The N and 0-acylations of C20-phytosphingosine anhydro base (Ia) , C20 -anhydro cere\u27brin (Ila) and C20 -cerebrin (IIIa) with longchain fatty acid chlorides have been examined. The reactions have been carried out in both quinoline and N,N-dimethyl formamide. The ceramides and ceramide esters thus obtained are represented by formulas I-III

B. Ries-Lešić

M . Proštenik

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CR 0 AT IC A CHEMIC A ACT A 32 (1960) 17 C CA-167 547 .435 :582 .. 282.23 :547 .298.1 :547 .398.1.26-11 Studies in the Sphingolipids Series. XIII.':· On the Ceramides and £eramide Esters of C20 -Phytosphingosine and Cio -Phytosphingosine Anhydro Base of Yeast B. Ries-Lesic and M. Prostenik Department of B i ochemistry, Institute »Ruder Bo!ikovit«, Zagreb, Croatia, Yugoslavia Received January 23, 1960 The N and 0-acylations of C20-phytosphingosine anhydro base (Ia), C20-anhydro cere'brin (Ila) and C20-cerebrin (IIIa) with long-chain fatty acid chlorides have been examined. The reactions have been carried out in both quinoline and N,N-dimethyl formamide. The ceramides and ceramide esters thus obtained are represented by formulas I-III. The long-chain fatty acid amides o.f sphingosine (ceramides) such. as .N-palmitoyl, N-stearoyl, N-lignoceryl and N-cerebronylsphingosine have been reported either as constituents of liver and spleen lipids or as hydrolysis products of sphingolipide preparations1• The existence in spleen of a sphingo-myelin in which the second hydroxy group of the sphingosine is esterified with a long-chain fatty acid has been assumed by Thannhauser and Reichel2. On the other hand, N-lignoceryl, N-cerebronyl, N-(2-hydroxyhexacosanoyl) and N-(2 ,3-dihydroxytetracosanoyl) derivatives of homologous C1 8 and C20-4-hydro-xydihydrosphingosines, i.e. c!B and Czo-phytosphingosines, have been isolated from yeast, corn, soybean and other plant lipids3- 6• Although some ceramides of the sphingosine and dihydrosphingosine series bave been prepared synthetically by Reichel and Thannhauser7 no synthetic work has been done yet on ceramides and ceramide esters of phytosphingosines. This problem seems to be of interest since one may suppose inter alia the occurrence of the ceramide esters in the native lipids. On the other hand, it was also desirable to work out a satisfactory method of approaching the synthesis of compounds of this type, particularly of cerebrin and anhydro ·{;erebrin. In our experiments the starting materials were yeast cerebrin (Hia) -purified through its tetraacetyl derivative -, anhydro cerebrin (Ha) and C20-phytosphingosine anhydro base (Ia). It has been established recently that the yeast bases represent a mixture of C18 and C20-homologues respectively6•8 •9 • The isolation procedure originally proposed by Reindel et al. yielded the C20-anhydro compound which was evidently not contaminated with a noticeable amount of the C18-compound8 •10• The C~0-compounds only were employed in this investigation. The synthesis of ceramides and corresponding esters of Ia, Ha and Hia was effected by two methods. According to Reichel and Thannhauser7 the N,0-acylation can be carried out in ether solution in presence of quinoline. * P aper XII : M. P r o s t e n i k , B. M a j h o f e r - 0 r e s c a n i n, M. M u n k -·we inert, and B. Rie s - L e s i c, Croat. Chem. Acta 32 (1960) 11. 18 B . RIES-LESIC AND M. PROSTENIK The ester group can then be selectively h ydrolyzed to yield the N-acyl compound. By this way mono~, di- and tetraacyl derivatives of Ila and Illa have been obtained. The acylation with oleyl chloride of Ila afforded the monooleyl ester (Ilb), Since Ila has two hydroxy groups the position of the acyl rest remains to be elucidated. An alternative method described recently by Weiss and Raizman11 using N,N-dimethylformamide as a solvent in presence of traces of pyridine appears· to be more suitable for the preparation of N-monoacyl derivatives. The amides and esters of long-chain fatty acids thus prepared are repre-sented by formulas I - III. I o I I CH3 (CH2)15-CH-CH-CH-CH2 , I l OR1 NHR2 a) R1 = R2 = H b) R1 = H, R2 = CH3(CH2)1 6CO c) R1 = CH3CO, R 2 = CH3(CH2)16CO d) R1 = H, R2 = CH3 (CH2)14GO e) R1 = H, R2 = CH3(CH2) 1 5CH(OH)CO I o ! II CH3 (CH2)15-CH-CH-CH-CH2 I I OR3 NH-OC-CH(CH2) 23 CH3 I OR4 a) Ra= R4 = H b) R 3 = H, R4 = CH3 (CH2) 1 CH = CH(CH2) 1CO or Ra= CH3 (CH2)1 CH = CH(CH2)1CO, R., .= H c) R3 = H, R4 = CH3(CH2) 1 6CO or . Ra = CH3(CH2)1 6CO, R4 = H d) Ra = R4 = CH3(CH2) 16CO III CH3 (CH2) 15-CH-CH-CH-CH20R; I I I OR5 OR5 NH-OC-CH(CH2) 23CH3 I OR5 a) R5 ==r ·H b) R5 = CH3(CH2) 16CO EXPERIMENTAL The melting points are uncorrected. N-Stearoyl C20-Phytosphingosine Anhydro Base (lb) A 400 mg. (1.22 mM) sample of C20-phytosphingosine anhydro base (Ia, m. P -88-890) prepared according to Reindel et ai.3 ,• was dissolved in warm dimethyl ON THE CERAMIDES AND CERAMIDE ESTERS 19 formamide (4 ml.). To this solution dry pyridine (0.1 ml.) and stearoyl chloride (333 mg.,, 1.G9 mM) in dimethyl formamide (1 ml.) were ·added. The reaction mixture was allowed to stand at room temperature for 1 hr. and then at .5° overnight. The separated crude crystahline product was filtered by suction (300 mg., m . p . 94-96°) and recrystallized four times from 950/o ethanol. Coloul'll.ess crystals, m . p . 109-110.50; [a]~ + 7.4o (c, 0.35 in chloroform). Anal. 6.272 mg. subst.: 17.59 mg. C02, 7.116 mg. H20 7.173 mg. subst.: 0.16'6 ml. N2 (Q1lO, 760 mm) CasH1sNOa (593.98) calc'd.: C 76 .83 ; H 1.2.72 ; N 2.350/o found: C 76.79; H 12.69 ; N 2.450/o Molecular W eight Determination 0.65 mg. subst.; 9.35 mg. camphor ; depression 4.20 calc 'd. : MW 5194; found : MW 662 0-Acetyl N-Stearoyl C20-Phutosphingosine Anhydro Base (le) N-Stearoyl anhydro b ase (200 mg.), dry pyridine (1 ml.) and acetic anhydride (0.5 ml.) were heated at 100° for 1 hr. After cooling the reaction mixture was poured into cold water (15 ml.) . The separated colourless solid was filtered, washed with water and dried. The m elting point was 88.5-89·.50 and r emained unchanged after crystallization from acetonitrile. [a]g -18.040 (c, 0.205 in chloroform). Anal. 6.260 mg. subst.: 17.28 mg. C02, 6.74 mg. H20 5.145 mg. subst.: o.n o ml. N2 (250, 739 mm) C40H;;N04 (636.02) calc'd.: C 75 .53; H 12.20 ; N 2..200/o found: C 75 .33 ; H 12.05 ; N 2.380/o Molecular Weight Determination 0.90 mg. subst. ; 11.2 mg. camphor; depression 5.10 calc'd .: MW 636; fonnd: MW 630 N-Palmitoyl C20-Phytosphingosine Anhydro Base (Id) The palmitoyl derivative was prepared from the anhydro b ase (400 mg., 1.22 mM, m.p. 88-890), p almitoyl chloride (300 mg., 1.09 mM), pyridine (0.1 ml.) and dimethyl formamide in the same way as described for the stearoyl compound. The crude product (400 mg., 58°io) m elted at 105-106.5°. One crystallization from ethanol gave colourless crystals, m. p . 110-110.50 ; [alf; + 10.20 (c, 0.127 in chloroform). Anal. 5.551 m g. subst. : 15.52 mg. C02. 6.34 mg. H20 7.431 mg. sub.st.: 0.177 ml. N2 (21°, 750 mm) C36H11N03 (565.92) calc'd.: C 76.40 ; H 12.67; N 2.470/o found: C 76.29 ; H 12.78 ; N 2.730/o N-(2-Hydroxystearoyl) C20-Phytosphingosine Anhydro Base (le) To a solution o·f anhydro base (0.98 g., 3 mM, m . p . 88-89°) in warm dimethyl formamide 2-acetoxystearoyl chloride (1 g. , 2.7 mM) and pyridine (0.39 ml.) were added. The reaction mixture w as k ept at 5° in the refrigerator overnight and then the crystalline precipita te filter ed by suction. The crude yellowish product (0.74 g ., m . p. 90-990 was dissolved in m ethanolic N KOH and allowed to stand at room temperature overnight. The solution was then poured into cold water, extracted with ether, the ether layer w ashed with water , dried over sodium sulphate and evaporated in v acuo to dryness. After crystallizaUon of the colourless crystalline residue (m. p. 96-97°) from ethanol the m elting point r aised to 106-107°; [aJi,2 + 6.06° (c, 0.325 in chloroform) . Anal. 5.637 mg. subst. : 15.50 mg. C02, 6.18 m g. H20 C3sH15N04 (609.98) calc'd .: C 74.82; H 12.390/o found : C 75 .01 ; H 12.270/o 20 B. RIES-LESIC AND M. PROSTENIK Distearoyl Anhydro Cerebrin (Ild) A sample of anhydro cerebrin (Ila, 4 g., 5.53 mM, m. p . 115.5-1160)3,4 was dissolved in warm freshly distilled quinoline (35 ml.) dried previously with solid potassium hydroxide. A solution of stearoyl chloride (12 ml.) in dry ether (40 ml.) wais then added dropwise in the course of 45 minutes with stirring. The reaction mixture was refluxed for 4 hrs . with continuous agitati-on. After that time the ·cooled solution was extracted with ether (400 ml.) , the ether layer washed suc-cessively with 2 N hydrochloric acid and water to the neutra l r eaction. The ether solution w as left to stand at room temperature overnight and the separated substance filtered by suction. The crude product (4.99 g., 72°/o, m. p. 65-66°) was recrystallized from a mixture of ether and methanol (2:1) and melted finally at 68-58.5°. To the original ether mother liquid t en volumes of methanol were added causing t he separation of an additional crop of crystals melting at the same temperature ; [a ) i; + 3.360 (c, 0.624 in chloroform). Anal. 6.787 mg. subst.: 19.56 mg. C02, 7.72 mg. H20 Cs2H159N00 (1255.1) calc'd .: C 78.47; H 12.770/o found: •C 78.62; H 12.720/o Monooleyl Anhydro Cerebrin (Ilb) To a solution of Ila (12.75 g., 3.8 mMJ in quinoline (20 ml.) oleyl chloride (2.66 g., 8 mM) dissolved in absolute ether (40 ml.) was added dropwise. The mixture was refluxed for 4 hrs. and then worked up as described above. From the ether-methanol solution 1.22 g. colourless substance, m. p. 46---480 separated. The melting point was unchanged after recrystallization from acetone. Anal. 7.370 mg. subst.: 21.01 mg. C02, 8.26 mg. H 20 8.410 mg. subst.: 0.118 ml. N 2 (24°, 746 mm) C64H 123N05 (986.62) calc 'd .: C 77.90; H 12.5·6; N 1.410/o found : C 77.80; H 12.55 ; N 1.58°/o Molecular Weight D etermination 1.45 mg. subst.; 12.45 mg. camphor; depression 4 50 calc'd.: MW 987; found : MW 1035 Monostearoyl Anhydro Cerebrin (Ile) The monooleyl compound (190 mg.) was dissolved in 960/o ethanol (20 ·ml.) and hydrogenated in presence of Adams' platinum catalyst (50 mg.) at room temperature and a t atmospheric pressure. After the theoretical amount of h ydrogen was taken up, the catalyst was removed by filtration and the solvent evaporated to yield 155 mg. colourless substance, m. p . 59-61°. After recrystaillization from acetone it melted at 6'4-65°. Anal. 9.215 mg. subst. : 26,26 mg. C02, 10.41 mg. H 20 8.885 mg. subst.: 0.-122 ml. N2 (25°, 739 mm) C64H125N05 (988.64) calc'd.: C 77 .74; H 12.74 ; N 1.410/o found: C 77.77; H 12.64; N l. ::>30/o Molecular Weight D etermination 1.05 mg. subst .; 13.05 mg. camphor; depress ion 3.6o calc'd.: MW 989 ; found: MW 897 Tetrastearoyl Cerebrin (IIIb) A sample of cerebrin (Illa, 0.,276 mM, m. p. 140°) 3·4 purified through its tetraacetyl derivative was dissolved in quinoline (15 ml.). Five milliliters of stearoyl chloride dis-solved ·in ether (20 ml.) were added dropwise to the agitated soiution of cerebrin. The reaction mixture w as worked up as described above. The ether solution furnished on standing 0.6' g. of a crude substance which was recrystallized first from acetone and then from ether-methanol (2:1) . The analyt_ical sample melted at 78-79°. Anal. 5.120 mg. subst. : 14.75 mg. C02, 5.75 mg. H 20 C 11sH 229N09 (1806.01) calc'd.: C 78.47 ; H 12.78°/o found: C 78.6,2; H 12.&7°/o ON THE CERAMIDES AND CERAMIDE ESTERS 21 Acknowledgment. We are indebted to Mrs. M. Munk-Weinert (Department of Chemistry, Medical Faculty, Univ. of Zagreb) for the microanalyses. REFERENCES 1. For references regarding these ceramides see B. Weiss and P. Ra i z man, J. Am. Chem. Soc. 80 (1958) 4657; see also G . V . Ma r in e t ti and E. Stotz, J . Am. Chem. Soc. 79 (1957) 145. 2. S . J. Than n ha user and M. Reiche 1, J. Biol. Chem. 135 (1940) 1. 3. F. Rein dell, Ann. 479 (1930) 76. 4. F. R e indel, A. Wei ckmann, S . Picard, K. Luber, and P. Turula, Ann. 544 (1940) 116. 5. T . 0 d a, J. Pharmac. Soc. Japan 72 (1952) 136, 141. 6. T. 0 d a and H. K am i ya, Chem. & Pharm. Bull. Japan 6 (1958) 682. 7. M . Reich el and S. J . Than n ha user, J. Biol. Chem. 135 (1940) 15. 8. M. Pros ten i k and N. Z. S tan a c e v, Ber. 91 (1958) 961. 9. C. C. Sweeley, Biochim. , Biophys. Acta 36 (1959) 268. rn. M. Pro s t e n i k, B. M a j ho fer - 0 r es c a n i n, M. Mun k - Wein er t, and B. R i es - L es i c, Croat. Chem. Acta 32 (1960) 11. 11. B. Weiss and P. Ra i z man, J. Am. Chem. Soc. 80 (1958) 4657. IZVOD Studije u redu sfingolipoida. XIII. 0 ceramidima i cernmidskim esterima C20-fitosfingozin a i C20-fitosfingozin anhidro haze iz kvasca B . Ries-Lesic i M. Prostenik Ispitane su mogucnosti N i 0-acil.acija C20-fitosfingozin anhidro baze (Ia), C20-anhidro cerebrina (Ila) i cerebrina (Illa) kloridima visih rnasnih kiselina i 2-hidroksi-kiselina. Acilacije su izvedene ili u kinolinu ili u N,N-dimetil- formamidu. Dobiveni amidi i esteri prikazani su formulama I - III. BIOKEMIJSKI ODJEL INSTITUT »RUDER BOSKOVIC« ZAGREB Primljeno 23 . sijeenja 1960. 

Studies in the Sphingolipids Series. XIII. On the Ceramides and Ceramide Esters of C20-Phytosphingosine and Cio-Phytosphingosine Anhydro Base of Yeast

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