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Applications of Langmuir-Blodgett Films and Langmuir Monolayers: Supported Catalysis and Membrane Models for Urinary Stone Formation

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Title:
Applications of Langmuir-Blodgett Films and Langmuir Monolayers: Supported Catalysis and Membrane Models for Urinary Stone Formation
Creator:
BENITEZ, ISA O. ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Calcium ( jstor )
Crystals ( jstor )
Lipids ( jstor )
Monomolecular films ( jstor )
Oxalates ( jstor )
Phospholipids ( jstor )
Phosphonic acids ( jstor )
Porphyrins ( jstor )
Precipitation ( jstor )
Rafts ( jstor )

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University of Florida
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University of Florida
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Copyright Isa O. Benitez. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/31/2005
Resource Identifier:
436098621 ( OCLC )

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APPLICATIONS OF LANGMUIRBLODGETT FILMS AND LANGMUIR MONOLAYERS: SUPPORTED CATALYSI S AND MEMBRANE MODELS FOR URINARY STONE FORMATION By ISA O. BENTEZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Isa O. Bentez

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To my mother, the woman I admire the most.

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ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Daniel Talham, for giving me the opportunity to perform my graduate research in his laboratory. Dan has been much more than a member of my supervisory committee; he has been a colleague and a friend over the past 5 years. Under his guidance I learned to perform research creatively and independently, trust my instincts and communicate my findings in a professional and effective manner. I am grateful for his infinite patience and understanding that made graduate school an enjoyable experience. Members of the Talham group have made enormous contributions to my graduate work. In particular I would like to acknowledge Christine Lee who took me under her wing when I was an overwhelmed first year and showed me the ropes of the lab, her project and became my friend. Her contributions to my career as a chemist did not stop when she left Gainesville as she aggressively campaigned my resume at Unilever which got me several interviews and my first job. I must also acknowledge Melissa Petruska and Jeffrey Culp who answered all my questions and gave me helpful advice during the first few years of my studies. Gail Fanucci, although already graduated from the group when I came to Gainesville, was always willing to share her ideas and expertise when she came to visit. It was Gail who encouraged me to look into lipids rafts as more accurate membrane models for the precipitation of calcium oxalate. Eduardo Prez has been a great friend over the past years and was always eager to help with anything from moving a desk to discussing experimental results. Finally, I thank Rnal Backov, Chen Liu, iv

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Sarah Lane, Franz Frye, Monique Williams and Justin Gardner, for making our research group an excellent working environment. I would like to thank Bruno Bujoli and Fabrice Odobel, our collaborators at the Universit de Nantes. Bruno, Fabrice and their graduate student Laurent Camus synthesized the porphyrins used in our supported catalysis project. They were also excellent hosts during my visits to Nantes where I caught a glimpse at how much work it takes to make phosphonic acid derivatized porphyrins and bipyridines. My friends Ivana, Janina, Tamara, Andy and Cira deserve special recognition. They are my family away from home. They listened to my talks, read my papers, heard my complaints, offered advice and celebrated with me every little accomplishment. I must thank my parents, Isa and Alfonso, and my sister Yaitza who have always supported my choices. I now realize that it must have been very hard to let a teenager leave her home and country in pursuit of a better education. I thank them for allowing my future be a priority. I could not have gotten this far otherwise. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES .............................................................................................................ix LIST OF FIGURES .............................................................................................................x ABSTRACT .....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Scope of the Dissertation..............................................................................................1 Lipids and Membranes.................................................................................................2 Membrane Models........................................................................................................6 Vesicles..................................................................................................................6 Langmuir Monolayers and Langmuir-Blodgett Films..........................................6 Phospholipid-protein films.............................................................................7 Phospholipid mixtures....................................................................................8 Brewster Angle Microscopy.......................................................................................12 Applications of Brewster Angle Microscopy.............................................................13 Phase Behavior of Langmuir Monolayers...........................................................13 Photochemical Isomerization..............................................................................15 Polymerization.....................................................................................................15 Gibbs Monolayers...............................................................................................16 2 BREWSTER ANGLE MICROSCOPY OF CALCIUM OXALATE MONOHYDRATE PRECIPITATION AT PHOSPHOLIPID PHASE BOUNDARIES...........................................................................................................22 Introduction.................................................................................................................22 Experimental Section..................................................................................................24 Materials..............................................................................................................24 Langmuir Monolayers.........................................................................................25 Brewster Angle Microscopy................................................................................25 Crystal Counting Procedures...............................................................................26 Scanning Electron Microscopy............................................................................26 Zeta Potential.......................................................................................................27 vi

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Results.........................................................................................................................27 Visualization of COM Growth at LC Langmuir Monolayers.............................27 Phospholipid Langmuir Monolayer Topographic Instabilities............................28 Using BAM to Quantify COM Growth at Monophasic Langmuir Monolayers.29 COM Growth at Single-Component Langmuir Monolayers with Phase Boundaries.......................................................................................................29 COM Growth at Phase-Separated Binary Phospholipid Mixtures......................30 Discussion...................................................................................................................32 Effect of Compressibility....................................................................................32 Effect of Phase Boundaries.................................................................................33 Expanded Phases.................................................................................................35 Conclusions.................................................................................................................36 3 PRECIPITATION OF CALCIUM OXALATE MONOHYDRATE AT MEMBRANE LIPID RAFTS MODELS...................................................................46 Introduction.................................................................................................................46 Lipid Rafts...........................................................................................................46 Cholesterol and Lipids.........................................................................................47 Lipid Raft Models................................................................................................48 Experimental Section..................................................................................................51 Materials..............................................................................................................51 Langmuir Monolayers.........................................................................................52 Atomic Force Microscopy (AFM).......................................................................52 Brewster Angle Microscopy (BAM)...................................................................53 Domain Area Calculation....................................................................................53 Crystal Counting Procedures...............................................................................53 Results.........................................................................................................................54 Sphingomyelin Monolayer..................................................................................54 Sphingomyelin/Dihydrocholesterol Monolayers.................................................55 POPC/Sphingomyelin/Dihydrocholesterol Monolayer.......................................55 POPC/Dihydrocholesterol Monolayer.................................................................57 Discussion...................................................................................................................58 Domain Formation at Binary and Ternary Lipid Mixtures.................................58 COM Precipitation at Phase Boundaries.............................................................60 Relevance to Membrane Lipid Rafts...................................................................61 Conclusions.........................................................................................................61 4 MONOLAYERS AS MODELS FOR SUPPORTED CATALYSIS: ZIRCONIUM PHOSPHONATE FILMS CONTAINING MANGANESE(III) PORPHYRINS......73 Introduction.................................................................................................................73 Experimental...............................................................................................................77 Materials..............................................................................................................77 Porphyrin Synthesis.............................................................................................77 Substrate Preparation...........................................................................................78 Film Formation....................................................................................................78 vii

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Film Characterization..........................................................................................79 Catalytic Oxidations............................................................................................79 Results.........................................................................................................................81 Optical Properties of the Porphyrins...................................................................81 Self-Assembled Monolayers...............................................................................81 Catalysis..............................................................................................................84 Characterization of the Catalyst Systems............................................................86 Heterogeneous and homogeneous reactions with 1.....................................86 Homogeneous reactions with 2....................................................................86 Discussion...................................................................................................................87 Comparing Homogeneous Reactions of 1 and 2.................................................88 Films of 1 vs the Homogeneous Reactions.........................................................89 Effect of Phosphonate..........................................................................................89 Monolayers as Models for Supported Catalysts..................................................90 5 SUMMARY................................................................................................................97 6 FUTURE WORK......................................................................................................100 Experimental.............................................................................................................101 Materials............................................................................................................101 POPC Vesicles...................................................................................................101 POPC/SM/Dihydrocholesterol Vesicles............................................................101 COM Precipitation.............................................................................................102 LIST OF REFERENCES.................................................................................................105 BIOGRAPHICAL SKETCH...........................................................................................116 viii

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LIST OF TABLES Table page 1-1 Structure of common glycerophospholipid head groups............................................3 1-2 Common alkyl tails found in glycerphospholipids....................................................3 1-3 Structures of amphiphiles in each chapter of this work.............................................4 1-4 Langmuir monolayers and thin films of phospholipid mixtures..............................12 2-1 Experimental conditions and COM formation for monolayers with phase boundaries................................................................................................................32 3-1 Expanded phase area to domain area ratios for the POPC/dihydrocholesterol monolayers with and without SM............................................................................61 4-1 Epoxidation of cyclooctene by PhIO and catalyzed by manganese porphyrins in monolayer films and in solution.a............................................................................85 ix

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LIST OF FIGURES Figure page 1-1 Singer and Nicolson fluid mosaic model of membranes, where lipids (open circles and black tails) are arranged as a fluid and random bilayer structure where proteins are embedded. 19 ...............................................................................18 1-2 Schematic representation of a Langmuir monolayer experiment.............................18 1-3 Schematic representation of the association of a GPI-linked protein to membranes...............................................................................................................19 1-4 Reflectivity of the air-water interface for polarizarizations p (in the plane of incidence) and s (perpendicular to this plane)..........................................................19 1-5 Schematic illustration of the principle behind Brewster Angle Microscopy...........20 1-6 Schematic representation of a pressure-area isotherm showing the possible phases present during the compression of an amphiphile........................................20 1-7 Compression isotherm of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) on a water subphase at 25 C and its corresponding BAM images.........................21 2-1 COM particles precipitated homogenously and used in zeta potential measurements...........................................................................................................38 2-2 BAM images of DPPC compressed to 30 mN/m over an RS 10 calcium oxalate subphase at 25.3 0.3 C.........................................................................................39 2-3 BAM images of DPPG over a room temperature RS 5 calcium oxalate subphase at (a) 27.5 mN/m and (b) 28.8 mN/m.......................................................................40 2-4 DPPC isotherm over an RS 10 calcium oxalate subphase at 25 C.........................41 2-5 Extent of COM precipitation at a DPPC monolayer (a) per unit area and (b) normalized to the trough area...................................................................................42 2-6 BAM image of DPPC compressed to 100 2 /molecule over an RS 5 calcium oxalate subphase at 25 C after 1 h (ST = 1/50 s)....................................................43 2-7 DPPC compressed to 5 mN/m over an RS 5 calcium oxalate subphase at 21.8 C after 16 h (ST = 1/120 s)..........................................................................................43 x

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2-8 Compression isotherm and BAM images (ST = 1/50 s) of a 1:1 DPPC/DMPC mixture over an RS 5 calcium oxalate subphase......................................................44 2-9 BAM images of a 1:1 DPPC/DMPC monolayer (ST = 1/50 s) held at 25 mN/m over an RS 5 subphase.............................................................................................45 2-10 BAM image of a 1:1 DPPC/DMPC monolayer held at 18 mN/m over an RS 5 calcium oxalate subphase at 21.8 C........................................................................45 3-1 Chemical structures of the raft forming species.......................................................63 3-2 Schematic representation of the membrane detergent extraction of lipid rafts........63 3-3 Schematic representation of physical state of lipid bilayers....................................64 3-4 Compression isotherm and BAM images (ST = 1/50 s) of a SM monolayer over an RS 5 calcium oxalate subphase...........................................................................65 3-5 BAM image of a SM monolayer (ST = 1/120 s) held at 32 mN/m over an RS 5 subphase at 25 C after 1.5 hours.............................................................................66 3-6 Compression isotherm and BAM images (ST = 1/50 s) of a 1:1 SM/ dihydrocholesterol monolayer over an RS 5 calcium oxalate subphase..................67 3-7 BAM images of a 2:1:1 POPC/SM/dihydrocholesterol monolayer held at 32 mN/m over an RS 5 subphase at 25 C...............................................................68 3-8 Distribution of the COM precipitated at the phase boundary, LE phase and LO domains of a 2:1:1 POPC/SM/dihydrocholesterol monolayer.................................69 3-9 AFM image of a 2:1:1 POPC/SM/dihydrocholesterol film transferred on mica after being held for 1 h at 32 mN/m.........................................................................69 3-10 AFM images of two 2:1:1 POPC/SM/dihydrocholesterol films transferred on mica after being held for 1 h at 32 mN/m................................................................70 3-11 BAM images of a 2:1 POPC/dihydrocholesterol monolayer (ST=1/50 s) held at 32 mN/m over an RS 5 subphase at 25 C...............................................................71 3-12 Distribution of the COM precipitated at the phase boundary and LE phase of a 2:1 POPC/dihydrocholesterol monolayer.................................................................71 3-13 AFM image of a 2:1: POPC/dihydrocholesterol film transferred at 32 mN/m on mica..........................................................................................................................72 4-1 Three-step deposition procedure for the formation of films of 1.............................92 xi

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4-2 Optical spectra of 1 as (a) 1 x 10 -6 M in CH 2 Cl 2 (b) monolayer film after deposition from ethanol/water and (c) the same film rinsed in methylene chloride for 30 minutes............................................................................................93 4-3 Optical spectra of (a) 2.4 x 10 -6 M solution of 2 in chloroform (b) solution of 2.4 x 10 -6 M of 2 with 1 x 10 -4 M ethylphosphonic acid and 1 x 10 -4 M triisobutyl amine in chloroform.................................................................................................93 4-4 ATR-FTIR spectrum of a film of 1 referenced to a zirconated LB layer in the region of the alkyl and phosphonate stretches.........................................................94 4-5 Extent of the epoxidation of cyclooctene with PhIO oxidant over 72 hours with catalysts: films of 1 (), 2 in solution () and no catalyst ()...............................94 4-6 Optical spectra of (a) the supernatant solution of an oxidation with a monolayer film of 1 after 72 hours, (b) a homogeneous reaction of 1 after 7 hours, (c) a film of 1 after 11 hours of reaction, and (d) a film of 1 immersed in the oxidative conditions after 10 minutes of tumbling...................................................95 4-7 Optical spectra of homogeneous reactions of 2 after (a) 1 hour and (b) 72 hours of tumbling...............................................................................................................95 4-8 Representation of the orientation of the porphyrin 1 in the monolayer films..........96 6-1 Representation of the preparation of lipid unilamellar vesicles.............................103 6-2 Schematic representation of two possible setups for the calcium depletion experiment in the presence of lipid vesicles...........................................................104 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy APPLICATIONS OF LANGMUIR-BLODGETT FILMS AND LANGMUIR MONOLAYERS: SUPPORTED CATALYSIS AND MEMBRANE MODELS FOR URINARY STONE FORMATION By Isa O. Bentez December 2004 Chair: Daniel R. Talham Major Department: Chemistry This dissertation looks at the precipitation of calcium oxalate monohydrate (COM) at phospholipid Langmuir monolayers with the aid of Brewster angle microscopy (BAM). COM crystals are monitored in situ with BAM as they appear as bright objects that are easily identified and quantified. Crystal precipitation was monitored at monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in liquid condensed (LC) and liquid expanded (LE) phases as well as biphasic LC/LE and LE/gas (G). COM appears preferentially at phase boundaries of the single-component monolayers. However, when an LC/LE phase boundary is created by two different phase segregated phospholipids, crystal formation occurs away from the interface within the LC phase. COM growth at phase boundaries is preferred only when there is molecular exchange between phases. Biologically relevant monolayers were prepared by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ Sphingomyelin (SM)/dihydrocholesterol in a 2:1:1 ratio where the SM/sterol components form domains equivalent to lipid rafts in xiii

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membranes. COM precipitation at these monolayers occurs exclusively at phase boundaries. An investigation of organized monolayer films of a manganese tetraphenylporphyrin used as supported oxidation catalysts is also discussed. Manganese 5,10,15,20-tetrakis(tetrafluorophenyl-4’-octadecyloxyphosphonic acid) porphyrin (1) has been immobilized as a monolayer film by a combination of Langmuir-Blodgett (LB) and self-assembled monolayer techniques. Film analysis shows that the films consist of non-interacting molecules anchored and oriented nearly parallel to the surface. The monolayers are stable to the solvent and temperature conditions needed to explore organic oxidations. The activity of films of 1 towards the epoxidation of cyclooctene using iodosylbenzene as the oxidant was compared to that of Manganese 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (2) and 1 under equivalent homogeneous conditions. The immobilized porphyrin 1 shows an enhanced activity relative to either homogeneous reaction. The main difference between 1 and 2 is the four alkyl phosphonate arms in 1 designed to incorporate the porphyrin within the films. The increased activity of immobilized 1 is a combination of the porphyrin structure, which prohibits the formation of -oxo dimers even in solution, and a change in conformation when anchored to the surface. The study demonstrates that careful monolayer studies can provide useful models for the design and study of supported molecular catalyst systems. xiv

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CHAPTER 1 INTRODUCTION Scope of the Dissertation Chapters 2 and 3 describe the use of lipid Langmuir monolayers as membrane models for the precipitation of calcium oxalate monohydrate (COM). The motivation for this project was initiated by the success of Whipps and Backov in our group at characterizing COM growth ex situ with SEM. 1-4 BAM is used here as a means of characterizing crystal formation in situ. Single-component and binary phospholipid monolayers showed that the precipitation can be observed and quantified and that phase boundaries play an important role in COM formation. More biologically relevant monolayers were prepared by mixing POPC, cholesterol and sphingomyelin. These lipids are commonly used as representative of those found in the outer leaflet of the human plasma membrane and are thought to be involved in the formation of lipid rafts. 5-14 The raft mixture at biologically relevant surface pressures forms cholesterol-sphingomyelin enriched domains whose phase boundaries are sites of COM precipitation. Chapter 4 of this dissertation describes the catalytic activity of a porphyrin immobilized at a Langmuir-Blodgett monolayer. The films are prepared by a well understood process, 15,16 involving the deposition of a monolayer of octadecylphosphonic acid (ODPA) on hydrophobic glass, followed by a reaction with zirconyl chloride. The reaction with Zr 4+ cross-links the ODPA acid groups, creating a reactive surface towards phosphonic acid containing molecules. This film was then used to deposit a phosphonic acid derivatized porphyrin prepared by our collaborators Bruno Bujoli and Fabrice 1

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2 Odobel at the Universit de Nantes, France. Spectroscopic characterization of the catalytic film indicated that the porphyrin molecules are non-interacting and lie nearly flat on the surface. The activity of this film towards the epoxidation of an alkene was then compared to the homogeneous reaction of an equivalent porphyrin with no acid groups. The remainder of this chapter is a literature review on the use of lipid monolayers as membrane models as well as the principle and use of Brewster angle microscopy. A review on mineralization at Langmuir monolayers and vesicles can be found in the dissertation by Scott Whipps. 17 A description of the optical properties, immobilization and catalytic properties of porphyrins is presented in the dissertation by Christine Lee. 18 Lipids and Membranes Lipids are molecules of biological origin soluble in organic solvents such as ether and chloroform and insoluble in water. 5 The major class of lipids present in membranes are glycerophospholipids, which are amphiphilic molecules with a polar head group and two non-polar hydrophobic tails. The structures of some common glycerophospholipid head groups are summarized in Table 1-1. Common glycerophospholipid aliphatic tails are described in Table 1-2. The carbon number in Table 1-2 (A:B) refers to the number of carbons of the aliphatic tail (A) and number of unsaturated bonds in that chain (B). It is possible that the alkyl tails of a glycerophospholipid differ in chain length and unsaturation; for example the 16:0-18:1 choline lipid can be referred to as palmitoyloleoylphosphatidylcholine or POPC. The names, abbreviation, and structures of all amphiphiles used in this work are summarized in Table 1-3.

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3 Table 1-1. Structure of common glycerophospholipid head groups. R1OOR2OOOPOXOStructure of X Name of phospholipid -OH Phosphatidic acid (PA) ON(CH3)3+ Phosphatidylcholine (PC) ONH3+ Phosphatidylethanolamine (PE) OOHOH Phosphatidylglycerol (PG) ONH3COO-+ Phosphatidylserine (PS) OHOHOHOHOHO Phosphatidylinositol (PI) Table 1-2. Common alkyl tails found in glycerophospholipids Carbon number Common name IUPAC name 16:0 Palmitoyl Hexadecanoic 18:0 Stearoyl Octadecanoic 18:1 Oleoyl 9-cis-Octadecenoic 20:0 Arachidoyl Eicosanoic 20:4 Arachidonoyl 5,8,11,14 (all -cis) Eicosatetraenoic 22:0 Behenoyl Docosanoic 24:1 Nervonoyl 15-cis-Tetracosenoic

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4 Table 1-3. Structures of amphiphiles in each chapter of this work. amphiphile abbreviation chapter structure Octadecylphosphonic acid ODPA 4 CH3(CH2)17PO3H2 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine DPPC 2 (CH3)3N+OPOO-OOHOR1OOR2 R 1 = R 2 = (CH 2 ) 14 CH 3 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPC 2 (CH3)3N+OPOO-OOHOR1OOR2 R 1 = R 2 = (CH 2 ) 12 CH 3 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] DPPG 2 OPOO-OOHOR1OOR2OHHO R 1 = R 2 = (CH 2 ) 14 CH 3 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE 2 H3N+OPOO-OOHOR1OOR2 R 1 = R 2 = (CH 2 ) 14 CH 3 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] DPPS 2 OPOO-OOHOR1OOR2HH3N+O-O R 1 = R 2 = (CH 2 ) 14 CH 3 Brain sphingomyelin SM 3 H3N+OPOO-OHNHOR(CH2)12CH3HOH R = mixture 46% (CH 2 ) 14 CH 3 and others. Saturated/unsaturated ratio=10 Dihydrocholesterol ___ 3 HOCH3HCH3HHH3C

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5 Table 1-3. Continued. amphiphile abbreviation chapter structure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC 3 (CH3)3N+OPOO-OOHOR1OOR2 R1= (CH2)14CH3R2=(H2C)7 (CH2)7CH3 Membrane lipids form bilayer structures where the polar head groups are oriented towards the aqueous face and the alkyl tails are secluded from the water. 5 In 1972 Singer and Nicolson exposed their fluid mosaic model of the cell membrane structure, where it is proposed that membrane proteins function as icebergs in a fluid sea of bilayer lipids, Figure 1-1. 19 The validity of the model has been shown in a number of experiments, of which that performed by Frye and Edidin has had a large impact. 20 In this work, proteins of human and mouse cells were labeled with different fluorescent markers and then fused to form a heterokaryon. The human and mouse components were segregated immediately following the fusion, but after 40 minutes at 37 C, the proteins were thoroughly mixed. Addition of protein synthesis inhibitors did not slow the mixing, but lowering the culture temperature to 15 C did. 20 This experiment shows that proteins diffuse through the fluid membrane, a process that can be slowed by lower temperatures. The Singer and Nicolson model remains largely relevant to this day, except that some lateral organization has been found among the membrane lipids. 6-14 These organized membrane lipid structures, called lipid rafts, are the subject of discussion in Chapter 3.

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6 Membrane Models Vesicles When lipids that form bilayers are hydrated and agitated, they tend to form multilamellar vesicles (MLVs) or liposomes, where a series of concentric lipid bilayers are separated by water. MLVs can then be further treated to obtain unilamellar vesicles consisting of a single bilayer. Small unilamellar vesicles (SUVs) with a diameter of 25-40 nm, large unilamellar vesicles (LUVs) with a diameter of 50-500 nm and giant unilamellar vesicles (GUVs) with a diameter of several microns have been prepared. 5 Membrane model studies can be carried out on the free standing unilamellar vesicles or in solid supported bilayers prepared by vesicle fussion. Free standing and supported bilayer vesicles provide little control on the molecular density and phase state of the lipids, which can be easily manipulated in Langmuir monolayers and Langmuir-Blodgett films. Therefore, the discussion on membrane models will focus on these. Langmuir Monolayers and Langmuir-Blodgett Films Langmuir monolayers are monomolecular films prepared at an interface, most often between air and water. The films are formed by amphiphilic molecules where the polar head group is immersed in the aqueous subphase and the alkyl tail remains in the air. They can be compressed by barriers and the state of the monolayer monitored by a Wihelmy balance, Figure 1-2. The monolayers can be transferred to a solid support in order to extend their applications as thin films, performing additional characterization not accessible at the air-water interface and to form multilayers. The transferred monolayer is called a Langmuir-Blodgett or LB film. An extensive review on the formation of these films can be found in the original papers by Irving Langmuir and Katherine Blodgett 21-24 as well as dissertations 18,25,26 and books 27-29 on the subject.

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7 It is easy to see that a Langmuir monolayer of an amphiphilic compound, Figure 1-2, closely resembles the structure of half of a membrane bilayer, Figure 1-1. This fact has been recognized early on, and studies have been carried out to determine how comparable Langmuir monolayers are to membranes and bilayers. Demel et al. studied the effect of a series of phospholipases on 14 C-labelled Langmuir monolayers of POPC, POPC/sphingomyelin and POPC/sphingomyelin/cholesterol. 30 Phospholipiases are a group of enzymes capable of hydrolyzing phospholipids at the acyl-ester position, the glycerophosphate bond or the head group. 31 It was found that the phospholipases studied that do not hydrolyze erythrocyte membranes cannot hydrolyze the monolayers at pressures above 31 mN/m. The phospholipases that can hydrolyze erythrocyte membranes can also hydrolyse the monolayers at pressures of above 31 mN/m. It was concluded that the packing of the erythrocyte membrane is comparable to surface pressures between 31 and 34.8 mN/m. In addition, Blume found by calorimetric and isobar (area versus temperature at constant surface pressure) experiments that at an approximate pressure of 30 mN/m monolayers of choline, ethanolamine, methyl ester and phosphatidic acid phospholipids were comparable to the bilayer of the same lipid. 32 It is therefore possible to use Langmuir monolayers and the transferred films to model membranes. Phospholipid-protein films It is known that proteins associate with membranes, as described by the model of Singer and Nicolson. 19 A number protein-lipid interactions have been studied in the literature with the use of Langmuir monolayers. 33 Of recent interest are glycosylphosphatidyliositol (GPI)-anchored proteins as it is believed that the GPI-anchor allows the protein to interact with lipid rafts. 6,34 These proteins interact with the

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8 membrane through a phosphatidylinositol group linked to two alkyl tails by a phosphodiester bond, Figure 1-3. The interactions of GPI-anchored proteins with membranes can be modeled by the use of phospholipid Langmuir monolayers. Ronzon et al. have studied the interaction of an alkaline phosphatase (AP)-GPI with phospholipid monolayers of 16:0-16:0 phosphatidylcholine (DPPC) and 16:0-16:0 phosphatidylserine (DPPS). 35 An increase in the molecular area in the isotherm of both phospholipids showed that the protein interacts with both monolayers. Infrared spectroscopy showed that while AP-GPI modifies the organization of the DPPS alkyl tails, it only interacts with the DPPC head group at low surface pressures. The protein perturbs the organization of the DPPC alkyl tails only at high pressures, 30 mN/m, which is not surprising considering that this pressure is biologically relevant. 30,32 The different lipid-protein interactions were attributed to the hydration layers induced by DPPC and DPPS. Ronzon et al. also investigated the effect of the alkyl tail unsaturation by studying the effect of the same AP-GPI protein in 18:0-18:0 phosphatidylcholine (DSPC) and 18:1-18:1 phosphatidylcholine (DOPC). 36 By monitoring the pressure increase of the monolayer upon addition of AP-GPI, it was found that the protein penetrates more effectively into the saturated DSPC monolayer. This finding is in agreement with the isolation of GPI-linked proteins with ordered rafts domains. 34 Phospholipid mixtures Membranes are always composed of a mixture of lipids that may include PE, PC, PS, sphingomyelin and cholesterol. 5 Membrane models often include Langmuir monolayers and LB films of two or more lipids to study their miscibility and/or phase separation. This section reviews some of the recent efforts towards preparing and characterizing mixed phospholipid films.

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9 Soletti et al. performed AFM studies on the effect of phospholipid unsaturation on the phase separation of LB films. 37 The DPPE/DOPE mixture exhibits formation of domains of higher height and whose area corresponds to the molar fraction of DPPE used. At the transfer pressure used, 40 mN/m, DPPE is expected to be in a condensed state whereas DOPE remains expanded due to the unsaturation of its alkyl tails. The difference in lipid packing clearly leads to phase segregation. The phase separation for the DPPC/DOPE monolayer was dependent on the molar ratio used, where DPPC in a molar fraction of 50% or above leads to segregation. Finally, the DPPC/DPPE monolayer did not induce any domain formation in the transferred film. 37 Dufrne et al. studied a 1:1 DSPE/DOPE LB monolayer and bilayer by AFM. 38 It can be anticipated from the discussion above that this film also shows phase separation of condensed DSPE domains in an expanded DOPE matrix. Imaging of the monolayer in air in contact mode revealed that film deformation can occur yielding higher than expected height differences between the expanded and condensed phases. It is thus postulated that, in addition to molecular length and tilt, mechanical properties of the different phases can affect the topographic contrast imaged by AFM. In solution, the DSPE/DOPE bilayer exhibits short-range repulsive forces, an additional factor affecting the domain contrast. 38 Badia et al. carried out a series of AFM studies of LB monolayers and bilayers and Langmuir-Schaeffer monolayers of the DPPC/DLPC mixture. 39-42 These lipids contain the same choline group but their alkyl tail lengths differ by 4 carbons. While DLPC cannot form a condensed phase at room temperature due to its short alkyl tails, DPPC can form closed packed domains. Therefore, phase segregation of these lipids is observed.

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10 AFM imaging of 1/1 and 1/3 DPPC/DLPC LB films shows phase separation with the DPPC condensed phase having the expected area and higher height difference from the DLPC phase. It was also found that the DPPC regions were arranged in parallel stripes due to the distortion of the condensed domains at the contact line between the substrate, subphase and air. The buildup of DPPC at the contact line is followed by a depletion region where DLPC is deposited, resulting in the stripe morphology. 39,40 The striped monolayer was later used to demonstrate the selective adsorption of human serum albumin (HSA) and human globulin (HGG) to the DLPC phase of the monolayer. 41 Although the DPPC/DLPC LB films provide an interesting method for patterning at the nanometer scale, they do not reflect the true morphology of the DPPC domains at the air-water interface. To study the topography of the phase segregated DPPC/DLPC films, Langmuir-Schaefer monolayers were prepared on alkylated gold substrates and studied with AFM. 42 In the Langmuir-Schaefer technique, a monolayer is spread at the air-water interface and a substrate is placed horizontally on the monolayer film. 28 The support can then be lifted away from the surface or, as in the case of the DPPC/DLPC monolayer, it can be pushed through the interface and kept in water to avoid monolayer reorganization. While no domain formation was observed for either lipid on the hydrophobic Au support, DPPC regions were imaged when mixed films were transferred at surface pressures above the mixture phase transition. The qualitative effect of the compression speed and surface pressure on DPPC domain morphology was investigated. 42 Mixtures containing lipids isolated from biological tissue have also been reported. DeWolf et al. prepared mixtures of DSPC and phosphatidylinositol (PI) extract from bovine liver. 43 The PI obtained is a mixture of saturated and unsaturated amphiphiles

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11 with varying alkyl tail lengths. This heterogeneity causes PI to remain in an expanded state whereas the fully saturated DSPC forms a condensed phase. Since PI is a relatively minor component of membranes, only 5% to 20% PI was used. Images of the DSPC/PI obtained with Brewster angle microscopy (BAM), a technique discussed in the section below, appear inhomogeneous suggesting phase separation. AFM of transferred LB films showed the condensed DSPC domains with the expected area for the lipid ratio used. Also, grazing incidence X-ray diffraction (GIXD) was used to obtain information on the alkyl tail organization in the fixed film. GIXD shows some minimal amount of PI present within the DSPC phase. 43 Phase separation can also be induced by factors other than the ability of lipids to form close-packed arrays. Ross et al. studied the calcium-induced domain formation in Langmuir monolayers and LB films of 4:1 DPPC/DPPS. 44 These lipids have identical alkyl tails but their head groups are zwitterionic (PC) or negatively charged (PS). The presence of calcium ions in the subphase interacts strongly with DPPS but not with DPPC, inducing phase separation. Since DPPS and DPPC have the same alkyl tails, topographic AFM measurements would yield no indication of lipid segregation. Instead, fluorescence microscopy of the DPPC/DPPS Langmuir monolayer, and TOF-SIMS, SEM and lateral force microscopy of the LB films clearly showed DPPS rich domains formed in the presence of calcium. 44 The work described above, in addition to other recent studies, is summarized in Table 1-4, which includes film composition, film type and method of detection. Membrane models containing lipid raft forming components (cholesterol and sphingolipids), are reviewed in the introduction of Chapter 3.

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12 Table 1-4. Langmuir monolayers and thin films of phospholipid mixtures. Lipids Film type Method(s) of detection Reference DPPC/DPPG and DPPC/DOPG Langmuir monolayer Free energy calculation from isotherm data 45 DPPE/DOPE, DPPC/DOPE and DPPE/DPPC LB AFM 37 DSPE/DOPE LB AFM 38 DPPC/DPPG, DPPC/DOPG and DPPC/POPG LB Fluorescence microscopy 46 Lipid Extract from Red Blood Cells Langmuir monolayer Fluorescence microscopy 47 DSPC/PI (extract from bovine liver) Langmuir monolayer and LB BAM, AFM, GIXD 43 DSPE/DOPE Langmuir monolayer AFM 48 DPPC/Ceramide 3 LB AFM 49 DPPC/DPPS LB Fluorescence microscopy, SEM, TOF-SIMS, AFM 44 Protein/phospholipids/neutral lipids extracted from calf surfactant Langmuir monolayer Fluorescence microscopy 50 DPPC/DLPC LB AFM 39,40,42 Brewster Angle Microscopy Brewster angle microscopy (BAM) has emerged as a widely used technique to image Langmuir monolayers in situ over the past 10 years. The first report of a home-built Brewster angle microscope appeared in 1991 by Hnon and Meunier. 51,52 Today highly sophisticated instrumentation is available commercially. The principle of operation is based on the Brewster angle, B , at which linearly polarized light directed

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13 towards a boundary of two media having different refractive indices will transmit completely from one medium to the other. The Brewster angle is given by B = tan -1 (n 1 /n 2 ) where n 1 and n 2 are indices of refraction of the media involved. The Brewster angle for the air-water interface is 53. During a typical BAM experiment at the air-water interface, a vertically polarized laser beam is directed towards the water surface and adjusted to the Brewster angle. A polarized (in the plane of incidence) laser source must be used as its reflectivity at the Brewster angle vanishes, Figure 1-4. 52 Under these conditions, a camera opposing the laser source will not detect any light, Figure 1-5. When a monolayer of a different refractive index is spread at the air-water interface a detectable change in the reflectivity of a factor of about 35 occurs, Figure 1-5. 52 This reflected light is then used to form an image of the monolayer. Although most BAM experiments are performed at the air-water interface, an air-solid interface can also be interrogated by use of the appropriate Brewster angle. 53 Applications of Brewster Angle Microscopy Phase Behavior of Langmuir Monolayers A very useful application of BAM is that it enables visual characterization of the phase behavior of monolayers at the air-water interface. As a Langmuir monolayer is compressed, it will exhibit distinct phases analogous to the gas, liquid and solid states in three-dimensional materials. At high MMA a disorganized Gas (G) phase is present, although practically a pure G phase is not observed as it requires very large trough areas. Compression of the monolayer will yield a liquid expanded (LE) phase where the molecules are still disorganized but in closer proximity and finally a liquid condensed

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14 phase (LC) where the amphiphiles are closed packed and uniformly oriented. These phases can be identified in an isotherm by sharp slope changes as shown in Figure 1-6. There can also be coexistence regions of G/LE and LE/LC phases which appear as plateaus in the isotherm, Figure 1-6. The shape of the isotherm pictured in Figure 1-6 is not necessarily observed for all surfactants, as coexistence regions might not occur or a LC phase might not form due to the inability of the molecules to close-pack. The presence or lack of these phases can be easily observed by BAM for any Langmuir monolayer at the air-water interface. The early studies on the monolayer visualization by BAM were carried by Hnon and Meunier 51 and Hnig and Mbius. 54 Hnon and Meunier give an extensive description of their home-built BAM as well as the first images of myristic acid in a G/LE and LE/LC coexistence. Shortly after that, Hnig and Mbius reported the reflectivity-area isotherms of arachidic acid and 14:0-14:0 phosphatidylethanolamine (DMPE). A clear correlation of reflectivity with monolayer density was observed. BAM images of DMPE were recorded, where formation and growth of LC domains at the LE/LC equilibrium were identified. 54 Detection of monolayer phases can also be performed with the commercial instrumentation available today, although images of better quality can now be obtained. Figure 1-7 shows the compression isotherm and corresponding BAM images of DPPC recorded in our laboratory by the commercial BAM2plus system (Nanofilm Technologie GmbH, Goettingen, Germany) over a water subphase at room temperature. Imaging at the LE/G, LE, LE/LC and LC regions show that these phases can be easily identified as dark gray for the G phase, gray for the LE phase and light gray for the LC phase, Figure 1-7. The interference fringes, most obvious

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15 in Figure 1-7c and d, are an artifact produced by the BAM optical components. The propeller-like domains in Figure 1-7c are typical for DPPC and have been observed by fluorescence microscopy. 55 Photochemical Isomerization BAM has been used to study the photochemical isomerization of an amphiphilic azobenzene derivative at the air-water interface. 56,57 The reflectivity of the trans-isomer is remarkably high, about 15 times higher than that of arachidic acid when they are both in an LC state. This is due to the higher thickness and refractive index of the trans azobenzene film. The trans isomer also has a much higher reflectivity than the cis isomer, and the kinetics of the cis-trans isomerization monitored by BAM show that the process is fully reversible. 56 Mixing of the azobenzene amphiphile with dimyristoylphosphatidic acid in a 1:1 ratio allows for the unconstrained isomerization and therefore the monolayer structure is retained during the process. 57 The reflectivity contrast for the mixed film between the cis and trans states is also quite high, although not as much as for the pure azobenzene derivative. In addition, just as in the case for the pure film, the isomerization is fully reversible. 57 Polymerization BAM can be helpful when monitoring the polymerization of an LB film. Mller and Riedel investigated the electron polymerization of an octadecyl methacrylate (ODMA) film on glass. 58 In the absence of an ATR-IR spectrometer to monitor the decrease of the C=C IR absorption, BAM was used to monitor the ODMA films before and after polymerization. Domains with differing alkyl tail tilt, which can be observed by the use of an analyzer, were eliminated after the treatment. It was also found that the

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16 films before treatment were rapidly destroyed after being exposed 20 min to vacuum, whereas the polymerized ODMA was stable. 58 BAM can also be used to monitor polymerization at the air-liquid and liquid-liquid interface. Uredat and Findenegg investigated the polymerization of 4,4’-isopropylidenediphenol-dimethacrylate (IDDMA) at the n-dodecane/water and the n-hexane/water interface. 59 When a solution of IDDMA in the hydrocarbon is brought in contact with water a monolayer of the monomer forms which is then polymerized by UV-radiation. BAM images shows progressively brighter films as the radiation proceeds indicating monolayer cross-linking followed by multilayer formation. 59 Britt et al. observed the polymerization of mixed amphiphilic diacetylenes with fluorescence microscopy and BAM. 60 The polymerization was carried out at the air-water interface and compared to the film supported on glass, hydrophobic glass, cadmium arachidate bilayers on glass and mica. Increased intensity was observed by BAM as the polymerization occurs. The effectiveness of the reaction varied with the support used as water>>mica>cadmium arachidate bilayer>(glass=OTS), where the bare glass and OTS supported films did not undergo any polymerization. The reaction efficiencies are explained in terms of monolayer mobility. 60 Gibbs Monolayers The adsorption of soluble amphiphiles at the air-water interface leads to the formation of Gibbs monolayers. Vollhardt et al. demonstrated the usefulness of BAM in the study of the adsorption layers with a series of acid amides. 61-63 The amphiphilic molecules were carefully designed so that they will have high surface activity, are soluble in water to a certain extent and are soluble in organic solvents that allow the preparation of Langmuir monolayers. Careful comparison of the Langmuir and Gibbs films by BAM

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17 showed for the first time that Gibbs monolayers are capable of undergoing a phase transition. The formation of LC domains in equilibrium with the LE phase can be observed as the adsorption progresses. 61-63

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18 Figure 1-1. Singer and Nicolson fluid mosaic model of membranes, where lipids (open circles and black tails) are arranged as a fluid and random bilayer structure where proteins are embedded. 19 subphase Figure 1-2. Schematic representation of a Langmuir monolayer experiment. Wilhelmy balance barrier monolayer

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19 O NH CH2 O P OO O Man3-GlcN HO OH O(CH2)nCH3 OH O P O OO ( )n( )n PhosphatidylinositolProtein Membrane bilayer( )2 Figure 1-3. Schematic representation of the association of a GPI-linked protein to membranes. Figure 1-4. Reflectivity of the air-water interface for polarizarizations p (in the plane of incidence) and s (perpendicular to this plane).

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20 B No reflection Reflection water air water air filmB Figure 1-5. Schematic illustration of the principle behind Brewster Angle Microscopy. LE + GLELE + LC LC Mean Molecular Area G Figure 1-6. Schematic representation of a pressure-area isotherm showing the possible phases present during the compression of an amphiphile.

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21 a LC LE LE bG LE c LC d Figure 1-7. Compression isotherm of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) on a water subphase at 25 C and its corresponding BAM images at the (a) G/LE coexistence, (b) LE phase, (c) LE/LC coexistence and (D) LC phase.

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CHAPTER 2 BREWSTER ANGLE MICROSCOPY OF CALCIUM OXALATE MONOHYDRATE PRECIPITATION AT PHOSPHOLIPID PHASE BOUNDARIES Introduction Calcium oxalate and calcium phosphate are the principal crystalline materials found in urinary stones. 64,65 The inorganic crystals are always mixed with an organic matrix composed of carbohydrates, lipids, and proteinaceous materials that account for about 2% of the total mass of the stones, although a much larger percentage of the total volume. 66,67 It has been shown that lipid matrixes induce the in vitro precipitation of calcium oxalate from metastable solutions. 68 In addition, there is evidence that calcium oxalate precipitation can be induced in vivo by renal epithelial cells 69,70 as well as in vitro by membrane vesicles isolated from renal brush-border membranes. 71,72 To better understand the process of stone formation, it is important to study interactions between the organic and crystalline components. Our group has previously performed a series of studies on calcium oxalate precipitation at an interface provided by phospholipid Langmuir monolayers that serve as models for the phospholipid domains within membranes. 1-4 It was observed that the Langmuir monolayers can effectively catalyze the precipitation of calcium oxalate monohydrate (COM) and that the identity of the monolayer has a strong influence on the rate of crystal formation. Negatively charged monolayers, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (DPPS), induce more extensive precipitation than the neutral monolayer 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 22

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23 (DPPC), implicating a mechanism whereby the calcium ions are concentrated at the interface promoting nucleation. 1,2 Consistent with this mechanism is the observation that a large majority of crystals produced had their calcium-rich (10-1) face oriented toward the monolayer. Experiments performed with DPPC and DPPG at different degrees of monolayer compression revealed that the COM number density more than doubles when the surface pressure is decreased from 20 mN/m to 0.1-0.3 mN/m. 2,3 This result suggested that more fluid monolayers have the ability to reorganize to accommodate and stabilize nucleating or growing crystals. To further investigate the effect of surface pressure on crystal formation, a series of phospholipids with a glycerol headgroup and alkyl tails of differing lengths and degrees of saturation (DPPG, dimyristoylphosphatidylglycerol (DMPG), palmitoyl-oleoylphosphatidylglycerol (POPG) and dioleoylphosphatidylglycerol (DOPG)) was used. 3 Within this series, DPPG is the only one able to form a liquid condensed (LC) phase at room temperature while all others remain in a liquid expanded (LE) phase due to a short alkyl tail (DMPG) or the presence of unsaturated bonds (POPG and DOPG). When COM growth was compared at the different phosphatidylglycerols held at high pressure, where DPPG is in an LC phase and the others are in an LE phase, crystallization was greatest at the DPPG layer. When the same series was compared at low pressure, where each is in an LE phase, COM formation was again greatest at DPPG. The results suggested that crystal nucleation is greatly enhanced if a monolayer has the potential to organize in a small area, although preorganization is not necessary. In these experiments, the same selectivity for the (10-1) face relative to the other COM faces remained nearly constant.

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24 These earlier studies also raised questions about the role of monolayer phase boundaries in the crystallization process. DPPG can exist as LC, LE, or in a coexistence region. These effects are difficult to quantify if crystallization is monitored ex situ with electron microscopy. Our group previously demonstrated that Brewster angle microscopy (BAM) can be used for in situ observation of COM crystals growing at Langmuir monolayers. 1,3 BAM has also been recently used to monitor calcium carbonate precipitation under fatty acid monolayers. 73 Advances in the commercial instrumentation now allow high-quality images of the monolayer and crystals located at the air-water interface. In this study, BAM is used to provide further quantitative evidence of the in situ observation of COM precipitated at phospholipid interfaces as well as show the spatial distribution of the crystal growth in monolayers where two phases are present. Two kinds of mixed-phase monolayers are studied, a single phospholipid in equilibrium at a phase change and a phase-separated binary mixture of different phospholipids. Experimental Section Materials All reagents were purchased from commercially available sources and used without further purification. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) (purity >99%) were purchased from Avanti Polar Lipids (Alabaster, AL). Sodium oxalate and tris(hydroxymethyl)aminoethane hydrochloride (Tris . HCl) were purchase from Aldrich Chemical Co. (Milwaukee, WI). Calcium chloride dihydrate and sodium chloride were obtained from Fisher Scientific (Pittsburgh, PA).

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25 Langmuir Monolayers A KSV Instruments (Stratford, CT) Langmuir-Blodgett system KSV 2000 was used in combination with a 700 cm 2 Teflon double-barrier trough. A paper Wilhelmy balance suspended from a microbalance was used to monitor the surface pressure. Subphases were prepared with pure water of resistivity of 18.0-18.2 M cm from a Barnstead (Boston, MA) NANOpure system. To prepare calcium oxalate subphases, two solutions of equal volume of 150 mM NaCl and 5 mM Tris . HCl were prepared and the appropriate amount of calcium was added to one and oxalate to the other to achieve relative supersaturation (RS) values of 5 or 10 (0.35 mM and 0.50 mM, respectively) once combined. The pH of both solutions was adjusted to 7.00 with an aqueous KOH solution. The solutions were combined, filtered through paper of fine porosity and slow flow rate, and used immediately. The subphase temperature was adjusted with a Fisher Scientific model 900 isotemp refrigerated circulator. The phospholipids were dissolved in 9:1 choroform/methanol and spread on the subphase. The monolayer was allowed to equilibrate undisturbed for 45 min and compressed at a rate of 3 mN/m/min with a maximum speed of 5 mm/min. Brewster Angle Microscopy BAM experiments were performed using a Nanofilm Technologie GmbH (Goettingen, Germany) BAM2plus system with the Langmuir-Blodgett trough described above. A polarized Nd:YAG laser (532 nm, 50 mW) was used with a CCD camera (572 x 768 pixels). The instrument is equipped with a scanner that allows an objective of nominal magnification of 10x or 20x to be moved along the optical axis, producing a focused image. For the 10x objective a laser power of 50% and maximum gain is used. A shutter timing (ST) of 1/50 s, 1/120 s or 1/1000 s is used to obtain maximum contrast

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26 between the monolayer and the COM crystals. For the 20x objective a laser power of 80%, maximum gain and ST of 1/50 s are always used. The distortion of the images due to the angle of incidence is corrected by image processing software provided by the manufacturer. The incident beam is set at the Brewster angle in order to obtain minimum signal before spreading the monolayer. A piece of black glass is placed at the bottom of the trough to absorb the refracted light beam that would otherwise cause stray light. The polarizer and analyzer are set at 0 for all experiments. The laser and camera are mounted on an x-y stage that allows examination of the monolayer at different regions. Crystal Counting Procedures For each crystal growth experiment, an area of 8.25 – 16.51 mm 2 was carefully analyzed by taking 36 72 BAM pictures of different areas of the monolayer with a 10x objective and ST of 1/120 s. Each experiment was repeated at least three times. The time was set to zero at the point where the monolayer reached the desired target pressure. To account for the different amounts of time required to reach different pressures, the crystal number density obtained at time zero for each experiment was subtracted from that obtained at all other times. Therefore the number densities reported correspond to crystals formed at the target pressure. Scanning Electron Microscopy Scanning electron microscopy (SEM) images were obtained with a Hitachi S-4000 FE-SEM at 6 kV. The sample of COM formed at a DPPC monolayer was prepared by transferring the film onto a piece of silicon wafer immersed in the subphase before the spreading of the monolayer. The wafer was placed at an angle of ca. 45 with respect to the water surface, and the upstroke speed was 8 mm/min. The film was dried at 40 C overnight and coated with gold prior to sample examination.

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27 Zeta Potential COM crystals were precipitated homogeneously as described elsewhere. 74 SEM of the precipitated particles confirmed their identity, Figure 2-1. When an electric field is applied to charged particles and their closely associated counter ions, they will move through the solution as an unit and the potential between this unit and the surrounding medium is called zeta potential (). The particle charge can be obtained from the following expression derived from the Poisson equation: = q /(4a(1+a/ D )) where q is the charge of the particle, a is the particle radious is the permittivity of vacuum time the relative permittivity and D is the Debye screening length. For the case of an aqueous solution, =( 8.85x10 -12 C 2 N -1 m -2 )(78.54). The Debye length can be approximated as D (3)/(C salt ) 1/2 where C salt is the molar concentration of the salt in the dispersing medium. The zeta potential on COM dispersed in an RS 5 subphase at room temperature was measured with a ZPi Zeta Reader (Zeta Potential Instruments, Inc., Bedminster, NJ). Results Visualization of COM Growth at LC Langmuir Monolayers COM crystals precipitating at Langmuir monolayers can be observed in situ by BAM, as demonstrated here for a phospholipid in an LC phase. A DPPC monolayer was spread on a subphase of RS 10 at 25.3 0.3 C, compressed, and held at 30 mN/m. The high RS value was chosen to ensure abundant COM precipitation, while the subphase temperature was controlled as it affects the lipid phase behavior. As shown by Figure 2-2a, the BAMplus instrument allows capture of highly focused pictures of COM crystals at their early growth stages, 1 h in this case. After a few hours, a distribution of crystal

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28 sizes can be observed indicating that COM nucleation and growth occur simultaneously, Figure 2-2b. After 16 h the crystals were abundant, large (ca. 30 m) and presented the elongated shape characteristic of COM grown at phospholipid interfaces, 1 Figure 2-2c. Since the COM crystals appear very bright, the image of Figure 2-2c was obtained by reducing significantly the light allowed to reach the camera, causing the normally white background associated with the LC phase of DPPC to appear black. The BAM laser and camera are mounted on an x-y stage over the trough to examine the monolayer at different regions showing that the crystal formation occurs evenly in the area observed (8.25 – 16.51 mm2). To confirm the identity of crystals observed by BAM, a similar film was prepared and held at 30 mN/m overnight and then transferred and examined by SEM. The morphology of the transferred crystals, Figure 2-2d, is consistent with COM formed under phospholipid monolayers in previous studies. 1-4 Therefore, it can be said with confidence that BAM provides images of COM precipitating at the air-water interface. Phospholipid Langmuir Monolayer Topographic Instabilities Although rarely observed in Langmuir monolayers, topographic instabilities are caused by packing defects at the LE to LC phase transition. 75 The instabilities can be readily identified by BAM as bright objects in the LE phase just prior to the transition to a pure LC phase. Figure 2-3 shows BAM images of DPPG over a calcium oxalate subphase before and after the emergence of the instabilities. Similar results were obtained with DPPS and dipalmitoylphosphatidylethanolamine (DPPE). Although topographic instabilities have also been observed for DPPC, 75 they were not detected under the present experimental conditions. The phenomenon, observed previously for a number of phospholipids by light scattering microscopy (LSM), 75 makes crystal detection

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29 ambiguous. Therefore, the studies reported here were restricted to monolayers containing DPPC. Using BAM to Quantify COM Growth at Monophasic Langmuir Monolayers COM crystals can be counted in situ to obtain the extent of precipitation as a function of time, allowing comparisons of the effects of different conditions. The isotherm of DPPC at room temperature on a subphase of RS 10 is shown in Figure 2-4, where the different lipid phases can be easily identified. At 25 C DPPC is in an LC state at pressures of 20 and 30 mN/m, whereas a pressure of 5mN/m yields an LE phase (Figure 2-4). Crystals were counted at these pressures. Crystal formation per unit area is largest at 20 mN/m and lowest at 5 mN/m, Figure 2-5 a. The monolayer assumes a larger area at low pressure than at high pressure for the same amount of lipid, so the data are corrected for the area of the monolayer (crystals/mm 2 x 2 /molecule) in Figure 2-5 b. After this correction, it can be seen that the number of COM crystals obtained at 30 and 5 mN/m is similar, but the number density at 20 mN/m is much larger. While the more fluid LC monolayer leads to increased COM formation, the number of crystals observed at the LE monolayer is lower and comparable to that of the LC monolayer at 30 mN/m. COM Growth at Single-Component Langmuir Monolayers with Phase Boundaries Although the crystal growth is uniform at single-phase LC or LE monolayers, this is not the case for monolayers in equilibrium between two phases. Our group has previously observed that COM forms at the phase boundary between gas analogous (G) and LE phases of a DPPG monolayer. 3 A similar observation is made for DPPC in LE/G coexistence. A DPPC monolayer was compressed on an RS 5 subphase to 100 2 /molecule, which is the smallest practical area where the LE/G equilibrium can be

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30 maintained. Under these conditions, crystals are observed exclusively at phase boundaries (Figure 2-6). COM crystals also appear preferentially at phase boundaries when LC and LE phases coexist, as has been observed by Vogel and co-workers using light scattering microscopy in combination with fluorescence microscopy. 76 Precipitation at LC/LE phase boundaries is also observed under the present experimental conditions. The LC/LE coexistence region was reached by compressing DPPC to 5 mN/m on a calcium oxalate subphase of RS 5 at 21.8 0.1 C. Although a pressure of 5 mN/m yields a pure LE phase at 25 C, the LE to LC phase transition occurs at a lower pressure at 21.8 C, allowing the LC/LE coexistence to be studied at the same pressure as the pure LE phase. BAM imaging clearly shows extensive crystal nucleation at phase boundaries after 16 h, Figure 2-7. COM Growth at Phase-Separated Binary Phospholipid Mixtures A phase boundary can also be created by mixing phospholipids that phase segregate. A 1:1 mixture of DMPC and DPPC at 21.8 0.1 C on an RS 5 subphase was chosen since, under these conditions, only DPPC can form an LC phase while DMPC remains in the LE phase upon compression. At high mean molecular areas, an LE phase containing both lipids is observed (Figure 2-8). Upon further compression, a critical pressure (15 mN/m) is reached where DPPC begins to form LC domains that appear as light gray islands in a LE matrix of darker gray color, Figure 2-8. At the early stages of growth, just above 15 mN/m, the LC domains are pure DPPC but the LE matrix contains both lipids (Figure 2-8 a). The mixture remains phase separated at all pressures higher than 15 mN/m as shown by Figures 3-8 b,c. A pressure of 25 mN/m was chosen to carry out the COM precipitation as the LC domains did not increase in size upon further

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31 compression, indicating complete lipid segregation so that at this point the LC phase is DPPC and the LE phase is DMPC. Crystals were observed to grow exclusively on the DPPC domains as shown in Figure 2-9. Even after 24 h the precipitation remains exclusive to DPPC. Although control experiments (not shown) confirm that DMPC is able to catalyze crystal formation as a pure LE monolayer, the crystal precipitation is preferred at the DPPC monolayer. It is noteworthy that for the phase-segregated binary mixture, although there is an interface between LC and LE phases, COM does not grow at the phase boundaries. This effect becomes quite apparent when Figures 2-5 and 2-7b are compared. Although both monolayers are held at the same temperature and subphase composition and both have LE/LC phase boundaries, the COM crystals form exclusively at the phase boundary in the single-component DPPC monolayer and away from it at the LC phase in the DPPC/DMPC binary mixture. The same binary mixture held at lower pressure gives different results. Below 25 mN/m, the DPPC is not fully compressed, so that at 18 mN/m the same mixture produces a monolayer with pure DPPC LC domains and a mixture of DPPC and DMPC in the LE matrix. Under these conditions, COM precipitation remains exclusive to the DPPC domains, but in this case, the crystal formation now occurs both at the phase boundary and away from it, Figure 2-10. In one experiment in which the crystals were counted, 40% of COM is at the boundaries while 60% is inside the LC domains after 11 h. Unlike the situation in Figure 2-9, where DPPC and DMPC are completely segregated, at 18 mN/m DPPC is in equilibrium between LC and the mixed DPPC/DMPC LE phase. This dynamic exchange between phases appears to be a condition for crystals to appear at the

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32 phase boundary. Table 2-1 summarizes and compares the findings obtained for monolayers with phase boundaries. Table 2-1. Experimental conditions and COM formation for monolayers with phase boundaries. monolayer conditions phase COM observed at single-component DPPC RS = 5, 100 (0 mN/m) and 25 C G/LE equilibrium G/LE phase boundary single-component DPPC RS = 5, 5 mN/m and 21.8 C LE/LC equilibrium LE/LC phase boundary 1:1 DMPC/DPPC RS = 5, 25 mN/m at 21.8 C LC DPPC and LE DMPC LC domains 1:1 DMPC/DPPC RS = 5, 18 mN/m at 21.8 C LE/LC equilibrium DPPC and LE DPPC LE/LC phase boundary and inside LC domains Discussion Effect of Compressibility The heterogeneous precipitation of oriented crystals at organic monolayers has been observed for a wide variety of organic and inorganic materials 1-4,77-86 and has been commonly attributed to a templating effect where the spatial distribution of the monolayer molecules matches closely the atomic coordinates of a particular face of the precipitating crystal, inducing nucleation of this plane. 77-79 Such a templating mechanism would truly be fortuitous, as even small deviations from commensurate lattice matching would greatly destabilize the template/crystal interaction and inhibit precipitation. Cooper et al. 83 were the first to recognize that oriented crystal nucleation under fluid monolayers can occur at higher rates compared to their close-packed analogues, in apparent contrast to a templating mechanism. Their experiments involved the precipitation of aspartic acid and asparagine monohydrate at monolayers of amphiphilic

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33 tyrosines. The higher compressibility of the monolayer allows it to reorganize to compensate for lattice mismatches of the overlayer, facilitating the crystal nucleation and growth. This concept is supported by the FTIR studies by Ahn et al. 84 where structural reorganization of the monolayer is observed as the growth of calcite occurs. The positive effect of the monolayer compressibility on the crystal number density and oriented growth has since been observed for other systems by our group 2-4 and others. 85,86 The results reported here showing that COM precipitation increases at a LC DPPC monolayer when the pressure is decreased from 30 to 20 mN/m add further support for the idea that the heterogeneous precipitation is a synergistic process whereby the lipid assembly reorganizes to accommodate the growing crystal. While the LE and G phases are even more compressible, it is not possible to make similar comparisons between phases as differences in lipid density and the presence of phase boundaries are also important factors. Effect of Phase Boundaries Studies of COM precipitation at DPPC monolayers with two phases in equilibrium reveal that crystal formation occurs preferentially at the phase boundary, either LC/LE (Figure 2-7) or LE/G (Figure 2-6). Similar results were seen previously for DPPG. 3 The preferential precipitation at LC/LE boundaries has also been observed by BAM for CaCO 3 under fatty acids 73 and by light scattering microscopy combined with fluorescence microscopy for calcium oxalate. 76 The role of phase boundaries in COM precipitation can be further explored by preparing monolayers of phase-segregated phospholipid mixtures. Phase-separated binary phospholipid mixtures have been more commonly examined in situ by fluorescence microscopy 46,87-89 and in transferred films by atomic force

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34 microscopy, 38,39,42,90 but it is also possible to observe the segregation by BAM. The COM precipitation at a 1:1 DMPC/DPPC mixture at 25 mN/m occurred exclusively at the DPPC domains (Figure 2-9). This result is consistent with earlier studies on individual phospholipids that showed that for the same functional headgroup, more crystals will form at the phospholipid that is able to achieve a LC state. 3 Nucleation is preferred at DPPC because even though DMPC is more fluid, it cannot organize as densely as DPPC under the current conditions. In contrast to the single component mixed-phase systems, the precipitation occurred within the LC domains and away from the interface at all times up to 24 h (Figure 2-9). At a surface pressure of 25 mN/m, the binary mixture is not in equilibrium and the lack of molecular exchange between the LC and LE phases seems to prevent the COM formation at the boundaries. This observation is supported by the fact that if a similar film is prepared at a lower pressure, where DPPC is in both phases and the LC and LE are in equilibrium, crystal precipitation is again observed at the phase boundaries (Figure 2-10 and Table 2-1). A remaining question is why COM formation is observed at phase boundaries. One possibility is that dipole/dipole interactions attract crystals to the boundaries where they attach. Nassoy et al. 91 has shown through experiments with latex beads suspended at pentadecanoic acid monolayers that a charged particle with a net dipole moment will be attracted to LC domains which act as macrodipoles. Vogel and co-workers have suggested this mechanism applies to COM growth at lipid monolayers. 76 While not observed directly by BAM, it is possible that COM nucleates at the LE phase and before it can be detected by BAM moves to the LC phase boundary. This mechanism requires that the particles are charged under the experimental conditions. The zeta potential of a

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35 particle is a measure of its effective charge, and that of calcium oxalate varies enormously (from +30 to -30 mV) depending on the dispersing medium. 92-94 The zeta potential of COM crystals precipitated homogeneously and dispersed in an RS 5 solution was determined to be 1 2 mV which has a large but typical error for these measurements. 92-94 This near null potential suggests that under the high ionic strength of these experiments, a mechanism where dipole interactions drive the crystals toward the interface might be attenuated. An alternative explanation for crystals appearing at phase boundaries is that extensive nucleation indeed occurs there. One expects the line tension of the two-dimensional phase boundary to contribute to the overall free energy gain upon precipitation. In addition, the LC phase boundary has a high density of lipid molecules with partial order, yet enough fluidity to allow reorganization of the lipids to effectively stabilize the nucleating crystal phase. For the phase-segregated binary mixtures (Table 21), the compliancy of the phase boundary is lost due to the lack of molecular exchange between phases, providing a kinetic barrier to nucleation. Once dynamic exchange of molecules between phases is reestablished (Figure 2-10), the compliancy of the phase boundary leads to nucleation. Expanded Phases Studies on expanded phases, LE and LE/G coexistence, were more difficult to quantify using BAM. The more fluid LE DPPC monolayer (5 mN/m) yields a lower crystal number density than the LC monolayer at 20 mN/m. A similar trend was observed by Cooper for asparagine monohydrate precipitation (although not for aspartic acid), where low monolayer pressures induced low crystal formation and medium pressures (5-20 mN/m) were optimal for the precipitation. 83 However, this is in contrast

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36 to previous studies in our group using DPPG and DPPC monolayers where low pressures corresponding to an LE/G coexistence yielded an increase in crystallization. The difference is likely a result of the in situ crystal monitoring used in the present study and the ex situ SEM analysis used previously. Once crystals obtain a certain size, their interaction with the LE DPPC monolayer is weak because the expanded phase does not provide enough intermolecular interactions at the interface to keep the crystals from sedimenting. Observation of the monolayer after 16 h shows abundant COM crystallization at the LC phase (Figure 2-2) but almost none at the LE phase. It appears that crystals forming at the LE monolayer grow to a critical size and then fall off, giving rise to the low number densities at 5 mN/m in Figure 2-5. In the previous study on DPPC and DPPG, slides placed under the monolayer for transferring the film detected these crystals. In contrast, long-term observations do not show sedimentation of crystals formed at LC phases. While crystal formation can be observed with BAM, it is difficult to quantify events at expanded phases. Conclusions BAM provides a useful tool to visualize and quantify the effect of changes such as pressure, subphase composition, and phospholipid identity on the COM number density. 1-3 BAM has the advantage of being a relatively inexpensive in situ technique, which does not require COM crystallization to be halted for quantification. It is shown here that BAM can be used to quantify the effect of surface pressure on COM formation as well as monitor the effect of phase boundaries on crystal formation. Experiments using DPPC confirm that compressibility plays an important role in the precipitation at LC monolayers. Crystal formation is enhanced at LC phases at lower surface pressure.

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37 Phase-separated binary phospholipid mixtures can also be monitored in situ with BAM, and it is shown that phase boundaries can play an important role in COM formation. Crystal precipitation is observed at phase boundaries when there is molecular exchange between the LE and LC phases, such as for DPPC held at 5 mN/m and 22 C where the LC and LE phases coexist. On the other hand, at segregated mixtures of two related phospholipids, DMPC and DPPC, COM growth was selective at the DPPC LC phase and not observed at the DMPC LE phase or at the phase boundary. If the conditions are changed so that the DPPC can exchange between the LE and LC phases, crystals again appear at the phase boundaries. The role of the phase boundaries is still not certain.

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38 Figure 2-1. COM particles precipitated homogenously and used in zeta potential measurements.

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39 (b) (a) Figure 2-2. BAM images of DPPC compressed to 30 mN/m over an RS 10 calcium oxalate subphase at 25.3 0.3 C after (a) 1 h (where the arrows indicate the precipitated COM, shutter timing (ST) = 1/120 s), (b) 3 h and 45 min (ST = 1/120 s), and (c) 16 h (ST = 1/1000 s). Since COM crystals in image c are very bright, this image was obtained by reducing significantly the light allowed to reach the camera, causing the background associated with the LC phase of DPPC to appear darker than images a and b. The scale bars for images a-c represent 100 m. (d) SEM image of COM crystals transferred from the air/water interface after forming under a DPPC monolayer held at 30 mN/m on an RS 10 subphase at 25 C after 14 h. (c) (d)

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40 a b Figure 2-3. BAM images of DPPG over a room temperature RS 5 calcium oxalate subphase at (a) 27.5 mN/m and (b) 28.8 mN/m. The arrow in image b points are a region with topographic instabilities (bright objects). The scale bars represent 100 m.

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41 Figure 2-4. DPPC isotherm over an RS 10 calcium oxalate subphase at 25 C. The monolayer was compressed at a rate of 3 mN/m/min with a maximum barrier speed of 5 mm/min.

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42 1234 010203040 Number of crystals/mm2Time (h)(a)01234 05101520 Number of crystals/molecule (x1012)Time (h)(b) Figure 2-5. Extent of COM precipitation at a DPPC monolayer (a) per unit area and (b) normalized to the trough area. The monolayers were held at () 30 mN/m, () 20 mN/m, and () 5 mN/m over an RS 10 calcium oxalate subphase at 25.3 0.3 C.

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43 Figure 2-6. BAM image of DPPC compressed to 100 2 /molecule over an RS 5 calcium oxalate subphase at 25 C after 1 h (ST = 1/50 s). The gas analogous phase is dark and the LE phase is gray. The arrows indicate COM crystals. The scale bar represents 100 m. Figure 2-7. DPPC compressed to 5 mN/m over an RS 5 calcium oxalate subphase at 21.8 C after 16 h (ST = 1/120 s). The dark background is the LE phase and the light gray is the LC phase. Crystals appear as bright spots at the LE/LC phase boundary. The scale bar represents 100 m.

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44 (a) (b) (c) Figure 2-8. Compression isotherm and BAM images (ST = 1/50 s) of a 1:1 DPPC/DMPC mixture over an RS 5 calcium oxalate subphase at surface pressures of (a) 17 mN/m, (b) 29 mN/m, and (c) 46 mN/m. The monolayer was compressed at a rate of 3 mN/m/min with a maximum barrier speed of 5 mm/min at 21.8 C. The scale bars represent 60 m.

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45 Figure 2-9. BAM images of a 1:1 DPPC/DMPC monolayer (ST = 1/50 s) held at 25 mN/m over an RS 5 subphase at 21.8 C after (a) 5 h and (b) 24 h. The dark gray background is DMPC, the light gray islands are DPPC, and the bright spots are COM. The arrows in image a point at crystals precipitating on the DPPC domains. Although the LC domains in image a are rounded, these fuse over time to give the monolayer in image b. The scale bars represent (a) 60 m and (b) 110 m. (a) (b) (a) (b) Figure 2-10. BAM image of a 1:1 DPPC/DMPC monolayer held at 18 mN/m over an RS 5 calcium oxalate subphase at 21.8 C after 11 h (ST = 1/120 s). The dark background is the LE matrix, and the gray is the LC DPPC. COM grows both at phase boundaries (a) and at LC domains (b). The scale bar represents 100 m.

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CHAPTER 3 PRECIPITATION OF CALCIUM OXALATE MONOHYDRATE AT MEMBRANE LIPID RAFTS MODELS Introduction Lipid Rafts The view that membranes have lateral organization in their lipid components is a relatively new one. 6-14 In 1997 Simons and Ikonen were the first to gather all existing evidence and propose the existence of membrane microdomains enriched in cholesterol and sphingolipids (Figure 3-1), termed lipid rafts. 6 In spite of the explosion of research in the lipid rafts field, the existence of membrane domains remains controversial to this day. 12,13 The most recognizable characteristic of lipid rafts is their insolubility in cold non-ionic detergents and hence the alternative terms detergent-resistant membranes (DRMs), detergent-insoluble glycolipid-enriched membranes (DIGs), glycolipid-enriched membranes (GEMs) and Triton-insoluble floating fraction (TIFF) have emerged. TIFFs were named after the first raft extraction from cells, where 1% Triton X-100 at 4 C was used to isolate cholesterol-sphingolipids rich domains, Figure 3-2. 34 In this study, cells expressing a glycosylphosphatidylinositol (GPI)-anchored protein, human placental alkaline phosphatase (PLAP), were extracted with the non-ionic detergent. Interestingly, PLAP is soluble right after its synthesis but becomes insoluble in a period of 3 hours. The insoluble fraction also contains a number of phospholipids, but it is enriched in 46

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47 cholesterol and sphingolipids. It is thought that PLAP associates with the raft domains after it is synthesized, which renders the protein detergent-insoluble. 34 Lipid rafts have been shown to be enriched in cholesterol and highly saturated sphingolipids, including glycosphingolipids. Glycosphingolipids have the same structure as sphigomyelins (Figure 3-1) except for the head group, which contains sugar moieties. The Simons and Ikonen model explains the detergent insolubility of lipid rafts by the strong interaction of sphingolipids and cholesterol, where the sterol molecules fill voids caused by the packing of glycosphingolipids whose head groups are larger than its alkyl tails. 6 Research in the area of cholesterol-sphingolipids and cholesterol-glycerolipids interactions support this hypothesis. 13 For example, Mattjus et al. found that monolayer mixtures of cholesterol, unsaturated sphingomyelin and unsaturated phosphatidylcholines are more succeptible to oxidation by cholesterol oxidase than the corresponding saturated analogues, indicating a stronger cholesterol-saturated lipid interaction. 95 In addition, Slotte et al. have shown that monolayers of phosphatidylcholines are much more succeptible to oxidation than the corresponding chain-matched sphingomyelins, which agrees with the observation that the insoluble lipid rafts are enriched in sphingolipids. 96 Cholesterol and Lipids Monolayer and bilayer lipids are in different physical states depending on their alkyl tail saturation, temperature and presence of cholesterol. Molecules with highly saturated alkyl tails at lower temperatures exist in a very organized state referred to by physical chemists as liquid condensed (LC), Figure 3-3a. Alternative nomenclature has emerged in the biology and biochemistry fields, and so the LC phase is also commonly referred to in the literature as gel, L and solid ordered (S o ). An increase in temperature will cause a transition of the LC phase to a more disorganized liquid expanded (LE)

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48 phase, Figure 3-3b. The LE phase can also exist at lower temperatures if the lipid alkyl tails are unsaturated, since kinks in the hydrocarbon chains prevent the formation of a closed packed array. Other names for the LE phase include liquid-crystalline, L and liquid-disordered (L d ). Addition of cholesterol to an LC array, will induce the formation of a liquid-ordered (LO) state, where the lipid tails are highly organized as in the LC state but exhibit the high lateral mobility of the LE state. 14 Lipid Raft Models The early models for lipid raft formation constitute monolayers and bilayers of binary lipid mixtures without cholesterol much like those recently reported by our group. 97 An example of this early work is that by Le Grimellec et al., where the topology of a supported phase separated bilayer mixture of 3:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/ 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was examined by atomic force microscopy (AFM). 98 In these bilayers the formation and evolution of the DPPC condensed domain was studied as the bilayer was cooled from 60 C to 23 C. The same group also performed AFM studies of a 1:1 DOPC/DPPC LB monolayer with the addition of 0.2 4.0 % of the ganglioside G M1 , a glycosphingolipid commonly used as a lipid raft marker. 99 G M1 was found at the condensed DPPC domains, supporting the hypothesis that this ganglioside associates with lipid rafts in membranes. In fact, the co-localization of G M1 with LC or LO domains in lipid raft models is a common finding. 100,101 Other examples of binary phospholipid mixtures are described in Chapter 1 (see Table 1-4). More accurate membrane lipid rafts models are given by monolayers, bilayers and vesicles containing molecules resembling the lipid raft composition. Common mixtures contain DOPC or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SM and

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49 cholesterol. 101-107 This makeup is based on lipid composition analysis of canine kidney MDCK cells and Triton insoluble DRMs. 34 An important study in this group was carried out by Dietrich et al. 102 where mixtures of DOPC/SM/cholesterol and POPC/SM/cholesterol were examined as monolayers, bilayers and giant unilamellar vesicles (GUVs). The supported films where detected by fluorescence microscopy by adding a small amount (less than 1%) of fluorescent probes that partitions preferentially into the condensed or expanded phases of the films. It was consistently found that ordered domains are present with the addition of SM. Interestingly, the ordered domains containing SM/cholesterol resist Triton X-100 extraction whereas the remaining expanded POPC or DOPC was readily eliminated by detergent incubation. The GUVs were imaged by adding Laurdan, a fluorescence-labeled lipid whose emission is different when it is in an LE or LO phase. The vesicles also show ordered domains that can be reversely formed and disrupted by temperature changes. 102 Samsonov et al. also prepared lipid raft supported bilayers. In this case, the expanded phase of choice was a mixture of 2:1 mixture of DOPC /1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) most likely to reflect the presence of glycerophospholipids with ethanolamine and choline head groups in the DRMs of MDCK cells. 34 The bilayers were monitored using a fluorescent probe, rho-DOPE, that preferentially partitions into the expanded phase. LO domains were observed when both SM and cholesterol were added to the DOPC/DOPE mixture in concentrations larger than 15%. Addition of only SM or cholesterol to the lipid mixture did not yield the phase separated bilayer. The authors discuss that domains observed are indeed in an LO and not LC phase since they are circular, rapidly merge and their shape is restored after

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50 deformation. They also show that the addition of cholesterol oxidase to the raft forming mixtures gradually causes the dissolution of the LO domains. Veatch and Keller prepared GUVs containing POPC, cholesterol and a saturated PC. 107 Vesicles with 0 to 55% cholesterol were imaged with fluorescence microscopy and a transition from an LC (0% cholesterol) to an LO (10-50% cholesterol) phase was observed. Concentrations of 50-55% cholesterol lead to uniform vesicles. Similar results were obtained when Langmuir monolayers of the same composition were imaged by fluorescence microscopy. When 2:1 egg PC/brain sphingomyelin was used in vesicles, the LO phase occurred between 20-40% cholesterol and the uniform vesicles were imaged at 45% cholesterol. 107 In addition to fluorescence microscopy, AFM has been used to characterize supported PC/SM/cholesterol monolayers and bilayers. 101,104 Milhiet et al. have shown that SM/POPC monolayers transferred at 30 mN/m at 2:1 and 4:1 ratios form condensed SM domains. 104 Addition of cholesterol increases the area occupied by the higher-height domains, suggesting that cholesterol interacts with the LC SM-enriched domains. Yuan et al. also showed the formation LO domains in monolayers and bilayers of 1:1:1 POPC/SM/cholesterol at monolayer pressures of 10, 15 and 30 mN/m. In addition, it was shown that G M1 in concentrations of 1-5% is preferentially located at the SM/cholesterol-rich phase. 101 It has been previously demonstrated that the precipitation of calcium oxalate monohydrate (COM) at Langmuir monolayers can be readily monitored with BAM and that phospholipid phase boundaries play an important role in COM formation. 97 COM appears exclusively at phase boundaries when a single-component 1,2-dipalmitoyl-sn

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51 glycero-3-phosphocholine (DPPC) monolayer at a phase change, either LC/LE or LE/gas (G), is prepared over a calcium oxalate subphase. The COM distribution is different if an LC/LE phase boundary not at equilibrium is prepared. A phase-separated binary mixture of 1:1 DPPC/1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), where the LC domains are DPPC and the LE domains are DMPC, induces COM formation away from the phase boundary and inside the LC region. If the lipid exchange between the LC/LE domains is restored, then COM precipitation again occurs at the boundaries. 97 In this work biphasic Langmuir monolayers at calcium oxalate subphases are prepared, where one phase is LO domains formed by the association of SM and dihydrocholesterol and the other phase is expanded POPC. Although these lipid mixtures have been intensely investigated in the literature by fluorescence microscopy and AFM, 101-106 no BAM studies on the raft forming monolayer have yet been reported. BAM shows that COM appears at phase boundaries of the raft domains. Experimental Section Materials All reagents were purchased from commercially available sources and used without further purification. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and brain sphingomyelin (SM) (purity >99%) were purchased from Avanti Polar Lipids (Alabaster, AL). Dihydrocholesterol, sodium oxalate and tris(hydroxymethyl)aminoethane hydrochloride (Tris . HCl) were purchase from Aldrich Chemical Co. (Milwaukee, WI). Calcium chloride dihydrate and sodium chloride were obtained from Fisher Scientific (Pittsburgh, PA).

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52 Langmuir Monolayers A KSV Instruments (Stratford, CT) Langmuir-Blodgett system KSV 2000 was used in combination with a 700 cm 2 Teflon double-barrier trough. A paper Wilhelmy balance suspended from a microbalance was used to monitor the surface pressure. Subphases were prepared with pure water of resistivity of 18.0-18.2 M cm from a Barnstead (Boston, MA) NANOpure system. To prepare calcium oxalate subphases, two solutions of equal volume of 150 mM NaCl and 5 mM Tris . HCl were prepared and the appropriate amount of calcium was added to one and oxalate to the other to achieve relative supersaturation (RS) values of 5 (0.35 mM) once combined. The pH of both solutions was adjusted to 7.00 with an aqueous KOH solution. The solutions were slowly combined drop wise over a 2 hour period and used immediately. The subphase temperature was adjusted with a Fisher Scientific Model 900 isotemp refrigerated circulator. The lipid/cholesterol mixtures were prepared as 9:1 chloroform/methanol solutions and spread at the air water interface. A solvent evaporation period of 15 min was followed by compression at a rate of 3 mN/m/min with a maximum speed of 5 mm/min. Atomic Force Microscopy (AFM) Langmuir monolayers of composition 2:1:1 POPC/SM/dihydrocholesterol were prepared as described above over a calcium oxalate subphase of RS 5. The monolayer was compressed to 32 mN/m and transferred to freshly cleaved mica (Ted Pella, Redding, CA) on the upstroke at a speed of 2 mm/min. The film was allowed to air dry for 2-4 hours and then imaged using a Multimode AFM with a Nanoscope IIIa controller (Digital Instruments, Santa Barbara, CA) and commercially available silicon cantilever probes (Nanosensors, Phoenix, AZ).

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53 Brewster Angle Microscopy (BAM) BAM experiments were performed using a Nanofilm Technologie GmbH (Goettingen, Germany) BAM2plus system with the Langmuir-Blodgett trough described above. A polarized NdYAG laser (532 nm, 50 mW) was used with a CCD camera (572 x 768 pixels). The instrument is equipped with a scanner that allows an objective of nominal magnification of 10x to be moved along the optical axis, producing a focused image. A laser power of 50% and maximum gain are used. A shutter timing (ST) of 1/50 s or 1/120 s is used to obtain maximum contrast between the monolayer and the COM crystals. The distortion of the images due to the angle of incidence is corrected by image processing software provided by the manufacturer. The incident beam is set at the Brewster angle in order to obtain minimum signal before spreading the monolayer. A piece of black glass is placed at the bottom of the trough to absorb the refracted light beam that would otherwise cause stray light. The polarizer and analyzer are set at 0 for all experiments. The laser and camera are mounted on an x-y stage that allows examination of the monolayer at different regions. Domain Area Calculation Domain areas for the sterol or LO domains of 1:1 and 2:1 POPC/dihydrocholesterol as well as 2:1:1 POPC/SM/dihydrocholesterol were estimated from BAM images using the free NIH software ImageJ. Area calculations were performed for pictures taken within 30 minutes of reaching 32 mN/m. The reported values are an average of at least 3 images and the errors represent standard deviations. Crystal Counting Procedures For each experiment 50-100 crystals were observed in-situ and assigned to the phase boundary, expanded phase or liquid ordered domain. A shutter timing (ST) value

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54 of 1/120 s was used for the 2:1:1 POPC/SM/dihydrocholesterol monolayer and an ST value of 1/50 s was used for the 2:1 POPC/dihydrocholesterol film. Each experiment was repeated at least three times. The time was set to zero at the point where the monolayer reached 32 mN/m. Results Sphingomyelin Monolayer Phase segregation is observed when a monolayer of sphingomyelin (SM) is compressed over a calcium oxalate subphase of RS 5. SM is a brain extract containing a mixture of lipids with varying alkyl tail lengths and degrees of unsaturation. It is reasonable to expect that the shorter and unsaturated SM components will not form a condensed phase, leading to phase segregation. The SM compression isotherm and corresponding BAM images are shown in Figure 3-4. At pressures below 7 mN/m, expanded and gas phases are observed, Figure 3-4a and 3-4b. Further compression causes LC domains to appear, which remain phase segregated from the expanded phase throughout the compression. Phase segregation of SM components has also been observed by AFM. 104 COM precipitation at a phase segregated SM monolayer can be observed. A SM monolayer was compressed and held at 32 mN/m, a pressure considered biologically relevant. 30,32 Crystal precipitation was observed exclusively associated with the LC SM, Figure 3-5. This result is expected. It is shown in Chapter 2 that a phase separated binary mixture will induce COM formation at the LC domains. 97 Due to the size and shape of the LC SM, it is not possible to determine with certainty whether COM forms at the LC/LE phase boundary or inside the domains. Nevertheless, careful observation of

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55 many images like those in the inset of Figure 3-5 suggests that crystals appear both at boundaries and inside LC regions. Sphingomyelin/Dihydrocholesterol Monolayers The addition of sterols to SM monolayers produces a sharp change in the phase behavior of SM. The condensing effect of cholesterol on lipids has been investigated, 95,96,103,108 and it is thought that the condensation of SM by cholesterol leads to the formation of membrane lipid rafts. 6-14 In this study dihydrocholesterol is used as it is resistant to air oxidation and its phase behavior in lipid/sterol mixtures are identical to that of cholesterol. 109,110 The compression isotherm and BAM images of a 1:1 SM/dihydrocholesterol over a calcium oxalate subphase of RS 5 is shown in Figure 3-6. It is apparent that the SM monolayer no longer forms phase segregated domains as in Figures 3-4 and 3-5. Instead, the addition of cholesterol induces the formation of a homogeneous liquid ordered (LO) phase, Figure 3-6. POPC/Sphingomyelin/Dihydrocholesterol Monolayer The LO domains of a 2:1:1 POPC/SM/dihydrocholesterol monolayer can be readily observed with BAM. In such monolayers, the LO SM/dihydrocholesterol will appear as light grey islands surrounded by darker grey LE POPC. The raft forming mixture was spread at a calcium oxalate subphase of RS 5 and compressed to 32 mN/m as described in the experimental section. Figure 3-7 shows that initially the SM/dihydrocholesterol domains are of a size in the order of 10-20 m. The raft domains merge to form larger circular domains, reaching sizes of several hundred microns within 30 minutes, Figure 3-7. This behavior is expected for LO domains and has previously observed for lipid raft membrane models. 103 In a control experiment, the formation and coalescence of LO domains with cholesterol showed identical behavior to the monolayer with

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56 dihydrocholesterol. The lipids rafts formed with cholesterol and dihydrocholesterol are similar to those characterized by AFM and fluorescence microscopy by others, 101-107 although the larger sizes observed here are likely due to the lack of monolayer support. COM precipitation is observed at the 2:1:1 POPC/SM/dihydrocholesterol monolayer as shown in Figures 3-7c and 3-7d. Most of the crystals were observed at phase boundaries between the LO domains and the LE phase (Figure 3-7d) although a significant number was observed at the expanded phase (Figure 3-7c). Figure 3-8 shows that about 80 % of crystals are observed at the phase boundaries, 20 % at the LE phase and a negligible amount is found inside the LO rafts. This distribution remained constant within the experimental uncertainty over 3 hours. It is surprising to find a significant COM precipitation at the LE POPC phase of a two-phase mixture. It has been previously shown that single-component and two-component lipid monolayers with LC/LE phase coexistence show COM precipitation at phase boundaries and/or inside the LC domains. 97 It is possible that COM appearing at the expanded phase is actually precipitating at phase boundaries of LO domains too small (below 1 m) to be detected by BAM. To determine whether sub-micron rafts exist, a 2:1:1 POPC/SM/dihydrocholesterol monolayer was held at 32 mN/m for 1 h and then transferred to mica. Figure 3-9 is an AFM image of the edge of a large LO domain. It is clear that, in addition to the large rafts, there are sub-micron domains present. AFM images of a similar film transferred immediately after reaching 32 mN/m (not shown) also have sub-micron rafts, suggesting that these small domains are present at all times. It is concluded that COM identified as precipitating at the expanded phase in Figure 3-8 by BAM is at the phase boundaries of small LO rafts.

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57 A distribution of domain sizes in biphasic lipid films has also been observed by Sanchez and Badia for the DPPC/DPLC mixture. 42 Two types of domains were observed for the mixed monolayer transferred as a Langmuir-Schaefer film; flower-like domains of several tenths of microns and a number of smaller sub-micron sized LC regions. Comparison of our film to that of Sanchez and Badia, 42 in addition to the same step height for the large and small rafts, lead us to believe that the sub-micro domains are not a substrate-mediated condensation of POPC. The BAM and AFM images of the 2:1:1 POPC/SM/dihydrocholesterol monolayer show similar LO raft morphology, although additional information can be gained by AFM. Figure 3-10a shows that, in agreement with BAM images of Figure 3-7, LO domains of sizes larger than 50 m are present in the film. An advantage of AFM is the ability to obtain the step difference between the LO domains and surrounding LE phase. Regardless of domain size, the step height was found to be 0.50 0.08 nm, a value consistent with other raft model work. 104,106 In addition, AFM shows the large domains to be porous, with the degree of porosity varying from sample to sample. Figure 3-10b is an AFM image of a film prepared under identical conditions to those of 3-10a. The scan of Figure 3-10b was taken inside a large LO domain, showing a smaller amount of pores. These holes likely form during transfer, particularly since COM is not observed inside the LO domains by BAM, where the pores would form abundant phase boundaries. POPC/Dihydrocholesterol Monolayer To gain further insight into the sterol/lipid interaction in our Langmuir monolayers, films containing POPC and dihydrocholesterol were prepared. Cholesterol and dihydrocholesterol have been shown to segregate from phospholipid monolayers at biologically relevant concentrations. 111-118 Although the miscibility transition tends to

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58 occur at low pressures for other lipid/sterol mixtures, 111-118 distinct sterol domains are observed by BAM for a 2:1 POPC/dihydrocholesterol monolayer at 32 mN/m over an RS 5 calcium oxalate subphase, Figure 3-11. A mixture of 2:1 POPC/cholesterol shows identical sterol domain formation. Separate BAM experiments (not shown) confirm that POPC does not form condensed domains, as expected for lipids with unsaturated alkyl tails. Therefore, the light grey domains in Figure 3-11 can be assigned to dihydrocholesterol segregated from the expanded POPC. COM precipitation was observed at the 2:1 POPC/dihydrocholesterol monolayer, Figure 3-11b. Most crystals appear at the phase boundary between the sterol domains and the expanded POPC, although a few were observed at the LE POPC. This observation was also made for a 2:1 POPC/cholesterol monolayer. Figure 3-12 shows that over a period of 3 hours, 90 % of crystals are observed at phase boundaries, 10 % at the expanded phase and none inside the sterol domains. In analogy to the 2:1:1 POPC/SM/dihydrocholesterol monolayer, an AFM image of a 2:1 POPC/dihydrocholesterol film shows sub-micron cholesterol regions where COM likely precipitates at phase boundaries, Figure 3-13. Discussion Domain Formation at Binary and Ternary Lipid Mixtures The first BAM investigation of a monolayer with lipid raft forming components is presented here. The unsaturated phospholipid/sphingomyelin/sterol mixture is a common theme in the recent membrane models literature. 101-107 Single-component and phospholipid binary mixtures have been previously used as membrane models to investigate COM precipitation. 97 In an effort to prepare more biologically relevant models, the raft mixture is used to look at COM formation.

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59 It is important to highlight the difference between the POPC/dihydrocholesterol Langmuir monolayers with and without SM, Figures 3-7 and 3-11 respectively. The first indication that these monolayers are different is that the sterol domains (Figure 3-11b) are darker than the LO sterol/SM domains (Figure 3-7b). The contrast in BAM is given by difference in monolayer reflectivity which is affected by monolayer thickness. Since dihydrocholesterol is expected to be shorter than the alkyl tail of most non-steroidal lipids, 10,119 it follows that the addition of brain SM, whose mayor component is the 18:0 lipid, will induce the formation of lighter grey LO domains. The different in thickness of the monolayer domains is also reflected in the crystal counting procedure for both monolayers (experimental section). A shutter timing (ST) value of 1/50 s is adequate for the COM quantification in the absence of SM. Nevertheless, in order to efficiently observe the COM precipitation at the monolayers with SM, the shutter timing (ST) has to be decreased to 1/120 s to reduce the domain brightness which obscures the small crystals growing at the phase boundaries. In addition, the step height obtained by AFM of the transferred film with SM is 0.50 0.08 nm whereas that of the monolayer with no SM is 0.23 0.05 nm, a direct indication of the presence of the longer SM alkyl tails. Finally, it is also observed that the merging of the SM/dihydrocholesterol domains occurs much faster than that of the dihydrocholesterol domains. Figure 3-7c shows that after 30 minutes, several of the LO domains are hundreds of microns in size whereas after 1 hour, the dihydrocholesterol regions remain mostly under 100 microns in size, Figure 3-11b. Although the Langmuir monolayers of POPC/dihydrocholesterol with and without SM may appear similar under BAM, there are differences that show SM is enriched in the sterol domains.

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60 COM Precipitation at Phase Boundaries COM precipitation occurs at the phase boundaries of the POPC/dihydrocholesterol monolayers with and without SM. It has been shown that COM appears at boundaries of biphasic monolayers, either single-component DPPC or binary DPPC/DMPC, when there is molecular exchange between the expanded and condensed phases. Phase segregated DPPC/DMPC films induce COM precipitation inside the LC DPPC regions and away from the phase boundary. 97 The immediate question that arises is whether the LO raft domains are truly phase segregated from the expanded POPC. I believe they are not. The mean molecular area (MMA) at 32 mN/m from compression isotherms for SM/dihydrocholesterol (Figure 3-6) is 35.5 2 and for POPC is 83.6 2 (isotherm not shown). For a 2:1:1 POPC/SM/dihydrocholesterol monolayer, the POPC area (A POPC ) would be 2.4 times larger than that of the LO domains (A LO ) if the raft mixture was phase-segregated. However, an estimate of the coverage of the LO regions yields an A POPC /A LO ratio of 1.4 0.3 (Table 3-1). This value indicates that a small amount of POPC is incorporated in the LO raft. Furthermore, the fact that POPC is both at expanded and ordered regions suggests that molecular exchange between phases can occur, inducing COM appearance at the boundaries. POPC is also incorporated into the sterol domains in binary phospholipid/sterol mixtures. The MMA of dihydrocholesterol at 32 mN/m is 33.2 2 (isotherm not shown). The expected A POPC /A sterol for a 2:1 and 1:1 POPC/dihydrocholesterol monolayer is 5.0 and 2.5 respectively. These predicted values are larger than the observed. The A POPC /A sterol ratio for the 2:1 monolayer is 2.2 0.3 and for the 1:1 monolayer is 0.3 0.1 (Table 3-1). At 32 mN/m dihydrocholesterol domains contain a significant amount of

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61 POPC, likely leading to molecular exchange and resulting in COM appearance at the phospholipid/sterol boundaries. Table 3-1. Expanded phase area to domain area ratios for the POPC/dihydrocholesterol monolayers with and without SM. Monolayer Theoretical POPC area to domain area ratio Measured POPC area to domain area ratio 2:1:1 POPC/SM/dihydrocholesterol 2.4 1.4 0.3 2:1 POPC/dihydrocholesterol 5.0 2.2 0.3 1:1 POPC/dihydrocholesterol 2.5 0.3 0.1 Relevance to Membrane Lipid Rafts It is proposed that lipid rafts in membrane models contain phospholipids in addition to being enriched in cholesterol and sphingolipids. The domain areas, as well as the COM formation at the phase boundaries, support this hypothesis. This finding should be of no surprise. The original raft isolation from cells with Triton X-100 by Brown and Rose contained a significant amount of phospholipids. 34 In this isolation, the ratio of phospholipids (PE, PC, PS and PI) to cholesterol/sphingomyelin in vesicles reconstituted from the insoluble fraction is 1.2. Our results agree with the cells extraction in that phospholipids can be partially incorporated into lipid rafts. Conclusions It has been shown that the lipid raft forming mixture 2:1:1 POPC/SM/dihydrocholesterol can be monitored by BAM as a Langmuir monolayer. This film prepared over a calcium oxalate subphase shows COM formation at phase boundaries of large LO domains (hundreds of microns in size). 20% of COM appears at sub-micron sized LO regions, which cannot be imaged with BAM but can be easily observed by AFM. The appearance of COM at phase boundaries and estimates of the

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62 area occupied by the LO domains suggest that some POPC is incorporated into the rafts at 32 mN/m.

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63 Choline head group (a) Sphingomyelin Fatty acid (chain length varies) HOHHH OOPOOO-N+OHNH Sphingosine backbone (c) Cholesterol Figure 3-1. Chemical structures of the raft forming species: (a) sphingomyelin, a representative sphingolipid, where the shaded region is the sphingosine backbone, the white box is the choline head group and the remaining part is a fatty acid and (b) cholesterol. raft Detergent, 4 C Insoluble raft fraction (cholesterol and sphingolipids) Soluble fraction (unsaturated phospholipids) Figure 3-2. Schematic representation of the membrane detergent extraction of lipid rafts.

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64 (a) (b) Figure 3-3. Schematic representation of physical state of lipid bilayers where (a) represents highly saturated lipids in an LC state and (b) represents a bilayer with unsaturated lipids in an LE state.

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65 (a) (b) (d) (c) Figure 3-4. Compression isotherm and BAM images (ST = 1/50 s) of a SM monolayer over an RS 5 calcium oxalate subphase at surface pressures of (a) 0 mN/m, (b) 5 mN/m, (c) 13 mN/m and (d) 40 mN/m. The monolayer was compressed at a rate of 3 mN/m/min with a maximum barrier speed of 5 mm/min at 25 C. The scale bars represent 100 m.

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66 Figure 3-5. BAM image of a SM monolayer (ST = 1/120 s) held at 32 mN/m over an RS 5 subphase at 25 C after 1.5 hours. The arrows point at crystals precipitating on the SM LC domains. The inset is a magnification of a region where COM is present. COM appears at the phase boundary and inside the LC domains. No crystals were observed at the expanded phases. The scale bar represents 100 m.

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67 (a) (b) Figure 3-6. Compression isotherm and BAM images (ST = 1/50 s) of a 1:1 SM/ dihydrocholesterol monolayer over an RS 5 calcium oxalate subphase at surface pressures of (a) 0 mN/m, (b) 23 mN/m. The monolayer was compressed at a rate of 3 mN/m/min with a maximum barrier speed of 5 mm/min at 25 C. The scale bars represent 100 m.

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68 (b) (a) (c) (d) Figure 3-7. BAM images of a 2:1:1 POPC/SM/dihydrocholesterol monolayer held at 32 mN/m over an RS 5 subphase at 25 C after (a) 0 min (ST = 1/50 s), (b) 10 min (ST = 1/50 s), (c) 30 min (ST = 1/120 s) and (d) 3.5 h (ST = 1/120 s). The dark background is POPC, the light gray islands are SM/dihydrocholesterol, and the bright spots are COM. The arrows point at crystals precipitating at the LE phase in c and at the phase boundaries in d. The scale bars represent 100 m.

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69 phase boundaryLE phaseLO domains 0255075100 % of crystalsLocation of crystals 1 h 2 h 3 h Figure 3-8. Distribution of the COM precipitated at the phase boundary, LE phase and LO domains of a 2:1:1 POPC/SM/dihydrocholesterol monolayer. (a) (b) Figure 3-9. AFM image of a 2:1:1 POPC/SM/dihydrocholesterol film transferred on mica after being held for 1 h at 32 mN/m. The image is a 15 x 15 m scan where the arrows point at (a) the edge of a large LO domain and (b) and example of a LO domain of sub-micron size.

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70 (a) Figure 3-10. AFM images of two 2:1:1 POPC/SM/dihydrocholesterol films transferred on mica after being held for 1 h at 32 mN/m. Image (a) is a 50 x 50 m showing a large LO domain. Image (b) is a 25 x 25 m scan of a different film taken inside a large LO domain. Note that the porosity of the LO domains in (a) is larger than that of (b). (b)

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71 (a) (b) Figure 3-11. BAM images of a 2:1 POPC/dihydrocholesterol monolayer (ST=1/50 s) held at 32 mN/m over an RS 5 subphase at 25 C after (a) 0 hour and (b) 1 hour. The dark background is POPC, the light gray islands are dihydrocholesterol, and the bright spots are COM. The arrows in (b) point at crystals precipitating at the LE phase and phase boundaries. The scale bars represent 100 m. phase boundaryLE phase 0255075100 % of CrystalsLocation of crystals 1 h 2 h 3 h Figure 3-12. Distribution of the COM precipitated at the phase boundary and LE phase of a 2:1 POPC/dihydrocholesterol monolayer.

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72 Figure 3-13. AFM image of a 2:1: POPC/dihydrocholesterol film transferred at 32 mN/m on mica. The image is an 8 x 8 m scan where the arrows point at examples of dihydrocholesterol domains of sub-micron size.

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CHAPTER 4 MONOLAYERS AS MODELS FOR SUPPORTED CATALYSIS: ZIRCONIUM PHOSPHONATE FILMS CONTAINING MANGANESE(III) PORPHYRINS Introduction There are several potential advantages to be derived from confining molecular catalysts at surfaces to form heterogeneous systems, including easier product purification and catalyst recovery. Common strategies of immobilizing molecular catalysts include incorporating them into organic polymers, 120-129 high surface area carbon, 130,131 and inorganic frameworks such as zeolites, 132-134 silica 135-140 or other metal oxides. 141,142 Immobilized catalysts can mirror their homogeneous behavior, or the process can alter a catalyst’s activity, perhaps even changing the mechanism whereby it operates. Detailed understanding of catalytic behavior at interfaces is clearly desirable, but studies of supported catalysts can be riddled with uncertainties. Typical high-surface area supports are often not uniform, giving variation in catalyst binding sites, aggregation, and orientation with respect to the surface. It is often even difficult to determine if the structure of the molecular catalyst has remained intact upon adsorbing it to a surface. If catalytic activity is observed with a supported system, it is frequently impossible to know what species is responsible for the catalysis. Therefore, procedures for studying molecular catalysts at a uniform and reproducible interface could enhance the understanding of their behavior in heterogeneous environments. In this study, we take advantage of a well-understood surface modification process 15,16,143-147 to compare the activity of a monolayer film of a surface-confined 73

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74 manganese porphyrin oxidation catalyst with its homogeneous behavior. Through careful molecular design and controlled deposition, monolayers with known surface coverage, molecular orientation, and mode of binding to the interface are obtained. Using this controlled surface for the example of alkene epoxidation, we have observed increased activity with the surface-confined catalyst relative to homogeneous reactions under the same conditions. In contrast to many supported catalysts, all of the catalytic complexes present in the monolayer films are fully accessible, thus allowing accurate determination of the ratio of active site to substrate that is necessary to evaluate the effect of immobilization on catalytic activity. The monolayer films also allow careful monitoring of the state of the catalyst during the course of the reaction so that different behavior of the homogeneous and supported systems can be quantified. In addition to providing insight into the specific example of immobilized manganese porphyrins, the results demonstrate the utility of well-characterized molecular monolayers as models for supported molecular catalyst systems. Methods for immobilizing metalloporphyrins in monolayer films frequently involve coordination bonds to the axial sites on the metal center. 148,149 However, under the conditions needed to catalyze organic transformations, a covalent linkage through the macrocycle ligand is preferred to free up the metal center to participate in the reaction and to guard against desorption that may result from labile coordination bonds. Our method of preparing monolayers of covalently confined molecules has been described previously, and is based on zirconium phosphonate linkages in combination with Langmuir-Blodgett and “self-assembled monolayer” adsorption. 15,16,143-147 It involves the initial formation a Langmuir-Blodgett monolayer of octadecylphosphonic acid

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75 (ODPA) on a hydrophobic support that after adsorption of a layer of Zr 4+ ions provides a well-defined surface for the subsequent deposition of molecules containing phosphonic acid groups (Figure 4-1). 15,16 The thin films are modeled after layered solid-state metal phosphonates, where organic layers separate continuous inorganic layers. 150-155 The practice of sequential adsorption of organophosphonates and tetravalent metal ions has frequently been used to build-up multilayered films. 155-159 However, the present study employs a single deposition cycle to afford a controlled monolayer. Films formed in this way are stable to solvents and reaction conditions commonly used for many organic transformations, 148 making it possible to use them in studies of supported catalysis. The manganese porphyrin used in this study is molecule 1. The fluorinated tetraphenylporphyrin has four alkylphosphonic acid arms intended for binding and orienting the porphyrin at the surface. Previous studies on the Pd 2+ 146 and free-base analogs 18 of 1 have shown that the ligand structure indeed leads to monolayer coverage with the porphyrin macrocycle oriented parallel to the surface. Another important feature is that the molecules do not aggregate on the surface. 146 The phosphonate groups bind strongly to the zirconated LB layer to reach a high surface coverage of non-interacting molecules. NNNN F F F F O(CH2)18PO3H2 F F F F F F F F F F F F H2O3P(H2C)18O Mn O(CH2)18PO3H O(CH2)18PO3H2(III)1

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76 n oxidation catalysts, and there has been a great since the early reports that an iron ze a number of alkenes and alkanes.160 The tly serve as oxidation catalysts unfortunately leaves them oxidative degradation of the ligand, medation of the metal center to form -oxo dimers. mobilization of the chromophore.172 Attemso orphyrin 1 was f the alkylphosphonate chains are employed in the immobilization. We find that the films of 1 NNNN F F F F F F F F F F F F F F F F F F F Mn FCl (III)2 Natural metalloporphyrins are knowdeal of investigation of synthetic analogs160-171porphyrin with iodosylbenzene could oxidiability of porphyrins to efficienas potential substrates. In addition totalloporphyrins deactivate through oxiOne way of avoiding deactivation is through im pts at immobilizing porphyrin catalysts within supports such as organic polymers, 122,173 silica, 122,173-176 zeolites, 177,178 and metal phosphonates 179,180 have albeen reported. The monolayer studies reported here represent an attempt to better understand the behavior of surface-confined porphyrins. The activity of a monolayer of pcarefully compared to its homogeneous reaction and that of catalyst 2, an equivalent molecule without the alkylphosphonic acid arms. Catalyst 2 was chosen for its ease opreparation and because its geometry is likely to mimic the one found in 1 once

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77 offer higher yields and have a longer lifetime than either of the catalysts 1 or 2 in solution. The p rincipal difference is that 2 deactivates through the formation of -oxo dimeres, , e m the . 0 from Wilmad Glass (Buena, NJ). Synthesis cid) rsit s. On the surface, molecule 1 cannot dimerize, but in solution the alkylphosphonic acid arms also protect the metalloporphyrin core from dimerization. However, 1 still shows increased catalytic activity in the monolayer relative to in solution. In both casthe molecule oxidatively degrades with time, but the monolayer deactivates more slowlyleading to a higher number of turnovers. Confining the bulky molecule to the surfacalters the conformation of the porphyrin, changing the steric environment around the metal center. This feature along with the density of the catalytic sites resulting fromonolayer deposition technique may also play a role in the observed increase in activityExperimental Materials Reagents were obtained from commercial sources and used as received unless indicated. Bicyclohexyl 99%, purchased from Acros (Pittsburg, PA), was purified by percolation through basic alumina. Teflon (fluoro(ethylene-propylene)) sheets were purchased from American Durafilm (Holliston, MA). Single crystal silicon wafers (1 0) were purchased from Semiconductor Processing Company (Boston, MA). Germaniumattenuated total-reflectance (ATR) crystals (45, 50mm x 10mm x 3mm) were purchased Porphyrin Manganese 5,10,15,20-tetrakis(tetrafluorophenyl-4’-octadecyloxyphosphonic aporphyrin (1) and manganese 5,10,15,20-tetrakis(pentafluorophenyl)-porphyrin (2). These compounds were prepared by Dr. Bruno Bujoli and Dr. Fabrice Odobel, Univede Nantes, France, according to published methods. 181-183

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78 Substrate Preparation Glass microscope slides were used as substrates for UV-vis and catalysis studies. These were cleaned by the RCA p rocedure184 and blown dry with a nitrogen stream. To c, they were immersed in a 5mM solution of OTS in bicyclohexyl for 2 e icated for 15 minutes each in e methanol/chloroform, and chloroform. The substrates were then s r, ton, on technique (Scheme 1).15,16 The aqueous subphase was 2.6 mM in CaCl2 adjusted to pH make them hydrophobi minutes and dipped in toluene for a few seconds. The slides were dried with a nitrogen stream and the process repeated. 185 Single crystal silicon wafers (1 0 0), wercut using a diamond glass cutter to 25 mm x 15 mm x 0.8 mm for XPS substrates. The substrates were cleaned using the RCA procedure then son methanol, 50/50 by volum onicated in a 2% OTS solution in hexadecane and chloroform (50/50 by volume) for two hours. Finally, the substrates were sonicated for 15 minutes each in chloroform, 50/50 by volume methanol/chloroform, and methanol. 186 Germanium attenuated total-reflectance (ATR) crystals were washed with hot chloroform by placing them in the thimble of a Soxhlet extractor. They were made hydrophobic by soaking them in the above OTS solution for an hour, and then rinsed again with hot chloroform. Film Formation KSV Instruments (Stratford, CT) Langmuir-Blodgett systems KSV 2000 and 3000 were used in combination with a homemade, double barrier Teflon trough for the LB filmpreparation. A platinum or filter paper Wilhelmy plate, suspended from a KSV microbalance, measured the surface pressure. Subphases were prepared with pure watewith a resistivity of 17.8-18.0 M cm -1 produced from a Barnstead NANOpure (BosMA) purification system. Zirconium phosphonate films were prepared using a three-step depositi

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79 7.8 with a 0.10 g/L KOH solution.143 A glass sample vial was placed in the subphase in the winto the in Zr4+. After at least 36 hours in the zirconium solution, the hydrorm Catal carbon tetrachloride, so 27.5 mg were diluted in 20 mL and sonicated for 30 min. After sonicating, 13 L of cyclohexanone, the internal standard, and 130 L of cyclooctene were added. The ell of the trough. ODPA was spread from a 0.3 mg/mL chloroform solution and compressed at 10 mm/min on the water surface to a target surface pressure of 20 mN/m. The hydrophobic substrate was dipped down through the monolayer surface and sample vial at 8 mm/min, transferring the ODPA layer. The substrate and the vial were then removed from the trough and an amount of zirconyl chloride was added to the vial tomake the solution ca. 3 mM philic substrate was removed from the vial and rinsed with water. To deposit the porphyrin layer, the hydrophilic substrate was submerged in a solution of 1 at about 10 -5 M in 9/1 ethanol/water, and the porphyrin was allowed to adsorb for 2 hours. The substrate was then rinsed with methylene chloride or chlorofofor 30 minutes to remove any physisorbed chromophores. UV-vis and ATR-IR were used to monitor the completeness of the deposition. Film Characterization Methods for attenuated total-reflectance FTIR, X-ray photoelectron spectroscopy and transmittance UV-vis experiments were the same as we have reported in the past. 16,146 ytic Oxidations Iodosylbenzene (PhIO) was synthesized from the diacetate precursor using NaOH. 187 Its purity was confirmed using catalyst 2 in a test reaction of the epoxidation of cyclooctene by PhIO where the published results were obtained. 179 The PhIO compound is only slightly soluble in

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80 mixture was stirred for ab out 1 min and solvent was added to complete 25 mL of solutiope m x 26 mm x 1 mm) with the adsorbed catalyst while the blank and contained a clean slide of the same dimensions. In all reactions, catalyureactions. For the reactiA) 0 mm was used tion evaporation of the solvent, the syring on. Samples of 1 mL of this mixture were used for the “blank, homogeneous” and “heterogeneous” runs in Teflon bags. The heterogeneous bag contained a microscslide (34 m homogeneous reactions st:oxidant:substrate ratios were near 1:5000:40000. For the homogeneous reactions, 10 L of a 0.1 mM (1 nmol) solution of 1 or 2 were added, while coverage on the glass slides was measured as 1.0 0.2 nmol for the heterogeneos ons with ethylphosphonic acid (EtPO 3 H 2 ), 5.2 L of 1.9 x 10 -4 M or 1.9 x 10 -5 M EtPO 3 H 2 were added for a 2:acid ratio of 1:1 or 1:10. The Teflon bags were made by sealing sheets with a Technaseal (Livermore, Cthermal impulse heat sealer. The sealed bags, with dimensions of approximately 4x 30 mm, were placed in a closed beaker and tumbled with a mechanical arm at ca. 100 rpm. Product concentrations were determined relative to cyclohexanone, whichas the internal standard. The gas chromatograph was a Hewlett Packard 5890 Series II equipped with a hydrogen flame ionization detector. A 0.4 L portion of the reacsolution was injected onto the 25 m, 0.33 m film thickness, 0.20 mm ID HP-5 column (Crosslinked 5% PH ME Siloxane). The column was held at 80 C for 1 min and then ramped at 5 C/min to a temperature of 95 C. A second ramp of 70 C/min was appliedto a temperature of 200 C for 1 min. In order to avoid e hole was sealed after each sampling with a self adhesive label of a size similar tothe bag. The average solvent loss for each reaction was less than 6%.

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81 After completion of the reaction, films were continuously rinsed with chloroform in a Sox hlet extractor for at least one hour to remove any remaining PhIO. Optical spectra of the 4-1-1 to olutions. The porph y rinsing with solvent filtered through basic alumina. Purposeful addition of chloride to a solutoes not change the optical spectrum. nic acid (EtPO3H2) and 1x10-4 M triisobutyl aminened to y slides were then recorded. Results Optical Properties of the Porphyrins The optical spectrum of 1 in a 1x10 -6 M methylene chloride solution shows a Soretband at 460 nm ( = 2.9 x 10 M cm) (Figure 4-2 a). This peak position is attributedintramolecular phosphonate binding to the Mn 3+ ion based on studies of the parent compound, 2. Chloroform solutions of 2 in the concentration range 10 -5 to 10 -8 M consistently exhibit the Soret band near 476 nm ( = 4.5 x 10 4 M -1 cm -1 ). Optical spectra reveal no tendency for the chromophore 2 to aggregate in these dilute s yrin is axially bound by chloride ligands either from the porphyrin synthesis or present in low concentration from the solvent. Chloride coordination can be significantlyreduced or eliminated in the film b ion of 2 d However, when 1 x 10 -4 M ethylphospho were added, the Soret band shifts to 462 nm (Figure 4-3). The amine is necessary to deprotonate the phosphonic acid and it was found not to bind the porphyrin in the absence of EtPO 3 H 2 . The peaks at 462 nm for 2 and at 460 for 1 are therefore assigaxial coordination of the Mn 3+ center by phosphonate. Self-Assembled Monolayers Monolayers of 1 were prepared by adsorbing the porphyrin from solution onto zirconated LB monolayers of octadecylphosphonic acid according to Figure 2-1. A ke

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82 to the process is that the LB layer generated after step 2 provides a flat, uniform surface allowing detailed analysis of the subsequently adsorbed porphyrin layer. The deposited porphyrin layer is clearly observed in optical spectroscopy. Films of 1 deposited from ethanol/water (9/1 mixture) show a max at ca. 463 – 464 nm (Figure 4-2 b), which corresponds to the peak in solutions of 1 and can be attributed to non-aggregated metalloporphyrins with coordinated phosphonate. Evidently, not all of the phosphonagroups on each porphyrin bind the surface. When self-assembled films of 1 are rinsed in methylene chloride or chloroform, the red shoulder asso te ciated with a change in the ligand ntly, indicating that chloride can displace phosphonate ligatio -ke of the porphyrin in the films can be determined from polarized absorbance of the Soret band, according to a procedure described by Mbius.188 Optical environment grows in significa n (Figure 4-2 c). However, as the films dry, the chloride ion is displaced by the phosphonate group. Additional evidence for porphyrin adsorption is seen in FTIR and XPS studies. AnATR-FTIR spectrum of a monolayer of 1, referenced to the zirconated LB layer, clearly shows the presence of the alkyl chains associated with the four alkylphosphonic acid substituents (Figure 2-4). Bands at 2926 cm -1 and 2855 cm -1 corresponding to the a (CH 2 ) and s (CH 2 ) modes, respectively, are consistent with a disordered, liquid-liarrangement of the alkyl substituents. The size of the porphyrin prohibits the alkyl substituents from organizing to any extent. A complex pattern centered near 1050 cm -1 , due to the phosphonate P-O stretches, is also observed. An XPS survey scan of the film shows the presence of Mn, F, O, and N, corresponding to the porphyrin, as well as Zr, Pand O from the metal phosphonate network. 18 The orientation

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83 spectrf e ase be a easuring the optical spectra of the porphn as the surface area per molecule in the absence of constverage of layers ive a were recorded using polarized light at an incident angle of 45 o . The ratio of sand p-polarized absorbance for the band at 464 nm corresponds to the porphyrin having an orientation where the ligand plane is tilted from the substrate normal in the range o72 o -76 o . No in-plane anisotropy was observed. Therefore the porphyrin molecules ararranged nearly parallel to the surface, as was found previously for the Pd 2+ and free-banalogs of 1. 146 The slight deviation from laying exactly parallel to the surface mayconsequence of the altered molecular geometry caused by one of the alkylphosphonate arms coordinating the metal center. The porphyrin coverage in the films was measured by etching the metal phosphonate layer from of the glass slides and m yrin in the resulting solutions. The films do not wash off with water or organic solvents because of the insolubility of the zirconium phosphonate network. However, zirconium phosphonates are soluble in HF, so films prepared on glass coverslips were rinsed off with a solution of 50% HF and water. Transmittance optical spectra of the glass slide after rinsing revealed that porphyrin 1 was entirely removed. Application of Beer’s law to the washings indicates a coverage of (61) x 10 -11 mol/cm 2 (average of 4 films). The measured coverage can be compared with that observed for a Langmuir monolayer of 1 on water. 18 A compression isotherm of 1 on a water subphase at room temperature shows an increase in surface pressure near a mean molecular area of 250 2 /molecule. This value can be take raints on the organization of the alkyl substituents and corresponds to a co6.6 x 10 -11 moles/cm 2 . Therefore, the observed coverage for the metal phosphonate is consistent with a model whereby the porphyrin molecules adsorb from solution to g

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84 a complete monolayer of non-interacting chromophores laying approximately parallel tthe surface. In order to show that 1 does not desorb from the film under the conditions used the catalysis studies, a slide with a monolayer of porphyrin was stirred in 1mL of CCl o in talyst on each slide. The small amount of catalyst imposed the condition of a smallesis.189,190 We adapted the approach to prepare reactors to hold the glass all solvent volume to wet the slides and eliminate solvent evapof the films, and for homogeneous reactions with 1 or 2, is reported in Figure 4-5 and Table 4-1. The 4 for 24 hours. Afterwards, the optical spectrum of the solvent did not show the presenceof 1. For the slide, a decrease of less than 10% in the area of the Soret band was observed. Catalysis We chose to investigate the catalytic properties of the films using the epoxidation of cis-cyclooctene with iodosylbenzene (PhIO) as an oxygen donor (1) The monolayer films were deposited to cover 17.7 cm 2 , resulting in approximately 1 nmol of ca O PhIO, catalyst (1) solvent volume, chosen to be 1 mL in this study. Significantly larger volumes would lead to low epoxide concentration and inaccurate detection. Thermally sealed Teflon bags, formed in the shape of the slides, served as reaction vessels for the small-volume reactions. Similar bags have been used by other groups for solvatothermal solid-state synth slides. The bags allow a sm ration during the course of the reaction. The extent of epoxidation as a function of time in the presence o

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85 yields are calculated relative to the limiting reagent, which is the oxidant in each case. Small variations in the amount of catalyst one slide to another were accou 2-5 s s in monolayer films and in solution.a eaction time (hr) Yield of epoxidedTurnover numbere present from nted for by normalizing the result for the amount of catalyst, determined from the area under the Soret band. Each point is an average of at least three runs and representative error bars, calculated at the 95% confidence level, are included. Figurealso shows the epoxide yield in the absence of catalyst (blank reaction). Homogeneoureactions containing 1 nmol of 1, 1 nmol of 2 with 1 nmol of EtPO 3 H 2 , and 1 nmol of 2with 10 nmol EtPO 3 H 2 were also monitored over time and their yields and turnovers after7 to 8 hours are also included in Table 4-1. Table 4-1. Epoxidation of cyclooctene by PhIO and catalyzed by manganese porphyrinCatalyst R Mono layer of 1 8 20 6% 893 268 1 Homogeneous b 7 4.9% 245 3.6 0.5% 180 25 b2 Homogeneous 2 Homogeneousb 8 2 Homogeneous with 1 nmol EtPO3H28 4.1% 205 Monolayer of 1 72 30 7% 1340 350 100 b with 10 nmol EtPO 3 H 2 7.5 6.1% 305 Blank c 8 0.5 0.2% 2 Homogeneousb 72 15 2% 750 Blank c 72 0.9 0.3% aMolar ratios of catalyst:PhIO:cyclooctene were 1:5000:40000 in CCl4. Uncertainties are based on standard deviations from at least 3 runs. b1nmol of catalyst used. cConditions identical to homogeneous reactions but no catalyst used. dCalculated using on PhIO as the limiting reagent. eCalculated as moles of cycloocte ne oxide produced over moles of catalyst.

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86 Characterization of the Catalyst Systems Heteroge neous and homogeneous reactionsThe optical spectra of the films used in oxidations were recorded after they were sorbed PhIO. The spectrave the same before use, with a ntly found near 464 nm (Fre 2-6 c), alteverage of 4 films) an 4% after 72 hours e of 3 films). Optical spectra of the supernatant reaction solutions show no rin (Figure 4-6 a). For compn, the spece solneous reaction of 1 shows similar degradation of the porphyrin, but at an even ster rate (Figure 4-6 b). As the spectra in Figure 4-6 a-c were taken after catalysis runs and after the slides have been removed from the reaction conditions, it is possible that they do not represent the state of the film during the course of the reaction. Therefore, a slightly different experimental setup was used to monitor the porphyrin film under the reaction conditions. A film of 1 was prepared on a thin glass coverslip and placed in an optical cuvette containing cyclooctene and PhIO in the same concentrations used in the quantitative runs. The cuvette was tumbled by a rotating arm and then stopped to record the electronic in Figure 4-6 d, is similar to the ex situ spectrum and no -oxo or other higher oxidation state porphyrin species are observed. Homogeneous reactions with 2 The optical spectra of the homogeneous reaction solutions of 2 showed at all times the Soret band shifted to 422 nm with a shoulder at 500 nm (Figure 4-7). This species has been identified in related porphyrins as a Mn(IV) -oxo dimer.191 The intensity of with 1 rinsed to remove ad ha bands as Soret band consiste igu though the in nsity is decreased by 70 10% after 11 hours (a d 99 (averag dissolved porphy ariso trum of th ution of a homoge fa spectra of the film while still immersed in the reaction conditions. The spectrum, shown

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87 the Soret band does not change even after 72 hours of reaction, suggesting that this species is stable under the o xidative reaction conditions. When EtPO3H2 is added to a solutie s is spectra. This result was expected since the n the periphery of the chromophore was designed to allowre 4-rly shows that the film catalyzes alkene epoxidation, demonstrating that monolayers prepared in this way can be used to investigate supported catalysis. To better understand the monolayer on of 2 in a 1:1 ratio, the optical spectrum taken after 8 hours is similar to that of threaction without the acid. On the other hand, a 10-fold excess of the acid causes a significant inhibition of the -oxo dimer with a Soret band at 478 nm. Discussion Analysis of how the catalyst 1 is organized in the zirconium phosphonate filmconsistent with a near monolayer coverage of non-interacting porphyrins, with their macrocycle plane oriented almost parallel to the substrate. A combination of optical spectroscopy, XPS, and FTIR spectroscopy confirms that 1 adsorbs to the surface, and the orientation of the molecule is supported by measurements of surface coverage together with dichroism of the optical position of the phosphonic acids o the porphyrin to stand like a table on the surface, and a similar monolayer arrangement was found for the Pd 2+ analogue of 1. 146 However, not all of the phosphonate arms of 1 bind to the zirconated surface, as the Soret band at 464 nm (Figu4-2 c) indicates that at least one arm participates in axial binding of the Mn 3+ center.Therefore, our view of the porphyrin on the surface is similar to that depicted in Figure 8, with molecules chemisorbed to the surface such that the porphyrin plane tilts slightly from parallel. The catalytic activity reported in Table 4-1 and in Figure 4-5 clea

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88 catalyst and its activity, homogeneous studies were performed using 1 and the related compther ns r than 4-1) n ains on 1 prevents the formation of -oxo dimers and a band at ca. 420 nm, ty ts ound 2 under conditions similar to those used in the heterogeneous studies. The homogeneous reaction conditions were not chosen to optimize reaction yields, but rato allow useful comparisons of the catalyst behavior. Therefore, homogeneous reactioused the same amount of catalyst, substrate, oxidant and solvent as were used with the monolayer reactions. It is for this reason that reaction yields reported here are lowethose of other studies using similar porphyrins but with larger catalyst:substrate ratios. 122,174,176,179,180 Comparing Homogeneous Reactions of 1 and 2 The activity of catalysts 1 and 2 are very similar after 7 to 8 hours (Tableresulting in ca. 200 turnovers. However, the mechanisms of deactivation of the molecules are very different. Porphyrin 2 forms a -oxo dimer shortly after the reactiostarts and perseveres over a period of 72 hours (Figure 4-7). Epoxidation continues over this time, but the rate of conversion is low. Inclusion of the sterically hindering alkyl phosphonate ch pical of these species, 191 is not seen in optical spectra of the reaction mixture. On the other hand, the molecule 1 has been completely destroyed after 7 hours as evidencedby the absence of any Soret band at all (Figure 4-6 b). The two homogeneous catalysgenerate epoxide at similar rates early on in the reaction, perhaps via the same mechanism, but then deactivate differently. Compound 1 is destroyed by the reaction conditions, while 2 forms the less active -oxo dimer.

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89 Films of 1 vs the Homogeneous Reactions The films of 1 s how a significant improvement in catalyst turnovers over the first igure 4-5). The turnover numbers are five tar to atives n ontinues parison with the homogeneous of the catalyst, and not only its modified structever, gives a slightly higher turnover. Optical spectra of these reaction solutions show that the few hours relative to the homogeneous reactions (F imes higher for the films relative to either 1 or 2 in solution after 8 hours. Similthe homogeneous reaction, the immobilized catalyst 1 does not form -oxo deriv(Figures 4-6 c and 4-6 d), as its structure combined with surface immobilization preventsdimer formation. Nevertheless, the films of 1 degrade during the reaction and there is no increase in the epoxide yield after about 30 hours. The degradation mechanism is probably the same as in the homogeneous reaction of 1, as optical spectra of the reactiosolutions are similar (Figures 4-6 a and 4-6 b). However, immobilization offers some protection towards porphyrin degradation since 30% of the porphyrin is still present on the film after 11 hours, as compared to the homogeneous reaction with 1 where no porphyrin is present in solution after 7 hours. In addition, the remaining film cto catalyze with the same rate of turnover (corrected for the amount of remaining porphyrin) as the starting film. This result, plus the com reactions of 2, clearly shows that immobilization ure, have an important influence on its activity. Effect of Phosphonate Another set of control reactions was performed to investigate the role of phosphonate. Ethylphosphonic acid was added to a homogeneous reaction with 2 to investigate the potential role of the phosphonate as a co-catalyst in the films. A molar ratio of 1:1 2:EtPO3H2 gives turnovers comparable to the reaction without the acid (Table 4-1). Increasing the amount of phosphonic acid to a molar ratio of 1:10, how

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90 1:1 ratio does not affect -oxo dimer formation, while a significant inhibition is observed when a 10-fold excess of the phosphonic acid is included. The phosphonic acid appearsto stabilize the porphyrin in the oxidation conditions, but the activity is still significantlyless than for the immobilized porphyrin. Monolayers as Models for Supported Catalysts While monolayers on flat surfaces have limited utility as practical catalysts dthe difficulty of achieving large surface areas, the present study shows that they can be useful tools in the study of heterogeneous reactions. An important feature is that a flat surface, prepared under controlled conditions, allows f ue to or uniform adsorption. Deposition ts in monolayers that contain essentially a single type of catalytic site. ture with s ed ere ight arise from the ability to organize molec of the catalyst then resul This feature of monolayers is important when attempting to correlate strucproperties. Chemical and structural characterization is easier if only a single species ipresent, which then allows catalytic activity to be correlated with a specific, characterizsite. These points are in contrast with the situation for high surface area supports, whdifferent surface features and a variety of pore shapes and sizes lead to a dispersion of catalyst environments. Activity must then be assigned to an ensemble of sites, making it difficult to correlate behavior with surface structure. The monolayer films can then serve as models for specific types of molecular sites on high surface-area supports. Changes inmolecular design and deposition procedures can be used to explore different molecular arrangements. It should also be noted that new phenomena m ular catalysts into controlled assemblies. The recent example of Tllner et al. showed that an LB film of an amphiphilic rhodium bipyridine complex presented a largely improved catalytic efficiency with respect to the same complex in solution. 192

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91 The authors attributed the increase to the influence of the assembly of sites at the interface. In the case of the monolayer films of 1, the observed increase in activity can be attributed to a combination of enhanced catalyst lifetime and the altered conformation that the molecule adopts upon adsorption. However, effects due to the local concentration of sites cannot be ruled out. Finally, monolayer deposition techniques provide special opportunities for arranging molecular components into “supramolecular assemblies” at solid supports. 193Organized structures can present different or enhanced properties compared to those of the individual molecules. For example, with respect to the present system, the deposition process might be extended to include surface-bound ligands to axially coordinate the porphyrin 1. The axial coordination would complement covalent linkage, but might alsobe used to direc t surface adsorption. In addition, axial ligands as cofactors can improve the ca talytic performance of the porphyrin and can also extend the array of useful oxidants.

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92 PO3H2PO3H2PO3H2POHPOHPOHPOHPO3H2PO3H2PO3H2Step1LB DepositionPhosphonic AcidLangmuir MonolayerOTS coated slideSample vial PO3H232323232PO3H2PO3ZrPO3ZrPO3ZrPO3ZrPO3ZrPO3ZrPO3ZrPO3ZrO3PO3PO3PO3PO3PZr4+H2OAdsorption of 1( )EtOH/H2O Figure 4-1. Three-step deposition procedure for the formation of films of 1. The film is present on both sides of the slide.

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93 Figure 4-2. Optical spectra of 1 as (a) 1 x 10-6 M in CH2Cl2 (b) monolayer film after deposition from ethanol/water and (c) the same film rinsed in methylene chloride for 30 minutes. Soret bands near 460 nm – 464 nm are assigned to phosphonate coordinated to the Mn3+ while the peak position at 478 nm is Figure 4-3. Optical spectra of (a) 2.4 x 10-6 M solution of 2 in chloroform (b) solution of 2.4 x 10-6 M of 2 with 1 x 10-4 M ethylphosphonic acid and 1 x 10-4 M triisobutyl amine in chloroform. 400500600700 attributed to Cl coordination 0.000.080.160.24 AbsorbanceWavelength (nm)ab476 462 400500600700 0.000.020.040.06 AbsorbanceWavelength (nm) 464 478 460 bca

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94 300027001200900 0.0000.0040.0080.012 AbsorbanceWavenumber (cm-1) 2855 2 926 t Figure 4-4. ATR-FTIR spectrum of a film of 1 referenced o a zirconated LB layer inregion of the alkyl and phosphonate stretches. the 010203040020406080Time (hr)% Yield Figure 4-5. Extent of the epoxidation of cyclooctene with PhIO oxidant over 72 hours with catalysts: films of 1 (), 2 in solution () and no catalyst ().

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95 Figure 4-6. Optical spectra of (a) the supernatant solution of an oxidation with a monolayer film of 1 after 72 hours, (b) a homoge neous reaction of 1 after 7 hours, (c) a film of 1 after 11 hours of reaction, and (d) a film of 1 immersed in the oxidative conditions after 10 minutes of tumbling. Figure 4-7. Optical spectra of homogeneous reactions of 2 after (a) 1 hour and (b) 72 hours of tumbling. 400500600700 0.00 0.020.040.06 AbsorbanceWavelength (nm)b 422 500400500600700 0.000.040.08 AbsorbanceWavelength (nm)abcd a

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96 NNNNOOOOPO3O3PPO3F4F4F4F4MnPOOHO Figure 4-8. Representation of the orientation of the porphyrin 1 in the monolayer films.

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CHAPTER 5 SUMMARY Langmuir monolayers have been used to examine the effect of an organic interface bservation and quantification of COM at an 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer. COM was observed in situ by Brestwer angle microscopy (BAM) as bright objects at monolayers in liquid condensed (LC) and liquid expanded (LE) phases as well as LC/LE and LE/Gas (G) equilibria. Within the LC monolayers, COM precipitation was largest at the more fluid monolayers, suggesting that the ability of the organic film to reorganize to accommodate the growing inorganic material is important. COM consistently appeared at the phase boundary of the LC/LE and LE/G DPPC. Although it is possible that the crystal formation occurs at the expanded phase and then is attracted to the boundary by dipole-dipole interactions, this mechanism seems unlikely due to the low zeta potential of COM dispersed in an RS 5 solution. It is also possible that the precipitation occurs at the phase boundary. At this point it is not possible to conclusively distinguish between these scenarios. COM precipitation at monolayers with phase boundaries was further explored by preparing a phase segregated binary mixture. A 1:1 DPPC/,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) mixture at 25 mN/m provided a monolayer where the LC domains are DPPC and the expanded phase is DMPC. In this case COM appears exclusively inside the LC phase and away from the boundaries. The same monolayer prepared at a lower pressure, where the LE phase contains a significant amount of both on the precipitation of calcium oxalate monohydrate (COM). Chapter 2 describes the o 97

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98 DPPC and DMPC, induces some precipitation at phase boundaries. It appears that the diminished molecular exchange of the film at high pressure prevents the COM from appearing at the DPPC/DMPC boundary. In an effort to prepare biologicambrane models I turned my attention membas ars at e at ace. In a periodthin ed as lly relevant me rane lipid raft microdomains, Chapter 3. A 2:1:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/brain sphingomyelin (SM)/dihydrocholesterol wprepared as a Langmuir monolayer. The raft domains enriched in SM and dihydrocholesterol were readily observed by BAM. In this case, 80% of COM appethe phase boundaries of large (hundreds of microns) raft domains. Sub-micron sized domains also exist and although they cannot be imaged by BAM, they can be readilyobserved by atomic force microscopy (AFM). It is likely that the 20% of crystals that appear at the expanded phase by BAM are precipitating at the phase boundaries of thsub-micron sized rafts. The work in Chapters 2 and 3 adds further support to the idea thmembranes can act as substrates for the precipitation of inorganic materials from supersaturated urine, forming urinary stones. Chapter 4 describes the incorporation of a manganese porphyrin at a zirconium phosphonate Langmuir-Blodgett film. The catalyst monolayer contains non-interacting porphyrin macrocyles whose plane is oriented nearly parallel to the film surf of 72 hours, the porphyrin activity is much higher within the thin film than in analogous homogenous reactions. The increased activity is due to the protection of the porphyrin against degradation and -oxo dimer formation when incorporated in the film. The work in Chapter 4 demonstrates that Langmuir-Blodgett films can be us

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99 models for supported catalysis, where activity can be correlated with an uniform array ocatalytic molecules. f

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CHAPTER 6 FUTURE WORK Bilayer vesicles will be used as additional membrane models to observe the precipitation of calcium oxalate monohydrate (COM). In these experiments the vesicles will be dispersed in an aqueous solution containing calcium and oxalate, and the inorganic crystal precipitation will be monitored by calcium depletion with a calcium electrode. The calcium electrode will be provided by Professor Laurie Gower at the Materials Science and Engineering Department, University of Florida. The preparation of large unilamellar vesicles (LUVs) is extensively described in the Avanti website (http://www.avantilipids.com/PreparationOfLiposomes.html, http://www.avantilipids.com/LUVET.html, http://www.avantilipids.com/extruder.html). All procedures in this chapter related to vesicle formation and Figure 6-1 were derived from the above websites. To prepare unilamellar vesicles, a dry film of the lipid is prepared (Figure 6-1a), swollen with an aqueous solution (Figure 6-1b), agitated to form multilamellar vesicles (MLVs) (Figure 6-1c), frozen/thawed and extruded to yield the LUVs. Small unilamellar vesicles (SUVs) can be formed by sonication of the MLVs suspension. To determine the effect of lipid rafts on COM precipitation, vesicles of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 2:1:1 POPC/sphingomyelin/dihydrocholesterol will be prepared. 100

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101 Experimental Materials All reagents will be purchased from commercially available sources and used without further purification. 1-Palmlycero-3-phosphocholine (POPC), and bipids ldrich e solvent e solution was frozen in an acetone/dry ice bath and thawed 5 times the eous solution of 150 mM NaCl, 5 mM Tris.HCl and 0.35 mM sodium oxalate and pH 7 was added and the solution was placed in a shaking incubator at 35-37 C for 1 hour. The solution was frozen in an acetone/dry ice bath and thawed 5 times. itoyl-2-oleoyl-sn-g rain sphingomyelin (SM) (purity >99%) were purchased from Avanti Polar L(Alabaster, AL). Dihydrocholesterol, sodium oxalate and tris(hydroxymethyl)aminoethane hydrochloride (Tris . HCl) were purchase from AChemical Co. (Milwaukee, WI). Calcium chloride dihydrate and sodium chloride werobtained from Fisher Scientific (Pittsburgh, PA). POPC Vesicles The solvent of a POPC chloroform solution (4 mg) was evaporated with a gentle N 2 stream. The dry film was placed under vacuum overnight to ensure complete evaporation. An aqueous solution of 150 mM NaCl, 5 mM Tris . HCl, 0.35 mM sodium oxalate and pH 7 at room temperature was added to the lipid film and then tumbled for 1hour in a rotating arm. Th and then extruded at room temperature through a 100 m membrane. The vesiclesize, determined by dynamic light scattering (DLS), was 136 2 nm (average of 5 measurements with standard deviation as error) POPC/SM/Dihydrocholesterol Vesicles A 2:1:1 POPC/SM/Dihydrocholesterol chloroform solution was prepared and solvent evaporated with an N 2 stream. The lipid film was placed under vacuum overnight. An aqu

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102 The vesicle extruder was pre-heated to 36 C since extrusion ought to be performed at s above the phase transition of the lipid mixture. The vesicles were extruded and thontamination with the organic mount of calcium solution can be and the Ca2+ depletion measured. Alternatively, the electrode can be place temperature eir size determined by DLS to be 159 1 nm. COM Precipitation COM formation induced by the lipid vesicles can be monitored by calcium depletion with a calcium electrode. To prevent electrode cmaterial, two possible experimental setups can be used, Figure 6-2. The solution of the extruded vesicles can be placed in a dialysis bag, Figure 6-2a. Once the bag is prepared and sealed in an oxalate solution, the appropriate a added drop wise d in a dialysis bag and immersed in the vesicle solution containing both calcium and oxalate ions.

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103 Figure 6-1. Representation of the preparation of lipid unilamellar vesicles. a. Dr y li p id fil m b . swellin g c. MLV SUV LUV Freeze/fracture sonication and extrusion

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104 Figure 6-2. Schematic representation of two possible setups for the calcium depletion experiment in the presence of lipid vesicles. Ca2+C2O42Calcium electrode Aqueous solution containing calcium and oxalate ions Dialysis bag containing LUVs a Ca2+C2O42-electrode Calcium Aqueous solution containing calcium ions, oxalate ions and LUVs Dialysis bag b

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BIOGRAPHICAL SKETCH Ia O. Bentez was born in Valencia, Venezuela, in 1977. She received a sNew Mexico, USA. he thn pursued her undergraduate studies at McGill University, Canada, where she majored in chemistry. At McGill she performed undergraduate research under the supervision of Professor Linda Reven and Professor Hanadi Sleiman in the area of solid state NMR. She started her graduate studies at the University of Florida in the fall of 1999 and joined Professor Daniel Talham’s research group shortly thereafter. Her research interests involve catalyst immobilization at monolayers and membrane models for urinary stone formation. She will join Unilever’s Research Center in Edgewater, NJ, in December 2004 and then Unilever’s Global Technology Center in Trumbull, CT, in January 2005. scholarship 1993 that allowed her to attend an international boarding high school, the Armand Hammer United World College of the American West, in S e 116