Lipoxygenase for Oxidative Functionalisation of Lignins

Paola Giannì, Heiko Lange, Claudia Crestini

Polyphenol Chemistry & Material Science Laboratorywww.polyphenols.weebly.com

Department of Chemical Sciences and TechnologiesUniversity of Rome ‘Tor Vergata’, Italy

COST Association EventPolytechnic School Of San Sebastian, University Of The Basque Country, San Sebastian, Spain, May 26 th/27th, 2015

COST Action FP1105

Principles of Cell Wall Assembly – Understanding and Implementation

Lignin Upgrade

Problems:

– wide heterogeneity for different botanical origins

– diversity due to the isolation processes

Objective: – Selective functionalisation of lignin to improve its compatibility and performance

in composite and copolymer materials through the development of sustainable alternative processes

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Until today, several ligninolytic enzymes have been used to oxidatively valorise lignin:

– Laccase

– Lignin peroxidase

– Horseradish peroxidase (HRP)

Mechanism of action: generation of phenoxy radicals at the terminal phenolic

groups

This implies undesirable reactions occur along with desirable ones

FOCUS: identification of non conventional enzymes in order to achieve

functionalisation of the lignin-backbone without background reactions

Lignin - Enzymatic Valorisation: State of the Art

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Lignin - A New Enzymatic Strategy:Lipoxygenase (LOX)

• Non-heme iron-containing dioxygenase that catalyses the addition of molecular oxygen to polyunsaturated fatty acids with a (Z,Z)-1,4-pentadiene structural unit to give an unsaturated fatty acid hydroperoxide

• LOX is able to generate both peroxide and carbon centered radicals

• LOX displays low substrate specificity

• => LOX for lignins?

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DIPPMPO22.2 ppm

carbon adduct25.7 ppm

DIPPMPO oxide

25.0 ppm

peroxo adduct17.3/17.0 ppm

N

P(OiPr)2

CH3

OHO

O

N

(iPrO)2PCH3

OH

O

O

O

C4H9 C6H12COOH

N

P(OiPr)2CH3

OH

O

C4H9 C6H12COOH

O

OH

O

OH

O

OH

O

OH

O

OH

O

OH

insertion of O2

O

O

O

O

O

OH

O

OHO

O

HO

HO

9-HpODE 13-HpODE

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13

H attraction

H abstraction

LOX Reaction Pathway

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LOX reaction pathwayLipoxygenase oxidation of linoleic acid

Previous studies have added more detailed informations on LOX mechanism of reaction upon a model substrate (LA)

• under air: oxygen-centred radicals are preferentially formed

• anoxic conditions: carbon-centred radicals are preferentially formed

• The formation of carbon-centred radicals offers new interesting possibilities for functionalisation

L. Zoia, R. Perazzini, C. Crestini, D. S. Argyropoulos; Bioorg. & Med. Chem. 2011, 19, 3022-3028.

Treatment Mn MwMWL 4400 24500MWL, Air, LA 8100 28600

•Lignin was treated with LOX in the presence of LA as an oxidation mediator

•GPC analysis of the reaction mixture showed increase of molecular weight

Lipoxygenase oxidation of black spruce Milled Wood Lignin (MWL)

LOX ACTS ON LIGNIN SUBSTRATES

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HO

O

MeO

HO

OH

OMe

HO

OH

O

OH

OMe

O

HOOH

OMeO

OHHO

MeO O

OH

OHMeO

HO

OOMeHO

MeO

O

OHHO

MeOO

HOOH

HO

MeO

HO

MeO

OH

O

OH

MeO

OHO

HO

OH

MeO

HO

OOMe

HO

MeO

O

OHHO

MeOO

HOOH

O

MeO

O

MeO

OH

O

OH

MeOOH

O OH

OMe

HO

MeO

OO

MeO

HO

HO

O

HO

O

MeO

OH

OMe

OHO

OH

HO

MeO

HO

MeOOH

OHO

MeO

OH

MeO

LOX ?YES!

LOX Reactivity with Lignin

Sample/Treatment Mn MwMWL 4400 24500MWL, Air, 30 min, LA 6800 27300MWL, Air, 12h, LA 8100 28600MWL, N2 1 atm, 30 min, LA 5100 24800

MWL, N2 1 atm, 12h, LA 5800 24800

MWL, Air, 12h, NO mediator 5300 27500

• In all cases an increase of lignin molecular weight occurred

• Oxidation of MWL increases with time

• MWL is significantly polymerized in the absence of oxygen

Carbon-centered radicals!

• Polimerisation of MWL also in the absence of the oxidation mediator

Data obtained via GPC analysis; mediator: linoleic acid (LA)

LOX Reactivity with Lignin:Screening of Reaction Conditions

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REACTION TIME

ABSENCE OF OXYGEN

NO MEDIATOR

The possibility to generate carbon centered radicals on lignin paves the way for innovative and selective functionalisation processes

Based on the results obtained, we have developed protocols for LOX-mediated lignin functionalisations allowing the realization of unprecedented lignin functionalisation motifs

BIOMIMETIC project GA n.°282945

PATENTS (AND PUBLICATIONS) PENDING

Lipoxygenase - Mediated Functionalization of Lignin

This work is funded under the European project

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Conclusions

• Lignin is a substrate for lipoxygenase

• Lipoxygenase oxidizes lignin undergoing polymerisation in the presence of

oxygen with or without linoleic acid as an oxidation mediator

• Lipoxygenase can be used under nitrogen as a carbon-centered radicals

generator in lignin

Lipoxygenase constitutes a promising non conventional enzyme for lignin

upgrade

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Acknowledgments

• Prof. Claudia Crestini• Dr Heiko Lange• Elisavet D. Bartzoka

• Dimitris S. ArgyropoulosNorth Carolina State UniversityRaleigh, North Carolina, United States

BIOMIMETIC project GA n.°282945

COST Action FP1105

Principles of Cell Wall Assembly – Understanding and Implementation

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