Fluorogenic phospholipid and metabolically stabilized inositol analogues as signal transduction probes

PLases (phospholipases) participate in a wide variety of cellular signals for healthy and diseased processes. PLA (phospholipase A), PLC (phospholipase C), and PLD (phospholipase D) enzymes cleave PLs (phospholipids) to give distinct, bioactive products. Fluorogenic substrate analogues offer the possibility of detecting PLase activity in itro and in living cells and tissues in real-time and with high sensitivity. Here, fluorogenic analogues of the PLs PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), and PS (phosphatidylserine) were synthesized as PLA substrates for determining the influence of PL head group modifications on cell signaling in vitro and in cells. The initial synthetic route to a fluorogenic analogue of PA used exclusively chemical transformations. Later, an enzyme-assisted synthetic route was employed, which included remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA2 and transphosphatidylation with a particular PLD. This enzyme-assisted synthesis allowed the PA analogue to be synthesized more efficiently than by purely chemical methods and also provided ready access to a variety of different head groups. The resulting fluorogenic Dabcyl- and BODIPY-containing PL analogues--DBPA, DBPC, DBPE, DBPG, and DBPS---were used to determine PLA2 kinetics in mixed micelle assays. DBPC was then used to determine the Xi(50) value of a common PLA2 inhibitor. Finally, the head group selectivity of a series of commercially available PLA2 enzymes was established using the DBPL substrates. For assaying PLD activity in vitro and in cells, a series of fluorogenic analogues of PC and LPC (lysophosphatidylcholine), including DDPB and lysoDDPB, were synthesized, again by an enzyme-assisted strategy. The analogues were evaluated as substrates for PLC, PLD, and lysoPLD (lysophospholipase D). DDPB was cleaved by PC-PLC and by bacterial, plant, and human PLD and represents the first direct fluorogenic substrate for mammalian-type enzymes. Inositol polyphosphates, products of PL hydrolysis by PLC, also mediate cell signaling. In the concluding chapter, metabolically stabilized inositol polyphosphate analogues are proposed that are designed to be long-lived agonists/antagonists at intracellular inositol polyphosphate binding sites. Synthetic studies toward these analogues are detailed, culminating in a new synthetic route to the stabilized inositol analogue inositol(1,4,5)tris(methylphosphonate).

FLUOROGENIC PHOSPHOLIPID AND MET ABOLICALL Y STABILIZED INOSITOL ANALOGUES AS SIGNAL TRANSDUCTION PROBES by Tyler Max Rose A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of .1 Doctor of Philosophy Department of Medicinal Chemistry The University of Utah August 2006 Copyright © Tyler Max Rose 2006 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Tyler Max Rose This dissertation has been read by each member of the following supervisory conunittee and by majority vote has been found to be satisfactory. J' /2 ~' .{~j~ -.. ~ .. - ... _-- ... ~~- . Jerald C. Hinshaw 11~A ----.~-~ Matthew K. Topham THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Tyler Max Rose in its final fonn and have found that (1) its fonnat, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is reM~l;!b~Sion t~The Grad~te:ChOi~~~ Date Gle~p D. restwich Chair: Supervisory Committee Approved for the Major Department Approved for the Graduate Council ~.....!J f. cO· ~-- · David S. Chapman Dean of The Graduate School ABSTRACT PLases (phospholipases) participate in a wide variety of cellular signals for healthy and diseased processes. PLA (phospholipase A), PLC (phospholipase C), and PLD (phospholipase D) enzymes cleave PLs (phospholipids) to give distinct, bioactive products. Fluorogenic substrate analogues offer the possibility of detecting PLase activity in vitro and in living cells and tissues in real-time and with high sensitivity. Here, fluorogenic analogues of the PLs PA (phosphatidic acid), PC (phosphatidy1choline), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), and PS (phosphatidylserine) were synthesized as PLA substrates for determining the influence of PL head group modifications on cell signaling in vitro and in cells. The initial synthetic route to a fluorogenic analogue of PA used exclusively chemical transformations. Later, an enzyme-assisted synthetic route was employed, which included remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA2 and transphosphatidylation with a particular PLD. This enzyme-assisted synthesis allowed the PA analogue to be synthesized more efficiently than by purely chemical methods and also provided ready access to a variety of different head groups. The resulting fluorogenic Dabcyl- and BODIPY -containing PL analogues -DBPA, DBPC, DBPE, DBPG, and DBPS- were used to determine PLA2 kinetics in mixed micelle assays. DBPC was then used to determine the Xj(50) value of a common PLA2 inhibitor. Finally, the head group selectivity of a series of commercially available PLA2 enzymes was established using the DBPL substrates. For assaying PLD activity in vitro and in cells, a series of fluorogenic analogues of PC and LPC (lysophosphatidylcholine), including DDPB and lysoDDPB, were synthesized, again by an enzyme-assisted strategy. The analogues were evaluated as substrates for PLC, PLD, and lysoPLD (lysophospholipase D). DDPB was cleaved by PC-PLC and by bacterial, plant, and human PLD and represents the first direct fluorogenic substrate for mammalian-type enzymes. Inositol polyphosphates, products of PL hydrolysis by PLC, also mediate cell signaling. In the concluding chapter, metabolically stabilized inositol polyphosphate analogues are proposed that are designed to be long-lived agonists/antagonists at intracellular inositol polyphosphate binding sites. Synthetic studies toward these analogues are detailed, culminating in a new synthetic route to the stabilized inositol analogue inositol(1,4,5)tris(methylphosphonate). v TABLE OF CONTENTS ABSTRACT ............................................................................................................... .iv LIST OF FIGURES .................................................................................................. viii LIST OF SCHEMES ..................................................................................................... x LIST OF ABBREVIATIONS ...................................................................................... xi ACKNOWLEDGMENTS ......................................................................................... xvii PART I. FLUOROGENIC PHOSPHOLIPID ANALOGUES ...................................... 1 CHAPTER 1. INTRODUCTION .................................................................................................... 2 Classification of Phospholipases ............................................................................... 4 Methods for Assaying Phospholipases .................................................................... 11 PLase Selectivity .................................................................................................... 20 Summary and Research Overview .......................................................................... 25 References .............................................................................................................. 26 2. A FLUOROGENIC PA ANALOGUE: rac-DBPA ................................................ 4O Introduction ............................................................................................................ 40 Results and Discussion ........................................................................................... 42 Experimental Details .............................................................................................. 51 References .............................................................................................................. 56 3. FLUOROGENIC ANALOGUES OF PA, PC, PE, PG, AND PS AS HEAD GROUP-SELECTIVE REPORTERS OF PLA ACTIVITy ........................ 59 Introduction ............................................................................................................ 59 Results and Discussion ........................................................................................... 61 Experimental Details .............................................................................................. 83 References .............................................................................................................. 99 4. DDPB AND LYSO-DDPB: FLUOROGENIC SUBSTRATES FOR PLD AND PC-PLC .............................................................................................. 1 05 Introduction .......................................................................................................... 105 Results and Discussion ......................................................................................... 108 Experimental Details ............................................................................................ 123 References ............................................................................................................ 138 PART II. INOSITOL(1,4,5)TRIPHOSPHATE ANALOGUES ................................ 143 5. APPROACHES TOWARD THE SYNTHESIS OF 5- AND 4,5-MET ABOLICALL Y STABILIZED INOSITOL( 1 ,4,5)TRIPHOSPHA TE ANALOGUES ..................................................................................................... 144 Introd ucti on .......................................................................................................... 144 Results and Discussion ......................................................................................... 145 Experimental Details ............................................................................................ 157 References ............................................................................................................ 172 Appendices A. TABLE OF COLLABORATING RESEARCH GROUPS .................................... 174 B. PUBLICATIONS LIST ........................................................................................ 181 vii LIST OF FIGURES Figure 1.1. Sites of PL cleavage catalyzed by each category of PLase .................................... 3 1.2. Methods for extracting the products of PLase reactions ...................................... 15 1.3. A dithioester PL analogue suitable for assaying PLA1 or PLA2 ........................... 17 1.4. The SIBLINKS substrate (a) and p-nitrophenylphosphorylcholine (b) ............... 17 1.5. An example of a coupled assay ........................................................................... 19 1.6. Structures offluorogenic substrates for PLA (a-c) or PLC (d) ............................ 21 1.7. Structures of the fluorogenic lysoPLD substrates FS-3 (a) and CPF4 (b) ............ 22 1.8. LUMI-PI, a chemiluminescent substrate for PI-PLC ......................................... 22 2.1. Fluorogenic assays of rac-DBPA with snake venom PLA2 •••••••••••••••••••••••••••••••• 45 2.2. Concentration-dependent, raw fluorescence increases upon treatment of rac-DBP A in PC vesicles with cobra venom PLA2 ••••••••••••••••••••••••• 46 2.3. The Inner Filter Effect ........................................................................................ 48 2.4. NIH3T3 cells that underwent a transfection protocol with mouse mPA-PLAIa plasmid were treated with water-soluble rac-DBP A .............................. 50 3.1. Concentration dependent hydrolysis of DBPC and DBPA by PLA2 ••••••••••••••••••• 70 3.2. Inhibition of bee PLA2 by thioether amide-PC ................................................... 72 3.3. Head group selectivities of commercially-available PLA2 .................................. 74 3.4. Fluorescence of DBPS incubated with either bee or cobra venom PLA2 ............. 77 3.S. Fluorescence of DBPC in a cell-based assay of stimulation of an adult mouse superior cervical ganglion neuron expressing cPLA2 but deficient in sPLA2 ............................................................................................................. 82 3.6. Cell-based PLA assays of the peritoneal mesothelial cell lines LP9 and LP3, with (+) or without (-) overnight FBS incubation (a-d) .............................. 84 4.1. Fluorescence evolution during 3 min incubation of either dimethylated DDPB (a)or monomethylated compound 12 (b) ............................................... 113 4.2. Fluorescence evolution during 3 min incubation of a) DDPB and b) Cl2-­DDPB in Triton mixed micelles with 1 U PLD or PLA2 from various sources ............................................................................................................. lIS 4.3. Fluorescence evolution during 3 min incubation of a) lysoDDPB and b) Cl2--1ysoDDPB in Triton nlixed micelles with 1 U PLD from various sources ............................................................................................................. 116 4.4. Fluorescence evolution during a 60 min incubation of a) DDPB, and b) lysoDDPB in Triton mixed micelles with 1 U PLD or PLA2 enzymes .............. 117 4.S. Fluorescence evolution during a 3 min incubation of DDPB or lysoDDPB in Triton mixed micelles with 1 U PC-PLC or PI-PLC from B. cereus or C. perfringens .................................................................................................. 118 4.6. Fluorescence evolution during a 3 min incubation of DDPB (circles), lysoDDPB (triangles), Cl2--DDPB (squares), or Cl2--1ysoDDPB (diamonds) in Triton mixed micelles with 1 U B. cereus PC-PLC .................... 120 4.7. Changes in fluorescence of FS-3, DDPB, lysoDDPB, Cl2--DDPB, and Cl2--1ysoDDPB in Triton mixed micelles upon incubation with FBS at 37°C. Inset: Increase in UV absorption upon incubation of pNP-TMP with FBS at RT ................................................................................................ 121 4.8. Fluorescence increases from DDPB in PE/PtdIns(4,S)P2Iipid vesicles incubated for 20 min at 37°C with 200 ng to 1 }lg GST -tagged, recombinant human PLDlb .............................................................................. 124 5.1. Proposed 5-stabilized Ins(l,4,S)P3 analogues ................................................... 146 ix LIST OF SCHEMES Scheme ................................................................................................................... Page 2.1. A schematic diagram illustrating the process of fluorescence dequenching ......... 41 2.2. Chemical synthesis of rae-DBP A ........... ........................................................... 43 3.1. Enzyme-assisted synthetic route to DBPC ......................................................... 62 3.2. Enzyme-assisted synthetic route to DBPA ......................................................... 64 3.3. Enzymatic reactions leading to DBPE, DBPG, and DBPS precursors ................ 65 3.4. Final synthetic steps to DBPG from 5 ................................................................ 67 3.5. Completion of DBPE from 6 .............................................................................. 68 3.6. Completion of DBPS from 7 .............................................................................. 69 4.1. Fluorescence dequenching of a model fluorogenic PL analogue by PLD .......... 107 4.2. Synthesis of di-dabcyl intermediate 5 .............................................................. 109 4.3. Synthesis of DDPB, lysoDDPB, and monomethylated product 12 .................... 110 4.4. Synthesis of C12-DDPB and C12-lysoDDPB .................. ............................... 112 5.1. First attempted synthesis of the 5-thiono-Ins(1,4,5)P3 analogue ....................... 147 5.2. Second attempted synthesis of the 5-thiono-Ins(1,4,5)P3 analogue ................... 149 5.3. First attempted synthesis of the 4,5-bisthiono-Ins(1,4,5)P3 analogue ............... 151 5.4. Attempted synthesis of the 4,5-bis(methylphosphonyl)Ins(1,4,5)P3 analogue ........................................................................................................... 152 5.5. Second attempted synthesis of the 4,5-bisthiono-Ins( 1 ,4,5)P3 analogue ........... 154 5.6. Synthesis of Ins(1,4,5)tris(methylphosphonate) ................................................ 155 AcCN ATX B. cereus BBPC Bn boc BSTFA Bz C. perfringens CDI CMC CNEt cPLA2 DAG DBPA DBPC DBPE DBPG LIST OF ABBREVIATIONS degrees celsius acetonitrile autotaxin Bacillus cereus bis-BODIPY -phosphatidylcholine benzyl tert-butyl carbamate N,O-bis-(Trimethylsilyl)trifluoroacetimide benzoyl Clostridium perfringens carbonyldiimidazole critical micelle concentration cyanoethyl cytosolic phospholipase A2 diacyl gl ycerol dabcyl-BODIPY phosphatidic acid dabcyl-BODIPY phosphatidylcholine dabcyl-BODIPY phosphatidylethanolamine dabcyl-BODIPY phosphatidylglycerol DBPL DBPS DBU DCC DDPB DIPEA DMAP DMF DMPM DMSO EDCI ESI EtOAc EtOH FBS FmocCI FRET GPI-PLD GPC GPCR GST h HBSS dabcyl-BODIPY phospholipid dabcyl-BODIPY phosphatidylserine Diaza( 1,3)bicyclo[S.4.0]undecane dicyclohexylcarbodiimide dabcyl-dabcyl-phosphatidyIBODIPY diisopropylethylamine N,N-dimethylaminopyridine N,N-dimethylformamide dimyristoylphosphatidylmethanol dimethylsulfoxide N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride electrospray ionization ethyl acetate ethyl alcohol fetal bovine serum 9-Fluorenyl methyl chloroformate fl uorescence resonance energy transfer glycosylated phosphatidylinositol-selective PLD glycerophosphorylcholine G-protein coupled receptor gl utathi one-S-transferase hour, hours Hank's Balanced Salt Solution xii HEPES HRMS Ins(1,4,5)P3 iPLA2 IPP5P iPr LBPA LCAT LPA LPC LPE LPG LPL LPS LRMS lysoDDPB lysoPLA lysoPLase lysoPLC lysoPLD MALDI mCPBA Me 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid high resolution mass spectroscopy inositol( 1 ,4,5)triphosphate intracell ular, Ca2 + -independent PLA2 inositol polyphosphate 5-phosphatase isopropyl lysobisphosphatidic acid lecithin-cholesterol acyl transferase lysophosphatidic acid lysophosphatidylcholine lysophosphatidylethanolamine lysophosphatidylglycerol lysophospholipid I ysophosphatidy lserine low resolution mass spectroscopy I yso dabcyl-dabcy I-phosphatidylBO D IPY lysophospholipase A I ysophospholi pase lysophospholipase C lysophospholipase D matrix-assisted laser desorption ionization meta-chloroperoxybenzoic acid methyl xiii MeOH MHz min MOMCI mPA-PLAt N. mossambica N. naja NaOMe NHS NMR NPP Oxo-M PA PAF-AH PA-PLAI PC PC-PLC PE PG PI-PLC PIPP piv PL n1ethanol megahertz minute, minutes methoxymethylchloride membrane-associated phosphatidic acid selective phospholipase Al Naja mossambica mossambica Naja naja naja sodium methoxide N-hydroxysuccinimide nuclear magnetic resonance nucleotide pyrophosphate/phosphodiesterase oxotremorine-M phosphatidic acid platelet activating factor acetyl hydrolase phosphatidic acid selective phospholipase Al phosphatidylcholine phosphatidylcholine-selective phospholipase C phosphatidylethanolamine phosphatidylglycerol phosphatidylinositol-selective phospholipase C proline-rich inositol polyphosphate 5-phosphatase pivaloyl phospholipid xiv PLA PLA1 PLA2 PLase PLB PLC PLD pNP-TMP PPTS PS PS-PLA1 Ptdlns(4,5)P2 p-TsOH pyr RFU RT S. cerevisiae S. chromofuscus scPLD SE s phospholi pase A phospholipase Al phospholipase A2 phospholipase phospholipase B phospholipase C phospholipase D para-nitrophenylthymidine monophosphate pyridinium para-toluene sulfonate phosphatidylserine phosphatidylserine-selective phospholipase Al phosphatidylinositol( 4,5)bisphosphate para-toluene sulfonic acid pyridine relative fluorescence units room temperature Streptomyces cerevisiae Streptomyces chromofuscus Streptomyces chromofuscus PLD succinimidyl ester second, seconds secreted phospholipase A2 temperature xv TBAF TBDPSCI t-BuOH TEA TEAB TEPC TFA THF TIPDSCl2 TLC TMSBr TNF TPSNT Tris u UV xyl tetrabuty lammoni urn fluoride tert-butylchlorodiphenylsilane tert-butanol triethylamine triethylammonium bicarbonate thioether amide phosphatidylcholine trifluoroacetic acid tetrahydrofuran 1,3-dichloro-l,1,3,3-tetraisopropyldisiloxane thin-layer chromatography trimethylsilylbromide tumor necrosis factor 2,4,6-triisopropylbenzenesulphonyl-3-nitro-l,2,4-triazole tris(hydroxymethyl)aminomethane units of enzyme activity ultraviolet xylylene xvi ACKNOWLEDGMENTS Many thanks go to my graduate advisor, Dr. Glenn D. Prestwich, for providing a supportive and stimulating environment in which to learn medicinal chemistry. I am also grateful to my graduate committee members, namely, Drs. Donald K. Blumenthal, Jerald C. Hinshaw, Jeanette C. Roberts, Eric W. Schmidt, and Matthew K. Topham, each of whom has provided valued advice and instruction over the course of my graduate studies. I gratefully acknowledge our many helpful and talented collaborators, who are listed by name in Appendix A. Special thanks go to Dr. Junken Aoki for providing nlouse mPA-PLAt plasmid~ to Drs. Rubing Zhao, Yen Bai, Michael J. Sanderson and Ann R. Rittenhouse for the cell images in Figure 3.5, along with the associated experimental details~ and, to Dr. Matt Hodgkin for providing Figure 4.8. I would also like to acknowledge Echelon Biosciences, Inc., an Aeterna Zentaris company, for contributing FS-3 as a gift, and for sharing instrument time and the time and expertise of many of its employees, including Drs. Li Feng, Colin G. Ferguson, PaulO. Nielsen, and Piotr W. Rzepecki. Thanks also to Ms. Marie Dippolito and to the many great colleagues who made lab life enjoyable and who frequently provided helpful advice and materials. I further wish to acknowledge the Center for Cell Signaling, a member of the Utah Centers of Excellence Program (1997-2002) and the NIH (Grants HL070231 and NS29632, and a Predoctoral Traineeship in Biological Chemistry (2002-03)) for financial support of this research. xviii PART I FLUOROGENIC PHOSPHOLIPID ANALOGUES CHAPTER 1 INTRODUCTION The PLase (phospholipase) megafamily of enzymes can be divided into four broad categories, designated PLA, PLB, PLC, and PLD. Each category of enzyme is responsible for catalyzing the hydrolysis of PLs, each in a separate location on the molecule. As summarized in Figure 1.1, PLA enzymes catalyze cleavage of either the sn-l (PLA1) or sn-2 (PLA2) acyl chains; PLB of both sn-l and sn-2 chains; PLC of the phosphodiester head group on the glyceryl side; and, PLD of the phosphodiester head group on the alcohol side. In each case, the products of PLase-catalyzed PL hydrolysis are important signal mediators in biological processes. For example, arachidonic acid liberated by PLA enzymes is a biosynthetic precursor for pain- and inflammation-inducing eicosanoids.1 LPLs (Lysophospholipids), the second product of PLA action, have a spectrum of biological activity, dependent on head group and acyl chain composition. The well­characterized LPA (lysophosphatidic acid), in particular, is known to promote cell growth and mitogenesis, among other effects.2 DAG (diacylglycerol), a PLC product, can activate downstream signaling enzymes like protein kinase C, a Ser/Thr kinase central to many signaling pathways, and which can be tumorigenic when overactivated.3 Similarly, PA resulting from PLD cleavage can mediate biological responses by activating proteins H phosphatide acid ~ "'~/ "'\ "'" + " phosp hat d ylcholi ne ~~H3 R3= ph asp hat d yleth ana Ia mine X'y-oH OH ph asp hat d ylgl ycerol OH HXXo,_." OH I _ OH OH phosp hat id yl in ositol ph osp hat id ylse rin e sn-l __ --".., o ' ••• 11 R O~ "f) PLA2 ;&-R~ / C!. .. b -Ra PLD sn-2 -----:.- PLC Figure 1.1. Sites of PL cleavage catalyzed by each category of PLase 3 through membrane recruitment, by modulating vesicle trafficking, or by serving as a precursor for the biosynthesis ofLPA or DAG.4 4 The structural features and subcellular localization of the PLase products playa large role in determining their ultimate biological effects. Therefore, discovering the substrate selectivities and spatiotemporal activation profiles of the parent PLase enzymes becomes essential to understanding what signaling processes the PLases are involved in and what interventions are most effective in nullifying their involvement in diseased signaling. The development of improved methods for probing these parameters is thus fundamental for studies of the PLase enzymes' contribution to health and disease, and is the purpose of the work presented here. This chapter introduces the currently recognized members of the PLase family, as defined by what is known about their structures, mechanisms, and biological origins. Commonly used methods for detecting PLase activity, and their advantages and disadvantages, will also be described, along with a discussion of features unique to PLase catalysis, including interfacial kinetic and substrate specificity considerations. Classification of Phospholipases Excluding traditional, nonsignaling, digestive lipases that have PLAl activity, there are of two types of PLAl' Type I and Type II.2,5 Both types contain amino acid sequence motifs common to alllipases, but Type I PLA1 enzymes share the most similarity to the broader lipase family.5 Type I PLA1 enzymes are extracellular (secreted) enzymes that may also be membrane-associated. They maintain a Ser-His-Asp catalytic triad and have 6 5 conserved cysteine residues.5 Disulfide bonds between the cysteine residues help stabilize the enzymes' tertiary structures and support a lid-like domain, a characteristic of most lipases that is thought to help govern substrate recognition. The lid of a Type I PLAl is shorter than that of a conventional lipase, which could explain the inability of PLAl to hydrolyze triacylglycerols.5 Type I PLAl enzymes include PS-PLA1 (phosphatidylserine-selective PLAl)6 and two kinds of membrane-associated PA(phosphatidic acid)-selective PLA1 enzymes (mPA-PLAl U and (3).7 PS-PLAl was initially isolated from activated rat platelets8 and later detected in mice and humans.9 Human platelets do not contain PS-PLAl, but it is found in kidney, heart, liver, lung, and testicular tissues.9 Likewise, mPA-PLAlu is widely distributed in human tissues, including testis, ovary, colon, kidney, lung, brain, and heart. lO mPA-PLA1(3 is found in testis and was shown to localize to the junction between the head and tail of sperm.7 Type II PLA1 enzymes are found intracellularly. They share little homology with other known lipases, lacking extensive disulfide linkages and thus the characteristic lipase lid domain.5 The catalytic residues of a Type II PLAl lack a conserved motif. Enzymes of this type include PA-selective PLAl (pA-PLAl),ll-13 KIAA0725,t4 and p125. 15,16 Type II PA-PLAl enzymes are found in bovine brain and testis. 11 KIAA0725 and p125 are found ubiquitously in human tissues. 14 PLAz enzymes are classified according to nucleotide sequence into 12 groups, many of which are subdivided further into lettered subgroups. 17, 18 A less formal classification method that is based on the biochemical properties of a PLAz is also used. In this system there are four broad categories: sPLAz (secreted PLAz), cPLAz (cytosolic, Ca2+-dependent), iPLA2 (intracellular, Ca2+-independent), and PAF-AH (platelet activating factor acetyl hydrolase). 6 sPLA2 isoforms constitute Groups I, II, III, V, IX, X, XI, and XII. These enzymes require Ca2+ and have no preference for arachidonic acid. They are low molecular weight (10 - 20 kDa) and have 5 - 8 disulfide linkages that stabilize their tertiary structures. 17, 18 PLA2 enzymes from digestive secretions and from reptile and insect venoms are from the sPLA2 category and were among the first PLA2 enzymes to be characterized. In the sPLA2 active site, histidine is responsible for activation and orientation of a cleaving water molecule, resulting in a functional pH range of 7 - 9. A proximal Asp residue ligates Ca2+ to form an oxyanion hole in the active site that helps stabilize the catalytic intermediate. Therefore, Ca2+ in mM concentrations is required for sPLA2 activation. I9 - 21 cPLA2 enzymes belong to Group IV, preferentially cleave arachidonate chains, and have higher (60+ kDa) molecular weights. 17, 18 The first cPLA2 was sequenced in 199122 and was shown to have a mechanism involving a catalytic serine.23 Later it was shown that the Group IV -A cPLA2 catalyzes acyl chain hydrolysis via a novel Ser-Asp catalytic dyad, in which a serine-acyl intermediate is formed with Asp acting as a general base.24 ,25 Both Group IV -A and -B cPLA2 enzynles bear C2 domains through which membrane recruitment can occur.26 ,27 Translocation correlates with binding of two Ca2+ by the C2 domain, though there is evidence that membrane binding and activation can also occur at low Ca2 + concentrations if levels of Ptdlns( 4,5)P2 (phosphatidylinositol( 4,5)bisphosphate) are increased.28 iPLA2 enzymes are from Group VI, have higher molecular weights (80 - 100 kDa), and no arachidonate preference. 17, 18 PAF-AHs comprise Groups VII and VIII, and 7 are enzymes of lower molecular weight (25-50 kDa) that preferentially cleave short chains (up to nine carbons) from the sn-2 position of PAF or other pLS.29 iPLA2 and PAF-AH enzymes, which exhibit no Ca2+ dependence, both contain lipase consensus motifs (Gly-X-Ser-X-Gly) in which the serine is catalytically active.30 Unlike other PLases, the Group VII-A PAF-AH only hydrolyzes monomeric substrates, as opposed to PL substrates that are aggregated into vesicles or micelles.31 PLBs have both PLase and lysoPLase (lysophospholipase) activity, resulting in cleavage of both acyl chains from a PL. Four unrelated gene families encoding PLB have been reported: bacterial,32 fungal,33 mammalian,34-36 and amebic?7 The bacterial and fungal PLB gene products encode proteins of ~ 600 amino acids. The enzymes are secreted, and probably playa role in infectivity and pathogenesis,33 though PLB gene SPO], from Streptomyces cerevisiae, appears to be required for meiosis.38 Mammalian PLB is a 1400 amino acid ectoenzyme associated with digestion in intestinal brush­borders, 35,39 but interestingly is also expressed in epidermis40 and in the epididymis, where the PLB product GPC (glycerophosphorylcholine) appears to be essential for sperm production.41 The most recently identified gene family was first identified in Dictyostelium, from which a 574 amino acid secreted PLB was isolated.37 A BLAST search revealed closely related genes in mammals, flies, and worms.37 The PLB enzymes bear the lipase consensus motif and appear to have a Ser-His­Asp catalytic triad that is responsible for activity,36.42 with two exceptions: 1) The sequence of yeast PLB bears similarities to Group IV -A cPLA2 that indicate both may operate by way of a Ser-Asp dyad mechanism.37 2) The Dictyostelium PLB lacks a G­X- S-X-G consensus motif, though a potential active site serine has been proposed.37 8 There are 11 mammalian PI-PLCs (phosphoinositide-selective PLCs), divided into four subgroups based on sequence alignment: PLC (3, y, (), and £.17 These enzymes cleave Ptdlns(4,5)P2 to give Ins(I,4,5)P3 (inositol(I,4,5)triphosphate) and DAG. All PI­PLC sequences contain X and Y domains that combine to form a catalytic site, and variously contain domains that regulate recruitment of the enzymes to membranes (i.e., PH and C2) and that govern protein-protein interactions (i.e., SH2 and RA).17 The PI­PLC catalytic mechanism43 proceeds through an intramolecular pentacoordinate phosphate intermediate formed by deprotonation of the inositol 2-hydroxyl by a general base followed by nucleophilic attack on the phosphorus. DAG is then released, followed by cleavage of the cyclic intermediate by an activated water molecule and release of Ins(I,4,5)P3 product. Distributed throughout skeletal muscle, heart, lung, liver, brain, blood, and glandular tissues, PI-PLCs have been studied in knockout mice, where roles for the enzymes in brain, visual, and immune function were indicated.44 PC-PLCs (phosphatidylcholine-selective PLCs), which catalyze formation of phosphocholine and DAG, have also been described in mammals45-47 and are suggested to be important components in apoptotic48 and immune49 ,50 signaling. A 40 kDa PC-PLC was identified using an antibody for Bacillus cereus PC-PLC and purified from human U937 cells,47 but no sequence data are yet available for mammalian PC-PLC. In contrast, PC-PLC enzymes from bacteria are well studied. The gram-positive bacteria B. cereus and Clostridium perfringens express related 29 kDa and 43 kDa enzymes that require Ca2+ and Zn2+.51 The C. perfringens enzyme is a toxic component in gas gangrene. 51 A crystal structure of B. cereus PC-PLC indicates that catalysis occurs via a nucleophilic water molecule that is activated by a Glu reside coordinated to Zn2+ in the active site.52 9 PLD enzymes cleave PLs to give PA and a free alcohol. They are found in mammals, plants, yeast, bacteria, and many other classes of organisms. PLDs share a conserved HKD motif, HxKx4Dx6GG/S, which contains residues essential for catalytic activity.5 Mammalian enzymes also contain PX and PH domains.53 The proposed catalytic mechanism54,55 begins with attack by histidine to form a covalent phosphate adduct. The alcohol head group is then released and water that has been activated by a second histidine Ii berates the PL adduct as PA. Most PLD enzymes are also, often preferentially, able to catalyze transphosphatidylation,56 where an alcohol takes the place of water as the cleaving nucleophile, resulting in PL head group exchange. Other phosphodiesterases that use PL substrates and do not maintain the HKD motif have also been identified.57 These non-HKD PLD usually use metal ions to catalyze PL hydrolysis directly57 and catalyze transphosphatidylation poorly.58,59 scPLD (Streptomyces chromofuscus PLD) is a prototypical non-HKD PLD. In plants, where PLD activity was first discovered, there are at least 12 PLD genes, which can be organized into five groups, a, (3, y, ~, and ~.60 Among other functions, plant PLD controls stromal opening.61 In mammals, two genes have been cloned so far that can be variously spliced to give PLD proteins (PLD1 and PLD2- splice variants are lettered).62,63 Almost all human tissues express at least one PLD isoform, as do most human cell lines, lymphocytes and leukocytes being the excepti on. 64 PLD activity has also been detected at virtually all cellular membranes, including at the nucleus, the plasma membrane, the ER, and the Golgi apparatus. 65 LysoPLases 10 LysoPLA cleaves an LPL to give a glycerylphosphate diester and a free fatty acid. Secreted lysoPLA enzymes in Pseudomonas aeruginosa66 and Legionella pneumophila67 , 68 are thought to increase the infectivity of the microorganisms as well as to protect them from the toxic effects of some LPLs. Mammalian enzymes have also been studied. A 95 kDa bovine brain lysoPLA with apparent structural similarity to cPLA2 and a preference for arachidonate chains may be involved in neuronal arachidonic acid signaling,69 and a 25 kDa human lysoPLA associated with the endoplasmic reticulum and nuclear envelope could be an important off switch of LPL signaling.70 LysoPLC cleaves an LPL to give monoacylglycerol and a phosphate monoester. A lysoPLC has been proposed to be involved in choline plasmalogen biosynthesis.71 Other human lysoPLCs have been identified72,73 and proposed to be involved in choline resorption, LPL metabolism, or signaling processes, though little is known about the signaling roles of the lysoPLC products. LysoPLD activity was recently discovered from ATX (autotaxin), a protein associated with increased cell growth and motility in tumor cells.74, 75 It is now known that ATX mediates these effects by producing LPA from LPLs,76 especially LPC (lysophosphatidylcholine), which is abundant in plasma77 and secreted by some cancer cell types.75 ATX is a secreted 125 kDa enzyme that is a member of the NPP (nucleotide pyrophosphate/phosphodiesterase) family, and is also known as NPP2.78 The NPP family currently consists of seven members in mammals (NPPl_7),78 but only NPP2 has 11 lysoPLD activity; NPP672 and NPP779 have lysoPLC activity. NPPs are metalloenzymes and are stucturally related to alkaline phosphatases.8o They require metal-chelating residues and an active site Thr for both lysoPLase and NPP catalytic activities, indicating that catalysis for both activities occurs at the same active site.81 The mechanism is thought to proceed through a covalent intermediate.8°-82 Methods for Assaying Phospholipases Interfacial catalysis and kinetics PLases are soluble proteins that catalyze hydrolysis of membrane-bound PL substrates. With few exceptions, PLases catalyze hydrolysis of monomeric PL substrates poorly. As the amount of PL is increased beyond its CMC (critical micelle concentration), an exponential increase in PLase activity is observed.83 As a result, in vitro analyses of PLases rely mainly on micellar or vesicular substrate matrices. Kinetic analysis of interfacial enzymes is different from that of enzymes having soluble substrates in several important ways.84 First, an initial interfacial binding step must be taken into account, where Kd is an interfacial binding constant. Second, the affinity of an interfacial enzyme for a substrate (Ks *), product (Kp*), or inhibitor (KI*) in its active site can be complicated by the membrane partitioning equilibrium constants of each ligand (KL')' Third, a suitable assay system should be used that allows dilution of substrate in a neutral, homogenous 2D matrix that does not compete for the enzyme active site or allosterically activate or inhibit the enzynle. PLase catalytic processivity proceeds by two methods.83 In scooting mode, an enzyme binds tightly to the 2D interface, hydrolyzing substrate on the surface and remaining bound thereafter. In hopping mode, an enzyme can move from one micelle or vesicle to another, transiently catalyzing hydrolysis at each location. Maintenance of a scooting mode is preferred because it greatly simplifies kinetic analysis by defining a single kinetic pathway and limiting the number of interfacial binding events to one. It also ensures that screened inhibitors only affect catalytic steps and not lipid binding. Common PL substrate carrier systems 12 Two carrier systems are commonly used to derive information about the kinetics of PLase reactions. In the first, vesicles are generated from the anionic PL analogue DMPM (dimyristoylphosphatidylmethanol), for which PLases have been shown to have high affinity.85 Forced to remain in scooting mode, the enzyme's primary kinetic parameters can be derived by systematically diluting substrate in the vesicles and fitting the rate data to the Michaelis-Menten equation. The binding step is considered separately. In this assay, it is important that the integrity of the vesicles remains uncompromised to avoid hopping behavior and that the enzyme concentration be low enough to ensure one or fewer enzymes per vesicle. DMPM is currently not commercially available and must be synthesized. In the second system, the catalytically inert detergent Triton X-IOO is used to generate mixed micelles containing PL substrate.86 It has been shown that rapid mixing between the micelles makes the entire substrate pool available to a given PLase, allowing a larger concentration of enzyme to be used than in the DMPM assay.87 A method for kinetic analysis of PLase on Triton mixed micelles has been published that takes into account rate dependence on both the mole fraction and bu1k concentration of PL.88 The micelle binding step is considered part of the kinetic analysis. Triton X-IOO has the advantage of being commonly used and commercially available, but some have disputed the use of detergent-based mixed micelles for obtaining primary kinetic data based on several of the kinetic model's underlying assumptions.84 13 Other assay systems have also been developed. In a monodisperse system, short­chain PLs are used at concentrations below their CMC.89 The assay avoids the issue of membrane binding by reducing the kinetic analysis to that of a soluble enzyme acting on a soluble substrate. However, most PLases do not readily react with monodisperse substrates and it has been suggested that most activity seen with monomeric substrates can be attributed to hydrolysis at air bubbles or reaction vessel interfaces.9o The assay is also not reflective of in vivo conditions, so inhibitors identified with this method may not be inhibitors in a native environment. Bile salts91 ,92 or preparations from egg yolk93 or cell membrane fractions94 have also been used to conveniently solubilize PL in PLase assays. The nonhomogenous nature of these carriers or their aggregates, though, calls into question the validity of these assays and their reproducibility compared with other methodologies.95,96 Emulsions or reversed micelles, in which PLases are dissolved in a minimal amount of water and vigorously stirred with PL dispersed in organic solvents are commonly used for preparative purposes in enzymatic organic syntheses. The system is less frequently used for PL assays97, 98 and little is known about its physical properties or applicability to living systems. Other assays include monolayers of PL at air-water or other interfaces,99 where PLase activity is dependent on surface pressure, and cross­linked PL dispersions,l°O which are useful for studying protein-lipid hydrophobic interactions and substrate segregation in PLase activity. Detection methods The topic of detection methods for PLases has been extensively reviewed. WI Only the most common methods will be discussed again here. 14 Extraction. Procedures for extracting PL or PLase products selectively from a reaction milieu have been developed by DolelO2 and by Bligh and DyerlO3 (Figure 1.2). The Bligh and Dyer technique is to simply extract all PL into an optimized organic phase. Solvents are then removed and the mixture of products and starting material analyzed by chromatographic methods. In the Dole extraction, only fatty acids are extracted; thus, the extraction solution can be assayed directly if radiolabeled acyl chains are employed. The Bligh and Dyer method is applicable to all PLase reactions, while the Dole extraction is limited to assaying PLA and PLB. Titrametry. Direct titration of the acidic products of PLase reactions104 was one of the first detection methods to be used because of its simplicity. Though straightforward and inexpensive, the method lacks sensitivity and reports PLase activity nonselectively. The assay is continuous and adaptable to all PLases, but will not work well at acidic pHs or in assay systems where protons may be released for reasons other than PLase activity_ Monolayers. The monolayer assay system employees a unique method of detection in which changes in PL surface pressure are monitored.105 Specialized equipment is required, but very small concentration changes can be detected. It also has the advantage of being continuous and of using unmodified PL. Monolayer assays have been adapted for PLA,I06 PLC,107 and PLD.108 Chromophores. One way to detect PLase reactions chromogenically is to use a pH indicator dye to detect fatty acid liberation.109 The pH indicator technique has been Bligh & Dyer Extraction Chromatography Dole Extraction Scintillation Counter/ Chromatography Figure 1.2. Methods for extracting the products of PLase reactions i--' VI 16 used as a variation on the titrametric method. In the case of PLC, a chromogenic phosphate indicator has been used to detect the amount of liberated phosphate following extraction of DAG product and PL starting materia1. 11o , 111 Chromogenic methods using unnatural substrates have also been developed. 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiobispyridine are both colorimetric reagents for detection of the free thiol that is liberated when PLA1l2 or PLC113 cleaves a thioester PL analogue (Figure 1.3). Hydrolysis of thioesters is catalyzed preferentially over oxyesters by PLA2• Other synthetic substrates for PLA 114, 115 and PLC116 have been developed that release a nitrophenyl chromophore upon substrate hydrolysis (Figure 1.4). These detection methods are especially useful for rapid, continuous PLase assays, and can be adapted for high-throughput screening. Coupled assays. Coupled assays monitor PLase activity indirectly with secondary or tertiary detection reagents. One example is the polarographic assay, 117 where unsaturated fatty acid PLA products are detected by following the consumption of O2 upon controlled free fatty acid oxidation with soybean lipoxygenase. In another example, CoA-coupled assaysllS enzymatically incorporate PLA­released fatty acids into acyl-CoA and then oxidize them with acyl-CoA oxidase. The peroxide released during the oxidation is harnessed by peroxidase to oxidatively couple 4-aminoantipyrine to 3-methyl-N-ethyl-N-(~-hydroxyethyl)aniline or to 2,4,6- tribromo-3-hydroxybenzoic acid, generating a new chromophore. In yet another coupled assay, PLA2 that are capable of cleaving plasmalogens, PLs in which the sn-1 acyl chain is replaced with an enol ether, may be tracked by treating the lysoplasmalogen product with lysoplasmalogenase.7 The resulting aldehyde 17 + H~ AAbs = 324 nm Figure 1.3. A dithioester PL analogue suitable for assaying PLAj or PLA2• After enzymatic cleavage, the free thiol reacts with 4,4'-dithiobispyridine to yield the chromophore 4-thiopyridine. b Figure 1.4. The SIBLINKS substrate (a)llS andp-nitrophenylphosphorylcholine (b).116 Both substrates produce the chromophore p-nitrophenol upon hydrolysis by PLA2 or PC-PLC, respectively. Spontaneous cyclization following PLl\2 cleavage releases p-nitrophenol in the case of the SIBLINKS substrate. is reduced with alcohol dehydrogenase, and the consumption of NADH is followed spectrophotometricall y. 18 The Amplex Red reagent has been used to develop coupled assays for many different kinds of enzymes. 119 In particular, commercial Amplex Red assays for PLD and PC-PLC are available (Molecular Probes/Invitrogen). In each case, choline is produced, either directly from PC by PLD or by subsequent hydrolysis of phosphocholine by alkaline phosphatase, for PC-PLC (Figure 1.5). Choline is oxidized to an acid by choline oxidase, giving hydrogen peroxide as a by-product. Amplex Red, in the presence of hydrogen peroxide and horseradish peroxidase, is converted into the highly fluorescent compound resorufin. Coupled assays can be continuous or discontinuous and have the advantage of conveniently linking PLase cleavage of unmodified PLs to the generation of a chromophore or fluorophore that can be readily detected spectrophotometrically. However, having multiple steps between PLase activity and the generation of a detectable signal also provides multiple checkpoints for false inhibition or false positives to occur. Therefore, the results from coupled assays should be viewed in light of possible confounding factors in the experimental design. Fluorophores. Fluorescently labeled PL analogues have been synthesized that can be used to detect PLase activity with high sensitivity. These assays are typically not as sensitive as those using radioactive substrates but do not carry the same health risks. Like the radioactivity assay, fluorescence assays are usually discontinuous, requiring extraction and/or chromatographic separations. However, phase transfer offluorescently labeled products from a self-quenching aggregate to a freely fluorescing monomeric state 19 -"~--~,-~ ~ k alkaline k PLC Ji'..... phosphatase ~ ~1-__________· __Hi f_:'_~_LD__~_ + __ -_-_-_-_~·_H_~~l + choline 1./ oxidase H~~ ------~~~ Jl, horseradish k peroxidase H + + H20 2 --~---... Amplex Red resorufin '\ 587 nm Figure 1.5. An example of a coupled assay. Amplex Red reagent is converted to fl uorescent resorufin in the presence of hydrogen peroxide and horseradish peroxidase. The hydrogen peroxide is a by-product of enzymatic oxidation of the choline that results from PLC or PLD cleavage of a PL. 20 using a lipid binding protein, like albumin, has also been used to develop continuous PLA assays.120 Continuous fluorescence displacement assays have also been developed for PLA,121 PLC,122 and PLD.123 In this assay, the large increase in fluorescence that occurs during the interaction of 11-(dansylamino)undecanoic acid with liver fatty acid binding protein decreases as fatty acids from the PLA reaction displace the labeled probe from the binding protein. When a phase transfer type of assay is used for inhibitor screening, it must be verified that the enzyme is being inhibited and not the phase transfer process. Fluorogenic substrates, which generate fluorescence upon cleavage by a PLase, have all the advantages of high sensitivity, rapidity, continuity, convenience, and scalability. In addition, because they require no additional reagents or separations for signal detection, they offer the unique advantage of being able to be used to monitor in vivo PLase activity in real time. Several different fluorogenic substrates have been produced for analysis of PLA (Figure 1.6),124-128lysoPLD (Figure 1.7)/29,130 and PI­PLC. 131 Fluorescence is generated either through the formation of a new fluorophore or by the dequenching of a fluorescent donor-acceptor FRET (fluorescence resonance energy transfer) pair upon enzyme cleavage. A novel chemiluminescent substrate for PI­PLC (LUMI-PI, Figure 1.8) has also been reported. 132 PLase Selectivity Undoubtedly, membrane composition plays an in1portant role in the binding affinity and resulting catalytic activity of an attacking PLase. In sPLA2 studies it was shown that the ability of an enzyme to bind PC vesicles correlated well with its ability catalyze fatty acid release from PC-rich membranes (i.e., the extracellular plasma membrane of most mammalian cells ).133 sPLA2 enzymes generally prefer vesicles with a 21 a b c d ~ ~ H ~ H OHO-OH Figure 1.6. Structures offluorogenic substrates for PLA (a-c) or PLC (d). Compound a, bis-BODIPY PC,127 can be cleaved by PLA1 or PLA2 and is an example of a self-quenching fluorophore. Compounds band c, BCll-DNPCg-PC134 and PED6,134, 135 maintain PLA2 selectivity with an sn-1 ether or with a head group-- appended fluorescence acceptor. In band c, BODIPY is quenched by DNP (2,4- dinitrophenol), which emits at a distinct wavelength. Compound d releases a fluorescein analogue upon phosphodiester hydrolysis by PI_PLC.131 22 a ~ycy~< Hof~~~~H b Figure 1.7. Structures of the fluorogenic lysoPLD substrates FS-3 (a),13() and CPF4 (b).129 In FS-3, fluorescein fluoroescence is quenched by the nonemitting quencher dabcyl. In CPF4, fluorescein quenches emission by the coumarin donor. ~ ~ H 7 H "'OH OH Figure 1.8. LUMI-PI, a chemiluminescent substrate for PI-PLC 23 significant anionic component, a phenomenon that has been explained using electrostatic arguments. 136, 137 For example, spiking PC vesicles with PS considerably increased the vesicle affinity and resulting catalytic activity of human Group IIA sPLA2.133 Additional studies showed that pure vesicles of PG were preferred over PS, which were preferred over PC, in concordance with a decrease in the anionic nature of the substrates. 138 Once bound with reasonable affinity to the lipid wall of its assigned cellular compartment, the PLase's intrinsic head group and fatty acyl chain affinities help determine what signaling products are generated. PLA2 enzymes are stereospecific for 2R substrates139, 140 and, surprisingly, also show specificity for the Rp isomer in PL analogues that are chiral at phosphorous.141 Among sPLA2 enzynles there is very little selectivity for saturated versus unsaturated sn-2 fatty acids.138 However, the cPLA2 enzymes have a pronounced preference for unsaturation at this position due to the increased depth to which they insert into the lipid bilayer, which allows protein interactions with the acyl chains to occur.142 Little is known about the influence of PL chain composition on PLC and PLD activity, but it has been reported that plant PLDs prefer PLs of medium chain length and have a slight preference for unsaturation. 143 PI-PLC also shows a preference for unsaturated chains in cell-based assays. 144, 145 PC-PLC and PLD demonstrate stereoselectivity for the glycerol backbone under certain conditions, a factor that is dependent on chain composition. l46 PC-PLC, PI-PLC, and PLD are also all stereospecific at phosphorous. 141 Head group composition plays a large role in PLC and PLD selectivity. PLCs hydrolyze either Ptdlns( 4,5)P2 or PC, though PC-PLC can also cleave PE and PS to lesser degrees. 147 Mammalian PLDs usually prefer PC over any other substrate,148 but there are also well-known PLD enzymes that specifically cleave N-acyIPE,149 or glycosylated PI (GPI_PLD).150 Plant PLDs are able to cleave a wide range of PL head groups. 143 24 Several methods have been used to determine PLA head group selectivities, which can vary among the enzyme family members. Generally, there is good agreement between the various assays in the order of head group preference, with the only differences being in the ratios of the relative rates. A very straightforward, though tedious, method for measuring PLA head group selectivity is to assay an equimolar mixture of all PLs in vesicles.138 LPL products are extracted and analyzed chromatographically by comparison with standards. Another method that has been used is to compare PLA activity with pyrene-Iabeled PLs in the crosslinked PL assay system. 151 Other head group selectivity measurements have been done using an array of PLs with different head groups in thioester,t52 titrametric, 153, 154 or continuous fluorescence displacement assays. 155 PLA head group preferences are important because the composition of the LPL and/or fatty acid products can determine which of a variety of signaling outcomes are initiated. For example, LPC is a likely signal for macrophage recruitment to apoptosing cells. l56 LPG (lysophosphatidylglycerol) may, among other, undiscovered functions, be a precursor for LBPA (lysobisphosphatidic acid), a molecule important in cell transport. I57 LPE (lysophosphatidylethanolamine) is thought to be an intermediate in the biosynthesis of endogenous cannabinoids. I58 LPS (lysophosphatidylserine) works synergistically with nerve growth factor to stimulate nerve differentiation159 and to release histamine from 25 mast cells. 160 Finally, LPA, produced either directly or via other LPL intermediates, is known to playa host of biological roles through receptors at both the plasma membrane and at the nucleus.2 Summary and Research Overview In this Introduction, the various classes of PLases found in humans have been described. Features of the enzymes that are important to their biological function and the methods that have been used for characterizing their activity in cells and in vitro were also reviewed. The evidence for the importance of the PLase enzymes in cell signaling and thereby in human health forms a vast and growing literature. The main focus of the work described here was to synthesize and evaluate fluorogenic reporter molecules suitable for assaying PLases in vitro and in living cells and tissues. Fluorogenic substrates provide advantages over other kinds of assay methods; these include, sensitivity, convenience, continuity of monitoring, and applicability to cell- and tissue­based assays. Part I outlays the synthesis and evaluation of fluorogenic PL analogues suitable for assaying PLases. First, substrates for PLA are described that contain a set of the most common PL head groups. Their synthesis and use in activity, inhibition, and head group selectivity assays in vitro and in living cells is documented. Next, the synthesis of fluorogenic substrates for PLC and PLD is described, along with data demonstrating their use in microtiter assays. Appendix A shows both the range of the collaborative interest that these fluorogenic probes for PLA, PLC, and PLD have generated, and the broad spectrum of PLase biochemistry to which they have been applied. 26 A secondary research focus involved synthetic studies toward analogues of inositol polyphosphates containing poorly hydrolysable phosphate mimics. And so, in Part II, synthetic studies toward metabolically stabilized Ins(1,4,5)P3 analogues as long-lived agonists and inhibitors in PtdIns( 4,5)P signaling pathways are discussed, culminating in the synthesis of Ins(1,4,5)tris(methylphosphonate) by a new synthetic protocol. References 1. Cook, J. A. Eicosanoids. Crit. Care Med. 2005,33, S488-S491. 2. Aoki, J. Mechanisms of lysophosphatidic acid production. Semin. Cell Dev. BioI. 2004, 15, 477-489. 3. Oliva, J. L.; Griner, E. M.; Kazanietz, M. G. PKC isozymes and diacylglycerol-regulated proteins as effectors of growth factor receptors. Growth Factors 2005, 23,245- 252. 4. Jenkins, G. M.; Frohman, M. A. Phospholipase D: a lipid centric review. Cell. Mol. Life Sci. 2005, 62, 2305-2316. 5. MUller, G.; Petry, S. Lipases and phospholipases in drug development: from biochemistry to molecular pharmacology; Wiley-VCH: Weinheim, 2004; p xvii, 336. 6. Aoki, J.; Nagai, Y.; Hosono, H.; Inoue, K.; Arai, H. Structure and function of phosphatidylserine-specific phospholipase AI' Biochim. Biophys. Acta 2002, 1582, 26- 32. 7. Hiramatsu, T.; Sonoda, H.; Takanezawa, Y.; Morikawa, R.; Ishida, M.; Kasahara, K.; Sanai, Y.; Taguchi, R.; Aoki, J.; Arai, H. Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A}s, mPA-PLA}alpha and mPA­PLAl beta. J. BioI. Chern. 2003, 278, 49438-49447. 8. Sato, T.; Aoki, 1.; Nagai, Y.; Dohmae, N.; Takio, K.; Doi, T.; Arai, H.; Inoue, K. Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family. J. BioI. Chem. 1997,272,2192-2198. 9. Nagai, Y.; Aoki, J.; Sato, T.; Amano, K.; Matsuda, Y.; Arai, H.; Inoue, K. An alternative splicing form of phosphatidylserine-specific phospholipase Al that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans. J. BioI. Chern. 1999,274, 11053-11059. 27 10. Sonoda, H.; Aoki, J.; Hiramatsu, T.; Ishida, M.; Bandoh, K.; Nagai, Y.; Taguchi, R.; Inoue, K.; Arai, H. A novel phosphatidic acid-selective phospholipase Ai that produces lysophosphatidic acid. J. Bioi. Chern. 2002,277, 34254-34263. 11. Higgs, H. N.; Glomset, J. A. Identification of a phosphatidic acid-preferring phospholipase Al from bovine brain and testis. Proc. Nati. Acad. Sci. U. S. A. 1994, 91, 9574-9578. 12. Higgs, H. N.; Glomset, J. A. Purification and properties of a phosphatidic acid-preferring phospholipase Al from bovine testis. Examination of the molecular basis of its activation. J. Bioi. Chern. 1996,271, 10874-10883. 13. Higgs, H. N.; Han, M. H.; Johnson, G. E.; Glomset, J. A. 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Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell 2003, 113, 717-730. 157. Shinozaki, K.; Waite, M. A novel phosphatidylglycerol-selective phospholipase A2 from macrophages. Biochemistry 1999,38, 1669-1675. 158. Sun, Y. X.; Tsuboi, K.; Okanl0to, Y.; Tonai, T.; Murakami, M.; Kudo, 1.; Ueda, N. Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D. Biochem. J. 2004, 380, 749-756. 159. Lourenssen, S.; Blennerhassett, M. G. Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells. Neurosci. Lett. 1998, 248, 77-80. 160. Kawamoto, K.; Aoki, J.; Tanaka, A.; Itakura, A.; Hosono, H.; Arai, H.; Kiso, Y.; Matsuda, H. Nerve growth factor activates mast cells through the collaborative interaction with lysophosphatidylserine expressed on the membrane surface of activated platelets. J. Immunol. 2002, 168,6412-6419. CHAPTER 2 A FLUOROGENIC PA ANALOGUE: rac-DBPA Introduction In this chapter, the chemical synthesis and in vitro evaluation of rac-DBPA, a PA analogue containing one member of a FRET (Fluorescence Resonance Energy Transfer) pair at the end of each acyl chain, is described. The sn-l acyl chain contains a nonfluorescing quencher molecule (dabcyl, also known as p-methyl red), and the sn-2 chain contains a fluorescent donor molecule (BODIPY, Molecular Probes, Inc.). In FRET, quenching occurs because the absorbance spectrum of the quencher overlaps the emission spectrum of the donor. Following hydrolytic removal of an acyl chain, as catalyzed by a PLA, the acyl chain containing BODIPY is separated fron1 the dabcyl chain such that FRET no longer occurs and an increased fluorescent signal can be detected. This process is illustrated in Scheme 2.1. No special modifications were made to deter hydrolysis of either acyl chain; thus, fluorescence amplification is expected upon hydrolytic dequenching by PLAl or PLA2. Because rac-DBPA was designed to be fluorogenic upon hydrolytic cleavage by PLA, it has advantages over other PLA assay methods, including radiometric,l titrametric,2 and chromatographic,3 which are time-consuming and often cannot be used to track continuous changes in enzyme activity. It is also an improvement over chromogenic PLA assays,4-6 which can be rapid and continuous, but are limited to in vitro o .t ...... ,."!' it · •....... ·.. . ···1/·:·. · FRET ~ \ \ + Scheme 2.1. A schematic diagram illustrating the process of fluorescence 41 dequenching. A fluorescence quencher, Q, nonradiatively accepts fluorescence from a fluorophore, F, via FRET in the shown model fluorogenic phospholipid analogue. Following cleavage by PLA2, the intermolecular distance increases to the point where FRET is no longer efficient and fluorescence is observed. For rac-DBPA: R = H, Q = dabcyl, and F = BODIPY. 42 applications. Although a number of fluorogenic substrates for PLA have been synthesized previously,7-11 all of these were based on a PC skeleton. However, PLAs that are selective for head groups other than PC are also known. In particular, many forms of PA-selective PLA1 have been reported,12-16 several of which appear to be important in the generation of signaling LPA.17 PLA2 enzymes also have head group preferences, and it has been suggested that the Group II sPLA2 has a preference for PA over other PL head groups, the significance of which is not yet clear. 18 rac-DBP A and compounds related to it are expected to be useful in tracking the localization of PLA activity in cells, most particularly in studies on PA-selective PLA. The probe may also be useful in rapid, high-throughput screens for discovery of isotype­selective PLA inhibitors. Results and Discussion rac-DBP A was synthesized in six steps, starting from sol ketal (Scheme 2.2). Solketal was treated with dimethylchlorophosphate and base, resulting in phosphorylation of the primary alcohol to give 1 in 540/0 yield. After acidic deprotection of the diol, the first acyl chain, containing the fluorescent quencher dabcyl (2), was installed by selective esterification of the primary alcohol, giving 3 with a 40% yield. A protected 6- aminoacyl group was similarly placed on the secondary alcohol (5) in 50% yield. Simultaneous deprotection of both the phosphate-protecting methyl groups and amine­protecting Boc group with a 3% solution of TMSBr, set up the molecule for the final reaction-attachment of the fluorophore, BODIPY FL-X, using either of two mildly basic aqueous conditions. The identity of the product was confirmed by NMR and MALDI-MS. a) (MeO)2POC1, DMAP, 1 Eq DCC, Hf _t-_B_UO_K_,_C_H2_C_12_. .... MeO- ~OH CH2CI2, O°C .. MeO_S:>~Q O-/. b) p-TsOH, MeOH Md'b OH 'it H Meif''b OH DMAP, 1 Eq DCC, CH2CI2, RT HO~C 4 5 1 HO~abcYI 2 a) TMSBr (3% in CHsCN) b) BODIPY FL-X/SE, 0.1 M NaHCOs, pH 8.4: DMF (3:1) OR: 0~ '(13.0 :M2 TE)A B, pH 8.3: r Scheme 2.2. Chemical synthesis of rac-DBP A 43 ~ 3 N~_ ~ rac-DBPA Q r 44 The newly-synthesized, purified rac-DBPA was assayed for its ability to generate a fluorescent signal upon lipolysis by a PLA. In this assay, liposomes were generated by sonicating PC, PE, and rac-DBP A in aqueous buffer containing CaCI2• To these liposomes was added a solution of PLA2 in the same buffer, and fluorescentreadings were taken at 37°C. The results of this assay (Figure 2.1) show that both 1 U and 5 U solutions of PLA2 caused evolution of a fluorescent signal that is significantly above baseline compared to rac-DBPA-containing liposomes without enzyme (Lipids Only) or PLA2 without lipids (Enzyme Only). Furthermore, the dependence of the fluorescence increase on the concentration of rac-DBP A was demonstrated by incubating increasing concentrations of the analogue, in liposomes, with 0.5 U of PLA2 (Figure 2.2). From the raw data shown in Figure 2.2, a lag time can be observed between the time of enzyme addition (0 s) and the beginning of fluorescence increase (120-160 s). This lag time has been observed frequently for PLA assays that use PL vesicles as the diluent. 19 When the rates of linear fluorescence increase for rac-DBP A cleavage in PL vesicles were plotted against rac-DBP A concentrations, the plots did not always fit Michaelis-Menten kinetics, and outliers were frequently observed. It was initially thought that competitive effects from the PLs used to generate the liposomes or from PL hydrolysis products created the perceived kinetic anomalies. To circumvent the use of other PL substrates for PLA2 enzymes in the assay, rac-DBP A was passed through an ion-exchange column, thereby converting it to a salt. Now water soluble, suspension of the analogue in vesicles or micelles was no longer required, and this monomeric form of rac-DBP A was submitted to hydrolysis with the same snake venom PLA2 as before. In this case, zones of small fluorescence increase were observed that did not correlate well 450 400 350 300 250 i! 200 a: 150 100 !II 50 II 0 ~~~* * .* ~I~. .'. 100 150 200 250 300 350 400 -50 Time (s) Figure 2.1. Fluorogenic assays of rae-DBP A with snake venom PLA2 45 +1 U PLA2 11.5 U PLA2 It. Lipids Only x Enzvme 46 ~ 100~-·",,,,,·,,,,.·,,,,,,,,·,,~·-,,,,·,,· .... ,,·,.,,, .. ·,,1 .... ,,·,,·,,·,,,,~~~~":"" .. ,, .. ,,·,,: .... · .. -~·k~~~~:~~T=~=~: ....... "" .. ,~ .. ""'."'.J a:: o 200 400 600 800 1000 1200 1400 Time (s) o 04 Figure 2.2. Concentration-dependent, raw fluorescence increases upon treatment of rac-DBPA in PC vesicles with cobra venom PLA2• A brief lag time is observed. 47 with the concentrations of rac-DBPA used. These results support the proposal that monomeric assays of interfacial enzyn1es only measure enzyme activity at surfaces within the assay system.20 In another approach, the water-soluble form of rac-DBPA was sonicated with Triton X-I 00 (reduced) detergent to form nuxed micelles and was assayed with PLA2, but no signal was observed. Thus, it is likely that the water-soluble form of the analogue does not appreciably partition into micelles. It is known that the signals of de quenched fluorescent groups can be attenuated at high concentrations because of intermolecular quenching by nearby molecules containing complementary fluorescence acceptors. This phenomenon is referred to as the Inner Filter Effect.21 In an attempt to determine the influence of the Inner Filter Effect on the signal generated by PLA2 cleavage of rac-DBP A, increasing concentrations of rac­DBPA were added to an enzyme-free, 1 !J.M solution of BODIPY FL-X in 95% MeOH(aq) (Figure 2.3). As shown, when the probes are dispersed freely in solution, the fluorescence of 1 JlM BODIPY is reduced by about 1 % for each 1 JlM of uncleaved rac­DBPA present. A determination of the Inner Filter Effect for products and substrates confined to vesicles or micelles was also attempted, but did not result in linear curves. If the Inner Filter Effect significantly affected the fluorescent signal, it would be expected to do so at low concentrations of product relative to substrate, giving a lower-than­expected V max or a Michaelis-Menten plot that tailed downward at increasing substrate concentrations. In this case, instead of systematic deviation at high concentration, non­hyperbolic scattering of the data points was observed, regardless of substrate concentration. Therefore, further examination of the Inner Filter Effect in vesicles or micelles was not pursued. 48 1.00 s= 9:: 0.95 = -0.0095x + Q 0 ~ 0.90 R2 = 0.9881 ::> u.. 0.85 «-ct: 0.80 a.: ~ 0.75 I (.') a\-s 0.70 + >a.-. 0.65 0 o 0.60 fQ. :u:>.. 0.55 a: 0.50 0 10 20 30 40 50 [rac-DBPAJ (J.1M) Figure 2.3. The Inner Filter Effect. In 95% MeOH(aq)' the ratio of the fluorescence of BODIPY FL-X plus unhydrolyzed rac-DBPA to the fluorescence 1 JiM BODIPY only (y-axis) decreases as the concentration of unhydrolyzed rac-DBPA (x-axis) is increased. Inset: the equation for a linear fit of the data. 49 Finally, rac-DBPA was applied to normal NIH3T3 cells and to ones that had been transiently transfected with a plasmid containing the gene for mouse mPA-PLAIa. Problems with low transfection efficiency and cell viability, most likely the result of high endotoxin levels because of poor plasmid purification, stymied efforts to obtain good comparisons of fluorescence increases of control vs. transfected cells, but confocal fluorescence microscopy of cells treated with the water-soluble form of rac-DBPA indicated that the probe was membrane-permeable, and that it distributed in the cytoplasm (Figure 2.4). In conclusion, a fluorogenic analogue of phosphatidic acid was synthesized from racemic starting materials in six steps. The analogue generated fluorescence with PLA2 in a concentration-dependent manner when sonicated into phospholipid vesicles. The nl0nomeric, water-soluble form of the substrate was not processed consistently or predictably by PLA2 and did not appear to insert into detergent micelles. An Inner Filter Effect was observed for free BODIPY in the presence of unhydrolyzed rac-DBPA in solution. And, treatment of cells with water-soluble rac-DBP A showed the probe was membrane-permeable. At this point, further experiments with rac-DBP A were suspended in favor of developing a new, more versatile, stereospecific route to fluorogenic analogues of both PC and PA. As discussed in Chapter 3, the source of deviations from Michaelis-Menten kinetics were re-examined with the new analogues by systematically varying the fraction of the probes in detergent micelles. Figure 2.4. NIH3T3 cells that underwent a transfection protocol with mouse mPA­PLAIa plasmid were treated with water-soluble rac-DBPA. The cells were subsequently washed and incubated in medium. As shown in this confocal image, the rac-DBP A was able to pass through the cell membranes and aggregate within the cells. 50 Experimental Details Synthesis of sn-glyceryl dimethylphosphate ester (1) 51 To solketal (0.25 g, 1.9 mmol) stirring in CH2Cl2 at 0 °C, dimethy1chlorophosphate (0.4 g, 2.9 mmol) was added, followed by dropwise addition of potassium t-butoxide (325 mg, 2.9 mmol). Mter 4 h, the reaction was poured into sat. NH4CI, and extracted with CH2CI2• The organic layer was washed with brine and dried with Na2S04• Following removal of solvent in vacuo, the crude compound was dissolved in MeOH (5 mL), to which was added p-TsOH (71 mg, 0.38 mmol), with stirring. After 35-40 min, the reaction was neutralized with sodium bicarbonate (32 mg, 0.38 mmol). Solvent was removed under vacuum, and compound 1 was isolated from Si02 (Flash Chromatography ASTM 230-400 Silica Gel) with 10% MeOH in CH2Cl2 (200 mg, 54% yield). ~H(400 MHz, CDCh): 4.07-4.00 (m, 2H), 3.85 (dt, J= 10, 1H, 5 Hz), 3.77-3.70 (m, 6H), 3.65-3.54 (m, 2H), 2.91 (s, 1H), 2.82 (s, IH). ~p(l62 MHz, CDCb): 2.6. Synthesis of 6-dabcyl aminohexanoic acid (2) 6-aminohexanoic acid (37 mg, 0.28 mmol) and dabcyl-SE (Molecular Probes. 100 mg, 0.27 mmol) were combined in a 50 mL round bottom flask. A 1:1 solution (4 mL, total) of water and 10(w/v)% DMAP in DMF was added to the flask and the suspension was vigorously stirred for 4 h. The resulting homogeneous solution was poured into 5% aqueous NaHS04 and extracted with CH2CI2• The extract was washed with brine and dried with anhydrous Na2S04• Organic solvents were removed under reduced pressure, and compound 2 (72 mg, 69% yield) was purified from a Si02 column with a stepwise gradient of 100% CH2Cl2 to 20% MeOH in CH2C12• ~H(MeOD): 7.92 (d, J= 8.4 Hz, 2H, dabcyl), 7.85 (d, J= 9.2 Hz, 2H, dabcyl), 7.84 (d, J= 8.4 Hz, 2H, 52 dabcyl), 6.83 (d, J= 9.2 Hz, 2H, dabcyl), 3.40 (t, J= 7.2 Hz, 2H), 3.10 (s, 6H, dabcyl), 2.32 (t, J= 7.4 Hz, 2H), 1.73-1.61 (m, 4H), 1.50-1.39 (m, 2H). Synthesis of l-Q-(6-dabcyl-aminohexanoyl)-sn-gJyceryl dimethyl phosphate ester (3) To a flask containing 6-dabcyl-aminohexanoic acid (51 mg, 0.13 mmol) and DCC (27.4 mg, 0.13 mmol) at 0 °C and under N2 was added CH2Cl2 (3 mL). COlllpound 3 (45 mg, 0.23 mmol) and DMAP (17.6 mg, 0.14 mlllol) were then added with stirring via cannula in 4 mL, total, CH2C12. After stirring overnight at 0 °C, the reaction was filtered, concentrated, and passed over a Si02 column with 100% ethyl acetate to give compound 4 (30 mg, 40% yield). &H(CDCb): 7.93-7.79 (m, 6H, dabcyl), 6.80-6.69 (d, J = 8.8 Hz, 2H, dabcyl), 4.26-4.02 (m, 4H), 3.82-3.74 (m, 6H), 3.47 (dt, J = 6.8, 6.0 Hz, 2H), 3.10 (s, 6H, dabcyl), 2.38 (t,J= 7.2 Hz, 2H), 1.75-1.60 (m, 4H), 1.50-1.38 (m, 2H). &p(CDCh): 3.1. Synthesis of 6-boc aminohexanoic acid (4) 6-aminohexanoic acid (2.84 g, 21.6 mmol) and tert-butyl dicarbonate (5.7 g, 25.9 mmol) were combined in a 500 mL round bottom flask. 50 mL ofTHF was added to the flask, followed by, with stirring, 50 mL saturated NaHC03(aq)' This solution immediately turned cloudy white, but was allowed to stir for 24 h. The solvents were removed under reduced pressure and the residue redissolved in 5% NaHS04• The resulting solution was extracted with ethyl acetate. The organic layer was dried with brine and Na2S04 and the crude product used without further purification. &H(CDCb): 11.14 (br s, IH), 3.05-2.83 (br m, 2H), 2.20 (t, J= 7.4 Hz, 2H), 1.51 (dt, J= 7.8,7.4 Hz, 2H), 1.42-1.27 (m, 2H), 1.30 (s, 9H), 1.27-1.17 (m, 2H). Synthesis of 1-0-(6-dabcyl-aminohexanoyl)-2-0-(6-boc­aminohexanoyl)- sn-glyceryl dimethyl phosphate ester (5) 53 To a solution of 4 (9.4 mg, 0.041 mmol), DCC (5.4 mg, 0.026 mmol), and DMAP (3.6 mg, 0.029 mmol) stirring in CH2Cl2 (3 mL) at RT and under N2, was added compound 3 (18 mg, 0.032 mmol) in CH2Cl2 (2 mL) via cannula. After 24 h, another treatment of 4 (9.2 mg mg, 0.04 mmol), DCC (8.6 mg, 0.04 mmol), and DMAP (4.6 mg, 0.04 mmol) was added in CH2Cl2 (1.5 mL) via cannula. After an additional 12 h, the reaction solution was concentrated under vacuum and compound 5 was isolated from a Si02 column using 100% ethyl acetate (11 mg, 500/0 yield). oH(CDCb): 7.87-7.76 (m, 6H, dabcyl), 6.69 (d, J= 8.8 Hz, 2H, dabcyl), 5.20-5.13 (br m, IH), 4.33-4.24 (m, 2H), 4.18-4.03 (m, 2H), 3.73-3.65 (m, 6H), 3.45-3.32 (m, 4H), 3.04 (s, 6H, dabcyl), 2.35- 2.21 (m,4H), 1.68-1.48 (br m, 8H), 1.46-1.16 (4H), 1.37 (s, 9H). op(CDCb): 2.1. Synthesis of 1-0-(6-dabcyl-aminohexanoyl)-2-0-(6- aminohexanoyl)-sn-glyceryl phosphatidic acid Boc-protected compound 5 (11 mg, 0.013 mmol) was treated with a 3(v/v)% solution ofTMSBr in CH3CN (900 JlL). After 15 min, the reaction was placed under high vacuum overnight and then stirred in 95% MeOH for 30 min. The MeOH was removed under reduced pressure, and the title compound was isolated from Si02 using 65:25:4 CH2CI2:MeOH:H20 (4 mg, 47% yield). oH(MeOD): 7.94 (d, J= 8.4 Hz, 2H, dabcyl), 7.85 (d, J= 9.2 Hz, 2H, dabcyl), 7.84 (d, J= 8.0 Hz, 2H, dabcyl), 5.29-5.21 (br m, 1H), 4.40-4.33 (dd, 54 12.0,4.0, 1H), 4.30-4.16 (m, 1H), 4.27-3.93 (m, 2H), 3.40 (t, J= 6.8, 2H), 3.10 (s, 6H, dabcyl), 2.96-2.86 (m, 2H), 2.42-2.33 (m, 4H), 1.74-1.59 (br m, 8H), 1.49-1.38 (4H). op(MeOD): 1.9. Synthesis of 1-0-( 6-dabcyl-aminohexanoyl)-2-0-( 6-( 6- (3-BODIPY -propanoyl)aminohexanoyl)aminohexanoyl)­sn- glyceryl phosphatidic acid (rac-DBP A) Procedure 1. To 1-0-(6-dabcyl-aminohexanoyl)-2-0-(6-aminohexanoyl)-sn-glyceryl phosphatidic acid (2 mg, 0.003 mmol) and BODIPY FL-X/SE (1.6 mg, 0.003 mmol) in DMF (100 pL) was added a 0.1 M solution of NaHC03 (pH=8.4, 300 pL). The reaction was stirred for 5 h, at which time the solvents were removed in vacuo. The product, rac-DBPA (1.3 mg), was isolated from Si02 using 65:25:4 CH2CI2:MeOH:H20 in 42% yield. UV-vis (65:25:4 CH2CI2:MeOH:H20): Amax (E) = 507 nm (12000 M-1cm- 1),488 nm (shoulder). Procedure II. To 1-0-(6-dabcyl-aminohexanoyl)-2-0-(6-aminohexanoyl)-sn-glyceryl phosphatidic acid (2 mg, 0.003 mmol) and BODIPY FL-X/SE (1.6 mg, 0.003 mmol) in DMF (200 pL) was added a 1.0 M solution of TEAB (triethylammonium bicarbonate) buffer (pH=8.3, 300 pL), made from freshly-distilled TEA. The reaction was stirred for 5 h, at which time the solvents were removed in vacuo. Product was visualized on TLC using 65:25:4 CH2CI2:MeOH:H20. oH(d-DMSO): 8.02 (d, J = 8.4 Hz, 2H, dabcyl), 7.80 (d, J 9.2 Hz, 2H, dabcyl), 7.77 (d, J= 10.4 Hz, 2H, dabcyl), 7.67 (s, 1H, BODIPY), 7.06 (d, 4.0, 1H, BODIPy), 6.82 (d, J= 9.2 Hz, 2H, dabcyl), 6.34 (d, J= 3.6, 1H, BODIPy), 6.26 (s, IH, BODIPy), 5.08-4.97 (br m, IH), 4.27-4.17 (br m, IH), 4.13-4.03 (br m, 3H), 3.77-3.68 (br m, 2H), 3.06 (s, 6H, dabcyl), 3.02-2.90 (br 55 m, 8H), 2.43 (s, 3H, BODIPY), 2.32-2.24 (br m, 4H), 2.22 (s, 3H, BODIPY), 2.04-1.97 (br m, 2H), 1.59-1.07 (18H). Op(CDCh): 3.7. HRMS (ESI): mlz 1059.4682; calcd: 1059.4699 (M+Nat. UV-vis (65:25:4 CH2CI2:MeOH:H20): ~ax (E.) = 504 nm (12000 M-1cm-1 ), 488 nm (shoulder). Fluorogenic assays of rac-DBPA with PLA2 A modified procedure7 was used as follows: A 100 pM liposomal suspension containing 65-70% PC, 25% PE, and 5-10% rac-DBP A was prepared by combining the lipids in a culture tube, evaporating the transfer solvents, and sonicating in 100 pM boric acid buffer containing 100 pM CaCl2 (pH=8.4). PLA2 (Naja mossambica mossambica venom, Sigma) was prepared at 1 U/pL or 0.1 U/pL in the same buffer. All solutions were allowed to equilibrate at 37 °C in a 96-well plate, then the liposomal and enzyme solutions were combined and fluorescence readings were taken (Aex=502 nm; Aem=525 nm) every 30 s for 10-15 min on a SpectraMax GeminiXS fluorescent plate reader. The assay demonstrating concentration-dependence on rac-DBPA was performed exactly as above, only using 0.1 U/pL PLA2 and by making dilutions of the rac-DBP A -containing liposomes to give concentrations of rac-DBP A from 2-20 pM. Transfection and labeling of NIH3T3 cells with rac-DBPA The mouse IT1PA-PLAla gene in the pCAGGS plasmid (1 pglpL) was donated by Dr. J. Aoki. The plasmid was amplified by transforming NovaBlue supercompetent cells (Novagen) and growing them in medium selective for ampicillin resistance. The plasmid was harvested using a QIAprep Spin Miniprep kit (Qiagen). Lipofectamine (Invitrogen) was used according to the manufacturer's protocol to transfect NIH3T3 cells that had 56 been plated at a density of 2 x lOS cells in a 35 mm glass-bottom culture dish (MatTek Corp.) and incubated overnight at 37 °CI5% CO2 in DMEM containing 10% PBS and antibiotics. Transfected cells were washed once with HBSS (Hank's Balanced Salt Solution), then incubated for 2 min with 1 pg rac-DBPA in 100 pL HBSS. The rac- DBPA solution was then removed and the cells were washed once with HBSS. DMEM containing PBS and antibiotics was then added to the cells, which were incubated again briefly before being imaged by confocal microscopy (Figure 2.4). References 1. Reynolds, L. 1.; Washburn, W. N.; Deems, R. A.; Dennis, E. A. Assay strategies and methods for phospholipases. Methods Enzymol. 1991, 197, 3-23. 2. Dennis, E. A. Kinetic dependence of phospholipase A 2 activity on the detergent Triton X-100. J. Lipid Res. 1973,14, 152-159. 3. Lister, M. D.; Deems, R. A.; Watanabe, Y.; Ulevitch, R. 1.; Dennis, E. A. Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid. J. BioI. Chem. 1988,263,7506-7513. 4. Cho, W.; Markowitz, M. A.; Kezdy, F. 1. A new class of phospholipase A2 substrates: kinetics of the phospholipase A2 catalyzed hydrolysis of 3-(acyloxy)-4- nitrobenzoic acids. J. Am. Chem. Soc. 1988, 110, 5166-5171. 5. Washburn, W. N.; Dennis, E. A. Novel general approach for the assay and inhibition of hydrolytic enzymes utilizing suicide-inhibitory bifunctionally linked substrates (SIBLINKS): exemplified by a phospholipase A2 assay. J. Am. Chem. Soc. 1990, 112,2040-2041. 6. Yu, L.; Dennis, E. A. Thio-based phospholipase assay. Methods Enzymol. 1991, 197,65-75. 7. Feng, L.; Manabe, K.; Shope, J. C.; Widmer, S.; DeWald, D. B.; Prestwich, G. D. A real-time fluorogenic phospholipase A(2) assay for biochen1ical and cellular activity measurements. Chem. BioI. 2002,9,795-803. 8. Hendrickson, H. S.; Hendrickson, E. K.; Johnson, L D.; Farber, S. A. Intramolecularly quenched BODIPY -labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging. Anal. Biochem. 1999,276, 27-35. 57 9. Hendrickson, H. S.; Rauk, P. N. Continuous fluorometric assay of phospholipase A2 with pyrene-Iabeled lecithin as a substrate. Anai. Biochem. 1981, 116, 553-558. 10. Meshulam, T.; Herscovitz, H.; Casavant, D.; Bernardo, J.; Roman, R.; Haugland, R. P.; Strohmeier, G. S.; Diamond, R. D.; Simons, E. R. Flow cytometric kinetic measurements of neutrophil phospholipase A activation. J. Bioi. Chem. 1992,267, 21465-21470. 11. Wichmann, 0.; Schultz, C. FRET probes to monitor phospholipase A2 activity. Chem. Commun. (Camh.) 2001, 2500-2501. 12. Higgs, H. N.; Han, M. H.; Johnson, G. E.; Glomset, 1. A. Cloning of a phosphatidic acid-preferring phospholipase Al from bovine testis. J. Bioi. Chem. 1998, 273,5468-5477. 13. Hiramatsu, T.; Sonoda, H.; Takanezawa, Y.; Morikawa, R.; Ishida, M.; Kasahara, K.; Sanai, Y.; Taguchi, R.; Aoki, J.; Arai, H. Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLAIalpha and mPA­PLAI beta. J. Bioi. Chem. 2003,278, 49438-49447. 14. Nakajima, K.; Sonoda, H.; Mizoguchi, T.; Aoki, J.; Arai, H.; Nagahama, M.; Tagaya, M.; Tani, K. A novel phospholipase Al with sequence homology to a mammalian Sec23p-interacting protein, p125. J. Bioi. Chem. 2002,277, 11329-11335. 15. Sonoda, H.; Aoki, J.; Hiramatsu, T.; Ishida, M.; Bandoh, K.; Nagai, Y.; Taguchi, R.; Inoue, K.; Arai, H. A novel phosphatidic acid-selective phospholipase Al that produces lysophosphatidic acid. J. Bioi. Chem. 2002, 277,34254-34263. 16. Tani, K.; Mizoguchi, T.; Iwamatsu, A.; Hatsuzawa, K.; Tagaya, M. p125 is a novel mammalian Sec23p-interacting protein with structural similarity to phospholipid­modifying proteins. J. Bioi. Chem. 1999,274,20505-20512. 17. Aoki, J. Mechanisms of lysophosphatidic acid production. Semin. Cell Dev. Bioi. 2004, 15, 477-489. 18. Snitko, Y.; Y oon, E. T.; Cho, W. High specificity of human secretory class II phospholipase A2 for phosphatidic acid. Biochem. J. 1997,321 ( Pt 3),737-741. 19. Brown, S. D.; Baker, B. L.; Bell, J. D. Quantification of the interaction of lysolecithin with phosphatidylcholine vesicles using bovine serum albumin: relevance to the activation of phospholipase A2• Biochim. Biophys. Acta 1993, 1168, 13-22. 20. Yu, B. Z.; Berg, O. G.; Jain, M. K. Hydrolysis of monodisperse phosphatidylcholines by phospholipase A2 occurs 011 vessel walls and air bubbles. Biochemistry 1999,38, 10449-10456. 58 21. Liu, Y.; Kati, W.; Chen, C. M.; Tripathi, R.; MolIa, A.; Kohlbrenner, W. Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal. Biochem. 1999, 267, 331-335. CHAPTER 3 FLUOROGENIC ANALOGUES OF PA, PC, PE, PG, AND PS AS HEAD GROUP-SELECTIVE REPORTERS OF PLA ACTIVITY Introduction All PLA reactions generate a free fatty acid and an LPL, and each product has the potential to mediate cellular responses. The widespread distribution of PLA enzymes in the human body and the bioactive nature of their cleavage products have implicated these enzymes in many human diseases, including autoimmunel and cardiovascular diseases,2 neurological disorders,3 and cancer.4 To explore the effects of particular PLA isozymes in cell physiology, it is important to understand their spatiotemporal activation, and substrate selectivities, including diacyl group selectivity, and head group selectivity. For example, PLA isozymes have been reported with selectivities for PA,5 PE,6, 7 PC,~l0 PS, 11,12 and PG,13, 14 but information regarding the biological significance of the head group selectivity is limited. Furthermore, the pathophysiology of PLA activity in various diseases has made these enzymes important targets for isoform-specific drug development. In this case, rapid, sensitive, high-throughput, and real-time fluorescence based activity assays are Reproduced in part with permission from ACS Chern. Bioi. 2006, 1,83-91. Copyright 2006 American Chemical Society. desirable which would be compatible with isolated enzymes or suitable for cell-based assays. 60 PLA activity has traditionally been monitored using radiometric, titrametric, or chromatographic endpoint analyses,15 all of which are time-consuming and often do not permit real-time monitoring. Chromogenic assays16 allow real-time monitoring of PLAs in vitro, but fail for in situ applications. Fluorogenic PLA probes based on a PC skeleton, have been developed by the Prestwich research group,17 as well as by others. 18,19 These fluorogenic probes provide rapid, sensitive, real-time monitoring of PLA activity in vitro and in situ. The synthesis and evaluation offluorogenic PLA probes with five different head groups-PA, PC, PE, PG, and PS - by enzyme-assisted organic synthesis are described here. Each fluorogenic substrate contains the same diacylglyceryl moiety, in which the sn-l acyl chain contains an attached fluorescence quencher (dabcyl) and the sn-2 acyl chain contains an appended BODIPY tluorophore. Intramolecular FRET to the dabcyl group quenches BODIPY fluorescence until PLA-mediated substrate cleavage; then, a fluorophore is released when the LPL and fatty acid moieties are separated and the intermolecular distance exceeds that required for efficient energy transfer. It is demonstrated here that these four DBPLs-specifically DBPA, DBPC, DBPE, DBPG, and DBPS-are suitable for in vitro monitoring of PLA activity, including applications for inhibitor screening and head group selectivity studies. 61 Results and Discussion The significance of head group selectivity among PLA enzymes is one aspect of their crucial roles in cell signaling that has not been studied in great detail. To address this unnlet need, fluorogenic PL analogue probes having different head groups were synthesized using an enzyme-assisted synthetic route. The resulting probes were validated using real-time, continuous in vitro assays. In addition, these substrates are being employed to identify spatiotemporal regulation of PLA activity in living cells. Enzyme-assisted synthesis of DBPC and DBP A The original synthesis of DBPC17 was modified to improve yields and permit access to a variety of PL head groups. The new enzyme-assisted synthetic route is shown in Scheme 3.1, and was based on a route used to prepare photoactive phosphatidic acid derivatives.20 Initial attempts to condense dabcyl-linked aminohexanoic acid (1) with PC-glycerol using DCC/DMAP conditions resulted in poor yields of a mixture of monoacyl and diacyl products. Other unsuccessful esterification conditions included use of Sc(OTf)3 as a catalyst,21 elevation of reaction temperature, and conversion of the fatty acid to an acid chloride. Finally, acyl imidazole chemistry, previously shown to be effective at acylating PC-appended glycerol,22 provided the desired diacyl product 2 in acceptable yield (Scheme 3.1). After removal of the sn-2 fatty acid with cobra venom and reesterification in high yield, the carbamate 4 was cleaved and the resulting primary amine was conjugated with CrBODIPY(FL), SE (Molecular Probes) in 78% yield to give DBPC as the final product. This new synthesis generates a DPBC that has a diacylglycerol moiety with more appropriately matched distances between the glyceryl backbone and the appended quencher and fluor. 62 ~H . ~Q cobra venom PLA2, . ~~ HO. ... '-- OH a) HOOC(CH2)5NH-dabcyl (1)~O-Y. .... Q \ boric acid buffer H0-y.. ... OH b) DMSO, DBU, 40°C ) ) -u carbonyldiimidazole, DMSO ~-u (pH 8.5), MeOH, 38 °C ~-u +/ + 2 + 3 H~ ~ b~ ~ r a) 20% TFA in CH2CI2) +H~~~ b) DMF:1.0 M TEAB, pH 8.4 (3:2), C5-BODIPV(FL), SE ~ 4 ~ OSPC H ~ Scheme 3.1. Enzyme-assisted synthetic route to DBPC 63 Treatment of intermediate 4 with peanut PLD yielded its PA analogue, which was deprotected with TFA and then condensed with the active ester Cs-BODIPY(FL), SE to give DBP A (Scheme 3.2). This revised route, using PLA2 and PLD as synthetic reagents, thus provided both DBPA and DBPC in eight steps overalL The combined yield for DBPC was 24%, an approximately lOO-fold increase over the totally synthetic route previously reported. I7 DBPA was synthesized in 8% overall yield, a 4-fold increase from the synthesis of rac-DBPA described in Chapter 1. Synthesis of DBPE, DBPG, and DBPS by transphosphatidylation In the presence of excess alcohol, PLD can preferentially catalyze the alcoholysis of phosphatidylcholine in a process called transphosphatidylation.23 - 25 It was therefore envisaged that transphosphatidylation of intermediate 4 might yield PE, PG, and PS analogues, which could be processed further to give DBPE, DBPG, and DBPS. In our hands, the use of anhydrous conditions defined earlier4 for transphosphatidylation gave little or no product regardless of nucleophile. Addition of nucleophile in buffer, or of buffer alone, to intermediate 4 in CHCl3 was required to drive the PLD reaction to completion. This procedure, a modification of earlier studies/3 was followed using ethanolamine, glycerol, and L-serine as nucleophiles. The method gave DBPG and DBPE precursors,S and 6, in good to excellent yields (Scheme 3.3). In the case of the L-serine reaction, the product distribution favored DBP A precursor upon buffer addition. Modifying the nucleophile to include variously protected forms of L-serine did not generate the desired DBPS precursor. Finally, a simple adjustment in the organic phase, from CHCl3 to EtOAc,26 allowed transphosphatidylation between unprotected L-serine and intermediate 4 (Scheme 3.3). Streptomyces sp. PLD(P) (Genzyme) was the only B a) Peanut PLD (240 U), pH 5.6 buffer:CHCl3 (7:3),40°C b) 20% TFA in CH2CI2 c) DMF:1.0 M TEAB, pH 8.5 (3:2), C5-BODIPY(FL), SE Scheme 3.2. Enzyme-assisted synthetic route to DBP A 64 PLD, CHCI3 ethanolamine (aq, pH 5.6), 4O-45°C PLD, EtOAc L-serine (aq, pH 5.4), 4O-45 QC 5 6 7 Scheme 3.3. Enzymatic reactions leading to DBPE, DBPG, and DBPS precursors 65 PLD that consistently catalyzed transphosphatidylation over hydrolysis. Other commercial PLDs gave either no reaction or PA analogues. 66 To eliminate the need for protection and deprotection of the phosphatidylethanolamine moiety, direct transphosphatidylation of DBPC was attempted with PLD using the same conditions as above. Unfortunately, these conditions resulted in decomposition of the starting material, with no DBPE product detected. With this knowledge, the routes shown in Scheme 3.4, Scheme 3.5, and Scheme 3.6 were used to convert precursors 5, 6, and 7 to final products DBPG, DBPE, and DBPS. Using the described semienzymatic synthesis, five fluorogenic PL analogues with different head groups were synthesized from a common intermediate (4) in two to five steps. Chemical methods for head group introduction from modified diacylglyceryl precursors gave unacceptably low yields. Furthermore, chemical introduction of head groups before incorporation of acyl chains was rejected as inefficient, as it would require separate synthetic routes for each probe. Transphosphatidylation by PLD provided a mild, effective way to rapidly introduce head group diversity in our PL probe design. In vitro activity assays with DBPC and DBPA In vitro enzyme assays with DBPC and DBPA produced linear fluorescence increases that were dependent on the concentrations of enzyme and probe. These assays also revealed that predictable tracking of enzyme activity is predicated on the amount of detergent or PL used as a carrier for the probe. Figure 3.1 shows that as the fraction of probe in Triton X-lOO micelles is decreased, the plots of initial velocity versus probe concentration become increasingly hyperbolic. At ~ 0.2 (w/w)% of DBPC in Triton (0.3 67 n 0 a) TFA:CH2CI2 (1 :1) rro H HO'\J O~O b) DMF:1.0 M TEAB, pH 8.4> HoJHO-"~ O~ (1:1), C5-BODIPV(FL), SE H H ~ ~~PG ()r ()r B H Scheme 3.4. Final synthetic steps to DBPG from 5 68 :J'~r FmocCI, dioxane: Hf~O~r H \) sat. NaHC03 (2:1) u 3+ -------.)r- Fmoc>J ~ JQ ~ ~ ~ ~c ~ ~ H~~~ H . Q"~ Fm H3~ --------------+)r- > b) DMF:1.0 M TEAS, pH 8.4 (1:1), C5-SODIPY(FL), SE ~ ~ ~ ~PE r r Scheme 3.5. Completion of DBPE from 6 b) DMF:1.0 M TEAS, pH 8.8 (3:1),1 Eq. C5-SODIPY(FL), SE Scheme 3.6. Completion of nBPS from 7 69 ) 70 a 2 + OBPC=2.2(w/w)% 0 OBPC=O.9(w/w)% • OBPC=O.6(w/w)% -:au(:./J:.) •o OOBBPPCC==OO..24((ww//ww))%% 0 -1 0 2 3 4 [OBPC](JiM) b 0.75 + OBPA= O.34(w/w)% 0 OBPA= O.15(w/w)% 0.50 • OBPA= O.08(w Iw)% -(/) ::J 0 OBPA= O.03(w/w)% u.. a: • OBPA= O.02(w/w)% 0.25 o.oo~~~~~::::::D=:~C=--. 0.00 0.25 0.50 0.75 1.00 1.25 1.50 [OBPA](JiM) Figure 3.1. Concentration dependent hydrolysis of nBPC (a) and nBPA (b) by PLA2• An increasingly hyperbolic curve is observed for plots of initial velocity (RFU (relative fluorescence U/s) versus probe concentration (pM) as the fraction of probe (either nBPC (a), or nBPA (b)) in Triton X-lOO is decreased. LysoMaxS PLA2 (0.3 U well-I) and bee venom PLA2 (0.5 U well-I), respectively, were used with nBPC and nBP A in these assays. 71 U/well LysoMaxS PLA2) and :s 0.03 (w/w)% of DBPA in Triton (O.S U/well bee venom PLA2), the data fit the Michaelis-Menten equation, and apparent V max and Km values can be determined at a given Xd• Regardless of whether the probes were dispersed in Triton micelles (Figure 3.1) or PL vesicles, a window of robust PLA activity was observed. Above a certain mole fraction of DBPL the reported enzyme activity stopped following Michaelis-Menten kinetics; at too Iowa mole fraction of DBPL, the fluorescent signal fell below detection limits. Mole fractions of probe beyond an upper limit presumably yield disrupted aggregation states that are resistant to enzyme catalysis, resulting in lower-than-expected signal generation. Inhibitor assay with DBPC The Triton mixed micelle assay, despite possible limitations for obtaining primary kinetic parameters,27 is widely used to obtain relative kinetic information in inhibitor screens, and is the carrier of choice for a well-developed chromogenic sPLA2 assay.28 Triton X-IOO has the advantages of being commercially-available and relatively inert, and is frequently used for isolation of membrane-associated proteins. As shown in Figure 3.2, Triton/DBPC micelles provided a suitable matrix to allow an XlSO) for thioether amide-PC29 inhibition of PLA2 to be quantified. Increasing concentrations of thioether amide-PC were sonicated into Triton/DBPC mixed micelles and assayed in duplicate with bee venom PLA2 (Figure 3.2). The resulting XlSO) was calculated to be 0.004, corresponding to a thioether amide-PC concentration of 2 pM. This result correlates with that of a previous IC(SO) measurement, also 2 pM, for thioether amide-PC inhibition of cobra venom PLA2• 29 0.75 0.50 !!2 :::J LL a: 0.25 O.OO+---r------.---.-----.--------. -4.5 -4.0 -3.5 -3.0 IOg(XTEPC) -2.5 -2.0 thioether amide-PC Figure 3.2. Inhibition of bee PLA2 by thioether amide-PC. The log of increasing 72 mole fractions of thioether amide-PC sonicated with 0.5 pM nBPC at Xd = 0.001 (0.2 (w/w)%) in Triton X-I 00 micelles is plotted versus initial velocities (n = 2) generated upon addition of 0.01 U/well bee venom PLA2• Calculated Xj(50) = 0.004, corresponding to [thioether amide-PC] = 2 pM. 73 Head group selectivity assays The completed fluorogenic probes DBPA, DBPC, DBPE, and DBPG were used to experimentally determine the head group selectivities of a sampling of commercial PLA2 enzymes in Triton mixed micelles. Each fluorogenic substrate was assayed with LysoMaxS, bee venom, cobra venom, bovine pancreas, S. violaceoruber, and Human Type V PLA2 in TritionX-lOO (reduced) micelles. When selectivity was expressed as a percentage of the slope of the analogue showing the most activity (Figure 3.3), several interesting trends became apparent. First, mammalian enzymes (bovine and human) preferred the PG headgroup, followed by PC>PE»PA. Second, the venom and bacterial enzymes preferred the PC headgroup, followed by PG>PE»PA. Third, only the venom and pancreatic enzymes significantly catalyzed DBPA hydrolysis. Previous reports on the head group selectivity of these or other closely related PLA2 enzymes, as determined using a variety of assay methods, are summarized in Table 3.1. The degree of agreement between the listed head group selectivity studies is noteworthy, considering the contrasting methods used. Since the PLA head group preferences are conserved from assay platform to assay platform in vitro, the same preferences might also legitimately extrapolate to living systems. These data validate the applicability of the DBPL fluorogenic assay for further biological studies and inhibitor discovery. In vitro assays with DBPS Completed after the other analogues had been synthesized and tested, DBPS was assayed separately as a substrate for two commercial PLA2 enzymes from bee and cobra ! a 120 -100 1j? 80 .;.;0.... . 60 ~ 40 !;J < 20 0 -20 c 120 100 ~ 80 ;;0.... 60 =..:.:. 40 <!;J 20 0 -20 1 00 -j-~