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Lipoxygenase: unprecedented carbon-centred lignin activation Paola Giannì, Heiko Lange, and Claudia Crestini ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04772 • Publication Date (Web): 18 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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ACS Sustainable Chemistry & Engineering

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Full research paper for publication in: ACS Sustainable Chemistry and Engineering

Lipoxygenase: unprecedented carbon-centred lignin activation

Paola Giannì, Heiko Lange and Claudia Crestini*

Department of Chemical Sciences and Technologies, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy * Corresponding author: [email protected]

Abstract The biotechnological conversion of lignocellulosic biomass is a pivotal aspect in the development of overall sustainable and environmentally friendly biorefinery and valorisation processes of non-fossil biomass. Lipoxygenase (EC 1.13.11.x), a non-cellulolytic oxidizing enzyme normally working on linoleic acid metabolism, is demonstrated to catalyse the oxidative functionalization of lignin. Lipoxygenase was used for structurally altering the most important types of technical lignins. Most noteworthy oxidation is achieved by activation via a radical mechanism leading to both oxygen- and carbon-centred radicals.. Since this activation takes also

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place on the lignin backbone, the ketyl and benzyl radicals generated can be used to functionalise lignins via formation of carbon-carbon bonds.

Keywords lignin, lipoxygenase, valorisation, carbon-centred radicals, exo-depolymerisation

Introduction Some of the most abundant renewable, non-fossil-based resources are still struggling to become a major resource pillar for the chemical industries.1 One of the most prominent examples of such an abundant but under-utilised natural resource is lignin,. Functionalisation of industrially isolated and commercially available high quality lignins intend to change or improve their inherent characteristics and performances for making them suitable materials for a wide range of downstream applications.2–5 Often these modifications run via utilisation and manipulation of phenolic hydroxyl groups. The aim is to either render isolated lignins suitable co-polymers or to degrade isolated lignins into monomeric building blocks as eventual starting materials for traditional chemical industries. Sustainable processes in these areas of valorisation of isolated lignins,6–11 but also in the biorefinery processes for lignin isolation itself,10–17 are based on the use of lignocellulosic and lignolytic enzymes; most commonly used are cellulases, laccases and peroxidases. However, these enzymatic treatments are used predominantly during


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the initial pre-treatment of biomass for easing separation of components, for the production of different fuel classes out of initial biomass degradation products or funnelling processes following thermal and / or chemical degradation of lignin10,12,13,17–19 Several types of oxidative enzymes, namely laccases, polyphenol oxidases, horseradish peroxidases have been widely reported to oxidatively valorise lignin via degradation.18,20–24 Huge scientific effort has been devoted at investigating the potentialities of lignolytic enzymes, i.e., mainly laccase and peroxidases, to depolymerize or alternatively polymerize lignin. From a less conventional point of view, the selective functionalization of lignin by use of oxidative enzymes as lignin backbone activators represents an innovative approach to lignin valorisation. Isolated lignins could in principle be oxidatively activated and functionalised25 also by targeting intrinsic functional groups that are part of the target groups for the lignocellulolytic enzymes. Lignin activation could be eventually achieved using non-conventional ‘lignincompatible enzymes’, arriving thus at a route for biotechnological functionalisation without the risk of degradation. In this context, our work has been focused at identifying potential lignincompatible enzymes for direct or mediated lignin oxidation and the development of an innovative enzyme-based approach for the functionalisation of the lignin-backbone, if possible without strongly competing background reactions that attack phenol groups and degrade the polymeric nature of the lignins. The alternative enzymatic approach described in this work is based on the use of lipoxygenase (EC 1.13.11.x) (LOX, Scheme 1), a non-heme iron-containing dioxygenase that naturally catalyses the addition of molecular oxygen to polyunsaturated fatty acids with a (Z,Z)-1,4pentadiene structural unit. Its model substrate is linoleic acid (LA), yielding an unsaturated fatty acid hydroperoxide under aerobic conditions.26–29 31P NMR-based spin trapping studies showed



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that this enzyme, depending on the chosen conditions , is able to generate alkyl and peroxide radicals in its natural substrate: under oxygen-containing atmospheres, oxygen-centred radicals are preferentially formed; under inert atmospheres and in degassed solvents, in contrast, carboncentred radicals dominate (Scheme 1).30

Scheme 1: Mechanism of action of lipoxygenase (insert: PDB 1LOX, iron centre highlighted in red 31) (EC 1.13.11.x) on its model substrate linoleic acid (LA), containing the typical (Z,Z)-1,4-pentadiene structural unit, to the oxidised products (±)9- hydroperoxy-10E,12Z-octadecadienoic acid, 9-HpODE, and (±)13-hydroperoxy9Z,11E-octadecadienoic acid 13-HpODE , respectively.30,32

This paper reports initial orienting results regarding the reactivity of LOX towards selected lignin model compounds and different isolated lignins, namely a well-characterised laboratoryproduced softwood milled wood lignin from Norway spruce (NS-MWL),33,34 a well characterised industrially produced grass organosolv lignin from wheat straw (WS-OSL)35–38 and a well-studied kraft lignin from mixed softwoods (SW-KL)39–43 (Figure 1). Linoleic acid


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was eventually added to generate a mediator system. The purpose of these incubations of lignins with LOX was to structurally modify lignin, aiming especially at an activation of carbon atoms.27,30

Materials and methods General: Softwood milled wood lignin (MWL) was isolated from Norway spruce wood as reported before34 by slight modification of Björkman’s procedure.33 Wheat straw organosolv lignin (WS-OSL) was produced via the BioligninTM process by CIMV (Compagnie Industrielle de la Matière Végétale), Levallois Perret, France.35 Softwood kraft lignin (SW-KL) was obtained by a modified Lignoboost process by FCBA, Grenoble, France.39,40 Lipoxygenase (EC 1.13.11.x, LOX), linoleic acid (LA), buffer salts and used solvents in the appropriate grades were purchased from Sigma Aldrich and used as received. Aqueous buffer solutions were prepared freshly on a weekly basis with the following specifications: i) carbonate buffer, 0.1 M, pH 9; ii) borate buffer, 0.1 M, pH 9; 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO) was prepared as described elsewhere,30 and was stored under argon at −78° C. Dimeric β-ether (β-O-4’) models I and II were synthesised largely following literature procedures.44–46 Test for LOX-activity: Enzyme activity was controlled following literature precedence:47 Briefly, LOX activity was determined by peroxidation of linoleic acid. The assay mixture contained 0.011% (v/v) linoleic acid, 178 mM borate buffer, pH 9, 0.01% (v/v) ethanol and a suitable amount of enzyme. Peroxidation of LA was followed by absorbance increase at λ = 234



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nm. Enzyme activity was expressed in units, with each unit being defined as causing an increase in A234 of 0.001 per minute at pH 9.0 at 25 °C with linoleic acid as the substrate (1 cm light path). Test for laccase activity of LOX preparation: Enzyme activity was controlled following standard literature precedence:48 Briefly, laccase activity was determined spectrophotometrically using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the substrate. The assay mixture contained 0.5 mM ABTS, 0.1 M sodium acetate pH 5 and a suitable amount of a highly concentrated LOX solution; the substrate oxidation was followed by an absorbance increase at λ = 420 nm for one minute (ɛ = 3.6 × 104 M−1 cm−1). LOX-mediated lignin modification: Depending on the desired reaction mode of the enzyme, buffer solutions were degassed, and the atmosphere in the reaction vessel was exchanged to nitrogen after addition of all components. In a typical reaction, 500 mg of lignin were suspended in 50 mL of carbonate or borate buffer (0.1 M) at pH 9. Approx. 25000 U of LOX were added, and the reaction mixture was stirred under the appropriate atmosphere at room temperature for the desired amount of time. Subsequent isolation of the lignin was achieved by acidifying the solution to pH 2-3, and isolation of the precipitate by centrifugation. After three sequential washes with slightly acidified water, the retained solids were freeze-dried prior to structural analyses. LOX-activity on dimeric lignin models: Approx. 15 µmol (1.0 equiv.) of the lignin model compound were accurately weighted and initially dissolved in 50 µL dimethyl sulfoxide (DMSO). This solution was diluted by 1300 µL carbonate buffer solution (0.1 M) at pH 9. 10 mol-% LA were added in form of 10 µL of a 0.3 M solution of LA in DMSO. LOX)was


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added at concentrations of 0.1 U/µmol in form of a 5 U/mL solution of LOX in carbonate buffer (0.1 M) at pH 9. Final reaction volumes were eventually adjusted with carbonate buffer (0.1 M) at pH 9 as to reach 2 mL, containing 5 % dimethylsulfoxide (DMSO). Reaction mixtures were stirred at 30° C for 24 h, before the reaction mixture was brought to pH 2 by addition of 2 N aqueous hydrochloride solution. The aqueous solution was subsequently extracted once with 1.5 mL of ethyl acetate. The organic phase was separated and dried over magnesium sulphate. Solids were pelleted by centrifuging, and an aliquot of the organic phase was subjected to GCMS analysis as described below. Reactions under anaerobic conditions were realised following the same protocol using degassed buffer solutions and working under a nitrogen atmosphere. GC-MS analysis: Mass spectrometric analyses were performed on aliquots of the reaction mixture after a quick acidic work-up (vide supra) before and after in situ silylation using N,Obis(trimethylsilyl)trifluoroacetamide in the presence of dry pyridine as reported before. Gaschromatographic separation of the samples using a Shimadzu GCMS QP2010 Ultra system (gas chromatograph GC2010 Plus, equipped with Shimadzu autosampler AOC20i) at 70 eV ionisation energy. A Supelco fused-silica capillary column SLBTM-5ms (30m long, 0.25 mm thick, 0.25 µm diameter) was used as stationary phase, He (UHP grade) as mobile phase; the system was operated in ‘linear velocity mode’ with a starting pressure of 100 kPa, 280° C injection temperature, and 200 °C interface temperature, running the temperature program: 50 °C start temperature for 1 min, 10 °C min˗1 heating rate, 280 °C final temperature for 15 min). System control and analyses were realised using Schimadzu analysis software package Labsolutions–GCMSsolution Version 2.61.



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GPC-analyses: Approx. 5 mg of lignin were acetobrominated for analysis,49 and analyses was performed as detailed before50 omitting the determination of correction factors, using a Shimadzu instrument consisting of a controller unit (CBM-20A), a pumping unit (LC 20AT), a degasser (DGU-20A3), a column oven (CTO-20AC), a diode array detector (SPD-M20A), and a refractive index detector (RID-10A) ), and controlled by Shimadzu LabSolutions (Version 5.42 SP3). Different set-ups comprising two or three analytical GPC columns (each 7.5 x 30 mm) in series were realized for analyses: in case of two columns Agilent PLgel 5 µm 10000 Å, followed by Agilent PLgel 5 µm 1000 Å, which in case of the three columns are followed by an Agilent PLgel 5 µm 500 Å. HPLC-grade THF (Chromasolv®, Sigma-Aldrich) was used as eluent (0.75 mL min˗1, at 40° C). Standard calibration was performed with polystyrene standards (Sigma Aldrich, MW range 162 – 5 x 106 g mol˗1). Analyses were run in duplicate. 31

P NMR analysis: In general, a procedure similar to the one originally published was

used.51,52 Approx. 30 mg of the lignin were accurately weighed in a volumetric flask and suspended in 400 µL of a solvent mixture of pyridine and deuterated chloroform (CDCl3) (1.6:1 v/v) the above prepared solvent solution. One hundred microliters of the internal standard solution, i.e., cholesterol at a concentration of 0.1 M in the aforementioned NMR solvent mixture, were added. 50 mg of Cr(III) acetyl acetonate were added as relaxation agent to this solution, followed by 100 µL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxa-phospholane (ClTMDP). After stirring for 120 min at ambient temperature, 31P NMR spectra are recorded on a Bruker 300 MHz or Bruker 700 MHz NMR spectrometer controlled by TopSpin software, using an inverse gated decoupling technique with the probe temperature set to 20° C. Typical spectral parameters for quantitative studies were as follows: 90° pulse width and sweep width of 6600 Hz. The spectra are accumulated with a delay of 15 s between successive pulses. Line


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broadening of 4 Hz was applied, and a drift correction was performed prior to Fourier transform. 256 scans were acquired. The maximum standard deviation of the reported data is 0.02 mmol g˗1, while the maximum standard error is 0.01 mmol g˗1.53,51 GC-MS-based spin-trapping experiments: As a variation of the procedure used for the GCMS-based study on LOX-reactivity on dimeric lignin models, 100 µL of solution of DIPPMPO in DMSO at a concentration of 50 mg/mL were added to the reaction mixture 30 min before the work-up according to the standard protocol was initiated. Aliquots of the crude products in ethyl acetate were subjected to GC-MA-based analysis as described before. 31

P NMR-based spin-trapping experiments: Adopting the general analysis strategy

successfully applied before,30 approx. 30 mg of lignin were weighted in a reaction vessel and suspended in 65 mL of carbonate buffer (0.1 M) at pH 9, which has been degassed in case reactions were supposed to be run under inert conditions. 13 µL of linoleic acid were added – eventually followed by a drop of 1.0 M NaOH to assist solubility, before approx. 0.5 mg of lipoxygenase were added. The suspension was stirred at room temperature for a defined period of time, before 5.0 mg of DIPPMPO were added. After additional 30 min of stirring, the reaction mixture was acidified to pH 3. The solids were isolated via centrifugation, washed, and freeze-dried. The combined aqueous phases were extracted three times with chloroform, and washed with brine. The combined organic phases were dried over sodium sulphate and concentrated in vacuo. An aliquot was dissolved in 600 µL of CDCl3 and subjected to 31P NMR spectroscopic analysis using the basic instrument settings as described above and changing the offset to the appropriate ppm region.



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Results and discussion The first step of the present study consisted in the assessment of the ability of LOX to catalyse the oxidative activation of lignin via radical generation. Different lignins present different structural elements that can be seen as very characteristic in terms of points of attack with respect to the development of novel protocols for the valorisation of lignins. These lignins, that conveniently serve as model systems, are: i) laboratory-produced milled wood lignin (MWL)33; ii) modern industrially available oligomeric organosolv lignin (OSL)35 and iii) traditional, abundantly industrially available kraft lignin (KL).39 The various functional motifs displayed by these lignins are shown on Figure 1; depending on the source of the lignin and the procedure used for its isolation, abundances of structural groups differ significantly, as recently demonstrated and regularly summarised.4,34,37,42,54,55 Most conveniently, as described elsewhere in details as well, monitoring changes in these groups using dedicated analytical methods allows insight in effectiveness and chemical natures of any valorisation.55–58


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Figure 1: Structural features of important types of lignin: (A) linear, oligomeric softwood milled wood lignin (e.g., NS-MWL) and organosolv lignins (e.g., WS-OSL);34 (B) mix of polymeric and oligomeric branched structures present in softwood kraft lignin (SW-KL).42 NB: The structures intend to give a conceptual overview of generally present groups; abundancies are not representative.



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Given the dense complexity of the different functional groups in different isolated lignins along the lignin backbones (Figure 1), different types of isolated lignins thus represent a priori a wide array of possible substrates for a LOX-mediated functionalisation, anticipating that the substrate specificity of LOX is eventually low enough to also directly activate lignin-inherent motifs like benzylic positions. Although lipoxygenase is susceptible, like other enzymes, to inhibition by phenolic extracts, i.e., tannins,26,29,59 only a negligible inhibiting effect was initially anticipated by parts of the structurally different lignins and the impurities contained in them. Nevertheless, this assumption was to be proven in the experiments.

LOX-activity on lignin model compounds In order to test for most specific structural motifs susceptible to oxidative activation by LOX itself or the LOX-LA mediator system, dimeric β-O-4’ model 1 as a representative structure for phenolic end-groups in lignin chains, and dimeric β-O-4’ model 2 representing the most abundant interunit bonding motif found in, e.g., softwood MWL and grass OSL were studied in GC-MS-based analysis.34,37,38 Results obtained in the screening experiments are summarised in Scheme 2 and Table 1.


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13 (A)

HO HO

O

lipoxygenase

O

O

O

O

O

O

O

conditions O

O

OH

O

OH

OH

1

HO

3

O

HO

HO

O

O

lipoxygenase

O

4

O

O O

O

O

O O

O

conditions O

5

6

Chemical Formula: C8H9O3• Chemical Formula: C10H13 O4• Exact Mass: 153.06 Exact Mass: 197.08

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

Chemical Formula: C23H38 NO8P•+ Chemical Formula: C21H34NO7P•+ Chemical Formula: C9H11O2• Exact Mass: 487.23 Exact Mass: 443.21 Exact Mass: 151.08

(C)

Chemical Formula: C8H9O2• Chemical Formula: C11H15O5• Exact Mass: 227.09 Exact Mass: 137.06 Chemical Formula: H• H OH

O O O

OH

Exact Mass: 1.01

O O

O

O

O OH

O

O

O

O

O O

N O

P

O

O

Chemical Formula: HO• Chemical Formula: C3H7• Exact Mass: 17.00 Exact Mass: 43.05

O

Chemical Formula: C9H11 O2• Exact Mass: 151.08

OH

O

O

O

O

O OH

O

O N

O N

O N

Chemical Formula: C17 H25NO5P2•+ Exact Mass: 354.15

O P O O

O P O O

O P O

Chemical Formula: C23H37NO9P2•+ Exact Mass: 502.22

OH

O

N P

O

Chemical Formula: C9H11O2• HO Exact Mass: 151.08 HO

Chemical Formula: C11H15 O5• Exact Mass: 227.09

OH

O

O O

HO HO

O

N P

O

O

HO HO

O O

P

O

O

N

Chemical Formula: C18H26NO8P3•+ Chemical Formula: C22 H35NO8P2•+ Exact Mass: 472.21 Exact Mass: 415.14 Chemical Formula: CH3O• Exact Mass: 31.02

Chemical Formula: C20H24O7 Exact Mass: 376.15

O

HO HO

O

N P

O

O

HO HO

O

O O

O

2

(B)

HO

O

O

Chemical Formula: C20H32 NO6P•+ Exact Mass: 413.20

O

Chemical Formula: C3H7O• Exact Mass: 59.05

Scheme 2: A: Conversion of phenolic and non-phenolic dimeric β-O-4’ lignin model compounds, 1 and 2, respectively, by lipoxygenase (LOX) and lipoxygenase-linoleic acid (LOX-LA) mediator system; conditions and detailed results are summarised in Table 1; B: Ketyl radical-based spin adducts using 5-diisopropoxy-phosphoryl-5methyl-1-pyrroline-N-oxide (DIPPMPO) as spin trapping reagent; and C: C-5 radical-based spin adducts using 5diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO) as spin trapping reagent. Given m/z-values in B and C represent indicative fragments observed in the GC-MS (EI)-based analysis.



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Table 1. Key results obtained in the LOX- and LOX-LA-mediated conversion of phenolic and non-phenolic dimeric β-O-4’ lignin model compounds, 1 and 2, respectively under standardised reaction conditions. entry

model

conditions a

compound

total conversion [%]

identified product (yield [%])

1 2 3

1 1 1

Air (blank) LOX, air LOX, air, LA

0 0 3

----3 (3.3), 4 (0.1), one unidentified (>0.1)

4 5 6

1 1 1

N2 (blank) LOX, N2 LOX, N2, LA

0 0 4

----3 (3.9), 4 (0.1)

7 8 9 10

2 2 2 2

Air (blank) LOX, air LOX, air, LA LOX (excess b), air, LA

0 0 0 3

------5 (1.2), 6 (0.6),two unidentified (0.8))

11 12 13 14

2 2 2 2

N2 LOX, N2 LOX, N2, LA LOX (excess b), N2, LA

0 0 0 1

------5 (0.5), 6 (0.3)

a: Reactions in 0.1 M carbonate buffer, pH 9, for 24 h at 30 °C, with LOX concentrations of 0.1 U per 1 µmol of substrate. b: LOX concentrations were increased to 10 U per 1 µmol of substrate.

LOX was able to oxidise both the phenolic and non-phenolic model compounds when used in the presence of LA as an oxidation mediator. Reactions occur under both normoxic and hypoxic conditions. Cleaner reactions have been observed under hypoxic conditions for both models. Based on literature reports regarding the stabilities of C-H bonds in the models used in this study,60,61 it was expected that LOX- and LOX-LA-mediated activation preferentially proceeds at the benzylic position present in 1 and 2 and eventually also at the phenolic groups in 1. An oxidative activation on the free phenolic moieties would give rise to oxidative coupling


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reactions. Low oligomeric structures such as tetramers resulting from direct radical coupling, or truly polymeric products of such oxidative polymerisation reactions are, however, not observable in the GC-MS-based analysis. A GPC analysis of the reaction mixtures has not been performed, given the overall low conversion under the chosen model study conditions. Isolation of the products and preparation of these products for the THF-based GPC-analysis would not have guaranteed that an absence of a signal indicative of higher molecular weights would actually indicate that they do not form during the reaction, since model studies can anyway have only an orientating character. It was thus decided to target the question of a potential polymerisation using a full lignin sample (vide infra). Activation in the benzylic position, would lead to identifiable oxidation products such as aryl ketones, in case no other reaction partners are offered. Careful comparative analyses revealed that incubation of dimeric models with the LOX-LA mediator system yields oxidised structures 3 and 5, (Scheme 2, Table 1), which can be traced back to an activation in benzylic position (Table 1, entries 3, 6, 10 and 14). Higher concentrations of LOX, and hence higher concentrations of activated mediator were needed, however, in case of the ‘blocked’ phenolic structure, hinting at significantly reduced kinetics for the observed oxidations in case of etherified phenols, i.e., structural motifs situated along the lignin chains. Interestingly, both lignin models, resembling lignin end groups and internal units, respectively, do not only undergo oxidation reactions at the benzylic position, but also in γposition to form aldehyde 6. Most interestingly, however, is the additionally observed oxidation of the aromatic ring carbons in both models: mass fragmentation analysis clearly indicates in case of both models 1 and 2, an oxidation of the G-type aromatic units, i.e., of the blocked phenolic groups (products 4 and 6 Scheme 2, Table 1).



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GC-MS analysis-based spin trapping experiments using 5-diisopropoxy-phosphoryl-5methyl-1-pyrroline-N-oxide (DIPPMPO)62,63 proofed useful in the past to identify various radical species.64,65 This technique was thus applied in this study to further understand the source of the observed oxidation of the benzyl alcohol, as well as the oxidation of the ring systems in the dimeric lignin model 2. In a slight variation of the experiments leading to the results presented in Table 1, DIPPMPO was added to the reaction mixture, and the isolated crude product mixtures were analysed by GC-MS. It was possible to identify on the basis of characteristic fragments a ketyl radical-based DIPPMPO-adduct as well as an adduct stemming from a C-5 radical (Scheme 2B). The latter C-5 adduct represents a precursor for the observed hydroxylation on the aromatics. The observed ketyl radical adduct, however, proofs the activation of the dimeric lignin model systems at the benzylic position under the chosen reaction conditions. It thus represents the fact that an unprecedented carbon-centred activation of lignins is possible using a LOX-based system.

LOX-activity on Norway spruce milled wood lignin In order to understand whether there is a fundamental interaction between lignins and LOX, or whether the LOX-LA mediator system is needed for an effective interaction between lignin and LOX, structurally very well understood Norway spruce (NS) MWL was incubated under various conditions with LOX alone and the LOX-LA system . Results are presented in Table 2 in form of GPC and 31P NMR spectroscopy data. GPC-data non-ambiguously reveal that the lignin samples polymerized under all reaction conditions, both in the presence and in the absence of LA as mediator, in normoxic or hypoxic


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conditions. This polymerization is more pronounced in the presence of LA, a result that indicates that the lignin modification by LOX benefits from a mediator, especially under non-inert conditions (Table 2, entries 4, 5, 7 and 8). Under an inert nitrogen atmosphere, polymerization is generally less pronounced according to GPC-data (Table 2, entries 7 and 8); loss in phenolic hydroxyl groups is, however, similar to the aerobic case. It thus can be speculated that the polymerisation, in this case runs to a significantly larger extend via carbon-centred radicals that eventually react not only in immediate radical polymerisation reactions, but also lead to a depolymerisation under concomitant formation of new radicals which in turn can either polymerise or further decompose. The observed polymerisation to various extends can be interpreted, in this case, as a sign of an interrupted exo-depolymerisation.



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18 Table 2. Structural key data obtained for NS-MWL before and after treatment with lipoxygenase (LOX) (approx. 5000 U LOX per 100 mg lignin).

entry

conditions

mass return [%]

1

NS-MWL

---

2a

NS-MWL ‘washed’

3b 4b 5b 6c

7b 8b

95

LOX, air, 12 h

88

LOX, air, LA, 0.5 h

86

LOX, air, LA, 12 h

84

LOX, air, LA, 12 h

85

LOX, N2, LA, 0.5 h

88

LOX, N2, LA, 12 h

90

Mn [Da]

Mw [Da]

4400

24500

---

---

5300

27800

6800

27300

8100

28600

7900

27500

5100

24800

5800

24800

aliphatic OH [mmol/g]

condensed phenolic OH [mmol/g]

guaiacyl phenolic OH [mmol/g]

p-hydroxyphenyl phenolic OH [mmol/g]

0.62

0.15

0.27

0.04

0.59

0.16

0.26

0.04

0.19

0.22

0.01

0.04

0.26

0.11

0.04

0.03

0.18

0.11

0.04

0.01

0.20

0.12

0.03

0.02

0.30

0.08

0.04

0.02

0.21

0.10

0.06

0.03

a: NS-MWL suspended in carbonate buffer 0.1 M, pH 9 and re-isolated after 12 h. b: Reactions conditions: carbonate buffer 0.1 M, pH 9, for 12 h at 25 °C. c: Reactions conditions: borate buffer 0.1 M, pH 9, for 12 h at 25 °C.

Most interestingly is the observed polymerization in the absence of linoleic acid, indicating a direct activity of LOX towards NS-MWL (Table 2, entries 1,2 and 3). This finding is not directly explained by the model studies discussed above, that did not reveal any direct activity, nor any polymerisation. It can be assumed, however, that, under the model study conditions, with the enzyme amount kept purposefully low in order to identify the primary reaction steps, conversion in the absence of LA was too low to be detected. Nevertheless, also in this case, LOX-mediated lignin polymerization does not seem primarily, nor exclusively to proceed through oxidative coupling of aromatic rings. Significant changes are here observed only for the


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19

aliphatic hydroxyl content and, to a lesser degree, for guaiacyl-type phenolic hydroxyl groups. Apart from a prematurely interrupted exo-depolymerisation mechanism, also an endodepolymerisation mechanism could then be postulated, that, due to less favourable kinetics, is taking place as a background reaction. This background depolymerisation might become the dominating reaction as soon as the vast majority of end-standing, i.e., phenolic β-O-4’ units are consumed by the exo-depolymerisation freeing additional enzymatic catalyst activity for the kinetically more demanding ‘internal’ cleavages. A significant hydroxylation of the aromatic units along the lignin chains, as it could have been expected based on the results obtained in the model studies, has not been observed. A significant hydroxylation of the aromatic moieties would have led to a notable increase of G-type phenolic hydroxyl groups. Such an increase is not oberseved, nor an invariance of the abundance of the G-type phenolic hydrocyl groups, that would eventually represent a compensation of the loss of G-type phenolics due to polymerisation by a backbone hydroxylation. In order to gain more detailed mechanistic insight and to further support the lignin modelbased speculations with respect to the nature of the various radical species involved in the LOXmediated lignin activation, spin-trapping experiments were performed. The same protocol used for the mechanistic investigation of LOX-activity on the model substrate LA was chosen, thus using also here as spin-trapping reagent DIPPMPO.62,63 The various spin-trapping experiments reveal generally a rather complex shift pattern when NS-MWL is incubated with LOX without the mediator. Radical species were basically identified in form of their DIPPMPO adducts on the basis of reported characteristic chemical shifts:66–69 i) C-5-carbon radicals (D, 27.1±0.2 ppm); ii) ketyl radicals (E, 26.8±0.3 ppm); iii) phenoxy radicals (F, 25.2±0.2 ppm); iv) benzyl radicals (G, 23.0±0.2 ppm); v) C-β-carbon radicals (H, 17.9±0.2 ppm); vi) peroxo-ketyl radicals (E,



Page 20 of 35

20

16.9±0.1 / 17.1±0.1 ppm) (Scheme 3). Radical occurences are summarised in Table 3; putative ways of their formation are shown in Scheme 3. ‘Monomeric’ species D was detected only once upon radical trapping after an extended incubation time, whereas all other species occurred in various relative amounts according to the reaction conditions. Detection of the crucial ketyl radical is generally more difficult due to the fact that the range of possible shifts has been reported to be relatively large, spanning over a 1 ppm range. Figure 2 shows a representative 31P NMR spectrum in which some of the discussed adducts are indicated next to shift regions reported before for radical species generated from lignin models.


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21 Table 3. Detailed results obtained in the DIPPMPO-based spin-trapping of radical species generated upon incubation of NS-MWL with LOX and the LOX-LA mediator system under various condistions; letter codes of spin adducts refer to the discussion in the main text, examples of mentioned species are depicted in Scheme 3.

entry

conditions a δlit [ppm] δis [ppm]

1

2

3 4 5 6

control LOX, LA, air, w/o NS-MWL, 12 h control LOX, LA, N2, w/o NS-MWL, 0.5 h

LOX, air, 12 h LOX, air, LA, 0.5 h LOX, air, LA, 12 h LOX, N2, LA, 0.5 h

D 27.1±0.2 n.n.

DIPPMPO spin adduct (rel. abundance [%]) b E F G 26.8±0.3 26.7±0.5

25.2±0.2 24.7±0.4

23.0±0.2 23.2±0.2

H

I

17.9±0.2 17.8±0.1

17.0±0.3 17.3±0.1

n.o.

n.o.

n.o.

n.o.

n.o.

3c

n.o.

n.o.

4d

n.o.

n.o.

n.o.

n.o. n.o. ~ 15 e n.o.

90 68 72 52

Lipoxygenase: unprecedented carbon-centred ... - ACS Publications

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