Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1017
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`

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`
`
`LLIPID BIOCHEMICAL PREPARATIONS
`
`edited by
`
`Li). Bergelson
`
`USSR Academy of Sciences, Shemyakin Institute of Bioorganic
`Chemisuy, Moscow, U.S.S.R.
`
`
`
`
`
`1980
`
`UMV. OF CONN.
`
`“a 51999
`
`ELSEVIERINORTH-HOLLAND BIOMEDICAL PREssx’J-Tili'z‘fi
`AMSTERDAM-NEW YGRK-OXFORD
`
`653.513", LlfiRARY
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`
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` *
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`AKER877ITC0079651
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`’
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`

`iv
`
`9 1980 ElsevierfNorth-Hofland Biomedical Press
`
`
`
`;
`
`All right reserved. No part of this publication may be reproduced, stored in a retrieval sys—
`tem, or transmitted,il any form or by any means, electronic, mechanical, photocppyingv,
`recording ur otherwise, \vilheut the prior permission of the copyright owner.
`1an 04443-801464
`
`V
`
`Prefau
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`might serve :
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`chemistry an
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`Preparing P“
`tory. The pr
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`Besides a
`cantributed 1
`by L reading 31
`V.I. Kulikov
`
`v.1! Shvets, ]
`Zvonkova. It
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`Published by:
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`ElsevieI/North-Holiand Biomedica} Press
`335 Jan van Galenstraat, PD. Box 211
`Amsterdam, The Netherlands
`
`Sake distributors for the USA and Canada:
`ElsevierfNorth-Holland, Inc.
`52 Vanderbilt Avenue
`New YOIKNJY. “1017, U.S.A.
`
`Library ofCongas: Cataloging in Publication Data
`.
`_
`Mam entry under tlfle:
`Lipid biochemical preparafions!
`
`Bibliography: p.
`Includes index.
`1. Lipids. 2. Extraction (Chemistry) 3. Lipids
`-- Analysis. I. Bergelson, LD.
`QPYS 11.546 574.19’293 80-12236
`ISBN 0-444-80146-4
`
`
`
`
`000003
`AKER877ITC00739652
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`

`

`
`
`Suwd, stored in a retrieval sys-
`iic, mechanical, photocopying,
`:pyright owner.
`
`Preface
`
`The present book is an outcome of the joint experience of the staff of the
`lipid Laboratory in the Shemyakin Institute of Bioorganic Chemistry acetami-
`lated in 15 years of working in the lipid field. Since the main occupation of the
`laboratory is the physica-chemicalstndy of the structure and functioning of
`lipids in cell membranes, our work depends strongly on the availability ofpure
`lipid substances. Such substances are constantly prepared in our laboratory and
`we thought it usefid to summarize our experience in the form of a hook that
`might serve as a practical guide for students as well as for experienced workers.
`The book consists of two parts, Part} is an introduction into preparative lipid
`chemistry and biochemistry. It also contains practical instructions for the prep—
`aration, purification and handling of lipid substances and describes the different
`approaches used in the partial synthesis of complex lipids as well as the tech-
`niques of purity control of lipid samples. Part I] contains detailed procedures for A
`preparing pure lipid substances, most of which have been tested in our laborar
`tory. The procedures are assembled in seven chapters covering the main lipid
`classess; in the eighth one we deal with the preparation of intennediates. Each
`chapter is opened by a short introductory survey (by Li). Bergelson) and
`presents also recommendations for the purity characterization and storage
`conditions of Lipids belonging to the given class.
`Besides the authors indicated in the title the followong colleagues have
`contnhuted to the book by submitting and testing some of the procedures and
`by reading and commenting upon the manuscript: V.V. Bezuglov, ML. Cirenina,
`V.I. Kuhkov, TJ. Iazmkina, L.F. Nikuiina, T.G. Pilipenko, VP. Shevchenko,
`VJ. Shvets, N.G. Timofeeva, AN. Ushakov, V.A. Vaver, V.I. Volkova and EN.
`Zvonkova. It is a pleausre to acknowledge their invaluable help and advice.
`
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`Introduction
`
`The contemporary science of lipids and related substances (lipidology) has
`developed mainly on the border between biochemistry, organic chemistry and
`physical chemistry. The long path. by which lipidology has achieved its con-
`temporary status was not straightDuring the first period which lasted about a
`century (from Chevreuille to Hilditch) the preparative approach dominated. In
`order to identify lipids and to determine their amounts it was necessary to
`isolate and purify the substances in quantity, to obtain derivatives and to
`measure their physical constants. In those days an analysis of a complex lipid
`mixture required years of tedious work: workers were fully occupied with
`extractions, evaporations, recrystallizations and distillations. Purif'cation was
`difficult to achieve and mostly incomplete leading the organic purists to call this
`type of work ‘Schmierchemie”. Slow progress began only in the early fifties
`with the appearance of different
`types of chromatography, counteremTent
`distribution and, other novel separation methods. However, only in the sixties
`a qualitative jump took place due to the advent of sensitive techniques such as
`thin-layer and gasJiquid chromatography, mass spectrometry, high performance
`liquid chromatography and their combinations. This resulted in a dramatic
`decrease in sample size and a concomitant increase of productivity. From kilo-
`grams used in the past, the amount of starting materials decreased tornilligams
`and the size of analytical samples reached the microgram and even the nanogram
`level. The time required for a fatty acid analysis shortened from several months
`to a few hours. New unprecedented possibilities opened before the lipidologist,
`who now could include in his studies microscopic objects of cellular biology.
`The preparation methods developed in the past seemed to have become almost
`useless.
`
`Recently, however, preparative lipid chemistry has received new stimuli. This
`
`is...”mm
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`may be explained by different reasons. 0n the one hand modern purification
`methods allow to obtain lipid preparations of high purity; on the other hand the
`need for such preparations has drastically increased. The new needs stem mainly
`from studies of biological membranes. A widely used approach in these studies is
`based on utilization of model membranes, Le. “black” bilayer membranes and
`lipid vesicles (liposornes). A prerequisite for such work is the availability of
`reasonablypure and standard lipids. Another field requiring pure lipid substances
`is the study of membrane-botmd enzymes. Frequently such studies include a
`reconstitution step in which solubilized and purified enzymes are interacted
`with well defined lipids. A new promising area of research opening exciting
`perspectives is based on the utilization of liposomes for therapeutic purposes,
`e.g. as drug carriers or iinrnunostirmdants. Besides, pure lipids of known struc-
`tare are of comse also used in traditional studies of the biosynthesis and break-
`down of lipid substances. Research in each of the above directions requires not
`only purified lipids of natural origin. which are mostly mixtures of a large
`number of Components differing in their fatty acid compositidn, but to an
`increasing extent mily individual lipid compounds (Le. individual molecular
`species) which can only be supplied by chemical synthesis or semisynfliesis.
`Besides, radioactive labeled lipids and modified lipids containing spin- and
`fluorescent labels or photoreactive groups are applied more and more.
`In response to increasing demands a number of companies started the produc-
`tion of lipids on a larger scale and at present the list of commercially available
`lipid products increases steadily. Nevertheless, many impertant substances are
`still not available. Besides, commercial preparations are frequently very expen-
`sire and insufficiently pure. Of course, the commercially manufactured simpler
`lipids such as rnethylpalmitate, tristearin, triolein and quite a few others may be
`obtained in a state of high purity. However, more complex lipids are more
`difficult to prepare and to purify. Consequently commercial preparations fre—
`quenfly contain impurities. A large number of cronmtercial phospholipids and
`glycolipids tested in the audacrsl laboratory proved to be contaminated with
`related or foreign substances which showed up as extra spots on thin-layer
`chromatograms.
`Often this is not the fault of the manufacturer became many lipids are so
`unstable that they cannot be stored and shipped under ordinary conditions.
`Therefore additional purification of. the preparations becomes necessary. Such
`purification frequently consumes so much time and materials that purchasing,
`commercial preparations is of no advantage.
`The purpose of this book is to present detailed instructions for the prepara-
`tion of pure lipids, in particularly their individual molecular species.
`
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`When
`methods
`Reliab
`had pars:
`SWPZI
`in an ave
`exist has
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`exotic st
`mostly o
`available
`Intent
`points 9f
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`equipmr
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`Scaling 1
`steps be:
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`
`ix
`
`r purification
`her hand the
`
`. stem mainly
`rese studies is
`mbranes and
`vailability of
`id substances
`ies include a
`re interacted ‘
`ning exciting
`tic pruposes.
`known struc-
`sis and break—
`
`5 requires not
`es of a large
`n, but to an
`tal molecular
`
`endsynthesis.
`ng spin- and
`re.
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`dithe produc-
`ially available
`ubstances are
`I very expen—
`turecl simpler
`fibers may be
`lids are more
`
`pamtions fre-
`pholipids and
`minated with
`
`on thinalayet
`
`' lipids are so
`y conditions.
`cessary. Such
`at purchasing
`
`r the prepara—
`
`When selecting procedures for the preparation of lipids from the wealth of
`methods described in the literature we followed four principles:
`Reliabilfw. Emphasis was placed on procedures with which the authors have
`had personal experience.
`Simplicity. Preference was rendered to simple procedures which can be used
`in an average biochemical laboratory. Only in cases where no simple approaches .
`exist have complicated methods such as total synthesis been included.
`Availability of starting mtm'als. We tried to avoid procedures depending on
`exotic starting materials. Accordingly, the isolation procedures presented use
`mostly organs of rats or cattle, common vegetables, cereals and some readily
`available microorganisms.
`Interchangeability of methods. Usually no single method is best from all
`points of view. Since the choice of method is determined not only by the nature
`of the lipid required but also by the experience of the worker and the available
`equipment more than one method has been included in many cases.
`It must be noted that not only newcomers but also experienced people
`acquainted with the analytical aspects of lipidology encounter difficulties when
`attempting toywork on a preparative scale. Usually the lately developed methods
`for isolating and purifying lipids are carried out on a micro- or semi-micro scale-
`Scaling up of such micromethods presents new unpredicted problems. Many
`steps become tedious, solvent volumes are unpleasantly large for handling,
`evaporation and solvent purification consume too much time and become
`dangerous, partitioning of solvent becomes difficrdtbecause stable emulsions are
`formed etc.
`0
`Taking into account such problems and the fact that those using lipids in
`their work may have only limited if any experience in handling lipid substances,
`we tried to select optimal scales and conditions and believed it important to
`draw attention to the small details which may determine the success of the
`experiment. A: the same time the authors hope that the present book will serve
`not only as a “collection of recipes”. We shall consider its purpose achieved if it
`would help the one or the other biochemist to step onto hitherto unknown land
`and to discover that “it can be done” also in the field of lipids.
`
`L33. Bergelson
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`lntroduction .....
`
`...........................
`
`vii
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`Perri. The preparation ofpure lipid substance:
`
`Chapter 1.1. Preparative extraction oflipids from naturgl‘sources ...... . .....
`1.1. General comments .................................
`1.2. Pracficalinstmcfiong................... ..... . .......
`1.2.1. Solvents ..................................
`1.2.2. Treatment of material prior to extraction . .
`.
`. . .
`. ........
`1.2-3. Exuacfive homogenization .......................
`1.2.4. Scpmtion of the organic and aqueous phases ............
`1.2.5. Removal of nonnlipid contaminants ...... '..... . ......
`1.2.6. Solventnmoval.
`1.2.7.
`Storage of lipid preparations .................... .
`.
`
`Chapter L2 Purification oflipids by column chromatography ..............
`2.1. General commons .......... . ......................
`2.2-
`Practical instructions ................................
`2.1.1. Column preparation ..........................
`2.2.2.
`Sample application and the elation of the columns
`.
`.
`. ......
`2..3.3 Control of the elution princes by micro-thin—Jaye: chromatog-
`raphy . . .................................
`2.3. Chromatography of lipids on column: of silicic acid .
`. . ...........
`23.1. Generaloomments.....-......-. ..............
`2.3.2.
`Practical instructions . i .........................
`2.4. Cinematography of lipids on cohunniof aluminium oxide . . .
`. .
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`. . .
`2.4.1. General comments ......... .
`., .............. .
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`2.4-2. Practical'instructions ........................ . . .
`2.5. Chromatography ofpolafiipifis on ion exchange cellulose: .
`. .
`. .
`. . .
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`2.5.1. General comments ............................
`2.5.2. Pmcficalinstmcfions...,.............. ..........
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`HDWMflMMMI—cm
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`15
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`xii
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`1.4. Hydro;
`1.4.1.
`1.4.2.
`,1,4.3‘_
`1.4.4.
`1.4.5.
`1.5. hosts;
`1.5.1-
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`Chapter 1.3. Preparative min-layer chromatography nf lipids ......... . ......
`3.1. General comments .................................
`3.2.
`Practical instructions ................................
`3.2.1 Prepmfionoftheplatea........................
`32.2 82mph appliufion ............................
`3.2.3. Development of cluomatograms and detection of the zones .
`.
`.
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`3.2.4.
`lsotafion of the separated fracuons
`.
`. . ..... .
`. ..... . . .
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`Chaptex 1.4. Use ufnon-chmmatographic methods inthe preparation of lipids
`4.1.
`Introduction ...................... ................
`4.2-.
`Selective extaction and precipitation ......................
`4-3.
`Solvent pattition ..................................
`4.4. Crystallization .
`.
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`37
`37
`40
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`1.6.
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`1.5.2.
`1.5.3,
`1.5.4.
`Spin-121
`1.6.1.
`1.6.2.
`1.6.3.
`17.12G4
`1.8.
`11-N-(2
`acid .
`1.8.1.
`1.8.2.
`
`Chapte: 11.2. Neu
`2.1.
`Inbred:
`2.2. Menogi
`2.2.1.
`2.2.2.
`2.2.3.
`2.2.4.
`2.3. Diglyct
`2.3.1.
`2.3.2.
`2-3-3-
`2.3.4.
`2-3—5;
`2.4. Triglyo
`2.4.1.
`2.4.2.
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`Chapter 1.5. Partial synthesis of complex lipids .......................
`iL hmmmmm .....................................
`5.2. Phosphatidylchofines ...............................
`5.3. Phosphzfidyiserihes ............ .
`.
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`......... .
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`. .
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`5.4V.
`Phosphatidylethanolamines ............................
`5.5. Phosphatidicacids....................I..............
`5.6. Conversion of phosphatidic acid into 0111:! phospholipids ..........
`5.7. Glyeofiyids .....................................
`5.7.1. Glycasyldiglycefides ...........................
`5.7.2. Hexosylceramkies
`........
`
`51
`51
`52
`57
`57
`58
`60'
`60
`60
`63
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`-
`65
`Chapter 1.6. Determination of purity and characterization oflipid substances .....
`65
`6.1. Introduction.................-.......... .........
`66
`6.2. Fattyacids................................ .....
`69,
`6.3. Mono-,di-andtriglyoefides.-..........................
`69'
`6.4. Phosphofipids. . .
`.
`. ...............................
`64 ..1 Detemfination of the nitrogen 01 ester group to phosphorus 13110.70
`64.2 Detennination quolarmoietiei ....................
`73
`6..43.Hyd101ysisbyphc$hulipases. .
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`6.5. Glycolipids ........ .
`..........
`74
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`Part H. Procedures
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`Chapter [1.1. Fatty acids ...................................
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`Chapter 113.019
`1.1.
`Introduction ....................................
`3-1-
`Introdv
`83
`1.2. DbTubexcnIosteatic acid ........ . ....................
`
`
`3'2" Natural
`86
`1.3; Pelyenoicadds.:.........T........................
`3-11.-
`86
`1.31. Methyl Imolenate. Preparation from linseed ofl ............
`
`
`322’
`88
`1.3.2. Methyl amdlidonate. Preparation from rat liver
`.
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`3'23“
`89
`1.3.3-
`'1'ritium—Iabeled mahidonic acid ............. . ......
`
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`1-3.4. Characterizaa‘on of the products and storage conditions ......
`90
`13113111111!
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`1.3;.
`...................
`90
`331
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`42
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`1.5.
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`1.6.
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`1.7.
`1.8.
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`2.4.
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`92
`. ..........
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`1.4. Hydroxy fatty acids ...................{.
`92
`1.4.1.
`2—Hydroxypalmific acid .........................
`93
`1.4.2.
`3-Hydroxysteafic add ..........................
`94
`.1113. Mycnlicacids................ ...............
`96
`1.4.5. Characterization of the products and storage conditions ......
`96
`1.4.5. Notes
`.
`..
`. ................................
`98
`Prostaglandins ................... . ...............
`98
`15.1.
`Prgstagiandin E2.Biosynthem's from arachidonic acid .
`.
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`101
`1.5.1.
`Prostaglandin Fm ‘.
`.
`.
`.
`.
`. .
`.
`. ...................
`103
`1.5.3. Characterizafionoftheproducts and storage conditions ......
`1.5.4. Notes ................................... 103
`Spin-labeled fatty acids ..............................
`104
`1.6.1.
`6-spiro—2'-(N-oxyHfi'dimeflIyloxazolidine)pahnitic acid .....
`105
`1.6.2.
`10-spiro-2'-(N-oxyl-4,4'«dimethyloxazolidine)palmitic acid . . .
`.
`.
`106
`1.6.3. Characterization of the products and storage conditions ......
`107
`12-(9-Anthry1}-’Il—trans~do&eoenoie said, a fluorescent labeled fatty acid .
`.
`108
`11-N-(2-Nitro4ozidopheny1hminoundecanoic acid, a photoreactive fatty
`acid ............................' .............
`109
`1.8.1. Characerizafion of the product and storage conditions
`. .
`.
`.
`.-
`.
`.
`110
`1.8.2. Note:.....................-.... .........
`111
`Chapter 11.2.Neutralglycerides
`.
`.
`. .................... .
`.
`.
`.
`.
`.
`.
`.
`113
`2.1.
`Introduction .-
`.
`. ................. . ........... .
`.
`. .
`113
`2.2. Monoglycerides .
`.
`..
`. .
`. ............................. 115
`2.2.1-
`3-Oleoy1-sn-glyoerol
`. .
`. .................,
`.
`. ......
`115
`2.2.2. DL—IO-ds—octadec-S’cnylglyeerol (DL-selachyl aloohol) ......
`1 16
`2.2.3. Characterization of the products and storage conditions ......
`117
`2.2.4.
`140125 ................................... 117
`2.3. Diglyoefides.
`. ................. .
`. .
`. .............. .
`118
`2.3.1.
`1,2-Diglyeerioes(1.2-diaey1-sn-glyoerols) from :53 lecithin .
`.
`.
`.
`.
`118
`2.3.2.
`1,34Distearoysglyeeml ..........................
`119
`2.3.3.
`1,2-Difinolenoyl—m—glyeerol ....................... 120
`2.3.4. Characterization of the products and storage conditions ......
`121
`2.3.5. Notes
`.
`.
`.
`.
`.
`.
`.
`. ........................... .122
`Triglycerides. 1501mmfrom rat liver ......... ..............
`122
`2.4.1. Characterization of the product and storage conditions . . .
`.
`.
`. .
`123
`2.4.2. Notes ..........
`124
`Chapter 11.3. Choline phospholipids ........... . ..... .
`.
`. .
`. .......
`125
`3.1.1nh‘oduction........... ......................... 125
`3.2. Natural 1eeithin mixmres ............................. 123
`3.2.1. Eggiecimm ..........
`......
`128
`3.23. Ratliver‘leeithin ......... . ..................-
`.
`129
`3.2.3. Characterization ofthe products and storage conditions . .....
`130
`3.2.4. Notes
`.
`.
`. ................................. 131
`Individuallecifllins......................~ .......... ’.
`132
`3.3.1.
`1,2—Dipalmitoyllecithin and Ifldioleoynedthjn. A partial syn-
`thesis.
`. ..........................,....... .
`132
`
`
`
`
`
`3.3.
`
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`
`3.3.2.
`
`
`lsolatiofi of a crude sample from rat
`1,2-Dipahnfitoyllecithin.
`lung .................................... 134
`1,2-Dilinoieoy11eeimin .
`.
`.
`. . ................ ;
`.
`.
`. .
`135
`3.3.3.
`1-Pa1mitoy1-2—oleoylledthin ................ , .......
`136
`3.3.4.
`3.3.5. Charactezizafion of the products and storage ennditions
`.
`.
`.
`.
`.
`.
`137
`3.3.6. Netes” ..................
`138
`Lysolecithins.............. ......................
`139
`3.4.1. Palmitoyl-Lwlysulecithin . ........... ,............ 139
`3.4.2.
`Paimitoyl—Lqfiwlysolecithin ........................ 140
`3.4.3.
`1-A}k-1'-eny1—sn~glycero-3éphosphoch01ine (plasmalogen, lysoleci-
`thin) .................................... 141
`3.4.4. Characterization of the products and storage conditions ......
`142
`3.4.5. Nutes ................................... 142
`l-Alk-l'enyl-I-palrnitoYl—sn-glyeerwB-phosphaehofine (plasmalugen lexi-
`
`4.3.
`
`Chapter I]
`5.1.
`5.2.
`
`E
`g
`E,
`
`k gé%
`
`5.3.
`
`5.4.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`3.4.
`
`3.5.
`
`3.6.
`
`3.7.
`
`3.8.
`
`3.7.2.
`
`-144
`Radinaefiwlaheledlecithins ......
`
`.7.......... _.
`.
`.
`.
`144
`.
`.
`.
`3.6.1-
`[N-“CI-lgnabeiediecithin . .
`
`3.6.1.1.Biosynthesis............./.......-......«144
`3.5.12. Chemical synthesis of [N-"CHQ—labeled phosphatidyl-
`
`145 .
`choiine fromeg lecithin ......... _. .
`.7 .......
`
`141
`.
`1,2-Di-{1-“C301eoynecithin ....... .
`.
`. . .
`.
`.
`. .
`. .
`.
`.
`.
`3.6.2.
`
`l,2-Di-{9,10-3H}steamyllecithin ........ . ............ 148
`3.6.3.
`
`3.6.4. Characterization of the products and storage condition; ......
`150
`
`3.65. Note:.................... ....... ....;...1.50
`Spin and fluorescent labeledlecithim . . ....................
`151
`
`3.7.1.
`1-Pa1mitoy1—2—(10-N-oxyM,4‘4hneihyloxazolidinenalnfitoyl-
`
`miglycero-B-phosphédmfine ...................... 151
`
`l-Paknitoyl—Z-[12«(9-anthryl)-1l—trans-dodeeenayll-snflyeero-S»
`
`phosphuchofine .............................. 153
`
`3.7.3- Chmcterizafian of the products and storage conditions ......
`154
`
`3.7.4. Notes ............................... . .
`.
`.
`154
`
`Sphhlgomyem ..... .
`. . ............ . .
`.
`. ........... 155
`
`3-8.1. Bovinebrainsphingomyelin.......................
`155
`
`3.3.2. DLZ-N-Steaxoyldmydrosyhmgomyefin. A chemical synthesis . .
`.
`157
`
`3.8.3. Characterization of the products and storage conditions ......
`159159
`
`3.8.4. Notes ..-.._...........-.. ...............
`
`
`
`
`. .......... »......
`.
`.
`Chapter 11.4.Aminophosphofipids . ......... .
`
`4.1.
`Introduction - .
`.
`.
`.
`.
`. ..............................
`161
`4.2.
`Phosphatidyleflmnom‘mes . . . .
`.
`. .
`.
`.
`.
`. .................
`165
`
`
`
`
`4.2.1. Natural phosphafidykfhanolamint mixtures .............
`165
`4.2.1.1. Isolation of phosphafidylethanolamine from egg yolk .
`. .
`165
`
`
`
`4.2.1.2. Branched-chain phosphafidyletlunulanine .........
`I, Chapter I!
`
`
`4.2.1.3. Phasphafidyiethanolamine
`(lam-l’enyl—lncyl‘m-giye—
`,6.1.
`
`
`ero—3-phosphoethano‘lzmine). Isolatian from calf bum .
`.
`4.2.1.4. Martian of the products and storage conditions
`.
`
`168
`169
`
`
`
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`
`IV
`
`4.2.2.
`
`4.2.3.
`
`5-3.
`
`5.4.
`
`
`
`
`17.2
`.y ......
`.
`4.2.1.5. Notes ...................... .
`from [at
`173
`.
`.
`.
`.
`.
`. .
`.
`Individual phosphafldylethmclamines .
`.
`.
`. .
`.
`.
`.
`.
`134
`______
`
`173
`4.2.2.1. 1,2-Distearoy1—sn—glycaro—S—phosphoeflunolnmine .
`.
`.
`. .
`135
`______
`
`4.2.2.2. 1-Pahnitny1—2—aleoymmacro-S-phosphaethanolamme.
`136
`______
`
`Achemicalsynthesis......................175
`137
`......
`
`4.2.2.3- Characterization of the productsand storage. conditions
`.
`17s
`133
`.
`.
`.
`.
`.
`.
`.
`
`4.2.2.4.N6135.......,................./.......
`176
`139
`.......
`{Ilia-adulation of labeled claim. acid in position 1 of phosphatidyl—
`139
`.......
`
`ethanalamm 17‘!
`14G
`.......
`4.2.3.1-Nates ...............................
`178
`n iysoleci-
`
`Lysophosphafidaikylethanolmnme
`(lalkyl-m-giyuero-B—phospho—
`4.2.4
`141
`.......
`
`ethanoiamine)....-......‘...................
`178
`142
`......
`4.3- Phosphafidylsezines................................
`179
`142
`.........
`
`Isolation of phosphatidylserhxe from bovine brain by silicic acid
`4.3.1.
`logen lem-
`chromatomphy ................. ; ...........
`180
`143
`.
`. . .
`.
`.
`.
`
`4.3.2.
`isolation of phosphafidylsen'ne from humanbrain”by DBMS-cellu-
`144
`.......
`
`loseculumnchmmatogmphy......................
`181
`....... 1“
`
`43.3.
`l3-Dipnlmitcy1-sn-glycem-3ahosphascfine. Enzyfnafic synthesis
`.......
`144
`
`{ram phosphafidylchofine .
`.
`. .
`. ..... . .............
`182
`msphatidyl—
`
`4.3.4. Characterization cfthe products and Sim-age conditions .
`.
`.
`.
`183
`.
`.’ .....
`14$
`
`4.3.5.Notes.,........-........................
`184
`.......
`147
`E
`.......
`I48
`
`Chapter 11.5.1’01yg1ycerophosphafifias andphowhafidic acid .............. 187
`.
`.
`. .
`.
`.
`150
`
`5.1. Introduction....................................
`187
`.......
`150
`5.2. Caxdiolipinfiiphosphafiflyiglyceml) .
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`189
`.......
`151
`
`5.2-1. Bovine heart mdioupin ......................... 139
`lmitoyl-
`
`5.2.2. Bacterial wdioiipin (Momma-ms Iysodeiktiw)
`. . .
`.
`. .
`. .
`.
`.
`191
`.......
`151
`
`5.2.3. CExaractexization ofthe products and storage conditions ......
`192
`g1yeam-3—
`
`5.2.4. Notes ......
`.....................
`192
`153
`Phosphatidyiglycerol ................................ 193
`u ______
`154
`
`5.3.1.
`Isolation ofphosphafidylglycerol fromspfimchlmvcs ........
`193
`154
`,
`.
`.
`_
`.
`,
`_ ,
`
`5.3.2.
`Isolation of phasihatidylglyceml framMiaacoccus Iymdeiktims .
`194
`________ 155
`
`5.3.3. Preparation of phosphafidyiglycemlby phaspholipase 1) catalyzed
`________
`155
`
`tranmefificafion ofeggleciflfin . .
`. . . . ..............
`195
`fitness;
`_
`_
`157
`5.3.4. Characterizationofthe productsmad storage mndifions ......
`196
`15 ______
`159
`
`53.5. 100253-. 197
`_______ 159
`
`Phosphafidic mid . . ................................ 198
`
`5.4.1. Prepmfian of phcyhatidic acid by phosphofipasen catalyzed
`161
`
`hydrolysisofeggleéflfin........................
`198
`"""" 161
`
`5.4.2.
`Synthesis of’1,Z-dipalmitobeL-afiycero3fihusphate .
`.
`.
`.
`.
`.
`.
`zoo
`""""" 165
`
`Synthesisof l‘palmitoyl-Lnlcoyl-m-glyuero-3-phosp’hate .
`.
`.
`. .
`.
`201
`5.4.3.
`““““ 165
`
`5.4.4. Chmcterizafion of the productsanfl storage condifiuns .
`. .
`.
`.
`.
`203
`““““ '
`
`5.4.5. Notes ............ . ....... .
`.
`. ............ 203
`‘33 “'1“ -
`-
`155
`
`
`,
`.
`. . .....
`165
`.
`Zfichffi‘Y"
`.
`‘u
`Chapter11.6. Inositokphosphafides , . .
`. . . ........................ 205
`
`“Him-- “38
`;
`6.1.
`lntroclucfiun.............................. ...... 205
`
`
`.
`169
`conditions
`
`s
`
`'
`-‘
`1
`‘
`
`,
`;
`
`.
`I
`:'
`’
`f
`9
`
`;
`E
`E
`'
`E
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`E
`E
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`
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`
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`
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`
`6.2.
`Isolation of phosphatidylinositol from baker's yeast .............. 207
`Chapter I
`6.3.
`l—D-l-O-{l-Palrnitoy1—2—oieoy1-m-glycero—3-phospho)—mya-inositoI. A chem-
`8.1.
`
`imlsynthesis.......................... ..........
`208
`31
`Preparation of phosphatidyl—fiflfinositol with Kloeckera brevi: .......
`213
`6.4.
`
`Phosphafidyfinositol phosphate (diphosphoinosifide) ............. 214
`6.5.
`
`Phosphatidyfinositol mannoside .
`.
`.
`. r.
`. .
`.
`.
`.
`.
`. ............. 216
`6.6.
`6.7. Characterization of the products and storage conditions ........... 217
`
`6.8. Notes .........
`219
`
`’
`
`’
`
`:
`
`33.
`
`3_4_
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`7.5.4.
`
`. .............. 223
`.
`.
`. ,.
`.
`Chapter 11.7. Glycoligids and related substances . .
`7.1.
`Introduction .................................... 223
`7.2. Ceramides (N-Acylsphingosines)
`. . .
`. . . .
`.
`.
`. ........ , ...... .
`227
`7.2.1. Preparation of cersmides from sphjngomyelin .
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`227
`7.2.2.
`ISO-benzoyloeramide
`.
`.
`. ....................... 228
`7.2.3. N‘ Stearoyl- and N«(DL-2«Hydroxypalmitoyl)DL—sphinynhte. A
`chemical synthesis ............................ 230
`7.2.4. Characterization of the products and storage conditions ......,
`231
`7.2.5. Notos...........-..... .................. 232
`7.3. Ccrebrosides and psychosines ........................... 233
`7.3.1. O-fi—D-Gahctosyfll .. Dceramirle ........... .
`.
`.
`. . . .
`.
`233
`7.3.2.. O‘p-D-Glucosylu -+ Doeramide .................... 235
`7.3.3.
`35S-Iabeled cerebmfide migrate ........... .
`.
`. .
`.
`.
`. .
`.
`236
`7.3.4.
`l-O-Galactosylsphingosinc (psychosine) ................ 238
`7.3.5. Characterization of the products and Storage conditions ......
`239
`7.3.6.. Notes ................................... 240
`7.4. Gangliosides .......... 241
`7.4.1.
`Isolation of total gangliosidcs from bovine brain .
`. ......... 241
`7.4.2. Hematosid: (monosialyflactosyl ceramide 6M3). flotation from
`ratiivex ....-.. ........................... 244
`L 7.4.3. Monosialylteu-ahexosyl oeramide GM; and disialyltetrahexosyl
`ccrunideGDll.................... ..........
`245
`7.4.4. Preparation of hitium labeied gnnglioside GD“ ........... 246
`7.4.¢.I. Catalytic addition of tritinm gas ........... .
`.
`.
`.
`246
`7.4.4.2. Periodate oxidation and reduction with trifiated borohy—
`dride ...................... .
`. .......
`7.4.5. Characterization of the products and storage conditions ......
`7.4.6. Notes .................................. .
`7.5. Glycosyldiglyoeriées.......-........
`.
`7.5.1. Mono— and digalactosydiglyerides. Isolation from wheatflour . .
`7.5.2.
`1,Z-Di-O-Pahnitoyl-S-O-rfi-Dfilamopyrmosyl-snglyoerol. A partial
`zynfltesis from plant galactosytdiglyoafides .............. 252
`7-5.3. Mono— amt dig‘lucosylfligtycefides. Isolation from Srrepromcazs
`drhcerflactz's................. ............... 254
`1,Z-Di-O—Palmitoyl-S-O-fi-D—glucopyranosyl-m-glycerol. A chemical
`synthesis ...... ............. .
`.
`.
`.
`...........
`7.5.5. Characterization of the: products and storage conditions ......
`7.5.6.
`.........
`
`
`
`g;
`§
`i
`3:.
`g
`i
`:2
`g
`:
`E
`1
`1
`
`,
`
`»
`
`3;
`
`3.5.
`
`3.7.
`3‘3_
`
`3,9_
`3‘19,
`3.31,
`
`Referenc
`
`Subject i
`
`
`
`
`
`
`
`
`247.
`248
`249
`250
`250
`
`256
`259260
`
`
`
`
`
`AKER877ITCOO739662
`
`

`

`..........
`. ..........................
`.
`Chapter 11.8. Miscollaneons products
`
`8.1.
`Inuoduction .............................. . .....
`263
`
`8.2.
`[14CJ-Unifoxmlylabeieé phospholipids from rat livex.
`.
`.
`.
`. ........ 265
`
`8.2.1. Characterization of the products and storage conditions ......
`267
`
`Sepharose linked lecithin .................. .
`. ........ 267
`
`8.3.1. Charactexization of the products and storage conditions ...... 269
`
`8.3.2. Notes............ ....................... 269
`8.4. Unsaturated acid chlorides .
`.
`.
`.
`.
`. ...................... 269
`
`3.4.1. Oleoyl chloride .............................. 269
`8.4.2. Linolenoyl chloride ......................... .
`.
`270
`
`8.4 .3. Characterization of the products and storage conditions ......
`271
`
`8.4.4. Notes ...................... , ............. 271
`1,2-Isnpmpylidens-sn-glyoerol ................... ' ....... 271
`
`8.5.1. Characterization of the product .................... 273
`85.2. Notes ..... . .......................... y.
`. . .
`273
`
`3-Trity1-sn-egcemi.
`.
`. . . ................... ,. .
`.
`.
`.
`.
`. L
`.
`273
`
`8.6-1. Notes.......-..............‘....{ ......... 274
`1,2-DfiokbanzylM-glyoetol ............................ 274
`
`8.7.1. Notes ................................... 278
`sn-Glycaro—B—phusphochofine mdmium chioride adduct .
`.
`. . .
`. .
`.
`.
`.
`.
`.
`278
`
`8.8.1. Characterization of the products and storage conditions ......
`280
`8.8.2. Notes ................................... 280
`1-Palmitoy1lohoyl-sn-glycetal-S-iodohydfin .....’ ............ 280
`
`8.9.1. Notes .................. . ................ 283
`8.10. 1D-2,3,4,5,6~Psnta-O-aaety1—myo-inositol .................... 283
`
`8.10.1. Notes ................................... 286
`
`8.11. 6,6‘-Di0—mycoloy1—czg’-D-h'ehalose (cord-factor) ........ ,....... 286
`
`8.11.1. Chamcterization of the product and storage conditions ....... 287
`
`8.11.2. Notes ................................... 287
`
`
`
`
`8.3.
`
`8.5.
`
`8.6.
`
`8.7.
`
`8.8.
`
`8.9.
`
`......
`
`241
`
`.,,.r..,..Ww...,.
`
`
`
`,..,mwwmwwmuwvmw«Mm
`
`
`References ........................................... 289
`
`..........
`......
`
`Subjectindex .......................................... 303
`
`..........
`
`..........
`
`.ac‘.....a
`
`..........
`
`0000014
`
`AKER877ITC00739663
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`

`

`Part I
`
`
`
`The preparation of pure lipid substances
`
`
`w“errwwmmmflnwsw¢(swampsmsw
`
`
`
`AKER877ITC00739664
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`

`

`
`
`CHAPTER 1.1
`
`Preparative extraction of