Chemistry and Physics of Lipids 15 (1975) 33-36 © North-Holland Publishing Company
A FACILE SYNTHESIS OF CERAMIDES Yasuo KISHIMOTO Eunice Kennedy Shriver Center for Mental Retardation at W.E. Fernald State School, Waltham, Mass. 02154; Department of Neurology, Massachusetts General Hospital Boston, Mass. 02114, USA
Received May 10, 1 9 7 5 ,
accepted August 5, 1975
A new simple procedure for the synthesis of ceramide from free fatty acid and sphingosine by oxidation-reduction condensation with triphenylphosphine and 2,2'-dipyridyldisulfide has been described. N-lignoceroyl and N-cerebronoyl sphingosine were synthesized with this procedure producing an overall yield of 74% from lignoceric acid and 64% from cerebronic acid.
I. Introduction Ceramide plays an important role in the metabolism of sphingolipids in mammalian tissues [1 ]. In the past, several procedures have been used for the synthesis of ceramides including the reaction of sphingosine with the appropriate fatty acid chloride [2, 3], N-hydroxysuccinimide esters of fatty acids [4], or free fatty acid in the presence of a mixed carbodiimide [5]. Mukaiyama et al. [6] published a new procedure for peptide synthesis using an oxidation-reduction condensation with triphenylphosphine and 2,2'-dipyridylsulfide. We have applied this procedure to the synthesis of ceramides and find this method to be superior in many respects to the methods previously employed.
II. Experimental A. Materials
Sphingosine (flee base) obtained from bovine brain was purchased from Serdary Research Laboratories, London, Ontario, Canada and lignoceric acid from Lachat Chemical Co., Chicago Heights, Ill. Both nonradioactive [1-14C] labelled cerebronic acid and [1-14C] lignoceric acid were synthesized in this laboratory [3]. O-Acetyl cerebronic acid was prepared according to HammerstfiSm [5]. Triphenylphosphine and 2,2'-dipyridyldisulfide (Aldithiol-2) were purchased from Eastman Kodak, Rochester, N.Y. and Aldrich Chemical Co., Milwaukee, Wis., respectively. Methylene chloride
34
K Kishimoto, Synthes&of ceramides
was obtained from Burdick and Jackson Lab, Inc., Muskegon, Mich., and used without further purification. Precoated thin-layer chromatographic plates were purchased from Analtech, Inc., Newark, Del.
B. Synthesis of N-lignoceroyl sph&gosine To a mixture of 12.5 mg lignoceric acid (34/~moles), 17.5 mg triphenylphosphine (67//moles), and 14.7 mg aldrithiol-2 (67 ~moles) was added 0.65 ml of methylene chloride solution containing 10.1 mg sphingosine (34//moles); this was stirred at room temperature for 5 hr or longer. The mixture became yellow immediately after mixing. The solvent was removed from the reaction mixture by a flow of nitrogen; the residue was redissolved in 1 ml of chloroform-methanol 2 : 1 and washed with water according to Folch, Lees, and Sloane-Stanley [7]. After washing, the lower layer was evaporated to dryness, and the residue was fractionated by preparative thin-layer chromatography on silica gel G plates using chloroform-methanol-acetic acid (90 : 2 : 8) as the solvent. The band of ceramide was located by spraying with methanol-water 1 : 1, scraped, and eluted with chloroform-methnaol 2 : 1 containing 5% water. The eluate was washed once [7] and evaporated to dryness.
C Synthesis ofN-cerebronoyl sphingos&e O-Acetyl D, L-cerebronic acid (13.9 rag, 33//moles) was reacted with an equimolar quantity of sphingosine in the presence of triphenylphosphine and aldrithiol-2 as described above. The product was subjected to mild alkaline methanolysis using 1 ml chloroform and 0.5 ml 0.2N methanolic NaOH [8] and then fractionated by thin-layer chromatography. N-D-cerebronoyl sphingosine and N-L-crebronoyl sphingosine were separated from each other under these chromatographic conditions [9] and eluted separately from the silica gel powder as described above. III. Results
Synthesis of lignoceroyl sphingosine was complete after 4.5 hr at room temperature as indicated by thin-layer chromatographic analysis of the reaction mixture (fig. 1). Further reaction for up to 24 hr did not change the appearance of the thinlayer chromatogram. The reaction product was isolated with an overall yield of 75% (16.2 mg N-lignoceroyl sphingosine obtained from 12.5 mg lignoceric acid and 10.1 mg sphingosine). The infrared spectrum of the product was in good agreement with the previously published spectrum [5]. In one experiment [1-14C] lignoceric acid containing 200,000 cm (specific activity 56 Ci/mole) was mixed with nonradioactive lignoceric acid and reacted as described in the Experimental Section. The thin-layer chromatogram of the reaction product was scanned by a Varian-Berthold radio scanner (Varian Aerograph). After 4.5 hr, nearly all radioactivity was detected with the spot of N-lignoceroyl sphingosine; a small amount of radioactivity
Y. Kishimoto, Synthesis of ceramides
A
3 12
B
3 12
C
21
35
D
3 12
Fig. 1. Thin-layer chromatograms of reaction products after a reaction time of 30 min (A), 1 hr (B), 2 hr (C), and 4.5 hr (D). In each chromatogram, lane 1 contains the reaction product; lane 2, lignoceroyl sphingosine, and lane 3, lignoceric acid. White spots on the origin are due to psychosine; the reagents gave a number of spots above that of ceramide. The plates coated with 0.25 mm thick silica gel G (2.5 cm × 10 cm) were developed with chloroform-methanol-acetic acid (90 : 2 : 8). Blue prints of the plate were prepared after visualization by bromothymol blue spray. was present with the unreacted lignoceric acid spot. This distribution o f radioactivity did not change even after a reaction time of 24 hr. Reaction o f free cerebronic acid with sphingosine under the same conditions was not as straightforward. Although most of the sphingosine disappeared from the reaction mixture after 5 hr, the yield of N-cerebronoyl sphingosine was low: 4.9 mg D-cerebronoyl sphingosine and 4.7 mg L-isomer were obtained from 13.1 mg cerebronic acid and 10.1 mg sphingosine. Examination o f the product after the addition of 330,000 cpm of [1-14C] D,L-cerebronic acid (specific activity 1 Ci/mole) in a similar fashion as described above showed that, in addition to the ceramides, three radioactive byproducts were present. All three moved farther than the ceramides under the given thin-layer chromatographic conditions and the Rf o f the most polar b y p r o d u c t was identical to that of lignoceroyl sphingosine. The amounts of radioactivity in these spots increased with reaction time while those in the cerebronoyl sphingosines decreased. The synthesis of N-cerebronoyl sphingosine was achieved by protecting the hydroxyl group of the cerebronic acid by an acetyl group'as shown previously for the synthesis of N-cerebronoyl sphingosine [5] and N-cerebronoyl psychosine [2] by other mechanisms. Starting from 13.9 mg acetylcerebronic acid (32.7/amole), 6.8 mg of N-D-cerebronoyl sphingosine (10.2/~moles) and 7.0 mg N-L-cerebronoyl sphingosine (10.6 ktmoles) were obtained; overall yield was 64%. Infrared spectra of b o t h ceramides agreed with those published [5].
36
Y. Kishimoto, Synthesis of ceramides
IV. Discussion This procedure for ceramide synthesis, using oxidation-reduction condensation of free fatty acid and sphingosine, has the advantages o f shorter reaction time and better yield over the method using carbodiimide as a coupling reagent [5]. By eliminating the extra step of derivatization, it also is better than other methods which require the derivatization of fatty acids either to fatty acid chlorides [2] or to N-hydroxysuccinimide esters [4]. These advantages are especially significant when synthesis o f small amounts of radioactive ceramide with high specific-activity is required.
Acknowledgements This work was supported in part by Grants NS-10741, NS-11899, HD-05515, and HD-04147 from National Institutes of Health, U.S. Public Health Service. The author is grateful to Dr. Robert lt.K. Chert for suggesting the use of the reagent and helpful discussion throughout this study. Skillful assistance by Helen O. Hincman is acknowledged.
References [ 1 ] P. Morell and P. Braun, J. Lipid Res. 13 (1972) 293 [2] K.C. Kopaczyk and N.S. Radin, J. Lipid Res. 6 (1965) 140 [3] M. tfoshi and Y. Kishimoto, J. Biol. Chem. 248 (1973)4123 [4[ D.E. Ong and R.N. Brady, J. Lipid Res. 13 (1972) 819 [5] S. Hammarstr~m, J. Lipid Res. 12 (1971) 760 [6] T. Mukaiyama, R. Matsueda and M. Suzuki, Tetrahedron letters (1970) 1901 [7] J. l:olch, M. Lees and G.H. Sloane-Stanley, J. Biol. Chem. 226 (1967) 497 [8] Y. Kishimoto, W.E. Davies and N.S. Radin, J. Lipid Res. 6 (1965) 525 [9] R.C. Arora, Y-N. Lin and N.S. Radin, Arch. Biochem. Biophys. 156 (1973) 77
A FACILE SYNTHESIS OF CERAMIDES Yasuo KISHIMOTO Eunice Kennedy Shriver Center for Mental Retardation at W.E. Fernald State School, Waltham, Mass. 02154; Department of Neurology, Massachusetts General Hospital Boston, Mass. 02114, USA
Received May 10, 1 9 7 5 ,
accepted August 5, 1975
A new simple procedure for the synthesis of ceramide from free fatty acid and sphingosine by oxidation-reduction condensation with triphenylphosphine and 2,2'-dipyridyldisulfide has been described. N-lignoceroyl and N-cerebronoyl sphingosine were synthesized with this procedure producing an overall yield of 74% from lignoceric acid and 64% from cerebronic acid.
I. Introduction Ceramide plays an important role in the metabolism of sphingolipids in mammalian tissues [1 ]. In the past, several procedures have been used for the synthesis of ceramides including the reaction of sphingosine with the appropriate fatty acid chloride [2, 3], N-hydroxysuccinimide esters of fatty acids [4], or free fatty acid in the presence of a mixed carbodiimide [5]. Mukaiyama et al. [6] published a new procedure for peptide synthesis using an oxidation-reduction condensation with triphenylphosphine and 2,2'-dipyridylsulfide. We have applied this procedure to the synthesis of ceramides and find this method to be superior in many respects to the methods previously employed.
II. Experimental A. Materials
Sphingosine (flee base) obtained from bovine brain was purchased from Serdary Research Laboratories, London, Ontario, Canada and lignoceric acid from Lachat Chemical Co., Chicago Heights, Ill. Both nonradioactive [1-14C] labelled cerebronic acid and [1-14C] lignoceric acid were synthesized in this laboratory [3]. O-Acetyl cerebronic acid was prepared according to HammerstfiSm [5]. Triphenylphosphine and 2,2'-dipyridyldisulfide (Aldithiol-2) were purchased from Eastman Kodak, Rochester, N.Y. and Aldrich Chemical Co., Milwaukee, Wis., respectively. Methylene chloride
34
K Kishimoto, Synthes&of ceramides
was obtained from Burdick and Jackson Lab, Inc., Muskegon, Mich., and used without further purification. Precoated thin-layer chromatographic plates were purchased from Analtech, Inc., Newark, Del.
B. Synthesis of N-lignoceroyl sph&gosine To a mixture of 12.5 mg lignoceric acid (34/~moles), 17.5 mg triphenylphosphine (67//moles), and 14.7 mg aldrithiol-2 (67 ~moles) was added 0.65 ml of methylene chloride solution containing 10.1 mg sphingosine (34//moles); this was stirred at room temperature for 5 hr or longer. The mixture became yellow immediately after mixing. The solvent was removed from the reaction mixture by a flow of nitrogen; the residue was redissolved in 1 ml of chloroform-methanol 2 : 1 and washed with water according to Folch, Lees, and Sloane-Stanley [7]. After washing, the lower layer was evaporated to dryness, and the residue was fractionated by preparative thin-layer chromatography on silica gel G plates using chloroform-methanol-acetic acid (90 : 2 : 8) as the solvent. The band of ceramide was located by spraying with methanol-water 1 : 1, scraped, and eluted with chloroform-methnaol 2 : 1 containing 5% water. The eluate was washed once [7] and evaporated to dryness.
C Synthesis ofN-cerebronoyl sphingos&e O-Acetyl D, L-cerebronic acid (13.9 rag, 33//moles) was reacted with an equimolar quantity of sphingosine in the presence of triphenylphosphine and aldrithiol-2 as described above. The product was subjected to mild alkaline methanolysis using 1 ml chloroform and 0.5 ml 0.2N methanolic NaOH [8] and then fractionated by thin-layer chromatography. N-D-cerebronoyl sphingosine and N-L-crebronoyl sphingosine were separated from each other under these chromatographic conditions [9] and eluted separately from the silica gel powder as described above. III. Results
Synthesis of lignoceroyl sphingosine was complete after 4.5 hr at room temperature as indicated by thin-layer chromatographic analysis of the reaction mixture (fig. 1). Further reaction for up to 24 hr did not change the appearance of the thinlayer chromatogram. The reaction product was isolated with an overall yield of 75% (16.2 mg N-lignoceroyl sphingosine obtained from 12.5 mg lignoceric acid and 10.1 mg sphingosine). The infrared spectrum of the product was in good agreement with the previously published spectrum [5]. In one experiment [1-14C] lignoceric acid containing 200,000 cm (specific activity 56 Ci/mole) was mixed with nonradioactive lignoceric acid and reacted as described in the Experimental Section. The thin-layer chromatogram of the reaction product was scanned by a Varian-Berthold radio scanner (Varian Aerograph). After 4.5 hr, nearly all radioactivity was detected with the spot of N-lignoceroyl sphingosine; a small amount of radioactivity
Y. Kishimoto, Synthesis of ceramides
A
3 12
B
3 12
C
21
35
D
3 12
Fig. 1. Thin-layer chromatograms of reaction products after a reaction time of 30 min (A), 1 hr (B), 2 hr (C), and 4.5 hr (D). In each chromatogram, lane 1 contains the reaction product; lane 2, lignoceroyl sphingosine, and lane 3, lignoceric acid. White spots on the origin are due to psychosine; the reagents gave a number of spots above that of ceramide. The plates coated with 0.25 mm thick silica gel G (2.5 cm × 10 cm) were developed with chloroform-methanol-acetic acid (90 : 2 : 8). Blue prints of the plate were prepared after visualization by bromothymol blue spray. was present with the unreacted lignoceric acid spot. This distribution o f radioactivity did not change even after a reaction time of 24 hr. Reaction o f free cerebronic acid with sphingosine under the same conditions was not as straightforward. Although most of the sphingosine disappeared from the reaction mixture after 5 hr, the yield of N-cerebronoyl sphingosine was low: 4.9 mg D-cerebronoyl sphingosine and 4.7 mg L-isomer were obtained from 13.1 mg cerebronic acid and 10.1 mg sphingosine. Examination o f the product after the addition of 330,000 cpm of [1-14C] D,L-cerebronic acid (specific activity 1 Ci/mole) in a similar fashion as described above showed that, in addition to the ceramides, three radioactive byproducts were present. All three moved farther than the ceramides under the given thin-layer chromatographic conditions and the Rf o f the most polar b y p r o d u c t was identical to that of lignoceroyl sphingosine. The amounts of radioactivity in these spots increased with reaction time while those in the cerebronoyl sphingosines decreased. The synthesis of N-cerebronoyl sphingosine was achieved by protecting the hydroxyl group of the cerebronic acid by an acetyl group'as shown previously for the synthesis of N-cerebronoyl sphingosine [5] and N-cerebronoyl psychosine [2] by other mechanisms. Starting from 13.9 mg acetylcerebronic acid (32.7/amole), 6.8 mg of N-D-cerebronoyl sphingosine (10.2/~moles) and 7.0 mg N-L-cerebronoyl sphingosine (10.6 ktmoles) were obtained; overall yield was 64%. Infrared spectra of b o t h ceramides agreed with those published [5].
36
Y. Kishimoto, Synthesis of ceramides
IV. Discussion This procedure for ceramide synthesis, using oxidation-reduction condensation of free fatty acid and sphingosine, has the advantages o f shorter reaction time and better yield over the method using carbodiimide as a coupling reagent [5]. By eliminating the extra step of derivatization, it also is better than other methods which require the derivatization of fatty acids either to fatty acid chlorides [2] or to N-hydroxysuccinimide esters [4]. These advantages are especially significant when synthesis o f small amounts of radioactive ceramide with high specific-activity is required.
Acknowledgements This work was supported in part by Grants NS-10741, NS-11899, HD-05515, and HD-04147 from National Institutes of Health, U.S. Public Health Service. The author is grateful to Dr. Robert lt.K. Chert for suggesting the use of the reagent and helpful discussion throughout this study. Skillful assistance by Helen O. Hincman is acknowledged.
References [ 1 ] P. Morell and P. Braun, J. Lipid Res. 13 (1972) 293 [2] K.C. Kopaczyk and N.S. Radin, J. Lipid Res. 6 (1965) 140 [3] M. tfoshi and Y. Kishimoto, J. Biol. Chem. 248 (1973)4123 [4[ D.E. Ong and R.N. Brady, J. Lipid Res. 13 (1972) 819 [5] S. Hammarstr~m, J. Lipid Res. 12 (1971) 760 [6] T. Mukaiyama, R. Matsueda and M. Suzuki, Tetrahedron letters (1970) 1901 [7] J. l:olch, M. Lees and G.H. Sloane-Stanley, J. Biol. Chem. 226 (1967) 497 [8] Y. Kishimoto, W.E. Davies and N.S. Radin, J. Lipid Res. 6 (1965) 525 [9] R.C. Arora, Y-N. Lin and N.S. Radin, Arch. Biochem. Biophys. 156 (1973) 77
