Horse radish peroxidase-catalysed oxidative coupling of methyl sinapate to give diastereoisomeric spiro dimers

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1994 Horse Radish Peroxidase-catalysed Oxidative Coupling of Methyl Sinapate to give Diastereoisomeric Spiro Dimers Harri Setala, Aarne Pajunen, llkka Kilpelainen and Gosta Brunow * Department of Chemistry, PO Box 6, SF-00014 University of Helsinki, Finland The oxidative coupling of methyl sinapate with H,O,/horse radish peroxidase at pH 4 in the presence of methanol gives dimeric spiro structures of a novel type. The crystal structures of two diastereoisomers have been determined. The coupling reaction shows some threo selectivity. Aryltetralins are an important group of natural lignans with a 1,2-dihydro- or tetrahydro-naphthalene skeleton. Many of these compounds have been observed to have biological and pharmacological effects.' The oxidative coupling of phenols catalyzed by peroxidases is a very attractive method for preparing phenolic dimers of this type from phenolic cinna- mates. The advantages of the enzymatic method are mild reaction conditions and fast reaction rates. The use of OH 6Hperoxidases in a preparatively useful manner is limited by the low selectivity of the oxidative coupling and the complexity of 1 2 (41%) the subsequent reactions. We have been investigating possible ways to enhance the selectivity of this reaction type, for example, by changing the pH or using organic co-s~lvents.~~~ In this paper we report how the addition of methanol changes the course of the oxidative dimerization of (E)-methyl sinapate giving two diastereoisomers of an interesting new spiro compound.In a previous investigation of the oxidative coupling of methyl sinapate published by Walli~,~ the reaction was carried out with ferric chloride in aqueous acetone. In our work the oxidant was hydrogen peroxide with horse radish peroxidase, HRP (EC 1.1 1.1.7), as a catalyst. The reactions were carried out at pH 4 because our previous work with ferulates have shown that low pH-values tend to favour the formation of dimers at the expense of polymeric prod~cts.~ When the reaction was carried out in aqueous acetone, compound 2 was observed as the only dimeric product (yield 41%) after acidification. Repeating the oxidation in aqueous methanol, compound 2 was a minor product, the main products being the diastereoisomeric spiro compounds 3a and 3b (Scheme 1).The structures of 3a and 3b were determined by X-ray crystallography. The form- ation of compound 2 was also observed in the work of Wallis and its formation can be understood as a prototropic rearrangement of an initially formed bisquinone methide 4 to a cinnamyl structure and subsequent cyclization of the other quinone methide onto the aromatic ring (Scheme 2).4 In formation of the spiro compounds 3a and 3b, we assume that the addition of methanol to the bisquinone methide 4 is a key step. We have previously observed that methanol at pH 4 reacts rapidly with quinone methides, even in aqueous solvent^.^ The next step can be formulated as an electrophilic attack by the protonated quinone methide on the aromatic ring to form the spiro compound (Scheme 2).Since it is unclear why five- membered ring formation is favoured in this case, the possible participation of phenoxy radicals cannot be ruled out. A possible mechanism where intermediate spiro compounds would result in the formation of 2 was ruled out by carrying out an acid hydrolysis of a mixture of 3a and 3b. In the dienone- phenol rearrangement only the oxygen-substituted side-chain migrated to give the isomeric dihydronaphthol 5 after elimination of methanol. 2 (14%) OH 3a (32%) 3b (17%) Scheme 1 26) The structure of the diastereoisomers 3a and 3b were determined by X-ray crystallography of the acetates and the assignments of the signals in the 'H and '3C NMR spectra were carried out using 2D NMR techniques (HMBC and HMQC).The trans configuration of the ester groups in the predominant dimer 3a shows that the radical coupling leading to the epbond has a stereospecificity consistent with the threo coupling observed with other 4-hydroxyphenylpropenes. Me0 V O M e 0)r' 4 H+/MeOHI Me Me;% C02Me HO C0,Me 0 Me0 0 Me0II Me0 OMe Me0 ' OMe 0 OH 3a HCVHPOI ?Me Me0 C02Me Me0QLMe OH 5 Scheme 2 Experimental General.-Horse radish peroxidase (EC 1.1 1.1.7) was from Serva, activity 277 U mg-' (purpurogallin method). Hydrogen peroxide, a 30% aqueous solution from Merck, was diluted to give a 3% solution before use. Silica gel 60 (0.040-0.063) for flash chromatography was from Merck.The preparative HPLC was performed with detection at 265 nm and a column (1 x 30 cm) with Silasorb 600 as an absorbent. Hexane-ethyl acetate was used as an eluent. The injection volume was 1 cm3. 'H and 13C NMR measurements were recorded with a Varian Unity 500 spectrometer with tetramethylsilane as internal standard. MS were recorded with a JEOL JMS-SX102 instrument. The melting points are uncorrected. (E)-Methyl Sinapate (Methyl 4-Hydroxy-3,5-dimethoxybenz-ylideneacetate) 1.-This compound was synthesised from vanillin. Bromination and methoxylation gave 5-methoxyvanil- lin (syringaldehyde).6 A subsequent Knoevenagel reaction with malonic acid and esterification with methanol-sulfuric acid gave the title compound.Oxidative Coupling of (E)-Methyl Sinapate in Aqueous Acetone.-Procedure (A).Methyl sinapate (0.40 g, 1.68 mmol) was dissolved in acetone (10 cm3) and a solution of buffer (0.02 mol drn-,, citrate-phosphate, pH 4; 40 cm3) was added to it. Hydrogen peroxide (0.85 cm3, 0.84 mmol) and aqueous HRP (1400 U; 1 cm3) were added during 10 min to the reaction mixture which was then stirred at room temperature for 1 h during which time it turned reddish brown. HCl (2 mol drn-,; 5 cm3) was added to the reaction mixture which, whilst being J. CHEM. SOC. PERKIN TRANS. 1 1994 stirred for 20 min at room temperature, turned yellowish. The mixture was extracted with ethyl acetate (x3) and the com- bined extracts were washed with 5% aqueous NaHCO,, water and brine, dried (Ma,SO,) and evaporated to dryness.The residue was acetylated with dry pyridine and acetic anhydride (1 :1) overnight at room temperature.' The products were separated using a short silica gel column (3 x 4 cm)eluting with hexane-AcOEt (1 : 1). The main fraction (351 mg) was purified by preparative HPLC (eluent: hexane-AcOEt, 1:1) to yield dimethyl 7-acetoxy- 1 -(4-acetoxy-3,5-dimethoxyphenyl)-6,8-dimethoxy-1,2-dihydronaphthalene-2,3-dicarboxylate (di-acetate of 2) which recrystallized from ethyl acetate-hexane (1 :4) as white crystals, m.p. 177-179 "C; 6,(500 MHz; CDC1,) 2.28 (3 H, S, 4'-OCOCH3), 2.33 (3 H, S, 7-OCOCH,), 3.64 (3 H, s, 8-OMe), 3.66 (3 H, s, 2-C0,CH3), 3.68 (6 H, s, 3',5'-OMe), 3.80 (3 H, s, 3-C02CH,), 3.86 (3 H, s, 6-OMe), 4.17 (1 H, d, J 1.1,2-H), 5.03 (1 H, S, 1-H), 6.23 (2 H, S, 2',6'-H), 6.76 (1 H, S, 5-H), 7.62 (1 H, s, 4-H); ~3~20.5 (OCOCH,), 39.3 (1-C), 45.6 (2-C), 52.l(3-CO2CH,), 52.6(2-CO2CH,), 56.1(3',5'-OMe), 56.2 (6-OMe), 61.4 (8-OMe), 104.1 (2',6'-C), 108 (5-C), 122.7 (9-C), 125.7 (343, 127.5 (4'-C), 129.8 (1'-C), 135.0 (7-C), 136.7 (4-C), 140.4(10-C), 151.0(8-C), 151.9 (6-C), 166.7 (3-CO,CH,), 168.1 (7-OCOCH,),168.7(4'-0COCH3)and172.0(2-CO,CH,);m/z 558 (M+, 24%), 516 (98), 499 (lo), 474 (69), 456 (15), 414 (loo), 383 (37), 355 (12), 320 (16), 289 (10) and 236 (10) (Found: M+, 558.1735.C28H30012 requires M, 558.1737). Further elution yielded a mixture of by-products (70 mg, 15%) and approximately 44% of the total yield was oligomeric material eluted from the silica gel column with EtOH-AcOEt (1 :1).Procedure (B).The reaction was performed in a similar way without the addition of HCl. The reddish brown reaction mixture turned slowly yellowish over 12 h. After this time the same work-up yielded the dimer 2 (1 52 mg, 32%). Oxidative Coupling of (E)-Methyl Sinapate in Aqueous Methanol.-Methyl sinapate( 1.44 g, 6.05 mmol) was dissolved in methanol (200 cm3) and a solution of buffer (0.02 mol drn-,, citrate-phosphate, pH 4; 450 cm3) was added to it. Hydrogen peroxide (3.84 cm3, 3.03 mmol) and aqueous HRP (6066 U, 10 cm3)were added during 15 min to the reaction mixture which was then stirred for 2 h at room temperature. The cloudy, yellowish reaction mixture was extracted several times with ethyl acetate and the compound extracts were washed with 5% aqueous sodium hydrogen carbonate, water and brine, dried (Na2S0,) and evaporated to dryness.The products were acetylated as above. Separation and Pur$cation of the Products.-The acetates of 2 and 3 were separated by preparative HPLC with hexane- ethyl acetate (1 :4) as eluent. The yield of dimer 2 was 238 mg (14%). The fraction containing the diastereoisomers 3a and 3b was then further fractionated with hexane-ethyl acetate (1 :9). Dimethyl 1-(4-Acetoxy-3,5-dimethoxyphenyl)-4,7,9-trirneth-oxy-8-oxospiro~4.5~deca-6,9-diene-2,3-dicarboxylate(Acetate of 3).-The yield of the acetates of 3 after HPLC was 49% including two diastereoisomers 3a (32%) and 3b (17%).Both of these were crystallised from diethyl ether-acetone mixture (9: I), 3a m.p. 195-196 "C and 3b m.p. 183-185 "C; 'H NMR and 13C NMR (3a in ['HJacetone) are shown in Table 1: 3a, m/z 548 (M+, 4%), 516 (42), 474 (25), 456 (49), 414 (loo), 383 (31), 372 (26), 355 (18), 336 (23) and 170 (16) (Found: M+, 548.1901. C27H32012 requires M, 548.1894); 3b, m/z 548 (M+, 30%), 516 (83), 474 (58), 456 (26), 414 (loo), 383 (40), 355 (13), 320 (15), 268 (17) and 170 (16) (Found: M', 548.1904. C27H32012 requires M, 548.1894). J. CHEM. SOC. PERKIN TRANS. 1 1994 Table 1 The assignment of 'H and I3Cchemical shifts for acetates of compounds 3a and 3b (500MHz, solvent [2H,]acetone, Jvalues are given in Hz) 3a 3b Assignment BH & 6, & 4'-OAc 4-OCH3 3-CH 9-OCH3 3-COzCH3 7-OCH3 3'-, 5'-OCH3 2-COzCH3 1 -CH 4-CH 2-CH 1 0-CH 6-CH 2'-, 6'-CH 5-C (spiro) 4'-c 2.16 (s) 3.38 (s) 3.48 (dd, J 8.8 and 3.6) 3.57 (s) 3.60 (s) 3.72 (s) 3.74 (s) 3.79 (s) 3.94 (d, J 12.1) 4.01 (d, J3.4) 4.04 (dd, J 12.0and 8.8) 6.04 (d, J 2.4) 6.28 (d, J2.4) 6.64 (s) 20.23 58.63 54.90 55.40 52.49 55.47 56.50 52.94 58.57 91.44 49.09 114.81 1 17.09 106.41 55.20 128.90 2.16 (s) 3.27 (s) 3.60 (dd, J 12.4 and 9.2 3.68 (s) 3.71 (s) 3.75 (s) 3.73 (s) 3.56 (s) 3.88 (d, J 11.9) 4.48 (d, J9.3) 4.33 (br d, J 12.1) 6.31 (d, J2.4) 6.42 (d, J 2.4) 6.65 (s) 20.21 59.91 50.99 55.64 52.49 55.56 56.44 52.34 56.50 91.24 47.15 113.32 120.13 106.51 55.40 128.88 1 '-c 135.18 135.28 9-c 152.24 153.76 3'-, 5'-c 152.36 152.29 7-c 152.49 153.18 2-COzCH3 173.66 172.97 3-COZCH3 173.91 173.43 8-0 175.47 175.98 Dimethyl 6-Acetoxy-l-(4-acetoxy-3,5-dimethoxyphenyl)-5,7-Structure analysis and reJinement.Positional parameters dime thoxy -1,2-dih ydr onap h thalene- 2,3-dicarboxy la te (Ace ta te were determined by direct methods using SHELXTL,' and of 5).-Compound 3 (100 mg, 0.18 mmol) was dissolved in were refined by full-matrix least-squares calculations in two dioxane (9 cm3) and 0.2 mol dm-, HCI (1 cm3) was added to the blocks with all non-hydrogen atoms treated anisotropically solution. The mixture was refluxed for 30 min after which it was using the weighting scheme w-' = a2(F)+ 0.0376F2. The extracted with ethyl acetate (2 x 50 cm3).The combined ex- hydrogen atoms were placed in calculated positions. The tracts were washed with aqueous sodium hydrogen carbonate, atomic scattering factors were those in the SHELXTL program. water and brine, dried (Na2S0,) and evaporated to dryness. The refinement converged at R = 0.135 (R, = 0.157). Frac- The residue was acetylated and purified by preparative HPLC tional atomic coordinates, bond lengths, angles and H-atom with hexane-ethyl acetate (1 :1) as eluent to give the diacetate of coordinates have been deposited at the Cambridge Crystal- 5as white crystals (EtOH), m.p. 186-187 "C; 6,(500 MHz) 2.30 lographic Data Centre.* (3 H, S, 4'-OCOCH,), 2.36 (3 H, S, 6-OCOCH,), 3.66 (3 H, S, 2-The crystal structure of 3a will be published separately. COZCH,), 3.69 (6 H, S, 3',5'-OCH,), 3.78 (3 H, S, 3-COzCH,), 3.79 (3 H, S, 7-OCH,), 3.88 (3 H, S, 5-OCH,), 4.04 (1 H, d, J2.6, 2-H), 4.68 (1 H, d, J2.6, 1-H), 6.23 (2 H, S, 2',6'-H), 6.53 (1 H, S, * For details see 'Instructions for Authors (1994),' J.Chem. Soc., 8-H) and 7.94 (1 H, s, 4-H); 6,20.5 (4',6-OC0CH3), 46.8 (C-1), Perkin Trans. I, 1994, Issue 1. 46.9 (C-2), 52.0 (3-CO2CH,), 52.6 (2-C02CH,), 56.1 (3'3'- OCH,), 56.2 (7-OCH3), 62.4 (5-OCH3), 104.4 (C-2',6'), 108.5 References(C-8), 118.6 (C-lo), 122.6 (C-3), 127.6 (C-4'), 131.1 (C-4), 132.1 1 F. D. Hostettler and M. K. Seikel, Tetrahedron, 1969, 25, 2325; (C-6), 135.5 (C-9), 140.2 (C-1'), 150.9 (C-5), 154.0 (C-7), 152.0 S. Sepelveda-Boza, S. Delhvi and B. K. Cassels, Phytochemistry,(C-3',5'), 166.9 (3-COzCH,), 168.4 (6-OCOCH,), 168.8 (4'- 1990,29,2357;A.J. Broomhead and P. M. Dewick, Phytochemistry, OCOCH,), 172.5 (2-COzCH3); m/z 558 (M', 49%), 516 (59, 1991,30, 1511. 2 P. Pietikainen and P. Adlercreutz, Appl. Microbiol. Biotechnol., 1990,499 (12), 474 (lo), 456 (51), 414 (loo), 383 (25), 320 (13), 195 33,455.(11) and 167 (13) (Found: M', 558.1741. C28H30012 requires 3 F. Chioccara,S. Poli, B. Rindone, T. Pilati, G. Brunow, P. Pietikainen M, 558.1737). and H. Setala, Acta Chem. Scad, 1993,47, 610. 4 A. F. A. Wallis, Aust. J. Chem., 1973, 26, 1571. Crystal Structure of 3b Acetate.-Crystal data. C27H32OI2, 5 K. V. Sarkanen and A. F. A. Wallis, J. Chem. Soc., Perkin Trans. I, M, = 548.54. Monoclinic, a = 11.818(5), b = 23.828(5), c = 1973, 1869. 11.016(5)A, p = 107.12(5)", V = 2965(2) A3 (by least-squares 6 P.S. Manchand, P. S. Belica and H. S. Wong, Synth. Commun., 1990, 20,2659.refinement on diffractometer angles of 16 automatically 7 K. Freudenberg and H. H. Hiibner, Chem. Ber., 1952,85, 1 181.centered reflections); F(000) = 1160, D, = 1.23 g ern-,, space 8 E. Adler, G. Brunow and K. Lundquist, Holzforschung, 1987, 41, group PZ,, 2 = 4, p(Mo-Ka) = 0.097 mm-', (Mo-Ka) = 199. 0.71069 A. White plates, recrystallized from methanol. 9 G. M. Sheldrick, SHELXTL PCTM Version 4.2, Siemens Analytical Data collection and processing. Data were collected on a X-Ray Instruments, Inc, USA, 1990. Nicolet P3 diffractometer using graphite monochromated Mo-Ka radiation and 0-28 scan type. Two standard reflections were monitored every 2 h and showed no significant deviation. 2874 Unique reflections were recorded (1.5 0 20°, Paper 3/07441B k h,k,l) but owing to the very poor quality of crystals only 1755 Received 20th December 1993 [F 5.0a(F)]were used in the refinement. Accepted 17th January 1994