L.D. BERGELSON EDITOR
`
`LIPID
`BIOCHEMICAL
`PREPARATIONS
`
`ELSEV|ER;"NORTH—HOLLAND
`
`Petition for Inter Panes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1017
`
`AKBM 1017
`
`AKER877IC00I739650
`
`

`
`
`
`LLIPID BIOCHEMICAL PREPARATIONS
`
`edited by
`
`Li). Bergelson
`
`USSR Academy of Sciences, Shemyakin Institute of Bioorganic
`Che11'1ist1'y, Moscow, U.S.S.R.
`
`
`
`
`
`1980
`
`ELSEVIERINORTH-HOLLAND BIOMEDICAL pREssH£4;:?‘§—:
`AMSTERDAM-NEW YORK-OXFORD
`
`1
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`AKER877lTC07651
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`iv
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`E) 1980 E1scvierfNorth-Holland Biomedical Press
`
`All right reserved. No part of this publication may be reproduced, stored in a retrieval sys-
`tem, or tranxmitted,.in any form or by any means. electronic. mechanical, photocgpying,
`recording an otherwise, without the prior pen-nission of the copyright owner.
`mm 0444401454
`
`;
`
`V
`
`Published by:
`
`Elsevierlfiorth-Holiand Biemedica} Press
`335 Jan van Galenstraat, PD. Box 211
`Amsterdam, The Netherlands
`
`Sake distributors for the U.S.A. and Canada:
`Elseviermorth-Ho]1and,Inc.
`52 Vanderbfit Avenue
`New Yur'k,N.Y. 10017, U.S.A.
`
`Library ofConyess cmloging in Pumimion Data
`_
`_
`Mam entry under trtle:
`Lipid biochemical preparations,
`
`Bmfiomphv: P-
`Includes index .
`1. Lipids. 2. Extraction (Chemisty) 3. Lipids
`-- Analysis. I. Bergelson, L.D.
`QP75 11.546 574.I9’293 SO-12236
`ISBN 0-444-80146-4
`
`
`
`>
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`Preface
`
`The present book is an outcome of the joint experience of the staff of the
`Lipid Lrboratory in the Shemyakin Institute of Bioorganic Chemistry accumu-
`lated in 15 years of working in the lipid field. Since the main occupation of the
`laboratory is the physico-chernicalstndy 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 useful 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, pmification and handling of Iipidsubstanccs 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 ,
`preparing pure lipid substances, most of which have been tested in our labora-
`tory. The procedures are assembled in seven chapters covering the main lipid
`classessg in the eighth one we deal with the preparation of intennediates. Each
`chapter is opened by a short introductory survey (by LJ). 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
`contrflruted to the book by submitting and testing some of the procedures and
`by reading and cornrnenting upon the manuscript: V.V. Bezuglov, M.L. Cirenina,
`V.I. Kulikov, TJ. Iazurkina, L.F. Nikulina, T.G. Pilipenlco, V1’. Shevchenko,
`VJ. Shvets, N.G. Timofeeva, A.N. 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 straight.—During 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 iipid
`mixture required years of tedious work: workers were fully occupied with
`extractions, evaporations, recrystallizations and distillations. Purificafion was
`difficult to achieve and mostly incomplete leading the organic purists to call
`type of work “Schmierchemie”. Slow progress began only in the early fifties
`with the appearance of different
`types of chromatography, countercurrent
`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 gas»Iiquid 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 to
`and the size of analytical samples reached the micrograrn 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 Iipidologist,
`who now could include in his studies microscopic objects‘ of cellular biology.
`The preparation methods developed in the past seemed to have become almost
`mics-s.
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`
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`Recently, however, preparative lipid chemistry has received new stimuli. This
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`AKER877|TC0739654
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`may be explained by different reasons. On the one hand modem 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, i.e. “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-bound 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 immunostimulants. Besides, pure lipids of known struc-
`tore are of course also used in traditional studies of the hiosynthesis and brealo
`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 composition, but to an
`increasing extent truly individual lipid compounds (i.e. individual molecular
`species) which can only be supplied by chemical synthesis or semisynfoesis.
`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 mnnber 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 important substances are
`still not available. Besides, commercial preparations are frequently very expen-
`sire and insufiiciently 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
`difficlilt to prepare and to purify. Consequently corrnnercial preparations fre-
`quenfly contain impurities. A large number of commercial phospholipids and
`glycolipids tested in the authors’. laboratory proved to be contaminated with
`related or foreign substances which showed up as extra spots on thin-layer
`cbromatograms.
`Often this is not the fault of the manufacturer because 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.
`
`“rhen
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`When selecting procedures for the preparation of lipids from the wealth of
`methods described in the literature we followed four principles:
`Reliability. 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 materials. 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 difficrrlties when
`attempting topwork 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 micrornethods 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 difficultbecause 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
`
`r purification
`her hand the
`
`. stem mainly
`rese studies is
`rnbranes and
`vailability of
`id substances
`ies include a
`re interacted ‘
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`sis and break-
`
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`es of a large
`u, but to an
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`
`emisynthesis.
`ng spin- and
`re.
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`d’tl1e produc-
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`ubstances are
`r‘ very expen-
`turecl simpler
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`lids are more
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`Preface .
`lntrn‘duction....... .
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`Part I. The preparation ofpure lipid substance:
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`Chapter 1.1. Preparative extraction oflipids from naturalxaources . . .
`1.1. General oamments . .
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`1.2.
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`1.2.1. Solvents . . . . .
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`1.2.2. Treannent of material prior to extraction . .
`1.2-3. Extractive homogenizafian . .
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`1.2.4. Separation of the arganic and aqueous phases
`1.2.5. Removalufnun-lipid cbntamirrams . .
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`1.2.6. Salventremoval
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`1.2.7.
`Storage of lipid preparations . .
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`by column rzhromatography . . . .
`Chapter L2. Purification of
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`2.1. General comments .
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`2.2.
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`2.1.1. Column preparation . . . .
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`2.2.2.
`Sample application and the elution of the columns
`2.3.3. Control of the elufion prone by micro-thin-layer t:hmruatog-
`raphy.... . . . .
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`2.3. Chromatography of lipids on column: of silicic acid .
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`Practical instructions. ;
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`2.4. Chromatography uflipids on cahrmniibfaluxrfizzium oxide . . .
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`2.4-2. Pncfical instructions . . . .
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`2.5. Chromatography ofpolarlipicis on ion exchange cellulose: .
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`2.5.2. Praciicalinstrnctions. . .,. . .
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`Chapter 1.6. Detennination of purity and characterization oflipid substances . .
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`6.1.
`lntroducfion .
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`6.2.
`Fatty acids .
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`6.3. Mono-, n'.ii- and triglycerides .
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`6.4 .1. Detennination of the nitrogen ox ester group to phosphorus ratio .
`6.4.2 Detennination ofpolannoietiei .
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`6.4.3.
`1-IydIo1ysisbyp11c$11o1ipa.ses .
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`6.5. Glycolipids . .
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`Chapter 1.3. Preparative thin-layer chromatography of lipids .
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`3.1. General comments . .
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`3.2.
`Practical instructions . .
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`3.2.1. Preparation of the plates
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`3.2.2. Sampk application .
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`3.2.3. Development of ehxomatogranxs and detection of the zones .
`3.2.4.
`Isolation ofthe separated fractions
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`Chaptex1.4.Useofnon-chromatographicmethadsintlxe preparation of lipids
`4.1.
`Introduction .
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`4.2-.
`Selective extaction and precipitation . . . . . .
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`4.3.
`Solvent partition .
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`4.4. Crystallization .
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`Chap1eti.S. Partial synthesis of complex lipids .
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`5.1.
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`5.2. Phosphatidylcholines
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`5.3.
`l’hosphztidy1seri11es
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`Phosphatidylethanolamines . .
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`5.5. Phosphatidic acids .
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`5.6. Conversion of phosphatidic acid into oiher phospholipids .
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`5.7. Glyeolipids .
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`5.7.2. Hexosyleeramkies .
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`Part H. Procedures
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`37
`37
`40
`40
`41
`42
`43
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`45
`45
`45
`46
`48
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`51
`51
`52
`57
`57
`58
`60'
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`63
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`65
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`66
`69,
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`70
`73
`74
`74
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`1.4. Hydro;
`1.4.1.
`1.4.2.
`1.4.3‘.
`1.4.4.
`1.4.5.
`Pmstag
`1.5.1-
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`1.5.
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`1.6.
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`1.7.
`1.8.
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`1.5.2.
`1.5.3,
`1.5.4.
`Spin-lai
`1.6.1.
`1.6.2.
`1.6.3.
`12-(9-A
`11-N-(2
`acid .
`1.8.1.
`1.8.2.
`
`Chaptezr 11.2. Neu
`2.1.
`Introdx:
`2.2. Menogi
`2.2.1.
`2.2.2.
`2.2.3.
`2.2.4.
`2.3. Diglyct
`2.3.1.
`2.3.2.
`3-3-3-
`2.3.4.
`2-3-5;
`2.4. Triglyo
`2-41-
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`Chapter [1.1. Fatty acids
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`1.1.
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`1.3;
`Polyenoip adds ._. .
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`1.3.1. Methyl linolenate. Preparation from linseed ofl .
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`1.3;. Notes
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`81
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`as
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`90
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`Chapter H3-Cho
`3-1-
`Intwdv
`3'2‘ Natutil
`3117
`31%
`3-2-3-
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`37
`1.4. Hydi-ax)’ fatty acids .
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`1.4.1.
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`1.4.2.
`3-Hydroxystearic acid .
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`. 1.4.3. Mycnlic acids .
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`1.4.4. Characterization of the products and storage coriditiens .
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`1.4.5. Notes
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`96
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`Prustagiandiiis
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`15.1.
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`1.5.2.
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`1.5.3. Charactexizatianofthepmducts and storage eonditimis .
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`1.5.4. Notes
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`103
`45
`Spin-labeled fatty acids .
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`104
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`1.6.1.
`6-spiIo—2'-(N-oxy1—4,4'-dimefliyloxazclidine)pa1mitic acid .
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`105
`1.5.2.
`10-spin}2'-(N-oxyl-4,4'-dimethyloxazolidiiie)palmitic acid . . .
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`1.6.3. Characterization of the products and storage conditions .
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`12-(9-AntI'uy1}-'11—tians-dodeeenévic acid, a fluurescent labeled fatty acid .
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`52
`11-N-(2-Nitru-4-ixzitlophenylhniixiorindecaziuic acid, a photoreactive fatty
`57
`acid .
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`109
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`1.8-1. Characerizatiun of the product and storage conditions
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`1.8.2. Note:
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`I11
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`60
`50
`Chapter I[.2.NeutIa1g1ycerides
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`113
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`2.1.
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`113
`53
`2.2. Monaglycerides .
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`115
`2.2.1-
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`115
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`2.2.2. DL-I-0-cis-uctadec-9'-enylglyceml (DI.-selachyl alcohol) .
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`2.2.3. Charzcterizafien of the products and storage conditions .
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`2.3. Diglyoezides.
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`2.3.1.
`1,2-Diglycerides (1.2-diacyl-an-glyeerols) from eg lecithin .
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`2.3.2.
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`2.3.3.
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`120
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`2.3.4. Chaxactexizafion of the products and storage ounditions .
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`.122
`Triglycerides. lsolafioufrum rat liver
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`122
`2.4.1. Characterization of the product and stnrage conditions . .
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`2.4.2. Notes
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`124
`Chapter IL3. Choline phospholipids .
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`125
`3.1.
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`125
`3.2. Natural Xeeitliin mixtures .
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`3.2.1. Eggiecithin .
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`3.2.2. Ratlivetleeitixin .
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`3.2.3. Characterization ofthe products and storage conditions .
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`3.2.4. Notes
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`Individual leeithins . .
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`132
`3.3.1.
`1,2—Dipa1:-nitoyllecithin and 1,2-dieleoyllecithin. A partial syn-
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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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`3.3.2.
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`3.4.
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`3.5.
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`3.5.
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`3.7.
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`lsolafiofi of a cnzde sample from rat
`1,2-Dipalmitoyllecithin.
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`1,2-Dilinolcoyllecithin .
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`1-Pa1mitoy1—2-olnoyllecfithili .
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`3.3.5. Chaxactezization of th: products and storage cnnditions
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`PaJn1itoy1-L-mlysulecithin .
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`3.4.4. Characterization of the products and storage conditions
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`3.5.12. Chemical synthesis of [N-”CH3}—1abeled, pnospamiayz.
`chaiine f1:om.eg1ecit11in .
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`3.6.4. Characterization of the products and storage condition;
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`3.7.1.
`1-Pavlmitoyl-2-(10-N-9xyI4,4‘-dhneihyloxnzolidine)-palnfitoyb
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`.m—-glycero-3-phosphocholine .
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`3.7.2.
`1-Palmitoyl-2-[12«(9-anthryl)-11—tIans-dodecennyll-smglycero-3~
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`phosphucholine .
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`3.8. Sphingomw-.]ins........ .
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`3.8.2. DI.~2-N-Steaxoyldihydrosphingomyelin.Achemical synthesis ., .
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`3.8.3. Characterization of the products and stoxzge conditions
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`3.8.4. News.....-............... . . . .
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`2
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`3
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`4,3.
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`Chapter 1]
`5.1.
`5.2.
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`5-3-
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`5.4.
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`Chapter I1.4..‘A.minophospha1ipids .
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`4.1.
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`4.2.
`Phosphatidyltgtbanuhxnines . . . .
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`4.2.1. Natural phosphafidylethanolamine mixtures .
`4.2.1.1. Isolaficm of phosphatidylethanolamine from egg yolk .
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`4.2.1.2. Branched-chain phasphafidylethanulagnine .
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`4.2.1.3. Phasphatidyiefltanolamine
`(lsalk-1'-enyl—2~ncy1-M-g1yc—
`16E
`:ro-3-phosphoethmoknfine). Isclatian from calf brain .
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`169
`4.2.1.4. Characterization of the products and storage ctmditions
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`6.1.
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`Individual phosphatidylethanolamines .
`173
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`4.2.2.1. 1,2-Distearoyl-an-glycero-3-phosphoethanolnmine .
`4.2.2.2. 1-Palmitoyi-2-oleoyl-an-glyooro-3-phosphoethanolamine.
`175
`Achemicalsynthesis..............‘........
`176
`4.2.2.3.Cham:te:ization of the products and storage conditions
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`176
`4.2.2.4.Notes........................./.......
`Inixoduction of labehacl oleic acid in position 1 of phosphatidyl-
`17‘!
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`4.2.3.1-NIJtBS . . .
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`Lysophosphatidaikylethanolamine
`(1~a1ky1-an-giyoero-3—p11os;:ho—
`178
`ethanoiamine)....-......L..................
`179
`Isolation of phosphatidylserine from bovjne brain by siiioic acid
`4.3.1.
`180
`chromatogmphy . .
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`isolation of phosphafidylsefine from humanburnby DEAE-oellw
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`181
`1oseculum:1chromatography......................
`1.2-Dipnlmitoyi-an-glycero-3—phosphasex-inc. Enzyrhafic synthesis
`43.3.
`182
`from phosphatidylcholine .
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`4.3.4. Characterization ofthe products and storage conditions .
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`Chapter Il.5.Po!yglyoerophosph1fiI1as anclphowhatidic acid .
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`5
`5.1. Introduction...-................................
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`J
`5.2. Caxdiolipinkiiphosphafirlylglycerol) .
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`5.2-1. Bovine heart caxdiolipin .
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`5.2.2. BactaIial¢::dio1ipix:(I:fi‘cracoccu:Iysodeikticu:)
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`5.2.3. Cfixanctexization ofthe products and storage conditions .
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`5.2.4. Notes
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`Phosplutidyiglycerol . .
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`5.3.1.
`Isolation ofphosphatidylglycerol from .spinat:h1eaves .
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`5.3.2.
`Isolation of phosphatidy1g1ycerolfram.MicrDcoccu§ Iymdeiktiau .
`194
`5.3.3. Preparation of phosphatidyiglycorolby phospholipase D mtalyzed
`transesterificafion ofegglecithin . .
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`5.3.4. Characterizationofthe productsmad storage conditions .
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`53.5. Notes...................................
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`Phosphafidic acid . . . . .
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`5.4.1. Preparation of phcsphatidic acid by phosphoiipasen catalyzed
`hydrolysisofegglexithin........................
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`5.4.2.
`Synthesis ofl,2-dip?}mitoy1~DL-afiycero—3-phosphate .
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`Synthesisof 1~pa1mxtoyl-2-o1coyl-m-gIyuer0-3~pho?11hate .
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`5.4.3.
`5.4.4. Chaxacterizafion of the productsanfl storago condmons .
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`5.4.5. Notes
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`ChapterII.6.Inos'1to}ph0sphafides . . .
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`6.1.
`Introduction . . . .
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`s
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`from rat
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`4.2.4
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`4.3.
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`5.3.
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`5.4.
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`

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`5.2.
`6.3.
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`Isolation ofphosphatidylinositol from baker's yeast .
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`207
`C,,ap.e,,
`1-D-1-O-{I-Palmitoyl-2-oieoyl-m-g1ycero—3-phospho)-rnyo-inositol. A chem-
`8_1'
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`3_2_
`ical synthesis .
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`Preparation of phospixyatidyrl-[3I—1}inosito1\vit11Kloeckera brevi: .
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`6.4.
`Phosphafidylinositol phosphate (diphosphoinositide) .
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`6.5.
`Phosphatidylfinositol mannoside .
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`6.6.
`6.7. Characterization of the products and storage comiitions
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`6.8- Notes
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`Chapter II.7.Glyoo!ipids and related substances . .
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`7.1.
`Introduction . .
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`7.2. Ceramides (N-Acylsphingosines)
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`7.2.1. Preparation of cersmides from sphingomyelin .
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`3-O~benzoyloeramide
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`7.2.3.
`.N— Stearoyl- and N-(DL-2-Hydroxypalmitoyl)-DI.-sphinganine. A
`chemical synthesis .
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`7.2.5. Notes
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`7.3. Cerehmsides and psychosines .
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`7.3.1.
`0-:3-D-Ga1?:ctosy1(I -» Dceramide .
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`7.3.2.. O~p-D-G1ucnsyl(1-+ 1)-eeramide
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`13.3.
`3 ‘S-Labeled cerebmsme sulfate .
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