laccase in organic synthesis and its applications
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Laccase in Organic Synthesis and Its Laccase in Organic Synthesis and Its ApplicationsApplications
Suteera WitayakranArt J. Ragauskas
Outline
Laccase in Organic SynthesisLaccaseLaccase application in organic synthesisThe synthesis of naphthoquinonesThe synthesis of benzofuran derivatives
Laccase in Fiber ModificationLaccase application in fiber modificationModification of linerboard softwood kraft pulp
Laccase
A multi-copper-containing oxidoreductase enzymeFound in plants and fungiIn fungi: function in pigment production, plant pathogenesis, detoxification, and delignificationImplicated in the synthesis of naturally occurring substancesCatalyze the oxidation of a variety of phenoliccompounds
Gianfreda, L.; Xu, F.; Bollag, J-M. Bioremediation Journal, 1999, 3, 1.Morozova, O. V.; et.al, Biochemistry (Moscow), 2007, 72, 1136.
LaccaseLignin
A phenolic polymer consisting of 3 different phenyl propane units
Oxidation of monomeric phenols has been shown to result in coupling to lignin macromolecule (Lund 2001)
CH
OH
CH
CH2OH
CH
OH
CH
CH2OH
OCH3
CH
OH
CH
CH2OH
OCH3H3CO
p-coumaryl alcohol coniferyl alcohol sinapyl alcohol
Laccase
COUPLING
Laccase
Laccase (ox)
H2O
O2
OH
OCH3
CH
CH
CH2OH
O
OCH3
CH
CH
CH2OH
O
OCH3
CH
CH
CH2OH
O
OCH3
CH
CH
CH2OH
O
OCH3
CH
CH
CH2OH
O
OCH3
CH
CH
CH2OH
Active Site of Laccase
Ribbon diagram of Trametes versicolor laccaseshowing the two channels leading to the T2/T3 cluster
Cu 2+
Cu 2+
OCu 2+
H
T ype 2
T ype 3
L accase trinuclear oxygen binding site
Three major steps of laccase catalysis:1. Type 1 Cu reduction2. Internal electron transfer3. O2 reduction at T2/T3 center
D3
D2
D1
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 PaperPulping: increase fiber bondingBleaching: laccase-mediator systemFiber 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 MethoxyphenolsPhenolso-diphenols and p-diphenolsAminophenolsPolyphenolsPolyaminesLignin-related molecules
Burton, S. G. Current Organic Chemistry, 2003, 7, 1317.
Laccase in Organic SynthesisMany studies reported Laccase-catalyzed reactions
The synthesis of actinocin and cinnabarinic acid
Oxidative coupling of hydroquinone and (+)-catechin
Oxidation of hydroxyl groups of sugar derivativesSynthesis of polymers
O
H
OHHH
HO
OH
OH
OHOH
OH
Catechin
O
H
OHHH
HO
OH
OH
OH
OH
HO
Laccase+
Laccase in Organic Synthesis
O
O
NH
HN
O
O
NH
And/Or
H2N R5
R5
R5
R5
R1 R3
R4
R1
R4
Acta Biochimica Polonica, 1959, 6, 399-409.J. Org. Chem. 2005, 70, 2002-2008.
GoalsTo 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 assayLaccase (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 studyBubble O2 for 30 mins before adding reagentsAdd 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 No reaction
The Effect of Laccase Dose
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.
The Effect of Laccase Dose on the Formation of Compound 3
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25
Reaction time (hr)
Yiel
d (%
)
500 U/ 1g subatrate1000 U/ 1g substrate2000 U/ 1g substrate4000 U/ 1g substrate
The Effect of Laccase Dose on the Formation of Compound 4
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25Reaction time (hr)
Yiel
d (%
)
500 U/ 1g subatrate1000 U/ 1g substrate2000 U/ 1g substrate4000 U/ 1g substrate
O
O
MeO
O
MeO
O
Proposed Reaction Pathway
+
O
MeO
O
O
MeO
O
Compound 3Compound 4
The Effect of Temperature
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.
The Effect of Temperature on the Formation of Compound 3
0102030405060708090
0 5 10 15 20 25
Reaction time (hr)
Yiel
d (%
)
25 °C 50 °C 70 °C
The Effect of Temperature on the Formation of Compound 4
0102030405060708090
100
0 5 10 15 20 25
Reaction time (hr)
Yiel
d (%
)
25 °C 50 °C 70 °C
O
MeO
O
O
O
MeO
Reaction of Hydroquinones and Dienes
2c
Laccase-generated quinones in naphthoquinonesynthesis via Diels-Alder reaction
• Published in Tetrahedron Letters 2007, 48, 2983-2987.
Proposed Reaction Pathway
Preliminary StudyTo Find the optimal condition
323 °C (2 h), RT1:155
83 °C (2 h), RT1:54
no product formed
60 °C1:103
10RT1:102
473 °C (2 h), RT1:101
Yield of 3 (%)Temperature 1 : 2(equiv.)
Entry
0% of 327% of
1:1 Chloroform/acetate buffer8
181:1 MeOH/acetate buffer7
151:1 Ethylene Glycol/acetate buffer 6
81:1 p-Dioxane/acetate buffer 5
0p-Dioxane4
255% Aqueous PEG 20003
18Water2
470.1 M Acetate buffer pH 4.51
Yield of 3 (%)SolventEntry
OHHO
OHOH Laccase
0.1M acetate buf fer pH 4.524 hours
OO
21 3
Reaction of a Various Catechols
OHOH
Laccase
0.1M acetate buffer pH 4.53 oC - RT, 24 hours
OO
2R1 R1
1:10
no product formed97% of quinone7
14and 15% of (96hr)
6
no product formed5
11and 32% of
4
283
572
471
Yield (%)CatecholEntry
OO
H3CO
OCH 3
O
O
OO
OHOH
OHOH
CH3
OHOH
CHO
OHOH
Cl
OHOHH3CO
OHOH
OHOH
Reaction of a Various Dienes
OHOH
R4
Laccase
0.1M acetate buffer pH 4.53 oC - RT, 24 hours
OO R3
R4
1 : 10
R3
CH3 CH3
R2
R5R5
R2
no product formed6
76 (R2 =H)
( 2 eq.)
5
77 (R2 = H)4
103
712
571
Yield (%)DieneEntry
OCH 3
OCH 3
OCH 3
OO
CH 3
Reaction of 1-Acetoxy-1,3-Butadiene with a Variety of 1,4-Benzohydroquinone
69Cl5
67Br4
81OCH33
75CH32
67H1
Yield (%)R1Entry
Conclusions of the Synthesis of Naphthoquinones
An efficient green chemistry synthesis of naphthoquinones
The use of safe, environment-benign solventThe 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
2 4
1
2
4
Laccase, 0.2eq. Sc(OTf)3
0.1M Phosphate Buffer pH 7.0
Preliminary Study
61:20.1 M Phosphate buffer pH 8.04
01:20.1 M Acetate buffer pH 4.53
641:20.1 M Phosphate buffer pH 7.02
461:10.1 M Phosphate buffer pH 7.01
Yield of 3a (%)1a:2a (equiv)Solvent/ pHEntry
OH
OH O O O OH
OH
O
+LaccaseSolvent
RT, 4 hours
1a 2a 3a
The Effect of Lewis Bases and Lewis Acids
The effect of Lewis bases
131: 2: 10.1 M Phosphate buffer pH 7.0
DABCO5
91: 2: 10.1 M Phosphate buffer pH 7.0
DMAP4
541: 2: 10.1 M Phosphate buffer pH 7.0
Pyridine3
401: 2: 0.50.1 M Phosphate buffer pH 7.0
Pyridine2
331: 2: 0.5WaterPyridine1
Yield of 3a(%)
1a: 2a:Lewis base(equiv)
SolventLewisbases
Entry
491: 2: 0.2CuCl26
711: 2: 0.2InCl3.4H2O5
721: 2: 0.2Yb(OTf)34
761: 2: 0.2Sc(OTf)3/ SDS3
741: 2: 0.2Sc(OTf)32
631: 2: 0.1Sc(OTf)31
Yield of 3a(%)
1a: 2a: Lewisacid (equiv)
Lewis acidEntry
The effect of Lewis acids
The Reaction of Catechols and 1,3-Dicarbonyl Compounds
3a (11%)2a1f: R1 = H, R2 = COOH10
3a (9%)2a1e: R1 = H, R2 = Cl9
No product formed2a1d: R1 = F, R2 = H8
No product formed2a1c: R1 = OMe, R2 = H7
3d (46%) (1 hr)2c1b6
3c (66%) (1 hr)2b1b5
3c (68%)2a1b: R1 = R2 = H4
3b (48%) (1 hr)2c: R3 = Me, R4 = Cl, R5 = OEt1a3
3a (79%) (1 hr)2b: R3 = R5 = Me, R4 = Cl1a2
3a (76%)2a: R3 = R5 = Me, R4 = H1a: R1 = Me, R2 = H1
Entry
Recycling of the catalytic system
513
622
761
Yield of 3a (%)Run
Proposed Mechanism
Laccase-Lipase Co-Catalytic System for the Cascade Synthesis of Benzofuran Derivatives
OHR1 OH
R2 R3
O O
H (Cl)
O
R1OH
OHR2
R3 O
Laccase, Lipase
Phosphate Buffer pH 7.01.5 - 4 hours, RT
Proposed pathway of laccase/lipase catalytic system
O HO H
1 a
OOLa cc as e
O O
L ipas e2 a
O HO
OO
O HO
OH OO
O
O HO H
O
O
O HO H
A ro m atiz at ion
3a
A ir
Reaction with a variety of lipases
41Lipase B Candida Antarctica (CALB)
58Lipase from Pseudomonas cepacia(Lipase PS)
60Lipase from Candida rugosa(Lipase CR)
33No lipase
Yield (%)
Lipase
62Lipase B Candida Antarctica(CALB)
60Lipase from Pseudomonas cepacia (Lipase PS)
47Lipase from Candida rugosa(Lipase CR)
53No Lipase
Yield (%)
Lipase
The Formation of the Product 3aOH
OH O O O OH
OH
O
+Laccase, (Lipase PS)
Phosphate Buffer pH 7.01a 2a 3aRT
The reaction of catechols and 1,3-dicarbonyl compounds
3a (8%)2a1e: R1 = H, R2 = Cl11
No product formed2a1d: R1 = F, R2 = H10
No product formed2a1c: R1 = OMe, R2 = H9
3d (66%)b2d1b8
3d (13%)2c1b7
3c (72%)b2b1b6
3c (60%)2a1b: R1 = Me, R2 = H5
3b (53%)b2d: R3 = Me, R4 = Cl, R5 = OEt1a4
3b (11%)2c: R3 = Me, R4 = H, R5 = OEt1a3
3a (51%)b2b: R3 = R5 = Me, R4 = Cl1a2
3a (58%) 2a: R3 = R5 = Me, R4 = H1a: R1 = R2 = H1
Entry
Recycling of the catalytic system OH
OHO O O OH
OH
O
+Laccase,
0.1M Phosphate buf fer pH 7RT, 1.5 hours
Lipase PS
1b 2b 3cCl
53
622
721
Yield of 3c (%)Run
Conclusions of the Synthesis of Benzofurans
An efficient green chemistry synthesis of benzofuranderivatives
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 ModificationPotential 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 ligninHuttermann: carbohydrate onto lignosulfonateLund: guaiacol sulfoanate onto ligninMai: acrylic compounds onto liniosulfonatesMai: 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
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 (Scallan).Fiber swelling results in increase:
Fiber flexibilityConformabilityFiber-fiber bonding
Scallan, A. M. Tappi J. 1983, 66, 73-75.Laine, J.; Stenius, P. Paperi ja Puu 1997c, 79, 257-266.
Water Drawn In
Water Drawn In
Fiber Wall External Solution
Grafting low-molecular-weight compounds onto lignin-rich fiber
Recently,Grönqvist et al. reported laccase-catalysed functionalisation of TMP with tyramineTwo-stage functionalisation method consists of:
Enzymatic activation of fiber surfaceRadical coupling between activated TMP and radicalised tyramine
R
OH
OMeOH3NH2C
OHTyramine
Lignin
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
HypothesisCarboxylic 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 pulpDetermine conditions where the laccase-facilitated grafting system was the most effective for modifying fibersEvaluate the effects of the laccase-facilitated grafting treatment on paper strength properties
ExperimentGeneral Procedure:
0.1M Phosphate Buffer pH 7.0
5% csc Linerboard Pulp Laccase (80U/g pulp)
Amino acid
H2N COOH
R
Stir for 4 hours Let it stand for 20
hoursFilter Wash with deionized
waterDetermine the acidic
group content by conductrometrictitration
Preliminary Experiment
To find the optimal condition for modifying fibersUse Glycine (4 mmol/5g pulp) as model amino acid
Optimal Condition: pH7.0 and RT
H2N COOH
HH
0.1
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.2
Control Pulp Lac Gly Lac/Gly pH4.5, RT
Lac/Gly pH7.0, RT
Lac/Gly pH 7.0, 45˚C
CO
OH
(me
q/g
)
Experiment with Various Amino Acids
To find amino acid that give the best yield of carboxylic contentTo find optimal amount of amino acid for modifying fibersTest with 7 different amino acids:
H2N CH C
H
OH
O
Gly
H2N CH C
CH2
OH
O
C
OH
OAsp
H2N CH C
CH2
OH
O
N
NH
His
H2N CH C
CH2
OH
O
CH2
CH2
NH
C
NH2
NH
Arg
H2N CH C
CH3
OH
O
Ala
Experiment with Various Amino Acids
0.165
0.17
0.175
0.18
0.185
0.19
0.195
0.2
0.205
0.21
Gly Phe Ser Asp His Arg Ala
COOH
(meq
/g)
8 mmol/ 5g pulp 12 mmol/ 5g pulp16 mmol/5g pulp
His gave the best yield of acid groupsOptimal amount is 16 mmol/ 5g pulp
Experiment with Various Amino Acids
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.2
0.21
Control PulpLac Gly Phe Ser Asp His Arg Ala
COO
H (m
eq/g
)
Control Pulp LacAmino 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 modifying fibers
Use Histidine (16 mmol/ 5g pulp) for this study
The optimal amount of laccase is 80 U/ 1g pulp
0.18
0.185
0.19
0.195
0.2
0.205
0.21
20 U 40 U 60 U 80 U 100 U
CO
OH
(m
eq
/g)
Activity of Laccase/ 1g pulp
Effect of Laccase Dose
Paper Strength PropertiesUse optimal condition to treat the fibers
5% csc Linerboard pulpLaccase (80U/1g pulp)Histidine (16 mmol/5g pulp)In phosphate buffer pH 7.0Room 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
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Control pulp Lac Lac/His
%N
itrog
en in
Han
dshe
ets
% Nitrogen in Handsheet
12
12.5
13
13.5
14
14.5
15
15.5
16
Control Pulp Lac Lac/His
Tear
Inde
x (m
N.m
2/g)
Tear Strength
5151.5
5252.5
5353.5
5454.5
5555.5
56
Control Pulp Lac Lac/His
Tens
ile In
dex
(N.m
/g)
Dry Tensile Strength
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
Control Pulp Lac Lac/His
Tens
ile In
dex
(N.m
/g)
Wet Tensile Strength
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 fibersLaccase/His treatment provided the best result in increasing acid groups.This treatment results in increasing of paper strength of handsheetsThis procedure is environmental friendly method for modifying lignin-rich fiber
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