- Email: [email protected]
Synthesis of a maradolipid without using protecting groups
Tetrahedron Letters 54 (2013) 2274–2276
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a maradolipid without using protecting groups René Csuk ⇑, Andrea Schultheiß, Sven Sommerwerk, Ralph Kluge Martin-Luther-Universität Halle-Wittenberg, Organische Chemie, Kurt-Mothes-Str. 2, D-06120 Halle, Saale, Germany
a r t i c l e
i n f o
Article history: Received 20 January 2013 Revised 21 February 2013 Accepted 22 February 2013 Available online 1 March 2013
a b s t r a c t A convenient route has been developed to synthesize 6-O-mono- and 6,60 -di-O-acyl symmetrical and unsymmetrical (un)-symmetrically trehalose derivatives from trehalose using a combination of enzymic and nonenzymic reactions. Thus, a typical maradolipid was accessed in two-steps. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Maradolipid Chemoenzymatic trans-esterification Trehalose
Introduction Primary mono- and diesters of a,a-trehalose (1, Fig. 1) are important components1–3 of the outer membrane of mycobacteria, and they have been isolated from the enduring larvae of the nematode Caenorhabdis elegans helping this organism to survive conditions of extreme desiccation.4,5 One of the most abundant components is an unsymmetric 6,60 -trehalose diester, 6-O-(13methylmyristoyl)-6’-O-oleoyltrehalose (2),5 and several routes have been devised to access this class of interesting compounds, also known as maradolipids. By and large different protecting groups have been used including the temporary protection of the hydroxyl groups with silyl or trityl groups or by converting the primary hydroxyl groups into halides or sulfonates before carrying out SN2 reactions employing carboxylate salts.2,6–11 Recently, an elegant approach utilizing a primary selective desilylation12 of persilylated trehalose has been reported by Knölker et al.13 Protecting-group-free strategies appear to be most elegant. Thus, in Grindley’s recent approach, an uronium-based coupling reagent has been used14 allowing the two-step synthesis of maradolipids without protecting groups in good yields.
for the straightforward synthesis of complex (un)-symmetrically substituted maradolipids remained unexploited. Thus, the reaction of trehalose dihydrate (1, Scheme 1) with vinyl oleate (3) in the presence of Novozyme 43516,17 for 2 days gave quite nicely the 6,60 -di-O-oleoyl-derivative 5; under the same conditions from 1 and 418 6,60 -di-O-13-methylmyristoyltrehalose 6 was obtained. Attempts to use this enzyme to obtain 6-O-monoacylated compounds 7 and 8 failed to give good yields. Reproducibly high yields of 7 and 8 were obtained, however, by using the commercially available enzyme Alcalase from Bacillus licheniformis.19 The reaction of 1 with a 1:1 mixture of the vinyl esters 3 and 4 in the presence of Novozyme 435 gave a mixture of monoacylated products 7 and 8 together with an unseparable mixture
Results and discussion The selective chemoenzymatic monoesterification of mono- and disaccharides has been known for a long time. Thus, the monoesterification of the primary hydroxyl group of 1 has been described a decade ago15 but the potential of these enzymic transformations ⇑ Corresponding author. Tel.: +49 (0) 345 5525660; fax: +49 (0) 345 5527030. E-mail address: [email protected] (R. Csuk). 0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2013.02.076
Figure 1. Structure of trehalose (1) and maradolipid (2).
R. Csuk et al. / Tetrahedron Letters 54 (2013) 2274–2276
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Scheme 1. Synthesis of mono- and sym. diacylated trehalose derivatives as well as of maradolipid 2.
of diacylated 2, 5, and 6. In addition, the Novozyme 435 or alcalase catalyzed acylations of 7 and 8 proceeded very slowly, and it was not possible to isolate significant amounts of maradolipid 2.20 As an alternative, monoacylated 7 and 8 were subjected to Mitsunobu reactions in hexamethylphosphorous triamide (HMPTA)21 to yield the unsymmetrically substituted maradolipid 222 in 69–71% yields. In summary, symmetrical 6,60 -di-O-acylated trehalose derivatives can be accessed from trehalose by using the enzyme Novozyme 435 in transesterification reactions employing the corresponding vinyl carboxylates whereas the corresponding monoesters can be obtained using the Alcalase from B. licheniformis. Unsymmetrically substituted diesters can be obtained by a twostep procedure of a chemoenzymatic monoacylation followed by a regioselective monoacylation either by a Mitsunobu reaction21 as here, or using uronium-based promotion of esterification.14 Thus, maradolipid 2 was obtained in an over-all yield of approx. 50% in a two-step sequence. Acknowledgment We like to thank Dr. D. Ströhl for measuring the NMR spectra and Mrs. J. Wiese for skillful experimental help in the analysis of the compounds.
References and notes 1. Noll, H.; Bloch, H.; Asselineau, J.; Lederer, E. Biochim. Biophys. Acta 1956, 20, 299–309. 2. Khan, A. A.; Chee, S. H.; McLaughlin, R. J.; Harper, J. L.; Kamena, F.; Timmer, M. S. M.; Stocker, B. L. Chembiochem 2011, 12, 2572–2576. 3. Hutacharoen, P.; Ruchirawat, S.; Boonyarattanakalin, S. J. Carbohydr. Chem. 2011, 30, 415–437. 4. Erkut, C.; Penkov, S.; Khesbak, H.; Vorkel, D.; Verbavatz, J. M.; Fahmy, K.; Kurzchalia, T. V. Curr. Biol. 2011, 21, 1331–1336. 5. Penkov, S.; Mende, F.; Zagoriy, V.; Erkut, C.; Martin, R.; Pässler, U.; Schuhmann, K.; Schwudke, D.; Gruner, M.; Mantler, J.; Reichert-Muller, T.; Shevchenko, A.; Knölker, H.-J.; Kurzchalia, T. V. Angew. Chem., Int. Ed. 2010, 49, 9430–9435. 6. Lederer, E. Chem. Phys. Lipids 1976, 16, 91–106. 7. Barry, C. S.; Backus, K. M.; Barry, C. E.; Davis, B. G. J. Am. Chem. Soc. 2011, 133, 13232–13235. 8. Sanki, A. K.; Boucau, J.; Umesiri, F. E.; Ronning, D. R.; Sucheck, S. J. Mol. Biosyst. 2009, 5, 945–956. 9. Nishizawa, M.; Garcia, D. M.; Minagawa, R.; Noguchi, Y.; Imagawa, H.; Yamada, H.; Watanabe, R.; Yoo, Y. C.; Azuma, I. Synlett 1996, 452–454. 10. Nishizawa, M.; Yamamoto, H.; Imagawa, H.; Barbier-Chassefiere, V.; Petit, E.; Azuma, I.; Papy-Garcia, D. J. Org. Chem. 2007, 72, 1627–1633. 11. Sarpe, V. A.; Kulkarni, S. S. J. Org. Chem. 2011, 76, 6866–6870. 12. Toubiana, R.; Das, B. C.; Defaye, J.; Mompon, B.; Toubiana, M. J. Carbohydr. Res. 1975, 44, 363–369. 13. Pässler, U.; Gruner, M.; Penkov, S.; Kurzchalia, T. V.; Knölker, H.-J. Synlett 2011, 2482–2486. 14. Paul, N. K.; Twibanire, J.-D. A.; Grindley, T. B. J. Org. Chem. 2013, 78, 363–369. 15. Raku, T.; Kitagawa, M.; Shimakawa, H.; Tokiwa, Y. J. Biotechnol. 2003, 100, 203– 208. 16. Liu, J.; Park, S. K.; Moore, J. A.; Cramer, S. M. Ind. Eng. Chem. Res. 2006, 45, 9107– 9114.
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17. John, G.; Zhu, G. Y.; Li, J.; Dordick, J. S. Angew. Chem., Int. Ed. 2006, 45, 4772– 4775. 18. Compound 4 was obtained by a sequence starting from methyl 11-iodoundecanoate that was transformed into the corresponding ylide (TPP, NaOEt in DMF) being reacted with isobutyraldehyde. The olefin was hydrogenated (H2, PtO2, 25 °C, 45 psi, EtOH) followed by a saponification (KOH in EtOH) and reaction of the 13-methyltetradecanoic acid with vinyl acetate (4 equiv) in the presence of Hg(OAc)2. 19. In a typical experiment, to a solution of the vinyl carboxylate (5.3 mmol) and 1 dihydrate (1.8 mmol) in acetone (20 ml) Novozyme 435 (0.28 g) was added, and the mixture was stirred at 45 °C until TLC (ethyl acetate/MeOH/water, 17:4:1) showed completion of the reaction (48–72 h). The mixture was filtered, the solvents were removed, and the residue was subjected to chromatography to afford the diesters. For the mono-acylations the Alcalase from Bacillus licheniformis was used. Selected analytical data: 5 mp: 154–155 °C, [a]D +6.4 (c 0.48, MeOH), MS (ESI, MeOH, LiCl): 877.6 (100%, [M+Li]+, 909.7 (44%, [M+MeOH+Li]+), 1H NMR (500 MHz, DMSO-d6): d = 5.34–5.26 (m, 4 H, CH'CH, H-15, H-16), 5.04 (d, J = 5.4 Hz, 2H, H-4), 4.87 (d, J = 4.9 Hz, 2H, H3), 4.80 (d, J = 3.6 Hz, 2H, H-1), 4.74 (d, J = 6.1 Hz, 2H, H-2), 4.21 (dd, J = 11.6, 1.5 Hz, 2H, H-6A), 4.01 (dd, J = 12.2, 5.3 Hz, 2H, H-6B), 3.90–3.83 (m, 2H, H-5), 3.53 (dd, J = 9.2, 4.9 Hz, 2H, H-3), 3.24 (ddd, J = 9.7, 6.1, 3.7 Hz, 2H, H-2), 3.10 (dd, J = 9.3, 5.5 Hz, 2H, H-4), 2.24 (t, J = 7.4 Hz, H-8), 2.02–1.92 (m, 8H, H-14A,B, H-17A,B), 1.56–1.40 (m, 4H,H-9A,B), 1.32–1.17 (m, 40H, CH2 (H(C-10)-H-(C13), H-(C18)–H-(C23)), 0.83 (t, J = 6.9 Hz, 6H, CH3, H-24) ppm; 13C NMR (125 MHz, DMSO-d6): d = 172.8 (C-7), 129.73 and 129.70 (C-15, C-16), 93.4 (C-1), 72.85 (C-3), 71.56 (C-2), 70.25 (C-4), 69.82 (C-5), 63.2 (C-6), 33.7 (C-8), 31.4 (C-22), 29.19, 28.93, 28.78, 28.67, 28.57, 28.54 (CH2, C-10–C-13, C-18–C-21), 26.69, 26.67 (C-14, C-17), 24.60 (C-9), 22.2 (C-23), 14.1 (C-24) ppm; analysis for C48H86O13 (871.19): C, 66.18; H, 9.95; found: C, 65.97; H, 7.02; 6 mp 163– 164 °C; [a]D +7.7 (c 0.4, MeOH), MS (ESI, MeOH, LiCl): 797.6 (100%, [M+Li]+, 814.5 (27%, [M+Na]+), 1H NMR (500 MHz, DMSO-d6): d = 5.04 (dd, J = 3.6, 1.7 Hz, H-4), 4.86 (d, J = 1.5 Hz, H-3), 4.81 (d, J = 3.6 Hz, 2 H, H-1), 4.75-4.72 (m, 2H, H-2), 4.22 (dd, J = 11.4, 1.5 Hz, 2H, H-6A), 4.01 (dd, J = 11.9, 5.5 Hz, 2H, H-
6B), 3.87 (m, 2H, C-5), 3.59-3.49 (m, 2H, H-3), 3.24 (ddd, J = 9.5, 5.5, 3.8 Hz, 2H, H-2), 3.15–3.05 (m, 2H, H-4), 2.25 (t, J = 7.3 Hz, 4H, H-8), 2.14–2.08 (m, 4H, H9), 1.54–1.39 (m, 6H, H-17, C-19), 1.19 (m, 28 H, CH2, H-(C-10)–H(C-16)), 1.15– 1.06 (m, 4H, H-18), 0.83 (dd, J = 6.6, 1.4 Hz, 12H, H-20, H-21) ppm; 13C NMR (125 MHz, DMSO-d6): d = 173.2 (C-7), 93.8 (C-1), 73.14 (C-3), 71.90 (C-2), 70.59 (C-4), 70.18 (C-5), 63.5 (C-6), 38.9 (C-18), 35.2 (C-9), 34.0 (C-8), 29.53, 29.50, 29.46, 29.44, 29.39, 28.87 (C-11–C-16), 27.8 (C-19), 27.2 (C-10), 24.9 (C-17), 22.98, 22.97 (C-20, C-21) ppm; analysis for C42H78O13 (791.06): C, 63.77; H, 9.94; found: C, 63.51; H, 10.03.; 7 mp 165–167 °C (lit. 166–168 °C: Ref. 13), [a]D 1.2 (c 0.32, MeOH), MS (ESI, MeOH): 605.2 (29%, [MH], 651.9 (100%, [M+HCO2]); NMR as reported [Ref. 13]; 8 mp: 112–114 °C, [a]D +6.6 (c 0.4, MeOH), MS (ESI, MeOH, LiCl): 573.3 (100%, [M+L]+, 589.2 (100%, [M+Na]+), 1H NMR (400 MHz, DMSO-d6): d = 5.02 (d, J = 5.4 Hz, 2 H, C-4, H-40 ), 4.84 (d, J = 3.7 Hz, 1H, H-1), 4.82 (d, J = 3.6 Hz, 1H, H-10 ), 4.75 (d, J = 4.7 Hz, 2H, H-3, H30 ), 4.65 (d, J = 6.1 Hz, 2H, H-2, H-20 ), 4.33 (t, J = 5.8 Hz, 1H, H-6’), 4.21 (dd, J = 11.5, 1.4 Hz, 1H, H-6), 4.01 (m, 1H, H-6), 3.92–3.81 (m, 2H, H-5, H-50 ), 3.68– 3.58 (m, 2H, H-3, H-30 ), 3.53 (m, 1H, H-60 ), 3.45 (dd, J = 11.8, 5.8 Hz, H-60 ), 3.23 (m, 2H, H-2, H-20 ), 3.15–3.07 (m, 2H, H-4, H-40 ), 2.25 (t, J = 7.4 Hz, 2H, H-8), 1.47 (tt, J = 13.1, 6.6 Hz, 2H, H-9), 1.37–1.28 (m, 1H, H-19), 1.19–1.23 (m, 16H, H-(C-10)–H-(C-17)), 1.15-1.08 (m, 2 H, H-18), 0.82 (d, J = 6.6 Hz, 6H, H-20, H21) ppm; 13C NMR (100 MHz, DMSO-d6): d = 173.5 (C-7), 93.60 (C-10 ), 93.48 (C1), 73.04 (C-30 ), 72.91 (C-3), 71.78 (C-50 ), 71.75 (C-20 ), 70.32 (C-2), 70.27 (C-40 ), 69.88 (C-4), 69.76 (C-5), 63.18 (C-6), 61.11 (C-60 ), 38.90 (C-18), 33.70 (C-8), 29.59, 29.31, 29.22, 29.17, 29.01, 28.94, 28.72 (C-11–C17), 29.00 (C-19), 26.9 (C-10), 24.4 (C-9), 22.7 (C20, C-21) ppm; analysis for C27H50O4 (566.68): C, 57.23; H, 8.89; found: C, 57.11; H, 8.98. 20. Using prolonged reaction times led to significant deacylation of the starting materials; the formation of traces of 2 was determined only by ESI-MS. 21. Jenkins, I. D.; Goren, M. B. Chem. Phys. Lipids 1986, 41, 225–235. 22. Selected analytical data for 2: colorless gummy solid; [a]D +7.2 (c 0.2, MeOH), 1H and 13C NMR as reported [refs 5 and 14], MS (ESI, MeOH): 837.6 (100%, [M+Li]+).
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a maradolipid without using protecting groups René Csuk ⇑, Andrea Schultheiß, Sven Sommerwerk, Ralph Kluge Martin-Luther-Universität Halle-Wittenberg, Organische Chemie, Kurt-Mothes-Str. 2, D-06120 Halle, Saale, Germany
a r t i c l e
i n f o
Article history: Received 20 January 2013 Revised 21 February 2013 Accepted 22 February 2013 Available online 1 March 2013
a b s t r a c t A convenient route has been developed to synthesize 6-O-mono- and 6,60 -di-O-acyl symmetrical and unsymmetrical (un)-symmetrically trehalose derivatives from trehalose using a combination of enzymic and nonenzymic reactions. Thus, a typical maradolipid was accessed in two-steps. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Maradolipid Chemoenzymatic trans-esterification Trehalose
Introduction Primary mono- and diesters of a,a-trehalose (1, Fig. 1) are important components1–3 of the outer membrane of mycobacteria, and they have been isolated from the enduring larvae of the nematode Caenorhabdis elegans helping this organism to survive conditions of extreme desiccation.4,5 One of the most abundant components is an unsymmetric 6,60 -trehalose diester, 6-O-(13methylmyristoyl)-6’-O-oleoyltrehalose (2),5 and several routes have been devised to access this class of interesting compounds, also known as maradolipids. By and large different protecting groups have been used including the temporary protection of the hydroxyl groups with silyl or trityl groups or by converting the primary hydroxyl groups into halides or sulfonates before carrying out SN2 reactions employing carboxylate salts.2,6–11 Recently, an elegant approach utilizing a primary selective desilylation12 of persilylated trehalose has been reported by Knölker et al.13 Protecting-group-free strategies appear to be most elegant. Thus, in Grindley’s recent approach, an uronium-based coupling reagent has been used14 allowing the two-step synthesis of maradolipids without protecting groups in good yields.
for the straightforward synthesis of complex (un)-symmetrically substituted maradolipids remained unexploited. Thus, the reaction of trehalose dihydrate (1, Scheme 1) with vinyl oleate (3) in the presence of Novozyme 43516,17 for 2 days gave quite nicely the 6,60 -di-O-oleoyl-derivative 5; under the same conditions from 1 and 418 6,60 -di-O-13-methylmyristoyltrehalose 6 was obtained. Attempts to use this enzyme to obtain 6-O-monoacylated compounds 7 and 8 failed to give good yields. Reproducibly high yields of 7 and 8 were obtained, however, by using the commercially available enzyme Alcalase from Bacillus licheniformis.19 The reaction of 1 with a 1:1 mixture of the vinyl esters 3 and 4 in the presence of Novozyme 435 gave a mixture of monoacylated products 7 and 8 together with an unseparable mixture
Results and discussion The selective chemoenzymatic monoesterification of mono- and disaccharides has been known for a long time. Thus, the monoesterification of the primary hydroxyl group of 1 has been described a decade ago15 but the potential of these enzymic transformations ⇑ Corresponding author. Tel.: +49 (0) 345 5525660; fax: +49 (0) 345 5527030. E-mail address: [email protected] (R. Csuk). 0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2013.02.076
Figure 1. Structure of trehalose (1) and maradolipid (2).
R. Csuk et al. / Tetrahedron Letters 54 (2013) 2274–2276
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Scheme 1. Synthesis of mono- and sym. diacylated trehalose derivatives as well as of maradolipid 2.
of diacylated 2, 5, and 6. In addition, the Novozyme 435 or alcalase catalyzed acylations of 7 and 8 proceeded very slowly, and it was not possible to isolate significant amounts of maradolipid 2.20 As an alternative, monoacylated 7 and 8 were subjected to Mitsunobu reactions in hexamethylphosphorous triamide (HMPTA)21 to yield the unsymmetrically substituted maradolipid 222 in 69–71% yields. In summary, symmetrical 6,60 -di-O-acylated trehalose derivatives can be accessed from trehalose by using the enzyme Novozyme 435 in transesterification reactions employing the corresponding vinyl carboxylates whereas the corresponding monoesters can be obtained using the Alcalase from B. licheniformis. Unsymmetrically substituted diesters can be obtained by a twostep procedure of a chemoenzymatic monoacylation followed by a regioselective monoacylation either by a Mitsunobu reaction21 as here, or using uronium-based promotion of esterification.14 Thus, maradolipid 2 was obtained in an over-all yield of approx. 50% in a two-step sequence. Acknowledgment We like to thank Dr. D. Ströhl for measuring the NMR spectra and Mrs. J. Wiese for skillful experimental help in the analysis of the compounds.
References and notes 1. Noll, H.; Bloch, H.; Asselineau, J.; Lederer, E. Biochim. Biophys. Acta 1956, 20, 299–309. 2. Khan, A. A.; Chee, S. H.; McLaughlin, R. J.; Harper, J. L.; Kamena, F.; Timmer, M. S. M.; Stocker, B. L. Chembiochem 2011, 12, 2572–2576. 3. Hutacharoen, P.; Ruchirawat, S.; Boonyarattanakalin, S. J. Carbohydr. Chem. 2011, 30, 415–437. 4. Erkut, C.; Penkov, S.; Khesbak, H.; Vorkel, D.; Verbavatz, J. M.; Fahmy, K.; Kurzchalia, T. V. Curr. Biol. 2011, 21, 1331–1336. 5. Penkov, S.; Mende, F.; Zagoriy, V.; Erkut, C.; Martin, R.; Pässler, U.; Schuhmann, K.; Schwudke, D.; Gruner, M.; Mantler, J.; Reichert-Muller, T.; Shevchenko, A.; Knölker, H.-J.; Kurzchalia, T. V. Angew. Chem., Int. Ed. 2010, 49, 9430–9435. 6. Lederer, E. Chem. Phys. Lipids 1976, 16, 91–106. 7. Barry, C. S.; Backus, K. M.; Barry, C. E.; Davis, B. G. J. Am. Chem. Soc. 2011, 133, 13232–13235. 8. Sanki, A. K.; Boucau, J.; Umesiri, F. E.; Ronning, D. R.; Sucheck, S. J. Mol. Biosyst. 2009, 5, 945–956. 9. Nishizawa, M.; Garcia, D. M.; Minagawa, R.; Noguchi, Y.; Imagawa, H.; Yamada, H.; Watanabe, R.; Yoo, Y. C.; Azuma, I. Synlett 1996, 452–454. 10. Nishizawa, M.; Yamamoto, H.; Imagawa, H.; Barbier-Chassefiere, V.; Petit, E.; Azuma, I.; Papy-Garcia, D. J. Org. Chem. 2007, 72, 1627–1633. 11. Sarpe, V. A.; Kulkarni, S. S. J. Org. Chem. 2011, 76, 6866–6870. 12. Toubiana, R.; Das, B. C.; Defaye, J.; Mompon, B.; Toubiana, M. J. Carbohydr. Res. 1975, 44, 363–369. 13. Pässler, U.; Gruner, M.; Penkov, S.; Kurzchalia, T. V.; Knölker, H.-J. Synlett 2011, 2482–2486. 14. Paul, N. K.; Twibanire, J.-D. A.; Grindley, T. B. J. Org. Chem. 2013, 78, 363–369. 15. Raku, T.; Kitagawa, M.; Shimakawa, H.; Tokiwa, Y. J. Biotechnol. 2003, 100, 203– 208. 16. Liu, J.; Park, S. K.; Moore, J. A.; Cramer, S. M. Ind. Eng. Chem. Res. 2006, 45, 9107– 9114.
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17. John, G.; Zhu, G. Y.; Li, J.; Dordick, J. S. Angew. Chem., Int. Ed. 2006, 45, 4772– 4775. 18. Compound 4 was obtained by a sequence starting from methyl 11-iodoundecanoate that was transformed into the corresponding ylide (TPP, NaOEt in DMF) being reacted with isobutyraldehyde. The olefin was hydrogenated (H2, PtO2, 25 °C, 45 psi, EtOH) followed by a saponification (KOH in EtOH) and reaction of the 13-methyltetradecanoic acid with vinyl acetate (4 equiv) in the presence of Hg(OAc)2. 19. In a typical experiment, to a solution of the vinyl carboxylate (5.3 mmol) and 1 dihydrate (1.8 mmol) in acetone (20 ml) Novozyme 435 (0.28 g) was added, and the mixture was stirred at 45 °C until TLC (ethyl acetate/MeOH/water, 17:4:1) showed completion of the reaction (48–72 h). The mixture was filtered, the solvents were removed, and the residue was subjected to chromatography to afford the diesters. For the mono-acylations the Alcalase from Bacillus licheniformis was used. Selected analytical data: 5 mp: 154–155 °C, [a]D +6.4 (c 0.48, MeOH), MS (ESI, MeOH, LiCl): 877.6 (100%, [M+Li]+, 909.7 (44%, [M+MeOH+Li]+), 1H NMR (500 MHz, DMSO-d6): d = 5.34–5.26 (m, 4 H, CH'CH, H-15, H-16), 5.04 (d, J = 5.4 Hz, 2H, H-4), 4.87 (d, J = 4.9 Hz, 2H, H3), 4.80 (d, J = 3.6 Hz, 2H, H-1), 4.74 (d, J = 6.1 Hz, 2H, H-2), 4.21 (dd, J = 11.6, 1.5 Hz, 2H, H-6A), 4.01 (dd, J = 12.2, 5.3 Hz, 2H, H-6B), 3.90–3.83 (m, 2H, H-5), 3.53 (dd, J = 9.2, 4.9 Hz, 2H, H-3), 3.24 (ddd, J = 9.7, 6.1, 3.7 Hz, 2H, H-2), 3.10 (dd, J = 9.3, 5.5 Hz, 2H, H-4), 2.24 (t, J = 7.4 Hz, H-8), 2.02–1.92 (m, 8H, H-14A,B, H-17A,B), 1.56–1.40 (m, 4H,H-9A,B), 1.32–1.17 (m, 40H, CH2 (H(C-10)-H-(C13), H-(C18)–H-(C23)), 0.83 (t, J = 6.9 Hz, 6H, CH3, H-24) ppm; 13C NMR (125 MHz, DMSO-d6): d = 172.8 (C-7), 129.73 and 129.70 (C-15, C-16), 93.4 (C-1), 72.85 (C-3), 71.56 (C-2), 70.25 (C-4), 69.82 (C-5), 63.2 (C-6), 33.7 (C-8), 31.4 (C-22), 29.19, 28.93, 28.78, 28.67, 28.57, 28.54 (CH2, C-10–C-13, C-18–C-21), 26.69, 26.67 (C-14, C-17), 24.60 (C-9), 22.2 (C-23), 14.1 (C-24) ppm; analysis for C48H86O13 (871.19): C, 66.18; H, 9.95; found: C, 65.97; H, 7.02; 6 mp 163– 164 °C; [a]D +7.7 (c 0.4, MeOH), MS (ESI, MeOH, LiCl): 797.6 (100%, [M+Li]+, 814.5 (27%, [M+Na]+), 1H NMR (500 MHz, DMSO-d6): d = 5.04 (dd, J = 3.6, 1.7 Hz, H-4), 4.86 (d, J = 1.5 Hz, H-3), 4.81 (d, J = 3.6 Hz, 2 H, H-1), 4.75-4.72 (m, 2H, H-2), 4.22 (dd, J = 11.4, 1.5 Hz, 2H, H-6A), 4.01 (dd, J = 11.9, 5.5 Hz, 2H, H-
6B), 3.87 (m, 2H, C-5), 3.59-3.49 (m, 2H, H-3), 3.24 (ddd, J = 9.5, 5.5, 3.8 Hz, 2H, H-2), 3.15–3.05 (m, 2H, H-4), 2.25 (t, J = 7.3 Hz, 4H, H-8), 2.14–2.08 (m, 4H, H9), 1.54–1.39 (m, 6H, H-17, C-19), 1.19 (m, 28 H, CH2, H-(C-10)–H(C-16)), 1.15– 1.06 (m, 4H, H-18), 0.83 (dd, J = 6.6, 1.4 Hz, 12H, H-20, H-21) ppm; 13C NMR (125 MHz, DMSO-d6): d = 173.2 (C-7), 93.8 (C-1), 73.14 (C-3), 71.90 (C-2), 70.59 (C-4), 70.18 (C-5), 63.5 (C-6), 38.9 (C-18), 35.2 (C-9), 34.0 (C-8), 29.53, 29.50, 29.46, 29.44, 29.39, 28.87 (C-11–C-16), 27.8 (C-19), 27.2 (C-10), 24.9 (C-17), 22.98, 22.97 (C-20, C-21) ppm; analysis for C42H78O13 (791.06): C, 63.77; H, 9.94; found: C, 63.51; H, 10.03.; 7 mp 165–167 °C (lit. 166–168 °C: Ref. 13), [a]D 1.2 (c 0.32, MeOH), MS (ESI, MeOH): 605.2 (29%, [MH], 651.9 (100%, [M+HCO2]); NMR as reported [Ref. 13]; 8 mp: 112–114 °C, [a]D +6.6 (c 0.4, MeOH), MS (ESI, MeOH, LiCl): 573.3 (100%, [M+L]+, 589.2 (100%, [M+Na]+), 1H NMR (400 MHz, DMSO-d6): d = 5.02 (d, J = 5.4 Hz, 2 H, C-4, H-40 ), 4.84 (d, J = 3.7 Hz, 1H, H-1), 4.82 (d, J = 3.6 Hz, 1H, H-10 ), 4.75 (d, J = 4.7 Hz, 2H, H-3, H30 ), 4.65 (d, J = 6.1 Hz, 2H, H-2, H-20 ), 4.33 (t, J = 5.8 Hz, 1H, H-6’), 4.21 (dd, J = 11.5, 1.4 Hz, 1H, H-6), 4.01 (m, 1H, H-6), 3.92–3.81 (m, 2H, H-5, H-50 ), 3.68– 3.58 (m, 2H, H-3, H-30 ), 3.53 (m, 1H, H-60 ), 3.45 (dd, J = 11.8, 5.8 Hz, H-60 ), 3.23 (m, 2H, H-2, H-20 ), 3.15–3.07 (m, 2H, H-4, H-40 ), 2.25 (t, J = 7.4 Hz, 2H, H-8), 1.47 (tt, J = 13.1, 6.6 Hz, 2H, H-9), 1.37–1.28 (m, 1H, H-19), 1.19–1.23 (m, 16H, H-(C-10)–H-(C-17)), 1.15-1.08 (m, 2 H, H-18), 0.82 (d, J = 6.6 Hz, 6H, H-20, H21) ppm; 13C NMR (100 MHz, DMSO-d6): d = 173.5 (C-7), 93.60 (C-10 ), 93.48 (C1), 73.04 (C-30 ), 72.91 (C-3), 71.78 (C-50 ), 71.75 (C-20 ), 70.32 (C-2), 70.27 (C-40 ), 69.88 (C-4), 69.76 (C-5), 63.18 (C-6), 61.11 (C-60 ), 38.90 (C-18), 33.70 (C-8), 29.59, 29.31, 29.22, 29.17, 29.01, 28.94, 28.72 (C-11–C17), 29.00 (C-19), 26.9 (C-10), 24.4 (C-9), 22.7 (C20, C-21) ppm; analysis for C27H50O4 (566.68): C, 57.23; H, 8.89; found: C, 57.11; H, 8.98. 20. Using prolonged reaction times led to significant deacylation of the starting materials; the formation of traces of 2 was determined only by ESI-MS. 21. Jenkins, I. D.; Goren, M. B. Chem. Phys. Lipids 1986, 41, 225–235. 22. Selected analytical data for 2: colorless gummy solid; [a]D +7.2 (c 0.2, MeOH), 1H and 13C NMR as reported [refs 5 and 14], MS (ESI, MeOH): 837.6 (100%, [M+Li]+).