J Nutr Sci Vitaminol, 61, 222–227, 2015 Chemical Synthesis of a Very Long-Chain Fatty Acid, Hexacosanoic Acid (C26:0), and the Ceramide Containing Hexacosanoic Acid Yoshinori YAMAMOTO, Toshimasa ITOH and Keiko YAMAMOTO* Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3–3165 Higashi-Tamagawagakuen, Machida, Tokyo 194–8543, Japan (Received December 24, 2014) Summary Hexacosanoic acid (C26:0) (1), a very long-chain fatty acid, is related to vari- ous diseases such as adrenoleukodystrophy (ALD), adrenomyeloneuropathy (AMN) and atherosclerosis. As the level of 1 higher than the normal is related to diseases above, hexa- cosanoic acid (1) and the ceramide 2, which contains 1, are thought to play an important role in various tissues. Hexacosanoic acid (1) is known to be a waxy solid and to be hard to dissolve in water as well as organic solvents. Due to this physical property, it is not easy to handle hexacosanoic acid (1) in a laboratory. Therefore, efficient chemical synthesis of the compounds 1 and 2 has not been reported. Here, we report a versatile synthetic method for hexacosanoic acid (1) and the ceramide 2 containing the fatty acid 1. Synthesis of hexa- cosanoic acid (1) was achieved by applying the coupling of two alkyl units as a key step. Ceramide 2 was efficiently synthesized by applying the reported procedure together with hexacosanoic acid (1) synthesized here. This synthetic strategy has an advantage of getting various carbon chain length fatty acids and their ceramides by using a variety of carbon chain units. This method is also applicable for large-scale synthesis. In addition, these com- pounds 1 and 2 are useful for investigation of details of these compounds related to diseases such as ALD and AMN. Key Words hexacosanoic acid, cerotic acid, ceramide, chemical synthesis, adrenoleuko- dystrophy Long-chain fatty acids are biogenic substances. They we report a useful synthetic method for hexacosanoic are part of signaling pathways, sources of energy, and acid (1) and ceramide 2 containing a hexacosanoic acid constituents of cell membranes. However, the physiolog- (Fig. 1). ical roles of some long-chain fatty acids, especially very MATERIALS AND METHODS long-chain fatty acids (VLCFA) such as hexacosanoic acid 1, remain unclear. General experimental procedures. All reagents were Hexacosanoic acid (C26:0) (1), which is also called purchased from commercial sources and used without cerotic acid, is related to adrenoleukodystrophy (ALD) further purification. Organic solvents used were dried by (1, 2), adrenomyeloneuropathy (AMN) (1), atheroscle- standard methods. All reactions were performed under rosis (3), coronary artery disease (4), aging (3, 5) and a nitrogen atmosphere. Silica gel (Kanto Chemical Sil- metabolic syndrome (6). As the level of 1 higher than ica gel 60, Spherical, 63–210 or 40–50 mm) was used the normal is related to the diseases above, hexaco- for column chromatography, and pre-coated silica gel 1 sanoic acid (1) and the sphingolipid containing 1 are 60F254 plates (0.25 mm, Merck) were used for TLC. H thought to play an important role in multiple tissues (5). NMR (300 MHz) and 13C NMR (75 MHz) were recorded Hexacosanoic acid is known to be a waxy solid and to be using a Bruker AV300 instrument in CDCl3 solution hard to dissolve in water as well as organic solvents. It with TMS or CDCl3 (d 7.26) and CDCl3 (d 77.23) as an has been reported that due to this physical property of internal standard, respectively. The chemical shifts are hexacosanoic acid, its accumulation causes a decrease given in d values. Splitting patterns are indicated as fol- the fluidity of cell membranes (3). Ceramide, which is a lows: s, singlet; d, doublet; t, triplet; q, quartet; m, multi- kind of sphingolipid, has a function as a second messen- plet. Mass spectra were recorded on a JEOL AccuTOF LC- ger, so it is suggested that the ceramide is also related to plus JMS-T100LP and JEOL MS700 spectrometer using those diseases. However, efficient chemical synthesis of NBA as the positive-ion FAB matrix. these compounds has not been reported. In this article, 1-Bromo-10-(methoxymethoxy)decane (3): To a solution of 10-bromo-1-decanol (2.01 g, 8.47 mmol) E-mail: [email protected] and N,N-diisopropylethylamine (3.08 g, 23.9 mmol) in Abbreviations: ALD, adrenoleukodystrophy; AMN, adre- CH2Cl2 (10 mL) at 0˚C was added chloromethyl methyl nomyeloneuropathy; EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2- ether (1.85 g, 22.9 mmol) and the mixture was stirred dihydroquinoline; TBAF, tetra-n-butylammonium fluoride; at 0˚C for 3 h. The reaction was quenched with 1 N VLCFA, very long-chain fatty acids. HCl and extracted with CH2Cl2. The organic layer was 222 Very Long Chain Fatty Acid 223 the solution was stirred at 30˚C for 2 h. After the addi- tion of saturated NaHCO3 aq. and saturated Na2S2O3 aq., the mixture was extracted with CH2Cl2. The organic layer was washed with water and brine, dried over MgSO4, and evaporated. The residue was chromato- graphed on silica gel (20 g, CH2Cl2/hexane, 1 : 30 and Fig. 1. Structures of hexacosanoic acid and the 1 then CH2Cl2) to give compound 6 (0.1679 g, 84%). 6 H ceramide containing hexacosanoic acid. NMR d 0.88 (3 H, t, J 56.7 Hz, -CH2-CH3), 1.16–1.38 (44 H, m, -CH2-), 1.59–1.70 (2 H, m,-CH2-CH2-CHO), 2.42 (2 H, dt, J 57.23, 1.85 Hz, -CH2-CHO), 9.77 (1 H, 13 washed with saturated NaHCO3 aq. and brine, dried over t, 1.86 Hz, -CHO). C NMR d 14.3, 22.3, 22.9, 29.4, MgSO4, and evaporated. The residue was flash-chroma- 29.6, 29.7, 29.81, 29.89, 29.93 (15 carbons), 32.2, tographed on silica gel (50 g, hexane/AcOEt, and then 44.1, 203.1. MS (ESI2) m/z: 379 [M-H]2. HRMS (ESI2) 1 12 1 16 80 : 1) to give compound 3 (1.78 g, 75%). 3 H NMR d calcd. for C26 H51 O1 379.3940, found 379.3947. IR 21 1.02–1.51 (12 H, m, -CH2-), 1.51–1.70 (2 H, m, -CH2- (CHCl3): 2926, 2854, 1724 cm . CH2-O-CH2-O-CH3), 1.70–1.98 (2 H, m, Br-CH2-CH2-), Hexacosanoic acid (1): To a solution of aldehyde 3.36 (3 H, s, -O-CH2-O-CH3), 3.41 (2 H, t, J56.9 Hz, 6 (0.0999 g, 0.262 mmol) and 2-methyl-2-butene Br-CH2-), 3.52 (2 H, t, J56.6 Hz, -CH2-O-CH2-O-CH3), (1.3 mL, 12.3 mmol) in tert-butyl alcohol (51 mL) was 13 4.62 (2 H, s, -O-CH2-O-CH3). C NMR d 26.3, 28.3, added a solution of sodium chlorite (0.22 g, 2.43 mmol) 28.9, 29.49, 29.50, 29.59, 29.9, 33.0, 34.1, 55.2, and sodium dihydrogenphosphate dihydrate (0.29 g, 67.9, 96.5. IR (neat): 2928, 2854, 1150, 1111, 1045, 1.82 mmol) in water (2.1 mL) dropwise over 4 min. 644, 563 cm21. The mixture was stirred at room temperature for 1 h. 1-(Methoxymethoxy)hexacosane (4): Hexadec- After volatile components were removed under vacuum, anyl magnesium bromide was prepared from Mg 2 N HCl was added. The mixture was extracted with (0.954 g, 39.2 mmol) and 1-bromohexadecane (8.16 g, CH2Cl2. The organic layer was washed with water and 26.7 mmol) in THF (22 mL). To a mixture of 3 (0.50 g, brine, dried over MgSO4, and evaporated. The residue 1.79 mmol), Li2CuCl4 (100 mM solution in THF, 5.3 was chromatographed on silica gel (1 g, MeOH/CH2Cl2, mL) and N-methylpyrrolidone (0.69 mL, 7.16 mmol) 1 : 15) to give compound 1 (0.0772 g, 74%). 1 1H was slowly added hexadecanyl magnesium bromide NMR d 0.88 (3 H, t, J56.72 Hz, -CH2-CH3), 1.14–1.40 (0.476 M, 7.6 mL, 3.62 mmol) at room temperature (44 H, m, -CH2-), 1.61–1.69 (2 H, m, -CH2-CH2-CHO), 13 under nitrogen. The deep purple solution was stirred at 2.36 (2 H, t, J57.5 Hz, -CH2-COOH). C NMR d 14.4, room temperature for 2.5 h. The reaction was quenched 22.9, 24.9, 29.3, 29.5, 29.6, 29.7, 29.8, 29.9 (15 car- 2 2 with saturated NH4Cl aq. and extracted with AcOEt. The bons), 32.2, 33.8, 177.8. MS (ESI ) m/z: 395 [M-H] . 2 12 1 16 organic layer was washed with water and brine, dried HRMS (ESI ) calcd. for C26 H51 O2 395.3889, found 21 over MgSO4, and evaporated. The residue was flash- 395.3903. IR (CHCl3): 2926, 2854, 1724 cm . chromatographed on silica gel (50 g, hexane/AcOEt, (E)-hexadec-2-enal (8): To a solution of oxalyl 1 80 : 1) to give compound 4 (0.623 g, 81%). 4 H NMR chloride (3.1 mL, 36.1 mmol) in CH2Cl2 (160 mL) d 0.88 (3 H, t, J56.5 Hz, -CH2-CH3), 1.14–1.47 (46 H, was added a solution of DMSO (5.1 mL, 71.9 mmol) m, -CH2-), 1.57–1.65 (2 H, m, -CH2-CH2-O-CH2-O-CH3), in CH2Cl2 (20 mL) at 278˚C. After being stirred for 3.36 (3 H, s, -O-CH2-O-CH3), 3.52 (2 H, t, J56.6 Hz, 10 min, a solution of alcohol 7 (7.19 g, 29.9 mmol) in 13 -CH2-O-CH2-O-CH3), 4.62 (2 H, s, -O-CH2-O-CH3).