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Laccase in Organic Synthesis and Its Applications

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Laccase in Organic Synthesis and Its Applications LaccaseLaccase inin OrganicOrganic SynthesisSynthesis andand ItsIts ApplicationsApplications Suteera Witayakran Art J. Ragauskas Outline Laccase in Organic Synthesis Laccase Laccase application in organic synthesis The synthesis of naphthoquinones The synthesis of benzofuran derivatives Laccase in Fiber Modification Laccase application in fiber modification Modification of linerboard softwood kraft pulp Laccase A multi-copper-containing oxidoreductase enzyme Found in plants and fungi In fungi: function in pigment production, plant pathogenesis, detoxification, and delignification Implicated in the synthesis of naturally occurring substances Catalyze the oxidation of a variety of phenolic compounds Gianfreda, L.; Xu, F.; Bollag, J-M. Bioremediation Journal, 1999, 3, 1. Morozova, O. V.; et.al, Biochemistry (Moscow), 2007, 72, 1136. Laccase Lignin A phenolic polymer consisting of 3 different phenyl propane units CH2OH CH2OH CH2OH CH CH CH CH CH CH OCH3 H3CO OCH3 OH OH OH p-coumaryl alcohol coniferyl alcohol sinapyl alcohol Oxidation of monomeric phenols has been shown to result in coupling to lignin macromolecule (Lund 2001) Laccase CH2OH CH CH OCH3 OH H2O Laccase O2 Laccase (ox) CH2OH CH2OH CH2OH CH2OH CH2OH CH CH CH CH CH CH CH CH CH CH COUPLING OCH3 OCH3 OCH3 OCH3 OCH3 O O O O O Active Site of Laccase D1 Type 2 Cu 2+ D3 Type 3 Cu 2+ Cu 2+ O H L accase trinuclear oxygen binding site Three major steps of laccase catalysis: 1. Type 1 Cu reduction 2. Internal electron transfer 3. O reduction at T2/T3 center D2 2 Ribbon diagram of Trametes versicolor laccase showing the two channels leading to the T2/T3 cluster Piontek, K.; Antorini, M.; Choinowski, T. J. Biol. Chem. 2002, 277, 37663. Burton, S. G. Current Organic Chemistry, 2003, 7, 1317. Applications of Laccase In Pulp and Paper Pulping: increase fiber bonding Bleaching: laccase-mediator system Fiber modifications In Organic Synthesis Other applications: detoxification, washing powders, removal of phenolic browning products from food products, treating environmental pollutants Laccases in Organic Synthesis Broad specificity for substrates Oxidation of variety of organic compounds Methoxyphenols Phenols o-diphenols and p-diphenols Aminophenols Polyphenols Polyamines Lignin-related molecules Burton, S. G. Current Organic Chemistry, 2003, 7, 1317. Laccase in Organic Synthesis Many studies reported Laccase-catalyzed reactions The synthesis of actinocin and cinnabarinic acid Oxidative coupling of hydroquinone and (+)-catechin OH OH OH OH OH OH HO HO O Laccase HO O + H OH H OH H H OH OH Catechin OH H H Oxidation of hydroxyl groups of sugar derivatives Synthesis of polymers Laccase in Organic Synthesis O R5 R1 R3 N R4 H H2N R5 O And/Or O H R5 R1 N N R4 R5 H Acta Biochimica Polonica, 1959, 6, 399-409. O J. Org. Chem. 2005, 70, 2002-2008. Goals To determine the potential use of laccase in chemical synthesis To develop green chemistry synthesis Green reagent: enzyme (laccase) Green solvent: water The Synthesis of Naphthoquinones One-pot synthesis of 1,4-naphthoquinones and related structures with laccase Published in Green Chemistry, 2007, 9, 475- 480. Enzyme Assay Enzyme assay Laccase (EC 1.10.3.2) from Trametes villosa was donated by Novo Nordisk Biochem, North Carolina. Laccase activity was determined by oxidation of 2,2’-azinobis-(3-ethylbenzyl thiozoline-6- sulphonate) (ABTS). The oxidation of ABTS is followed by an absorbance increase at 420 nm. Enzyme activity is expressed in units (U = mmol of ABTS oxidized per minute). Bourbonnais, R.; Leech, D.; Paice, G. M. Biochimica et Biophysica Acta 1998, 1379, 381. General Reaction Procedure Preliminary study Bubble O2 for 30 mins before adding reagents Add ¼ of the laccase (250 U/ 1g substrate) each at the beginning of each hour of the first 4 hours of the 24-hour reaction. No laccase Æ No reaction The Effect of Laccase Dose The Effect of Laccase Dose on the Formation of Compound 3 The Effect of Laccase Dose on the Formation of Compound 4 O O 500 U/ 1g subatrate 80 MeO MeO 500 U/ 1g subatrate 80 1000 U/ 1g substrate 70 1000 U/ 1g substrate 2000 U/ 1g substrate 70 2000 U/ 1g substrate 4000 U/ 1g substrate 60 4000 U/ 1g substrate 60 O O ) 50 % ) 50 ( % 40 ( 40 Yield Yield 30 Yield 30 20 20 10 10 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Reaction time (hr) Reaction time (hr) The quantitative study of 3 and 4 was measured by 1H- NMR spectroscopy using tetrafluorobenzaldehyde as an internal standard. The more laccase used, the more products generated. Proposed Reaction Pathway O MeO + O O MeO O Compound 4 Compound 3 The Effect of Temperature The Effect of Temperature on the Formation of Compound 3 The Effect of Temperature on the Formation of Compound 4 O MeO 25 °C 50 °C 70 °C 25 °C 50 °C 70 °C O 90 100 MeO 90 80 80 70 O 70 60 ) % 60 50 O ( 50 40 Yield Yield Yield (%) Yield 40 30 30 20 20 10 10 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Reaction time (hr) Reaction time (hr) At 100 oC: no reaction Compound 4 When the temperature increased, the yield increased. Compound 3 When the temperature increased, the converted rate of 3 increased. At low temperature, the major product is the Diels-Alder adduct. Reaction of Hydroquinones and Dienes 2c Laccase-generated quinones in naphthoquinone synthesis via Diels-Alder reaction • Published in Tetrahedron Letters 2007, 48, 2983-2987. Proposed Reaction Pathway Preliminary Study To Find the optimal condition OH O OH O Laccase 0.1M acetate buffer pH 4.5 132 24 hours Entry 1 : 2 Temperature Yield of 3 (%) Solvent Yield of 3 (%) (equiv.) Entry 1 0.1 M Acetate buffer pH 4.5 47 1 1:10 3 °C (2 h), RT 47 2 Water 18 2 1:10 RT 10 3 5% Aqueous PEG 2000 25 4 p-Dioxane 0 3 1:10 60 °C no product formed 5 1:1 p-Dioxane/acetate buffer 8 6 1:1 Ethylene Glycol/acetate buffer 15 4 1:5 3 °C (2 h), RT 8 7 1:1 MeOH/acetate buffer 18 5 1:15 3 °C (2 h), RT 32 OH 8 1:1 Chloroform/acetate buffer 0% of 3 HO 27% of Reaction of a Various Catechols Entry Catechol Yield (%) OH 1 OH 47 OH OH 2 57 CH3 OH O OH OH O OH Laccase 3 28 R1 0.1M acetate buffer pH 4.5 R 2 1 O CH o OH 3 C - RT, 24 hours O OCH 3 10 : 1 4 H3CO OH 11 O and 32% of H3CO OH OH 5 no product formed Cl OH OH 14 O 6 and 15% of O (96hr) OH OH O no product formed O 7 97% of quinone Reaction of a Various Dienes Entry Diene Yield (%) 1 57 2 71 OH R2 O R2 OCH 3 OH R O R 3 Laccase 3 3 10 OCH 3 R4 0.1M acetate buffer pH 4.5 R4 OCH 3 CH R CH3 R5 3 oC - RT, 24 hours 3 5 4 77 (R2 = H) 1 : 10 O O CH 3 5 76 (R2 =H) ( 2 eq.) 6 no product formed Reaction of 1-Acetoxy-1,3-Butadiene with a Variety of 1,4-Benzohydroquinone Entry R1 Yield (%) 1 H 67 2 CH3 75 3 OCH3 81 4 Br 67 5 Cl 69 Conclusions of the Synthesis of Naphthoquinones An efficient green chemistry synthesis of naphthoquinones The use of safe, environment-benign solvent The use of nonhazardous oxidizing agent This reaction system can yield naphthoquinones up to 80% Reactivity and selectivity depend on the exact structure of the starting hydroquinone and diene. The Synthesis of Benzofurans Cascade Synthesis of Benzofuran Derivatives via Laccase Oxidation-Michael Addition Published in Tetrahedron, 2007, 63, 10958- 10962. 1 1 Laccase, 0.2eq. Sc(OTf)3 2 0.1M Phosphate Buffer pH 7.0 4 2 4 Preliminary Study OH O OH OH OO Laccase + Solvent OH RT,4 hours 1a 2aO 3a Entry Solvent/ pH 1a:2a (equiv) Yield of 3a (%) 1 0.1 M Phosphate buffer pH 7.0 1:1 46 2 0.1 M Phosphate buffer pH 7.0 1:2 64 3 0.1 M Acetate buffer pH 4.5 1:2 0 4 0.1 M Phosphate buffer pH 8.0 1:2 6 The Effect of Lewis Bases and Lewis Acids The effect of Lewis bases The effect of Lewis acids Entry Lewis Solvent 1a: 2a: Yield Entry Lewis acid 1a: 2a: Lewis Yield of 3a bases Lewis base of 3a acid (equiv) (%) (equiv) (%) 1 Sc(OTf)3 1: 2: 0.1 63 1 Pyridine Water 1: 2: 0.5 33 2 Sc(OTf)3 1: 2: 0.2 74 2 Pyridine 0.1 M Phosphate 1: 2: 0.5 40 buffer pH 7.0 3 Sc(OTf)3/ SDS 1: 2: 0.2 76 3 Pyridine 0.1 M Phosphate 1: 2: 1 54 buffer pH 7.0 4 Yb(OTf)3 1: 2: 0.2 72 4 DMAP 0.1 M Phosphate 1: 2: 1 9 buffer pH 7.0 5 InCl3.4H2O 1: 2: 0.2 71 5 DABCO 0.1 M Phosphate 1: 2: 1 13 buffer pH 7.0 6 CuCl2 1: 2: 0.2 49 The Reaction of Catechols and 1,3- Dicarbonyl Compounds Entry 1 1a: R1 = Me, R2 = H 2a: R3 = R5 = Me, R4 = H 3a (76%) 2 1a 2b: R3 = R5 = Me, R4 = Cl 3a (79%) (1 hr) 3 1a 2c: R3 = Me, R4 = Cl, R5 = OEt 3b (48%) (1 hr) 4 1b: R1 = R2 = H 2a 3c (68%) 5 1b 2b 3c (66%) (1 hr) 6 1b 2c 3d (46%) (1 hr) 7 1c: R1 = OMe, R2 = H 2a No product formed 8 1d: R1 = F, R2 = H 2a No product formed 9 1e: R1 = H, R2 = Cl 2a 3a (9%) 10 1f: R1 = H, R2 = COOH 2a 3a (11%) Recycling of the catalytic system Run Yield of 3a (%) 1 76 2 62 3 51 Proposed Mechanism Laccase-Lipase Co-Catalytic System for the Cascade Synthesis of Benzofuran Derivatives OH R1 R OH OO OH 1 Laccase, Lipase O R R R2 2 3 Phosphate Buffer pH 7.0 H(Cl) OH 1.5-4hours,RT R 3 O Proposed pathway of laccase/lipase catalytic system O O O OH O OH OH O Laccase 2a Air Lipase 1a O O OH OH O OH OH OH Aromatization O O HO O O O 3a Reaction with a variety of lipases Lipase Yield Lipase Yield (%) (%) No lipase 33 No Lipase 53 Lipase from Candida rugosa 60 Lipase from Candida rugosa 47 (Lipase CR) (Lipase CR) Lipase from Pseudomonas cepacia 58 Lipase from Pseudomonas cepacia 60 (Lipase PS) (Lipase PS) Lipase B Candida Antarctica 41 Lipase B Candida Antarctica 62 (CALB) (CALB) The Formation of the Product 3a OH OH OH OO O Laccase, (Lipase PS) + Phosphate Buffer pH 7.0 OH 1a 2aRT O 3a The reaction of catechols and 1,3- dicarbonyl compounds Entry 1 1a: R1 = R2 = H 2a: R3 = R5 = Me, R4 = H 3a (58%) b 2 1a 2b: R3 = R5 = Me, R4 = Cl 3a (51%) 3 1a 2c: R3 = Me, R4 = H, R5 = OEt 3b (11%) b 4 1a 2d: R3 = Me, R4 = Cl, R5 = OEt 3b (53%) 5 1b: R1 = Me, R2 = H 2a 3c (60%) 6 1b 2b 3c (72%)b 7 1b 2c 3d (13%) 8 1b 2d 3d (66%)b 9 1c: R1 = OMe, R2 = H 2a No product formed 10 1d: R1 = F, R2 = H 2a No product formed 11 1e: R1 = H, R2 = Cl 2a 3a (8%) Recycling of the catalytic system OH OO O OH OH Laccase, Lipase PS + Cl 0.1M Phosphate buffer pH 7 OH RT, 1.5 hours 1b 2b O 3c Run Yield of 3c (%) 1 72 2 62 3 5 Conclusions of the Synthesis of Benzofurans An efficient green chemistry synthesis of benzofuran derivatives using a catalytic system of laccase and Sc(OTf)3 in surfactant aqueous medium.
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