Chemoenzymatic Synthesis of Enantiopure Structured Triacylglycerols

Abstract

A highly efficient chemoenzymatic method for the synthesis of enantiopure ABC-type asymmetrically structured triacyl­glycerols has been developed starting from enantiopure (S)-solketal and involving two lipase steps.

Key words

asymmetrically structured triacylglycerols - regioselectivity - lipase - eicosapentaenoic acid - acyl migration

References and Notes

• 1a Miura S. Ogawa A. Konishi H. J. Am. Oil Chem. Soc.  1999,  76:  927 
• 1b Fomuso LB. Akoh CC. J. Am. Oil Chem. Soc.  1998,  75:  405 
• 1c Christensen MS. Höy C.-E. Becker CC. Redgrave TG. Am. J. Clin. Nutr.  1995,  61:  56 
• 2 Halldorsson A. Magnusson CD. Haraldsson GG. Tetrahedron  2003,  59:  9101 
• 3a Haraldsson GG. Hjaltason B. In Structured and Modified Lipids   Gunstone FD. Marcel Decker; New York: 2001.  p.313-350  
• 3b Hjaltason B. Haraldsson GG. In Modifying Lipids for Use in Food   Gunstone FD. Woodhead Publishing; Cambridge UK: 2006.  Chap. 4. p.56-79  
• For reviews, see:
• 4a Stansby ME. In Fish Oils in Nutrition   Stansby ME. van Nostrand Reinhold; New York: 1990.  Chap. 10. p.268-288  
• 4b Nettleton JA. Omega-3 Fatty Acids and Health   Chapman and Hall; New York: 1995. 
• 4c Lands WEM. Fish, Omega-3 and Human Health   2nd ed.:  AOCS Press; Champaign IL: 2005. 
• 5 Haraldsson GG. In Handbook of Industrial Biocatalysis   Hou CT. CRC Press, Taylor and Francis Group; Boca Raton: 2005.  Chap. 18. p.18-1-18-21  
• 6a Berger M. Laumen K. Schneider MP. J. Am. Oil Chem. Soc.  1992,  69:  955 
• 6b Aha B. Berger M. Jakob B. Machmüller G. Waldinger C. Schneider MP. In Enzymes in Lipid Modification   Bornscheuer UT. Wiley-VCH; Weinheim: 2000.  p.100-115  
• 6c Irimescu R. Furihata K. Hata K. Iwasaki Y. Yamane T. J. Am. Oil Chem. Soc.  2001,  78:  285 
• 6d Irimescu R. Iwasaki Y. Hou CT. J. Am. Oil Chem. Soc.  2002,  79:  879 
• 6e Kawashima A. Shimada Y. Yamamoto M. Sugihara A. Nagao T. Komemushi S. Tominaga Y. J. Am. Oil Chem. Soc.  2001,  78:  611 
• 8 Kato Y. Fujiwara I. Asano Y. J. Mol. Catal. B: Enzym.  2000,  9:  193 
• 9a Wang Y.-F. Wong C.-H. J. Org. Chem.  1988,  53:  3127 
• 9b Guanti G. Banfi L. Basso A. Bevilacqua E. Bondanza L. Riva R. Tetrahedron: Asymmetry  2004,  15:  2889 
• 9c Halldorsson A. Thordarson P. Kristinsson B. Magnusson CD. Haraldsson GG. Tetrahedron: Asymmetry  2004,  15:  2893 
• 9d Ransac S. Rogalska E. Gargouri Y. Deveer AMTJ. Paltauf F. de Haas GH. Verger R. J. Biol. Chem.  1990,  265:  20263 
• 9e Theil F. Lemke K. Ballschuh S. Kunath A. Schick H. Tetrahedron: Asymmetry  1995,  6:  1323 
• 10a Rogalska E. Ransac S. Verger R. J. Biol. Chem.  1990,  265:  20271 
• 10b Villeneuve P. Pina M. Montet D. Graille J. Chem. Phys. Lipids  1995,  76:  109 
• 10c Villeneuve P. Pina M. Graille J. Chem. Phys. Lipids  1996,  83:  161 
• 10d Lang DA. Dijkstra BW. Chem. Phys. Lipids  1998,  93:  115 
• 10e Rogalska E. Cudrey C. Ferrato F. Verger R. Chirality  1993,  5:  24 
• 10f Lang DA. Mannesse MLM. de Haas GH. Verheij HM. Dijkstra BW. Eur. J. Biochem.  1993,  254:  333 
• 10g Uzawa H. Nishida Y. Ohrui H. Meguro H. Biochem. Biophys. Res. Commun.  1990,  168:  506 
• 10h Uzawa H. Noguchi T. Nishida Y. Ohrui H. Meguro H. Biochim. Biophys. Acta  1993,  1168:  253 
• 11a Chandler IC. Quinlan PT. McNeill GP. J. Am. Oil Chem. Soc.  1998,  75:  1513 
• 11b Iwasaki Y. Yamane T. J. Mol. Catal. B: Enzym.  2000,  10:  129 
• 12 Piyatheerawong W. Yamane T. Nakano H. Iwasaki Y. J. Am. Oil Chem. Soc.  2006,  83:  603 
• 13 Compton DL. Vermillion KE. Laszlo JA. J. Am. Oil Chem. Soc.  2007,  84:  343 
• 15a Kodali DR. Tercyak A. Fahey DA. Small DM. Chem. Phys. Lipids  1990,  52:  163 
• 15b Bloomer S. Adlercreutz P. Mattiasson B. Biocatalysis  1991,  5:  145 
• 15c Fureby AM. Virto C. Adlercreutz P. Mattiasson B. Biocat. Biotrans.  1996,  14:  89 
• 16 Laszlo JA. Compton DL. Vermillion KE. J. Am. Oil Chem. Soc.  2008,  85:  307 
• 19 Vilcheze C. Bittman R. J. Lipid Res.  1994,  35:  734 
• 21 Reese CB. Yan H. J. Chem. Soc., Perkin Trans. 1  2001,  1807 

7

Novozym 435, CAL-B; a gift from Novozyme A/S (Bagsvaerd, Denmark).

14

Sigma-Aldrich (Steinheim, Germany): (S)-(+)-1,2-O-isopropylidene-sn-glycerol of 98% purity and 99% ee.

17

[α]_D ^²0 +5.5 (c 20, CHCl₃), identical to that in ref. 18.

18

Aldrich Chemical Catalog, (R)-(+)-benzyloxy-1,2-propanediol, 99%: [α]_D ^²0 +5.5 (c 20, CHCl₃).

20

Procedure for the Preparation of 1- O -Octadecanoyl- sn -glycerol [( S )-4]
To a solution of 3-O-benzyl-sn-glycerol [(R)-3, 1.497 g, 6.73 mmol] and vinyl stearate (3.827 mg, 12.3 mmol) in CH₂Cl₂ (8.2 mL) was added immobilized CAL (99 mg). The resulting suspension was stirred at r.t. for approx. 70 min when TLC monitoring (EtOAc-PE, 1:1) indicated a complete reaction. The lipase preparation was separated by filtration and the solvent removed in vacuo on a rotary evaporator. The resulting residue was dissolved in THF (30 mL) without further purification followed by addition of n-hexane (70 mL) and Pd/C catalyst (370 mg). The reaction was performed in a PARR reactor under hydrogen pressure (5 bar) during which the product precipitated. When the reaction had proceeded to completion (about 2 h), THF was added until all the product had been dissolved. The catalyst was separated off by filtration by the aid of Celite and the solvent removed in vacuo on a rotary evaporator. The residue was redissolved in minimum amount of THF and a fourfold volume of n-hexane added. The resulting mixture was allowed to stand at r.t. overnight to afford the product as white crystals (2.057 g, 5.73 mmol) in 85% overall yield; mp 59.5-60.7 ˚C. [α]_D ^²0 +2.43 (c 6, THF). ^¹H NMR (400 MHz, CDCl₃): δ = 4.21 (dd, J = 11.6, 4.8 Hz, 1 H, OCOCH₂), 4.15 (dd, J = 11.6, 6.0 Hz, 1 H, OCOCH₂), 3.96-3.90 (m, 1 H, CH₂CHCH₂), 3.72-3.67 (m, 1 H, CH ₂OH), 3.63-3.57 (m, 1 H, CH ₂OH), 2.52 (d, J = 5.2 Hz, 1 H, CHOH), 2.35 (t, J = 7.6 Hz, 2 H, CH₂COO), 2.08 (t, J = 6.0 Hz, 1 H, CH₂OH), 1.65-1.59 (m, 2 H, CH ₂CH₂COO), 1.36-1.18 (m, 28 H, CH₂), 0.88 (t, J = 6.5 Hz, 3 H, CH₂CH ₃). ^¹³C NMR (100 MHz, CDCl₃): δ = 174.3, 70.3, 65.2, 63.3, 34.1, 31.9, 29.7 (5 C), 29.6 (2 C), 29.4 (2 C), 29.3, 29.2, 29.1, 24.9, 22.7, 14.1. FT-IR: 3150-3600 (br, OH), 2918 (vs, CH), 2849 (vs, CH), 1735 (vs, C=O) cm^-¹. HRMS (APCI): m/z calcd for C₂₁H₄₂O₄ - OH: 341.3050; found: 341.3042 amu.

22

Procedure for 3- O -Decanoyl-1-octadecanoyl- sn -glycerol [( S )-5]
Immobilized Candida antarctica lipase (45 mg) was added to a 10 mL round-bottom flask containing a mixture of 1-octadecanoyl-sn-glycerol [(S)-4, 203 mg, 0.566 mmol] and vinyl decanoate (168 mg, 0.849 mmol) dissolved in THF (2 mL). The resulting suspension was stirred at r.t. for 2-3 h or until TLC monitoring (EtOAc-PE, 1:1) indicated a complete reaction. The lipase preparation was removed by filtration and the solvent removed in vacuo on a rotary evaporator to afford pure 3-O-decanoyl-1-octadecanoyl-sn-glycerol [(S)-5, 246 mg, 0.480 mmol] as a white crystalline material in 85% yield after recrystallization from MeOH; mp 53.4-54.0 ˚C; [α]_D ^²0 +0.02 (c 10, CH₂Cl₂). ^¹H NMR (400 MHz, CDCl₃): δ = 4.20-4.06 (m, 5 H, CH₂CHCH₂), 2.47 (d, J = 3.6 Hz, 1 H, OH), 2.34 (t, J = 7.6 Hz, 4 H, CH₂COO), 1.68-1.59 (m, 4 H, CH ₂CH₂COO), 1.36-1.18 (m, 40 H, CH₂), 0.87 (t, J = 6.8 Hz, 6 H, CH₃). ^¹³C NMR (100 MHz, CDCl₃): δ = 173.9 (2 C), 68.4, 65.1 (2 C), 34.1 (2 C), 32.0, 31.9, 29.7 (5 C), 29.6 (2 C), 29.5 (2 C), 29.4 (3 C), 29.3 (2 C), 29.1 (2 C), 24.9 (2 C), 22.7 (2 C), 14.1 (2 C). FT-IR: 3300-3600 (br, OH), 2914 (vs, CH), 2849 (vs, CH), 1732 (vs, C=O), 1708 (vs, C=O) cm^-¹. HRMS (APCI): m/z calcd for C₃₁H₆₀O₅ - OH 495.4408; found: 495.4412 amu.

23

Procedure for 3- O -Decanoyl-2-eicosapentaenoyl-1-octadecanoyl- sn -glycerol [( S )-2]
To a solution of 3-O-decanoyl-1-octadecanoyl-sn-glycerol [(S)-5, 100 mg, 0.195 mmol] and EPA as free acid (66.0 mg, 0.218 mmol) in CH₂Cl₂ (2 mL) were added DMAP (20 mg, 0.16 mmol) and EDAC (50 mg, 0.26 mmol). The resulting solution was stirred on a magnetic stirrer hotplate at r.t. for 12 h. The reaction was disconnected by passing the reaction mixture through a short column packed with silica gel by use of Et₂O-CH₂Cl₂ (10:90). Solvent removal in vacuo afforded the pure product as a yellowish oil (141 mg, 0.177 mmol, 91% yield). [α]_D ^²0 +0.16 (c 10, CH₂Cl₂). ^¹H NMR (400 MHz, CDCl₃): δ = 5.42-5.33 (m, 10 H, =CH), 5.32-5.23 (m, 1 H, CH₂CHCH₂), 4.29 (dd, J = 12.0, 4.4 Hz, 2 H, CH ₂CHCH ₂), 4.14 (dd, J = 12.0, 6.0 Hz, 2 H, CH ₂CHCH ₂), 2.86-2.78 (m, 8 H, =CCH₂C=), 2.34 (t, J = 7.6 Hz, 2 H, CH₂COO in EPA), 2.31 (t, J = 7.6 Hz, 4 H, CH₂COO), 2.14-2.04 (m, 4 H, =CCH ₂CH₂ and =CCH ₂CH₃), 1.70 (quint, J = 7.6 Hz, 2 H, CH ₂CH₂COO in EPA), 1.64-1.55 (m, 4 H, CH ₂CH₂COO), 1.34-1.20 (m, 40 H, CH₂), 0.97 (t, J = 7.6 Hz, 3 H, CH₃ in EPA), 0.88 (t, J = 6.8 Hz, 6 H, CH₃ in C₁₈). ^¹³C NMR (100 MHz, CDCl₃): δ = 173.3 (2 C), 172.6, 132.0, 128.9, 128.8, 128.6, 128.3, 128.2, 128.1, 128.0, 127.8, 127.0, 69.0, 62.1 (2 C), 34.0 (2 C), 33.6, 31.9, 31.8, 29.7 (5 C), 29.6 (2 C), 29.5, 29.4 (2 C), 29.3 (2 C), 29.2 (3 C), 29.1, 26.5, 25.6 (3 C), 25.5, 24.8 (2 C), 24.7, 22.7, 22.6, 20.5, 14.3, 14.1 (2 C). FT-IR: 3013 (s, CH), 2925 (vs, CH), 2854 (vs, CH), 1745 (vs, C=O) cm^-¹. HRMS (APCI): m/z calcd for C₅₁H₈₈O₆ + NH₄: 814.6919; found: 814.6913 amu.