Phospholipids and Glycolipids

Chapter 21

Phospholipids and Glycolipids R. H. G I G G

The phospholipids (phosphatides) and glycolipids are often classified as "complex lipids" to distinguish them from the "simple lipids" like glycerides, waxes and steroids. They occur naturally in microorganisms and in plant and animal tissues, usually in the form of complexes with proteins (lipoproteins) which have to be degraded before extraction with organic solvents is possible. (For reviews of methods of extraction see W. M. Sperry, Methods ofbiochem. Analysis, 1955, 2, 83; N. S. Radin, ibid., 1958, 6, 163; C. Entenman, Methods in Enzymology, Academic Press, New York, 1957, Vol. 3, p. 299; H. Wagner and P. Wolff, Fette u. Seifen, 1964, 66, 425; M. Kates, "Techniques in Lipidology", in Laboratory Techniques in Biochemistry and Molecular Biology, eds. T. S. and E. Work, Elsevier, Amsterdam, 1972, Vol. 3, p. 267.) The extracted lipids can be separated into various classes by solvent fractionation procedures or preferably by chromatography on silicic acid and analysed by thin-layer chromatography (t.l.c.) or paper chromatography (for reviews see J. J. Wren, J. Chromatog., 1960, 4, 173; H. K. Mangold, J. Amer. Oil Chemists' Soc., 1961, 38, 708; 1964, 41, 762; O.S. Privett et al., ibid., 1964, 41,371 ; G. Rouser et al., ibid., 1965, 42, 215; H. Jatzkewitz, Z. physiol. Chem., 1964, 336, 25; L. J. Morris, J. Lipid Res., 1966, 7, 717; G. V. Marinetti, ibid., 1962, 3, 1 ; 1965, 6, 315; "Lipid Chromatographic Analysis", Vol. 1, Dekker, New York, 1967; S. R. Eder, Fette u. Seifen, 1972, 74, 519; W. W. Christie, "Lipid Analysis", Pergamon, Oxford, 1973). The most abundant phospholipids and glycolipids are derivatives of glycerol or sphingosine (and similar bases). In the glycerol-containing lipids, fatty acids are esterified to one or two of the hydroxyl groups of glycerol and in the sphingosine-containing lipids the fatty acid is joined by an amide linkage to the amino group of the sphingosine base. Each lipid class contains a spectrum of different fatty acids and hence it is not usually possible [349]

350

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

to isolate a chemically homogeneous compound from natural mixtures. Some success has been achieved in-the separation and analysis of each lipid class either by direct chromatography on silicic acid impregnated with silver nitrate or by a preliminary enzymic hydrolysis followed by chromatography of derivatives of the diglycerides or ceramides produced (for reviews see C. V. Viswanathan, Chromatog. Reviews, 1969, 11, 153; O. Renkonen, Adv. Lipid Res., 1968, 5, 329). The phospholipids and glycolipids are important constituents of biological membranes and the interaction of proteins and lipids in these structures is under intensive study. Many membrane-bound enzymes lose their activity when separated from phospholipids and this activity can be regenerated on the addition of exogenous phospholipids (for review see R. Coleman, Biochim. Biophys. Acta, 1973, 300, 1). Artificial membranes ("liposomes") can also be formed from mixtures of phospholipids in aqueous media and the properties of these resemble natural membranes in some respects (for reviews see A. D. Bangham, Progr. Biophys. Mol. Biol., 1968, 18, 29; Bangham et al., in "Methods in Membrane Biology", Vol. 1, ed. E. D. Korn, Plenum, New York, p. 1; see also S. Batzri and E. D. Korn, Biochim. Biophys. Acta, 1973, 298, 1015). General references (a) Phospholipids and ylycolipids : H. Wittcoff, "The Phosphatides", Reinhold, New York, 1951; H. J. Deuel, "The Lipids", Vols. 1-3, Interscience, New York, 1951-1957; J. A. Lovern, "The Chemistry of Lipids of Biochemical Significance", Methuen, London, 1957; D. J. Hanahan, "Lipide Chemistry", Wiley, New York, 1960; K. Bloch, "Lipide Metabolism", Wiley, New York, 1960; J. Asselineau, "Les Lipides Bact6riens", Hermann, Paris, 1962; J. C. Dittmer, "Distribution of Phospholipids" (p. 231) and R. M. C. Dawson, "Metabolism of Phospholipids" (p. 265), in "Comparative Biochemistry", Vol. 3, Academic Press, New York, 1962; eds. Dawson and D. N. Rhodes, "Metabolism and Physiological Significance of Lipids", Wiley, Colchester, 1964; G. B. Ansell and J. N. Hawthorne, "Phospholipids", Elsevier, Amsterdam, 1964; D. Chapman, "The

Structure of Lipids by Spectroscopic and X-Ray Techniques", Methuen, London, 1965; ed. G. Schettler, "Lipids and Lipidoses", Springer, Berlin, 1967; K. P. Strickland, "The Biogenesis of the Lipids", in "Biogenesis of Natural Compounds", Pergamon, Oxford, 1967, p. 103; W. M. O'Leary, "The Chemistry and Metabolism of Microbial Lipids", World, 1967; E. J. Masoro, "Physiological Chemistry of Lipids in Mammals", Saunders, London, 1968; Chapman, "Introduction to Lipids", McGraw Hill, New York, 1969; D. Shapiro, "Chemistry of the Sphingolipids", Hermann, Paris, 1969; ed. J. M. Lowenstein, Methods in Enzymology, Vol. 14, Academic Press, New York, 1969; "Lipids", eds. M. Florkin and E. H. Stotz, "Comprehensive Biochemistry", Vol. 18, Elsevier, Amsterdam, 1970; "Lipid Metabolism", ed. Grossi-Paoletti, "Synthesis and Use of labelled Lipids", Karger, Basle, 1970; ed. S. J. Wakil, "Lipid Metabolism", Academic Press, New York, 1970; C. Hitchcock and B. W. Nichols, "Plant Lipid

GENERAL REFERENCES

351

Biochemistry", Academic Press, New York, 1971 ; M. I. Gurr and A. T. James, "Lipid Biochemistry: an Introduction", Chapman and Hall, London, 1971; eds. B. W. Volk and S. M. Aronson, "Sphingolipids, Sphingolipidoses and Allied Disorders", Adv. in Exp. Med. and Biol., Vol. 19, Plenum, New York, 1972; eds. J. B. Stanbury, J. B. Wijngaarde and D. S. Fredrickson, "The Metabolic Basis of Inherited Diseases", 3rd Edn., McGraw Hill, London, 1972; G. J. Nelson, "Blood Lipids and Lipoproteins", Wiley, Chichester, 1972; eds. J. Ganguly and R. M. S. Smellie, Biochem. Soc. Symposium No. 35, "Current Trends in the Biochemistry of Lipids", Academic Press, New York, 1972; eds. Ansell, Dawson and Hawthorne, "Form and Function of Phospholipids", Elservier, Amsterdam, 1973; eds. A. R. Johnson and J. B. Davenport, "Biochemistry and Methodology of Lipids", Wiley-Interscience, New York, 1971; ed. R. Wood, "Tumor Lipids", Amer. Oil Chemists Soc., 1973; ed. J. A. Erwin, "Lipids and Biomembranes of Eukaryotic Microorganisms", Academic Press, New York, 1973; H. Brockerhoff and R. G. Jansen, "Lipolytic Enzymes", Academic Press, New York, 1974; R. L. Jackson and A. M. Gotto, "Phospholipids in Biology and Medicine", New England J. Med., 1974, 290, 24, 87; Progress in the Chemistry of Fats and Other Lipids, Vols. 1-15, 1952-1974, Pergamon, Oxford; Advances in Lipid Research, Vols. 1-12, 1963-1974, Academic Press, New York; Annual Reviews of Biochemistry, Vols. 1-43, 1932-1974. For reviews on the synthesis of phospholipids see E. Baer, Trans. roy. Sc~c. Canad., 1966, 4, 181; A. J. Slotboom and P. P. M. Bonsen, Chem. Phys. Lipids, 1970, 5, 301; V. I. Shvets, Uspekhi Khim., 1971, 40, 625; Jensen and D. T. Gordon, Lipids, 1972, 7, 611; G. H. de Haas et al., Chem. Phys. Lipids, 1973, 11, 295. For reviews on the chemistry and biochemistry of the lipids of micro-organisms see Kates, Ann. Reviews Microbiol., 1966, 20, 13; M. lkawa, Bacteriol. Reviews, 1967, 31, 54; N. Shaw, ibid., 1970, 34, 365; Adv. applied Microbiol., 1974, 17, 63; W. J. Lennarz, Accounts Chem. Res., 1972, 5, 361; G. A. Thompson and Y. Nazawa, Ann. Reviews Microbiol., 1972, 26, 249; H. A. Blough and J. M. Tiffany, Adv. Lipid Res., 1973, 11,267 - - lipids in viruses; J. Lenard and R. W. Compans, Biochim. Biophys. Acta, 1974, 344, 51 membrane structure of lipid containing viruses; P. F. Smith, Ann. New York Acad. Sci., 1973, 225, 22 - - lipids of mycoplasmas. For a review of plant lipids see A. A. Benson, Ann. Reviews Plant Physiol., 1964, 15, 1. For other reviews see R. J. Rossiter, "Metabolism and Function of Phospholipids and Sphingolipids", Clin. Chem., 1965, 11, 171;Dawson, "Metabolism of Animal Phospholipids and their turnover in Cell Membranes", Biochem. Soc., Essays in Biochemistry, 1966, 2, 69; R. O. Brady, "Lipidoses", Biochimie, 1972, 54, 723; H. van den Bosch, L. M. G. van Golde and L. L. M. van Deenen, "Dynamics of Phosphoglycerides", Ergebnisse der Physiologie, 1972, 66, 13; L. D. BergeIson, "Diol Lipids", Fette u. Seifen, 1973, 75, 89. 31p n.m.r, spectra of phospholipids, T. O. Henderson et al., Biochemistry, 1974, 13, 623; G. K. Radda et al., Biochim. Biophys. Acta, 1975, 375, 186. (b) Membranes: J. L. Kavanau, "Structure and Function in Biological Membranes", Vols. 1-2, Holden-Day, San Francisco, 1965; ed. Chapman, "Biological Membranes", Academic Press, New York, 1968; eds. L. Bolis and B. A. Pethica, "Membrane Models and the Formation of Biological Membranes", North Holland, Amsterdam, 1968; ed. R. M. Dowben, "Biological Membranes", Little Brown, Boston, Mass., 1969; eds. E. Tria and A. M. Scanu, "Structure and Functional Aspects of Lipoproteins in Living

352

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Systems", Academic Press, New York, 1969"J. R. Villanueva and F. Ponz, "Membranes: Structure and Function", Academic Press, New York, 1970; ed. L. A. Manson, "Biomembranes", Vol. 1, Plenum, New York, 1971" eds. G. W. Ritcher and D. G. Scarpelli, "Cell Membranes", Williams and Wilkins, Churchill/Livingstone, London, 1971" eds. D. F. H. Wallach and H. Fischer, "Dynamic Structure of Cell Membranes", Springer, Berlin, 1972" ed. M. L. Hair, "The Chemistry of Biosurfaces", Vols. 1 and 2, Dekker, New York, 1972; M. K. Jain, "The Bimolecular Lipid Membranes", Van NostrandReinhold, New York, 1972; ed. D. E. Green, "Membrane Structure and its Biological Applications", Ann. New York Acad. Sci., 1972, Vol. 195; "Reviews of Biomembranes" volumes of Biochim. Biophys. Acta; eds. C. B. Anfinsen, R. F. Goldberger and A. N. Schechter, "Current Topics in Biochemistry. Membranes and Lipids", Academic, New York, 1972; Chapman and Wallach, "Biological Membranes", Vol. 2, Academic Press, New York, 1973; ed. Wallach, "Plasma Membranes" Dynamic Perspectives, Genetics and Pathology", Springer, Berlin, 1973" ed. C. F. Fox, Biochemistry of Cell Walls and Membranes", Univ. Park Press, Baltimore, 1974; C. Tanford, "The Hydrophobic Effect" Formation of Micelles and Biological Membranes", Wiley-Interscience, New York, 1973" S. J. Singer, "The Molecular Organisation of Membranes", Ann. Rev. Biochemistry, 1974, 43, 805" R. A. Capaldi, "A Dynamic Model of Cell Membranes", Scientific American, 1974, 230, 26" M. S. Bretscher, "Membrane Structure: Some General Principles", Science, 1973, 181,622.

1. Phospholipids and glycolipids based on glycerol ( a ) Acyl derivatives of glycerophosphoric acids

The glycerophosphoric acids are described in Chapter 18 (see p. 19). They exist in two optically active forms

CH2OH I HO-C-H I

CH2OPO(OH) 2

L-Glycerol 3-phosphate (sn-glycer,ol 3-phosphate)

CH20H I H-C-OH I CH2OPO(OH)2 D-Glycer,oI 3-phosphate (sn-glycer'ol 1-phosphate)

In the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature on the nomenclature of lipids (see Europ. J. Biochem., 1967, 2, 127; 1970, 12, 1), glycerol should be written in the following way

1CH2OH HO--2C- H 3(~H2OH and the numbering of the carbon atoms ["stereospecific numbering", Csn")] is as shown. Thus L-glycerol 3-phosphate becomes sn-glycerol 3-(dihydrogen phosphate) and D-glycerol 3-phosphate becomes sn-glycerol 1-(dihydrogen phosphate).

1

GLYCEROPHOSPHORICACIDACYLDERIVATIVES

353

Both optical isomers have been synthesised from the corresponding optically active isopropylideneglycerols (E. Baer and H. O. L. Fischer, J. biol. Chem., 1939, 128,491; 1940,135,321 ; Biochem. Prep., 1952, 2, 31). The glycerophosphatides are derivatives ofsn-glycerol 3-phosphate (see Baer and D. Buchnea, J. Amer. chem. Soc., 1959, 81, 1758). Since both glycerol 1- and 2-phosphates are isolated in the hydrolysis of natural glycerophosphatides it was thought for many years that derivatives of both compounds were present in lipid mixtures. However, the isolation of optically active lecithins and the demonstration of the formation of glycerol 1- and 2-phosphates by acid and alkaline hydrolysis of pure synthetic optically active lecithins and cephalins has shown that glycerol 2-phosphate is an artefact produced during hydrolysis and it is now considered that derivatives of glycerol 2-phosphate do not exist in natural phosphatides (see Baer et al., ibid., 1956, 78, 236). The intermediate in the migration of the phosphate group is a cyclic phosphate ester which has been identified as a hydrolytic product of glycerol phosphatides under certain conditions (B. Maruo and A. ,4. Benson, J. biol. Chem., 1959, 234, 254; see also L. Kuge! and M. Halmann, J. Amer. chem. Soc., 1967, 89, 4125) and these cyclic phosphate esters of glycerol have been synthesised (Buchnea, Lipids, 1973, 8, 289). 1-Fluoro-l-deoxy-sn-glycerol 3-phosphate and its enantiomer have been synthesised and their biological activities investigated (T. P. Fondy et al., J. med. Chem., 1974, 17, 697; W. J. Lloyd and R. Harrison, Arch. Biochem. Biophys., 1974, 163, 185). Phosphonic acid analogues of sn-glycerol 3-phosphate have also been synthesised and their effects in biological systems studied (R. Engel et al., Antimicrobial Agents and Chemotherapy, 1973, 4, 467; J. med. Chem., 1972, 15, 1074). (i) Phosphatidic acids CIH202 CR R1C O 2 - C - H CH2OPO(OH) 2

Phosphatidic acids are diacyl derivatives of sn-glycerol 3-phosphate and were first isolated from cabbage and spinach leaves, being formed by enzymatic hydrolysis of other phospholipids during the isolation procedure (see D. J. Hanahan and I. L. Chaikoff, J. biol. Chem., 1948, 172, 191). Phospholipase D (EC 3.1.4.4), from carrot root, hydrolyses egg and yeast lecithins to phosphatidic acids (M. Kates, Canad. J. Biochem. Physiol., 1956, 34, 967; M. J. Coulon-Morelec and M. Faure, Bull. Soc. Chim. biol., 1967, 49, 273, 279). The amount of phosphatidic acids in fresh tissue is small (see G. Hiibscher, Biochim. Biophys. Acta, 1961, 52, 571; J. H6lzl,

354

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Biochem. Z., 1965, 341, 168; J. Polonovski et al., Bull. Soc. Chim. biol., 1965, 47, 851) but they play an important role in the biosynthesis of triglycerides and phospholipids (see E. P. Kennedy, Harvey Lectures, 1962, 57, 143; S. J. Wakil, Ann. Review Biochem., 1962, 31,369). They are synthesised biologically by the acylation of sn-glycerol 3-phosphate in microsomes and mitochondria (see M.-S. Liu and K. J. Kako, Biochem. J., 1974, 138, 11), by the phosphorylation of diglycerides (see Y. Chang and Kennedy, J. biol. Chem., 1967, 242, 516; R. A. Pieringer et al., ibid., p. 2719; P. R. Vagelos et al., ibid., pp. 4459, 4466; Hiibscher et al., Europ. J. Biochem., 1967, 3, 70) and from dihydroxyacetone phosphate (B. W. Agranoff and A. K. Hajra, Proc. Nat. Acad. Sci., 1971, 68, 411; R. Reiser et al., Biochem. biophys. Res. Comm., 1971, 44, 1279). The biosynthesis of phosphatidic acids in the thyroid gland is stimulated by thyrotropin (P. B. Schneider, J. biol. Chem., 1972, 247, 7910). The dephosphorylation by the enzyme phosphatidate phosphatase (EC 3.1.3.4) gives diglycerides (see S. C. Jamdar and H. J. Fallon, J. Lipid Res., 1973, 14, 517). Several syntheses of these compounds have been reported (for reviews see E. Baer, Progr. Chem. Fats and Lipids, 1963, 6, 48; P. E. Verkade, Bull. Soc. chim. Fr., 1963, 1993; L. L. M. van Deenen and G. H. de Haas, Adv. Lipid Res., 1964, 2, 168; P. P. M. Bonsen and De Haas, Chem. Phys. Lipids, 1967, 1, 100; A. J. Slotboom and Bonsen, Chem. Phys. Lipids, 1970, 5, 301; R. Aneja and A. P. Davies, ibid., 1974, 12, 39; R. P. Evstigneeva et al., Zhur. obshcheT Khim., 1973, 43, 1617). The phosphorylation of 1,2-diO-acyl-sn-glycerols with phosphoryl chloride is a useful method for the preparation of both saturated and unsaturated phosphatidic acids. The related dialkyl derivatives of 2,3-dihydroxypropylphosphonic acid have also been synthesised (A. F. Rosenthal et al., Rec. Trav. chim., 1964, 83, 1273; J. chem. Soc., 1965, 7345; J. Lipid Res., 1966, 7, 779), and the effect of these compounds on the enzyme phosphatidate phosphatase (EC 3.1.3.4) which converts phosphatidic acids into diglycerides investigated (Rosenthal and S. Ching-Hsien Han, Biochim. Biophys. Acta, 1968, 152, 96). Diacyl derivatives of optically active 2,3-dihydroxypropylphosphonic acid ("phosphotidic acids") have also been synthesised (Baer and Basu, Canad. J. Biochem., 1970, 48, 1010). The action of diazomethane on cephalins and phosphatidylserines gives the dimethyl esters of the phosphatidic acids (see D. M. Brown and G. O. Osborne, J. chem. Soc., 1957, 2590; H. D. Crone, Biochim. Biophys. Acta, 1964, 84, 665; O. Renkonen, ibid., 1968, 152, 114). The X-ray spacings of a series of optically inactive synthetic phosphatidic acids have been investigated (T. H. Bevan, D. A. Brown and T. Malkin, J. chem. Soc., 1962, 3495).

1

GLYCEROPHOSPHORIC ACID ACYL DERIV.ATIVES

355

Phosphatidic acids are unstable compounds being hydrolysed in moist air or in solution in hydroxylic solvents (J. Olley, Chem. and Ind., 1954, 1069). The synthetic phosphatidic acid containing two oleic acid residues is a viscous oil soluble in ethanol, acetone, chloroform, ether and benzene but insoluble in water. Dimyristoylphosphatidic acid, m.p. 61.5-62.5~ [0c]24 +4.3 ~ (c, 10 in CHCIa). Dipalmitoylphosphatidic acid, m.p. 70-71~ [0C]D24+ 4.0~(C, 5 in CHCI3). Distearoylphosphatidic acid, m.p. 75.5-76~ [0c]24 + 3.8~(c, 9 in CHCI3). A pyrophosphate ("pyrophosphatidic acid") formed from two molecules of phosphatidic acid has been isolated from Cryptococcus neoformans and has been synthesised (T. Itoh and H. Kaneko, J. Biochem., Tokyo, 1974, 75, 1291).

(ii) Lysophosphatidic acids Lysophosphatidic acids are monoacyl derivatives of sn-glycerol-3-phosphate and are formed enzymically by the action of phospholipase D on lysolecithins (C. Long et al., Biochem. J., 1967,. 102, 216, 221 ), by the acylation of dihydroxyacetone phosphate (Hajra and Agranoff, J. biol. Chem., 1968, 243, 3542; Hajra, 1974, 9, 502) and sn-glycerol 3-phosphate (R. G. Lamb and H. J. Fallon, J. biol. Chem., 1970, 245, 3075) and by phosphorylation of monoglycerides (R. Paris and G. Clement, Proc. Soc. exp. Biol. Med., 1969, 131,363). The biosynthesis in rat-liver mitochondria and the specificity of different fatty acids in this process has been investigated (L. N. W. Daae, Biochim. Biophys. Acta, 1972, 270, 23). The enzymic acylation of synthetic lysophosphatidic acids has been studied (W. Stoffel et al., Z. physiol. Chem., 1966, 347, 94, 102; 1967, 348, 882; H. Okuyama and W. E. M. Lands, J. biol. Chem., 1972, 247, 1414; see also Van Deenen et al., Chem. Phys. Lipids, 1967, 1, 317) and the inhibition of this reaction by chlorophenoxyisobutyrate and fl-benzalbutyrate (which are hypolipidemic agents) studied (Lamb and Fallon, J. biol. Chem., 1972, 247, 1281). ( iii) Cytidine diphosphate diglyceride

Cytidine diphosphate diglyceride,

NH2

CI H202C R R1C02-C- H 0

O/N~ N

0 ii CH20- OH P-O-P-OC~ I OH I ''~O. ~ I

II

1

1

HO OH is formed biologically from phosphatidic acid and cytidine triphosphate (H. T. Hutchison and 3. E. Cronan, Biochim. Biophys. Acta, 1968, 164, 606; R. E. McCaman and W. R. Finnerty, J. biol. Chem., 1968, 243, 5074;

356

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

J. R. Carter, J. Lipid Res., 1968, 9, 748). It is the intermediate in the biosynthesis of phosphatidylserine, phosphatidylinositol, phosphatidylglycerol phosphate and cardiolipin. A chemical synthesis has been described in detail (Agranoffand W. D. Suomi, Biochem. Prep., 1964, 10, 47). A deoxycytidine diphosphate diglyceride is readily synthesised in rat-liver mitochondria in the presence of deoxy CTP (H. van den Bosch et al., Biochim. Biophys. Acta, 1971, 239, 234; 1972, 260, 380) and is used in the synthesis of other phospholipids in the same way as CDP-diglyceride (see also C. R. H. Raetz and Kennedy, J. biol. Chem., 1973, 248, 1098). (b) Acyl derivatives of sn-glycerol 3-(2-aminoethyl phosphate) and its N-methyl derivatives (i) Acyl derivatives of sn-glycerol 3-(2-aminoethyl phosphate)

(1) sn-Giyceroi 3-(2-aminoethyl phosphate), (glycerylphosphorylethanoiamine), CH2OH I

HO-C -H

O

II C H20- P-OCH2'CH2N H2 I

I

OH

is a degradation product of the phosphatidylethanolamines, being formed by mild alkaline hydrolysis (Dawson, Biochem. J., 1960, 75, 45)or by hydrogenolysis with lithium tetrahydridoborate (J. E. Bakke and R. A. Clayton, Biochem. biophys. Res. Comm., 1961, 5, 176). The synthetic compound (Baer and H. C. Stancer, J. Amer. chem. Soc., 1953, 75, 4510) forms a crystalline monohydrate, m.p. 86-87~ [0r z6 --2.9 ~ (C, 7.6 in H 2 0 ) a n d is soluble in methanol, glacial acetic acid and water. It has also been prepared by the catalytic hydrolysis of beef-brain plasmalogens (S. J. Thannhauser et al., J. biol. Chem., 1951, 188, 423) and occurs in the free state in various animal tissues (see Dawson, Biochem. J., 1955, 59, 5; G. Schmidt et al., J. biol. Chem., 1955, 212, 887; J. L. Gamble, Biochim. Biophys. Acta, 1963, 74, 130). An N-methyl derivative of this compound (Y. Kodaira and T. Mukaiyama, J. org. Chem., 1966, 31, 2903) and the related sn-glycerol 3-(2aminoethylphosphonate) (Baer and G. R. Sarma, Canad. J. Biochem., 1967, 45, 1755) have been synthesised. Fatty acid amides of glycerylphosphorylethanolamine have been detected in the hog stomach (B. L. Slomiany et al., Biochim. Biophys. Acta, 1973, 316, 35).

1

sn-GLYCEROL3-(2-AMINOETHYLPHOSPHATE)

357

(2) Cephalins, phosphatidylethanolamines, CH202CR I

RICO2--C--H I

0 II

C H20-- P-OCH2.CH2NH I

OH

are diacyl derivatives of sn-glycerol 3-(2-aminoethyl phosphate) and occur in many plant and animal tissues. They are also common constituents of bacterial lipids where, together with glycosyldiglycerides, they are the major components of the neutral lipid fraction. An interrelationship between phosphatidylethanolamines and glycosyldiglycerides in bacterial membranes has been suggested (J. Baddiley et al., Biochim. Biophys. Acta, 1971, 249, 651). They are best isolated from natural lipid mixtures by chromatography on silicic acid (see C. H. Lea et al., Biochem. J., 1955, 60, 353; T. Sakagami, J. Biochem., Tokyo, 1959, 46, 1617; C. Long and D. A. Staples, Biochem. J., 1961, 80, 557) or on diethylaminocellulose (G. Rouser, Biochem. Prep., 1967, 12, 73) although solvent fractionation procedures have been used (C. R. Scholfield and H. J. Dutton, ibid., 1957, 5, 5; E. Klenk and H. Dohmen, Z. physiol. Chem., 1955, 299, 248) (for a review of properties see D. C. Robins, J. Pharm. Pharmacol., 1963, 15, 701). Natural cephalins contain a spectrum offatty acids resulting in a mixture of related compounds which have been analysed by thin-layer chromatography on plates impregnated with silver nitrate (S. M. Hopkins et al., Biochim. Biophys. Acta, 1968,164,272) or after hydrolysing with phospholipase C to give diglycerides (L. M. G. van Golde and Van Deenen, Chem. Phys. Lipids, 1967, 1, 157; O. Renkonen, Adv. Lipid Res., 1968, 5, 329; B. 'J. Holub and A. Kuk. is, Lipids, 1969, 4, 466), or by the thin-layer chromatography of N-acyl-Omethyl derivatives (R. Sundler and B. Akesson, J. Chromatog., 1973, 80, 233; see also S. K. F. Yeung and Kuksis, Canad. J. Biochem., 1974, 52, 830). Phospholipase A removes the 2-acyl group specifically to give 1-O-acylsn-glycerol 3-(2-aminoethyl phosphate) ("lysocephalins") (Van Deenen et al., Rec. Trav. chim., 1963, 82, 469). Several methods have been described for the synthesis of cephalins with two identical fatty acyl groupings (for reviews see De Haas and Van Deenen, Adv. Lipid Res., 1964, 2, 168; Verkade, Bull. Soc. chim. Fr., 1963, 199"3; Baer, Progr. Chem. Fats and Lipids, 1963, 6, 31; Malkin and Bevan, ibid., 1957, 4, 97). The synthesis of cephalins with two different acyl groups has been achieved by starting from 1,2-di-O-acyl-sn-glycerols containing two different acyl groups (Baer and Buchnea, Canad. J. Biochem. Physiol., 1961, 39, 1471; N. A. Preobrazhenskii et al., Zhur. obshcheT Khim., 1963, 33, 2876, 2880; 1965, 35, 84, 554; F. R. Pfeiffer et al., J. org. Chem., 1969,

358

PHOSPHOLIPIDS

AND

GLYCOLIPIDS

21

34, 2795), by the reaction of 2,3-diacyloxy-l-iodo- or -1-bromo-propanes with the silver salts of derivatives of 2-aminoethyl phosphate (F. J. M. Daemen et al., Rec. Trav. chim., 1963, 82, 487; J. D. Billimoria and K. O. Lewis, J. chem. Soc., C, 1968, 1404; Daemen, Chem. Phys. Lipids, 1967, 1,476; J. S. Chadha, ibid., 1968, 2, 415), by acylation of sn-glycerol 3-(2phthalimidoethyl phosphate)(Baer et al., Biochemistry, 1963, 2, 1227; 1964, 3, 975; R. Aneja et al., Biochim. Biophys. Acta, 1969, 187, 439) or by the condensation of phosphatidic acids with derivatives of ethanolamine (I. Barzilay and Y. Lapidot, Chem. Phys. Lipids, 1969, 3, 280; 1971, 7, 93; S. Rakhit et al., Canad. J. Ch'em., 1969, 47, 2906; Aneja et al., Tetrahedron Letters, 1969, 4183) or aziridine (Aneja et al., Chem. Phys. Lipids, 1969, 3, 286) and by the acylation of sn-glycerol 3-(2-N-tritylaminoethyl phosphate) (idem, Biochim. Biophys. Acta, 1969, 187, 579). (For a review see Slotboom and Bonsen, Chem. and Phys. Lipids, 1970, 5, 301). Cephalins labelled with 14C, 32p and 3H have been prepared by mixing three individually prepared cephalins each containing one label (Billimoria et al., Chem. Phys. Lipids, 1974, 12, 327). Cephalins are soluble in chloroform and ethanol and are slightly soluble in acetone. Synthetic cephalins with short-chain acyl groups are water-soluble (Baer and T. Grot, Canad. J. Biochem. Physiol., 1960, 38, 859" J. Maurukas et al., Biochemistry, 1963, 2, 397). Diazomethane converts cephalins into the dimethyl esters of phosphatidic acids (see D. M. Brown and G. O. Osborne, J. chem. Soc., 1957, 2590; H. D. Crone, Biochim. Biophys. Acta, 1964, 84, 665). Various physical properties of cephalins and thin films of cephalins have been studied (D. Chapman et al., Trans. Farad. Soc., 1966, 62, 2607" Proc. roy. Soc, A, 1966, 290, 115" Biochim. Biophys. Acta, 1966, 120, 148" P. J. Quire1 and Dawson, Biochem. J., 1969, 113, 791" R. P. Rand et al., Chem. Phys. Lipids, 1971, 6, 333). The n.m.r, spectra of phosphatidylethanolamines (H. Richard et al., FEBS Letters, 1974, 45, 136)and the X-ray analysis of the crystalline structure of racemic dilauroylphosphatidylethanolamine (P. B. Hitchcock et al., Proc. Nat. Acad. Sci., 1974, 71, 3036) have been studied. Distearoylcephalin, m.p. 173-175~ [~]D24 + 6.0~ 2-oleoyl-l-stearoylcephalin, m.p. 189_190o, [~]20 + 6.1 o (c, 8 in CHCI3)" 1-oleoyl-2-stearoylcephalin, m.p. 186~ [~]D20 + 6.0~ (C, 6 in CHCI3). Related compounds based on 2-aminoethylphosphonic acid,

(HO)2 PCH2"CH2NH2 o

in place of 2-aminoethyl phosphate have been synthesised (Baer and H. Basu, Canad. J. Biochem., 1968, 46, 351; D. L. Turner et al., Lipids, 1968, 3, 234; A. F. Rosenthal and M. Pousada, Rec. Trav. chim., 1965, 84, 833; Baer, Canad. J. Biochem., 1974, 52, 570) and natural "phosphonolipids" of this structure have been detected in the lipids of Tetrahymena pyriformis

1

sn-GLYCEROL3-(2-AMINOETHYLPHOSPHATE)

359

(C. R. Liang and H. Rosenberg, Biochim. Biophys. Acta, 1966, 125, 548; M. Sugita and T. Hori, J. Biochem., Tokyo, 1971, 69, 1149) in the larvae of Musca domestica (R. E. Bridges and J. Ricketts, Nature, 1966, 211, 199) and in rumen protozoa (Dawson and P. Kemp, Biochem. J., 1967, 105, 837; T. Tamari et al., Biochimie, 1973, 55, 1311). Methods for the separation of 2-aminoethyl phosphate and 2-aminoethylphosphonic acid have been described (A. J. de Koning, J. Chromatog., 1971, 59, 185). Phosphonic acid analogues of cephalin containing ethylene glycol instead of glycerol have been synthesised (Baer and H. Basu, Canad. J. Biochem., 1968, 46, 1279; for a review of lipids based on ethylene glycol see L. D. Bergelson, Progr. Chem. Fats and Lipids, 1969, 10, 241). Other compounds related to the cephalins, containing 1,3-dihydroxypropane and 1,6-dihydroxyhexane in place of glycerol (I. Hara et al., Bull. Soc. chim. biol., 1964, 46, 895; 1968, 50, 1501; F. R. Pfeiffer et al., J. med. Chem., 1971, 14, 491), ethylene glycol in place of glycerol (see Daemen et al., Rec. Trav. chim., 1962, 81,348), 1,2-dihydroxypropane in place of glycerol (Baer et al., Canad. J. Biochem., 1968, 46, 69), 2-amino-2-methylpropan-l-ol in place of ethanolamine (Baer and G. V. Rao, J. biol. Chem., 1963, 238, 1941, see also Canad. J. Biochem., 1964, 42, 1547) and 3-aminopropan- 1-ol or 2-amino- 1methylethan-l-ol in place of ethanolamine (T. Muramatsu and Hara, Bull. Soc. chim. Fr., 1971,3335, 3338) have also been synthesised. N-Acetyl derivatives of the cephalins have been isolated from cattle and human brain (H. Debuch and G. Wendt, Z. physiol. Chem., 1967, 348, 471) and from bovine erythrocytes (M. Matsumoto and M. Miwa, Biochim. Biophys. Acta, 1973, 296, 350) and some N-acylcephalins have been isolated from seed lipids (see Dawson et al., Biochem. J., 1969, 114, 265; Aneja et al., Biochem. biophys. Res. Comm., 1969, 36, 401) and have also been synthesised (J. J. Wren and D. S. Merryfield, J. chem. Soc., 1964, 6251). Synthetic derivatives of cephalins containing amino acids joined to the amino group in amide linkage (G. I. Samokhvalov et al., Zhur. obshcheT Khim., 1966, 36, 1905; 1967, 37, 320, 2452; 1968, 38, 1495; Zhur. org. Khim., 1969, 5, 391) and an N-glucosylcephalin (Billimoria and Lewis, Chem. and Ind., 1968, 1731) have also been synthesised. An N-(1-carboxyethyl) derivative of phosphatidylethanolamine CH202 CR I R1CO2- C-H O I

II

CH3 I

C H20 IPOCH2"CH2NH -CHI OH CO2H

is present in the lipids of rumen protozoa (Dawson and Kemp, Biochem.

360

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

J., 1969, ll3, 555; Dawson et al., ibid., 1971, 123, 97) and a compound with this structure has been synthesised (S. K. Pavanaram and H. Flehmig, Experientia, 1972, 28, 498). The cephalins are synthesised biologically by a reaction between a 1,2-diO-acyl-sn-glycerol and cytidine diphosphate ethanolamine (E. P. Kennedy and S. B. Weiss, J. biol. Chem., 1956, 222, 193) by acylation of lysocephalins (I. Merkl and W. E. M. Lands, ibid., 1963, 238, 905; P. R. Turkki and J. L. Glenn, Biochim. Biophys. Acta, 1968, 152, 104) and by decarboxylation ofphosphatidylserine (Kennedy, J. biol. Chem., 1974, 249, 3079; M. lshinaga and M. Kito, Europ. J. Biochem., 1974, 42, 483; T. Suda and M. Matsuda, Biochim. Biophys. Acta, 1974, 369, 331). A base-exchange mechanism between phospholipids is also thought to occur (see J. N. Kanfer et al., J. biol. Chem., 1972, 247, 7153; J. Lipid Res., 1972, 13, 468; G. Porcellati et al., J. Neurochem., 1973, 20, 1167; M. O. Marshall and M. Kates, FEBS Letters, 1973, 31, 199; A. Nilsson et al., ibid., 1974, 43, 303; T. Taki et al., Jap. J. exp. Med., 1973, 43, 87). Phosphatidylethanolamine is an essential component of some enzyme systems (A. Endo and L. Rothfield, Biochemistry, 1969, 8, 3508) and is hydrolysed by phospholipase C to give diglycerides under certain conditions (see T. Takahashi and H. H. O. Schmidt, Chem. Phys. Lipids, 1968, 2, 220). Artifacts are sometimes produced in the isolation of cephalins, using acetone, due to condensation of the amino group with mesityl oxide present as a contaminant in the acetone (Wendt and Debuch, Z. Physiol. Chem., 1968, 349, 789; T. Yamakawa et al., J. Biochem., Tokyo, 1971, 70, 341)i The amino group of cephalin also reacts with glutaraldehyde, which is used as a fixative for tissues, resulting in cross-linking of cephalins to protein in the tissues (R. Gigg and S. Payne, Chem. Phys. Lipids, 1969, 3, 292). (3) Lysocephalins are monoacyl derivatives of sn-glycerol 3-(2-aminoethyl phosphate) (for review of properties see Robins, J. Pharm. Pharmacol., 1963, 15, 701). They have been isolated from human serum (U. K. Misra, Biochim. Biophys, Acta, 1965, 106, 371) and from Calanus plumchrus (S. Patton et al., ibid., 1972, 270, 479) and cause rupture of the red blood cells (see M. Jeannet and A. H~'ssig, Vox Sanguinis, 1964, 9, 113). 1-O-Acyl-sn-glycerol 3-(2-aminoethyl phosphate) is formed by the action of phospholipase A on cephalins (see K. Saito and K. Sato, J. Biochem., Tokyo, 1968, 64, 293) and this enzyme is present in serum (J. Polonovski et al., Bull. Soc. Chim. biol., 1967, 49, 1751) and many other tissues. Both 1-O- and 2-O-acyl derivatives of sn-glycerol 3-(2-aminoethyl phosphate) have been synthesised (Van Deenen et al., Rec. Trav. chim., 1963, 82, 469; Billimoria and Lewis, J. chem. Soc., C, 1968, 1404; F. R. Pfeiffer et al., J. med. Chem., 1971,

1

sn-GLYCEROL3-(CHOLINEPHOSPHATE)

ACYLDERIVATIVES

361

14, 493). A lysophospholipase (EC 3.1.1.5) from Penicillium notatum cleaves lysocephalin to give sn-glycerol 3-(2-aminoethyl phosphate) (Saito and Sato, loc. cit.). (ii) Phosphatidyl-N-methylethanolamines (I) and-N,N-dimethylethanolamines (II) CH202CR I

R1CO2-C - H I

CH202CR I

O

R1CO2-C - H

II

CH20 -- P - O C I

I

H2,CH2NH M e

O II

CH20 -P-OCH2.CH2NMe2

OH

I

OH

(I)

(If)

These compounds have been isolated from micro-organisms (M. O. Hall and J. F. Nyc, J. Lipid Res., 1961, 2, 321; B. J. Crocken and Nyc, J. biol. Chem., 1964, 239, 1727; H. Goldfine, Biochim. Biophys. Acta, 1962, 59, 504) and their presence suggests that they are intermediates in the biosynthesis of lecithins (see Kennedy, Federation Proc., 1961, 20, 934; G. A. Scarborough and Nyc, J. biol. Chem., 1967, 242, 238). Compounds of both types have been synthesised with saturated acyl groups (Baer and Pavanaram, J. biol. Chem., 196 l, 236, 1269, 2410; D. Shapiro and Y. Rabinsohn, Biochemistry, 1964, 3, 603; Preobrazhenskii et al., Zhur. obshcheT Khim., 1965, 35, 550; Aneja et al., Tetrahedron Letters, 1969, 4183). Methyllabelled phosphatidyl-N,N-dimethylethanolamine has also been prepared from phosphatidylethanolamine (D. LeKim and H. Betzing, Z. physiol. Chem., 1973, 354, 1490). These lipids accumulate in the liver and lungs of rats fed on N-methylethanolamine (S. L. Katyal and B. Lombardi, Lipids, 1974, 9, 81) and in tissue culture cells in the presence of the bases (P. R. Va#elos et al., Proc. Nat. Acad. Sci., 1974, 71, 4072). A phosphonic acid analogue of the N-methyl compound and its deacylated derivative have been synthesised (Baer and Pavanaram, Canad. J. Biochem., 1970, 48, 979, 988). Monoacyl derivatives of glycerol 3-(2-dimethylaminoethyl phosphate) have also been synthesised (Van Deenen et al., Chem. Phys. Lipids, 1967, 1, 317). (iii) Acyl derivatives of sn-glycerol 3-(choline phosphate) sn-Giycerol 3-(choline phosphate), OH CIH2 O

HO-C-H

I II (~ C H20 - P - O C H2.C H2N M e 3

362

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

is prepared by the hydrolysis of natural lecithin with mercuric chloride as a catalyst, with alkaline hydroxylamine, with mild alkali or enzymically (see H. Brockerhoff and M. Yurkowski, Canad. J. Biochem., 1965, 43, 1777; G. V. Marinetti, Biochemistry, 1962, 1,350; S. Okui et al., J. pharm. Soc., Japan, 1964, 84, 1206; Chadha, Chem. Phys. Lipids, 1970, 4, 104). The hydrogenolysis of lecithin with lithium tetrahydridoaluminate produces a pure product without racemisation (see De Haas and Van Deenen, Tetrahedron Letters, 1960, 7). The structure has been confirmed by synthesis (Baer and Kates, J. Amer. chem. Soc., 1948, 70, 1394; J. Maurukas and C. V. Holland, J. org. Chem., 1961, 26, 608). It occurs in the free state in various tissues (see R. M. C. Dawson, Biochem. J., 1955, 59, 5; G. Schmidt et al., J. biol. Chem., 1955, 212, 887). sn-Glycerol 3-(choline phosphate) is a hygroscopic, white, crystalline compound, m.p. 142.5-143~ [0t]D24 --2.89 ~ (C, 4.6 in H20), soluble in methanol, ethanol and water and forms a crystalline complex with cadmium chloride (CaH2oO6NP-CdCI2"3H20). It is an important intermediate for the synthesis of lecithins. The related compound sn-glycerol 3-(2-trimethylammonio-ethylphosphonate) has also been synthesised (Baer and R. Robinson, Lipids, 1967, 2, 194, 286; Canad. J. Biochem., 1967, 45, 1747). Oxidation of sn-glycerol 3-(choline phosphate) with periodate followed by reduction with tetrahydridoborate gives ethylene glycol (choline phosphate) (K. K. Yabusaki and M. A. Wells, Biochim. Biophys. Acta, 1973, 296, 546).

(I) Lecithins, phosphatidyicholines, C H202CR I R1CO2--C-H O C H20- P-OCHiCH2NMe 3 are diacyl derivatives of sn-glycerol 3-(choline phosphate) and occur in many animal and plant tissues and rarely in bacteria (see Goldfine and P.-O. Hagen, J. Bacteriol., 1968, 95, 367). They are best prepared from natural lipid mixtures by chromatography on alumina and silicic acid columns (see D. N. Rhodes and C. H. Lea, Biochem. J., 1957, 65, 526; D. J. Hanahan et al., J. biol. Chem., 1957, 228, 685; M. Marsh and R. T. Holzbach, Clin. Chim. Acta, 1973, 43, 87) or by thin-layer chromatography (K. H. Slotta, Monatsh., 1966, 97, 1723). Lecithins isolated from tissues contain a spectrum of fatty acids and are thus usually mixtures of related compounds although a dipalmitoleoyl-lecithin has been isolated from baker's yeast (Hanahan and M. A. Jayko, Biochem. Prep., 1955, 4, 12), and dipalmitoyl-lecithin is present in the larval form of the cat tapeworm (A. Lesuk and R. J. Anderson, J. biol. Chem., 1941, 139, 457) and in brain and spleen (S. J. Thannhauser et al., ibid., 1948, 172, 135). Dipalmitoyl-leci-

l

sn-GLYCEROL3-(CHOLINE

PHOSPHATE)ACYL

DERIVATIVES

363

thin is also the major component of the surface-active constituents of the lung and the ratio of lecithin to sphingomyelin in amniotic fluid is a useful index of fetal lung development (see J. M. Vereyken et al., Biochim. Biophys. Acta, 1972, 260, 70; L. Sarkozi et al., Clinical Chem., 1972, 18, 956; C. R. Whitfield, Brit. med. J., 1972, 2, 85; M. E. Avery et al., Scientific American, 1973, 228, 74; R. J. Mason, Amer. Review Resp. Diseases, 1973, 107, 678; C. Warren et al., Clin. Chim. Acta, 1973, 44, 457; R. J. King, Federation Proc., 1974, 33, 2238). The biosynthesis of dipalmitoyl-lecithin in lung has been studied (J. C. Beck et al., Canad. J. Biochem., 1973, 51, 1581; E. G. Tombropoulos, Arch. Biochem. Biophys., 1973, 158, 911). Much information about the composition of the fatty acid radicals in the 1- and 2-positions of natural lecithins has been obtained by enzymic hydrolysis of the 2-acyl group with phospholipase A2 (EC 3.1.1.4) (see G. H. de Haas et al., Biochim. Biophys. Acta, 1962, 65, 260; L. L. M. van Deenen et al., ibid., 1963, 67, 295; A. F. Robertson and Lands, Biochemistry, 1962, 1,804; N. H. Tattrie and J. R. Bennett, Canad. J. Biochem. Physiol., 1963, 41, 1983) and it has been shown that saturated fatty acyl groups occur mainly in the 1-position. A kinetic study of the hydrolysis of 1,2-dibutyryl-sn-glycerol 3-(choline phosphate) by the phospholipase AE from Crotalus adamanteus has been reported (Wells, Biochemistry, 1972, 11, 1030) and the activity of phospholipase A2 towards reversed micelles of lecithins in ether has been studied (idem, ibid., 1974, 13, 4921, 4928, 4937). The partial separation of natural lecithins into fractions containing different fatty acids has been achieved by thin-layer chromatography (see G. A. E. Arvidson, J. Lipid Res., 1967, 8, 155; F. Nakayama and S. Kawamura, Clin. Cfiim. Acta, 1967, 17, 53; J. Tinoco et al., Lipids, 1967, 2, 479) by chromatography of the mercuric acetate adducts on Sephadex LH-20 (Kin9 and J. A. Clements, J., Lipid Res., 1970, 11,381)and by counter-current distribution (F. D. Collins, Chem. Phys. Lipids, 1967, 1, 91). Other analyses of lecithins have been achieved by hydrolysis with phospholipase C (EC 3.1.4.3) to give diglycerides which are separated by thin-layer chromatography or converted into the acetates and separated by gas-liquid chromatography. The fatty acid distribution in the separated glyceride fractions has been determined by hydrolysis with pancreatic lipase (EC 3.1.1.3) (see Van Deenen et al., ibid., 1967, 1, 282; O. Renkonen, Biochim. Biophys. Acta, 1968, 152, 114; Lipids, 1968, 3, 191; O. S. Privett and L. J. Nutter, ibid., 1967, 2, 149; A. Kuksis and L. Marai, J. Lipid Res., 1969, 10, 25, 141; K. A. Devor and J. B. Mudd, ibid., 1971, 12, 396). The molecular species in normal liver and in hepatoma (L. D. Bergelson et al., Chem. Phys. Lipids, 1974, 12, 132) and in the mitochondria and endoplasmic reticulum of guinea pig liver (where they are identical

364

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

thus indicating a transfer of lipids between these two structures) (J. G. Parkes and W. Thompson, J. biol. Chem., 1973, 248, 6655) have also been studied. A combination of gas chromatography and mass spectrometry has also been used to study the molecular species of lecithins (K. Hasegawa and T. Suzuki, Lipids, 1973, 8, 631; T. Curstedt and J. Sjovall, Biochim. Biophys. Acta, 1974, 360, 24). The composition of the fatty acids esterified at the 2-hydroxy group of glycerol in lecithins of animal tissues can be altered by changing the fatty acid composition of the dietary lipids (Van Deenen et al., Biochim. Biophys. Acta, 1968, 152, 84; C. Pudelkewicz and R. T. Holman, ibid., p. 340) due to enzymic deacylatiQn to a lysolecithin followed by reacylation (see below). Several methods have been devised for the total synthesis of lecithins containing two identical fatty acyl radicals (for reviews see De Haas and Van Deenen, Adv. Lipid Res., 1964, 2, 168; Verkade, I1 Farmaco, Ed. Sci., 1968, 23, 149; Baer, Progr. Chem. Fats and Lipids, 1963, 6, 31; Malkin and Bevan, ibid., 1957, 4, 97; A. J. Slotboom and P. P. M. Bonsen, Chem. Phys. Lipids, 1970, 5, 301; Slotboom et al., ibid., 1973, 11,295). The lecithins with two identical fatty acids have also been prepared by the direct acylation of glycerol 3-(choline phosphate) with acid chlorides in amine-free, dry solvents (Tattrie and C. S. McArthur, Canad. J. Biochem. Physiol., 1957, 35, 1165), or with the tetraethylammonium salt of a fatty acid in the presence of the fatty acid anhydride (E. C. Robles et al:, Rec. Tray. chim., 1967, 86, 762; Biochim. Biophys. Acta, 1969, 187, 520; D. T. Gordon and R. G. Jensen, Lipids, 1972, 7, 261) and by acylation of the cadmium chloride complex of glycerol 3-(choline phosphate) with acid chlorides in pyridine (E. Baer and D. Buchnea, Canad. J. Biochem. Physiol., 1959, 37, 953; F. Kb'gl et al., Rec. Trav. chim., 1960, 79, 661). A by-product in this reaction has been identified as an acyl derivative of a chlorodeoxyglycerol 3-(choline phosphate) and the synthesis of this compound has been investigated in detail (R. Aneja et al., Chem. Phys. Lipids, 1973, 11, 89). The phosphorylation ofa 1,2-di-O-acyl-sn-glycerol with phosphoryl chloride followed by esterification with a choline salt (Baer and A. Kindler, Biochemistry, 1962, 1, 518; N. A. Preobrazhenskii et al., Zhur. obshcheT Khim., 1964, 34, 3983) and the esterification of a phosphatidic acid with choline in the presence of triisopropylbenzenesulphonyl chloride in pyridine (R. Aneja and J. S. Chadha, Biochim. Biophys. Acta, 1971, 248, 455) have also been used for the synthesis of lecithins with two identical fatty acids. For the preparation of lecithins with two different fatty acids the following route has been used (De Haas and Van Deenen, Tetrahedron Letters, 1960, 7; Bennett and Tattrie, Canad. J. Biochem. Physiol., 1961, 39, 1357; 1963, 41, 1983; Hanahan and H. Brockerhoff, Arch. Biochem. Biophys., 1960, 91,326; Slot-

1

sn-GLYCEROL3-(CHOLINEPHOSPHATE) ACYLDERIVATIVES 365

boom et al., Chem. Phys. Lipids, 1973, 11,295): CH2OH HO-C-H O

CH202CR acylate I ~-- RCO2--C-H O

C H20-P -O CH2"CH2N M e 3

C H20-P-OC H2"CH2NMe

C H202CR C H202CR I acylate I HO-C-H O -~ R1CO2--C- H O lipase A I ii (~) I ii C H20 - P-OC H2"CH2NMe3 C H20- P-OC H2-CH2N Me3

Phospho-

Totally synthetic lecithins containing two different fatty acid radicals (saturated and unsaturated) have also been prepared via 2,3-diacyloxy- ]-iodopropane (De Haas and Van Deeaea, Rec. Trav. chim., ]96], 80, 95 ]). The synthesis'of the related compound C H202CR I RCO2-C-H O C H20-P--CH2"CH2N Me 3 derived from 2-trimethylammonio-ethylphosphonic acid instead of choline phosphate (A. F. Roseathal and M. Pousada, Biochim. Biophys. Acta, 1968, 164, 226; Baer et al., Canad. J. Biochem., 1967, 45, 1783) and the syntheses of analogues based on ethylene glycol rather than glycerol (Baer, J. biol. Chem., ]953, 75, 5533; Bergelsoa et al., Izvest. Akad. Nauk, 1973, 410; K. K. Yabusaki and M. A. Wells, Biochim. Bioph.ys. Acta, 1973, 296, 546) as well as the ethylene glycolphosphonic acid analogue (Baer and R. Robinson, Canad. J. Biochem., ]968, 46, 1273) have been described. The synthesis of an analogue based on propylene glycol rather than glycerol (Baer et al., ibid., 1968, 46, 49; L. D. Bergelson et al., Izvest. Akad. Nauk, 1969, ]784) and of isomers of lecithin phosphorylated on the 2-hydroxyl group of glycerol (De Haas and Van Deeaea, Biochim. Biophys. Acta, 1964, 84, 469; O. Westphal et al., Ann., 196?, 709, 226; Bergelsoa et al., Zhur. org. Khim., 1974, 10, ]398) have also been described. The de-N-methylation of lecithin with sodium benzenethiolate followed by remethylation with

[a4C]methyl iodide (IV. Stoffel et al., Z. physiol. Chem., 1971, 352, 1058) and the biosynthesis in potato slices in the presence of [14C]acetate (T. Galliard, Biochim. Biophys. Acta, 1972, 260, 541) and in germinating soya beans in the presence of [14C]glycerol (S. Parthasarathy and J. Ganguly, ibid., 1973, 296, 62) (see also M. Sugano and M. Yamamoto, Ag. and biol. Chem., 1974, 38, 1255)and the methylation of phosphatidylethanolamine

366

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

(D. L e K i m and H. Betzing, Z. physiol. Chem., 1973, 354, 1490) have been

used for the preparation of labelled lecithins. Demethylation of lecithin to N,N-dimethylphosphatidylethanolamine followed by combined gas chromatography and mass spectrometry has been used for the analysis of lecithins (K. A. Karlsson et al., Biochim. Biophys. Acta, 1973, 326, 174). Various lecithin analogues have been synthesised and tested as potential inhibitors of phospholipase A2 (Bonsen et al., Chem. Phys. Lipids, 1972, 8, 199; Biochim. Biophys. Acta, 1972, 270, 364). The biosynthesis oflecithins proceeds by at least two routes, one involving a reaction between cytidine diphosphate choline and a 1,2-di-O-acyl-sn-glycerol and the other by direct methylation of phosphatidylethanolamine (see E. P. Kennedy, Federation Proc., 1961, 20, 934; D. Rehbinder and D. M. Greenberg, Arch. Biochem. Biophys., 1965, 109, 110; R. L. Lester et al., J. biol. Chem., 1969, 244, 3419; Arch. Biochem. Biophys., 1973, 158, 401; D. L. Young and F. Lynen, J. biol. Chem., 1969, 244, 377; Stoffel et al., Z. physiol. Chem., 1973, 354, 437). The acylation of lysolecithins is also an important biological route to lecithins (see below and see also T. Akino et al., Biochim. Biophys. Acta, 1971, 248, 274; Tohoku J. exp. Med., 1972, 108, 133; M. A. Trewhella and F. D. Collins, Biochim. Biophys. Acta, 1973, 296, 51). The exchange of phosphatidylcholine biosynthesised in the endoplasmic reticulum with mitochondria takes place via a specific exchange protein (for a review see K. W. A. Wirtz, ibid., 1974, 344, 95). Lecithins are only slightly soluble in acetone and form emulsions with water. Those containing unsaturated fatty acid groupings have higher solubilities in organic solvents than the saturated compounds. Some synthetic lecithins with short-chain acyl groups (e.g. hexanoyl) are water-soluble (Baer and V. Mahadevan, J. Amer. chem. Soc., 1959, 81, 2494). The micellar weights of the series of dioctanoyl- to didodecanoyl-lecithins have been studied (W. J. Pugh and L. Saunders, J. Pharm. Pharmacol., 1974, 26, 286). Many physical studies on lecithins and thin films of lecithins have been reported (see D. Chapman et al., Chem. Phys. Lipids, 1967, 1, 389, 445; J. mol. Biol., 1967, 30, 551; 1969, 40, 19; Biochem. J., 1967, 105, 401; Biochim. Biophys. Acta, 1966, 120, 148; M. Galdston and D. O. Shah, ibid., 1967, 137, 255; R. A. Demel et al., ibid., 1972, 266, 26; J. P. M. Janson et al., Chem. Phys. Lipids, 1972, 9, 147; J. mol. Biol., 1972, 67, 75). The mass spectrometry (R. A. Klein, J. Lipid Res., 1971, 12, 628), IH and 13C nuclear magnetic resonance (J. C. Metcalfe et al., J. chem. Soc., Perkin II, 1972, 1441 ; Biochemistry, 1972, 11, 1416; FEBS Letters, 1972, 23, 203; Biochemistry, 1973, 12, 1650; J. G. Batchelor et al., Biochem. biophys. Res. Comm., 1972, 48, 70; R. J. Kostelnik and S. M. Castellano, J. Magnetic Res., 1973, 9, 291; B. A. Cornell et al., Chem. Phys. Lipids, 1974, 13, 183), 3~p n.m.r. (D. G. Davis, Biochem. biophys. Res. Comm., 1972, 49, 1492; T. O. Henderson et ai., Biochemistry, 1974, 13, 623), 23Na n.m.r. (N. O. Persson et al., Chem. Phys. Lipids, 1974, 12, 261), and laser Raman spectroscopy (R. Mendelsohn, Biochim. Biophys. Acta, 1972, 290, 15) have been investigated.

1

sn-GLYCEROL3-(CHOLINE PHOSPHATE)ACYL DERIVATIVES 367

The lecithins find important uses in the food, plastic and pharmaceutical industries due to their properties as protective colloids, emulsifying agents and antioxidants. At the present time their biological significance as components of membranes is not known although they are essential components of certain enzymic reactions of the electron-transport chain in mitochondrial oxidations (see R. Coleman, Biochim. Biophys. Acta, 1973, 300, 1; S. Fleischer et al., J. biol. Chem., 1973, 248, 2549, 2556; Biochem. biophys. Res. Comm., 1974, 58, 309; C. S. Hexter and R. Goldman, Biochim. Biophys. Acta, 1973, 307, 421; H. M. Menzel and G. H. Hammes, J. biol. Chem., 1973, 248, 4885). A lecithin-type phospholipid isolated from the larvae of the blowfly (Phormia regina) after feeding carnitine, contains a fl-methylcholine instead of a choline residue (see L. L. Bieber et al., J. biol. Chem., 1969, 244, 630). An analogue of lecithin containing ~-methylcholine in place of choline has been synthesised (Baer and B. C. Pal, Canad. J. Biochem., 1967, 45, 309). Rats fed on N-ethyl-N,N-dimethylethanolamine or N,N-diethyl-Nmethylethanolamine incorporate these compounds into lecithin-like lipids (0. E. Bell and D. R. Strength, Arch. Biochem. Biophys., 1968, 123, 462). Distearoyl-lecithin, m.p. 230-231~ [~] 25 + 6.2:~.2-oleoyl- 1-stearoyl-lecithin, m.p. 237~ [~]Dz~ + 6.0~(C, 10 in CHCI3); 1-oleoyl-2-stearoyl-lecithin, m.p. 233-235~ [~]2o + 6.0~ (c, 10 in CHCI3).

(2) Lysolecithins are monoacyl derivatives of sn-glycerol 3-(choline phosphate) (for review of properties see N. Robinson, J. Pharm. Pharmacol., 1961, 13, 321); they occur naturally in many tissues. 1-O-Acyl-sn-glycerol 3-(choline phosphate) is readily prepared by the deacylation of lecithin with phospholipase A2 (see above). Lysolecithins can also be obtained by alkaline hydrolysis of lecithins (G. V. Marinetti, Biochemistry, 1962, 1,350; D. S. Merryfield and J. J. Wren, J. Pharm. Pharmacol., 1971, 23, 695). During the extraction of phospholipids, lysolecithins can be produced by enzymic hydrolysis of lecithins (see R. Letters and B. K. Snell, J. chem. Soc., 1963, 5127) or by acid hydrolysis of cholinecontaining plasmalogens. The position of the acyl group on the lysolecithins produced in this way is uncertain due to the ease of acyl migration (see L. L. M. van Deenen et al., Rec. Trav. chim., 1963, 82, 469; Biochim. Biophys. Acta, 1965, 106, 315, 326). Catalytic oxidation of the free hydroxyl group in lysolecithins with molecular oxygen and platinum occurs without acyl migration (0. W. Thiele, Europ. J. Biochem., 1968, 5, 540). 2-O-Acyl-snglycerol 3-(choline phosphate) has been prepared from natural phosphatidal cholines by hydrolysis of the vinyl ether linkage with iodine in an aqueous medium (H. Eibl and W. E. M. Lands, Biochemistry, 1970, 9, 423) or by the action of pancreatic lipase on natural lecithins (Van Deenen et al., Chem.

368

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

Phys. Lipids, 1970, 4, 15, 30). Syntheses of both 1-O-acyl- and 2-O-acyl-snglycerol-3-(choline phosphate) and various isomers and derivatives have been reported (Van Deenen et al., ibid., 1967, 1, 317; O. Westphal et al., Ann., 1967, 709, 231,240, 244; 1973, 1439). The enzymic acylation of lysolecithins occurs in many tissues and is an important biological route to the lecithins (see Lands et al., Biochim. Biophys. Acta, 1967, 144, 605; J. Lipid Res., 1968, 9, 12; W. Stoffel et al., Z. physiol. Chem., 1967, 348, 882; Van Deenen et al., Biochim. Biophys. Acta, 1967, 144, 613; 1968, 152, 84; M. Abe and T.'Akino, Tohoku J. exp. Med., 1973, 110, 167). The enzyme acyl CoA:monoacylphosphatidylcholine acyl transferase catalyses the incorporation of an unsaturated fatty acid preferentially on the 2-hydroxyl group of glycerol in 1-O-acyl-sn-glycerol 3-(choline phosphate) and a saturated fatty acid on the 1-hydroxyl group of glycerol in 2-O-acyl-sn-glycerol 3-(choline phosphate) ( Van Deenen et al., ibid., 1968, 152, 694; 1969, 176, 632; K. Waku and Lands, J. Lipid Res., 1968, 9, 12; S. Yamashita et al., Europ. J. Biochem., 1973, 38, 25). The enzymic hydrolysis of lysolecithins by phospholipase D to give lysophosphatidic acids (C. Long et al., Biochem. J., 1967, 102, 216, 221) and the enzymic hydrolysis of lysolecithins in rat liver to give sn-glycerol 3(choline phosphate) (Van Deenen et al., Biochim. Biophys. Acta, 1968, 164, 215) have been studied in detail. Lysolecithins cause rupture of the red blood cell membranes (see F. C. Reman et al., Chem. Phys. Lipids, 1969, 3, 221) and this effect depends on the chain length of the fatty acyl groups present. They may also be involved in cell fusion (J. A. Lucy et al., Nature, 1970, 227, 810, 815; 1975, 253, 194). The micelles formed by lysolecithins in aqueous solution have been studied in connection with their binding of certain hormones (A. B. Schneider and H. Edelhoch, J. biol. Chem., 1972, 247, 4986). A plasma enzyme, phosphatidyl choline: cholesterol acyl transferase (EC 2.3.1.26) transfers the fatty acyl group on the 2-position of glycerol in lecithin to cholesterol to give cholesterol esters and lysolecithin (for reviews see E. Gjone et al., Scand. J. Clin. Lab. Invest., 1974, 33, 191; 1974, Supplement No. 137; Biochim. Biophys. Acta, 1973, 326, 406; J. A. Glomset and K. R. Noruum, Adv. Lipid Res., 1973, 11, 1).

1

PHOSPHATIDYLSERINES

369

(c) Acyl derivatives of sn-glycerol 3-(L-serine phosphate) and other amino acid phosphates sn-Glycerol 3-(L-serine phosphate),

CH2OH J

HO-C -H I

O

NH 2

II

I

C H 2 0 - P - O C H 2 - 1 CI -CO2H OH H

has been obtained by the hydrolysis of phosphatidylserine (Dawson, Biochem. J., 1960, 75, 45)and by synthesis (Baer et al., J. Amer. chem. Soc., 1959, 81, 2166). It is a slightly hygroscopic, white solid (monohydrate) which is water-soluble, [0~] 24 + 4.5~(c, 10 in N HCI)' [~]~4 _ 2.0~ (c, 10 in H20). ( i) Phosphatidylserines

CH202CR I RIco2-C-H 0 NH2 I II I CH20-P-OCH2-CI-CO2H I

OH

H

Phosphatidylserines are diacyl derivatives of sn-glycerol 3-(L-serine phosphate) and were first identified in brain lipids (see J. Folch, J. biol. Chem., 1949, 177, 497) and are the major lipid components of the Gram-negative anaerobic bacterium Megasphaera elsdenii where they exist mainly in the plasmalogen form (L. M. G. van Golde et al., Biochim. Biophys. Acta, 1973, 326, 314). They are best prepared from natural lipid mixtures by chromatography (see H. Sanders, Biochim. Biophys. Acta, 1967, 144, 485) or chromatography followed by counter-current distribution (H. Jensen et al., Arch. Biochem. Biophys., 1960, 89, 91). A useful thin-layer chromatography system for the identification ofphosphatidylserine in the presence of other phospholipids has been described (H. D. Kaulen, Anal. Biochem., 1972, 45, 664). The natural compounds contain a mixture of fatty acyl groups. Phosphatidylserines with saturated acyl radicals (Baer and J. Maurukas, J. biol. Chem., 1955, 212, 25; Malkin et al., J. chem. Soc., 1957, 3086; D. M. Brown and P. R. Hammond, ibid., 1960, 4232) and unsaturated acyl radicals (D. L. Turner et al., J. Lipid Res., 1964, 5, 616; J. med. Chem., 1966, 9, 771 ; G. H.de Haas et al., Rec. Trav. chim., 1964, 83, 99; Preobrazhenskii et al., Zhur. obshcheT Khim., 1967, 37, 1454; R. P. Evstigneeva et al., ibid., 1973, 43, 193) have been synthesised (for reviews see De Haas and

370

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Van Deenen, Adv. Lipid Res., 1964, 2, 167; A. J. Slotboom and P. P. M. Bonsen, Chem. Phys. Lipids, 1970, 5, 301). Dipalmitoylphosphatidyl-[14C]serine (D. Rehbinder and D. M. Greenberg, Ann., 1965, 681, 182) and the corresponding compounds from ethylene glycol instead of glycerol (see De Haas and Van Deenen, loc. cit., p. 198) have also been synthesised. An N-acylphosphatidylserine has been isolated from sheep erythrocytes (G. Nelson, Biochem. biophys. Res. Comm., 1970, 38, 261). Phosphatidylserines are anticoagulants (see Turner et al., loc. cit.; E. E. Nishizawa and J. F. Mustardt, Brit. J. Haematol., 1971, 20, 45; V. Zitko et al., Canad. J. Biochem., 1970, 48, 1318) and the anticoagulant activity of the ethylene glycol analogues has also been studied (Turner et al., Lipids, 1972, 7, 680). Phosphatidylserines are also thought to be essential components of some enzyme systems (R. Whittam et al., J. Physiol., 1972, 220, 353; 1973, 230, 467; B. Roelofsen and Van Deenen, Europ. J. Biochem., 1973, 40, 245). Electron microscopy and other methods have been used to study the physical structure of phosphatidylserines in aqueous systems (S. Eins, Chem. Phys. Lipids, 1972, 8, 26; H. Hauser and M. C. Phillips, J. biol. Chem., 1973, 248, 8585; D. Atkinson et al., Biochim. Biophys. Acta, 1974, 339, 10; H. Diringer, Z. physiol. Chem., 1973, 354, 577). The acidic nature of this lipid leading to its interaction with calcium ions (S. Ohnishi and T. Ito, Biochem. biophys. Res. Comm., 1973, 51, 132)and vasopressin (D. Bach and I. R. Miller, Biochim. Biophys. Acta, 1974, 339, 367) results in membrane changes. The biosynthesis of phosphatidylserines from cytidine diphosphate diglycerides and L-serine bacteria, and by an exchange of free serine with the ethanolamine portion of phosphatidylethanolamine in mammalian tissue, has been described (see J. N. Kanfer and E. P. Kennedy, J. biol. Chem., 1964, 239, 1720; Raetz and Kennedy, ibid., 1974, 249, 5038; T. Taki and M. Matsumoto, Jap. J. exp. Med., 1973, 43, 219; W. D. Marggraf and F. A. Anderer, Z. physiol. Chem., 1974, 355, 1299). A monoacyl derivative ofsn-glycerol 3-(L-serine phosphate) (lysophosphatidylserine) has been prepared by the enzymatic hydrolysis of brain phosphatidylserine (L. Rathbone et al., Biochem. J., 1962, 83, 498). (ii) Phospholipids containin 9 other amino acids Various phospholipids containing amino acids other than serine have been described (see H. K. Mitchell et al., J. biol. Chem., 1957, 229, 131; A. J. de Koning, Biochim. Biophys. Acta, 1964, 84, 467; P. Proulx, Canad. J. Biochem., 1965, 43, 523). A phosphatidylhydroxyproline (Baer and A. Zschocke, J. biol. Chem., 1961, 236, 1273; Turner et al., Lipids, 1968, 3, 228), a phosphatidylthreonine (Baer and F. Eckstein, J. biol. Chem., 1962, 237, 1449; J. W. Moore and M. Szelke, Tetrahedron Letters, 1970, 4423),

1

PHOSPHATIDYLINOSITOLS

371

a phosphatidylserylglycylglycine (Baer et al., J. biol. Chem., 1957, 228, 181) and a phosphatidyltyrosine (Preobrazhenskii et al., Zhur. org. Khim., 1968, 4, 971) have been synthesised. A phosphatidylcarnitine has been isolated from embryonic tissue (M. A. Mehlman and G. Wolf, Arch. Biochem. Biophys., 1963, 102, 346) and its presence in bovine brain is also indicated (Proulx, Nature, 1963, 200, 1210). (d) Acyl derivatives of sn-glycerol 3-(L-myoinositol 1-phosphate) and related compounds sn-Glycerol 3-(L-myoinositoi 1-phosphate), CH20 H

H

'

H

HO-CI-H OIi _ H OH C H20- PI - O ---.,...~ ~ . . ~ OH OH H H

is a degradation product of phosphatidylinositol and is best prepared by hydrolysis with an alkaline solution of hydroxylamine (see D. M. Brown et al., J. chem. Soc., 1961, 3774). The cyclohexylammonium salt has m.p. 124-127~ [ o ~ 3 ~20 8~ - 13.2~ (c, 5 5 in H20). On alkaline hydrolysis it gives a mixture of 1- and 2-glycerophosphoric acids and myoinositol 1- and 2-phosphates. The racemic material has been synthesised (Brown et al., ibid., 1959, 3547) and the related racemic glycerol 1-(myoinositol 2phosphate) has also been prepared (J. H. Davies and T. Malkin, Nature, 1959, 184, 789; R. B. Ellis and J. N. Hawthorne, ibid., p. 790). Recent synthetic work (see below) has provided intermediates for the preparation of optically active material. It has been isolated from the rat prostate gland (M. E. Tare et al., J. biol. Chem., 1968, 243, 2424) and accumulates extracellularly in cultures of Saccharomyces cerevisiae (W. W. Angus and R. L. Lester, Arch. Biochem. Biophys., 1972, 151,483). Methods for the gas chromatography of sn-glycerol 3-(L-myoinositol 1-phosphate) have been described (see T. J. Cicero and W. R. Sherman, Anal. Biochem., 1973, 54, 32). (i) Phosphatidylinositols C H2O2C R R1CO..-C-H ' o I II C H 2 0 - PI - O OH

H

H

H

OH

~ 2

OH H

H

Phosphatidylinositols, the diacyl derivatives of sn-glycerol 3-(Lmyoinositol 1-phosphate) are present in many animal and plant tissues (for reviews see J. N. Hawthorne, J. Lipid Res., 1960, 1, 255; Vitamins

372

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

and Hormones, 1964, 22, 57; Hawthorne and P. Kemp, Adv. Lipid Res., 1964, 2, 127; B. A. Klyashchitskii et al., Russ. Chem. Reviews, 1969, 38, 345; Symposium papers in Ann. New York Acad. Sci., 1969, 165, 509-819). Cleavage with 98~ acetic acid at 100 ~ (J. Lecocq et al., Bull. Soc. chim. Fr., 1964, 2313) or enzymatic cleavage (H. Brockerhoff, Arch. Biochem. Biophys., 1961, 93, 641) gives a 1,2-di-O-acyl-sn-glycerol. Mild hydrolysis with alkaline hydroxylamine solution gives sn-glycerol 3-L-myoinositol 1phosphate) which on oxidation with sodium metaperiodate followed by the action of phenylhydrazine gives L-myoinositol 1-i~hosphate (see Brown and B. F. C. Clark, J. chem. Soc., 1963, 1475; Nature, 1962, 194, 1081; C. E. Ballou and L. 1. Pizer, J. Amer. chem. Soc., 1960, 82, 3333). LMyoinositol 1-phosphate can also be obtained by the alkaline hydrolysis of phosphatidylinositols (Ballou, Biochem. Prep., 1962, 9, 99). The action of sodium metaperiodate on phosphatidylinositols gives phosphatidic acids which on alkaline hydrolysis give sn-glycerol 3-phosphate (Brown et al., J. chem. Soc., 1961, 3774). Racemic and fully optically active phosphatidylinositols containing unsaturated and saturated fatty acids have been synthesised (see R. P. Evstigneeva et al., Tetrahedron, 1973, 29, 331 ; Zhur. obshcheT Khim., 1974, 44, 2595; J. G. Molotovsky and L. D. Bergelson, Chem. Phys. Lipids, 1973, 11, 135; Zhur. org. Khim., 1973, 9, 2421). Phosphatidylinositols have some function in mitochondrial contraction (A. L. Lehninger et al., J. biol. Chem. 1964, 239, 2011). They are synthesised biologically from inositol and cytidine diphosphate diglycerides (see J. A. Benjamins and Agranoff J. Neurochem., 1969, 16, 513; A. J. Holub and A. Kuksis, Lipids, 1972, 7, 78). The biosynthesis of phosphatidylinositols in cells is markedly enhanced by external stimuli, e.9. electrical, hormonal and by the action Of phytohaemagglutinins (for review see E. G. Lapetina and R. H. Michell, FEBS Letters, 1973, 31, 1). The increased production of inositol 1,2-cyclic phosphate which is a product of the catabolism of phosphatidylinositols (N. Clarke and R. M. C. Dawson, Biochem. J., 1972, 130, 229; 1973, 134, 59; N. Freinkel and Dawson, Nature, 1973, 243, 535; Michell and Lapetina, Biochem. J., 1973, 131, 433; M. A. Koch and H. Diringer, Biochem. biophys. Res. Comm., 1974, 58, 361; A. J. Majumder, ibid., 1974, 60, 133) results from the rapid turnover of the phosphatidylinositols under these conditions (Lapetina and Michell, loc. cit.). A protein which transfers phosphatidylinositols between biomembranes has also been described (F. Possmayer, Brain Res., 1974, 74, 167; Van Deenen et al., J. biol. Chem., 1974, 249, 6382). Other inositols (e.9. scyllo-inositol and isomytilitol) are also incorporated into yeast and fungal phospholipids (J. Deshusses, Experientia, 1974, 30, 592). Methods for the isolation of ptiosphatidylinositols from plant sources

1

PHOSPHATIDYLINOSITOLS

373

have been described (see W. E. Trevelyan, J. Lipid Res., 1967, 8, 281; G.. Colacicco and M. M. Rapport, ibid., 1967, 8, 513; H. E. Carter and E. J. Weber, Lipids, 1966, 1, 16). Their solubility properties depend on the nature of the cations associated with them (Carter and Weber, loc. cit.; Rapport et al., Biochemistry, 1969, 7, 1692). The molecular species of natural phosphatidylinositols have been investigated by thin-layer chromatography on silver nitrate impregnated media ("argentation chromatography") (T. Shimoja et al., Biochim. Biophys. Acta, 1970, 210, 343; Tohoku J. exp. Med., 1970, 101,289; Holub and Kuksis, J. Lipid Res., 1972, 12, 510, 699; M. G. Luthra and A. Sheltawny, Biochem. J., 1971, 126, 1231) and a thin-layer chromatography system for separating phosphatodylinositols from phosphatidylserine and other phospholipids has been described (It. D. Kaulen, Anal. Biochem., 1972, 45, 664). Phosphatidylinositols containing [3H]myoinositol have been prepared from the yeast Kloeckera brevis by growing it in the presence of tritiated inositol (Ballou et al., Lipids, 1970, 5, 463). Bilayers ("liposomes") of phosphatidylinositols (prepared by sonicating aqueous emulsions), and the effect of pH changes on these, have been studied by electron.microscopy (M. C. Wills et al., Biochim. Biophys. Acta, 1971, 241,483). A monoacyl glycerol 3-L-myoinositol 1-phosphate) (lysophosphatidylinositol) has been isolated from pigeon pancreas lipids and can also be produced by the action of phospholipase A on phosphatidylinositols (see R. W. Keenan and L. E. Hokin, ibid., 1964, 84, 458). The enzymatic acylation of lysophosphatidylinositol has also been demonstrated (see R. R. Baker and W. Thompson, J. biol. Chem., 1973, 248, 7060). ( ii) Phosphate esters of phosphatidylinositols Polyphosphoinositides were first shown to be present in brain lipids by J. Folch (ibid., 1949, 177, 505) who isolated a lipid fraction containing

CH202CR I R1C02-- C- I"1 O _ H O / 7 ~ ' ~ I II CH20-P-O

O PO3H2

I

OH

CH202CR I R1C02 - C-HI OIi CH20 -P--I OH

OPO3H2 HO~oPO3H2 O~"~.~/I~~OH

374

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

inositol and phosphorus in the ratio of 1:2 Cdiphosphoinositide"). This material, however, is a mixture of mono- and di-phosphate esters of phosphatidylinositols for which the following structures have been proposed after investigations of the inositol phosphates released on hydrolysis (see Brockerhoff and Ballou, ibid., 1961, 236, 1907; L. Hb'rhammer et al., Naturwiss., 1961, 48, 103; J. C. Dittmer, and Dawson, Biochem. J., 1961, 81, 535; Hawthorne et al., ibid., 1963, 88, 125; Brown and J. C. Stewart, Biochim. Biophys. Acta, 1966, 125, 413; see also "Cyclitols and Phosphoinositides", ed. H. Kindl, Pergamon, Oxford, 1966). For methods of preparation and separation oi" these compounds see P. Mandel et al., Bull. Soc. Chim. biol., 1967, 49, 1285; G. Hauser and J. Eichberg, Biochim. Biophys. Acta, 1973, 326, 201 ; Hawthorne et al., J. Chromatog., 1973, 87, 267). These phosphate esters are formed biologically by stepwise phosphorylation of phosphatidylinositols (see M. Colodzin and E. P. Kennedy, J. biol. Chem., 1965, 240, 3771; R. M. Saunders and Ballou, Biochemistry, 1966,5, 352; Hawthorne et al., Biochem. biophys.Res. Comm., 1966, 22, 370; Biochim. Biophys. Acta, 1968, 164, 238) and they have a rapid metabolic turnover but their physiological significance is unknown (see W. Thompson and Dawson, Biochem. J., 1964, 91, 233; Eichberg and Hauser, Biochim. Biophys. Acta, 1967, 144, 415; M. Chang and Ballou, Biochem. biophys. Res. Comm., 1967, 26, 199; Hawthorne et al., J. Neurochem., 1967, 14, 1013; A. Sheltawny and Dawson, Biochem. J., 1969, 111, 147, 157; C. G. Huggins et al., Arch. Biochem. Biophys., 1972, 149, 146). The subcellular distribution of these compounds (Hauser and Eichberg, Biochim. Biophys. Acta, 1973, 326, 210; M. G. Luthra and Sheltawny, Biochem. J., 1972, 128, 587) and of the enzymes which metabolise them in brain (Sheltawny et al., ibid., p. 579) have been investigated. They act as receptor substances for certain arboviruses (IV. Frisch-Niggemeyer, Acta Virol., 1971, 15, 119) and have been implicated in the active transport of cations (see J. T. Buckley and Hawthorne, J. biol. Chem., 1972, 247, 7218). These compounds are also present in the yeast Saccharomyces cerevisiae (R. L. Lester and M. R. Steiner, J. biol. Chem., 1968, 243, 4889; J. Bact., 1972, 109, 81; Biochim. Biophys. Acta, 1972, 260, 82). The molecular species of brain polyphosphoinositides have been studied (W. Thompson et al., J. Lipid Res., 1970, 11,558) and a satisfactory system for thin-layer chromatography has been described (E. Gonzalez-Sastre and J. Folch-Pi, ibid., 1968, 9, 532). Phosphatidylinositol monophosphate and diphosphate have been synthesised (N. A. Preobrazhenskii et al., Zhur. obshcheT Khim., 1969, 39, 2463; Evstigneeva et al., ibid., 1974, 44, 2573; Doklad. Akad. Nauk S.S.S.R., 1970, 195, 848) and the mass spectrometry of the natural polyphosphoinositides investigated (T. J. Cicero and W. R. Sherman, Biochem. biophys. Res. Comm., 1971, 42, 428).

1

PHOSPHATIDYLGLYCEROLS

375

( iii) Mannosides of phosphatidylinositols Phosphatidylinositols joined to either one, two or five molecules of mannose have been isolated from the phospholipids of Mycobacterium tuberculosis (var. boris, strain BCG) (see Ballou, E. Vilkas and E. Lederer, J. biol. Chem., 1963, 238, 69). In the monomannoside, which is also found in Propionibacterium shermanii (P. Brennan and Ballou, Biochem. biophys. Res. Comm., 1968, 30, 69), the mannose is joined to the 2-hydroxyl group of the inositol and in the dimannoside the mannose molecules are joined by a-glycosidic linkages to the 2- and 6-hydroxyl groups of the inositol (see formula p. 371 for numbering of inositol) (Ballou and Y. C. Lee, Biochemistry, 1964, 3,682; J. biol. Chem., 1964, 239, 1316). The pentamannoside resembles the dimannoside but contains an extra three molecules of mannose joined in a chain to the mannose on the 6-position (Ballou and Lee, Biochemistry, 1965, 4, 1395). The monomannoside is the only species present in Corynebacterium aquaticum (G. K. Khuller and D. Brennan, Biochem. J., 1972, 127, 369) and it is probably not synthesised biologically from phosphatidylinositol as is the monomannoside from Mycobacterium smegmatis (K. Takayama and E. L. Armstrong, FEBS Letters, 1971, 18, 67). The dimannoside is synthesised biologically from phosphatidyl inositol by donation ofmannose from guanosine diphosphate mannose (Brennan and Ballou, J. biol. Chem., 1967, 242, 3046; Takayama and D. S. Goldman, Biochim. Biophys. Acta, 1969, 176, 196). Mono- and di-acyl derivatives of phosphatidylinositol dimannoside are also present in Mycobacteria and Corynebacteria (see Brennan and D. P. Lehane, Lipids, 1971,6, 401 ; Khuller and D. Subrahmanyan, Experientia, 1968, 24, 851). The serological activities of the various phosphatidylinositol mannosides and their acyl derivatives have been investigated (M. C. Pangborn and J. A. McKinney, J. Lipid Res., 1966, 7, 627; Pangborn, Ann. New York Acad. Sci., 1968, 154, 133; Khuller and Subrahmanyan, Immunochemistry, 1971, 8, 251; Indian J. Med. Res., 1972, 60, 1794) and their subcellular distribution in Mycobacterium tuberculosis studied (D. S. Goldman, Amer. Review Respiratory Dis., 1970, 102, 543). For the chromatographic separation of these compounds see Subrahmanyan et al., J. Chromatog., 1974, 94, 342. ( e) Acyl derivatives of sn-glycerol 3-(sn-glycerol 1-phosphate) and related compounds (i) Phosphatidylglycerols

CH202CR I

R1CO2-C-H [

O

H

II

I

CH20-P-OC H2-CI-CH2OH I OH

OH

376

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

(I) Phosphatidylglycerols were first discovered as major components of plant chloroplast phospholipids (A. A. Benson and B. Maruo, Biochim. Biophys. Acta, 1958, 27, 189) and have since been isolated from mammalian and bacterial tissues (for reviews see M. G. Macfarlane, Adv. Lipid. Res., 1964, 2, 91; P. Plackett et al., Austral. J. exp. biol. Med., 1969, 47, 171), and are major components together with dipalmitoyl-lecithin of the pulmonary surfactant (R. C. Pfleger et al., Lipids, 1972, 7, 492; Chem. Phys. Lipids, 1972,9,51 ;S.A. Rooney et al., Biochim. Biophys. Acta, 1974,360, 56; M. Hallman and L. Gluck, Biochem. biophys. Res. Comm., 1974, 60, 1). Phosphatidylglycerol is an intermediate in the biosynthesis of lipoteichoic acids (L. Glaser and B. Lindsay, ibid., 1974, 59, 1131; L. I. Emdur and T. H. Chiu, ibid., 1974, 59, 1137). Spinach leaf phosphatidylglycerol has the structure 1,2-di-O-acyl-sn-glycerol 3.-(sn-glycerol 1-phosphate) as shown by investigations on the glycerophosphoric acids obtained after enzymatic hydrolysis on either side of the phosphate ester groups (E. Haverkate and L. L. M. van Deenen, Biochim. Biophys. Acta, 1964, 84, 106; 1965, 106, 78). 1,2-Di-O-acyl-sn-glycerol 3-(sn-glycerol 1-phosphates) have been synthesised (R. M. Saunders and H. P. Schwarz, J. Amer. chem. Soc., 1966, 88, 3844; P. P. M. Bonsen et al., Chem. Phys. Lipids, 1966, 1, 33). The biosynthesis of phosphatidylglycerols proceeds through an intermediate phosphate ester of phosphatidylglycerol (see N. Z. Stanacev et al., Biochim. Biophys. Acta, 1969, 176, 650; M. O. Marshall and M. Kates, ibid., 1972, 260, 558). The biosynthesis in E. coli is inhibited by 3,4-dihydroxybutyl- 1-phosphonate (C. S. Shopsis et al., J. biol. Chem., 1974, 249, 2473). A phosphatidylglycerol phosphate has been synthesised (Bonsen and De Haas, Chem. Phys. Lipids, 1967, 1,100). A compound related to phosphatidylglycerol which contains threobutane2,3-diol in place of glycerol has been isolated from Actinomyces olivaceus (L. D. Bergelson et al., Biochim. Biophys. Acta, 1973, 337, 29; Doklady Akad. Nauk, 1973, 211,722). The effect of calcium ions on aqueous dispersions of phosphatidylglycerol has been inverstigated (Van Deenen et al., Biochim. Biophys. Acta, 1974, 339,432). (2) Amino acid esters of phosphatidylglycerols. Amino acid esters of phosphatidylglycerols occur in the lipids of Gram-positive bacteria (for review see Bonsen et al., Chem. Phys. Lipids, 1967, 1, 83). An ornithinyl ester of phosphatidylglycerol is present in Mycobacterium 607 (Khuller and Subrahmanyan, J. Bacteriol., 1970, 101,654) and amino acid esters of phosphatidylglycerols may be present in Gram-negative bacteria (0. B. Sinha and W. L. Gaby, J. biol. Chem., 1964, 239, 3668). Some amino acid esters of phosphatidylglycerols have been synthesised (Baer and K. V. J. Rao,

1

PHOSPHATIDYLGLYCEROLS

377

Canad. J. Biochem., 1966, 44, 899; Bonsen et al., Biochemistry, 1967, 6, 1114; Bergelson et al., Izvest. Akad. Nauk, 1967, 925, 2321, 2498; Chem. Phys. Lipids, 1968, 2, 1; Preobrazhenskii et al., Zhur. org. Khim., 1968, 4, 1157; Evstigneeva et al., ibid., 1972, 8, 965). The biosynthesis of the lysine ester of phosphatidylglycerol from lysyl-sRNA has been studied and ~he results indicate that the lysine is esterified to the primary hydroxyl group of the phosphatidylglycerol (W. J. Lennarz et al., Biochemistry, 1967, 6, 2307; J. biol. Chem., 1968,243, 3088, 3096). The effect of various antibiotics on the biosynthesis of lysyl phosphatidylglycerol has been studied (B. H. Hebeler et al., Antimicrob. Agents and Chemotherapy, 1973, 4, 346). Esters of both D- and L-alanine with phosphatidylglycerol occur in Mycoplasma laidlawii (W. L. Koostra and P. F. Smith, Biochemistry, 1969, 8, 4794). A glycoside of glucosamine with phosphatidylglycerol is present in Bacillus megaterium and Pseudomonas ovalis. In B. megaterium, two isomers are present with glucosamine linked by/~-glycosidic linkages to the 2- or 3position of the glycerol and both of these isomers have been synthesised (see J. A. F. op den Kamp et al., Bio-organic Chem., 1971, 1, 174). (3) Acyi derivatives of phosphatidylglycerols. Diacyl derivatives of phosphatidylglycerols ("bis-phosphatidic acids") have been synthesised (E. Baer and D. Buchnea, Arch. Biochem. Biophys., 1958, 78, 294) and a partially deacylated bis-phosphatidic acid has been isolated from rabbit lung (D. R. Body and G. M. Gray, Chem. Phys. Lipids, 1967, 1, 424), from the tissues of a patient with.Niemann-Pick's disease (G. Rouser et al., Lipids, 1968, 3, 287; H. Debuch et al., Z. physiol. Chem., 1971, 352, 280; Arch. Neurol., 1972, 27, 45), from Salmonella typhimurium, where the acyl group is on the primary hydroxyl group of phosphatidylglycerol (R. W. Olsen and Ballou, J. biol. Chem., 1971, 246, 3305) from rat and human liver (J. R. Wherrett and S. Huterer, Lipids, 1973, 8, 531) and E. coli (P. Proulx et al., Biochim. Biophys. Acta, 1973, 326, 355). A bis-phosphatidic acid and a partially deacylated derivative have been isolated from cultured baby hamster kidney (BHK) cells. A lyso compound was also prepared by hydrolysis of synthetic bis-phosphatidic acid. Investigation of the products from the alkaline hydrolysis of the partially deacylated bis-phosphatidic acid from BHK cells indicated that the structure was a bis(3-acyl-sn-glycerol) 1-phosphate (O. Renkonen et al., Chem. Phys. Lipids, 1974, 13, 11, 178) and not the expected bis(1-acyl-sn-glycerol) 3-phosphate. A partially deacylated bis-phosphatidic acid also accumulates in the tissues of patients who take the coronary vasodilatator "Coragil" (4,4-diethylaminoethoxyhexestrol) (see K. Kasama et al., Lipids, 1974, 9, 235; T. Taketomi et al., Jap. J. exp. Med., 1974, 44, 133) where it accumulates in secondary lysosomes (Wherret and Huterer, loc. cit.).

378

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

( ii) Diphosphatidylglycerols. Cardiolipins

CH202CR I R1CO2-C-H O I

II

H I

CH202CR 3 I R2CO2--C-H O I II

CH20-P-OCH2-?-CH20-P-O--CH I

OH

OH

I

OH

2

Cardiolipins are present in animal (particularly heart and liver--see H. Yabuuchi and J. S. O'Brien, J. Neurochem., 1968, 15, 1383) and plant tissues (M. J. Coulon-Morelec and R. Douce, Bull. Soc. chim. biol., 1968, 50, 1547; J. Holzl, Biochem. Z., 1965,341, 168) and in bacteria including Mycobacteria (see Subrahmanyan, Canad. J. Biochem., 1964, 42, 1195; Y. Akamatsu and S. Nojima, J. Biochem., Tokyo, 1965, 57, 430). In animal tissues they are concentrated in the inner membranes of mitochondria (D. L. Schneider, Progr. in Surface and Membrane Sci., 1974, 8, 209) closely associated with cytochrome oxidase (see F. L. Crane et al., Biochem. biophys. Res. Comm., 1970, 39, 822; Biochim. Biophys. Acta, 1971, 226, 42). The location of cardiolipins in mitochondrial membranes has been studied by immunological methods (A. L. Lehninger et al., J. biol. Chem., 1971, 246, 7526; K. Aho et al., Clin. exp. Immunol., 1973, 14, 573) but it has been shown that antisera produced against cardiolipins also react with phosphatidylinositol, phosphatidylglycerol and phosphatidylglycerol phosphate (M. Guarnieri, Lipids, 1974, 9, 692). The various methods for the isolation of cardiolipins from beef heart have been reviewed (J. Eichber9 and J. D. Burnham, J. Lipid Res., 1970, 11, 386; S. A. Courtade and J. M. McKibbin, Lipids, 1971, 6, 260). For reviews on cardiolipins see M. G. Macfarlane, Adv. Lipid Res., 1964, 2, 91; Biochem. J., 1964, 92, 12C; Nojima, J. pharm. Soc. Jap., 1964, 84, 1037). Weak acid hydrolysis of cardiolipins liberates diglycerides (M.-J. CoulonMorelec, Compt. rend., 1962, 255, 2687) and glycerol diphosphate. Acid catalysis also leads to isomerisations of the cardiolipin molecule (D. E. Brundish et al., Biochem. J., 1967, 104, 205). The above configuration for cardiolipins has been established by degradative studies of the deacylated material (J. LeCocq and Ballou, Biochemistry, 1964, 3, 976) and confirmed by comparison with synthetic deacylated material (P. Plackett, Australian J. Chem., 1964, 17, 101). The mass spectrum of the trimethylsilyl ether of the deacylated material has been studied (T. J. Cicero and W. R. Sherman, Biochem. biophys. Res. Comm., 1971, 43, 451). A snake venom enzyme removes two fatty acids to give a lysocardiolipin (G. V. Marinetti, Biochim. Biophys. Acta, 1964, 84, 55) and a phospholipase D, isolated from Hemo-

1

CARDIOLIPINS

379

philus parainfluenza, which is specific for cardiolipin, hydrolyses it to phosphatidic acid and phosphatidylglycerol (L. Astrachan, ibid., 1973, 296, 79). The asymmetry of the cardiolipin molecule responsible for this enzymic result as well as for the non-equivalence of the phosphorus atoms, as observed by 31p n.m.r, spectroscopy, has been discussed in detail (G. L. Powell and J. Jacobus, Biochemistry, 1974, 13, 4024). The predominant fatty acids of natural cardiolipins are linoleic and linolenic acids. The fatty acid composition of the cardiolipins from various animal organs has been investigated (H. Wagner et al., Z. Naturforsch., 1966, 21b, 755) and the molecular species present in beef heart cardiolipins have been studied (Crane et al., Biochem. biophys. Res. Comm., 1970, 40, 1102). Unusually long-chain fatty acids (i.e. C2o-C30) are present in the cardiolipins and phosphatidylglycerols of the alcoholophilic (i.e. will tolerate concentrations of ethanol > 20?/0) Lactobacillus heterohiochii (K. Uchida, Biochim. Biophys. Acta, 1974, 369, 146). Cardiolipins are synthesised biologically in Escherichia coli, Micrococcus lysodeikticus and Staphylococcus aureus from two molecules of phosphatidylglycerol according to the following equation:

2 phosphatidylglycerol --* cardiolipin

+

glycerol

(C. B. Hirschber9 and Kennedy, Proc. Nat. Acad. Sci., 1972, 69, 648; S. A. Short and D. C. White, J. Bacteriol., 1972, 109, 820; N. Z. Stanacev et al., Canad. J. Biochem., 1973, 51, 747; E. Tunaitis and J. F. Cronan, Arch. Biochem. Biophys., 1973, 155, 420; cf the acid-catalysed isomerisations of Brundish et al., loc. cit.).

In the mitochondria of rat liver a different mechanism occurs and cardiolipins are synthesised from phosphatidylglycerol and cytidine diphosphate diglyceride (Stanacev et al., Biochem. biophys. Res. Comm., 1972, 47, 1021; Canad. J. Biochem., 1973, 51, 274, 286; K. Y. ttostetler et al., Biochim. Biophys. Acta, 1972, 260, 380, 507; J. B. Davidson and Stanacev, Canad. J. Biochem., 1974, 52, 936). Cardiolipins have been chemically synthesised (De Haas et al., Biochim. Biophys. Acta, 1966, 116, 114; Saunders and Schwarz, loc. cit.) and the products compared with the natural materials. These compounds are used together with cholesterol and lecithins as antigens in the serodiagnosis of syphilis (A. E. Wilkinson, Brit. med. J., 1972, 2, 573; D. J. M. Wright and O. Doniach, Proc. roy. Soc. Med., 1971,64, 419). Some analogues possessing similar serological properties have been synthesised (see K. lnoue and Nojima, Chem. Phys. Lipids, 1969, 3, 70; Nojima et al., Jap. J. exp. Med., 1968, 38, 251 and for a review of the antigenicity of cardiolipins see Rapport and L. Graf, Progr. in Allergy, 1969, 13, 273).

380

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

The physical structures formed by various salts of cardiolipins have been studied (R. P. Rand and S. Sengupta, Biochim. Biophys. Acta, 1972, 255, 484; D. Hegner et al., ibid., 1973, 307, 452). (f) Glycosyl derivatives of diacylglycerols

These lipids are major components of the lipids of plants and microorganisms (for reviews see N. Shaw, Bacteriol. Reviews, 1970, 34, 365; P. S. Sastry, Adv. Lipid Res., 1974, 12, 251). An interrelationship between the phospholipid and glycolipid content of bacteria'l membranes has been suggested (J. Baddiley et al., Biochim. Biophys. Acta, 1971, 249, 651 ; Nature, 1974, 249, 268; J. gen. Microbiol., 1974, 83, 415). The synthetic work in this field by the Russian workers has been reviewed (R. P. Evstigneeva et al., Chem. Phys. Lipids, 1973, 10, 267). ( i) Galactosyl-diglycerides

HO CH2OH

O~ CH2 HO~o

CH2O2CR 1 I R CO2-~-H

HO~/'t.,,.,~...~ OC H2 OH A digalactosyl-diglyceride of the above structure and the corresponding monogalactosyl-diglyceride were first isolated from wheat flour lipids and the structures established by methylation studies (Carter et al., J. Lipid Res., 1961, 2, 223). These or similar lipids are also the major components of the acetone-soluble lipids in grasses and other forage plants (R. O. Weenik and F. B. Shorland, J. Sci. Food and Agric., 1961, 12, 34, 39). Galactosylglycerol lipids have also been found in other green plants and micro-organisms (for reviews see A. Rosenberg, Science, 1967, 157, 1191; Sastry and M. Kates, Biochemistry, 1964, 3, 1271, 1280)and in mammalian brain (M. G. Rumsby, J. Chromatog., 1969, 42, 237; Rumsby and R. J. Rossiter, J. Neurochem., 1968, 15, 1472; R. A. Pieringer et al., J. biol. Chem., 1971, 246, 5688, 5695). Chromatographic methods for the isolation and analysis of these compounds have been described (H. W. Gardner, J. Lipid Res., 1968, 9, 139; P. G. Roughan and R. D. Batt, Anal. Biochem., 1968, 22, 74; V. A. de Stefanis and J. G. Ponte, Biochim. Biophys. Acta, 1969, 176, 198; M. A. B. Maxwell and J. P. Williams, J. Chromatog., 1968, 35, 223).

1

GALACTOSYL-DIGLYCERIDES

381

A trigalactosyl-diglyceride is present in some plants and has been isolated from potato tubers (T. Galli,rd, Biochem. J., 1969, 115, 335) and spinach (D. E. Webster and S. B. Chang, Plant Physiol., 1969, 44, 1523) and from Mycoplasma pneumoniae (P. Plackett et al., Aust. J. exp. Biol. Med., 1969, 47, 171). The serological relationship between the plant glycolipids and Mycoplasma pneumoniae glycolipids has been studied (G. E. Kenny and R. M. Newton, Annals New York Acad. Sci., 1973, 225, 54). A monogalactosyl diglyceride acylated on the 6-position of galactose with a fatty acid has been isolated from wheat flour (D. V. Myhre, Canad. J. Chem., 1968, 46, 3071) and spinach leaves (E. Heinz and A. P. Tulloch, Z. physiol. Chem., 1969, 350, 493) and a monoacyl derivative of a digalactosyl-diglyceride has been isolated from leaf homogenates (Heinz et al., ibid., 1974, 355, 612) these being formed by enzymic transacylation during the isolation procedure. Galactosyl-diglycerides (see P. ,4. Gent and R. H. Gigg, J. chem. Soc., Perkin I, 1975, 364) and galactosyl-glycerols have been synthesised (B. Wickberg, Acta Chem. Scand., 1958, 12, 1187). Partial syntheses of galactosyl-diglycerides by reacylation of the deacylated natural materials have also been described (Heinz, Biochim. Biophys. Acta, 1971, 231, 537; C. Critchley and Heinz, ibid., 1973, 326, 184). A glycerol galactofuranoside has been isolated from the lipids of Bacteroides symbiosus (R. E. Reeves et al., Biochemistry, 1964, 3, 1248) and from strains of Mycoplasma (Plackett, ibid., 1967, 6, 2746). The structures of the six major galactosyl-diglycerides from Bifidobacterium bifidum var. pennsylvanicus have been established. Mono-, di- and tri-galactosyl-diglycerides contained only galactopyranoside residues joined in fl-linkages to the 2-position of adjacent galactose residues. Acylated galactosyl-diglycerides contained galactose residues in the furanose form (J. H. Veerkamp, Biochim. Biophys. Acta, 1972, 273, 359; 1974, 348, 23). A phosphorylated galactofuranosyl-diglyceride of the following structure and the corresponding monoacyl derivative are also present in the same organism (Veerkamp and F. W. van Schaik, ibid., 1974, 348, 370): CH202C R RC02 t H O O~CH 2 OH HO

H I

CH20-P-OCH2-CI-CH2OHI OH

OH

3-O-(fl-D-Galactofuranosyl)-sn-glycerol has been synthesised and found iden-

382

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

tical with the material isolated from B. symbiosus (H. F. G. Bering et al., Acta Chem. Scand., 1967, 21, 2083). Some separation of the molecular species of monogalactosyl-diglycerides from Chlorella vulgaris has been achieved by argentation thin-layer chromatography (B. W. Nichols and R. Moorhouse, Lipids, 1969, 4, 311) and the distribution of fatty acids in these lipids from various sources has been studied (R. Safford and Nichols, Biochim. Biophys. Acta, 1970, 210, 57; R. O. Arunga and W. R. Morrison, Lipids, 1971, 6, 768; G. Auling et al., Z. physiol. Chem., 1971, 352, 905). Mono- and di-galactosyl-diglycerides are synthesised in the brain and in plants from a 1,2-di-O-acyl-glycerol and UDP-galactose (Pieringer et al., J. Neurochemistry, 1974, 22, 479; Renkonen and Bloch, J. biol. Chem., 1969, 244, 4899; Mudd et al., ibid., 1968, 243, 1588; J. Lipid Res., 1969, 10, 623). In plants these lipids are biosynthesised and located in the chloroplast membrane (J. F. G. Wintermans, Plant Physiol., 1974, 53, 496; Biochim. Biophys. Acta, 1974, 352, 245.; R. Douce, Science, 1974, 183, 852). Enzymes responsible for the hydrolysis of these lipids have also been investigated (see P. J. Helmsing, Biochim. Biophys. Acta, 1969, 178, 519). Pancreatic lipase specifically removes the fatty acid from the 1-position of monogalactosyl-diglycerides (M. Noda and N. Fujiwara, ibid., 1967, 137, 199; Heinz, ibid., 1971, 231, 537) and the galactosyl-2-O-acylglycerol thus produced can be reacylated by plants to give galactosyl-diglycerides (Nichols et al., ibid., 1971, 239, 509). A deficiency ofa fl-galactosidase for the hydrolysis of the monogalactosyldiglyceride in Krabbe's disease has been described (D. A. Wenger et al., Biochem. biophys. Res. Comm., 1973, 53, 680). The physical behaviour of these lipids in an aqueous medium has been studied (Nichols et al., Biochim. Biophys. Acta, 1973, 311, 531). Related compounds based on ethylene glycol rather than glycerol have also been isolated (Bergelson et al., Doklad. Akad. Nauk, 1974, 214, 1448). The mass spectrum of acetates of mono- and di-galactosyl-diglycerides have been studied (Heinz et al., Z. Naturforsch., 1973, 28C, 499). (ii) Glucosyl-diglycerides A monoglucosyi-diglyceride with a//-glycosidic linkage is present in Pseudomonas rubescens(S. G. Wilkinson, ibid., 1968, 164, 148) and the corresponding compound with an a-linkage is present in Pneumococcus Types I and XIV, Streptococci, Mycoplasma laidlawii strain B and Pseudomonas diminuta (see N. Shaw and J. Baddiley, Nature, 1968, 217, 142; Shaw et al., Biochem. J., 1968, 107, 329; W. Fischer and W. Seyferth, Z. physiol. Chem., 1968, 349, 1662; Wilkinson, Biochim. Biophys. Acta, 1969, 187, 492). A diglucosyl-

1

GLUCOSYL-DIGLYCERIDES

383

diglyceride with the following structure sn-diglyceride (3 -~- 1)L-glucopyranose(2 ~ 1)D-glucopyranose is also present in Mycoplasma (Shaw et al., loc. cit.) and in Streptococcus hemolyticus (I. lshizuka and T. Yamakawa, J. Biochem., Tokyo, 1968, 64, 13). A heptose (D-glycero-mannoheptose) containing pentaglycosyl-diglyceride, with a structure based on this diglucosyl-diglyceride, is present in Acholeplasma modicum (P. F. Smith et al., J. Bacteriol, 1974, 118, 898). The four isomers of glycerol(1 ~ 1)D-glucopyranose and the four isomers of glycerol(1 ~- 1)D-glucopyranose(x ~- 1)Dglucopyranose (where x = 2, 3, 4 or 6) (Brundish and Baddiley, Carbohydrate Res., 1968, 8, 308) and a diglucosyl-diglyceride (Evstigneeva et al., Zhur. org. Khim., 1972, 8, 2277) have been synthesised. The glucosyl-diglycerides are biosynthesised in Mycoplasma and in Streptococcus faecalis from a 1,2-di-O-acylglycerol and UDP-glucose (Smith, J. Bacteriol., 1969, 99, 480; R. A. Pieringer, J. biol. Chem., 1968, 243, 4894). A phosphorylated derivative of a diglucosyi-diglyceride with the following structure. CH202CR I R1C02--" C - - H

I

CH20 - -

a

D - g l u c o p y r a n o s e (2 ~ 6 O II

1)D-glucopyranose OH I

O-P-O-CH2~ "CH2OH OH H

and its diacyl derivative have been isolated from Streptococci and Mycoplasma laidlawii strain B (see R. T. Ambron and Pieringer, ibid., 1971, 246, 4216; Shaw et al., Biochem. J., 1972, 129, 167; Fischer, Biochim. Biophys. Acta, 1973, 296, 527; Smith, ibid., 1972, 280, 275). For detailed structural work on these compounds see Fischer et al., Biochim. Biophys. Acta, 1973,306, 353. Dephosphorylation of the diacyl derivative with hydrogen fluoride gives a diglyceride and diglucosyl-diglyceride (N. Shaw and A. Stead, Biochem. J., 1974, 143, 461). The compound is biosynthesised from diglucosyl-diglyceride and phosphatidyl-glycerol or cardiolipin (Pieringer, Biochem. biophys. Res. Comm., 1972, 49, 502). A related lipid which is a tetraacyl derivative of the following structure is present in Pseudomonas diminuta (Wilkinson and M. E. Bell, Biochim. Biophys. Acta, 1971, 248, 293):

384

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

CH2OH I

HO-C-H I

Q

CH20

D-glucose

I+ O

CH20 H ,

HO-C-H II I O-P-O--CH 2 I

OH

Before the structure of the phosphorylated derivative of diglucosyl-diglyceride had been established it was termed "phosphatidyi-glucose". For a review see Shaw and Stead, FEBS Letters, 1972, 21,249. Various phosphate diesters of diglycerides and glucose (H. M. Verheis et al., Biochim. Biophys. Acta, 1970, 218, 97; Evstigneeva et al., Zhur. obshcheT Khim., 1972, 42, 1405; 1974, 44, 414) and galactose (N. A. Preobrazhenskii et al., ibid., 1970, 40, 915; 1971, 41,446) have been synthesised. (iii) Other glycosyl diglycerides A lipid of the following structure 1,2-di-O-acyl-sn-glycerol (3 ,a. 1)o-glucopyranose(2
containing glucose and galactose has been isolated from Pneumococci and Streptococci (see Shaw and Baddiley, Nature, 1968, 217, 142; Shaw et al., Biochem. J., 1968, 107, 491) and a lipid with the following structure is present in Lactobacillus casei (Shaw et al., loc. cir.):

1,2-di-O-acyl-sn-glycerol(3 ~ 1)D-glucose (2 ~ 1)D-galactose(6 ~ 1)D-glucose

sn-Glyceroi(3- .~ 1)D-glucose (2 .~ 1)D-galactose has been synthesised (D. E. Brundish et al., J. chem. Soc., C, 1966, 521) and shown to be identical with the material obtained by deacylation of the lipid from the pneumococci. A dimannosyl-diglyceride and a mannosyl-diglyceride are present in Micrococcus lysodeikticus and the sugars are linked in the following way: sn-glycerol(3 ~ l)D-mannopyranose(3 ~ l)D-mannopyranose

The biosynthesis of this lipid has been investigated (W. J. Lennarz and B. Talamo, J. biol. Chem., 1966, 241, 2707). A similar lipid is present in Microbacterium lacticum (Shaw, Biochim. Biophys. Acta, 1968, 152, 427) and in Corynebacterium aquaticum (G. K. Khuller and P. Brennan, Biochem. J., 1972, 127, 369). An unsaturated mannosyl-diglyceride has been synthesised (Evstigneeva et al., Zhur. org. Khim., 1972, 8, 2271). A lipid containing glucuronic acid with the following structure

1

PLANT SULPHOLIPID

385

1,2-di-O-acyl-sn-glycerol(3 g-- 1)D-glucuronicacid is present in Pseudomonas rubescens (Wilkinson, Biochim. Biophys. Acta, 1968, 164, 148) and the s-isomer of this compound and another glucuronic acid containing lipid of the following structure:

1,2-di-O-acyl-sn-glycerol(3 ~ 1)D-glucuronic acid(4 & 1)D-glucose are present in Pseudomonas diminuta (Wilkinson, ibid., 1969, 187, 492). The biosynthesis of glucuronosyl-diglycerides present in a halophilic bacterium (N. Stern and A. Tietz, FEBS Letters, 1971, 19, 217) and in Pseudomonas diminuta (J. M. Shaw and Pieringer, Biochem.. biophys. Res. Comm., 1972, 46, 1201) have been studied. The mass spectrum of a glucuronosyl-diglyceride present in Actinomycetes has been studied (L. D. Bergelson et al., Khim. prirod. Soed., 1973, 704). A galacturonic acid containing glycolipid with the following structure: diglyceride ~L galacturonic acid (4 ~L 1)glucose I acyl group (on 2 or 3 position)

is present in Streptomyces LA 7017 (Bergelson et al., Chem. Phys. Lipids, 1970, 4, 181; Khim. prirod. Soed., 1970, 664). A glycoside of N-acetylglucosamine with a diglyceride has been identified in Streptococcus hemolyticus (I. lshizuka and Yamakawa, Jap. J. exp. Med., 1969,39, 321)and a glucosaminyl diglyceride has been isolated from Bacillus megaterium (P. J. R. Phizackerley et al., Biochem. J., 1972, 126, 499). A glycosyl-diglyceride with the following structure (or an isomer): diglyceride-glucose(2 ~- 1)N- 15-methylhexadecylglucosamine (6 ~ 1)galactos.e(2 +- 1)galactofuranose

is the major lipid of the extreme thermophile (i.e. will grow in hot springs at ,-~ 80 ~ C) Flavobacterium thermophilium (M. Oshima and Yamakawa, Biochemistry, 1974, 13, 1140).

(iv) Plant sulpholipid

CH2SO3H

R'COC--H O.

CH 2

A lipid with the above structure, containing a sulphonic acid derivative of 6-deoxy-D-glucose C6-sulphoquinovose") is a major constituent of the

386

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

lipids of photosynthetic tissue of plants (A. A. Benson, Adv. Lipid Res., 1963, 1, 387; J. S. O'Brien and Benson, J. Lipid. Res., 1964, 5, 432; W. E. Klopfenstein and J. Shigley, ibid., 1967, 8, 350; Roughan and R. D. Batt, Anal. Biochem., 1968, 22, 74; A. Radunz, Z. physiol. Chem., 1969, 350, 411; T. H. Haines, Progr. Chem. Fats and Lipids, 1971, 11,297). A deacylated compound with this structure has been synthesised (M. Miyano and Benson, J. Amer. chem. Soc., 1962, 84, 59; see also J. Lehmann and Benson, ibid., 1964, 86, 4469). The metabolism of the plant sulpholipid in higher plants (R. F. Lee and Benson, Biochim. Biophys. Acta, 1972, 261, 35) and in Chlorella pyrenoidosa (M. G. Wolfersberger and Pieringer, J. Lipid Res., 1974, 15, 1) has been studied. The fatty acid distribution in plant sulpholipids has been investigated (A. P. Tulloch et al., Z. physiol. Chem., 1973, 354, 879).

(g) Phospholipids derived from glycerol ethers (i) Plasmalogens, phosphatidal derivatives H H CH2OC=C-R

R2

I

R1CO2-CI - H

O II

2

CH20-P-OCH2R I OH

=

--CH2NH 2 (~ -- C H 2 N M e 3 -- C H ( N H 2 ) ' C O 2 H

The plasmalogens are naturally occurring 2-O-acyl-l-O-alk-l'-enyl-sn-glycerol 3-(2-aminoethyl,-choline or-serine phosphates) and are referred to as phosphatidal compounds (e. g. phosphatidal ethanolamines) to distinguish them from the phosphatidyl compounds. (For reviews see O. W. Thiele, Z. klin. Chem., 1964, 2, 33; E. Klenk and H. Debuch, Progr. Chem. Fats and Lipids, 1963, 6, 1; F. Snyder, ibid., 1968, 10, 287; ed. Snyder, "Ether Lipids: Chemistry and Biology", Academic Press, New York, 1972; Snyder and C. Piantadosi, J. pharm. Sci., 1970, 59, 283; M. M. Rapport and W. T. Norton, Ann. Rev. Biochem., 1962, 31, 103; Y. Nakazawa and K. Waku, J. Jap. biochem. Soc., 1972, 44, 287). Their presence in tissues was first indicated by histochemical studies (for reviews of the histochemistry of plasmalogens see J. Y. Terrier and E. R. Hayes, Stain Technol., 1961, 36, 265; S. H. Broderson and Hayes, Histochemie, 1968, 16, 97) due to the colour reactions of the long-chain aldehydes released on mild acid hydrolysis. Compounds containing bound long-chain aldehydes were later isolated from muscle phospholipids (R. Feulgen and Th. Bersin, Z. physiol. Chem., 1939, 260, 217) and brain phospholipids (S. J. Thannhauser et al., J. biol. Chem., 1951, 188, 417) by mild alkaline hydrolysis of the total

1

PLASMALOGENS,

PHOSPHATIDALDERIVATIVES

387

phospholipid mixture in order to decompose the phosphatidyl compounds. It was later realised that the compounds thus obtained were not identical with the natural materials (see G. B. Ansell and J. M. Norman, Biochem. J., 1953,55, 768)since the 2-acyl group has been removed during the alkaline treatment (see Rapport and R. E. Franzl, J. biol. Chem., 1957, 225, 851). The presence of the bound aldehyde in the form of an 0q/3-unsaturated ether was confirmed by hydrogenation of the double bond and isolation of O-alkylglycerols after hydrolysis (see G. V. Marinetti et al., J. Amer. chem. Soc., 1959, 81,861; Debuch, Z. physiol. Chem., 1959, 317, 182) and by a specific iodination of the enol-ether double bond (see Rapport and N. F. Alonzo, J. biol. Chem., 1960,235, 1953). The presence of the unsaturated ether linkage in the 1-position was confirmed by the isolation of 1-O-alkylglycerols after hydrogenation and hydrolysis, and was indicated also by the removal of the acyl group with phospholipase A which removes only 2-acyl groups from other phospholipids (see L. L. M. van Deenen et al., Biochem. biophys. Res. Comm., 1960, 3, 287). The plasmalogens (like other vinyl ethers) are rapidly hydrolysed by mercuric chloride (see Norton, Nature, 1959, 184, 1144)and this method ofhydrolysis has been used for the analytical determination of plasmalogens (see R. W. Keenan et al., Anal. Biochem., 1968, 23, 555). The reaction of methylmercuric chloride with plasmalogens has been suggested as a mechanism for the neurotoxicity of mercury compounds (H. J. Segall and J. M. Wood, Nature, 1974, 248, 456). Other methods for the determination of the plasmalogens depend on the estimation of the 4-nitrophenylhydrazones formed from the aldehydes liberated on acid hydrolysis (see W. J. Ferrell et .al., Lipids, 1969, 4, 278) and on an iodination reaction specific for vinyl ether linkages (C. V. Viswanathan et al., Fettte u. Seifen, 1969, 71, 618; 1968, 70, 858) as well as by gas chromatography of liberated aldehydes or their derivatives. Since the diacyl phospholipids (phosphatidyl compounds), plasmalogens (phosphatidal compounds) and saturated ether lipids (see below) occur together in tissues, their separation and analysis present considerable difficulties. Plasmalogens free from phosphatidyl compounds but still contaminated with saturated ether lipids have been prepared from natural phospholipid mixtures by preferential enzymic (E. L. Gottfried and M. M. Rapport, J. biol. Chem., 1962, 237, 329; H. Debuch et al., Z. physiol. Chem., 1973, 354, 1265) or alkaline hydrolysis (O. Renkonen, Acta Chem. Scand., 1963, 17, 634; Ansell and S. Spanner, J. Neurochem., 1963, 10, 941; M. F. Frosolono and M. Marsh, Chem. Phys. Lipids, 1973, 10, 203) to remove the phosphatidyl compounds. Chromatographic techniques have been used to investigate the mixtures

388

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

after suitable modifications ofthe original lipids (for review see Viswanathan, Chromatog. Rev., 1968, 10, 18; see also Renkonen, J. Lipid Res., 1968, 9, 34; L. A. Horrocks, ibid., p. 469; P. Wood et al., ibid., 1969, 10, 128; Yiswanathan et al., J. Chromatog., 1968, 38, 267; 1968, 35, 113; R. M. Broekhuyse, Biochim. Biophys. Acta, 1968, 152, 307; Wood and Snyder, Lipids, 1968, 3, 129; K. L. Su and H. H. O. Schmidt, ibid., 1974, 9, 208). The double bond of the ~,fl-unsaturated ether has the cis-configuration (Norton et al., J. Lipid Res., 1962, 3, 456; H. R. Warner and W. E. M. Lands, J. Amer. chem. Soc., 1963, 85, 60). Hydrogenolysis of the plasmalogens with lithium tetrahydridoaluminate gives 1-O-alk-f'-enylglycerols (G. A. Thompson and P. Lee, Biochim. Biophys. Acta, 1965, 98, 151; W. J. Baumann et al., Z. physiol. Chem., 1968, 349, 1677). Optical rotatory dispersion studies have confirmed the absolute configuration of these as 1-O-alk-l'-enyl-snglycerols (J. C. Craig et al., Tetrahedron, 1966, 22, 175). Mild alkaline hydrolysis of the plasmalogens gives lysoplasmalogens (Thannhauser et al., J. biol. Chem., 1951, 188, 417; Rapport et al., J. org. Chem., 1967, 32, 3998) which rearrange readily to cyclic acetals under acidic conditions (see Baumann et al., J. Lipid Res., 1972, 13, 640): CH2OCH =CHR I HO-C-H O I

II

CH20--P-OCH2R 1 I O.H

9

~~--

RCH2"CH

/ O - ~ H2 \O--C-H

O I II C H20 ~P--OCH2R 1 I OH

Cyclic acetals of this type have been synthesised (M. J. Egerton and T. Malkin, J. chem. Soc., 1953, 2800; G. K. Chacko and E. G. Perkins, J. org. Chem., 1967, 32, 1623; Baumann, ibid., 1971, 36, 2743). Several syntheses of 1-O-alk-l'-enylglycerols have been reported and these can be converted into plasmalogens (for reviews see R. H. Gigg, in "Ether Lipids: Chemistry and Biology", ed. Snyder, Academic Press, New York, 1972, p. 87;Slotboom and Bonsen, Chem. Phys. Lipids, 1970, 5, 301; Snyder and Piantadosi, J. pharm. Sci., 1970, 59, 283; Preobrazhenskii, "Recent Developments in the Chemistry of Natural Compounds", 1971, 4, 10, Akademiai Kiado, Budapest; and also see R. P. Evstigneeva et al., Zhur. org. Khim., 1970, 6, 2412; 1971, 7, 657, 660, 957; 1972, 8, 721, 1166, 1171, 2516; 1973, 9, 2487; 1974, 10, 185, 876; Tetrahedron Letters, 1971, 4605; Khim. prirod. Soed., 1971, 825). Alk-l-enyl ethers of ethylene glycol have also been synthesised (see J. K. G. Kramer and H. K. Mangold, Chem. Phys. Lipids, 1969, 3, 176; 1970, 4, 332; L. D. Bergelson et al., Izvest. Akad. Nauk, S.S.S.R., 1971, 1102) and obtained by degradation of natural 1-O-alk-l'-enyl-sn-glycerols (Baumann et al., Z. physiol. Chem., 1968, 349, 1677). The mass spectra

1

PLASMALOGENS,

PHOSPHATIDAL

DERIVATIVES

389

of esters of these glycol ethers have been studied (Kramer et al., Lipids, 1971, 6, 492). Lipids containing the alk-l-enyl ethers of ethylene glycol occur naturally in high concentrations in the lipids of the starfish, Distolasterias nipon(Bergelson et al., Chem. Phys. Lipids, 1971, 7, 75; Khim. prirod. Soed., 1970, 657) and are also present in rat liver (Bergelson et al., Doklad. Akad. Nauk, S.S.S.R., 1972, 205, 477). Both phosphatidal cholines and phosphatidal ethanolamines occur in mammalian heart (Spanner, Nature, 1966, 210, 637) and in spermatazoa (see V. G. Pursel and E. F. Graham, J. Rep. Fert., 1967, 14, 203; A. Darin-Bennett et al., ibid., 1974, 41, 471; P. J. Quinn and I. G. White, Aust. J. biol. Sci., 1967, 20, 1205) and phosphatidal ethanolamine is the major ethanolamine-containing phospholipid of the white matter of brain (J. S. O'Brien et al., J. Lipid Res., 1964, 5, 329; Horrocks and G. B. Ansell, Biochim. Biophys. Acta, 1967, 137, 90; Horrocks and G. Y. Sun, in "Research Methods in Neurochemistry", Vol. 1, eds. N. Marks and R. Rodnight, Plenum, New York, 1972, p. 223; for a method of isolation from bovine white matter see Frosolono and Marsh, loc. cit.). Phosphatidal serine has also been isolated from brain (Klenk and P. Bb'hm, Z. physiol. Chem., 1951, 288, 98), and, together with phosphatidal ethanolamine, is a major component of the phospholipids of Megasphera elsdenii (L. M. G. van Golde et al., Biochim. Biophys. Acta, 1973,326, 314; 1974, 348, 361). Plasmalogens are also present in oysters (J. Sampugna et al., Lipids, 1972, 7, 339), in the slime mould Physarum polycephalum (P. Comes and H. Kleinig, Biochim. Biophys. Acta, 1973, 316, 13), in adipose tissue (Snyder et al., Lipids, 1972, 7, 766) and probably in plants (see H. K. Mangold, in "Ether Lipids: Chemistry and Biology", p. 399). Only the anaerobic bacteria contain appreciable quantities ofplasmalogens (see H. Goldfine and P-0. Hagen, in "Ether Lipids: Chemistry and Biology", p. 329; Hagen, J. B Bacteriol, 1974, 119, 643; G. K. Khuller and Goldfine, J. Lipid Res., 1974, 15, 500). The enzymatic synthesis of phosphatidal choline from a 1-O-alk-l'-enyl2-O-acyl-sn-glycerol has been demonstrated (J. I(. Kiyasu and Kennedy, J. biol. Chem., 1960, 235, 2590; J. N. Cumings et al., Biochim. Biophys. Acta, 1968, 152, 629). A considerable amount of evidence now indicates that phosphatidal ethanolamine is synthesised biologically by dehydrogenation of the corresponding saturated ether lipid (see below) (see F. Snyder et al., J. biol. Chem., 1972, 247, 5442; Arch. Biochem. Biophys., 1974, 161, 402; F. Paltauf et al., J. biol. Chem., 1973, 248, 1609; 1974, 249, 2661) and the stereochemistry of this dehydrogenation reaction has been investigated (W. Stoffe! and D. Lekim, Z. Physiol. Chem., 1971,352, 501). A dehydrogenation reaction of the lyso derivative of the saturated ether lipid is also indicated (H. Fiirniss and Debuch, ibid., 1972, 353, 1377). This dehydrogena-

390

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

tion reaction is thought not to occur for the synthesis of phosphatidal choline in the brain (H. H. O. Schmidt et al., Biochim. Biophys. Acta, 1972, 270, 317; Paltauf, ibid., 1972, 260, 352). The acylation of lysophosphatidal ethanolamine in brain has been studied (V. Natarajan and Sastry, FEBS Letters, 1973, 32, 9; Snyder et al., Biochim. Biophys. Acta, 1973, 326, 26). High concentrations of highly unsaturated fatty acids (e.g. docosahexenoic acid) occur in phosphatidal ethanolamines of nervous tissue (see G. K. Chacko et al., ibid., 1972, 280, 1) and of kidney S. K. F. Yeung and Kuksis, Canad. J. Biochem., 1974, 52, 830). A base-exchange reaction for the biosynthesis of plasmalogens has also been considered (G. Porcellati et al., FEBS Letters, 1972, 27, 116). The enzymatic hydrolysis of the enol-ether bond has also been investigated (see M. Matsumoto et al., Jap. J. exp. Med., 1967, 37, 355; E. Yavin and S. Gatt, Europ. J. Biochem., 1972, 25, 431,437; Stoffel and G. Heimann, Z. physiol. Chem., 1974, 355, 651). Plasmalogens are cleaved more slowly than the diacylphospholipids (phosphatidyl compounds) by phospholipase A (H. Woelk and H. Debuch, Z. physiol. Chem., 1971, 352, 1275; Woelk, Biochem. biophys. Res. Comm., 1974, 59, 1278; Woelk and K. Peiler-Ichikawa, FEBS Letters, 1974, 45, 75; Z. Neurologie, 1974, 207, 319) and similar reduced rates are observed in the hydrolyses with phospholipases C and D (K. Waku and Y. Nakazawa, J. Biochem., Tokyo, 1972, 72, 149). The vinyl ether grouping also occurs in the neutral lipids of the triglyceride type (for reviews see R. H. Gigg and J. Gigg, J. chem. Soc., C, 1968, 2030; Snyder, Progr. Chem. Fats and Lipids, 1969, 10, 287) and in a sphingolipid (N. K. Kotchetkov et al., Biochim. Biophys. Acta, 1963, 70, 716; Biokhimiya, 1964, 29, 570). Alk-l-enyl ethers of cholesterol have also been synthesised (Evstigneeva et al., Zhur. org. Khim., 1974, 10, 975). (ii) Saturated ether lipids The presence, in natural products, of phospholipids corresponding to the plasmalogens but containing a saturated alkyl ether chain in the 1position was first shown by the isolation of the deacylated ethanolaminecontaining compound from egg-yolk lipids (H. E. Carter et al., J. biol. Chem., 1958, 232, 681) although neutral lipids (analogous to triglycerides), containing ether linkages, had been observed previously. These phospholipids have since been isolated from several other sources (see Snyder, Progr. Chem. Fats and Lipids, 1969, 10, 287; Schmidt et al., Biochim. Biophys. Acta, 1969, 187, 208; Wood et al., ibid., 1969, 176, 641; Wood and Snyder, Arch. Biochem. Biophys., 1969, 131,478; C.-R. Liang and K. P. Strickland, Canad. J. Biochem., 1969, 47, 85 and for a historical review see Debuch and P. Seng, in "Ether Lipids: Chemistry and Biology", p. 1). Compounds

1

SATURATED

ETHER

LIPIDS

391

of this type corresponding to lecithin and cephalin have been synthesised CH20(CHa)nC H3 I

R CO2-CI - H O rN II H2 C H 2 0 - P - O C H i C H 2I OH I, NMe3

(T. H. Bevan and T. Malkin, J. chem. Soc., 1960, 350; O. Westphal et al., Ann., 1967, 709, 226, 234; Chacko and D. J. Hanahan, Biochim. Biophys. Acta, 1968, 164, 252; for reviews see Baumann, in "Ether Lipids: Chemistry and Biology", p. 51;Paltauf, Chem. Phys. Lipids, 1973, 11, 270) and the corresponding derivatives containing phosphonate esters CI H20 (C H2)nCH3 RCO2-C-H

O

C H20 - P--C H2-CH2 I OH

LNMe3

have also been synthesised (Chacko and Hanahan, Biochim. Biophys. Acta, 1969 176, 190; E. Baer and H. Basu, Canad. J. Biochem., 1972, 50, 988). Extensive investigations on the biosynthesis of the glycerol ether lipids of this type have shown that the ether linkage is formed by the condensation of long-chain alcohols with dihydroxyacetone phosphate and the mechanism of this reaction has been investigated (see S. J. Friedber9 et al., Biochemistry, 1972, 11, 297; 1973, 12, 1100; Snyder et al., J. biol. Chem., 1972, 247, 2944; Paltauf, Biochim. Biophys. Acta, 1972, 260, 345; A. Radominska-Pyrek and Horrocks, J. Lipid Res., 1972, 13, 580; Snyder et al., Biochem. biophys. Res. Comm., 1973, 53, 350). 1-O-Alkyldihydroxyacetone phosphate derivatives (Snyder et al., Biochemistry, 1971, 10, 1417; C. Piantadosi et al., J. pharm. Sci., 1973, 62, 320) and 1-O-alkyl-(and acyl-)dihydroxyacetone 3-(2aminoethyl phosphate) derivatives have been synthesised (Piantadosi et al., J. pharm. Sci., 1972, 61, 607, 971). Enzymic cleavage of 1-O-alkylglycerols has also been studied (Snyder et al., J. biol. Chem., 1972, 247, 3923; Biochim. Biophys. Acta, 1973,316,259). For a review of the biosynthesis and biodegradation of ether bonds see Snyder, Adv. Lipid Res., 1972, 10, 233. Glycerol thio-ethers have also been isolated from human heart (W. J. Ferrell, Lipids, 1973, 8, 234). Lipids containing saturated alkyl chains on the 1- and 2-positions of glycerol (i.e. containing no acyl groups) have also been isolated from natural lipid extracts (G. V. Marinetti et al., J. Amer. chem. Soc., 1959, 81, 861; M. Popovic, Z. physiol. Chem., 1965, 340, 18; Horrocks and Ansell, loc. cit.) and the diether analogues of phosphatidic acids (M. Faure et al., Bull. Soc. chim. biol., 1968, 50, 1561) and of lecithin and cephalin have been

392

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

synthesised (N. Z. Stanacev et al., J. biol. Chem., 1964, 239, 410; M. Faure et al., Compt. rend., 1963, 257, 2187; 1965, 260, 3700; Bull. Soc. chim. biol., 1968, 50, 1561; Chacko and Hanahan, Biochim. Biophys. Acta, 1968, 164, 252; P. J. Thomas and J. H. Law, J. Lipid Res., 1966, 7, 453; J.-S. Chen and P. G. Barton, Canad. J. Biochem., 1970, 48, 585). The corresponding phosphonates and phosphinates (shown below) have also been synthesised and the inhibition of phospholipid degrading enzymes by some of these has been studied (see Baer et al., J. biol. Chem., 1965, 240, 44; Canad. J. Biochem., 1967, 45, 317; 1970, 48, 184; A. F. Rosenthal et al., Biochim. Biophys. Acta, 1968, 164, 226; 1970; 218, 213; 1971, 239, 248; 1972, 260, 369; Lipids, 1969, 4, 37; J. Lipid Res., 1971, 12, 277; Lipids, 1974, 9, 77)" CH2OR I RO-C -H O ~'NH2 I II CH20-P-CH2.CH 2Me 3 I OH Me 2

CH2OR I RO-C-H O I II

CH2OR I RO-C-H O

CH2OR I RO-C-H

CH2~P-OCH2.CH I

OH

2-

t;H2

tNMe3

O II (~ C H2,C H2-- IP- C H2.C H2N Me I

C H2-- P - C HiC H2NMe 3

O| C H20 R

C H20 R

I

RO-C-H l

I

0

RO-C-H

if

I

~

CH2.CH2- IP-CH2"CH2-CH2NMe 3 Oe

0

~l

e

CH 2- P--CH2.CH2.CH2NMe 3 ~e

The best characterised naturally occurring dialkyl ether lipids are the diether analogues of phosphatidylglycerol and phosphatidylglycerol phosphate which t'.ave been isolated from Halobacterium cutirubrum (M. Kates et al., Biochemistry, 1966, 5, 4092; 1967, 6, 3329; Kates and A. J. Hancock, Biochim. Biophys. Acta, 1971, 248, 254; for review see Kates, in "Ether Lipids: Chemistry and Biology", p. 351). The phosphatidylglycerol phosphate analogue has the following structure: O OH CH3 I

II

I

I OH

I H

CH20-P--OCH2--C-CHaOR I

( C H3 )2C H-C H2,C H2( C H2,C H ,C H2. C H 2 )3- O - C - H

CH 3 I

R= - P ( O ) (OH) 2

C H20 (C H2,C H2-C H.CH 2 )3C H2,C H2.C H (C H3)?.'

1

SATURATED ETHER LIPIDS

393

showing a derivative of sn-glycerol 1-phosphate which has the opposite configuration to that present in other known naturally occurring phospholipids. The corresponding sulphate (R = SO3H) has also been identified in this bacterium and synthesised (Hancock and Kates, J. Lipid Res., 1973, 14, 422, 430). Osmometric, microscopic, e.s.r, and dilatometric studies of liposomes prepared from these lipids have been made (Kates et al., Biochim. Biophys. Acta, 1974, 352, 202; Biochemistry, 1974, 13, 4906, 4914). Similar lipids are also present in another extremely halophilic bacterium Sarcina morrhuae (G. C. Woodrow et al., Aust. J. biol. Sci., 1973, 26, 787) and in thermophilic, acidophilic bacteria (M. de Rosa et al., Chem. Comm., 1974, 543; Smith et al., J. Bacteriol., 1974, 119, 106; Biochim. Biophys. Acta, 1974, 360, 217). The phytanyl ether groups have the 3R,7R,11R configuration and are biosynthesised from mevalonic acid (Kates et al., Canad. J. Biochem., 1968, 46, 971; 1970, 48, 63, 69; Biochim. Biophys. Acta, 1970, 202, 206). Both the phosphatidylglycerol analogue and the phosphate have been synthesised (C. N. Joo and Kates, ibid., 1969, 176, 278; see also Kates et al., Chem. Phys. Lipids, 1972, 8, 32) and the corresponding analogues of phosphatidic acid and cytidine diphosphate diglyceride containing phytanyl ether groups have also been prepared (Kates et al., Canad. J. Biochem., 1971, 49, 275). A sulphate ester of a glycolipid has also been isolated from Halobacterium cutirubrum (Kates et al., Biochim. Biophys. Acta, 1967, 137, 213). It has the following structure: galactose 3-sulphate(1 ~ 6)mannoset(1 -~ 2)glucose(12~ 1)2',3'-di-O-phytanyl-sn-glycerol (Kates and P. W. Deroo, J. Lipid Res., 1973, 14, 438).

A further ether containing sulphoglycolipid ("seminolipid"), 2-O-acyl-l-Oalkylglycerol 3-(fl-galactose 3'-sulphate) is the major glycolipid of animal testis and spermatozoa (T. Yamakawa et al., J. Biochem., Tokyo, 1973, 73, 77; 1974, 75, 1241; 1974, 76, 221; Jap. J. exp. Med., 1973, 43, 435; M. J. Kornblatt et al., Canad. J. Biochem., 1974, 52, 689). The biosynthesis of this lipid has been studied Handa et al., J. Biochem., Tokyo, 1974, 75, 77; Murray et al., Biochem. biophys. Res. Comm., 1973, 55, 179). It is a substrate for the enzyme arylsulphatase A (A. L. Fluharty et al., ibid., 1974, 61, 348).

394

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

2. Phospholipids and glycolipids based on sphingosine and related bases. Sphingolipids (a) Sphingosine bases and ceramides (i) Sphingosine bases The chemistry of these bases has been reviewed (see K. A. Karlsson, Lipids, 1970, 5, 878; Chem. Phys. Lipids, 1970, 5, 6; M. Prostenik, ibid., 1970, 5, 1; W. Stoffel, ibid., 1973, 11,318). C H2OH

C H2O H

H-C-NH 2

H-C-NH 2

H-C -NH 2

H-C-NH 2

H-C-OH

H-C-OH

H-C--OH

H-C-OH

I

I

I

I

I

I

CH II HC

I

I

(CH2) n I

CH 3 I

C H2OH I

I

H-C--OH

C H2OH I

I

H-C-OH

I

I

(CH2) n

(CH2) 3

I

(CH2)n

I

CH 3

I

CH3 Sphingosines

Dihydrosphingosines

Phytosphingosines

4-Sphingenines

Sphinganines

4 D-Hydroxysph inganines

CH II HC I (CH2/8,

I

CH 3 Dehydrophytosphingosine

(1) Sphingosine, 4-sphingenine, D(+)-erythro-2-amino-l,3-dihydroxytrans-4-octadecene. The name sphingosine refers specifically to the Cx 8 compound (n= 12 in the formula). It serves also as the generic name for the homologues which occur together in natural products. The I.U.P.A.C.I.U.B. Commission on Biochemical Nomenclature recommends the name 4-sphingenine for this compound. Sphingosine is present as a component of many natural lipids and these are particularly abundant in nervous tissue. It was first isolated from a hydrolysate of brain lipids (J. L. W. Thudichum, J. prakt. Chem., 1882, 25, 19) and the correct structure was finally established by H. E. Carter and coworkers (for review see C. A. Grob, Record. chem. Progr., 1957, 18, 55). Two syntheses of racemic sphingosine and its stereoisomers have been reported (D. Shapiro et al., J. Amer. chem. Soc., 1958, 80, 1194; D. Shapiro, "Chemistry of the Sphingolipids', Hermann, Paris, 1969; R. P. Evstigneeva et al., Zhur. org. Khim., 1972, 8, 1799; Grob and F. Gadient, Helv., 1957, 40, 1145). The method of Grob which is outlined below has been extended to the synthesis of a range of homologues (E. F. Jenny and J. Druey, ibid., 1959, 42, 401; N. A. Preobrazhenskii et al., Zhur. org. Khim., 1970, 6, 58):

2

SPHINGOSINE BASES

CH3(C H2)12C=C'C H O +

base

~

395

C H3(C H2)12C = C - C HOH I CHNO 2 I

HOCH2.CH 2NO 2

CH20 H H I

CH3(CH 2)12 C = C - C H O I CHNHAc I

CH2OH

/ ,.,, Na/Et~JH ~

CH3(CH2) 1 2 . C = C - C H O H I I H CHNHAc I

CH2OH

Cis- and trans-isomers of the optically active base have been synthesised from D-glucose (E. J. Reist and P. H. Christie, J. org. Chem., 1970, 35, 4127) and the optically active base has also been synthesised from L-serine (H. Newman, J. Amer. chem. Soc., 1973, 95, 4098). Radioactively labelled sphingosines have also been synthesised (A. E. Gal, J. Lab. Cmpds., 1967, 3, 112; W. Stoffel and G. Sticht, Z. physiol. Chem., 1967, 348, 1561). The related compound, 2-acetamido-1-hydroxy-4-octadecen-3-one has been synthesised (R. C. Gaver and C. C. Sweeley, J. Amer. chem. Soc., 1966, 88, 3643; Evstigneeva et al., Zhur. org. Khim., 1974, 10, 32) and its function as an intermediate in the biosynthesis of sphingosine studied (see below). A method for the conversion of the threo-isomers of sphingosine into the erythro-isomer has been investigated (A. Ya. Veinber9 et al., ibid., 1966, 2, 337) and the X-ray analysis of triacetylsphingosine has been described (A. M. O'Connell and I. Pascher, Acta Cryst. B, 1969, 25, 2553). Sphingosine is best obtained from natural sources by the basic hydrolysis of ceramides, isolated directly from beef lung lipids (C. L. Tipton, Biochem. Prep., 1962, 9, 127) or prepared from cerebrosides by a mild degradative method (Carter et al., J. Lipid. Res., 1961, 2, 228) or prepared from sphingomyelin by the action of phospholipase C (see p. 427). The sphingosine prepared in this way contains homologues and dihydrosphingosine as shown by g.l.c, of derivatives (Sweeley and E. A. Moscatelli, ibid., 1959, 1, 40; Sweeley, Biochim. Biophys. Acta, 1959, 36, 268). The hydrolysis of sphingolipids in acidic methanol produces some methyl ethers of sphingosine and products due to allylic rearrangements (see B. Weiss, Biochemistry, 1964, 3, 1288; T. Taketomi and N. Kawamura, J. Biochem., Tokyo, 1972, 72, 189). Several methods are available for the chromatography of the sphingosine bases on paper or thin-layers (see C. Michalec, J. Chromatog., 1968, 34, 375; 1969, 41, 267) on columns of silica gel (Y. Barenholz and S. Gatt, Biochim. Biophys. Acta, 1968, 152, 790) and by gas chromatography and identification by mass spectrometry (see Sweeley et al., Biochemistry,

396

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

1969, 8, 1811; Karlsson et al., Acta Chem. Scand., 1969, 23, 3597; S. Ando and T. Yamakawa, J. Biochem., Tokyo, 1971, 70, 335; K. Krisnangkura and Sweeley, Chem. Phys. Lipids, 1974, 13, 415). Methods are also available for the estimation of sphingosine and related bases in lipid samples by colorimetric methods (see C. J. Lauter and E. G. Trams, J. Lipid Res., 1962, 3, 136; L. Coles and G. M. Gray, ibid., 1970, 11, 164; K. Saito, J. Biochem., Tokyo, 1960, 47, 573; A. N. Siakotos et al., Lipids, 1971, 6, 254; A. Kisic and M. M. Rapport, J. Lipid Res., 1974, 15, 179; S. Roseman et al., Anal. Biochem., 1974, 58, 571). ~3C N.m.r. spectra of sphingosine and related compounds have also been recorded (see Stbffel et al., Z. physiol. Chem., 1972, 353, 1962; Proc. Nat. Acad. Sci., 1974, 71, 3696). A sphingosine homologue with a chain length of 20 carbon atoms ("icosisphingosine", "gangliosphingosine") is a major component of the sphingosine bases of the gangliosides (p. 422) (see K. Sambasivarao and R. H. McCluer, J. Lipid Res., 1964,5, 103; N. Z.Stanacev and E. Chargaff Biochim. Biophys. Acta, 1965, 98, 168; H. P. Schwarz et al., J. Neurochem. 1967, 14, 91) and the various isomers of this compound have been synthesised (B. Majhofer-Orescanin and M. Prostenik, Croat. Chem. Acta, 1962, 34, 161). Sphingosines with 14, 16 and 17 carbon atoms in the chain have also been detected in animal tissues (see M. Popovic, Biochim. Biophys. Acta, 1966, 125, 178; Sweeley et al., ibid., 1969, 187, 157; 1970,202, 195; Moscatelli and K. M. Gilliland, Lipids, 1969, 4, 244; D. G. Cornwell et al., J. Lipid Res., 1969, 10, 445) and 15-methyl-C~6- and 17-methyl-Cxa-sphingosines are present in a protozoan, in bovine kidney and in Bacteroides melanino9enicus (see Carter and Hirschberg, Biochemistry, 1968, 7, 2296; Sweeley et al., Biochim. Biophys. Acta, 1969, 187,527). An unsaturated derivative of sphingosine D-erythro-2-amino-l,3-dihydroxyoctadeca-trans-4-cis-14diene is a major component of the sphingosine bases of the serum sphingomyelins from various animals (Karlsson, Acta Chem. Scand., 1967, 21, 2577; Sweeley et al., Biochemistry, 1968, 7, 2609; O. Renkonen and E. L. Hirvisalo, J. Lipid Res., 1969, 10, 687) and in the cerebrosides of human serum (Renkonen, Biochim. Biophys. Acta, 1970, 210, 190). A related lipid with unsaturation at the 4- and 8-positions is present in oyster glycolipids (A. Hayashi and T. Matsubara, ibid., 1971, 248, 306; R. K. Hammond and Sweeley, J. biol. Chem., 1973, 248, 632). Sphingosine as well as cis- and trans-isomers of a Ca unsaturated sphingosine have been isolated from wheat flour lipids (R. Laine and Renkonen, Biochemistry, 1973, 12, 1106). A C2o-sphingosine with unsaturation at the 4- and l 1-positions is present in scorpion phospholipids (Sweeley et al., Biochim. Biophys. Acta, 1970, 202, 195). For the pharmacological properties of sphingosine see E. Hecht and Shapiro, Science, 1957, 125, 1041.

2

SPHINGOSINE

BASES

397

Triacetylsphingosine, m.p. 103-105~ [t~3D32 -9.4 ~ (c 1.3 in CHCI3). Tribenzoylsphingosine, m.p. 121.5-123.5~ [~]22 _ 11.2~ (2) Dihydrosphingosine, sphinganine, occurs together with sphingosine as a constituent of the sphingolipids in many animal tissues (see E. Okuhara and M. Yasuda, J. Neurochem., 1960, 6, 112). It is also present in many plant lipids (Sweeley and Moscatelli, J. Lipid Res., 1959, 1, 40; Carter et al., J. biol. Chem., 1961, 236, 1912) and the triacetate is produced in the culture liquors of the yeast Hansenula ciferri (for a review see F. H. Stodola et al., Bacteriol. Reviews, 1967, 31, 194). Many syntheses of the racemic compound have been reported (see Baer, Progr. Chem. Fats and Lipids, 1963, 6, 31; Shapiro and T. Sheradsky, J. org. Chem., 1963, 28, 2157; K. Sisido et al., ibid., 1964, 29, 2783; Preobrazhenskii et al., Zhur. org. Khim., 1966, 2, 2184; 1967, 3, 1340; 1968, 4, 532; 1970, 6, 665, 1413; Izvest Akad. Nauk S.S.S.R., 1972, 1445; Evstigneeva et al., Zhur. org. Khim., 1973, 9, 1137; 1974, 10, 878) and the optically active base has been prepared from D-glucose (Reist and Christie, J. org. Chem., 1970, 35, 3521). A stereospecific synthesis of D-threo-sphinganine has also been reported (Newman, ibid., 1974, 39, 100; Chem. Phys. Lipids, 1974, 12, 48). The corresponding 1,2,3trihydroxyoctadecane (B. Palameta and Prostenik, Tetrahedron, 1963, 19, 1463) and a C2o-dihydrosphingosine (Majhofer-Orescanin and Prostenik, ibid., 1961, 12, 56; Preobrazhenskii et al., Zhur. org. Khim., 1968, 4, 207; Stoffel and A. Scheid, Z. physiol. Chem., 1969, 350, 1593) have also been synthesised. The C2o-dihydrosphingosine is also present in human and bovine brain (Karlsson, Acta Chem. Scand., 1964, 18, 565; K. Sambasivarao and R. H. McCluer, J. Lipid Res., 1964, 5, 103). Labelled dihydrosphingosine has also been synthesised (Stoffel and Sticht, loc. cit.). The deamination of dihydrosphingosine with nitrous acid gives predominantly 1-hydroxyoctadecan-3-one (B. Weiss and R. L. Stiller, J. org. Chem., 1966, 31, 2023). Derivatives of 2-amino-l-hydroxyoctadecan-3one and its homologues have been synthesised (see idem, ibid., 1970, 35, 3543; Stoffel et at., Chem. Phys. Lipids, 1972, 8, 10; Newman, ibid., 1974, 12, 48; Evstigneeva e.t al., Zhur. org. Khim., 1973, 9, 483; 1974, 10, 32, 879; Y. Kishimoto and M. T. Mitry, Arch. Biochem. Biophys., 1974, 161, 426) and their function as intermediates in the biosynthesis and degradatiofi of sphingosine and dihydrosphingosine studied (see below).

Triacetyldihydrosphingosine, m.p. 100-101~ [oc]2~ + 18~ (CHCI3). Tribenzoyldihydrosphingosine, m.p. 146-148~ [e]g2 _ 30.2~ (c, 2.34 in CHCI3).

(3) Phytosphingosine (Cerebrin base), 4D-hydroxysphinganine, D-( -t- )ribo-2-amino-l,3,4-trihydroxyoctadecane, was first isolated from the mush-

398

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

room Amanita muscaria (J. Zellner, Monatsh., 1911, 32, 133) and was subsequently isolated from yeasts, moulds and plant seeds (see Carter et al., J. biol. Chem., 1954, 206, 613; Prostenik and L. Gospoeic, Bull. Sci. Acad. R.S.F. Yugoslav., 1965, 10, 317). Recently it has been found in animal tissues and is particularly abundant in kidney lipids (see K. A. Karlsson et al., Biochim. Biophys. Acta, 1968, 152, 230, 798; Acta Chem. Scand., 1968, 22, 1361; Carter and Hirschberg, loc. cit.; K. Puro and A. Keranen, Biochim. Biophys. Acta, 1969, 187, 393; G. Schmidt et al., ibid., 1968, 31, 137; W. R. Morrison, FEBS Letters, 1971, 19, 63). Its origin in mammalian tissue sphingolipids is considered to be from dietary plant sources (G. Assmann and Stoffel, Z. physiol. Chem., 1972, 353, 971; see however Morrison, Biochim. Biophys. Acta, 1973, 316, 98; Karlsson et al., ibid., 1973, 316, 317, 336). The arrangement of the hydroxyl groups and the amino group was established independently by Carter et al., loc. cit. and by T. Oda (J. pharm. Soc. Japan, 1952, 72, 142) and the configuration of the optical centres was established by degradative studies (Carter and H. S. Hendrickson, Biochemistry, 1963, 2, 389). Phytosphingosines from different sources can have chain-lengths of 18 or 20 carbon atoms (see Stanacev and M. Kates, Canad. J. Biochem. Physiol., 1963, 41, 1330) and a 19-methyl derivative of Cz0-phytosphingosine has been isolated from Crithidiafasciculata (Carter et al., Biochem. biophys. Res. Comm., 1966, 22, 316). A 16-methyl derivative of C18-phytosphingosine and C19-phytosphingosine are present in the sulphatides of the rectal salt gland of the spiny dogfish (Karlsson et al., Biochim. Biophys. Acta, 1973, 306, 307). The tetra-acetate of C~8-phytosphingosine is produced extracellularly by the yeast Hansenula eiferri and the enzymic changes responsible for this accumulation have been studied (see Stodola et al., loc. cit.; S. Gatt et al., Biochim. Biophys. Acta, 1971, 248, 458; 1973, 306, 341). This is a good source of phytosphingosine (see Weiss and Stiller, Lipids, 1970, 5, 782). Free phytosphingosine is present in the yeast Candida intermedia (IFO 0761) (A. Kimura et al., Ag. and biol. Chem., 1974, 38, 1263). Racemic Cta-phytosphingosines have been synthesised (Stanacev and Prostenik, Croat. Chem. Acta, 1957, 29, 107; Sisido et al., J. org. Chem., 1969, 34, 3539; 1970, 35, 350) and phytosphingosines with the correct optical configuration have been synthesised from sphingosine (Prostenik et al., Tetrahedron, 1965, 21,651; Ber., 1966, 99, 3480; Weiss, Biochemistry, 1965, 4, 686; J. org. Chem., 1965, 30, 2483). from D-glucosamine and D-galactose (Gi99 et al., J. chem. Soc., C, 1966, 1872, 1876, 1879) and by resolution of racemic synthetic material (Prostenik et al., Chem. Phys. Lipids, 1971, 7, 135). An unsaturated derivative of phytosphingosine ("dehydrophytosphingosine"). D-( +)-ribo-2-amino-l,3,4-trihydroxy-trans-8-oetadeeene is present in

2

SPHINGOSINEBASES

399

many plant seeds (see Carter and Hendrickson, loc. cit.; Prostenik and Majhofer-Orescanin, Naturwiss., 1961, 48, 500) and the cis-isomer of this compound is the major phytosphingosine base of hay and oilseed concentrate (Morrison et al., Chem. Phys. Lipids, 1973, 11, 99). The acetylenic analogue of this compound has been synthesised (Gi99 et al., J. chem. Soc., C, 1966, 1882). In plants the phytosphingosines are components of cerebrosides and phytoglycolipids which on acid hydrolysis give phytosphingosine and an anhydro derivative containing a tetrahydrofuran ring formed by dehydration between the 1- and 4-hydroxyl groups (see Y. Kishimoto et al., Biochemistry, 1974, 13, 3992). N-Benzoyl-C18-phytosphingosine, m.p. 135-136 ~ [ ~ ] D "3t- 5~ "Acetone derivative", m.p. 114-116 ~ N-Benzoyl-tri-O-acetyl-phytosphingosine, m.p. 79-80 ~ (4) The biosynthesis of the sphingosine bases. Palmitoyl-CoA is the precursor of the long chain and serine provides carbon atoms 1 and 2 (D. Sprinson and A. Coulon, J. biol. Chem., 1954, 207, 585). In cell-free preparations of the yeast Hansenula ciferri and in animal tissues these two compounds are condensed in the presence of pyridoxal phosphate to give 2-amino-l-hydroxy-3-oxo-octadecane. In the presence of TPNH this is reduced to dihydrosphingosine (E. E. Snell et al., ibid., 1969, 244, 491; N. S. Radin et al., ibid., 1970, 245, 335, 342; W. Stoffel et al., Z. physiol. Chem., 1968, 349, 1637). Sphingosine is then formed from dihydrosphingosine by a stereospecific desaturation at C~4) and C~5)(Stoffel et al., ibid., 1971, 352, 1531; 1973, 354, 169; 1974, 355, 91). Other workers consider that the 2-amino-l-hydroxyoctadecan-3-one is desaturated to give the corresponding 4-ene which is then converted into sphingosine (or that the 3-oxo-4-ene derivative may arise from trans-2-hexadecenoyl-CoA) (see M. Nakamo and Y. Fujino, Biochim. Biophys. Acta, 1973, 296, 457; Agr. and biol. Chem., 1973, 37, 2429; R. K. Hammond and Sweeley, J. biol. Chem., 1973, 248, 632). The direct conversion of N-acylsphinganine to N-acylsphingenine has also been demonstrated (D. E. Ong and R. N. Brady, ibid., 1973, 248, 3884) and the biosynthesis of sphingosine in Bacteroides melaninogenicus (M. Lev and A. F. Milford, Arch. Biochem. Biophys., 1973, 157, 500) and in blood platelets (Stoffel et al., Z. physiol. Chem., 1973, 354, 1311) has also been studied. For the biosynthesis of phytosphingosine, dihydrosphingosine is hydroxylated stereospecifically directly to phytosphingosine (Stoffel and E. Binczek, Z. physiol. Chem., 1971, 352, 1065; A. J. Polito and Sweeley, J. biol. Chem., 1971, 246, 4178). The biodegradation of dihydrosphingosine proceeds through the 1-phosphate and this is oxidised to the 3-oxo derivative which is cleaved to give hexadecanal (subsequently oxidised to palmitic acid) and 2-aminoethyl phosphate. The stereospecificity of solvent proton addition to carbon-2 ofsphinganine 1-phosphate in this degradation has been studied (G. J. Schroepfer et al., J. Amer. chem. Soc., 1974, 96, 939). Phytosphingosine similarly gives 2-hydroxypalmitic acid and 2-aminoethyl phosphate and sphingosine is converted via the 1-phosphate to hexadecenal (Stoffel et al., Z. physiol. Chem., 1969, 350, 63, 1233; 1970, 351, 1041; 1972, 353, 965; 1973, 354, 562; R. W.

400

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Keenan and B. Haegelin, Biochem. biophys. Res. Comm., 1969, 37, 888). The 2aminoethyl phosphate produced is used in the biosynthesis of phosphatidylethanolamine (A. Nilsson et al., Biochim. Biophys. Acta, 1973, 306, 460; Stoffel, Molecular and Cell Biochem., 1973,1,147). For reviews on the biosynthesis and biodegradation of the sphingosine bases see Stoffel (Chem. Phys. Lipids, 1970, 5, 139), Snell et al., (ibid., p. 116), S. Gatt (ibid., p. 235) and J. Kanfer (ibid., p. 159). The 1-phosphate esters ofthe sphingosine bases have been prepared enzymically (Stoffel et al., Z. physiol. Chem., 1970, 351, 635; G. J. Schroepfer, J. biol. Chem., 1970, 245, 3084; Keenan and L. Holloman, Biochim. Biophys. Acta, 1972, 270, 383). Stoffel and K. Bister (Z. physiol. Chem., 1973, 354, 169) have studied the metabolism of all four isomers of radioactively labelled 2-amino-l,3-dihydroxyoctadecane, in vivo, in the rat and have shown that all are readily converted into ceramides: C HeOH

C H20 H

H-C-NH 2

H-C-NH 2

I

I

H-C-OH I

R D-(+) e r y t h r o -

I

I

HO-C-H I

R L-(-) threo-

C HzO H I

H2N-C --H I

H-C-OH I

R D-(+) t h r e o -

C H20 H I

H2N-C-H I

HO--C--H I

R L-(-)erythro-

R =-(CH2)14CH 3

The desaturation ofdihydrosphingosine to sphingosine occurred with dihydrosphingosine and not with the 3-oxo derivative and only the D-(+)-erythro- and L-(--)-threoisomers were desaturated. Only these two isomers were precursors of sphingomyelin and cerebrosides and these products contained mainly the unsaturated bases. All four isomers were converted into the 1-phosphate esters but only the D-(+)-erythro-1phosphate was converted into hexadecanal and 2-aminoethyl phosphate. Dihydrosphingosine 1-phosphonate has been prepared. It is cleaved to hexadecanal and aminoethyl phosphonate by the sphingosine 1-phosphate lyase. The phosphonate acts as a competitive inhibitor of this enzyme (Stoffel and M. Grol, Chem. Phys. Lipids, 1974, 13, 372). (5) Sphingine, D-2-amino-l-hydroxyoctadecane, CHa(CH2)lsCH(NH2)CH2OH, m.p. 84-89 ~ [ct] 27 - 5 . 5 ~ The diacetyl derivative of sphingine was first characterised as a product of the hydrogenation of triacetylsphingosine (H. E. Carter and C. G. Humiston, J. biol. Chem., 1951, 191, 727) being formed by the hydrogenolysis of the allylic acetoxy group. Racemic sphingine, and [1-14C]sphingine (D. E. Sunko and M. Prostenik, J. org. Chem., 1953, 18, 1523; Prostenik et al., Croat. Chem. Acta, 1959, 31, 41) and the N-stearoyl derivative (R. P. Evstigneeva et al., Zhur. org. Khim., 1973, 9, 2010) have been synthesised. A C20-sphingine has also been synthesised (M. Munk-Weinert and M. Prostenik, ibid., 1960, 32, 197). Diacetylsphingine, m.p. 108-109~ [~]o27 + 22.5~(CHCIa). N-Acetylsphingine, m.p. 101103~ [~]O 27 -}- 12.0~

2

CERAMIDES

401

( ii) Ceramides Ceramides are long-chain N-acyl derivatives of the sphingosine bases. The ceramides of sphingosine are present in some tissues (see K. Samuelsson, Biochim. Biophys. Acta, 1969, 176, 211; E. Klenk and R. T. C. Huang, Z. physiol. Chem., 1969, 350, 373; W. Krivit and S. Hammarstrb'm, J. Lipid Res., 1972, 13, 525; J. Reisch et al., Naturwiss., 1972, 59, 364) and those from beef lung have been used for the preparation of sphingosine (C. L. Tipton, Biochem. Prep., 1962, 9, 127). They can also be prepared by the degradation of the cerebrosides (Carter et al., J. Lipid Res., 1961, 2, 228) and gangliosides (Klenk and Huang, Z. physiol. Chem., 1969, 350, 1081), by synthesis from sphingosine (Weiss and P. Raizmann, J. Amer. chem. Soc., 1958, 80, 4657; D. E. Ong and Brady, J. Lipid Res., 1972, 13, 819; see also Hammarstrb'm, ibid., 1971, 12, 760; L. Schmid et al., Monatsh., 1967, 98,373,802; 1969,100, 1849) and by the enzymic hydrolysis of sphingomyelin with phospholipase C (see p. 427). The ceramides of phytosphingosine Ccerebrins") are present in yeasts, moulds and fungi (see H. Wagner and W. Zofcsik, Biochem. Z., 1966, 344, 314). The composition of the cerebrins from from Saccharomyces cerevisiae has been investigated in detail (Prostenik et al., Chem. Phys. Lipids, 1973, 11, 83). Many methods for the analysis of ceramides have been described (Z. Kolman and C. Michalec, Z. physiol. Chem., 1967, 348, 723; Huang, ibid., 1971, 352, 1306; R. M. Bradley, Biochim. Biophys. Acta, 1965, 106, 417; V. Groom and M. Sribney, J. Lipid Res., 1965, 6, 220; E. C. Horning et al., Chem. Phys. Lipids, 1969, 3, 1) and in particular mass spectrometry has been extensively used (Hammarstrb'm, Europ. J. Biochem., 1970, 15, 581; J. Lipid Res., 1970, 11, 175; B. Samuelson et al., ibid., 1970, 11, 150; Chem. Phys. Lipids, 1970, 5, 44). The t.l.c. (see Karlsson and I. Pascher, J. Lipid Res., 1971, 12, 466) and infrared spectra (Evstigneeva et al., Zhur. org. Khim., 1971, 7, 2132) of ceramides and derivatives have also been studied. The crystal structure of N-tetracosanoylphytosphingosine has been studied by X-ray methods (B. Dahlen and Pascher, Acta Cryst., 1972, B28, 2396). The properties of monolayers of various ceramides have been studied (Stoffel et al., Chem. Phys. Lipids, 1974, 13, 466). The biosynthesis of ceramides from sphingosine and long-chain fatty acids has been studied (see Y. Fujino and S. Ito, Biochim. Biophys. Acta, 1968, 152, 627; E. Yavin and S. Gatt, Biochemistry, 1969, 8, 1692; M. D. Ullman and Radin, Arch. Biochem. Biophys., 1972, 152, 767; Barenholz and Gatt, Biochem. biophys. Res. Comm., 1969, 35, 676; Y. Fujino, Agr. and biol. Chem., 1972, 36, 1983). Enzymes which degrade ceramides to fatty acids and sphingosine bases have also been studied (Nilsson, Biochim. Biophys. Acta, 1969, 176, 339; Yavin and Gatt, loc. cit.). An enzyme convert-

402

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

ing ceramide to ceramide 1-phosphate has also been studied (E. G. Schneider and E. P. Kennedy, J. biol. Chem., 1973, 248, 3739). Ceramides accumulate in tissues in Farber's disease, a lipogranulomatosis (see Samuelson et a/., Adv. Exp. Med. Biol., 1972, 19, 533) due to the deficiency of a ceramide-cleaving enzyme (H. W. Moser et al., Science, 1972, 178, 110). Liquid chromatography of benzoylated ceramides has confirmed the excess present in Farber's disease (R. H. McCluer et al., J. Lipid Res., 1974, 15, 223). The fatty acid composition of the free ceramides from the kidney and cerebellum of a patient with this disease have been analysed (M. Sugita et al., Lipids, 1973, 8, 401). (b) Glycosides of ceramides

The glycosides ofceramides (for reviews see C. I. Samokhvalov et al., Uspekhi Khim., 1966, 35, 2072; C. Michalec, Chem. Listy, 1968, 62, 427; E. Martensson, Progr. Chem. Fats and Lipids, 1969, 10, 365; W. Gielen, Chimia, 1971, 25, 81;H. Wiegandt, Adv. Lipid Res., 1971, 9, 249; M. G. Uzbekov, Vop. Med. Khim., 1971, 17, 339; J. Kiss, Adv. Carbohydrate Chem., 1969, 24, 381; eds. B. W. Volk and S. M. Aronson, "Sphingolipids, Sphingolipidoses and Allied Disorders", Adv. exp. med. Biol., 1972, Vol. 19), which are described in the following sections, are present in many tissues and general methods for their extraction and separation have been described (see G. M. Gray, Chem. Phys. Lipids, 1967, 1,368; Biochim. Biophys. Acta, 1967, 144, 511; 1966, 116, 532; Martensson, ibid., 1966, 116, 296, 521; Sweeley, J. Lipid Res., 1967, 8, 621; K. Suzuki and G. C. Chen, ibid., 1967, 8, 105; G. Tsch6pe, Z. physiol. Chem., 1973, 354, 1291). The quantitative isolation of glycosphingolipids after acylation and separation from phospholipids by chromatography on Florisil followed by deacylation (T. Saito and S. Hakomori, ibid., 1971, 12, 257) and the high pressure liquid chromatography of benzoylated derivatives of glycosphingolipids on the nanomole scale (McCluer et al., Biochim. Biophys. Acta, 1972, 270, 565; J. Lipid Res., 1973, 14, 611) have been described. The oligosaccharide portion can be released from the lipid portion by ozonolysis of the ethylenic bond of the sphingosine residue and the oligosaccharide analysed by gas chromatography (see M. Ohashi and T. Yamakawa, ibid., 1973, 14, 698). The products obtained on oxidative ozonolysis have also been coupled to glass beads or agarose for immunological studies (Hakomori et al., J. biol. Chem., 1974, 249, 4460). Many of the glycosides of ceramides are antigenic and the immunological properties of these have been investigated (for reviews see Brady, J. Amer. Oil Chemists' Soc., 1966, 43, 67; Michalec, Chem. Listy, 1967, 61, 1342;

2

MONOGLYCOSYL-CERAMIDES

403

M. M. Rapport and L. Graf, Progr. in Allergy, 1969, 13, 273). Some of them accumulate in tissues in certain hereditary metabolic disorders ("sophingolipidoses") due to the reduced activity of degradative enzymes (for reviews see Brady, Ann. Rev. Med., 1970, 21, 317; Chem. Phys. Lipids, 1970, 5, 261; Angew. Chem., intern. Edn., 1973, 12, 1; Progr. in Medical Genetics, 1972, 8,225; Federation Proc., 1973, 32, 1660; Scientific American, 1973, 229, 88; Adv. Enzymology, 1973, 38, 293; eds. H. G. Hers and F. van Hoof, "Lysosomes and Storage Diseases", Academic Press, New York, 1973; H. R. Sloan, Chem. Phys. Lipids, 1970, 5, 250; J. S. O'Brien, New Engl. J. Med., 1971, 284, 893; Federation Proc., 1971, 30, 956; eds. S. M. Aronson and B. W. Volk, "Inborn Errors of Sphingolipid Metabolism", Pergamon, Oxford, 1967; eds. Bernsohn and 6rossman, "Lipid Storage Diseases, Enzymatic Defects and Clinical Implications", Academic Press, New York, 1971). Prenatal diagnosis of some of these disorders is now possible (A. Milunsky and J. W. Littlefield, Ann. Reviews Med., 1972, 23, 57) and enzyme replacement therapy in these disorders is being studied (see eds. J. M. Tager, G. J. M. Hooghwinkel and W. Th. Daems, "Storage Diseases. Enzymic Therapy in Lysosomal Storage Diseases", North Holland, Amsterdam, 1974; Brady et al., Life Sciences, 1974, lfi, 1235; Chem. Phys. Lipids, 1974, 13, 271). For a review of similar disorder in animals see R. D. Jolly and W. F. Blakemore, Veterinary Record, 1973, 92, 391. Many of the enzymes responsible for the metabolism of the ceramide glycosides have been investigated (for reviews see Michalec, Chem. Listy, 1967, 61, 1612; P. Morell and P. Braun, J. Lipid Res., 1972,13,293; B. Czartovyska, Postepy Biochemii, 1971, 17, 3; L. Svennerholm et al., J. biol. Chem., 1974, 249, 4132, 4157). The mass spectrometry (G. Dawson and Sweeley, J. Lipid Res., 1971, 12, 56; Karlsson, Biomedical Mass Spectrometry, 1974, 1, 49; R. W. Ledeen et al., Chem. Phys. Lipids, 1974, 13, 429) and chemical ionisation mass spectrometry (S. P. Markey and D. A. Wenger, Chem. Phys. Lipids, 1974, 12, 182) of ceramide glycosides and other physical studies (e.9. n.m.r., X-ray analysis etc.) on ceramie glycosides in model membrane systems have been studied (E. Oldfield and D. Chapman, FEBS Letters, 1972, 21, 303; K. A. Karlsson et al., Chem. Phys. Lipids, 1972, Svennerholm 152). Tritium-labelled glycosylceramides have been prepared by adding 3H to the unsaturated centre of the sphingosine base (J. L. DiCesare and Rapport, ibid., 1974, 13, 447). C H 2 0 - f l - hexapyranoside /

(i) Monoglycosyl-ceramides and derivatives H + NHCOR (1) Cerebrosides. H- t- O H (CH2) 12 I

CH 3

404

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

The cerebrosides are monoglycosyl-ceramides and are components of many tissues particularly of the nervous system (for reviews of brain cerebrosides see E. Klenk, Progr. Chem. Fats and Lipids, 1969, 10, 411 ; M. Sh. Promyslov and G. M. Popova, Vop. Med. Khim., 1972, 18, 227). They were discovered by J. L. W. Thudichum ("The Chemical Constitution of the Brain", Balli~re, Tindall and Cox, London, 1884 m facsimile edition with introduction by D. L. Drabkin, Archon Books, Hamden, Conn., 1962). Both sphingosine and dihydrosphingosine occur in these lipids and a 3~ure dihydrosphingosine-containing cerebroside has been isolated from human brain (E. Okuhara and M. Yasuda, J. Neurochem., 1960, 6, 112). The major sugar component of normal human brain cerebroside is galactose and the ratio of galactose to glucose in brain cerebrosides during the maturation of the brain has been studied (Y. Kishimoto et al., J. Neurochem., 1973, 21, 709). "Galactocerebrosides, are also present in yeasts (H. Wagner and E. Fiegert, Z. Naturforsch., 1969, 24b, 359). The major cerebroside of normal human kidney, spleen, liver and serum contains glucose ("glucocerebrosides") (E. and L. Svennerholm, Nature, 1963, 198, 688; J. Polonovski and M. Petit, Bull. Soc. chim. biol., 1963, 45, 111; A. Makita, J. Biochem., Tokyo, 1964, 55, 269; A. Wagner, Clin. Chim. Acta, 1964, 10, 175) and glucocerebrosides are also present in brain (K. Nishimura and T. Yamakawa, Lipids, 1968, 3, 262). The separation of gluco- and galacto-cerebrosides can be achieved by chromatography on borate-impregnated media (E. L. Kean, J. Lipid Res., 1966, 7, 449; O. M. Youn9 and J. N. Kanfer, J. Chromatog., 1965, 19, 611; C. Michalec, J. Neurochem., 1966, 16, 1552). In Gaucher's disease there is an accumulation ofglucocerebrosides in the spleen (see H. Rosenber9 and E. Chargaff, J. biol. Chem., 1958, 233, 1323; H. E. Carter et al., J. Lipid Res., 1961, 2, 228; N. G. Kennaway and L. I. Woolf, ibid., 1968, 9, 755). Cerebrosides containing phytosphingosines and glucose occur in human kidney (Karlsson and E. Martensson, Biochim. Biophys. Acta, 1968, 152, 230), and are the major species in bovine kidney cerebrosides (Karlsson et al., ibid., 1973, 306, 317) in wheat flour lipids (Carter et al., J. biol. Chem., 1961, 236, 1912)and in plant leaves (P. S. Sastry and M. Kates, Biochim. Biophys. Acta, 1964, 84, 231; S. Ito and Y. Fujino, Canad. J. Biochem., 1973, 51, 957). Glucocerebrosides are also present in sponges and starfish (see L. R. Bjb'rkman et al., Comp. Biochem. Physiol., 1972, 43B, 409; F. J. Schmitz and F. J. McDonald, J. Lipid Res., 1974, 15, 158) and in yeasts (S. Roseman et al., J. biol. Chem., 1972, 246, 4266; M. Prostenik and C. Cosovic, Chem. Phys. Lipids, 1974, 13, 117)and have also been obtained by degradation of the gangliosides (see pp. 419 et seq). A method for the preparation of cerebrosides from bovine spinal cord lipids has been described in detail (N. S. Radin and J. R. Brown, Biochem. Prep., 1960,

2

MONOGLYCOSYL-CERAMIDES

405

7, 31) and other methods for the isolation of cerebrosides from brain lipids have been described (see E. Mehl and H. Jatzkevitz, Naturwiss., 1963, 50, 227; K. Bernhard et al., Helv., 1962, 45, 1298). Natural cerebrosides contain a variety of fatty acyl groups (see J. S. O'Brien and G. Rouser, J. Lipid Res., 1964, 5, 339; J. H. Menkes et al., ibid., 1966, 7, 479; J. F. Mead et al., Lipids, 1966, 1, 449; Svennerholm, Brain Res., 1973, 55, 413). The distribution of molecular species of monoglycosyl-ceramides in the bovine digestive tract has been investigated in detail (Karlsson et al., Biochim. Biophys. Acta, 1974, 348, 232). Various methods for the hydrolysis of the cerebrosides prior to g.l.c. of the fatty acids have been investigated (see Moscatelli, Lipids, 1972, 7, 268). ct-Hydroxy-fatty acids (see P. Capella et al., ibid., 1968, 3, 431) and very long-chain fatty acids (i.e. C2o-C24 ) are characteristic components of the cerebrosides. Some galactocerebrosides containing an individual fatty acid have been described and these have been given special names, e.g. cerebron (phrenosine) contains cerebronic acid (2-hydroxylignoceric acid); kerasin contains lignoceric acid (tetracosanoic acid); nervone contains nervonic acid (tetracos-cis-15-enoic acid) and oxynervone contains 2-hydroxynervonic acid. Methods for the chromatographic identification and separation of the various cerebrosides are available (see N. K. Kotchetkov et al., Biochim. Biophys. Acta, 1962, 60, 431; 1963, 70, 716; G. J. M. Hooghwinkel et al., Rec. Tray. chim., 1964, 83, 576; S. Yokoyama and Yamakwa, Jap. J. exp. Med., 1964, 34, 29; Klenk et al., Z. physiol. Chem., 1965, 342, 187; 1967, 348, 1061; 1968, 349, 653; 1969, 350, 1598; Progr. Chem. Fats and Lipids, 1969, 10, 411; E. Coles and J. L. Foote, J. Chromatog., 1969, 39, 229; K. and B. Samuelsson, Biochem. biophys. Res. Comm., 1969, 37, 15; A. J. Acher and Kanfer, J. Lipid Res., 1972, 13, 139). The mass spectra of derivatives of several cerebrosides have been recorded (see Karlsson et al., Chem. Phys. Lipids, 1972, 9, 89, 230; J. Lipid Res., 1972, 13, 169; Biochim. Biophys. Acta, 1972, 270, 260; 1973, 306, 317) and the 220 MHz p.m.r, ofacetylated galactocerebrosides have been studied M. Martin-Lomas and D. Chapman, Chem. Phys. Lipids, 1973, 10, 152). A microchemical assay of galactocerebrosides has been described (H. S. Maker et al., Anal. Biochem., 1974, 61,471). The position of the monosaccharide on Ctl ~ of sphingosine has been determined by hydrogenolysis of fully acetylated galactocerebrosides and subsequent hydrolysis to give sphingine (H. E. Carter and F. L. Greenwood, J. biol. Chem., 1952, 199, 283; J. Kiss et al., Helv., 1960, 43, 2198). Enzymatic studies (see Rosenberg and Chargaff, loc. cir.) and chemical studies (P. Stoffyn, Angew. Chem., intrn. Edn., 1965, 4, 160) indicate a fl-glycoside linkage. Hydrolysis of the cerebrosides by base gives galactosyl-sphingosines ("psy-

406

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

chosines", see p. 408). Oxidation of the sugar residue by periodate followed by reduction with sodium tetrahydridoborate and mild acid hydrolysis gives ceramides (Carter et al., J. Lipid Res., 1961,2, 228). Galactocerebrosides containing dihydrosphingosine (Kiss, Chimia, 1959, 13, 115; D. Shapiro and H. M. Flowers, J. Amer. chem. Soc., 1959, 81, 2023), sphingosine (idem, ibid., 1961, 83, 3327) and phytosphingosine (I. Pascher, Chem. Phys. Lipids, 1974, 12, 303) have been synthesised. Several derivatives of the galactocerebrosides (substituted on the galactose) have been prepared (G. I. Samokvalov et al., Zhur. obshche'f Khim., 1970, 40, 1908, 2737, 2742, 2745; 1974, 44, 899). Two routes for the biosynthesis of galactocerebrosides have been described; one involving the glycosidation of sphingosine to give psychosine which is then acylated to give a cerebroside and the other by direct glycosidation of ceramide with UDP-hexoses (P. Morell et al., J. Lipid Res., 1972, 13, 293; J. biol. Chem., 1973, 248, 8240; Roseman et al., ibid., 1973, 248, 1388). The latter route is considered to be the more likely natural route in view of the fact that psychosine can be acylated with acyl-CoA nonenzymically (see S. Hammarstr6m, FEBS Letters, 1972, 21,259; see however J. A. Curtino and R. Caputto, Biochem. biophys. Res. Comm., 1974, 56, 142; S. S. Raghaven et al., ibid., 1974, 58, 99). Synthetic inhibitors of the UDP-galactose: ceramide galactosyl transferase (EC 2.4.1.62), particularly N-bromoacetyl-DL-erythro-2-amino-3-phenyl-l,3-propanediol, have been studied (R. C. Arora and N. S. Radin, Biochim. Biophys. Acta, 1972, 270, 254; see P. Mandel et al., ibid., 1974, 334, 309 for a purification of this enzyme). The galactocerebroside (and sulphatide) content of the brains of particular strains of mice with genetic defects ("quaking" and "jimpy" mice) are considerably reduced (see E. L. Hogan et al., J. Neurochem., 1972, 19, 307, 2435; N. Baumann et al., ibid., 1973, 20, 753; Morell and E. Constantino-Ceccarini, Lipids, 1972, 7, 266; Mandel et al., Brain Res., 1972, 42, 147; J.-M. Matthieu et al., ibid., 1973, 55, 403) due to a defect in myelin maturation and those present in the "quaking" mice are deficient in long-chain fatty acids due to the lack of a fatty acid elongating enzyme (K. Bloch et al., Science, 1973, 182, 497; Baumann et al., Biochimie, 1973, 55, 1473). An enzyme which cleaves glucocerebrosides to glucose and ceramides is present in tissues (see Kanfer, J. biol. Chem., 1965, 240, 609; R. O. Brady et al., ibid., 1973, 248, 5256; S. Gatt, Biochim. Biophys. Acta, 1967, 137, 192; N. S. Radin et al., J. Lipid Res., 1966, 7, 379) and the reduced activity of this enzyme in Gaucher's disease causes the accumulation of glucocerebrosides, in the spleen (Brady et al., J. Clin. Invest., 1966, 45, 1112; M. W. Ho et al., Proc. Nat. Acad. Sci., 1971, 68, 2810; Amer. J. Human Genetics, 1972, 24, 37; Kanfer et al., Adv. Exp. Med. and Biol., 1972, 19, 225; Radin, ibid., p. 475; Biochem. J., 1973, 131, 173). Enzyme replacement therapy

2

MONOGLYCOSYL-CERAMI

DES

407

(by the injection of the enzyme glucosyl ceramide fl-glucosidase) for the treatment of Gaucher's disease, has been investigated (Brady et al., New England J. Med., 1974, 291, 989). N-Hexyl-l-O-fl-glucosylsphingosine is an inhibitor of this enzyme (Radin et al., J. Lipid Res., 1973, 14, 133; J. biol. Chem., 1974, 249, 4638). The bilayer nature of these lipid deposits in Gaucher's spleen has been shown by physical methods (R. E. Lee et al., Arch. biochem. Biophys., 1973, 159, 259). A lysosomal fl-galactosidase in brain degrades galactocerebrosides (Radin et al., Chem. Phys. Lipids, 1970, 5, 178; Lipids, 1973, 8, 732; D. A. Wenger, Chem. Phys. Lipids, 1974, 13, 327) and the reduced activity of this enzyme in Krabbe's disease (globoid cell leukodystrophy) leads to a relative increase of galactocerebrosides and degeneration of myelin (see K. Suzuki et al., Adv. exp. med. Biol., 1972, 19, 487; J. biol. Chem., 1974, 249, 2105, 2109; Lipids, 1971, 6, 433; G. Dawson, ibid., 1973, 8, 154). Galactocerebroside degrading enzymes are also present in the intestine (A. Nilsson, Biochim. Biophys. Acta, 1969, 187, 113). Synthetic inhibitors and stimulators of this enzyme (galactosyl-ceramide fl-galactosidase) have been studied (Radin et al., Arch. biochem. Biophys., 1973, 156, 77; Adv. exp. med. Biol., 1972, 19, 475). Cerebrosides in which the hydroxyl group on the Ct3) of sphingosine or a hydroxyl group of the galactose is acylated with a fatty acid (see Klenk and J. P. Lohr, Z. physiol. Chem., 1967, 348, 1712; N. K. Kotchetkov et al., Doklad. Akad. Nauk S.S.S.R., 1966, 167, 346; Radin et al., J. Lipid Res., 1968, 9, 27; H. Singh, ibid., 1973, 14, 41; M. I. Horowitz et al., Biochim. Biophys. Acta, 1972, 280, 383; M. Kubota and T. Taketomi, Jap. J. exp. Med., 1974, 44, 145) or the Ct3) of sphingosine is alkylated with an ~,flunsaturated carbon chain ("sphingoplasmalogens") (Kotchetkov et al., Biokhimiya, 1964, 29, 570) have also been described. 3-O-Alkyl derivatives ofcerebrosides have been synthesised (Kotchetkov et al., Izvest. Akad. Nauk, 1970,411). Galactocerebrosides can be specifically labelled with tritium at the Ct6) of galactose by first oxidizing with galactose oxidase and then reducing with sodium [3H]tetrahydridoborate (Arora and Radin, J. Lipid Res., 1972, 13, 86). A ceramide galacturonate has also been prepared by this route by oxidation of the intermediate aldehyde with hypoiodite (Kanfer, Lipids, 1972, 7, 653). A xylose-containing cerebroside has been identified in the salt gland of the herring gull (K. A. Karlsson et al., J. Lipid Res., 1972, 13, 169). Antibodies to galactocerebrosides have been prepared (see J. M. Fry et al., Science, 1974, 183, 540; C. R. Airing et al., Immunochem., 1974, 11, 475; N. A. Greyson et al., Immunology, 1974, 26, 743; S. Leibowitz

408

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

et al., ibid., 1974, 27, 1117; 1975, 28, 213; J. Tremblay et al., J. Neurochem., 1974, 23, 315) and the reactions of these against nervous tissue studied in relation to neurological disease. (2) Psychosine. 1-O-fl-o-Galactopyranosyl-sphingosine is a hydrolytic product of galactocerebrosides and is best obtained by the action of barium hydroxide (H. E. Carter and Y. Fujino, J. biol. Chem., 1956, 221, 879) or potassium hydroxide in butanol (T. Taketomi and T. Yamakawa, J. Biochem., Tokyo, 1963, 54, 444; Radin, Lipids, 1974,.9, 358) and isolation as the sulphate (m.p. 190~with decomp.; [ t ~ ] a - - 15.8~in pyridine). Psychosine (D. Shapiro et al., J. Amer. chem. Soc., 1964, 86, 4472) and 1-O-fl-D-galactopyranosyl-phytoshhingosine (Pascher, Chem. Phys. Lipids, 1974, 12, 303) have been synthesised. The haemolytic activity of psychosine is similar to that of lysolecithin (Taketomi and K. Nishimura, Jap. J. exp. Med., 1964, 34, 255). Psychosine has been implicated in the ,biosynthesis of the cerebrosides but it has been shown that psychosine is acylated non-enzymically by long-chain acyl-CoA (S. Hammarstrb'm, FEBS Letters, 1972, 21, 259). The biosynthesis of psychosine has been investigated (V. L. Friedrich and Hauser, J. Neurochem., 1973, 20, 1131)and the deficiency of a galactosidase which degrades psychosine has been demonstrated in Krabbe's disease (T. Miyutake and Suzuki, J. Neurochem., 1974, 22, 231; Biochim. Biophys. Acta, 1974, 337, 333; D. A. Wenger et al., Proc. Nat. Acad. Sci., 1974, 71, 854). The corresponding derivative from glucocerebrosides, 1-O-fl-D-glycopyranosylsphingosine, has been prepared by the hydrolysis of glucocerebrosides (J. S. Erickson and Radin, J. Lipid Res., 1973, 14, 133) and the biosynthesis of this compound from sphingosine and UDP-glucose in rat brain microsomes has been described (J. A. Curtino and R. Caputto, Lipids, 1972, 7, 525). This compound has also been isolated from a spleen in a case of Gaucher's disease (Kanfer et al., J. Lipid Res., 1974, 15, 484). (3) Cerebroside suiphates; suiphatides. These compounds were first characterised as constituents of brain lipids (G. Blix, Z. physiol. Chem., 1933, 219, 82; for reviews see I. H. Goldberg, J. Lipid Res., 1961, 2, 103; P. Stoffyn, J. Amer. Oil Chemists' Soc., 1965, 43, 69; T. H. Haines, Progr. Chem. Fats and Lipids, 1971, 11, 297, and for methods of isolation see E. Martensson, Biochim. Biophys. Acta, 1966, 116, 521). Sulphatides have also been isolated from kidney and from avian and dog fish salt gland where they have been implicated in sodium transport (Karlsson et al., ibid., 1969, 176, 429; 1973, 306, 307; Europ. J. Biochem., 1974, 46, 243) and from hog gastric ~ucosa (M. I. Horowitz et al., Biochim. Biophys. Acta, 1974, 348, 388). Methylation studies have indicated that they are galactocerebrosides sul-

2

NEUTRAL OLIGOGLYCOSYL-CERAMIDES

409

phated on the C(3) of galactose (Yamakawa et al., J. Biochem., Tokyo, 1962, 52, 226; 1964, 55, 87, 365; P. and A. Stoffyn, Biochim. Biophys. Acta, 1963, 70, 218) and this is also indicated by their stability towards periodate oxidation (M. A. Wells and J. C. Dittmer, J. Chromatog., 1965, 18, 503). The sulphate ester is stable to alkaline conditions but is readily removed by acidic (e.g. 0.05 N hydrogen chloride in methanol at 20 ~ for 1 h; P. and A. Stoffyn, Biochim. Biophys. Acta, 1963, 70, 107; Kiss, Helv., 1967, 50, 1423) or enzymic hydrolysis (E. Mehl and H. Jatzkewitz, Z. physiol. Chem., 1963, 331,292; 1964, 339, 260)to give galactocerebrosides. Sulphatides derived from the cerebrosides, kerasin and phrenosine have been detected (Jatzkewitz, ibid., 1960, 320, 134; P. and A. Stoffyn, loc., cit.). A dihydrosphingosine-containing sulphatide (Flowers, Carbohydrate Res., 1966, 2, 371) and cerebrosides sulphated on the C(6) position of galactose (see Jatzkewitz and G. Nowoczek, Ber., 1967, 100, 1667) have been synthesised. Derivatives of O-galactose 3-sulphate have also been synthesised (Stoffyn, J. org. Chem., 1967, 32, 4001; Jatzkewitz and Nowoczek, loc. cit). The enzymic synthesis of sulphatides from galactocerebrosides and adenosine 3'-phosphate-5'-phosphosulphate has been studied (D. F. Farrell and G. M. McKhann, J. biol. Chem., 1971, 246 4694; Stoffyn et al., J. Lipid Res., 1971, 12, 318; F. B. Jungawala, ibid., 1974, 15, 114) and an enzymic synthesis of psychosine sulphate has also been described (J.-L. Nussbaum and Mandel, J. Neurochem., 1972, 19, 1789). The Golgi apparatus of rat kidney is a site of biosynthesis of sulphatides (B. Fleischer and F. Zambrano, J. biol. Chem., 1974, 249, 5995). A biosynthetic preparation of asS-labelled sulphatide has been described (A. L. Fluharty et al., Lipids, 1974, 9, 865). Cerebroside sulphates accumulate in tissues in metachromatic leucodystrophy (see M. J. Malone et al., J. Neurochem., 1966, 13, 1033, 1037) due to the deficiency of a lysosomal sulphatase (aryl sulphatase A) which normally degrades them to cerebrosides (see M. T. Porter et al., Biochim. Biophys. Acta, 1972, 258, 769; Science, 1971, 172, 1263; A. A. Farooqui and B. K. Bachhawat, J. Neurochem., 1973, 20, 889; M. Sugita et al., J. Lipid Res., 1974, 15, 227). Anti-sulphatide antibodies have been prepared (see S. Hakomori, J. Immunology, 1974, 112, 424; Methods in Enzymology, 1972, 28, 232). Sulphatides have been determined spectrophotometrically (E. L. Kean, J. Lipid Res., 1968, 9, 319) and analysed by mass spectrometry (Karlsson et al., Biochem. biophys. Res. Comm., 1969, 37, 22). (ii) Neutral oligoglycosyl-ceramides (1) Diglycosyi-ceramides. A diglycosyl-ceramide, ("cytolipin H", lactosyiceramide) of the following structure

410

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

ceramide( ~- 1) D-glucose(4 ~- 1) D-galactose

has been isolated from spleen, milk, serum, erythrocytes, kidney and liver and from a human epidermoid carcinoma (see M. M. Rapport et al., J. biol. Chem., 1962, 237, 1056; W. R. Morrison and L. M. Smith, Biochim. Biophys. Acta, 1964, 84, 759; E. and L. Svennerholm, Nature, 1963, 198, 688; E. Martensson, Acta Chem. Scand., 1963, 17, 2356; J. Polonovski and M. Petit, Bull. Soc. chim. biol., 1963, 45, 111; A. Wagner, Clin. Chim. Acta, 1964, 10, 175; A. Makita and T. Yamakawa, J. Biochem., Tokyo, 1962, 51, 124; 1964, 55, 365). It is also known as a degradation product of the gangliosides (see p. 418). The assignment of the lactose configuration to the carbohydrate moiety has been confirmed by comparison with synthetic material (D. Shapiro et al., Chem. Phys. Lipids, 1966, 1, 54; J. B. Hay and H. M. Gray, ibid., 1969, 3, 59; H. M. Flowers, Carbohydrate Res., 1966,2, 188; 1'967, 4, 42). The radiochemical decomposition oflactosylceramide tritiated on the Ct6) position of galactose resulting in the production of galactose has been studied (J. N. Kanfer et al., Lipids, 1973, 8, 238). Lactosyl-ceramide accumulates in the brain in gargoylism and in globoid leucodystrophy (Taketomi and Yamakawa, Jap. J. exp. Med., 1967, 37, 11; J. E. Evans and R. H. McCluer, J. Neurochem., 1969, 16, 1393; G. Dawson, J. Lipid Res., 1972, 13, 207). The enzymic hydrolysis S. Gatt and Rapport, Biochem. J., 1966, 101, 680; N. S. Radin et al., Brain Res., 1969, 14, 497) and enzymic synthesis by the donation of galactose from UDP-galactose to a glucocerebroside (J. Hildebrandt and C. Hauser, J. biol. Chem., 1969, 244, 5170) and the immunochemical properties of lactosylceramide (Shapiro et al., Europ. J. Biochem., 1967, 2, 79; Yamakawa et al., Israel J. Med. Sci., 1967, 3, 577; S. Razin et al., Proc. Soc. exp. Biol. Med., 1971, 138, 404; R. Arnon and D. Teitelbaum, Chem. Phys. Lipids, 1974, 13, 352; L. Grafand Rapport, ibid., 1974, 13, 376) have been studied. A digalactosyl-ceramide has been isolated from human and mouse kidney (Makita and Yamakawa, J. Biochem., Japan, 1964, 55, 365; G. M. Gray et al., Chem. Phys. Lipids, 1968, 2, 147; Biochem. biophys. Res. Comm., 1970, 38, 520, 527; Sweeley et al., J. Lipid. Res., 1969, 10, 188) and from a diseased brain (Gatt and E. R. Berman, J. Neurochem., 1963, 10, 43). A digalactosyl-ceramide also accumulates in the kidney in Fabry's disease (which is due to the deficiency of an ~-galactosidase) and the following structure has been proposed for this lipid (Y.-T. Li et al., Biochim. Biophys. Acta, 1972, 260, 88): ceramide(1 g-- 1)D-galactose(4 d-- 1)D-galactose

The biosynthesis of a lipid with this structure in the rat kidney has been

2

NEUTRAL OLIGOGLYCOSYL-CERAMIDES

411

studied (L. Svennerholm et al.,J, biol. Chem., 1974, 249, 4132). A digalactosylceramide has been isolated from pig pancreas (H. Debuch, Z. physiol. Chem., 1974, 355, 725)and digalactosyl-ceramides accumulate in the kidneys of female mice treated with testosterone (Gray, Biochim. Biophys. Acta, 1971, 239,.494; see also Y.-N. Lin and Radin, Biochem. J., 1973, 136, 1125). Digalactosyl-ceramides (containing dihydrosphingosine) with fl(1 ~ 3) and fl(1 ~ 4) linkages between the galactose molecules have been synthesised (Flowers, Carbohydrate Res., 1967, 5, 126; Hay and Gray, loc. cit.). A glucosyl-mannosyl-ceramide is present in Corbicula sandai (T. Hori et al.,J. Biochem., Tokyo, 1968, 64, 125) and a similar lipid with the following structure: mannose(1 ~ 4)glucose(1 ~ 1)ceramide has been isolated from wheat flour (R. A. Laihe and O. Renkonen, Biochemistry, 1974, 13, 2837). A diglycosyi-ceramide sulphate has been isolated from kidney and brain and hog gastric mucosa and has the following structure (P. Stoffyn et al., Biochim. Biophys. Acta, 1968, 152, 353; M. I. Horowitz et al., ibid., 1974, 348, 388): ceramide (1 g- 1)glucose (4 g-- 1)galactose 3-sulphate (2) Triglycosyl-ceramides. A triglycosyl-ceramide of the following structure: ceramide 1(1 g-- 1) D-glucose(4 g-- 1)D-galactose(4 ~ 1)D-galactose accumulates in tissues (particularly kidney) in Fabry's disease (Sweeley and B. Klionsky, J. biol. Chem., 1963, 238, PC 3148; T. Miyatake and T. Ariga, J. Neurochem., 1972, 19, 1911)and is present in smaller quantities in normal kidney (Sweeley et al., Chem. Phys. Lipids, 1970, 4, 393), spleen, liver and serum (E. and L. Svennerholm, loc. cir.) where it occurs as a catabolite of globoside (see below). It is also the major ceramide glycoside of thymus and blood leucocytes (G. M. Levis and M. Kesse-Elias, Lipids, 1974, 9, 651). The or-configuration of the terminal galactose residue has been confirmed by many methods (see Y.-T. Li and S.-C. Li, J. biol. Chem., 1971, 246, 3769; J. T. R. Clarke et al., ibid., 1971, 246, 5563; M. Philippart et al., Biochem. biophys. Res. Comm., 1971, 43, 913). This glycolipid has been identified as the pk antigen of human erythrocytes (M. Naiki and D. M. Marcus, ibid., 1974, 60, 1105). It is biosynthesised from lactosyl-ceramide by the transfer of galactose from UDP-galactose (Hildebrandt and Hauser, loc. cir.; L. Svennerholm et al., ibid., 1974, 249, 4132). An enzyme which cleaves the terminal galactose from this triglycosyl ceramide to give lactosylceramide has been detected in various tissues (Brady et al., ibid., 1967,

412

P H O S P H O L I P I D S AND G L Y C O L I P I D S

21

242, 1021) and a deficiency of this enzyme is the cause of Fabry's disease (Sweeley et al., J. biol. Chem., 1973, 248, 2461, 2471; Biochem. biophys. Res. Comm., 1973, 53, 1317; Arch. biochem. Biophys., 1973, 158, 297; M. W. Ho, Biochem. J., 1973, 133, 1; I. Kano and Yamakawa, J. Biochem., Tokyo, 1974, 75, 347). Enzyme therapy for Fabry's disease by the action of human 0t-galactosidase A has been considered (see P. D. Snyder et al., Biochim. Biophys. Acta, 1974, 350, 432). The mass spectra of this triglycosylceramide (and other glycosylceramides) have been investigated (Sweeley and G. Dawson, Biochem. biophys. Res. Comm., 1969, 37, 6; W. Stoffel and P. Hanfland, Z. physiol. Chem., 1973, 354, 21) and a related compound (containing//-linkages) has been synthesised (D. Beith-Halahmi and Flowers, Carbohydrate Res., 1968, 8, 340). A sulphated derivative of the natural ceramide trihexoside (sulphated on the 3-position of the terminal galactose) is present in hog gastric mucosa (Horowitz et al., Biochim. Biophys. Acta, 1974, 348, 388). A related trihexosyl-ceramide present in rat tissue has the following structure (Stoffyn et al., ibid., 1973, 306, 283; H. Arita and J. Kawanami, J. Biochem., Tokyo, 1974, 76, 1067): ceramide(1 ,0- 1)glucose(4 d-- 1)galactose(3 ~ 1)galactose

It differs from the other ceramide trihexoside only in the linkage position of the terminal galactose and is related to the corresponding tetraglycosylceramide of rat tissue (see below). Studies on the biosynthesis of these two ceramide trihexosides in rat tissue has shown (idem, ibid., 1974, 360, 174) that in kidney the (1 ~ 4) linked compound is formed whereas in other tissues only the (1 ~ 3) linked compound is found. A triglyeosyi-eeramide of the following structure: ceramide(1 g-- 1) D-glucose(4 ~- 1) D-galactose(4 d-- 1)N-acetylgalactosamine

has been isolated from a diseased human brain (see Y. Z. Frohwein and S. Gatt, Biochemistry, 1967, 6, 2783) and accumulates in tissues in Sandhofs disease which is due to the deficiency of N-acetyl-fl-hexosaminadases A and B which would normally degrade it to lactosyl-ceramide. This triglycosyl-ceramide ("asialo Tay-Sachs ganglioside") is a normal catabolite of Tay-Sachs ganglioside (see below) (see R. J. Desnick et al., Adv. Exp. Med. and Biol., 1972, 19, 351; E. H. Kolodny, ibid., p. 321) and has been synthesised (Shapiro et al., Chem. Phys. Lipids, 1973, 10, 28). It is also the major glycolipid of the guinea pig red blood cell membrane (Y. Seyama and T. Yamakawa, J. Biochem., Tokyo, 1974, 75, 837; M. Basu et al., Biochem. biophys. Res. Comm., 1974, 60, 1097).

2

NEUTRAL OLIGOGLYCOSYL-CERAMIDES

413

A triglycosyl-ceramide of the following structure has been isolated from wheat flour (Laine and Renkonen, Biochemistry, 1974, 13, 2837): mannose(1 A 4)mannose(1 A 4)glucose(1 -~ 1)ceramide The bovine blood group J substance is thought to be a phosphate diester of a triglycosyl-ceramide (containing glucose and galactose) and a di-Oacylglycerol (Horowitz et al., Immunochemistry, 1971, 8, 719; 1972, 9, 1067; 1973, 10, 145; see also O. W. Thiele et al., Europ. J. Biochem., 1971, 22, 294, 396; Fette und Seifen, 1972, 74, 161; Vox Sanguinis, 1973, 25, 317). (3) Tetraglycosyl-ceramides. A tetraglycosyl-ceramide ("gioboside", "cytolipin K" with the following structure is the major glycolipid of human erythrocytes ceramide(1 ~- 1) D-glucose(4 4!. 1) D-galactose(4 ~- 1) D-galactose

N-acetylgalactosamine ( Yamakawa et al., Jap. J. exp. Med., 1965, 35, 201 ; J. Biochem., Tokyo, 1971, 69, 625; S. Hakomori et al., J. biol. Chem., 1971, 246, 2271; J. Kawanami and T. Tsuji, Chem. Phys. Lipids, 1971, 7, 49). A similar compound is also present in human kidney, spleen, serum and liver (E. and L. Svennerholm, loc. cit.; Martensson, loc. cit.; Yamakawa et al., J. Biochem., Tokyo, 1964, 55, 202; Makita et al., ibid., 1966, 60, 502; Kawanami, ibid., 1967, 62, 272; Rapport et al., Arch. Biochem. Biophys., 1964, 105, 431; Biochim. Biophys. Acta, 1967, 137, 409) and in hog erythrocytes (Yamakawa et al., Jap. J. exp. Med., 1968, 38, 135). The biosynthesis of this compound by a fl-N-acetylgalactosaminyl transferase from embryonic chicken brain by adding N-acetylgalactosamine to a triglycosyl ceramide has been studied (S. Basu et al., J. biol. Chem., 1973, 248, 1778; see also A. Makita et al., Biochim. Biophys. Acta, 1974, 337, 92 for the biosynthesis in guinea pig kidney). Globoside accumulates in tissues in Sandhoff's disease. This is an autosomal recessive inborn error of metabolism due to the deficiency of the normal N-acetyl-fl-hexosaminadases A and B and is thus related to TaySach's disease (see below)(Desnick et al., loc. cit.; Kolodny, loc. cit.; Sweeley et al., J. Lipid Res., 1972, 13, 128). Globoside has been identified as the P antigen of human erythrocytes (Naiki and Marcus, Biochem. biophys. Res. Comm., 1974, 60, 1105). The N-acetylgalactosamine in this lipid can be labelled in the C~6~ position with tritium after oxidation with galactose oxidase followed by reduction

414

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

with sodium [3H]tetrahydridoborate (Y. and K. Suzuki, J. Lipid Res., 1972, 13, 687). The structures formed by globoside in aqueous systems have been investigated (L. Pinteric et al., Biochim. Biophys. Acta, 1973, 298, 630). Ozonolysis of peracetylated globoside followed by oxidation with hydrogen peroxide and alkaline hydrolysis gave a derivative of globoside which has been coupled to Sepharose and used as an affinity absorbent for purification of the anti-globoside antibody (Hakomori et al., Federation Proc., 1973, 32, 483, Abstract No. 1468). A tetraglycosyl-ceramide of the following structure ("cytolipin R") has been isolated from rat lymphosarcoma (M. M. Rapport et al., J. Lipid Res., 1972_, 13, 519; Arita and Kawanami, loc. cit.). ceramide( ~- 1) D-glucose(4 g-- 1) D-galactose(3 ~ 1) D-galactose

N-acetylgalactosamine and differs from globoside in' the galactose-galactose linkage. This lipid appears to be unique to rat kidney tissue (Hakomori et al., ibid., p. 657) since globoside is common to human erythrocyte, monkey kidney, horse spleen and bovine kidney. The immunochemistry of this glycolipid has been studied (Rapport et al., ibid., p. 119; J. Membrane Biol., 1970, 3, 280). The enzymic synthesis of a tetraglycosyl-ceramide of the following structure: ceramide(1 ~- 1) D-glucose(4 g- 1) D-galactose

o-galactose(1 -~ 4)N-acetylglucosamine has been studied with rabbit tissue (M. and S. Basu, J. biol. Chem., 1972, 247, 1489). It is an intermediate in the biosynthesis of the pentaglycosyl-ceramide which is the major glycolipid of rabbit erythrocytes (see below). It is also present in normal human erythrocytes and other tissues (B. Siddiqui and S. Hakomori, Biochim. Biophys. Acta, 1973, 330, 147; J. R. Wherrett, ibid., 1973, 326, 63; T. Yamakawa et al., J. Biochem., Tokyo, 1973, 73, 893; Svennerholm et al., J. biol. Chem., 1973, 248, 2634) and has been prepared by the degradation of the major ganglioside of human peripheral nervous tissue which is a monosialyl derivative of this compound (see below). A tetraglycosyi-ceramide containing three molecules of galactose and one molecule of glucose has been isolated from Neurospora crassa (R. L. Lester et al., ibid., 1974, 249, 3388).

2

NEUTRAL OLIGOGLYCOSYL-CERAMIDES

415

(4) Pentaglycosyi-ceramides. The major glycolipid of sheep and goat erythrocytes ("Forssman antigen") is a pentaglycosyl-ceramide of the following structure and is also present in the tissues of the horse and of some other animals: ceramide(1 ~- 1)glucose(4 ~- 1)galactose(4 ~ 1)galactose

N-acetylgalactosamine(1 -~ 3)N-acetylgalactosamine (see W. C. Boyd, "Fundamentals of Immunology", 4th Edn., Interscience, New York, 1966, p. 196; Siddiqui and Hakomori, J. biol. Chem., 1971, 246, 5766; Hakomori et al., Arch. biochem. Biophys., 1973, 155, 464; S. C. Kinsky et al., Biochemistry, 1969, 8, 4149; Naiki et al., Jap. J. exp. Med., 1972, 42, 205; B. A. Fraser and M. F. Mallette, Immunochem., 1973, 10, 745; 1974, 11,581; H. J. Rapp and J. Borsos, J. Immunology, 1966, 96, 913; S. Ando and Yamakawa, Chem. Phys. Lipids, 1970, 5, 91; T. Taketomi et al., J. Biochem., Tokyo, 1974, 75, 197; Makita et al., Z. physiol. Chem., 1973, 354, 1149). The immunochemistry of this glycolipid in artificial lipid membranes Cliposomes") has been studied (Kinsky et al., Biochemistry, 1971, 10, 2574; Biochim. Biophys. Acta, 1972, 265, 1; J. Immunology, 1974, 112, 1949). The Forssman antigen is also present in dog intestine (J. M. M c K i b b i n et al., Biochemistry, 1966, 5, 435; Anal. Biochem., 1972, 45, 608; Sweeley et al., Federation Proc., 1973, 32, 484, Abstract No. 1478). The mass spectrum of the Forssman antigen from horse kidney has been studied (K. A. Karlsson, J. biol. Chem., 1974, 249, 4819) and its biosynthesis in guinea pig kidney has been studied (Makita et al., Biochim. Biophys. Acta, 1974, 337, 92; Biochem. biophys. Res. Comm., 1974, 56, 177). A pentaglycosyl-ceramide of the following structure has been isolated from human erythrocyte membranes (Hakomori et al., J. biol. Chem., 1974, 249, 1022. ceramide(1 ~ 1)glucose(4 ~- 1)galactose(3 ~ 1)N-acetylglucosamine

galactose(1 -~ 3) galactose The major glycolipid of rabbit erythrocytes is a pentaglycosyl-ceramide of the following structure (Hakomori et al., Arch. Biochem. Biophys., 1973, 155, 464; M. and S. Basu, J. biol. Chem., 1973, 248, 1700; see also Yamakawa et al., J. Biochem., Tokyo, 1968, 64, 205):

416

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

ceramide(1 -~- 1)D-glucose(4~- 1)D-galactose(3-~- 1)N-acetylglucosamine

D-galactose(1 -~ 3)D-galactose The enzymic synthesis of this glycolipid by an ct-galactosyl transferase from rabbit bone marrow has been studied (M. and S. Basu, loc. cir.). This pentaglycosyl-ceramide shows immunological cross-reaction with the human blood group B substance due to the similarity of the configuration and linkages of the terminal carbohydrates. (5) Fucose-containing oligoglycosyi-ceramides. The fucose-containing glycolipids of human erythrocytes responsible for blood group activity (A, B, H and Le antigens) have been investigated by Hakomori and coworkers (Biochemistry, 1968, 7, 1279; Chem. and Phys. Lipids, 1970, 5, 96; Biochim. Biophys. Acta, 1970, 202, 225; Biochem. biophys. Res. Comm., 1972, 49, 1061; see also I. Liotta et al., Vox Sangufnfs, 1972, 22, 171; Hakomori and A. Kobata, in "The Antigens", Vol. 2, ed. M. Sela, Academic Press, New York, 1974, p. 80; G. Pardoe, Ann. N.Y. Acad. Sci., 1974, 234, 239). A variety of complex oligoglycosyl-ceramides all terminating in the following sequence (which is the blood group A determinant) were found in the blood group A active glycosyl-ceramides:

N-Acetylgalactosamine

67..

Galactose ( 1~ 4 ) N-Acetylglucosam ine L-Fucose

c~L~/~/rJ~ Ceramide

A pentaglycosyi-ceramide, containing fucose, of the following structure has also been isolated from human erythrocyte membranes and shows blood group H specificity (Hakomori et al., Biochemistry, 1973, 12, 656): ceramide(1 ~- 1)D-glucose(4 d- 1)o-galactose (3 ~- 1)N-acetylglucosamine

L-fucose(1 -~ 2) D-galactose Another fucose-containing pentaglycosyl-ceramide (which also contains phytosphingosine) of the following structure has been isolated from a human adenocarcinoma (H.-J. Yang and Hakomori, J. biol. Chem., 1971, 246, 1192):

2

NEUTRAL OLIGOGLYCOSYL-CERAMIDES P

Phytosph ingosine -D-glucose ( 4 - - - ~atty acid

1)

P

D-galactose ( 3 - - - - /

1)

417

N - A c e t y l g lucosa m ine

P

k.~ ~

L-fucose

D-galactose

J. Koscielak and his coworkers (Europ. J. Biochem., 1973, 37, 214; FEBS

Letters, 1974, 42, 101) have also described fucose-containing oligoglycosylceramides containing 20-40 sugar residues, attached to the ceramide, responsible for B1, H1 and H ll blood group activities. The terminal sugars of these compounds are similar to those described for the glycopeptide blood group substances (see W. T. J. Morgan, Bull. Inst. Pasteur, 1974, 72, 131). Other methods for the extraction of these glycolipid blood group antigens have been described (Tanner et al., Biochem. J., 1974, 138, 381; Europ. J. Biochem., 1974, 45, 31; A. Fiori et al., J. Chromatog., 1973, 84, 335). A blood group B glycolipid of the following structure has been found to accumulate in the pancreas of a patient with Fabry's disease (deficiency of an ~-galactosidase)(Wherrett and Hakomori, J. biol. Chem., 1973, 248, 3046): f~ f~ P

Ceramide (1---1)glucose(4--~-l)galactose(3--~l)N-Acetylglucosamine

Galactose Ratio ( 1 - - - 3 ) : ( 1 - - - 4 ) = 4;1

~/ Fucose

x~Tj Galactose

Other fucose-containing glycosyl-ceramides with blood group activity are present in human and dog intestine (McKibbin et al., Anal. Biochem., 1972, 45, 608; J. Immunology, 1973, 110, 1037), hog stomach mucosa (M. I. Horowitz et al., J. biol. Chem., 1974, 249, 1225; Biochim Biophys. Acta, 1974, 326, 224; Europ. J. Biochem., 1974, 43, 161) and in the slime mould Dictyostelium discoideum (0. Westphal et al., ibid., 1974, 48, 89). The fucolipids are altered in virally transformed and tumour cells due to the deficiency of glycosyl transferases (see S. Steiner and J. L. Melnick, Nature, 1974, 251, 717; Hakomori, Adv. Cancer Res., 1973, 18, 265; R. 0. Brady and P. H. Fishman, Biochim. Biophys. Acta, 1974, 355, 121). A deficiency of an c~-L-fucosidase results in the accumulation of fucosecontaining glycosyl-ceramides (and other fucose-containing macromolecules) in human tissues ("fucosidosis") (J. S. O'Brien et al., J. exp. Med.,

418

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

1972, 136, 197; J. Lab. Clin. Med., 1972, 79, 164; V. Patel et al., Science, 1972, 176, 426; G. Dawson, Adv. Exp. Med. and Biol., 1972, 19, 395; I. Matsuda et al., Tohoku J. exp. Med., 1973, 109, 41). (iii) Glycosyl-ceramides containin9 sialic acids. Ganyliosides Gangliosides were first discovered in the brain of a child who died from infantile amaurotic idiocy (E. Klenk, Z. physiol. Chem., 1935, 235, 24) and were later shown to occur in smaller quantities in normal brain (for reviews see Klenk and H. Debuch, Ann. Rev. Biochem., 1959, 28, 58; L. Svennerholm, J. Lipid Res., 1964, 5, 145; R. Ledeen, J. Amer. Oil Chemists' Soc., 1966, 43, 57; H. Wiegandt, Ergebnisse der Physiol., 1966, 57, 190; Angew. Chem. intern. Edn., 1968, 7, 87; Klenk, Progr. Chem. Fats and Lipids, 1969, 10, 411; J. Sci. Ind. Res., 1968, 27, 78; Chem. Phys. Lipids, 1970, 5, 193; R. H. McCluer, ibid., p. 220; Ledeen, ibid., p. 205; and for methods of extraction from brain see J. Folch-Pi, Adv. Exp. Med. Biol., 1972, 19, 63; T. P. Carter, Lipids, 1973, 8, 537; E. G. Brunngraber and V. A. Zibon, ibid., 1974, 9, 641; L. S. Wolfe, in "Research Methods in Neurochemistry", Vol. 1, eds. N. Marks and R. Rodnight, Plenum, New York, 1972, p. 233). The gangliosides are water-soluble compounds which on hydrolysis give sphingosine bases, fatty acids, glucose, galactose, N-acetylgalactosamine and N-acetylneuraminic acid (sialic acid) (C.C.C., 2nd Edn., Vol. I F, p. 300) (for a review of the chemistry and biochemistry of this carbohydrate see R. Schauer, Angew. Chem. intern. Edn., 1973, 12, 127). In aqueous solutions the gangliosides form micelles with molecular weights of ~ 30000 (see G. R. Dutton and S. H. Barondes, J. Neurochem., 1972, 19, 559; I. Raveglia and N. E. Ghittoni, J. Chromatog., 1971, 58, 288) above the critical micelle concentration at -~ 10- 4 M (150/~g/ml). Below this concentration they exist as monomers and can be dialysed (see J. N. Kanfer and C. Spielvogel, J. Neurochem., 1973, 20, 1483). Above this concentration they also migrate as micelles on polyacrylamide gel electrophoresis (Wiegandt, Anal. Biochem., 1974, 60, 382). T.l.c. resolves bovine and normal human brain gangliosides showing the presence of at least five closely related compounds (see McCluer et al., Biochim. Biophys. Acta, 1966, 116, 279; L. Svennerholm, J. Neurochem., 1963, 10, 613; K. Suzuki, ibid., 1965, 12, 969; V. H. MacMillan and Wherret, ibid., 1969, 16, 1621; L. S. Wolfe et al., Canad. J. Biochem., 1964, 42, 1057; D. van den Eijnden, Z. physiol. Chem., 1971,352, 1601), and they can also be separated by column chromatography on DEAE-cellulose (C. C. Winterbourn, J. Neurochem., 1971, 18, 1153) or silica gel (G. Tettamanti et al., Biochim. Biophys. Acta, 1973, 296, 160). The four major gangliosides of normal brain all give sphingosine, fatty acid, glucose, galactose and N-acetylgalactosamine in the molar ratio of

2

CONTAINING

SIALIC ACIDS

419

1" 1" 1"2" 1, respectively on acid hydrolysis. One of these gangliosides affords one molar proportion of sialic acid ("monosialoganglioside", "G MI "), two of them provide two molar portions of sialic acid ("disialogangliosides", "Go~a" and "Garb") and the fourth, three molar portions of sialic acid ("trisialoganglioside", "GT"). The trisialoganglioside and both disialogangliosides are converted into the monosialoganglioside by the enzyme neuraminadase ("receptor destroying enzyme", "R.D.E." which is present in the influenza virus and Vibrio cholerae ~ see A. Rosenberg et al., Biochemistry, 1973, 12, 1858). A neuraminadase from Clostridium perfringens will hydrolyse also the sialic acid from monosialoganglioside in the presence of bile salts (D. A. Wenger and S. Wardell, J. Neurochem., 1973, 20, 607)" ceramide(1 g- 1) o-glucose(4 g-- 1) D-galactose(3 ~ 2)N-acetylneuraminic acid

MONOSIALOGANGLIOSIDE

N-acetylgalactosamine

t

1

B

D-galactose Monosialoganglioside accumulates in the brain in an inherited disorder known as "generalised gangliosidosis" (Svennerholm, Biochem. J., 1969, 111, 5P; O'Brien et al., Science, 1969, 163, 946; Brady et al., Biochim. Biophys. Acta, 1970, 210, 193; Wolfe et al., Adv. Exp. Med. Biol., 1972, 19, 373; T. Abe and R. Okada, Jap. J. exp. Med., 1972, 42, 543) due to the deficiency of a fl-galactosidase. Monosialoganglioside is the specific receptor for cholera and tetanus toxins (see Wiegandt et al., Europ. J. Biochem., 1974, 48, 103; Van Heyningen, Science, 1974, 183, 656; Nature, 1974, 249, 415; Bull. Inst. Pasteur, 1974, 72, 433; L. Svennerholm et al., Medical Biology, 1974, 52, 229; see also P. Cuatrecasas et al., Biochemistry, 1973, 12, 3547, 3558, 3567, 3577, 4253; Proc. Nat. Acad. Sci., 1974, 71, 4224). The carbohydrate sequence of the monosialoganglioside has been confirmed by mass spe~ztrometry (Karlsson et al., Chem. Phys. Lipids, 1974, 12,271). The fifth ganglioside which is present in small amounts in normal brain but accumulates in Tay-Sach's disease affords equimolar proportions of th six basic constituents on hydrolysis. Dilute acid hydrolysis of any of the four major gangliosides of normal human brain rapidly removes the sialic acid to give a common tetraglycosylceramide ("asialoganglioside")

420

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

ceramide(1 d- 1) D-glucose(4 ~ 1) D-galactose(4 d- 1)N-acetylgalactosamine ASIALOGANGLIOSIDE

(i)fl D-galactose

The positions of the sugars in the asialoganglioside were indicated by partial acid hydrolysis (S. Bogoch, Biochem. J., 1958, 68, 319; Svennerholm, Biochem. biophys. Res. Comm., 1962, 9, 436; R. Kuhn and H.. Egge, Ber., 1963, 96, 3338). Partial acid hydrolysis also gave a crystalline 3-O-(fl-D-galactosido)N-acetylgalactosamine and a disaccharide containing galactose and Nacetylgalactosamine in which galactose had the free reducing group (E Klenk et al., Z. physiol. Chem., 1962, 330, 140; Kuhn and Wiegandt,Ber., 1963, 96, 866; Svennerholm, loc. cit.). These and related oligosaccharides have been synthesised for comparative purposes (D. Shapiro et al., Chem. Phys. Lipids, 1970, 5, 80; J. org. Chem., 1969, 34, 2652; 1970, 35, 229, 1464, 2436; 1971, 36, 832). Hydrolysis of the gangliosides with acetic anhydride, acetic acid and sulphuric acid ("acetolysis") gave oligosaccharides still containing sialic acids and these were hydrolysed with acid and their structures determined (Kuhn and Wiegandt, loc. cit.; see also G. A. Johnson and McCluer, Biochim. Biophys. Acta, 1964, 84, 587). Complete methylation of the asialoganglioside and monosialoganglioside and isolation of the individual methylated sugars after acid hydrolysis gave the linkage positions of the various sugars. P O Ceramide(l~---1)D-glucose(4---1)

D-galactose(3~--2)N-acetylneuraminic

N-acetylga lactosamine

acid

N-acotylneuraminic acid

DISIALOGANGLIOSIDE GDlb

D-galactose

C e r a m i d e (1---1 ) D - g a l a c t o s e ( 4 - ~ - I ) D - g a l a c t o s e ( 3 ~ - - - 2 ) N - a c e t y l n e u r a m i n i c acid

N- ac e t y l g a I ac t o s a m i ne

DISIALOGANGLIOSIDE G Dla

D - g a l a c t o s e ( 3 ~ - - 2 ) N - a c e t y l n e u r a m inic acid

2

CONTAINING

SIALIC ACIDS

421

Methylation studies of the two disialogangliosides showed that the linkage of the extra sialic acid molecule was as shown in the formulae (Kuhn and Egge, loc. cit.; Klenk and W. Gielen, Z. physiol. Chem., 1963, 330, 218; Klenk and W. Kunau, ibid., 1964, 335,275). The mass spectra of a disialoganglioside has also been studied (K. A. Karlsson et al., Biochemistry, 1974, 13, 3643). The trisialoganglioside (GT) contains an extra sialic acid in each of positions occupied by those in the two disialogangliosides (Kuhn and Wiegandt, Z. Naturforsch., 1963, 18b, 541): P P Ceram ide( 1 ----1 ) D-glucose(4 --,-- 1) D-galactose( 3 ~-- 2)N- acetylneuraminic acid TRISIALOGANGLIOSIDE

GT

/9

N-acetylgalactosamine

N-acetylneuraminic acid

N-acetylneuraminic acid (2---,,-3) D-galactose The anomeric configuration of the sialic acid linkages (~) is the same in each position (R. T. C. Huan9 and Klenk, Z. physiol. Chem., 1972, 353, 679). The biosynthesis of trisialoganglioside has been studied (R. Caputto et al., Biochem. biophys. Res. Comm., 1974, 59, 1). The ganglioside which accumulates in Tay-Sach's disease ("Tay-Saeh's ganglioside"), lacks the terminal galactose of a monosialoganglioside (Svennerholm, J. Neurochem., 1963, 10, 613; Klenk et al., Z. physiol. Chem., 1963, 334, 186; 1964, 335, 275; Makita and Yamakawa, Jap. J. exp. Med., 1963, 33, 361; Ledeen and K. Salsman, Biochemistry, 1965, 4, 2225):

ceramide(1 ~- 1) D-glucose(4 ~- 1) D-galactose(3 ~ 2)N-acetylneuraminic acid

TAY-SACH'S GANGLIOSIDF GM2 N-acetylgalactosamine The accumulation of this ganglioside in the hereditary disease is due to the deficiency of a specific fl-N-acetylhexosaminadase (see Brady et al., Adv. Exp. Med. Biol., 1972, 19, 277; J. S. O'Brien, Federation Proc., 19"73, 32, 191; J. F. Tallman, Chem. Phys. Lipids, 1974, 13, 292). Together with the corresponding sialic acid free derivative Casialo Tay-Sach's ganglioside") and globoside it also accumulates in Sandhoff's disease which is due to a general deficiency of fl-N-acetylhexosaminadases (K. Sandhoff and Jatzkevitz, Adv. Exp. Med. Biol., 1972, 19, 305; E. H. Kolodny, ibid., p. 321; R. J. Desnick et al., ibid., p. 351).

422

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

The enzymic hydrolysis of monosialoganglioside to Tay-Sach's ganglioside (S. Gatt, Biochim. Biophys. Acta, 1967, 137, 192) and the reverse enzymic process (J. A. Dain et al., ibid., 1971, 231, 385; Biochem. J., 1970, 118, 247), the enzymic synthesis of disialoganglioside from monosialoganglioside (Caputto et al., ibid., 1971, 125, 1131; S. Roseman et al., J. biol. Chem., 1968, 243, 5804; M. C. M. Yip, Biochim. Biophys. Acta, 1973, 306, 298) and of GDIaby addition of galactose to a precursor lacking galactose (Brady et al., J. biol. Chem., 1972, 247, 2322) have been studied. (For other studies on the biosynthesis of the gangliosides see M. Holm and Svennerholm, J. Neurochem., 1972, 19, 609; Holm, ibid., p. 623; J. L. di Cesare and Dain, ibid., p. 403; Yip, Biochim. Biophys. Acta, 1972, 273, 374; H. J. F. Maccioni et al., FEBS Letters, 1972, 23, 136; T. W. Keenan et al., J. biol. Chem., 1974, 249, 310; and for reviews see Caputto and Maccioni, Molecular and Cellular Biochem., 1974, 4, 97; P. H. Fishman, Chem. Phys. Lipids, 1974, 13, 305.) Tay-Sach's gangliosides radioactively labelled in the sialic acid residue or in the N-acetylgalactosamine residue have been prepared and the metabolism of these compounds by a neuraminadase and a hexosaminadase studied (Brady et al., J. biol. Chem., 1972, 247, 7570; J. Labelled Comp., 1972, 8, 483). Considerable alterations in the ganglioside composition of cells which have been transformed by oncogenic viruses and chemical carcinogens, due to the absence of specific glycosyl transferases in the transformed cells, have been observed (for reviews see Hakomori, Adv. Cancer Res., 1973, 18, 265; Brady and Fishman, loc. cit.; Brady et al., J. biol. Chem., 1975,

250, 55). The oligosaccharide portion of the gangliosides can be separated from the ceramide portion by ozonolysis followed by basic hydrolysis (H. Wiegandt and G. Baschang, Z. Naturforsch., 1965, 20b, 164) or by the action of osmium tetroxide and sodium periodate followed by basic hydrolysis (S. Hakomori, J. Lipid. Res., 1966, 7, 789). Stearic acid is the major fatty acid of brain gangliosides from various sources (see Y. Kishimoto and N. S. Radin, ibid., 1966, 7, 141) and the sphingosine bases are composed mainly of a mixture of C18- and C2o-sphingosines (see K. Sambasivarao and R. H. McCluer, ibid., 1964, 5, 103; E. Klenk et al., Z. physiol. Chem., 1970, 351,335; 1971, 352, 106). Several other minor gangliosides have been described (see Klenk et al., ibid., 1967, 348, 149, 1261; 1968, 349, 288; H. Seifert, Klin. Woch., 1966, 44, 469; Tettamanti et al., Nature, 1965, 206, 192; Biochim. Biophys. Acta, 1964, 84, 756; McCluer et al., ibid., 1966, 116, 279). A disialopentaglycosyl-ceramide of the following structure, which is resistant to the neuraminadase of Vibrio cholerae has also been isolated from human brain (L. Svennerholm et al., J. biol. Chem., 1973, 248, 740):

2

CONTAINING SIALIC ACIDS

423

ceramide(1 g- 1) D-glucose(4 g- 1) D-galactose(3~ 2)N-acetylneuraminicacid

N-acetylgalactosamine

N-acetylgalactosamine(1 ~ 4) D-galactose(3 ~- 2)N-acetylneuraminicacid Gangliosides are also present in extraneural tissues (for reviews see T. Yamakawa, in "Lipoide", Springer, Berlin, 1966; Wiegandt, Chem. Phys. Lipids, 1970, 5, 198; Z. physiol. Chem., 1973, 354, 1049). Those of the bovine adrenal medulla (Ledeen et al., Biochemistry, 1968, 7, 2287; K. Puro, Biochim. Biophys. Acta, 1969, 187, 401; Acta Chem. Scand., 1970, 24, 13), retina (S. Handa and R. M. Burton, Lipids, 1969, 4, 205; Svennerholm et al., Biochim. Biophys. Acta, 1972, 280, 356), plasma (R. K. Yu and Ledeen, J. Lipid Res., 1972, 13, 680; Adv. Exp. Med. Biol., 1972, 19, 77), and spleen (Wiegandt and H. W. Biicking, Europ. J. Biochem., 1970, 15, 287) and rabbit muscle (R. Caputto et al., J. Lipid Res., 1973, 13, 810) have been investigated. The spleen ganglioside which is also present in erythrocytes and is the major ganglioside of peripheral nervous tissue contains N-acetylglucosamine and has the following structure n = 1 (Wiegandt and Biicking, loc. cit.; Svennerholm et al., J. biol. Chem., 1973, 248, 2634; Medical Biology, 1974, 52, 240; Ando, J. Biochem., Tokyo, 1973, 73, 893; Hakomori and Siddiqui, Biochim. Biophys. Acta, 1973, 330, 147; Wherret, ibid., 1973, 326, 63; Koscielak, Europ. J. Biochem., 1973, 37, 214). The neuraminadase from Vibrio cholerae converts this ganglioside into the neutral tetraglycosyl-ceramide which is also a normal constituent of tissues" P P Ceramide(1 ----1 ) D-glucose(4---1 ) D-galactose

N-acylneuraminic acid ( 2 - - - 3)D-galactose(1----4 )

N-acetylglucosamin n

A related lipid containing extra sugars (n= 2) has also been isolated from human spleen (Wiegandt, Europ. J. Biochem., 1974, 45, 367). The major ganglioside ("hematoside", GM3) from canine and equine erythrocytes (see Klenk and G. Padberg, Z. physiol. Chem., 1962, 327, 249) which is also found in normal human spleen, liver, brain, plasma and erythrocytes (Svennerholm, Acta Chem. Scand., 1963, 17, 860; Ando, J. Biochem., Tokyo, 1973, 73, 893; Kuhn and Wiegandt, loc. cit., R. V. P. Tao and Sweeley, Biochim. Biophys. Acta, 1970, 218, 372) has the following structure:

424

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

ceramide(1 g-- 1) D-glucose(4 g-- 1) D-galactose HEMATOSIDE GM3

N-acylneuraminic acid

The N-acyl group of the sialic acid residue may be glycolyl or acetyl in the hematoside from horse and dog (Handa and Yamakawa, Jap. J. exp. Med., 1964, 34, 293) and is acetyl in the human spleen and brain glycolipids. Hematoside has been deacylated to give a "lysohematoside" (T. Taketomi and N. Kawamura, J. Biochem., Tokyo, 1970, 68, 475). A derivative of hematoside, in which the neuraminic acid is substituted with an O-acetyl group, is also present in horse erythrocytes (T. Saito and Hakomori, Biochemistry, 1969, 8, 5082). The enzymic synthesis of Tay-Sach's ganglioside from hematoside has been demonstrated (Roseman et al., Federation Proc., 1966, 25, 587). A fatal sphingolipidosis resulting in an accumulation of hematoside and an absence of higher gangliosides has been observed. Since hematoside is an intermediate in the biosynthesis of higher gangliosides it has been suggested that this is the first example of a sphingolipidosis with a defect in biosynthesis rather than degradation (Brady et al., New Eng. J. Med., 1974, 291,929; Science, 1975, 187, 68). The mass spectrum ofhematoside has been studied (Karlsson et al., Chem. Phys. Lipids, 1974, 12, 271). An inner ester of N-glycolyl-hematoside of the following structure is readily formed in acetic acid solution by esterification of the carboxyl group of the sialic acid residue with the 2-hydroxy group of galactose (McCluer and J. E. Evans, Adv. Exp. Med. Biol., 1972, 19, 95): HO CH20H

0 0

CH20 H ~\L~O O H O ~ . . ~ ~ ~ 0 C H2 HO H+NHCOR H---~-OH

H20H \ OH NHCO.CH20H

R

A ganglioside ("disialohematoside", GD3) from cat erythrocytes has a similar structure to that of hematoside but contains an extra sialic acid residue joined to the 8-position of the sialic acid already present (Handa, Jap. J. exp. Med., 1965, 35, 331):

2

SPHINGOPHOSPHOLIPIDS

425

Ceramide(1 ~- 1) D-glucose(4 ~ 1) D-galactose oI

IA, GD3

2

sialic acid (2 -~ 8)sialic acid A similar ganglioside in human brain acts as a serotonin acceptor (W. Gielen, Z. Naturforsch., 1968, 23b, ll7; see also G. I. Samokhvalov et al., Vopros. Med. Khim., 1972, 18, 477) and is also the major ganglioside present in butter milk (Huang, Biochim. Biophys. Acta, 1973, 306, 82), bovine mammary gland an milk (Keenan, ibid., 1974, 337, 255) and bovine retina (M. Holm and J. E. Mansson, FEBS Letters, 1974, 38, 261). Uncharacterised gangliosides are present in sea urchin gametes (Y. Isono and Y. Nagai, Jap. J. exp. Med., 1966, 36, 461; G. P. Smirnova, Carbohydrate Res., 1969, 9, 366; N. K. Kochetkov et al., Doklady Chem., 1970, 192, 360; 193, 486; Biochim. Biophys. Acta, 1973, 326, 74) and in the starfish Distolasterias nippon (Kochetkov et al., Biokhimiya, 1970, 35, 775; Doklady Akad. Nauk., 1973, 208, 981) and tetra- and penta-sialogangliosides are present in the brain of the cod fish (I. Ishizuka and Wiegandt, Biochim. Biophys. Acta, 1972, 260, 279). The gangliosides occur in the brain in nerve endings (see Wiegandt, J. Neurochem., 1967, 14, 671; E. G. Lapetina et al., Biochim. Biophys. Acta, 1967, 135, 33) but their function is unknown. The circular dichroism of several gangliosides has been recorded (A. L. Stone and E. H. Kolodny, Chem. Phys. Lipids, 1971, 6, 274). (c) Sphingophospholipids. Derivatives of ceramide 1-phosphate (i) Sphingomyelin, ceramide 1-(choline phosphate) 0 C H20- P - O C H2.CH2NMe 3 H NHCOR H~OH R

(I) Sphingomyelin was first discovered in brain by J. L. W. Thudichum in 1884 and occurs in many animal tissues. Acid hydrolysis liberates sphingosine, fatty acids, choline and choline phosphate. Serine and serine phosphate can be obtained from sphingomyelin after oxidation and hydrolysis showing that the choline phosphate is linked to the primary hydroxyl group of slahingosine (see E. Stotz et al., J. Amer. chem. Soc., 1953, 75, 310, 313).

426

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Dihydrosphingosine (Thannhauser and N. F. Boncoddo, J. biol. Chem., 1948, 172, 141 ; Y. Fujino and T. Negishi, Agric. and biol. Chem., 1962, 26, 126), C x6- and C17-homologues of sphingosine (K. A. Karlsson, Acta Chem. Scand., 1964, 18, 2395; C. Michalec and Z. Kolman, Naturwiss., 1966, 53, 254; M. Popovic, Biochim. Biophys. Acta, 1966, 125, 178; W. R. Morrison, ibid., 1969, 176, 537) are also present in natural sphingomyelins. The major long-chain base of the sphingomyelin from the honey bee, Apis mellifera, is a C~4-homologue of sphingosine (Karlsson et al., ibid., 1972, 270, 117). A C~4-~5 unsaturated derivative of sphingosine is a major component of serum sphingomyelins (see p. 396) and phytosphingosine is present in kidney sphingomyelins (Karlsson and G. O. Steen, ibid., 1968, 152, 798). Long-chain fatty acids (C22and C24) are common in natural sphingomyelins (see Svennerholm et al., ibid., 1966, 125, 178; W. A. Spencer and R. Schaffrin, Canad. J. Biochem., 1964, 42, 1659; Morrison, loc. cir.). Thin-layer and paper chromatography usually resolve natural sphingomyelins into two distinct bands depending on the chain lengths of the fatty acids present (Michalec and Kolman, J. Chromatog., 1967, 31, 636, 640; O. Minari et al., J. Biochem., Tokyo, 1968, 64, 275; P. Mandel et al., J. Neurochem., 1972, 19, 831; G. Soula et al., Biochimie, 1974, 56, 131). Methods for the isolation of sphingomyelins from the lipids of bovine red blood cells (D. J. Hanahan, Biochem. Prep., 1961, 8, 121), bovine heart lipids (Rapport and B. Lerner, J. biol. Chem., 1958, 232, 63), human and rat brains (K. Bernhard et al., Helv., 1963, 46, 601) and human plasma (Sweeley, J. Lipid Res., 1963, 4, 402) have been described in detail. Three routes for the enzymic synthesis of sphingomyelins have been described, by (i) reaction between ceramides and cytidine diphosphate choline; (ii) acylation of sphingosine 1-(choline phosphate) (see Y. Fujino et al., J. biol. Chem., 1968, 243, 4650; Biochim. Biophys. Acta, 1968, 152, 428; M. Sribney et al., Canad. J. Biochem., 1973, 51, 1498). Recent research indicates however hat sphingosine 1-(choline phosphate) is not acylated to sphingomyelin directly but is degraded to choline phosphate and sphingosine which is reacylated to give a ceramide. The choline phosphate is incorporated into lecithin (P. Stoffel and G. Assmann, Z. physiol. Chem., 1972, 353, 65); (iii) biosynthesis of sphingomyelin by the transfer of choline 1phosphate from lecithin to ceramide has also been described (see H. Diringer and M. A. Koch, Z. physiol. Chem., 1973, 354, 1661; M. D. Ullman and Radin, J. biol. Chem., 1974, 249, 1506; W.-D. Marggral and F. A. Anderer, Z. physiol. Chem., 1974, 355, 803). Sphingomyelins accumulate in the tissues (particularly spleen) in Niemann-Picks' disease, an heriditary disease due to the deficiency of an enzyme which normally degrades sphingomyelins to ceramides and choline phos-

2

SPHINGOPHOSPHOLIPIDS

427

phate (see P. B. Schneider and Kennedy, J. Lipid Res., 1967, 8, 202; S. Gatt et al., J. biol. Chem., 1966, 241, 3731; R. O. Brady et al., Science, 1967, 155, 86; Biochem. biophys. Res. Comm., 1969, 34, 582; S. Fowler, Biochim. Biophys. Acta, 1969, 191, 481; O. Giardini et al., Clin. Chim. Acta, 1972, 42, 15). Prenatal diagnosis of this condition is possible by measuring the activity of this enzyme in cultured cells from amniotic fluid (Brady et al., Amer. J. Human Genetics, 1971,23, 533). Sphingomyelins also accumulate in the eye during differentiation and ageing and the sphingomyelinase of the lens (J. H. Veerkamp et al., Biochim. Biophys. Acta, 1973, 306, 329) and the molecular species of sphingomyelins in the lens (Karlsson et al., Exp. Eye Research, 1974, 19, 477) have been studied. Sphingomyelins are also cleaved by phospholipase C from Clostridium perfringens and this method of hydrolysis has.been useful for determining the different species of ceramides present in natural sphingomyelins (Hay and Gray, Chem. Phys. Lipids, 1969, 3, 59; B. and K. Samuelsson, J. Lipid Res., 1969, 10, 47; E. L. Hirvisalo and Renkonen, ibid., 1970, 11, 54; I. Pastan et al., J. biol. Chem., 1968, 243, 3750; Karlsson, Acta Chem. Scand., 1968, 22, 3050). An enzyme which cleaves sphingomyelins to ceramide 1phosphates has also been described (C. Michalec et al., Biochim. Biophys. Acta, 1967, 144, 180) and the hydrolysis of sphingomyelins in "liposomes" by a sphingomyelinase has been studied (Gatt et al., FEBS Letters, 1973, 30, 281). Specific antibodies to sphingomyelin have been prepared by conjugating the lipid to proteins (D. Shapiro et al., Immunochem., 1973, 10, 735; R. Arnon and D. Teitelbaum, Chem. Phys. Lipids, 1974, 13, 352). Sphingomyelins are white crystalline substances, slightly soluble in cold alcohol and pyridine, insoluble in acetone and ether and form emulsions with water. They form complexes with cadmium chloride. Both sphingomyelin and dihydrosphingomyelin have been synthesised (see Shapiro et al., Chem. Phys. Lipids, 1968, 2, 223; "Chemistry of the Sphingolipids", Hermann, Paris, 1969; N. A. Preobrazhenskii et al., Zhur. org. Khim., 1968, 4, 210; see also R. P. Evstigneeva et al., ibid., 1973, 9, 31; 1974, 10, 1621; H. Alpes, Chem. Phys. Lipids, 1974, 13, 109). The hydrolysis of sphingomyelins under acidic and basic conditions has been studied (A. J. de Koning and K. B. McMullan, Biochim. Biophys. Acta, 1965, 106, 519). Sphingomyelins have been converted into ceramide 1-(2-N,N-dimethylaminoethyl phosphate) by reaction with sodium benzenethiolate and the N,N-dimethyl compound remethylated with [14C]methyl iodide to give labelled sphingomyelins (Stoffel et al., Z. physiol. Chem., 1971, 352, 1058). The instability of these radioactive sphingomyelins has been investigated (Kanfer et al., Chem. Phys. Lipids, 1973, 10, 149). The physical structures of films (R. A. Long et al., Biochem. biophys. Res. Comm., 1971, 45, 167) and aqueous dispersions

428

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

(G. G. Shipley et al., J. Lipid Res., 1974, 15, 124) of sphingomyelins have been studied.

(2) Sphingosine l-choline phosphate), H

H

O ii i~ CH3(C H2) 2C::= C - C -- C --C H 2 0 - P - OC H2.CH2N Me 3 I I I 6E ) H OH NH 2 I

H

I

I

is obtained by the acid hydrolysis of sphingomyelin (Dawson, Biochem. J., 1958, 68, 357; H. Kaller, Biochem. Z., 1961, 334, 451; 1962, 335, 518; Taketomi and Yamakawa, Jap. J. exp. Med., 1967, 37, 423). It has been synthesised (Shapiro et al., Chem. Phys. Lipids, 1967, 1, 183) and its metabolism in rat liver has been studied (Stoffel and Assmann, Z. physiol. Chem., 1972, 353, 65). It has been attached to a Sepharose column and the product used in the technique of affinity chromatography for the purification of the enzyme sphingomyelinase (H. R. Sloan et al., Adv. Exp. Med. and Biol., 1972, 19, 175). ( ii) Ceramide 1-(2-aminoethyl phosphonate). "Ceramide ciliatine" H

H

H

I

I

I

0 II C Ha(C H 2)12C = C - C -- C --C H20 - P - C H2.C H2N H 2 I

I

I

H

OH NH

I

OH

I

COR

This compound, related to sphingomyelin but containing 2-aminoethyl phosphonic acid in place of choline phosphate occurs in the lipids of the sea anemone, Anthopleura elegantissima (G. Simon and Rouser, Lipids, 1967, 2, 55) and Metridium senile (Karlsson et al., Biochim. Biophys. Acta, 1974, 337, 204), of the shellfish Corbicula sandai (Hori et al., J. Biochem., Tokyo, 1967, 62, 67), of tureen protozoa (Dawson and Kemp, Biochem. J., 1967, 105, 837), of Haliotis midae (De Konin9, J. Sci. Food Agric., 1966, 17, 460) and in the water snail Lymnea stagnalis (C.-R. Lian9 and K. P. Strickland, Canad. J. Biochem., 1969, 47, 85) and in invertebrate nervous tissue (Y. Komai et al., Biochim. Biophys. Acta, 1973, 316, 271). The gas chromatography-mass spectrometry of oyster ceramide ciliatine has been studied (T. Matsubara and A. Hayashi, Biochim. Biophys. Acta, 1973, 296, 171). This compound and its dihydro derivative have been synthesised (E. Baer and G. R. Sarma, Canad. J. Biochem., 1969, 47, 603). It is hydrolysed by phospholipase C (Hori et al., J. Biochem., Tokyo, 1968, 64, 533; K. Saito and K. Mukoyama, Biochim. Biophys. Acta, 1968, 164, 596). An N-methyl derivative of ceramide ciliatine has been isolated from the marine shell-fish Turbo

2

SPHINGOPHOSPHOLIPIDS

429

cornutus and from the marine snail Monodonta labio (see A. Hayashi and F. Matsuura, Chem. Phys. Lipids, 1973, 10, 51) and the molecular species of the T..cornutus lipid have been investigated (Hayashi et al., J. Biochem., Tokyo, 1973, 74, 49). A similar compound has also been isolated from Tetrahymema pyriformis (C. V. Viswanathan and A. Rosenber9, J. Lipid Res., 1973, 14, 327) and an N-methyl-N-acyl-derivative is present in Corbicula sandai (Hori and I. Arakawa, Biochim. Biophys. Acta, 1969, 176, 898) and a galactosyl derivative is present in Turbo cornutus (Hayashi and Matsuura, ibid., 1971, 248, 133). ( iii ) Ceramide 1-( 2-aminoet hyl phosphate), "Sphingoethanolamine" H I

H I

H I

0

II C H3(CH2 )2C = C - C --C - C H 2 0 - P - O C H2.C H2N H2 I I I I H OH NH OH I

COR

This lipid was first isolated from the pond snail, Heterogen longispira (see Hori et al., Biochim. Biophys. Acta, 1968, 152, 211) and has subsequently been identified in rumen protozoa and in the blowfly, Calliphora erythrocephala (Dawson and Kemp, Biochem. J., 1968, 106, 319) in Bacteroides melaninogenicus (D. C. White et al., J. Lipid Res., 1969, 10, 528; J. Bacteriol., 1970, 101, 84) and Bacteroides ruminicola (J. E. Kunsman and D. R. Caldwell, Applied Microbiol., 1974, 28, 1088), in the pupae of the green-bottle fly (Hori et al., J. Biochem., Tokyo, 1968, 64, 123) in a marine bivalve (Hori et al., ibid., 1973, 73, 191) and in chicken and rat liver (M. Sribney et al., Canad. J. Biochem., 1972, 50, 166) where its biosynthesis from ceramide and CDP-ethanolamine was also studied. The biosynthesis of this lipid in B. melaninogenicus (M. Lev and A. F. Milford, Biochem. biophys. Res. Comm., 1971, 45, 358) and in the rumen protozoon Entodinium caudatum (T. E. Broad and Dawson, Biochem. J., 1973, 134, 659) have also been studied. This lipid is hydrolysed by phospholipase C (Hori et al., J. Biochem., Tokyo, 1968, 64, 533.; Saito and Mukoyama, loc. cir.). The racemic dihydro derivative and its N,N-dimethyl derivative have been synthesised (Shapiro et al., Chem. Phys. Lipids, 1968, 2, 223; E. N. Zvonkova et al., Zhur. obshche]" Khim., 1970, 40, 942) and the N,N-dimethyl derivative can also be obtained by degradation of sphingomyelin (see p. 427).

(iv) Ceramide 1-(glycerol 1-phosphate) H H H 0 I I I II CHa(CH2)12C=C-C--C--CH20-P-OCH2-CH(OH)-CH2R I I I I H OH NH OH I COR.

430

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

A lipid with this structure (R = OH) and the corresponding phosphate R = OPO(H)2 are are present in the lipids of Bacteroides melaninogenicus and Acholeplasma axanthurn (White et al., loc. cit." Lipids, 1970, 5, 56; Plackett et al., J. Bacteriol., 1970, 104, 798) and a related compound (R = NH2 or the isomer of this compound) containing 3-aminopropane-l,2diol is reported to be present in the bacterial fractions of sheep rumen (Kemp et al., Biochem. J., 1972, 130, 221). A racemic ceramide 1-(glycerol 1-phosphate) has been synthesised (R. P. Evstigneeva et al., Zhur. org. Khim., 1973, 9, 2490).

(v) Phytoglycolipids Phytoglycolipids are sphingolipids isolated from plant seed lipids after mild alkaline hydrolysis. On further hydrolysis they give phytosphingosines, fatty acids, phosphoric acid, inositol, glucuronic acid, glucosamine, arabinose, mannose, galactose and (from some seed lipids) fucose (H. E. Carter et al., J. Amer. Oil Chemists' Soc., 1958, 35, 355; 1962, 39, 107; F. Aylward and B. Nichols, J. Sci. Food and Agric., 1961, 12, 645; H. Wagner et al., Z. Naturforsch., 1969, 24b, 922; for review see Carter, in "Lipoids", Springer, Berlin, 1966). Alkaline hydrolysis of the phytoglycolipid from corn produces ceramide phosphates, oligosaccharide phosphates and oligosaccharides indicating a heterogeneous nature. The oligosaccharides have been separated by ionexchange chromatography to give tri~octasaccharides. Dilute acid hydrolysis of the tetra~octasaccharides gives the crystalline trisaccharide (containing inositol, glucuronic acid and glucosamine) which is a common component of all plant seed phytoglycolipids (Carter et al., J. biol. Chem., 1958, 233, 1309; 1964, 239, 743; Biochemistry, 1964, 3, 1103). The alkaline hydrolysis used in the degradation of the phytoglycolipid also causes some isomerisation of the glucuronic acid to iduronic acid (idem, Biochemistry, 1969, 8, 389). When the trisaccharide is treated with nitrous acid the glucosamine is removed to give a disaccharide containing inositol and glucuronic acid and treatment of the other oligosaccharides with nitrous acid gives a trisaccharide containing inositol, glucuronic acid and mannose. The structure of the tetrasaccharide isolated by chromatography has been established by oxidation with periodate and methylation studies (idem, ibid., 1969, 8, 383) and the following partial structure represents the phytoglycolipids from corn. Both phytosphingosine and dehydrophytosphingosine are present in phytoglycolipids (Carter and H. S. Hendrickaon, Biochemistry, 1963, 2, 389). Phytoglycolipids are also present in the leaves of the bean Phaseolus vulgaris

3

MISCELLANEOUS

431

glucuronic acid (4~--1) glucosamine

~ H H H

I I C H3(C H2)3CI - - C I

O

I II C-OH 20--P-O I

OH OH NH I

I

HO

^

aOO ~~O:::)H

arabinose H

I

OH

galactose"

mannose

COR (Carter and J. L. Koob, J. Lipid Res., 1969, 10, 363). A related compound which contains all the component sugars except glucosamine has been found in seed lipids after countercurrent distribution of the total inositolcontaining lipids (Carter and A. Kisic, J. Lipid Res., 1969, 10, 356) and a related glycolipid, "mycoglycolipid" containing only inositol and mannose in the oligosaccharide portion has been isolated from yeasts (Wagner and Zofcsic, Biochem. Z., 1966, 346, 343). Several other inositol-containing sphingolipids have been isolated from Saccharomyces cerevisiae and one of these is reported to have the following structure (S. Steiner and R. L. Lester, J. Bacteriol., 1972, 109, 81 ; K. Tyorinoja et al., Biochem. J., 1974, 141,133):

O II

ceramide--(O--P--O--inositol)2-- mannose I OH A related compound (lacking the mannose) is also present in Neurospora crassa (Lester et al., J. biol. Chem., 1974, 249, 3388) and ceramide 1-(inositol phosphate) also occurs in yeast (idem, ibid., 1974, 249, 3395).

3. Miscellaneous phospholipids and glycolipids* Several glycolipids (some containing phosphoric acid) which do not contain sphingosine bases or glycerol have been isolated from micro-organisms (for reviews see J. H. Law, Ann. Reviews Biochem., 1960, 29, 131; Lederer, Adv. Carbohydrate Chem., 1961, 16, 207; Angew. Chem., 1964, 76, 241; Chem. Phys. Lipids, 1967, 1,294; J. Asselineau, "Les Lipides Bact6riens", Hermann, Paris, 1962; M. Kates, Adv. Lipid Res., 1964, 2, 17). They are mainly fatty acid esters of sugars or glycosides of long-chain alcohols. *see also Rodd's, C.C.C. Vol. I F, p. 712 et seq.

432

PHOSPHOLIPIDS

(I) Rhamnolipid,

AND GLYCOLIPIDS

O-CI H-CH2-C O2CI H - C

/

( CI H2 )6 CH 3

21

H2"C O2H

( C H 2 )6 I CH 3

o

HO

OH

contains two molecules of L-rhamnose and two molecules of D-3-hydroxydecanoic acid (linked as shown in the formula) and has been isolated from the culture medium of Pseudomonas pyocyanae (S. Bergstr6"m et al., Arch. Biochem. Biophys., 1946, 10, 165)and Pseudomonas aerueinosa (F. G. Jarvis and M. J. Johnson, J. Amer. chem. Soc., 1949, 71, 4124; J. R. Edwards and J. A. Hayashi, Arch. Biochem. Biophys., 1965, 111,415). (2) Cord factor is present in virulent forms of Mycobacterium tuberculosis and is so named because of its association with the aggregation of these organisms in long cords. Methylation and periodate oxidation studies have shown that cord factor contains two molecules of mycolic acid (for the structure of the mycolic acids see Rodd's C.C.C. Suppl. Vol. I C/D, 1973, p. 307, ed. M. F. Ansell) esterified to the 6 and 6' positions of trehalose (H. Noll et al., Biochim. Biophys. Acta, 1956, 20, 299) and this structure has been confirmed by synthesis (T. Gendre and Lederer, Bull. Soc. chim. Fr., 1956, 1478; G. Brocherd-Ferrdol and J. Polonsky, ibid., 1958, 714). A similar material containing corynomycolic acid and corynomycolenic acid has been isolated from Corynebacterium diphtheriae (T. Ioneda et al., Biochem. biophys. Res. Comm., 1963,13, 110; Lederer et al., Europ. J. Biochem., 1967, 1, 353;2, 460). Similar materials are also present in Nocardia strains (I. Yano et al., J. gen. Applied Microbiol., 1971, 17, 329) and contain corynomycolic and nocardic acids. Those present in Nocardia asteroides and rhodochrons have been investigated by mass spectrometry (Lederer et al., Chem. Phys. Lipids, 1970, 4, 375). A 2,3,6,6'-tetra-acyl-2'-sulphate of trehalose is found in Mycobacterium tuberculosis Strain H37Rv (M. B. Goren, Biochim. Biophys. Acta, 1970, 210, 116, 127; Lipids, 1971, 6, 40; Biochimie, 1973, 55, 559, 569, 575; Infection and Immunity, 1974, 10, 733). An octa-ester of trehalose with phleic acid (hexatriaconta-4,8,12,16,20-pentaenoic acid) is the major lipid of a series found in Mycobacterium phlei (C. P. Asselineau et al., Europ. J. Biochem., 1972, 28, 102). Synthetic analogs of cord factor, e.g. 6,6'-dimycolylsucrose have been synthesised (see M. Kato and J. Asselineau, ibid., 1971, 22, 364; Biochemistry, 1971, 10, 72) and their toxicity compared with cord factor. Antibody production to cord factor complexed

3

MISCELLANEOUS

433

with methylated bovine serum albumin has been studied (Kato, Infection and Immunity, 1972, 5, 203; 1973, 7, 9, 14). (3) Ascarosides have been isolated from the eggs of the parasitic worm Parascaris equorum and separated into three fractions. They are glycosides of ascarylose (3,6-dideoxy-L-arabinohexose)with long-chain alcohols (see C. Fouquey et al., Bull. Soc. Chim. biol., 1962, 44, 69; P. F. Jezyk and D. Fairbairn, Comp. Biochem. Physiol., 1967, 23, 691,707). (4) Mycosides.MycosideB is one of a class of glycolipids from the bovine strain of Mycobacterium tuberculosis (D. W. Smith et al., Ann. N.Y. Acad. Sci., 1957, 69, 145). It is a mixture of homologous compounds differing in the nature of the fatty acid constituents. The main component contains one molecule of 3-O-methyl-D-rhamnose joined by a fl-glycoside linkage to the phenolic hydroxyl group of a phenolic methoxyglycol. The alcohol groups are esterified to palmitic acid and C29-mycocerosic acid (for review see Lederer, Chem. Phys. Lipids, 1967, 1,294). Mycoside G (M. GastambideOdier, Europ. J. Biochem., 1973, 33, 81) contains a 2-O-mycolyl-3-O-methyl6-deoxypyranoside residue. (5) Torulopsis glycolipids have been isolated from the lipids of species of the yeast Torulopsis (see Tulloch et al., Canad. J. Chem., 1968, 46, 345, 3337, 3727; J. org. Chem., 1972, 37, 2868). The major components are the lactone (shown below) and the corresponding hydroxy acid, which are derivatives of sophorose glycosidically linked to L- 17-hydroxyoctadecanoic acid:

CH2OAc Ho~O HO~ O - C - H 0 CH2OAc /

0

O=C

Me,

0

(C H2)15

A similar compound based on 13-hydroxydocosanoic acid is present in Candida bogoriensis (see H. W. Esders and R. J. Light, J. Lipid Res., 1972, 13, 663; J. biol. Chem., 1972, 247, 1375). (6) Ustilagie acid is a mixture of acylated derivatives of the glycoside of cellobiose with di- and tri-hydroxyhexadecanoic acids (see D. E. Eveleigh et al., ibid., 1964, 239, 839) produced by the corn smut fungus Ustilago maydis. A further compound produced by this organism is the tetra-acyl derivative of a mannosylerythritol with predominantly saturated (C12, C14

434

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

and C16) fatty acids (A. L. Fluharty and J. S. O'Brien, Biochemistry, 1969, 8, 2627)" CH20H

H~I~OH

CH202CR

H

0

OH

O_CH 2

RC 0 2 ~

The deacylated material has been synthesised (P. A. J. Gorin and A. S. Perlin, Canad. J. Chem., 1961, 39, 2474; see also A. Liptak et al., Z. Naturforsch., 1973, 28C, 352) and the locations of the acyl groups in both of these glycolipids have been studied (Gorin et al., Carbohydrate Res., 1970, 13, 235). A glycolipid from Aspergillus niger has been identified as a glycoside of glucose with trans-2-hydroxyoctadecenoic acid (P. Brennan et al., Biochemistry, 1972, 11,226,7). Glycosides of glucose and galactose with 1,3,25trihydroxyhexacosane and 1,3,25,27-tetrahydroxyoctacosane have been isolated from algae (T. A. Bryce et al., Phytochemistry, 1972, 11, 295; F. Lambein and C. P. Wolk, Biochemistry, 1973, 12, 791). (7) Diaeylmyoinositol mannoside is present in the membrane lipids of Propionibacterium strains (C. Prottey and Ballou, J. biol. Chem., 1968, 243, 6196; Shaw and F. Dinglinger, Biochem. J., 1969, 112, 769): CH202CR

H O ~

J

9 HO~

HO

(8) Acyl derivatives of glucose. 3,4,6-Tri-O-acyl-fl-D-glucopyranose is the major glycolipid of Mycoplasma sp., strain J. (Smith and W. R. Mayberry, Biochemistry, 1968, 7, 2706) in which the major fatty acids are oleic and linoleic acids. A 3,4,6-tri-O-acetyl-2-O-dodecanoylglucose is present in Streptococcusfaecalis (K. Welsh et al., Biochem. J., 1968, 107, 313). 6-O-Acylglucoses (in which the acyl group is corynomycolic acid) are major components of the lipids of Corynebacterium diphtheriae and Mycobacterium smegmatis (Brennan et al., Europ. J. Biochem., 1970, 13, t17; FEBS Letters, 1970, 8, 322).

3

MISCELLANEOUS

435

(9) Arabinose mycolates. Esters of arabinose with mycolic acid are present in the glycolipids of Mycobacterium tuberculosis and marianum (E. Vilkas et al., Biochim. Biophys. Acta, 1969, 192, 49; Bull. Soc. chim. Biol., 1970, 52, 145; M. Bruneteau and G. Michel, Chem. Phys. Lipids, 1968, 2, 229) and in Nocardia brasiliensis (M. A. Laneelle and J. ,4sselineau, FEBS Letters, 1970, 7, 64). (10) 6-O-Methylglucose-containing lipipolysaccharide of Mycobacterium phlei. The complete structure of a polysaccharide from this bacterium containing 6-O-methyl-D-glucose has been established. Four glycolipids based on this carbohydrate backbone have been isolated. Each one contains 3 mols. of acetic, 1 mol. of propionic, 1 mol. of isobutyric and 1 mol. of octanoic acids esterified to the carbohydrate chain and they differ in having either 0, 1, 2 or 3 mols. of monoesterified succinic acid residues on the carbohydrate. The locations of the various acyl groups on the carbohydrate and the biosynthesis have also been studied (Ballou et al., J. biol. Chem., 1972, 247, 8129; 1973, 248, 7118, 7126; Biochem. J., 1973, 132, 329). (11) Polyisoprenoid lipid intermediates. Lipids of the following structures, containing undecaprenol or decaprenol (and related alcohols), were shown in 1965 to be intermediates in the biosynthesis of various bacterial polysaccharides e.g. the mucopeptide of cell walls, the bacterial O-antigens and mannans. Subsequently similar lipids have been shown to be intermediates in the biosynthesis of plant polysaccharides and mammalian glycopeptides (for review see W. J. Lennarz and M. G. Scher, Biochim. Biophys. Acta, 1972, 265, 417; F.. W. Hemming, in: MTP Internat. Rev. Sci., Biochem. (1973) Ser. I, Vol. 4, p. 39). Me I

h,,le-C=CH2.CH2

CH~.C=CH2.C H2

0

0

II

I

II

CH2--C=CH.CH2-O-P-O-P-O - s u g a r s

--8or9

O@

O@

O or

II

--O-P-O--sugars I| 0

The pyrophosphate intermediate is utilised in the synthesis of the core polysaccharide and the O-antigen of Salmonella (see A. Wright, J. Bacteriol., 1971, 105, 927; Robbins et al., J. biol. Chem., 1969, 244, 5436) and for mucopeptide synthesis (see J. L. Strominger et al., ibid., 1972, 247, 5107, 5113, 5116, 5123; J. Bacteriol., 1972, 112, 1302, 1306). The antibiotic bacitracin inhibits certain reactions in this process (see D. R. Storm, Ann. N.Y. Acad. Sci., 1974, 235, 387). The monophosphate is utilized in adding the

436

PHOSPHOLIPIDS

AND GLYCOLIPIDS

21

glucose side-chains to the O-antigen of Salmonella (K. and H. Nikaido, J. biol. Chem., 1971, 246, 3912) and in mannan synthesis in M. lysodeikticus (Lennarz et al., Proc. Nat. Acad. Sci., 1968, 59, 1313; R. Sentandreu and J. O. Lampen, FEBS Letters, 1972, 27, 331). An ester between dolichol and a sugar pyrophosphate is probably the donor in mammalian glycoprotein biosynthesis (see L. F. Leloir et al., Biochim. Biophys. Acta, 1972, 270, 529; Carbohydrate Res., 1973, 26, 393; W. C. Breckenridge and L. S. Wolfe, FEBS Letters, 1973, 29, 66). Polyisoprenol esters of sugar pyrophosphates (C. D. Warren and R. W. Jeanloz, Biochemistry, 1972, 11, 2565; Carbohydrate Res., 1973, 30, 257; 1974, 37, 252; Proc. Nat. Acad. Sci., 1974, 71, 5022) and polyisoprenol ester of glycosyl phosphates (Warren and Jeanloz, Biochemistry, 1973, 12, 5031, 5038; J. biol. Chem., 1974, 249, 6372) have been synthesised. (12)Lipopolysaccharides of Gram-negative bacteria. The endotoxins of Gram-negative bacteria, which in higher animals result in fever, shock and death have been characterised as lipopolysaccharides. These compounds are made up of three distinct parts: (i) the polysaccharide "O-antigen" which contains hexoses and sugars characteristic of each bacteria, (ii) a "core polysaccharide" which contains hexoses, hexosamines, heptoses and oxodeoxyoctanoates ("KDO") and (iii) a covalently bound lipid ("lipid A") (for reviews see O. Liideritz, Angew. Chem. internat. Edn., 1970, 9, 649; Liideritz et al., in "Microbial Toxins", eds. G. Weinbaum, S. Kadis and S. J. Aft, 1971, 4, 145; D. A. Reaveley and R. E. Burge, Adv. Microbial Physiol., 1972, 7, 1; LfMeritz et al., J. Infectious Diseases, 1973, 128, S17): Lipid A -- core polysaccharide -- O-antigen The lipid A portion can be separated by hydrolysis and it alone exhibits all of the endotoxic activity (idem, Europ. J. Biochem., 1971, 19, 143; 1971, 22, 218; 1972, 31,230). The "lipid A" portion of Salmonella lipopolysaccharide has the following unit structure and the units are possibly linked by pyrophosphate bridges (idem, ibid., 1972, 28, 166; 1969, 7, 370; 1971, 21, 355; see also C. Lugowski and E. Romanowska, ibid., 1974, 48, 81, 319 for a similar structure for the lipid A of Shigella sonnei) (see p. 437). Mutant bacteria whose lipopolysaccharide lacks the "core polysaccharide" portion and contains only "lipid A" and KDO have been used for structural investigations (see S. ,4. Rooney and H. Goldfine, J. Bacteriol., 1972, 111, 531). Extensive investigations on the structures of the "core polysaccharides" and "O-antigens" of various bacteria have been carried out (see reviews above).

3

MISCELLANEOUS

437

CH20R

o

o

c

0

0

CO

NH

I

I

IH2 CHOH I

(? H2)10 CH 3

0|

CO I

CH2

R = -CO(CH2)IoCH 3 -CO(C H2)I4CH3 and -CO-CH2-CH (C H2)IOCH 3 02C'(C H2)12CH3

I

CHOH I

(C H2)10 I

CH 3

KDO-KDO I KDO I core polysaccharide-O-antigen

(13) Pahutoxin is a secretion of the boxfish, Ostracion lentiginosus (which is toxic to other fish) and has the following structure"

C H3(CH2)12"CIH'C H2"C02C H2"C H2NMe3 02C'CH 3 Analogues of this compound, possessing similar toxic properties, have been synthesised (D. B. Boylan and P. J. Scheuer, Science, 1967, 155, 52). (14) An ornithine-containing lipid of various bacteria has the following structure (J. L. Brooks and Benson, Arch. Biochem. Biophys., 1972, 152, 347; Bergelson et al., Khim. prirod Soed., 1972, 145; W. Knoche and J. M. Shively, J. biol. Chem., 1972, 247, 170; Wilkinson, Biochim. Biophys. Acta, 1972, 270, l; Minnikin, FEBS Letters, 1974, 43, 257). CH 2

(9 H2)3C H'CO 2 | H3N(C

--2C'C ? H (OH)'(CH2)8c/H - \ C H (C H2)5. C H 3

NHCO'CH2"CH (C H2)12C H 3

(15) Chiorosulpholipids. These lipids, which are chlorinated derivatives (with up to 6 chlorine atoms) ofdisulphates ofdocosane-l,14-diol and tetracosanr 1,15-diol have been isolated from the phytoflagellate Ochromonas danica (see T. H. Haines, in MTP Internat. Rev. Sci., Biochem. Ser. I, 1973, Vol. 4, p. 271 ; Annual Rev. Microbiol., 1973, 27, 403; C. L. Mooney and Haines,

438

PHOSPHOLIPIDS AND GLYCOLIPIDS

21

Biochemistry, 1973, 12, 4469; J. Elovson, ibid., 1974, 13, 2105, 3483). A further lipid which is an ether between a diacyl glycerol and N,N,N-trimethylhomoserine has also been isolated from O. danica (A. F. Brown and Elovson, ibid., 1974, 13, 3476).