Lipoxygenase for Oxidative Functionalisation of Lignins Paola Giannì, Heiko Lange, Claudia Crestini Polyphenol Chemistry & Material Science Laboratory Department of Chemical Sciences and Technologies University of Rome ‘Tor Vergata’, Italy COST Association Event Polytechnic School Of San Sebastian, University Of The Basque Country, San Sebastian, Spain, May 26 th /27 th, 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 2

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 3

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? 4

DIPPMPO 22.2 ppm carbon adduct 25.7 ppm DIPPMPO oxide 25.0 ppm peroxo adduct 17.3/17.0 ppm LOX Reaction Pathway 5 LOX reaction pathway Lipoxygenase 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,

TreatmentMnMw MWL MWL, Air, LA 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 6 LOX ? YES! LOX Reactivity with Lignin

Sample/TreatmentMnMw MWL MWL, Air, 30 min, LA MWL, Air, 12h, LA MWL, N 2 1 atm, 30 min, LA MWL, N 2 1 atm, 12h, LA MWL, Air, 12h, NO mediator 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 7 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.° PATENTS (AND PUBLICATIONS) PENDING Lipoxygenase - Mediated Functionalization of Lignin This work is funded under the European project 8

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 9

Acknowledgments Prof. Claudia Crestini Dr Heiko Lange Elisavet D. Bartzoka Dimitris S. Argyropoulos North Carolina State University Raleigh, North Carolina, United States BIOMIMETIC project GA n.° COST Action FP1105 Principles of Cell Wall Assembly – Understanding and Implementation 10