to the inhibition effect. Assuming the percentage of EspB secretion in the positive control is 100%, a graph of inhibition of EspB secretion was obtained (Fig. 2.4). Fig.2.4 Bioassay result of fractions 16-35 and 53-62from LH-20 3 1 If we regard 0-20% as 'Very good", 21-40% as "good", 41-60% as "average", 61-80% as "poor" and 81+% as "none", then fractions 27-33 have promising biological activity. After combination of fractions 27-33 into F, a further bioassay was performed to test the optimal concentration of inhibition. 0.5 ug of glycolipid F was diluted in 20 uL of DMSO. 1 uL, 2 uL, 4 uL and 8 uL of the extract were put into 200 ul of bacterial culture separately. With the same biochemical test as above, a curve of percentage of EspB secretion via different concentration was obtained in Fig. 2.5. A stable inhibition effect was observed at ~ 0.25 jag/mL. 3 - — ^ — 0 -I r—— ' 0.125 0.25 0.5 1 ug/mL Fig. 2.5 Bioassay result of F at different concentrations 2.3 Chemical degradation of the glycolipids The presence of long aliphatic chains linked to the polar heads results in glycolipids being amphiphilic compounds. The glycolipids were strongly retained on reversed-phase material such as C-18 because of the hydrophobic nature of aliphatic chains. On the other hand, they were also strongly retained on silica gel owing to their sugar moiety, so that a 32 normal phase chromatographic separation did not work in this case. When subjected to many kinds of HPLC separation, the glycolipids produced foams and emulsions and resulted in broad peaks. Present as complex mixtures of homologues in the extract, the glycolipids have a similar polar moiety and alkyl chains, which makes the isolation of pure compounds very difficult, or even impossible.3 Actually, glycolipids were first purified by column chromatography only in the 1990's. 4 ' 5 As a general rule, determination of the structure of the saccharides and the non-polar aglycons are performed separately after chemical degradation.6' 7 Acid methanolysis can cut glycolipids into monosaccharides and aglycons. The structures of aglycons can be determined by NMR and MS. And a well-established chemical procedure for characterization of the sugar chain starts with the identification of the monosaccharides units followed by quantitative analysis of the obtained methyl glycosides using chromatographic methods. The subsequent step of the investigation is the identification of the position of the inter-unit linkages and sugar-aglycon bond. Methanolysis in acid was first applied to decompose glycolipid F. F (60 mg) was added into 10 mL of anhydrous methanol with 0.2 mL CH3COCI and the mixture was stirred at 70 °C for 2 hours. The reaction was worked up by putting it on a rotavap to remove the methanol. The residue was partitioned between H 2 0 and Et 2 0; the aglycon (FYO, 25 mg) went into the organic phase and the methyl glycosides (FYA, 30mg) went into the aqueous phase. 33 2.4 Aglycons 2.4.1 Isolation of the aglycons The Et20 soluble fraction (FYO, 25mg) was loaded on a normal phase silica gel column and eluted with a gradient system of Hexanes/EtOAc (5% EtOAc: 95% Hexanes —> 100% EtOAc). One major component and one minor component were observed by T L C analysis. The more polar major component was fractionated further by reversed phase HPLC eluting with 88% MeOH : 12% H2O. Three components (shown in Fig.2.6) were purified and named FYOP1, FYOP2 and FYOP3 respectively. « . M '' :'*.'«" IO^M it'.ti ' it'.tt u.m ' » : » -: u.«e «e^ o« ' «»:«« ».'«« »»:» ia'.m Minutes Fig. 2.6 HPLC analysis of the three aglycons ofFYOPl, FYOP2 andFYOP3 3 4 2.4.2 Structure determination of FYOP2 by N M R and MS The structures of the aglycons were determined by analysis of their 'H NMR, 1 3 C NMR, 2D NMR and mass spectrometry data; and they were also confirmed by synthesis of oxime derivatives. The electrospray ion mass spectrum (ESI MS) of the three aglycons offered very intense base peaks of [M+Na]+ m/z at 562, 576 and 590, indicating their molecular weights are 539, 553 and 567, corresponding to the formulae C32H61O5N, C33H63O5N, and C 3 4 H 6 5 0 5 N , respectively. The molecular formulae require three sites of unsaturation. The aglycons are homoldgues with different numbers of methylenes, which was apparent from the striking similarity of their NMR spectra. Thus, analysis of FYOP2 (6) gave a common structure elucidation of these homologues. 26 o 6 The 'H NMR spectrum of FYOP2 had three singlets at 8 4.11 ppm, 3.71 ppm and 3.05 ppm assigned to H-3, H- l and H-4. Each one has a small shadow peak (at 8 4.02 ppm, 8 3.76 ppm and 8 2.95 ppm, respectively), which comes from the rotamers or conformers of the amide bond (Fig. 2.7). From the H M Q C data, the corresponding carbons in 1 3 C NMR spectrum were easily assigned — C-3, C- l and C-4 were at 8 49.0 ppm, 8 52.5 ppm and 8 36.5 ppm, respectively. Three carbonyl carbons were found at 8 169.5, 172.0 and 212.5 35 13 ppm in the C spectrum, assigned to an ester, an amide, and a ketone. The carbons signals for the minor rotamer were too weak to observe. Fig. 2.7 Comformers of the aglycons There are three methylenes, H-6, H-17 and H-19, next to carbonyl carbons in this structure. Their triplet signals in 'H NMR spectrum are overlapped at 5 2.35 ppm. H-6 has a small shadow triplet peak at 8 2.22 ppm due to the hindered rotation of the amide bond. In the COSY NMR spectrum, strong correlations were shown between H-7 and H-6, and H-16/20 and H-17/19. Thus, H-7 is assigned to 8 1.62 ppm and H-16 and H-20 are assigned to 8 1.55 ppm in ! H NMR spectrum. 36 Fig. 2.8 !H and 13C NMR spectrum ofaglycon FY0P2 recorded in CDCl3 at 400 MHz 37 O Fig. 2.9 2D COSY spectrum of aglycon FYOP2 recorded in CDCl3at 400 MHz 38 HI andH3 (ppm) 3.2 2.4 4 . 6 -H3-C4 ° ° o ' : H4-C3 H26-C27 : • H3-C2 | c 4 H 1 - C 2 H6-C5 § « • f • H3-C5 H4-C5 H17/19-C18 ' 1 ' ' - 1 1 -r 1 1 1 r — 1 — ~ -(ppm) K120 160 200 1.6 0.8 Fig. 2.10 2D HMBC spectrum of aglycon FY0P2 recorded in CDCl3 at 400 MHz 3 9 Lots of useful information was obtained from the H M B C spectrum. The H-3 (8 4.02 ppm) protons show correlations to the N-methyl carbon C-4 (8 36.5 ppm) and to the two carbonyl carbons C-2 (8 169.5 ppm) and C-5 (8 172.0 ppm). H- l (8 3.71 ppm), the methoxy protons, has only one correlation to the carbonyl C-2, which means this methoxyl group is at one end of the molecule and belongs to an ester group. H-4 (8 3.05 ppm) shows correlations to C-3 (8 49.0) and to C-5 (8 172.0 ppm). ' The methine H-27 connected to the only hydroxyl carbon is too broad to be assigned in ! H NMR spectrum. While the signal of the carbinol methine C-27 was found in 1 3 C spectrum at 8 72.5 ppm. In the H M B C spectrum, there is a weak correlation between this carbon with the methyl H-26 (8 0.85 ppm), which indicates the proximity of the methyl branch and the hydroxyl. From the integration, six protons were assigned to 8 0.85 ppm in ! H NMR spectrum. Thus, two methyl groups with overlapped lH signals must be present. Based on these data and literature precedent,8 the structure for aglycon FYOP2 was proposed to be (6) and the NMR spectrum was assigned as shown in table 2.1. The positions of the hydroxyl (C-27) and ketone (C-17) functionalities in the chain were hard to decide from the NMR data. Thus, data from mass spectrometry fragments were essential for further refinement of the structure. 40 Table 2 . 1 NMR data for aglycon FYOP2 recorded in CDCl3 at 400 MHz Carbon No. 1 3 C N M R (8 ppm) *H N M R (8 ppm) H M B C Correlation C- l 52.5 H- l 3.71,3.76 (s)* --C-2 169.5 — H - l , H-3 C-3 49.0 H-3 4.11,4.00 (s) H-4 C-4 36.5 H-4 3.05, 2.95 (s) H-3 C-5 172.0 — H-3, H-4, H-6 C-6 33.0 H-6 2.35, 2.22(s) H-7 C-7 24.7 H-7 1.63 H-6, H-8 C-8-C-15 -29.5 H-8~ H-16-1.23 — C-l6, C-20 23.8 H-l6, H-20 1.54 H-l7/19, H-l5/21 C-17, C-19 42.8 H-17H19 2.35 H- l 6/20 C-18 212.5 — H17/19, H- l 6/20 C-21 -C-24 -.29.5 H-21-H-24-1.23 — C-25 38.1 H-25 1.42 H-26 C-26 22.5 H-26 0.85 H-24, H-25, H-27 C-27 72.5 H-27-3.58 (br)¥ H-26 C-28 ~ C-32 -29.5 H-28-H-32-1.23 — C-33 13.8 H-33 0.85 H-32 * s — small peak, br ;— broad peak 41 i-9 3 I ,.Pp,1,rr.pTTT.p.pp,l>.,,|.).|.(.|»P^^ S S g 8 S § 8 8 5 8 1 s | , | . . | . r .).[ l p | , V , . | . t . | . n . j l | l 1 . , . | .J, | . , 1 T W .p, r . | , r | t , , , . | . | , | , | , ( | (T,^^, 8 8 8 B 8 8 ? 8 8 S Fig. 2.11 Low resolution EI MS spectrum of the aglycon FYOP2 Table 2.2 Analysis of high resolution EIMS fragments ofFYOP2 42 Mass Spec. Data (m/z) Atomic Composition Proposed Fragments 553.47085 C 3 3 H 6 3 0 5 N Molecular ion peak 535.46020 C 3 3 H 6 1 0 4 N O 0 I 0 ~ \ _ J - ^ C 6 H 13 + 468.36970 C27H50O5N 26 r 0 L 0 1 0 + 27 OH 433.40506 C29H53O2 26 0 O OH + 341.25661 C 9 H 3 5 O 4 N 0 1 0 -1 + 9 326.23313 C18H32O4N 1 . 0 1 0 284.22257 C 1 6 H 3 o 0 3 N O 0 1 J + 145.07390 C 6 H n 0 3 N 0 ^ Y N 5 X 6 1 1 0 1 + 104.07093 C4H10O2N 0 ' + 43 Fig. 2.11 is the low resolution electron impact mass spectrum (EIMS), which revealed the fragment peaks of the aglycon FYOP2. The analysis of the proposed fragments from high res. EIMS in table 2.2 gave possible positions of the ketone and secondary alcohol functionalities in 6. A pair of peaks at m/z 284.22257 (Ci6H3o03N) and 341.25661 (C19H35O4N) were attributed to McLafferty rearrangements occurring on both sides of the ketone functionality, which located the ketone at C-18.9 A peak at m/z 468.36970 was assigned to an a cleavage adjacent to the secondary alcohol, which had to be at C-27. 1 0 2.4.3 Structure confirmed by oxime formation An oxime formation reaction was performed on aglycon FYOP2 1 1 to double confirm the positions of the two functionalities. Aglycon 6 was added to 10 mL MeOH together with hydroxylamine hydrochloride (NH 2 OH • HC1) and sodium acetate (CH3C02Na). The mixture was stirred at room temperature for 1 hour. The oxime product 7 was obtained in good yield. 2 7 ^ C 6 H 1 3 The crude oxime was partitioned between Et 2 0 and H 2 0 . The organic fraction was loaded onto a silica gel chromatography column and eluted with a gradient system (20 % EtOAc : 80 % Hexanes to 80 % EtOAc : 20 % Hexanes). The purified oxime product (7, 1.9 mg) was collected from fractions 5-7. The high resolution EIMS of the oxime was recorded at 200°C and compared with the spectrum of FYOP2. 44 Table 2.3 Analysis of the MS fragments of the oxime Mass Spec. Data (m/z) Atomic Composition Proposed Fragments 568.48153 C S S H M O J N ! M o l e c u l a r i o n p e a k 552.48564 C 3 3 H 6 4 0 4 N 2 0 ^ ^ i ^ ^ T ^ T CeH,l+ 0 ' N H O H J 467.38455 C 2 7 H 5 i 0 4 N 2 0 ^ Y Y ^ r ^ 0 ' N H 6 O H J 340.27350 C19H36O3N2 h - r ^ r T O 1 N H 325.24938 C i 8 H 3 3 0 3 N 2 [ r - V Y ^ ' 1 L O 1 N H -+ The fragments of the oxime paralleled the fragments of FYOP2. However, the group -C=NH was present in the fragments at m/z 340.27350 and m/z 325.24938, which indicated the original ketone was closer to the N-methyl glycine fragment. Thus, the sequence of the alcohol and ketone groups was identified. 2.4.4 Structures of the other two aglycons The *H NMR spectrum (Fig. 2.12) of the other two aglycons, FYOP1 and FYOP3, indicated structures closely related to FYOP2. FYOP3 (9) has a longer carbon chain with one more methylene after the hydroxyl group. FYOP1 (8) has only one methyl as indicated 45 by the integration of the [ H NMR spectrum, which means a straight carbon chain without any branches. 2.5 Structure elucidation of the sugars in the pachymosides 2.5.1 Determination of glucose The aqueous soluble portion (FYA) from the product of methanolysis was studied further. Analysis of the lH NMR spectrum of F Y A revealed the presence of methyl glycosides. Characteristic NMR features included a series of doublets at 8 3.97 - 4.57 ppm, assigned to anomeric protons. Four major doublets (at 8 3.98, 4.03, 4.52 and 4.57 ppm, respectively) indicate a mixture of four methyl glycosides. The downfield two doublets had smaller coupling constants (~ 3.58 and 3.67 Hz) and the upfield two had larger coupling constants (-7.79 and 7.24 Hz), indicating the difference between axial - axial coupling and axial -equatorial coupling (Fig.2.13). 46 47 4 8 As shown in Fig. 2.13, the a glycoside has a weak coupling between the axial H-2 and the equatorial anomeric H - l ; while the P glycoside has a strong coupling between the axial H -2 and the axial anomeric H - l . Thus, there appeared to be two kinds of sugars in the glycolipids. Each one would have formed two anomers after the methanolysis. From the integration, the ratio of these two sugars was about 2 : 1. The 1 3 C NMR spectrum also showed two pairs of anomeric carbons at 8101.5 and 105.5 ppm. It is difficult to isolate glycosides due to the polar - O H groups. Acetylation of the alcohols was performed in order to get nonpolar acetyl glycopyronosides.12 After removing solvents, F Y A (20 mg) was added to 9 mL of anhydrous pyridine and 3 mL of acetic anhydride, followed by adding ~1 mg of DMAP as a catalyst. The mixture was stirred at room temperature overnight, and the product was partitioned between H2O and Et20. The organic soluble fraction was then loaded onto a normal phase silica gel chromatography column and eluted with a gradient system of Hexanes : EtOAc ( 90% hexanes : 10% EtOAc to 100%) EtOAc). Silica gel T L C analysis using visualization with a vanillin spray guided the pooling of chromatography fractions. Fractions 31-38 were pooled for HPLC isolation. Isocratic normal phase HPLC was used, eluting with 31% EtOAc : 69% Hexanes and monitored with a refractive index (Rl) detector. Fig. 2.14 shows two neighboring peaks FYA1 (13 mg) and FYA2 (4 mg). *H NMR spectrum of the two components are shown in Fig. 2. 15. 49 FYA1 I Fig. 2.14 HPLC spectrum of the separation of FYA1 and FYA2 There is only one methoxy peak at 8 3.38 ppm in ! H N M R spectrum, but two anomeric carbons at 8 96.8 ppm and 8 97.2 ppm in 1 3 C N M R spectrum of F Y A 1 , which means two methyl glycosides with overlapped - O M e ' H N M R signals. Two series o f acetyl methyls can be observed: 8 1.97/1.99/2.04/2.07 ppm and 8 1.94/2.02/2.05/2.10 ppm. The former belongs to the major component and the latter belongs to the minor component. The doublet at 8 4.92 ppm is from the anomeric proton H - l because it only couples to H-2 in the C O S Y , while other protons have at least two correlations. The multiplet at 8 3.95 ppm comes from H-5, which couples to H-4 and the two methylene protons H-6 and H-6 The C O S Y correlations (Fig. 2.17) of the major component were assigned via the pathway Fig. 2.15 'HNMR spectrum ofFYAl (top) and FYA2 (bottom) 51 of 5 4.92 -> 4.86 -> 5.44 -> 5.03 -> 3.95 -> 4.07/ 4.22 ppm. Thus, these protons were shown to be H - l , H-2, H-3, H-4, H-5 and H-6/H-6' by the coupling sequence.13 Analysis of the coupling constants in the *H NMR spectrum gave a clear picture of the relative stereochemistry. Table 2.4 lists the coupling constant data for the protons in FYA1. Table 2.4 Assignment of the coupling constant of the FYA1 major component Proton H-l H-2 H-3 H-4 H-5 H-6 H-6' No. (d-d) (d-d) (d-d) (d-d-d) (d-d) (d-d) Coupling 3.4 10.4 10.2 10.1 10.4 12.5 12.5 Constant 3.4 4.6 4.6 2.4 J (Hz) 2.4 Thus, the relative configuration of this major component was determined as methyl 2,3,4,6-tetra-O-acetyl-a-glucopyranoside (10). Fig. 2.16 methyl-tetra-O-acetyl-a-glucopyranoside (10) has three a-a couplings 52 Fig. 2.17 COSY spectrum ofFYAl recorded in CDCl3 at 400 MHz 53 There are two 1-methoxy peaks at 5 3.48 and 3.49 ppm in the ! H NMR spectrum of FYA2, and also two anomeric carbons at 8 101.6 ppm and 5 102.1 ppm in the 1 3 C NMR spectrum, which indicates two methyl glycosides. Two series of acetyl methyls were also observed: 8 1.98/2.00/2.03/2.06 ppm and 8 1.96/2.02/2.04/2.13 ppm. The former belongs to the major component and the latter belongs to the minor component. As described above, protons were assigned via the COSY correlation sequence of 8 4.40 4.96 ->• 5.18 ->• 5.07 -> 3.67 -> 4.25/4.13 ppm. Thus, these protons were shown to be H - l , H-2, H-3, H-4, H-5 and H-6/H-6' in sequence. Analysis of the coupling constants in 1 13 the H NMR spectrum revealed the relative stereochemistry. Table 2.5 lists the coupling constant data for the protons in FYA2. Table 2.5 Assignment of the coupling constants of the FYA2 major component Proton H-l H-2 H-3 H-4 H-5 H-6 H-6' No. (d-d) (d-d) (d-d) (d-d-d) (d-d) (d-d) Coupling 8.0 9.4 9.6 9.9 9.9 12.3 12.3 Constant 8.0 9.4 9.6 4.7 4.7 2.5 J(Hz) 2.5 Thus, the relative configuration of the major component was found to correspond to methyl 2,3,4,6-tetra-O-acetyl-P-glucopyranoside (Fig. 2.18). u 4 OAC Fig. 2.18 Methyl 2,3,4,6-tetra-0-acetyl-/3-glucopyranoside has four a-a couplings 54 To confirm the glucose structure, the ! H NMR spectrum of standard methyl 2,3,4,6-tetra-0-acetyl-a/p-glucopyranoside was compared with the spectrum of FYA1 and FYA2. Commercial standard L-glucose (20 mg) was dissolved in 10 mL methanol with 0.2 mL of CH3COCI and stirred at 70 °C for 2 hours. The solution was then put on a rotavap to remove the solvent. After evaporation, the residue was partitioned between H2O and Et20. The aqueous soluble fraction was dried and acetylated with acetic anhydride and pyridine together with the catalyst DMAP overnight. The dried product was again partitioned between H2O and Et20 and the organic soluble fraction was then loaded onto a normal phase silica gel chromatography column and eluted with a gradient system of Hexanes : EtOAc ( 90% hexanes : 10% EtOAc to 100% EtOAc). The standard methyl 2,3,4,6-tetra-O-acetyl-glucopyranoside anomers were made and their *H N M R spectra (Fig. 2.19, upper) were compared with Fig. 2.15. All the peaks are aligned with the major component of FYA1 and FYA2, confirming the proposed structure. 2.5.2 Determination of galactose The minor component in FYA1 and FYA2 is another monosaccharides. To determine the structure of this sugar, the same NMR analysis was used which involved analysis of coupling constants and COSY correlations. In FYA1, analysis of the COSY correlations of the minor component revealed the pathway 5 4.96 —> 5.12 —> 5.31 - » 5.41 - » 4.16 ppm. Thus, these protons were shown to be H - l , H -2, H-3, H-4 and H-5 in sequence. Their coupling constants were analyzed and tabulated in Table 2.6. H-6 and H-6' were hard to assign because their peaks were overlapped. 55 Fig. 2.19 lH NMR spectrum of the standard methyl 2,3,4,6-tetra-O-acetyl-L-glucopyranoside (top) and methyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside (bottom), recorded in CDCls at 400 MHz 56 Table 2.6 HNMR spectrum assignment of the minor component FYA1 FYA2 Proton Chemical Coupling Proton Chemical Coupling No. shift 8 (ppm) constant J (Hz) No. shift 8 (ppm) constant J (Hz) HI (d) 4.96 3.6 Hl(d) 4.37 7.9 H2 (dd) 5.12 10.9 3.6 H2 (dd) 5.17 7.9 10.6 H3 (dd) 5.31 10.9 3.4 H3 (dd) 5.00 ^10.6 3.4 H4 (dd) 5.41 ~ H4 (dd) 5.37 3.4 1.0 H5 (ddd) 4.16 0.9 6.6 H5 (ddd) 3.88 1.0 6.7 In FYA2, the COSY correlations of the minor component were hard to see. Nevertheless, some peaks of the FYA2 lH spectrum can still be analyzed. The most obvious are the doublet at 8 4.37 ppm, which is H - l , and a doublet of doublets (with another small doublet) at 8 3.88 ppm, which may be H-5. A doublet (with another small doublet) at 8 5.37 ppm, a doublet of doublets at 8 5.17 ppm, a doublet of doublets at 8 5.00 ppm and some other peaks at around 8 4.15 ppm. While, due to the principle that neighboring protons have the same coupling constant, the assignment of H- l to H-5 were still done and results are in Table 2.6 To confirm this monosaccharide, a sample of standard D-galactose was used to compare their 'H NMR spectrum. Authentic methyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside was made from commercial D-galactose. The product contained both a and P anomers. From the coupling constant of H - l , a product (Fig. 2.20) is the major component. Its ! H NMR spectrum (Fig. 2.19, bottom) proved that galactose was the minor monosaccharide component of the pachymosides mixture. 57 O A c ? A l 11 Fig. 2.20 Methyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside (11) has only one a-a coupling 2.6 Absolute configuration of the monosaccharides It's impossible to get the absolute configuration of monosaccharides by NMR or MS. A useful method is chiral gas chromatography (chiral GC), which may separate enantiomers and diastereoisomers. Due to the cyclic hemiacetal formation, there is equilibrium between pyranose forms and open chain forms for monosaccharides. Sodium boron hydride (NaBH4) in water reduces the open chain aldehyde form in this equilibrium;14 all the monosaccharides were reduced to polyalcohol alditols, although only a small amount of the open chain form is present at any time. The aditols can be acetylated into nonpolar alditol peracetates while still retaining their stereochemistry. Fig. 2.21 is an example of D and L-glucose, which can be reduced and then acetylated for chiral GC separation. The standard D- and L- monosaccharides were 58 thus made and compared with pachymoside monosaccharides by their retention times on a chiral GC column. Acid hydrolysis of pachymosides was performed in water and HC1. The product was partitioned between water and EtOAc. The aqueous soluble fraction was glucitol and galacitol. These aditols was then acetylated into alditol peracetates by pyridine and DMAP, and separated into glucose fraction and galactose fraction by normal phase chromatography. Commercial D-glucose, L-glucose and D-galactose were also made into acetylated alditols as standards for the chiral GC analysis. H A c 2 0 , pyridine A c { > _ D-glucopyranose , 0 H D M A P , rt, 16 hrs CH2OH D-glucitol (an alditol) H-H,COAC —OAc —H -OAc -OAc H2COAC Acetylated D-glucitol HO-HO-3H OH L-glucopyranose HO--H HO--OH N a B H 4 H -rt. 1 hr -H HO--H HO-CH,OH L-glucose CH2OH CH2OH L-glucitol (an alditol) AcO-_ 0 H A c 2 0 , pyridine H _ -H D M A P , rt, 16 hrs A c Q _ AcO-H2COAC - H -OAc -H -H H2COAC Acetylated L-glucitol Fig. 2.21 Reduction and acetylation of the monosaccharides D- and L glucose 59 About 7 ug of the alditol peracetates were injected into the GC each time. The sample were eluted in helium, and detected by flame ionization detector (FID). The acetylated glucitol from the pachymosides was found to have the same retention time as the standard D-glucose product, not the L-glucose product. And the GC peak of the acetylated galacitol from the pachymosides had a retention time identical with the standard D-galactose product. Fig. 2.22 is the GC spectrum of D-glucitol peracetate (12), L-glucitol peracetate (13) and D-galacitol peracetate (14). The monosaccharides in the pachymosides were thus shown to be D-glucose and D-galactose. 60 CH 2 OAc -OAc "A trill ii ill i ill ill 11 ii Ii II II II i nm i AcO-AcO-AcO-AcO-AcO-AcO-H--OAc -OAc CH 2 OAc 12 CH 2OAc -OAc -H CH 2OAc 13 CH2OAc -OAc -OAc CH2OAc 14 Fig. 2.22 GC spectrum of alditol per acetates. The retention times are 28.3 min. for L-glucitolperacetate (top, 12), 28.7 min. for D-glucitolperacetate (bottom left, 13) and 30.7 min. for D-galacitolperacetate (bottom right, 14), respectively 61 2.6 References 1. W. Fresenius, J. F. K. Huber, E. Pungor, G. A. Rechnitz, W. Simon and T. S. West, Spectral data for structure determination of organic compounds, Springer- Verlag, Berlin Heidelberg, 1989 2. A. Gauthier and B. BrFinlay, ASM News, 2002, 68, 383 3. V. Costantino, E. Fattorusso and A. Mangoni, Bioactive Compound from Natural Sources, 2001, chapter 14, 556 4. H. Kubo and M . Hoshi, J. Biochem., 1990, 108,193 5. V. Costantino, E. Fattorusso, A. Mangoni, M . Aknin and E. M . Gaydou, Liebigs Annalen der Chemie, 1994, 1181 6. C. C. Sweeley and R.V. P. Tao, Meth. Carbohyd. Chem., 1972, 6, 8 7. V. Costantino, E. Fattorusso, A. Mangoni, M . D. Rosa and A. Ianaro, Bio. & Med. Chem. Lett., 1999, 271 8. F. Ingaki, S. Tate, H. Kubo and M . Hoshi, J. biochem., 1992, 112, 286 9. R. J. Fessenden and J. S. Fessenden, Organic chemistry, Willard Grant Press, Boston, Massachusetts, 2 n d edition, 1982, 942 10. R. Goobes, A. Rudi, Y. Kashman, M . Ilan and Y.Loya, Tetrahedron, 1996, 52, 7921 11. D. E. Williams, K. S. Craig, B. Patrick, L. M . Mchardy, R. van Soest, M . Roberge and J. R. Andersen, J. Org. Chem., 2002, 67, 245 12. R. Linlington; M . Robertson; A. Gauthier; B. B. Finlay; R. v Soest and R. J. Andersen, Org. Lett., 2002, 4, 23 13. J. Duus, C. H. Gotfredsen and K. bock, Chem. Rev., 2000, 100, 4589 14. M . Abdel-Akher, J. K. Hamilton and F. Smith, J. Am. Chem. Soc, 1951, 73, 4691 62 Chapter 3 Isolation and Structure Determination of Pachymoside A As mentioned in chapter 2, isolation of the natural glycolipids F, was difficult owing to the polar hydroxyls. To get the total structure of the glycolipid, peracetylation is necessary for further isolation. A peracetylated glycolipid, pachymoside A methyl ester, was isolated by HPLC after peracetylation and its structure was elucidated by analysis of NMR data in conjunction with the knowledge of monosaccharide and aglycon components. To determine the position of hydroxyls in the saccharides of natural pachymoside A, 1 3 C labeling was used and followed by HPLC isolation and NMR measurement. 1 3 C acetic anhydride was used to peracetylate the natural glycolipids with the same condition as normal peracetylation. The naturally unacetylated hydroxyl groups with 1 3 C acetyls after the labeling increase the C-H correlation by 100 times in the H M B C spectra, which helped us to determine these naturally unacetylated positions. Thus, the natural structure of pachymoside A was revealed and offered us a prospective method to determine the structures of other pachymosides in the extract. This part of work was finished with the help and cooperation of Dr. William Zimmerman and Dr. Kaoru Warabi, whose experience in synthesis and polysaccharide structures elucidation was essential to figure out the structure of pachymoside A. 63 3.1 Isolation of pachymoside A methyl ester As mentioned in the previous chapter, the pachymosides are partially acetylated at some of the monosaccharide hydroxyls. To facilitate purification, the molecules were made more lipophilic via acetylation using the same method mentioned in chapter 2. Natural extract pachymosides were reacted with acetic anhydride, pyridine, and DMAP at room temperature overnight, followed by partioning between H2O and Et 2 0. The organic soluble portion was injected onto reverse phase HPLC and eluted with 7 : 4 1- PrOH and H2O: Seven fractions, A, B, C, D, E, F and G were collected in sequence, as shown in Fig. 3.1. Fraction A was collected and then reinjected in the same HPLC system for final purification. The final product of A weighed 4 mg. ESI MS was used to determine the molecular weight of A. An intense base peak at m/z 2335 corresponded to [M+ Na]+, while a peak at m/z 1178 was assigned to [M+2Na]++. From this data, we concluded that the molecular weight of fraction A was around 2311. This compound is pachymoside A methyl ester (15). 3.2 Structure elucidation of pachymoside A methyl ester To get sharper peaks, J H and COSY spectrum of the acetylated fraction A were obtained at 800 MHz in C 6 D 6 (Fig. 3.2). 1 3 C , H M Q C and H M B C NMR data were also recorded at 500 MHz. Fig. 3.1 HPLC spectrum of the acetylated pachymosides. Fractions A to G were collected in sequence(top) and HPLC spectrum of purification offraction A (bottom) 65 Fig. 3.2 ! H NMR spectrum of pachymoside A (15) methyl ester recorded in C^D^ at 800 MHz 66 5 . 6 5 . 4 5 . 2 5 . 0 4 . 8 4 , 6 4.4 4 . 2 4 . 0 3 . 8 3 . 6 3 . 4 3 . 2 F I (ppm) Fig. 3.3 COSY spectrum of pachymoside A methyl ester (15) recorded in Q D Q - at 800MHz 67 Integration of the ! H NMR revealed three protons at 8 0.96 ppm, which is a methyl from the aglycon part. Thus, the aglycon in fraction A was proved to be FYOP1— the smallest one with only a straight carbon chain. The anomeric H- l proton resonances were identified due to their doublet coupling and their correlations to anomeric carbons in the HMQC. They are at 8 4.78, 4.67, 4.61, 4.56, 4.31 and 4.21 ppm respectively. Their coupling constants are all about 7-8 Hz, indicating six P glycosidic linkages. Starting from these six H- l resonances, all the monosaccharide protons (except for acetate resonance) were identified via COSY correlations and coupling constant analysis.1 Checking the coupling constants of H-4, four of them (monosaccharides A, B, C and D) have a-a coupling with H-5 (J -10 Hz), which indicates glucose, while the other two (monosaccharides A ' and B') have a-e coupling (J -3.4 Hz), which indicates galactose.2 Table 3.1 gives the assignments of all these protons. 1 3 C NMR resonances were then assigned by H M Q C correlations. And the links between monosaccharides were obtained by three bond H M B C correlations between protons and carbons.3'4 Proton A2, A4, B2 and B-4 were found to link with anomeric carbons A ' l , B l , B ' l and C l , respectively. Anomeric protons B1 ,C1 ,D1 ,A'1 and B ' l were found to link with carbons A4, B4, D2, A'2, B'2, respectively. The proton A l also showed a three-bond H M B C correlation to an aglycon carbon at 8 80.2 ppm, which is the carbinol methine. 68 Table 3.1 HNMR spectrum assignment of the saccharide protons HI d H2 dd H3 dd H4 dd H5 ddd H6 dd H6' dd (8 ppm) (8 ppm) (8 ppm) (8 ppm) (8 ppm) (8 ppm) (8 ppm) A 4.67 3.97 5.50 3.73 3.82 4.76 4.95 B 4.31 3.62 5.29 3.50 3.08 4.10 4.35 C 4.21 5.18 3.89 5.00 3.51 4.11 4.44 (not clear) (not clear) D 4.56 5.15 5.32 5.18 3.05 4.32 3.88 A v 4.78 5.56 5.20 5.54 3.49 4.25 4.27 B' 4.61 5.54 5.11 5.42 3.33 4.50 3.80 Thus, the NMR data shows that glucose A links to the aglycon hydroxyl with a 1 -glycoside bond. 5 Galactose A ' links to glucose A with a 1,2' -glycoside bond and glucose B links to A with a 1,4' -glycoside bond. Galactose B' to glucose B, glucose C to B, and glucose D to C are 1,2', 1,4' and 1,3' links respectively. All of them are p glycoside linkages as mentioned above. The structure of the pachymoside A methyl ester was revealed as 15. 69 R= 1JCOCH3 R' = COCH3 15 3.3 C Labeling for determination of acetylated positions To complete the structure of this natural product, the naturally acetylated positions have to be determined. 1 3 C labeling technology was used to discriminate acetylated and unacetylated monosaccharide hydroxyls in the natural products. The process of peracetylation and isolation were performed on the mixture of pachymosides. The only difference was that the reagent acetic anhydride was replaced by 1 3 C labeled acetic anhydride ( 1 3 C carbonyl). The peracetate methyl ester product was again purified by HPLC, and the same peak as fraction A was obtained for NMR. Because the pure C has much stronger carbon signals in NMR (100 times greater than natural abundance carbons), the H M B C correlations between 1 3 C acetylated carbons and 70 monosaccharides protons are much stronger than natural acetyl groups, as shown in Fig. 3.4. Thus, these strong signals indicate naturally unacetylated hydroxyls. The structure of natural glycolipid pachymoside A from marine sponge P. johnstonia was thus determined (16). 71 # it o 10 - D . •2 < M D ' lX\ 1 1 1 1 I 1 1 1 1 I 1 ' ' ' I ' i ' i I ' ' ' ' I i i i i I i i i i i i i i i i i i i i J H . C H «-< ri rH ^ | _j © H I GO h o ID CN tn in in Fig. 3.4 Enhanced HMBC spectrum of13C labeled pachymoside A methyl ester in CtyD6 at 500 MHz. 72 3.4 Significance of the result and future research Pachymoside is a newly found glycolipid family without any literature reports. Combining with the novel biochemical model of type III secretion inhibition, pachymosides are leads for development into antimicrobial drugs. The structures of pachymosides are related to another glycolipid family, the erylusamines.6 Erylusamines A - E (17 -21) are IL-6 receptor antagonists isolated from the marine sponge Erylus placenta. Erylusamines are pentose tetrasaccharides of long-chain dihydroxyketo-fatty acid amide with N, Af-dimethyl-1, 5-pentanediamine. This alkyl residue shares the common structure of amide, ketone and hydroxyl with our newly found pachymoside aglycons (6, 8 and 9), but has a diamine end and one more hydroxyl. It is known that the bioactivities of most glycolipids come from their ceramide part as well as the stereochemistry of the glycosyl linkage. Thus, the close ceramide structures between pachymosides and erylusamines may suggest similar bioacitivities. Erylusamine A (17) Ri =CH 2 CH 2 CH 3 , R 2 =H Erylusamine B (18) R, =CH 2 CH 2 (CH 3 ) 2 , R 2 =H Erylusamine C (19) Ri =CH 2 CH 2 (CH 3 ) 2 , R 2 =Ac Erylusamine D (20) Ri =CH 2 CH 2 C H 2 C H 3 , R 2 =Ac Erylusamine E (21) Rj =CH 2 CH 2 C H 2 C H 2 C H 3 , R 2 =Ac 73 It is not clear if it is pachymoside A, any other member in this family or all of them that is bioactive in the type III secretion inhibitor bioassay. As novel antimicrobial glycolipids, the isolation and structure elucidation of other members in this family are still the subject of ongoing research. 74 3.5 References 1. P. K. Agrawal, Phytochemitry, 1992, 31, 3307 2. J. Duus, C. H. Gotfredsen and K. bock, Chem. Rev., 2000, 100, 4589 3. A. Bax and M . F. Summers, J. Am. Chem. Soc, 1986, 108, 2093 4. S. Hakomori, J. Biochem., 1964, 55, 205 5. V. Costantino, E. Fattorusso and A. Mangoni, Liebigs Annalen der Chemie, 1995, 1471 6. N. Sata, N. Asai, S. Matsunaga and N. Fusetani, Tetrahedron, 1994, 50, 1105 75 Chapter 4 Experimental 4.1 General procedures Reversed phase and normal phase thin layer chromatography (TLC) was carried out on Whatman MKC18F and Kieselgel 20 F254 plates, respectively. Visualization was detected by viewing under ultraviolet radiation (UV) with wavelength of 254 nm and heating after spraying with a vanillin reagent, or dipping in a eerie sulphate reagent (for aglycons only). Normal phase column chromatography was carried out on Merck silica gel G60 (230-400 mesh). Reversed phase column chromatography was performed using Sep-pak vac C18 cartridges (35 cc). Sephadex LH-20 (bead size 25-100 (j.) was used for size exclusion chromatography. The sizes of the columns were varied according to the amount the sample being fractionated. High performance liquid chromatography (HPLC) separations were performed on one of two possible systems using either a normal phase Alltech Econosil silica column or reversed-phase Whatman Partisil 10 ODS-3 Magnum column. The first system consisted of a Waters 515 HPLC pump controller with a Waters 2487 dual absorbance detector. The second system consisted of a Waters 600E HPLC pump controller equipped with a Perkin-Elmer LC-25 RI detector. For gradient elution, Millennium™ 2000 chromatography software was used in a personal computer interface. The solvents used for extraction, T L C and column chromatography were Fisher reagent grade. HPLC solvents were Fisher HPLC grade which were filtered and degassed prior to 76 use. All other solvents, reagents and standards were reagent or commercial grade and were used without further purification. Except for the peracetyl pachymoside A, which was performed on an 800 MHz NMR spectrometer, all the *H and 2D NMR spectra were collected on either a Bruker AMX-500 (500 MHz) or a Bruker AV-400 (400 MHz) spectrometer equipped with a 5 mm probe. 1 3 C NMR spectra were recorded on a Bruker AM-400 (100.5 MHz) spectrometer equipped with a 5 mm probe. NMR spectra were recorded using deuterated chloroform (CDCI3), deuterated methanol (CD 3OD), hexadeutereobenzene (C6D6) and hexadeuterio-dimethyl sulfoxide (DMSO-rfg). Chemical shifts (8) are given in parts per million from tetramethylsilane (8 0 ppm) and spectral data was calibrated to the deuterated solvents used (CDCI3: 8 7.24 'H NMR, 8 77.0 1 3 C NMR; CD 3 OD: 8 3.30 *H NMR, 8 49.5 1 3 C NMR; C 6 D 6 : 8 7.15 'H NMR, 8 128.0 , 3 C NMR; DMSO-cfo 8 2.49 *H NMR, 8 39.5 1 3 C NMR). Low and high resolution electron impact (EI) mass spectra were recorded on a Kratos MS 50 spectrometer at 70 eV and 180 or 250°C. FABMS data were collected on a Kratos Concept IIHQ hybrid mass spectrometer with cesium ion secondary ionization and a magnetic sector mass analyzer. The mass spectrometric analyses were performed by the UBC chemistry Mass Spectrometry Laboratory. Low resolution electron spray ionization (ESI) mass spectra were recorded on a Bruker-HP EsquireLC_00085 mass spectrometer with an iontrap detector at 250°C. 77 Gas chromatography (GC) was performed on Hewlett-Packard 5890 series II plus gas chromatograph, equipped with a flame ionization detector and a chiral select 1000 (3-Dex-390 column. The chemicals used for synthesis were from Aldrich or Sigma. Anhydrous chemical were used directly without further drying. All reactions were protected by Argon. 4.2 Isolation Pachymosides A concentrated greenish methanol extract of P. johnstonia was partitioned between 300 mL water and 3 x 300 mL EtOAc. The organic soluble fraction (857 mg) was dissolved in 3 mL MeOH and loaded onto a Sephadex LH-20 column and eluted in 100% MeOH. The fractions obtained from this size-exclusion separation were collected and submitted for type III secretion inhibitor bioassay. A single peak of activity was observed in fractions 27-33. Aglycons (6, 8, 9) The diethyl ether soluble fraction (FYO, 25mg) from the methanolysis was loaded on a normal phase silica gel column and eluted with a gradient system of Hexanes/EtOAc (5%EtOAc: 95%Hexanes ->• 100%EtOAc). Eluting with 20% EtOAc : 80% Hexanes, one major component and one minor component were observed by T L C analysis. The more polar major component was fractionated further by reversed phase HPLC eluting with 2 mL/min of 88% MeOH : 12% H 2 0 (monitored with 206.0 nm detector wavelength). Three 78 components, FYOP1 (8), FYOP2 (6) and FYOP3 (9), were collected at 22, 26 and 35 minutes, respectively. The NMR and MS data of the aglycons are shown in Tab. 2.1 and Tab. 2.2 Formation of oxime (7) The crude oxime product from aglycons FYOP2 (6) was partitioned between Et20 and H2O. The organic fraction was dissolved in 0.3 mL Hexanes and loaded onto a silica gel chromatography column. The sample was eluted with a gradient system of EtOAc/Hexanes (20 % EtOAc : 80 % Hexanes -> 80 % EtOAc : 20 % Hexanes). T L C analysis in 20% EtOAc : 80% Hexanes guided to the collection of the purified oxime (7) product from fractions 5-7. The MS data of the oxime is shown in Tab.2.3 Acetylated glucose and galactose glycosides The water soluble fraction (FYA, 30mg) from the methanolysis was further acetylated. The product (20 mg) was loaded onto a normal phase silica gel chromatography column and eluted with a gradient system of Hexanes : EtOAc ( 90% hexanes : 10% EtOAc to 100% EtOAc). Silica gel T L C analysis using visualization with a vanillin spray guided the pooling of chromatography fractions. Fractions 31-38 were pooled for HPLC isolation. Isocratic normal phase HPLC was used, eluting with 31% EtOAc : 69% Hexanes and monitored with a RI detector. Acetylated methyl a-glucopyranoside/galactopyranoside and methyl P-glucopyranoside/galactopyranoside were collected at 67 and 80 minutes respectively. \ 79 Acetylated pachymoside A methyl ester (15) Acetylated pachymosides were partitioned between H2O and Et.20. The organic soluble portion was injected into reversed phase HPLC and eluted with isocratic 64% 1-PrOH : 36%) H2O (monitored by 202.0 nm detector wavelength). 4 mg of acetylated pachymoside A was collected at 65 minutes. Fraction B, C, D, E, F and G were also collected for future study. The NMR data is shown in Tab.3.1 4.3 Synthesis Methanolysis of the pachymosides To a room temparature (20 °C), stirred solution of CH3COCI (0.20 mL, 1.2 mmol) in anhydrous methanol (10 mL), was added pachymosides (fraction F from extract of P. johnstonia, 100 mg, 0.049 mmol). After the solution had been stirred with reflux for 2 hours, the reaction was worked up by putting on a rotavap to remove the MeOH. The residue was partitioned between H2O and Et_0; the aglycon (FYO, 25 mg, 0.045 mmol) went into the organic phase and the methyl glycosides (FYA, 30mg. 0.15 mmol) went into the aqueous phase. Oxime formation of aglycon To a room temperature (20 °C ), stirred solution of hydroxylamine hydrochloride (NH2OH • HCI, 1 mg, 0.014 mmol) and sodium acetate (CH 3 C0 2 Na, lmg 0.012 mmol) in 10 mL MeOH, was added the aglycon FYOP2 (6) (3 mg, 0.005 mmol). After the solution had been stirred at room temperature for 1 hour, After the reaction was worked up by putting on a rotavap to remove the MeOH, the crude product was partitioned between H2O and 80 Et20 and the organic extract was purified by column chromatography to obtain 1.9 mg of oxime (7) (0.0034 mmol). The yield of the reaction was 68%. Acetylation of mono glycopyranosides To a room temperature (20 °C), stirred solution of 4-dimethylamino pyridine (DMAP, ~1 mg), and acetic anhydride (3 mL, 30 mmol) in anhydrous pyridine (9 mL), was added the mixture of glycopyranosides (FYA, 20 mg, 0.11 mmol) from methanolysis. After the solution had been stirred at room temperature of 16 hours, pyridine and acetic anhydride was removed under reduced pressure. The crude product was partitioned between H2O and Et20 and the organic extract was purified by column chromatography to obtain methyl 2,3,4,6-tetra-O-acetyl-glucopyranoside/galactopyranoside (mixtures of a and P anomers, 33 mg, 0.09 mmol). The yield of the reaction was 81%. Acetylation of standard monosaccharides To a room temperature (20 °C), stirred solution of CH3COCI (0.20 mL, 1.2 mmol) in anhydrous methanol (10 mL), was added commercial standard L-glucose or D-galactose (both 20 mg, 0.11 mmol). After the solution had been stirred with reflux for 2 hours, the reaction was worked up by putting on a rotavap to remove the MeOH. The residue was partitioned between H 2 0 and Et 2 0. To a room temperature (20 °C), stirred solution of 4-dimethylamino pyridine (DMAP, ~1 mg), and acetic anhydride (3 mL, 30 mmol) in anhydrous pyridine (9 mL), was added the aqueous fraction from the next step. After the solution had been stirred at room temperature of 16 hours, pyridine and acetic anhydride was removed under reduced 8 1 pressure. The crude product was partitioned between H2O and Et20 and the organic extract was purified by column chromatography to obtain standard methyl 2,3,4,6-tetra-<9-acetyl-glucopyranoside/galactopyranoside (mixtures of a and P anomers). The total yields of the reaction were 72% to glucopyranoside and 55% to galactopyranoside. Hydrolysis of pachymosides To a room temperature (20 °C), stirred solution of water with 3 -5 drops of concentrated hydrochloride (HC1), was added pachymosides extract (40mg, 0.020 mmol). After the solution had been stirred with reflux for 1 hour, the reaction was worked up by putting on a rotavap to remove the water. The residue was partitioned between H2O and Et20, and the aqueous fraction is monosaccharides part (glycose and galactose) Reduction and acetylation of monosaccharides To a room temperature (20 °C), stirred solution of sodium boron hydride (20 mg, 0.53 mmol) in MeOH (20 mL), was added 40 mg monosaccharide (glucose or galactose, 0.22 mmol). After the solution had been stirred at room temperature for 1 hour, the reaction was worked up by putting on a rotavap to remove the MeOH. And the aditol products (glucitol or galacitol) were obtained. To a room temperature (20 °C), stirred solution of 4-dimethylamino pyridine (DMAP, ~1 mg), and acetic anhydride (3 mL, 30 mmol) in anhydrous pyridine (9 mL), was added the aditols (glucitol or galacitol) from the next step. After the solution had been stirred at room temperature of 16 hours, pyridine and acetic anhydride was removed under reduced pressure. The crude product was partitioned between H2O and Et20 and the organic extract 82 was purified by column chromatography to obtain the aditol peracetates. For D-glucitol and L-glucitol peracetates (12 and 13), ! H NMR (solvent: CDC13): HI 8 3.95 (J=12.1, 6.1 Hz), HI' 8 4.28 (J=12.1, 4.0 Hz), H2 8 5.15 (J= 4.1 Hz), H3 and H4 8 5.35 (J= 5.2 Hz), H5 8 4.93 (J= 4.1 Hz), H6 8 4.08 (J= 12.3, 5.3 Hz), H6' 8 4.16 (12.4, 3.6 Hz). For D-galacitol peracetate (14), lH NMR (solvent: CDC13): HI and H6, 8 4.25 (J= 11.6, 4.8Hz), H2 and H5, 8 5.26 (12.1Hz), H 3 and H4, 8 5.32 (multiplet). Peracetylation of pachymoside The procedure is totally the same as the acetylation of mono glycopyranosides mentioned above. 

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