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

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

OCH3

H2O Laccase

O2 Laccase (ox)

CH2OH CH2OH CH2OH CH2OH CH2OH

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

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

R5 R1 R3

N R4 H H2N R5 O And/Or

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 MeO MeO 500 U/ 1g subatrate 1000 U/ 1g substrate 1000 U/ 1g substrate 2000 U/ 1g substrate 2000 U/ 1g substrate 4000 U/ 1g substrate 80 4000 U/ 1g substrate 80 70 O O ) 70 % ) ( 60 %

( 60 50

Yield Yield 50

40 Yield 40 30 30 20 20 10 10

0 0 0 5 10 15 200 25 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

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

MeO

90 100 O 90 80 )

% 80

70 ( O 70 60 60 50 Yield Yield Yield (%) Yield 50 40 40 30 30 20 20 10 10 0 0 0 5 10 15 200 25 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 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. using a catalytic system of laccase and lipase PS in an aqueous medium.

The yield of the products from reaction depended on both the reactivity of catechols and β-dicarbonyl compounds. Catechols with moderate reactivity yield benzofuran products in excellent yield.

This catalytic system of laccase and Sc(OTf)3 could be recycled and reused for two additional runs, with only a minor drop in product yields. Laccase in Fiber Modification Potential tools for the modification of lignin-rich fiber

Activation of surface lignin to enhance auto adhesion of fiberboards (Felby et al.)

Grafting a variety of substrates onto lignin Huttermann: carbohydrate onto lignosulfonate Lund: guaiacol sulfoanate onto lignin Mai: acrylic compounds onto liniosulfonates Mai: acrylamide onto lignin in the presennce of organic peroxide

Kenealy, W. R.; Jeffries, T. W. Wood Deterioration and Preservation: Advances In Our Changing World. American Chemical Society, Washington, 2003, 210-239. Grafting low-molecular-weight compounds onto lignin-rich fiber

Chandra and Ragauskas grafted 4-hydoxybenzoic acid and Gallic acid to high kappa pulps. Increasing of carboxylic acid groups, tensile strength and burst strength of the resulting paper.

COOH

HO OH

Gallic acid

Biotechnol. Prog. 2004, 20, 255-261. The effect of acidic groups on the properties of fibers

Acid groups can

cause fiber swelling Water Drawn In (Scallan). Fiber swelling results in increase: Water Drawn In Fiber flexibility Conformability Fiber-fiber bonding

Fiber Wall External Solution

Scallan, A. M. Tappi J. 1983, 66, 73-75. Laine, J.; Stenius, P. Paperi ja Puu 1997c, 79, 257-266. Grafting low-molecular-weight compounds onto lignin-rich fiber

Recently,Grönqvist et al. reported laccase- catalysed functionalisation of TMP with tyramine Two-stage functionalisation method consists of: Enzymatic activation of fiber surface Radical coupling between activated TMP and radicalised tyramine

R Lignin

H3NH2C O OMe

OH Tyramine OH R= Lignin

Grönqvist, S. et al. Holzforschung, 2006, 60, 503-508. Modification of Linerboard Softwood Kraft Pulp Modification of Linerboard Softwood Kraft Pulp with Laccase and Amino Acids

Hypothesis Carboxylic acid groups can improve fiber- fiber bonding. Introduce acid groups to lignin-rich fiber by the addition reaction of laccase-oxidized fiber with amino acids Objectives

Evaluate the feasibility of a system utilizing laccase to graft amino acid with high kappa kraft pulp Determine conditions where the laccase- facilitated grafting system was the most effective for modifying fibers Evaluate the effects of the laccase-facilitated grafting treatment on paper strength properties Experiment

General Procedure:

Stir for 4 hours Let it stand for 20 hours 5% csc Linerboard Pulp Filter Laccase (80U/g pulp) Wash with deionized Amino acid water R Determine the acidic group content by H2N COOH conductrometric 0.1M Phosphate titration Buffer pH 7.0 Preliminary Experiment

To find the optimal condition for modifying fibers

Use Glycine (4 0.2 ) mmol/5g pulp) as 0.19 model amino acid 0.18 0.17 H H 0.16 0.15

H2N COOH COOH (meq/g 0.14 0.13 0.12 Optimal Condition: 0.11 pH7.0 and RT 0.1

Control Pulp Lac Gly Lac/Gly Lac/Gly Lac/Gly pH4.5, RT pH7.0, RT pH 7.0, 45˚C Experiment with Various Amino Acids To find amino acid that give the best yield of carboxylic content To find optimal amount of amino acid for modifying fibers Test with 7 different amino acids:

O O

H2NCHC OH H2NCHC OH

H CH2 Gly CH2 Arg

CH O 2 O NH H2NCHC OH O H2NCHC OH C NH CH2 H2NCHC OH CH2 His Asp NH2 CH N 3 C O Ala NH OH Experiment with Various Amino Acids

0.21

0.205

0.2

0.195

0.19

0.185

0.18

COOH (meq/g) 0.175

0.17

0.165 Gly Phe Ser Asp His Arg Ala 8 mmol/ 5g pulp 12 mmol/ 5g pulp 16 mmol/5g pulp

His gave the best yield of acid groups Optimal amount is 16 mmol/ 5g pulp Experiment with Various Amino Acids

0.21

0.2

0.19

0.18

0.17

0.16

COOH (meq/g) 0.15

0.14

0.13 Control PulpLac Gly Phe Ser Asp His Arg Ala

Control Pulp Lac Amino acid Lac/Amino acid

Laccase/amino acid- treated pulp gave highest yield of COOH. Effect of Laccase Dose

To find the optimal laccase dose for

Effect of Laccase Dose modifying fibers

0.21

0.205

0.2 Use Histidine (16

0.195 COOH(meq/g) mmol/ 5g pulp) for this 0.19 study 0.185

0.18 20 U 40 U 60 U 80 U 100The U optimal amount of Activity of Laccase/ 1g pulp laccase is 80 U/ 1g pulp Paper Strength Properties Use optimal condition to treat the fibers 5% csc Linerboard pulp Laccase (80U/1g pulp) Histidine (16 mmol/5g pulp) In phosphate buffer pH 7.0 Room Temperature

Make 3g handsheets of treated pulp to measure strength properties and compare with handsheets of control pulp and laccase-treated pulp Paper Strength Properties

% Nitrogen in Handsheet Dry Tensile Strength

0.14 56 0.12 55.5 55 0.1 54.5 0.08 54 53.5 0.06 53 0.04 52.5 %Nitrogen in in %Nitrogen Handsheets

Tensile Index Tensile Index (N.m/g) 52 0.02 51.5 0 51 Control pulp Lac Lac/His Control Pulp Lac Lac/His

Tear Strength Wet Tensile Strength

16 3 15.5 2.9 2.8 15 2.7 14.5 2.6 14 2.5 13.5 2.4 13 2.3 Tear Index (mN.m2/g) Tensile Index (N.m/g) 12.5 2.2 12 2.1 Control Pulp Lac Lac/His 2 Control Pulp Lac Lac/His SEM of Handsheets

Control Laccase Laccase/His

Lac/His-treated fibers collapse more and bond better than control and laccase-treated fibers. Conclusions

Laccase/amino acids treatment results in an increase in carboxylic acid groups of fibers Laccase/His treatment provided the best result in increasing acid groups. This treatment results in increasing of paper strength of handsheets This procedure is environmental friendly method for modifying lignin-rich fiber