Semisynthetic galactolipids of plant origin

BIOCHIMICA

BBA

ET BIOPHYSICA

537

ACTA

55860

SEMISYNTHETIC

GALACTOLIPIDS

OF PLANT

ORIGIN

E. HEINZ

Botaksches (Received

Institut, December

lJzzioevsit%t K&S, zpd,

5 Kdln-Linden&al,

Gyvhojstvasse

15 (Germany)

1970)

SUMMARY

Starting with natural lipids, mono- and digalactosyl diglycerides were prepared having identical or different acyl groups attached to C-I’ and C-z’ of the glycerol part of the molecules. This was achieved by the following reaction sequence : substitution of the hydroxyl hydrogens of the galactosyl residues by O-(I-methoxyethyl) groups, removal of the acyl groups with sodium methoxide, reesterification with new fatty acids and hydrolysis of the protecting groups by boric acid. The acyl residues included acetyl, palmitoyl, palmitoleoyl, stearoyl and oleoyl groups.

INTRODUCTION

Mono- and digalactosyl diglycerides, which are the predominant lipids of green plant tissuesl, have a slow turnover in mature leaves233,whereas they are subject to a rapid metabolism once the cell structure has been damaged. By now several enzymatic reactions are known by which the acyl residues of the galactolipids are transferred to various acceptors. If water or alcohols act as acceptors, free fatty acids or the corresponding fatty acid esters, respectively, are formed concomitantly with deacylated galactosyl glycerol and digalactosyl glycero14-6. If monogalactosyl diglyceride is the acceptor, the formation of acyl galactosyl diglyceride is observed’, a substance, which has also been isolated from wheat flours*g. For the purification and a more detailed investigation of the acyl galactosyl diglyceride-forming enzyme, labelled galactolipids with known fatty acids in definite positions should be useful substrates. Therefore this paper describes a procedure by which various semisynthetic mono- and digalactolipids can be prepared starting from the natural lipids. RESULTS

AND DISCUSSION

The plant galactolipids as well as the majority of the other glycerolipids are derivatives of I,z-di-O-acyl-sm-glycerol lo. After deacylation they give 3-O-p-n-galactopyranosyl-sn-glycerol I and 3-0-(6-0-a-D-galactopyranosyl-~-D-galactopyranosyl)sn-glycerol I111312. The chemical synthesis of glycerylgalactosides has been described by WICKBERG~~, laying special emphasis on the preparation of the desired configuration Biochim

Biophys.

Acta,

231 (1971) 537-544

of the glycerol has been

moiety.

recently

The

complete

described

by

synthesis

!VIXRLI

of monogalactosyl POMERAXZ

AND

I,O-di-O-acyl-2,5-0-methylene-r>-mannitol

with

glycerides group.

having

The

two

saturated

compounds

which

were

CHz--OR,

obtained

resulted and

in yields

ITS

in monogalactosyl

one unsaturated

d:i-

fatty

of 2% had no optical

acy:

rotation.

I'

i

R20-CH

2'

I

6 R@-Cti2 5

and

or one saturated

diglycerides

14. Their stniA1esis skmed

3

p2 0

R3*

4

Oas

0

3

7 1 2 ORa

I. Formulae of galactolipids

Es.

II 111 IV V VII VII VIII IX X XI XII XIII XIV XV XVI

R, R, R, R, R, R, R, K, R, R, R, R, R, R, R,

Apart of

R, := R, = H, R, = a-D-galactopyranosyl radical R, = long chain acyl radical, R, = R, == W R, F-=long chai~r acyl radical, X, = R, = r-methoxyethyl radical R, =: Hz R, = R, = r-mcthoxyethyl radical R, = acetyl radical, R, = R, = H R, = palmitoyl radical, R, = R, = K R, := oleoyl radical, R, = K, = H R, = long chain acyl radical, R, = H, I& = cc-n-galactcpyranosyl radical R, == a&>-l, R, = H, R, = ct-u-gal~c.ctop~ranos3r~ radical R, := oleoyl radical, R, = H, R, = n-n-galactopyranos?jI radical olcoyl radical, X, = H, R, = R, = ;-methoxyethyl radxal oleoyl radical, R, = palmitoleoyl radical, R, = R, =- H stearoyl radical, R, = palmitoyl radical, 23, = R, c II oleoyi radical, R, = pabnitokuyl radical, 33, = E-I, R, = cr-D-g-alactop!rranosyl rad palmitoyl radical, X, = acetyl radical, R, = K, = I-r

= = = = = = = = = = = = = = =

from

forming

&ceroi,there

protect

the desired

hydroxyl

can be taken

and C-2’

of glycerol

be critical

in preparing

described

here

acetyl

found for the natural above

by using

involves

acyl

The hydrogenated vinyl

ether

hand

cores

glycolipid

in the presence

this

acetyl

residues and

at C-I’

rotations

method

(for example,

at

might

and C-2’. The method diglycerides

galactolipids,

which

is an acyl hydrogens

of the acyl residues,

of the protecting

resi-

groups

procedure

the difficulties

basically

of the hydroxyl

ac$

in accordance

circumvents

used to

are acetyl

chain

deacylation

a.t C-2’

groups

usually

long

digalactosyl

of the natural The

removai

hydrolysis

groups

mono-

configuration

the blocking

removing

The procedure

O-acyl-3’-O-P-u-galactopyranosyl-svl-glycerol methyl

protecting

and optical

: substitution

groups,

acids and finally

with

quantities.

steps

and the proper

of removing

extensively

groups

the ~~ydro~~~li~

the following

These

to synthesize

compounds.

by Q-(I-methoxyethyl) fatty

groups. off without

compounds

in sufficient

bond

problem

I*--16. On the other

was used

and unsaturated

be prepared

anomeric

is the additional

the sugar

dues which C-i’

and their derivatives.

having

with

these

described can easily

exchange

and

of the galactose

reesterification

with new

groups.

monogalactosyl

111) was

of ~-tol~~~esulfonic

&glyceride,

acetalated acid.

by

I’$-&

reaction

‘The reaction

with

was com-

SEMISYNTHETIC

GALACTOLIPIDS

539

plete in about 15 min as seen by thin-layer chromatography and infrared spectroscopy which showed the formation of a more hydrophobic product having no free hydroxyl groups with the presumed structure of I’$-di-0-acyl-3’-0-[z,3,4,6-tetraO-(r-methoxyethyl)-~-D-galactopyranosyl]-sn-glycerol IV. 0-(I-Methoxyethyl) groups are stable to alkali but can be split off under very mild acidic conditions, as has been described by several authors for analytical and synthetic means17-20.Subsequently the acyl residues in IV were removed with sodium methoxide, and the product 3’-O[2,3,4,6-tetra-O-(~-methoxyethyl)-~-~-galactop~anosyl]-s~-glycerol V, which is the key intermediate for the subsequent syntheses, was isolated by silicic acid column chromatography. It was acylated with 2 moles of fatty acid chloride at room temperature in pyridine. The blocking groups were removed by heating the product with boric acid under unhydrous conditions1s~21,and the liberated galactosyl diglycerides were isolated by silicic acid column chromatography. In this way, monogalactosyl diglycerides were prepared having two acetyl VI, palmitoyl VII or oleoyl VIII groups. The overall yields were zo-33%. With digalactosyl diglycerides (r’,2’-di-O-acyl-3’-0-[6-O-cc-n-galactopyranosyl/I-n-galactopyranosyll-sn-glycerol IX) as starting material, digalactosyl diglycerides with two acetyl X or oleoyl XI groups were synthetsized in the same way, the yield being 18 and 24 %, respectively. To make sure that the final hydrolysis by boric acid did not cause acyl migration, the synthesized lipids were investigated by NMR spectroscopy. Although the NMR spectra of the synthesized compounds had the same signals as those of the natural hydrogenated lipidP, the spectrum of I’$-di-0-acetyl-3’-O-/5n-galactopyranosyl-s2z-glycerol VI (Fig. za) was selected to demonstrate that the acyl residues are in fact linked to the glycerol hydroxyl groups at C-I’ and C-z’. For this purpose, use is made of the fact that in NMR spectra of glycerides the signals of carbinol protons are shifted downfield after acylation of the corresponding hydroxyl group2’3324. The most downfield signal in the spectrum of VI is the multiplet at 5.52 ppm attributed to the tertiary proton at C-2’ of glycerol 22.Its downfield position demonstrates that one of the two acyl groups is linked to C-z’. On the other hand the remaining glycerol protons at C-I’ and C-3’ are difficult to be sorted out since they absorb in the same region as the ring protons of the galactose. Therefore the location of the second acyl group had to be deduced by excluding positions to which it was not linked. The multiplet at 5.52 ppm and the doublet of the anomeric proton at 4.70 ppm are not superimposed by any other signals, which would have been the case, had any of the sugar hydroxyl groups been acylated 22~25. In addition VI shows two acetate signals of equal height at I.96 and I.98 ppm, whereas the spectrum of the completely acetylated compound (galactosyl glycerol hexaacetate) gives rise to five acetate signals of different height spread between I.97 and 2.14 ppm when recorded in pyridine. Therefore a random distribution of the acetyl groups in VI would cause the appearance of more than two acetate signals. This altogether is evidence for the location of the second acyl residue at C-I’ of glycerol. Similar considerations apply to the spectra of the synthetic and natural digalactosyl diglycerides, which gave identical spectra. The most downfield signal was the multiplet at 5.53 ppm due to H-2’. The doublet adjacent to it at 5.43 ppm is attributed to the anomeric proton of the second galactosyl residue being in cc-galactosidic linkage.

Galacfosyl &glycerides with~ two diffeve?lt acyl poups The galactolipids most useful for studyin g the forming

enzyme

and other aspects

of galactolipid

acyl

galactosyl

metabolism

diglyceride-

are those having

two

different unsaturated acyl residues linked to C-I’ and C-Z’, which were synthesized in the followingway. V was reacted at -20’ w&h I mole of oleoyi chloride as described by DE HAAS AND VAN DEE?;EN~~ for the synthesis of lysophospholipids. After 2 h thin-layer chromatography showed a heavy spot corresponding to monoacylated V with the presumed structure of XII and minor spots due to the starting material and diacylated removal

V = IV. Before of the products

after purification with palmitoleoyl

continuing

just mentioned

the synthesis,

XII

had to be purified since the

is possible at this stage only. Therefore

only

by column chromatography XII was acylated at room temperature chloride. The product was hydrolyzed with boric acid and column

gave I’-Q-oleoyi- 2’-Q-palmitoleoyl-3’-O-~-~-gaiactopyranosyi-sn-glyRlethanolysis and gas-liquid chromatography of the resulting methyl

chromatography cerol XIII.

esters gave nearly equimolar amounts of palmitoieic and oleic acid. The hydrogenated product XIV gave a NMR spectrum identical to that of the hydrogenated natural monogalactosyl diglyceride, thus showing the attachment of the acy! groups at C-I’ and C-2’. The positional distribution of the individual fatty acids was investigated by hydrolysis with pancreatic lipase, which specifically splits esters of primary alcoholsx7. The lyso-compound formed during the enzymatic hydrolysis and separated from the reaction products by thin-layer chromatography contained paimitoleic and oleic acid in a ratio of 97 : 3 (ref. 28). This demonstrates

that XIII

acid distribution to a great extent. By the same method XV was prepared carrying oleate at C-I’ and palmitoleate

has the desired fatty

a digalactosyl at C-2’.

diglyceride

As an independent approach for the investigation of the specificity of the low temperature acylation, I’-O-palmitoyl-, ~‘-O-acetyl-3’-O-P-u-galactopyranosyl-sit-glgrcerol XVI was synthesized. The NMR spectrum of this compo*und in pyridine (Fig. zbj showed only one acetate signal at 1.98 ppm, whereas VI gave two acetate signals. Had XVI contained any appreciable amount of a compound carrying the acyl groups in exchanged positions, i.e. the acetyl group at C.-I’, then the 1\TNIRspectrum should have shown two acetate signals. The methods described here will be used to prepare labelled and doubly labelled galactolipids

and should be applicable

to other glycosyl

digiycerides

as well.

EXPERIMENTAL Matevials an.d methods Natural mono- and digaiactosyl diglycerides III and IX were isolated from lyophilized leaves of Sinapis alba and .§pi,utacia oleracea and hydrogenated as described before+zg. III had sintering point (s.p.) gII9z.5” and m.p. 15I.j~I53.5’, [a]~~~-z.28” m.p. 1622163”, (c 5.6 in pyridine), reported 122~ and 149’ (ref. 30) and s.p. Ioo-Io2°, gave 3’-O-B-u-galactopyranosyl-sn-glycerol I [CC]n25-I. 97” (ref. 22). Deacylation (c 2.2 in water); reported melting points vary with m.p. 137’ and [a]+ -8.12’ between I32 and 142’ (refs. II, 13, 14, zg-33), and reported rotations [E];D vary between+3.77 and -7.7” (refs. 8, II, 13, 14, zg, 33). Hydrogenated IX was precipitated from chloroform-methanol-ether mixtures which removed some contaminants which were difficult to remove by silicic acid column chromatography. It had [R]n”’ 137~8’

SE~ISY~THETIC GALACTOLIPIDS

541

(G 1.35 in pyridiae) and gave after deacylation 3‘-0-(6-0-~-D-gala~top~anosyl-~-Dgalactopyranosyl)~sn-glycerol II with m.p. 187-188” and [cc-O]21 -+88.8” (c 1.2 in water); reported m.p. 182-198~ and [a]n between f86.4 and i--88” (refs. 30, 34-36). Before acetalation the lipids were dried in vacua over P,O,. Oleic and palmitoleic acid were from Roth, Karlsruhe, Germany. The acid chlorides were prepared with SOCI, and purified by distillation. Pyridine and methylene chloride were dried by distillation from CaH, and P,O,, respectively. Optical rotations were measured with a Zeiss Lichtelektrisches Prazisionspolarimeter o.oo~‘, and NMR spectra were recorded with a Varian HA IOO spectrometer using tetramethylsilane as internal standard. For thin-layer chromato~aphy, Kieselgel G (Merck) and for column c~omato~aphy, Kieselgel o.oj-0.2 mm (Merck) was used. Compounds were detected by charring after spraying with 50% aqueous H,SO,. ~~icroanalyses were made by Mikroanalytisches Labor A. Bernhard& Elba&, Germany. Acetalation of glycolipids with methyl viny,? ether The procedure is described for III only, but the same quantities and solvent systems both for thin-layer chromatography and column chromatography were used for IX, except where stated otherwise. Hydrogenated III (3-g g) and&-toluenesulfonic acid (40-80 mg) were suspended in methylene chloride (40 ml) at IO’. Methyl vinyl ether condensed at -20~ was added dropwise with stirring. The lipid usually was completely dissolved after 15 min. Thin-layer chromatography with chloroformmethanol (98 : 2, v/v) showed complete transformation into the substituted product running close to the solsent front. Excess methyl vinyl ether was blown off by bubbling nitrogen through the reaction mixture, which subsequently was washed once with NaHCO, solution and once with water. After drying with Na,SO, and solvent removal, the product did not show any absorption in the infrared at the 36oo-cm-1 region. Yields were nearly quantitative. Deacylation

of acetalafed glycoli$ids

The acetalated product IV was dissolved in 8 vol. of sodium me&oxide (0.03 M) in methanol and kept for 30 min at 40’. Thin-layer chromatography in chloroformmethanol (9: I, v/v) showed a series of minor spots and one major product. The reaction mixture was extracted twice with light petroleum, and then the pH was adjusted to pH 7.5 by the addition of HCI. After solvent evaporation and removal of NaCI by filtration, the mixture was fractionated by column chromatograph~r. V was eluted with cl~lorofo~-l~ethanol (TOO:1.5, v/v) and obtained in yields varying from 40 to 80%. V, C,,H,,O,, required C, 51.84; II, 8.70. Found: C, 51.70; II, 8.61%. Reacylation and boric acid hydrolysis V was dissolved in pyridine (5-25 ml) and mixed with a slight excess (2 moles/ mole V) of the desired acyl chloride dissolved in methylene chloride (S-IO ml) or with acetic anhydride (for preparation of the diacetate). After standing overnight, the reaction mixture was diluted with light petroleum and washed with NaHCO, solution and water. After drying over Na,SO, the solvents were removed after the addition of isopropanol which was added to remove pyridine. The product was dried &z ~~~~~ over P*O,. For the preparation of the diacetates, the reaction mixture after acetyla-

tion was not extracted with SaHCQ, solution (because of the water solubility of compounds) but was evaporated to dryness after the addition of isopropanol. The dried products were mixed with an equal amount of boric acid (dried over P,O,) and refluxed for I h in trimethyl borate (j-10 ml). After removal of trimethyl borate in

the rotatory evaporator,

the reaction mixture was heated for 1 h at So” (ref. 24)~ The

residue was taken up in chloroform (or chloroform-methanol, I : I, v/v), freed from excess boric acid by filtration and extracted once with water. After drying over Na,SO, and solvent removal, the products were checked by thin-layer chromatographyin

chloroform-methanol (S5 : 15~v/v) for III and chloroform-methanol (0; : 25, v/v) for IX. Only one major spot corresponding to the desired product was detected.

Final pwi$cation

by silicic

acid

The reaction mixture column

chromatography.

colwm

obtained

c72vovmatoglfaphy

after boric acid hydrolysis

was fractionated

VI was eluted with chloroform-methanol

(95:5,

v/v)

by and

crystallized from methanol-ether, VII and VIII were eluted with chloroformmethanol (96 : 4, v/v). The final overall yields in the preparation of VI, VII and VIII were 20+33~/~. X was eluted with chloroform-methanol with

chloroform-methanol

(94 : 6, v/v).

Th e overall

(S3 : x7, v/v)! yields

XI was e!uted

were IS%

and 240/c,

respectively. VI,m.p. 1~)2-143~, [a]~~~ - 3.19’ (c 5.3 in pyridine). C,,H,,O,, required C, 46.15; H, 6.56. Found: C, 46,25; II. 6.64%. VII, s.p. gr-g3’, m.p. I~Z--154' (reported m.p. 56”, ref. 14), [a]~“’ -z.o;c’ (c

4.7 in pyridine).

C,,H,,O,,

required C, 67,36; H, 10.76. Found:

C, 67.45; H, no.SS%.

(c 5.6 in pyridine). C,,H,,O,, required C, 69.02; H, 1c.55. VIII, [a]n 21 -2.27’ Found: C, 68.92; II, 10.50%. X, [~l]# 170.3” (c 0.8 in pyridine). C,,H,,O,j required C, 45.60; II, 6.45~ Found: C, 4543; H, 6.61%. XI, [cc]+ f3S.g” Found:

(c 4.2 in pyridine).

C,,W,,O,,

required

C, 64Jo;

II, 9S1~

C, 64.58; H, 9.77%.

Galactolipids with diffeqepzt acylgqoqk at C-I’ and C-2’ The procedure is described for XV only, but essentially the same quantities (after correction for the lower molecular weight of the derivatives of III) and solvent systems both for thin-layer chromatography and silicic acid column chromatography were used for III, except where stated otherwise. Acetalation and deacylation of IX (g g) gave the intermediate

product

(i.e. II with the seven hydroxyl

groups of ihe

galactose residues substituted by O-(r-methoxy ethyl) groups, 4.3 g), which was obtained by column chromatography after elution with chloroform-methanol (roe : 1.5, v/v). The material was dissolved in pyridine (30 ml) and cooled to -20~. A soiution of oleoyl chloride (1.7 g) in methylene chloride (35 ml) also cooled to -20~ was then added dropwise with stirring over a period of 2 h, during which the reaction mixture was kept at -20~. After 3 h, thin-layer chromatography (9s: 2, v/v) showed one major product. After dilution with chloroform the reaction mixture was cnce extracted with water, dried over Na,SO, and then freed from solvents after the addition of isopropanol. The monoacylated compound (4.0 g) was obtained by column chromatography using light petroleun-acetone (75 : 25, v/v) for elution. The corresponding compound XII derived from III was eluted with light petroleum-acetone Biodti~~.

Biophys.

A&Z,

231 (1971)

5X7-544

SEMISYNTHETIC

GALACTOLIPIDS

543

(9 : I, v/v). The product was dissolved in pyridine (25 ml) and mixed with palmitoleyl chloride (1.4 g) dissolved in methylene chloride (IO ml). After standing overnight the reaction mixture was diluted with chloroform, once extracted with water and dried over Na,SO,. Solvent removal gave a product (4.5 g) which was hydrolyzed with boric acid (4.5 g) as described. Column chromatography with chloroform-methanol (g4:6, v/v) gave XV (2.0 g). XIII (2.3 g after starting with IO g of III) was obtained by elution with chloroform-methanol (96 : 4, v/v). Hydrogenation of XIII gave XIV. XVI was prepared in the same way and eluted from SiO, columns with chloroformacetone (2 : I, v/v), the yield being 0.6 g after starting with 5.5 g of III. XIII, [cI]D~~-2.04’ (c 3.5 in pyridine). C,,H,,O,, required C, 68.40; H, 10.41. Found: C, 68.19; H, 10.40%. XIV, s.p. 93-94’, m.p. 15~154’, [a] Dzl -2,24’ (c 3.8 in pyridine). C,,H,,O,, required C, 68.04; H, 10.89. Found: C, 68.04; H, II.oo~/~. XV, [a]nzl +37.1’ (c 1.1 in pyridine). C,,H,,O,, required C, 64.16; H, 9.67. Found: C, 64.02; H, 9.59%. 4.17’ (c 4.5 in pyridine). C,,H,,O,, required C, 60.65; H, 9.43. XVI, [E]D21 Found: C, 60.53; H, 9.39%. Lipase hydrolysis After hydrolysis of XIII (zoo mg) with pancreatic lipase (EC 3.1,1.3) the reaction products were analysed after separation by thin-layer chromatography. The details are given in ref. 28.

I

I

5.0

,

4.0

I

30

I

2.0

+pm Fig. 2. Nuclear magnetic resonance spectra of (a) 1’,2’-di-O-acetyl-3’-0-B-D-galactopyranosyl-s%glycerol VI and (b) I’-O-palmitoyl-2’-O-aCetyl-3’-O-~-D-galaCtOp~anOSyl-sn-glyCerOl XVI in pyridine. In the region of 1.8~2.3 ppm the amplitude is reduced as compared to the low field part of the spectrum. The region of o-1.8 ppm is omitted. Biochim.

Biophys.

Acta,

231 (1971) 537-544

Mopzogalnctosyl diglycer&cs. The spectra of VI and XVI are reproduced pertially in Fig. 2. The following signals were assigned in the spectra of VII, IX and XIV

dissolved

in pyridine.

Signals

derived from acyl groups:

terminal

-C

at 0.88 ppm; bulk of internal -CH,- at 1.28 ppm; a-CH,-, triplet at 2.33 ppm. Other signals: H-r, doublet at 4.73 ppm (j 8 cycles/set); H-z’, multiplet at 5.53 ppm. Digalactosyl diglycevides. H-z’, multiplet at 5.53 p-pm; anomeric proton of R1 (a-galactosidic linkage), doublet at 5.43 ppm (J 3 cycles/set), partially superimposed on H-2’. X gave in pyridine only one acetate signal at I.97 ppm. ACKNOWLEDGENENTS

The anthor is indebted these investigations,

to Prof. Dr. H. Reznik

gestions,

to Prof. Dr. WT. Menke and Mr. R. 3. Rirtz

to Mrs.

I. Wortmann

Deutsche

for his generosity

to Drs. P. A. J. Gorin and S. S. Bhattacharjee for experimental

assistance.

for recording This

work

in supporting for helpful sug-

NMR spectra

and

was supported

by

Forschungsgemeinschaft.

REFERENCES P. G. ROUGHAX ANI) R. D. BATT, Phytochemistvy, 8 (1969) 363. A. TR~MOLI~RES ANI) P. M~ZLI~K, Compt. Rend. SW. D, 267 (1968) 1039. P. G. ROUGHAN, B&hem. J,, 117 (1970) I. P. S. SASTRY AND M. KATES, Biochemistvy, 3 (1961) 1280. P. J. HELMSING, B&him. Biophys. Acta, 178 (1969) jrg. 9 (1970) I 725. 2 T. G-ALLIARD, Phytochemistvy, 7 E. HEINZ, B~lochim. Biophys. Acta, 144 (1967) 333. s D. V. MYRHE, Cuvz.jT.Chem., 46 (1968) 3071. AND W. R. MORRISON, J. Chvomatog., 47 (1970) 277. 9 T. A. CLAYTON, T. A. MACMURR~Y of Glycerol According to CBN Recommendations, European .i. Biochmz., 2 IO Nomenclature (1967) 127. H.E.CARTER,R. A.HENDKYAXD N.Z.STANACEV,J.L~P~~ Rns., 2(1g61j 223. II AND A. A. BENSOX, J. Am.Chem. Sot., 84(1962) j7. I2 M. MIYAXO I3 B. WICI~BERG, Acta Chem, Stand., 12 (1958) 1187. 14 H. P. WEHRLI AN'D Y. POMERAXZ, Chem. Phys. Lipids, 3 (1969) 357. P.M. BONSEN, J. A. IF.OP DEN KAMP, L.L.M.Var; DEESEX, Biochem.J., 108 =5 M. J.GuRR,P. (1968) 211. I6 H.M. VERHEIJ, P. F. SXIT~, P. P.M. ~ONSEN ANC L. L.M. VAN DEENEK, Biochim.Biophys, Acta, 218 (1970) 97. S. S. BHATTACHARJEE AND 6. G. PBREKH, Stiivke, 18 (x966) 131. I7 M.L. WOLFROM, Res., 8 (1968) I. I8 A. N. DE BELDER AND B. NORRMBN, Cwbohydvate I9 J. GIGG AXD R. GIGG, J. Chem. Sot., (:967) 431. 20 S. S. BHATTACHARJEE, R. H. HASXI~S AXED P. A. J. GORIN, Carbohydvate Res., 13 (1970) 23j. 21 J. B. MARTIN, J. em. Chem. SOL, 15 (1953) 5452. Physiol.Chem., 3jo(Ig69) 493. 22 E.HEINZ AND A.P.TuLLocH,Z. J. Chem. Sac., (1963) 13r. 23 D. CHAPMAN, AND K. K. CARROLL, J.Lipid Res., 7 (1966) 277. 24 B. SERD~REVICH APED A.H1~~,Cun.J.Chel?z.,46(1968) 2485. 25 A. P.TULLOCH 26 G. H. DE HAAS AND L. L. M. VAN DEENEN, Biochim.Biophys. dcta, 106(rg65) 315. 27 B.ENTRESSANGLES,H.SARIAND P.DESNUELLE, Biochim.Biophys.Acta, 125(1966) 597. 28 G. AULII\TC~, B. C. DAS AND A. P. TULLOCH, in preparation. 29 E. HEINZ, Biochim.Biophys. Acta, 144 (Ig67) 321. 30 P. S. SASTRY AND M. K~~~~,Bioche+~istvy,3 (1964) 1271. 3I C. R. SNIITH AND J. A. WOLFF, Lipids, I (x966) 123. 32 J. M. STEIM, Biochim. Biopkys. Acta, 144 (1967) 118. 33 3. E. BRUNDISH AND J. BADCILEY, Carbohydrate Res., 8 (1968) 308. 34 3. E. CARTER, R.H.McCLUER AND E.D. SLIFER,]. Am.Chem. Sot., 78(I956) 3735. 35 B. WICKBERG, ActaChem. Stand., 12(Ig58) 1183. 36 B. UIIBAS, Can.J. Chem., 46 (1968) 49. I 2 3 4

Biochim.

Biophys.

Acfa,

231 (1971)

537-544