Mendeleev Communications Electronic Version, Issue 6, 1998 (pp. 207–248) A simple preparation of -hydroxydienoic fatty acids with double-bond positional isomerism Igor V. Ivanov,*a,b Galina I. Myagkovaa and Hartmut Kühnb a M. V. Lomonosov State Academy of Fine Chemical Technology, 117571 Moscow, Russian Federation. Fax: +7 095 434 8711; e-mail: myagkova@httos.mitht.msk.ru b Institute of Biochemistry, University Clinics Charité, Humboldt University, D-10115 Berlin, Germany A set of new regioisomeric w-hydroxydienoic fatty acids has been prepared via a three-step procedure involving the methylation of w-carboxydienoic fatty acids followed by the NaBH4 reduction of a mixture of the resulting monomethyl esters and HPLC separation.Lipoxygenases are lipid peroxidising enzymes which oxygenate polyenoic fatty acids containing a (1Z,4Z)-pentadienoic system to their corresponding 1-hydroperoxy-(2E,4Z)-derivatives.1 Mammalian 15-lipoxygenase (LOX) is unique among other LOX due to its ability to oxidise polyunsaturated fatty acids incorporated in complex membrane lipids.2,3 Although the first X-ray structure of mammalian 15-LOX has recently been solved, the structural features of the substrate binding cage are not completely understood.4 Based on the theory1,4–7 of ‘hydrophobic binding cavity’ we suggested that the incorporation of a polar (hydroxy) or evenly charged (carboxy) group at the w-position of a fatty acid molecule could hinder the proper substrate alignment at the substrate binding site of 15-LOX.In order to prove this suggestion and test the substrate specificity of LOX we synthesised a set of isomeric w-carboxydienoic fatty acids.8,9 To avoid total synthesis in the preparation of w-hydroxydienoic acids we propose an original three-step procedure which provides access to both possible regioisomeric w-hydroxy acids (4a–e and 5a–e) from dicarboxylic acid 1a–e (Scheme 1).The methylation of dicarboxylic acid 1a–e with a small molar excess of diazomethane in diethyl ether leads to a mixture of two monomethyl esters, 2a–e and 3a–e.The sequential reduction of this mixture with an ethanol solution of sodium borohydride produced two w-hydroxy acids 4a–e and 5a–e.† Both isomeric products were eluted in reverse phase HPLC as a single peak when the chromatogram was developed with the solvent system methanol–water–acetic acid (85:15:0.1, v/v) at a flow rate of 1 ml min–1.However, when the mixture of w- hydroxydienoic acids was analysed by normal phase HPLC the isomeric compounds, 4a–e and 5a–e, were resolved (Figure 1). In carrying out the isomer separation we prepared both product isomers with a degree of purity exceeding 99%. The yield of the entire preparation procedure calculated for the mixture of 4 and 5 varied between 61–65%.The analysis of the ratio of isomeric products 4a–e and 5a–e (Scheme 1) shows that the methylation of dienedicarboxylic acids 1b–e with diazomethane occurs without any significant preference for either one or the other carboxylic group. Although the data observed were reproducible and the ratio of isomeric products 4a–e and 5a–e, and hence, that of monomethyl esters 3a–e and 4a–e in the starting mixture varied slightly, compound 1a failed to follow the trend.Whether methylation of one of the carboxylic groups has any preference or the predominant formation of one of the methyl esters in the case of 1a was accidental, should be studied further. † In a typical experiment, the dicarboxylic acid 1a–e (0.10 mmol) dissolved in 1 ml diethyl ether was methylated with a small molar excess of diazomethane (0.12 mmol).The ether was removed by argon flow and 7 ml of a 0.5 M ethanol solution of sodium borohydride (the solution was prepared as described by Brown et al.,10 and the concentration of borohydride was determined by the hydrolysis method) was added to the residue at room temperature under argon.The reaction mixture was stirred for 6–16 h at 24 °C. After the organic solvent was evaporated under vacuum, the reaction mixture was quenched with 2 ml of water and then acidified to pH 3. The fatty acid derivatives were extracted twice with 5 ml of ethyl acetate, the combined organic extracts were concentrated under vacuum and the resulting products 4 and 5, preliminary purified by RP-HPLC, were separated by normal phase HPLC.w 4a:5a = 3:17 4b:5b = 1:1 4c:5c = 3:2 4d:5d = 2:3 4e:5e = 1:1 Scheme 1 CO2H HO2C n m CO2Me HO2C n m CH2N2 CO2H MeO2C n m 2a–e 3a–e i, NaBH4, EtOH ii, HPLC separation CO2H HO n' m' OH HO2C m' n' 1a n = 3, m = 8 1b n = 3, m = 7 1c n = 6, m = 7 1d n = 5, m = 7 1e n = 5, m = 6 4a n' = 9, m' = 3 4b n' = 8, m' = 3 4c n' = 8, m' = 6 4d n' = 8, m' = 5 4e n' = 7, m' = 5 5a n' = 4, m' = 8 5b n' = 4, m' = 7 5c n' = 7, m' = 7 5d n' = 6, m' = 7 5e n' = 6, m' = 6 aOnly the early eluting compound in SP-HPLC (Figure 2) was analysed. A Nucleosil 100-7 column (250×5 mm, 7 mm particle size; Machery and Nagel, Germany) with the solvent system hexane–isopropanol–acetic acid (100:10:0.1, v/v) was used, flow rate 1 ml min–1.The oxygenated products were analysed by GC-MS as the corresponding trimethylsilylmethyl ester derivatives. b(C) is the minor intensity fragmentation. Table 1 Analytical data for the oxygenated w-hydroxydienoic fatty acid isomers.a,b w-Hydroxyacid Oxygenation product Retention time/min Key ion fragmentation, m/z 4a (n' = 9, m' = 3) 5.84 470 (M+), 255 (A), 317 (B) 4b (n' = 8, m' = 3) 6.57 456 (M+), 255 (A), 303 (B) 4c (n' = 8, m' = 6) 6.72 498 (M+), 355 (C), 297 (A), 303 (B) 4d (n' = 8, m' = 5) 7.98 484 (M+), 355 (C), 283 (A), 303 (B) 4e (n' = 7, m' = 5) 8.63 470 (M+), 341 (C), 283 (A), 289 (B) 5a (n' = 4, m' = 8) 13.78 470 (M+), 325 (A), 247 (B) 5b (n' = 4, m' = 7) 12.63 456 (M+), 311 (A), 247 (B) 5c (n' = 7, m' = 7) 7.58 498 (M+), 341 (C), 311 (A), 289 (B) 5d (n' = 6, m' = 7) 9.28 484 (M+), 327 (C), 311 (A), 275 (B) 5e (n' = 6, m' = 6) 9.00 470 (M+), 327 (C), 297 (A), 275 (B) COOMe TMSO OTMS n' m' A B CMendeleev Communications Electronic Version, Issue 6, 1998 (pp. 207–248) Unfortunately, neither 1H NMR spectra nor GC-MS data of the trimethylsilylmethyl ester provided sufficient information to localise the position of the double bond system in products 4 and 5, especially when the difference between n' and m' is less then 2 carbon atoms.‡ To solve this problem the w-hydroxylated fatty acids 4a–e and 5a–e were oxygenated to their corresponding hydroperoxy derivatives in the presence of 2,2'-azobis- (amindiopropane)hydrochloride which induces peroxy radical mediated lipid peroxydation.§,11 This procedure involves a double bond conjunction associated with a Z–E isomerisation of the double bond shifted.HPLC analysis of the reaction mixture indicated the formation of four oxygenation products (Figure 2) containing conjugated diene chromophores with absorbance maxima between 230 and 235 nm (inset). The UV spectra of the fraction eluting early from the oxygenated products, with absorbance maxima at 235 nm, indicated the geometry,12 and the GC-MS data in Table 1 indicated the position of the double bonds.The data observed confirmed the position of the double bond system in compounds 4a–e and 5a–e. This work was supported in part by a DAAD fellowship awarded to I. V. Ivanov (A/96/28729), a grant from the Russian Foundation for Basic Research (grant no. 96-03-327-68a) awarded to G. I. Myagkova and I. V. Ivanov and a grant awarded by the Deutsche Forschungsgemeinschaft [Ku 961(2-2)] to H. Kühn. References 1 W. D. Lehman, Free Radical Biol. Med., 1994, 16. 241. 2 H. Kühn and A. Brash, J. Biol. Chem., 1990, 265, 1454. 3 T. Schewe, W. Halangk, C. Hiebsch and S. M. Rapoport, FEBS Lett., 1975, 60, 149. 4 S. A. Gillmor, A.Villasenor, R. Fletterick, E. Sigal and M. F. Browner, Natur. Struct. Biol., 1997, 4, 1003. 5 Q.-F. Gan, M. F. Browner, D. L. Sloane and E. Sigal, J. Biol. Chem., 1996, 271, 25412. ‡ All the compounds 4 and 5 gave satisfactory analytical data and were characterised by 1H NMR spectroscopy and GC-MS analysis. 4a (n' = 9, m' = 3): 1H NMR (CDCl3) d: 1.25–1.35 (m, 12H, CH2), 1.56–1.70 (m, 4H, 3-CH2 and 17-CH2), 2.04 (m, 2H, 10-CH2), 2.09 (m, 2H, 4-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 7-CH2), 3.67 (t, 2H, 18-CH2, J 2.5 Hz), 5.30–5.38 (m, 4H, CH=CH).GC-MS, m/z: 382 (M+), 367 (M+ – Me), 351 (M+ – MeO). 4b (n' = 8, m' = 3): early eluting product in normal phase HPLC (see Figure 1); 1H NMR (CDCl3) d: 1.30–1.40 (m, 10H, CH2), 1.55–1.65 (m, 4H, 3-CH2 and 16-CH2), 2.04 (m, 2H, 10-CH2), 2.10 (m, 2H, 4-CH2), 2.37 (t, 2H, CH2COO, J 7 Hz), 2.77 (m, 2H, 7-CH2), 3.67 (t, 2H, 17-CH2, J 2.5 Hz), 5.35–5.42 (m, 4H, CH=CH).GC-MS, m/z: 368 (M+), 353 (M+ – Me), 337 (M+ – MeO). 4c (n' = 8, m' = 6): 1H NMR (CDCl3) d: 1.30–1.40 (m, 16H, CH2), 1.60 (m, 2H, 3-CH2), 1.65 (m, 2H, 19-CH2), 2.07 (m, 4H, 7-CH2 and 13-CH2), 2.37 (t, 2H, CH2COO, J 7 Hz), 2.79 (m, 2H, 10-CH2), 3.67 (t, 2H, 18-CH2, J 2.5 Hz), 5.35–5.45 (m, 4H, CH=CH).GC-MS, m/z: 410 (M+), 395 (M+ – Me), 379 (M+ – MeO). 4d (n' = 8, m' = 5): 1H NMR (CDCl3) d: 1.30–1.40 (m, 14H, CH2), 1.60 (m, 2H, 3-CH2), 1.66 (m, 2H, 18-CH2), 2.06 (m, 2H, 12-CH2), 2.10 (m, 2H, 6-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 9-CH2), 3.64 (t, 2H, 19-CH2, J 2.5 Hz), 5.30–5.40 (m, 4H, CH=CH).GC-MS, m/z: 396 (M+), 381 (M+ – Me), 365 (M+ – MeO). 4e (n' = 7, m' = 5): early eluting product in normal phase HPLC (see Figure 1); 1H NMR (CDCl3) d: 1.30–1.40 (m, 12H, CH2), 1.60 (m, 2H, 3-CH2), 1.65 (m, 2H, 17-CH2), 2.07 (m, 4H, 6-CH2 and 12-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 9-CH2), 3.65 (t, 2H, 18-CH2, J 2.5 Hz), 5.30–5.40 (m, 4H, CH=CH). GC-MS, m/z: 382 (M+), 367 (M+ – Me), 351 (M+ – MeO). 5a (n' = 4, m' = 8): early eluting product in normal phase HPLC (see Figure 1); 1H NMR (CDCl3) d: 1.25–1.35 (m, 12H, CH2), 1.59 (m, 2H, 3-CH2), 1.72 (m, 2H, 17-CH2), 2.05 (m, 2H, 9-CH2), 2.14 (m, 2H, 15-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 12-CH2), 3.67 (t, 2H, 18-CH2, J 2.5 Hz), 5.30–5.38 (m, 4H, CH=CH). GC-MS, m/z: 382 (M+), 367 (M+ – Me), 351 (M+ – MeO). 5b (n' = 4, m' = 7): 1H NMR (CDCl3) d: 1.30–1.40 (m, 10H, CH2), 1.57 (m, 2H, 3-CH2), 1.73 (m, 2H, 16-CH2), 2.05 (m, 2H, 8-CH2), 2.15 (m, 2H, 14-CH2), 2.37 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 11-CH2), 3.68 (t, 2H, 17-CH2, J 2.5 Hz), 5.30–5.38 (m, 4H, CH=CH). GC-MS, m/z: 368 (M+), 353 (M+ – Me), 337 (M+ – MeO). 5c (n' = 7, m' = 7): early eluting product in normal phase HPLC (see Figure 1); 1H NMR (CDCl3) d: 1.30–1.40 (m, 16H, CH2), 1.57 (m, 2H, 3-CH2), 1.64 (m, 2H, 19-CH2), 2.05 (m, 4H, 8-CH2 and 14-CH2), 2.37 (t, 2H, CH2COO, J 7 Hz), 2.79 (m, 2H, 11-CH2), 3.64 (t, 2H, 20-CH2, J 2.5 Hz), 5.30–5.40 (m, 4H, CH=CH).GC-MS, m/z: 410 (M+), 395 (M+ – Me), 379 (M+ – MeO). 5d (n' = 6, m' = 7): early eluting product in normal phase HPLC (see Figure 1); 1H NMR (CDCl3) d: 1.30–1.40 (m, 14H, CH2), 1.57 (m, 2H, 3-CH2), 1.65 (m, 2H, 18-CH2), 2.05 (m, 4H, 8-CH2 and 14-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.79 (m, 2H, 11-CH2), 3.64 (t, 2H, 19-CH2, J 2.5 Hz), 5.30–5.40 (m, 4H, CH=CH). GC-MS, m/z: 396 (M+), 381 (M+ – Me), 365 (M+ – MeO). 5e (n' = 6, m' = 6): 1H NMR (CDCl3) d: 1.30–1.40 (m, 12H, CH2), 1.59 (m, 2H, 3-CH2), 1.66 (m, 2H, 17-CH2), 2.05 (m, 4H, 7-CH2 and 13-CH2), 2.35 (t, 2H, CH2COO, J 7 Hz), 2.78 (m, 2H, 10-CH2), 3.65 (t, 2H, 18-CH2, J 2.5 Hz), 5.30–5.40 (m, 4H, CH=CH).GC-MS, m/z: 382 (M+), 367 (M+ – Me), 351 (M+ – MeO). § The w-hydroxylated acids 4a–e or 5a–e (0.01 mmol) were incubated at 40 °C for 2 h with 100 mmol of 2,2'-azo-bis(2-amindiopropane)- hydrochloride in 2 ml of 0.1 M borate buffer (pH 9.0) containing 10% (v/v) of methanol.Then the reaction was stopped and hydroperoxides were reduced to the corresponding hydroxides by addition of an equimolar amount of sodium borohydride. The mixture was acidified to pH 3.5, the fatty acids were extracted with 2 ml of ethyl acetate, concentrated under vacuum and analysed by normal phase HPLC and GC-MS as their thrimethylsilylmethyl ester derivatives [the carboxylic group was methylated with diazomethane, the hydroxylic groups were silylated with bis(trimethylsilyl)trifluoroacetamide in pyridine]. Absorbance at 210 nm Retention time/min 5c 4c 4e 5e 4b 5b 16.3 17.2 16.9 18.4 18.8 20.5 15 20 20 20 Figure 1 SP-HPLC separation of the products formed by NaBH4 reduction of a mixture of monomethyl esters of 1c (I), 1e (II) and 1b (III).[The compounds were separated on an analytical Nucleosil 100-7 column (250×5 mm, 7 mm particle size; Machery and Nagel, Germany) with the solvent system hexane–isopropanol–acetic acid (100:2:0.1, v/v), flow rate 1 ml min–1.] I II III Absorbance at 235 nm Retention time/min 13-OH(Z,E) 20 200 240 Absorbance l/nm (E,E) (Z,E) 17.7 19.5 22.9 24.2 13-OH(E,E) 9-OH(E,E) 9-OH(E,Z) Figure 2 Analytical SP-HPLC of non-enzymatically oxidised 17-hydroxy- (9Z,12Z)-heptadecadienoic acid 5b [this HPLC run was performed with the solvent system hexane–isopropanol–acetic acid (100:7.5:0.1, v/v)].(E,Z)Mendeleev Communications Electronic Version, Issue 6, 1998 (pp. 207–248) 6 S. Bongräber, R. J. Kuban, M. Anton and H. Kühn, J. Mol. Biol., 1996, 264, 1145. 7 H. Kühn, H. Sprecher and A. R. Brash, J. Biol. Chem., 1990, 265, 16300. 8 I. V. Ivanov, N. V. Groza, G.M. Malchenko, G. I. Myagkova and T. Schewe, Bioorg. Khim., 1997, 23, 519 (Russ. J. Bioorg. Chem., 1997, 23, 481). 9 I. V. Ivanov, N. V. Groza, H. Kühn and G. I. Myagkova, Bioorg. Khim., 1998, 24, 454 (Russ. J. Bioorg. Chem., 1998, 24, 398). 10 N. C. Brown, S. Narasimhan and Y. M. Choi, J. Org. Chem., 1988, 47, 4702. 11 C. D. Ingram and A. R. Brash, Lipids, 1998, 23, 340. 12 V. W. Bowry and R. Stocker, J. Am. Chem. Soc., 1993, 115, 6029. Received: Moscow, 3rd August 1998 Cambridge, 29th September 1998; Com. 8/06230G