An Introduction to Model Compounds of Lignin Linking Motifs; Synthesis and Selection Considerations for Reactivity Studies

Abstract The development of fundamentally new valorization strategies for lignin plays a vital role in unlocking the true potential of lignocellulosic biomass as sustainable and economically compatible renewable carbon feedstock. In particular, new catalytic modification and depolymerization strategies are required. Progress in this field, past and future, relies for a large part on the application of synthetic model compounds that reduce the complexity of working with the lignin biopolymer. This aids the development of catalytic methodologies and in‐depth mechanistic studies and guides structural characterization studies in the lignin field. However, due to the volume of literature and the piecemeal publication of methodology, the choice of suitable lignin model compounds is far from straight forward, especially for those outside the field and lacking a background in organic synthesis. For example, in catalytic depolymerization studies, a balance between synthetic effort and fidelity compared to the actual lignin of interest needs to be found. In this Review, we provide a broad overview of the model compounds available to study the chemistry of the main native linking motifs typically found in lignins from woody biomass, the synthetic routes and effort required to access them, and discuss to what extent these represent actual lignin structures. This overview can aid researchers in their selection of the most suitable lignin model systems for the development of emerging lignin modification and depolymerization technologies, maximizing their chances of successfully developing novel lignin valorization strategies.


Introduction to Lignin
To make our chemical industry sustainable, renewable carbon resources that can be applieda ss ubstitutes for finite fossil ones are required. The most abundant renewable source of carbon globally,a part from CO 2 ,i sl ignocellulosic biomass, which includes wood and agricultural residues.T hese materials have therefore been identified as potentialr enewable substitutes for fossil resources. [1,2] There have been many new developments for the conversion of lignocellulosic materials towards chemical products and fuels. However, most of these, such as second-generation bioethanol or furanics, extract value solely from the carbohydrate component, with the lignin component being treated as an undesired residue. Similarly,t he more established paper industry focuseso nh igh-quality cellulose, which inherently leads to the generation of al arge volumeo fl ow-value lignin as ab y-product. Apart for some niche applicationso fl ignosulfonate,t hese lignin residues are burned as al ow-value fuel, whichi su sed to generate process heat. However,f rom as ustainabilitya nd an economic perspective more efficient resource utilization would be desirable. [3] Therefore, value-extraction from the lignin fraction of lignocellulosicb iomass has become am ajor focus area. This includes the developmento fn ew fractionation methodsa sw ell as many elegant new catalytic methodologies for the depolymerization or modificationo fl ignin to generatee merging ligninderived chemicalp roducts. [4,5] Such efforts are essential for providing additional revenue streamsf or bio-refineries to boost their overall economic viability and competitiveness.T o generate value from the lignin biopolymer,i ts highly complex chemicalstructure needs to be understood and dealt with.
Approximately 450 million years ago, the first plants began to deposit lignin in their cell walls. This lignin evolved to play a key role in the defense of plants against pathogens and herbivores while also facilitating nutrient transportation anda cting as as upportive structure. This allowed for an increase in the size of plants and contributed to their dominance of the terrestrial environment. [6] The evolution of lignin biosynthesis has resulted in the formation of ah ighly complex, amorphous aromatic polymer consisting of phenylpropanoid subunits linked by ab road variety of CÀOa nd CÀCb onds. These originate, for the most part, from the combinatorial radical coupling of the monolignols: p-coumaryl alcohol (1), coniferyl alcohol (2), and sinapyla lcohol (3) ( Figure 1b ottomr ight). [3,7,8] These three main monolignols provide aromatic units with differentn umbers of methoxy substituents referredt oa sp-hydroxyphenyl (H), guaiacyl( G), and syringyl (S), respectively.T he coupling reactions lead to ac omplex network of which an illustrative chemicalr epresentation showing the major linking motifs discussedi nt his Reviewi sp rovided in Figure 1.
In planta,l ignin has ah ighly complex structure that varies significantly between plant species andd epends on plant age and numerous environmental factors. [9] When lignin is separated from the cellulosic and hemicellulosic fractions of plant biomass, the structurei nvariably becomes even more complexa s all isolation procedures induce chemicalm odifications in the structure. This complexity itself poses significant analytical challenges that are furthere xacerbated by lignin's high molecular weight. The primary strategy to mitigate these difficulties The development of fundamentally new valorization strategies for lignin plays av ital role in unlocking the true potential of lignocellulosic biomass as sustainable and economically compatible renewable carbon feedstock. In particular, new catalytic modification and depolymerizations trategies are required. Progress in this field, past and future,r elies for al arge part on the application of synthetic model compounds that reduce the complexity of workingw ith the lignin biopolymer.T his aids the development of catalytic methodologies and in-depth mechanistic studies and guides structuralc haracterization studies in the lignin field. However, due to the volumeo fl iterature and the piecemeal publication of methodology,t he choice of suitable lignin model compounds is far from straight forward, especially for those outside the fielda nd lacking a background in organic synthesis. For example, in catalytic depolymerization studies, ab alance between synthetic effort and fidelity compared to the actual lignin of interest needs to be found. In this Review,w ep rovide ab road overview of the model compounds availablet os tudy the chemistryo ft he main native linking motifs typically found in lignins from woody biomass, the synthetic routesa nd effort required to access them, and discuss to what extent these represent actual lignin structures. This overview can aid researchers in their selection of the most suitable lignin modelsystems for the development of emerging lignin modification and depolymerization technologies, maximizing their chances of successfully developing novel lignin valorization strategies.
is by the use of model systems to study the lignin structure and reactivity.T hese model systems have been extensively developeda nd used, rangingf rom monoaromatic compounds to diaromatic linking motif modelc ompounds, oligomeric model systems, up to fully synthetic dehydrogenation polymer (DHP) lignins. Although the selection of an appropriate model compound can be very important for the success or failure of a study,a nd its translation to real lignin chemistry is often ad ifficult choice as the literature on the topic is scattered and no comprehensive comparison of synthetic methods to access the compounds exists. This Review is aimed at providing this much neededo verview by covering the types of native lignin linking motif model systems that have been developed and providing ad iscussiono nt he different synthetic methodologies that can be used to access them.
Many studies make use of phenol,a nisole, or guaiacola s model compounds representing just the oxygenated aromatic motif. These model compounds can be useful when considering, for example, catalystd evelopment for hydrodeoxygenation studies, where removal of aromatic substituents is the limiting step. [10][11][12][13][14][15] This Review,h owever,f ocuseso nt he study of lignin linking motif models and so will not be addressing the use of monomeric models. Thus,t his overview will start with dimericm odel compounds that contain one linking motif and is sectioned according to the type of motif. Furthero n, larger models tructures bearing multiple linking motifs are also discussed. Finally,s ome general guidelines and considerations are provided for the selectiono ft he right modelc ompound for the type of research being undertaken, balancing the synthetic effort required against the fidelity of the model compounds. This should ultimately facilitate research studies to have the maximum impact in the fieldo fl ignin research.

Model compound naming
To follow discussions on lignin linking motifs and respective model compounds, it is important to understand the associated nomenclature. For the basic phenylpropanoid units that form lignin, shown in Figure 1, the carbon atoms in the aromatic rings are numbered 1-6, startinga tt he carbon atom attached to the propyl chain. The propyl chain is then most commonly numbered using the Greek letters a, b,a nd g,s tarting at the carbon atom next to the aromatic ring, or,a lternatively, by continuing the numerical sequence 7, 8, and 9( the former will be used throughout this Review). Extending this to linking motifs, in most cases, the nomenclatureu sed describes the bond formed during the key radical-radical coupling step [16] but not the subsequentb ondsf ormed during trappingo ft he resultingq uinone methides. Thus, the b-O-4' motif can be understoodt oc onnect the b carbon atom of one propylc hain to an oxygen atom at the 4p osition of another aromatic unit. The prime (')h ere denotes that the atom is from the second coupling unit;h owever,t his descriptor is often omitted (as it is for the remainder of this review). Similarly,t he terms b-5' and b-b' describet he motifs generated via coupling between the b-position on one unit and the 5-or b-positions on another unit. As these descriptors do not include all bonds formed during the couplingp rocess, they are inherently ambiguous; however, b-O-4' is usually used to describe arylglycerol-b-aryl ethers, b-5' for phenylcoumarans, and b-b' for resinols ( Figure 1). For composite linking motifs such as dibenzodioxocins and spirodienones that involve the connectiono fm ore than two phenylpropanoid units, the system outlined above becomes impractical, and so naming follows the type of ring structure that is formed. For example, the 8-membered ring of dibenzodioxocins contains 5-5, a-O-4, and b-O-4 bonds. Movingt om odel compounds these naming conventions are typicallyr etained, providing direct insight into the linking motif being modelled. It is important to note that the most commonly used namesf or the linking motifsa re described here;h owever,o ther names are sometimes used in literature.

General applicationofl ignin model compounds
Lignin model compounds are used for many reasons, but the primary ones being the study of structure and reactivityo f lignin on al evel of detail that is difficultt oa ttain using lignin itself given its complexity and high molecular weight. Whilst there are clear benefits to using low molecular weightm odel compounds, there are also limitations, as summarized below.
The benefits of the useofm odel compounds are: -s implification of the complexm ixtures of products obtained from depolymerization reactions for easeo fa nalysis -u se of avarietyo fm odel compounds of varying complexity allows development of adetailed understanding of the reaction mechanismsfor degradation or modification -t he fate of individual linking motifs can be studied in isolation or simple combinations -s tructuralfeatures formed via the modification of lignin linking motifs can be used to confirm the formation of new motifs in lignin The limitations of lignin modelcompounds are: -l ack of the full complexity and variations of the chemical structure in different lignins -d ifferent impurities than those found in isolated lignin streams -t he solubility constraints of isolated lignin polymers are not fully replicated -t he 3D environment created by the lignin polymer is not well represented -t he complexity of product streamsand possible separation technology required are not replicated There are many examples of the use of model compounds to study lignin. Recently,t he main focus for model compound use has become the development of novel catalytic conversion methodologies. Here, however,t he possibility frequently arises that ad egradation/modification system that works efficiently in am odel system may fail to be effective when applied to lignin. An example of this is the elegant hydrogen neutral Ru-Xantphos-catalyzed lignin CÀOc leavage methodology for the depolymerization of model b-O-4 motifs developed by Nichols et al. [17] This methodology performed excellently on the initially tested simple dimeric and even polymericl ignin b-O-4 model systems, which lacked g-carbinol groups, but upon application of the methodology to higher-fidelity b-O-4 model systems bearing a g-carbinolg roup, asf ound in lignin, the method provedi neffective. It was shown that the catalyst was deactivated via chelation of the Ru centerb yt he oxidized g-carbinol and the a-alcoholg roups, resulting in ac atalytically inactive acyl-enolate complex.T he g-carbinolg roup was not represented in the selected model compounds forthe initial study,highlightingt he importance of the lignin-model choice. [18] This also demonstrates that the better the model system can reflect the actual chemical structure of lignin, the more chance of successfult ranslation of the chemistry to real lignin. However,a s is discussed later,t his is balanced by the investments in time, effort, and expertise required to obtain the appropriate model compound.
Given the complexity of lignin, there is aw ide range of different model compounds that have been utilized to study its chemistry.A si nt he above example, studies most frequently employ modelso ft he b-O-4 linking motif as it is almostu niversally the most commonly occurring structural unit across native lignins in various differentt ypes of biomass (Table 1). For other linkages their abundance is significantly lower and more variable. Therefore, the b-O-4 linking motif is often selected for the development of new catalytic lignin depolymerization/modification methodologies. [19] Although it is the most obvious choice, it is important to note that the high abundance of b-O-4 linking motifs does not typicallyh old true for technical lignins as b-aryl-ethers can be significantly degraded during the fractionation process. This leads to the formation of am uch wider varietyo fd ifferent linking motifs that are often of the CÀCt ype and hard to degrade selectively. [3] Such an array of structures is typically hard to capturei nm odel compounds and therefore, the use of appropriate model compounds becomes more problematic. [20,21] Model compounds that represento ther native lignin linking motifs are often used to study the effect of chemical processing on the lignin structure as aw hole or for structurale lucidation purposes. [20,[22][23][24] In the remainder of this Review,t he types of model compounds and synthetic methodologies to access these are provided based on the most common native linking motifs provided in Figure 1a nd Ta ble1.A dditionally,f urther discussion on model compound selectioni sp rovided to conclude this Review. The b-O-4 linking motif is the mosta bundant linking motif in native lignin ( Figure 2) and is undoubtedly the most often replicated one in the literature. Consequently,awide variety of modelc ompounds, with differing levels of resemblance to the native b-O-4 motif in lignin, have been used to study this motifs'r eactivity.T he simplest b-O-4 Type Am odel is (2-phenoxyethyl)benzene,w here R 1 = R 2 = H, is often used as am odel compound as it is commercially available. [26][27][28][29][30][31][32] Variationso nb-O-4 Type Am odels with different substitution patterns on the aromatic rings can be readilys ynthesized via Williamson ether synthesis-type reactions using (2-bromoethyl)benzene derivatives containing the appropriate substituents on the aromatic ring with the desired phenol. [33] b-O-4 Type Am odels, however, lack both the a and g hydroxyl groups present in the native b-O-4 motif, which resultsi ns ignificantly different reactivity. Most studies have thus turned to b-O-4 Type Ba nd b-O-4 Type Cm odels, which incorporate the benzylic hydroxyl group at the a position. b-O-4 Type Aa nd b-O-4 Type Bm odelsc an be grouped as being C 6 -C 2 compounds (C 6 of the aromatic ring and the C 2 of the ethyl chain) and are distinct from the C 6 -C 3 b-O-4 Type Cc ompounds, which incorporatet he g-carbinol group (ÀCH 2 OH). b-O-4 Type Cc ompoundsa re the most representative models of the b-O-4 linking motif. Also note that the inclusion of the g carbon atom leads to the addition Table 1. Abundancies of some of the primary lignin linking motifs in softwoods, hardwoods, and grasses along with the monolignol ranges. Values quoted for lignin linking motifs are for abundance per 100 C 9 units.D ata taken from reviewa rticles. [3,25] LigninL inking motif [%] 5-5 [ b-O-4 Type Bm odel compounds are readily accessible in highy ield via the synthetic route shown in Scheme1a. Couplingo fa2-bromoacetophenone (4) with ap henol derivative( 5)u sing ab ase (typically K 2 CO 3 ,f or example in acetone) generates the ketoether intermediate 6,w hich is readily reduced using, typically, NaBH 4 to obtain the b-O-4 Type B model compounds. Where the desired bromoacetophenone startingm aterials are not commercially available, they can be accessed from the parenta cetophenone via bromination,f or example, by reacting with Br 2 in chloroform, ether,o re thanolf ollowed by purification by recrystallization. [34][35][36] The use of phenolic protecting groups such as benzyl (OBn) [34,35] or acetate [37][38][39] on the acetophenone prior to bromination allows access to phenolicm odels. In thesec ases, the conditions used for the brominations hould be chosen or modified accordingly;f or example, N 2 sparging( to remove HBr) can be beneficialw hen OBn groups are present [40] whereas acetate protecting groups preclude the use of alcoholic solvents. Syringyl-type acetophenones can be more challenging to selectively brominate than other analogues and therefore reagents such as CuBr 2 ,p yridine (Py)·Br 3 or 4-dimethylaminopyridine (DMAP)·Br 3 have been used as alternative brominating agents offerings uperior chemoselectivity. [41,42] Conveniently,c ompounds such as 6 and b-O-4 Type Bm odels tend to be crystalline solids allowing for straightforward purification by recrystallization, enabling large-laboratory-scale synthesis by anyone with basic chemistry training and equipment.  [46] b) Generalized route to accessing b-O-4 Type Cmodel compounds developed by Nakatsubo et al. [55] This route has been widely used and developed further by manyr esearchers. [45,50,58,80] An important consideration prior to discussing the synthesis of b-O-4 Type Cm odel compoundsi st hat these compounds contain two stereocenters, resulting in two diastereomers and four enantiomers of b-O-4 Type Cm odel exist. The two diastereomers, anti (alternatively termed erythro)and syn (alternatively termed threo), are shown in Figure 3. In native lignin the ratio between the diastereomers is controlled by the selectivity of the addition of water to the quinone methide duringt he lignification process. In general, this has been shown to yield a % 1:1r atio of diastereomers in softwood lignins and closert o % 3:1i nh ardwood lignins, with Su nits favoring the formation of anti isomers. [16,43] The synthesis of diastereomerically pure and mixtures of diastereomers of b-O-4 Type Cm odel compound have been developed(see below). Aselectivesynthetic route to enantiomerically pure b-O-4 Type Cm odel compounds has also been developed. This nine-step route (not discussed in detail here) involvingm ultiple protection/deprotection steps can be used to access the target compoundsi nm oderate-to-good yields. [44,45] Adler et al. and later also others developed am ethodology to access b-O-4 Type Cm odel compounds from the intermediate 6 (Scheme 1b,r eferred to as the Adler methodh enceforth). [46,47] This involves carrying out an aldol reaction between formaldehyde and 6 to generate 7,u sing K 2 CO 3 as ab ase. To day,1 ,4-dioxane is the most common solventf or performing this reaction and in our experience it is beneficial in limiting the formation of potential dehydration products. It should be noted, however,t hat the propensity of compounds to undergo dehydration appears to be highly substrate dependent.R ecently,n ew conditions have been reported using catalytic amountso fK OH in 1,4-dioxane/water giving improved yields with significantly reduced reactiont imes. [48] Deuteration of the b-position protons can be achieved by treating compounds such as 6 with K 2 CO 3 in D 2 O, subsequent aldol reactionw ith formaldehyde using an [D 6 ]acetone/EtOD solvent mixture resulted in a b-deuterated compound 7. [49,50] Such compounds can be very useful for mechanistic studies. The synthesis of b-O-4 Type Cm odel compounds is completed by reduction of the ketone group to give 7,t ypicallyu sing NaBH 4 .T he choice of reducing agent as well as solvents election during the reductions tep has been shown to affect the diastereomeric ratios of the resultant b-O-4 Type Cm odel compounds. The use of NaBH 4 in 50:50 H 2 O/methanolc an produce up to 86:20 syn/anti ratios while the use of iPrOH as solventp roduces 36:64 syn/anti. For the production of more anti-enriched products, LiAlH 4 in THF can be used to achieveu pt o2 5:75 syn/ anti. [51] Deuteriuml abeling of the a-position can be achieved by replacing NaBH 4 with NaBD 4 andu sing aT HF/D 2 Os olvent system during the reduction step. [49] Partly as ar esult of being mixtures of diastereomers, b-O-4 Type Cm odels compounds are typicallys omewhat harder to purify andh andle than b-O-4 Type Bm odelsa st hey are often obtained as sticky pastes or oils that occasionally crystallize on longtime standing after rigorous purification and drying.T he Adler methodology is particularly valuable in the synthesis of models bearing the G-G type substitution pattern for both phenolic and non-phenolic models. [52] The ready availability and low cost of the required startingm aterials and the fact that all intermediate compounds can be purified by recrystallization meansG -G, and to al esser extent G-S, b-O-4 Type Cm odelsc an be accessed on a multigram scale in am atter of days. Although less wells uited to the large-scale synthesis of S-H/G/S b-O-4 Type Cm odels, this methodology remains exceptionally valuablef or the synthesis of g-functionalized and more elaborate models. For example, g-acylated (e.g., p-hydroxybenzoate,c oumarate, ferulate, acetate) modelsa re commonly synthesized via this methoda sw ell as tricin-containing models. [53,54] Asecond commonly used route to b-O-4 Type Cmodel compounds was developed by Nakasubo et al. (henceforth the Nakasubo method), outlined in Scheme 1b. [55] Thisr oute involves the generation of an aryloxyesters uch as 9 from the reaction of ac hloro-or bromoacetate 8 (a potent lachrymator) with the desired phenol 5.T hisc an be achieved by reacting the two components in refluxing acetone with K 2 CO 3 ,g iving the desired ester in generally high-to-quantitativey ields without the need for purification. [56][57][58][59] Compounds of the type 9 are then reactedwith abenzaldehyde derivative 10 under aldol reaction conditions (À78 8C, lithium diisopropylamide, (LDA) in dry THF) to form the ester product 11.N otably,t his reaction can be carried out in one pot withoutn eedingt op reform the ester enolate, as is commonly practiced, [58] simplifying the reaction. G-G esters of type 11 can be purified by precipitation from diethyl ether in good yield;h owever,t his is less efficient with S-S-type esters, andc olumn chromatography is usually required to achieve good yields. Reduction of the ester in 11 gives access to b-O-4 Type Cm odel compounds; this can be achieved by using LiAlH 4 or NaBH 4 . [45,58] Di-g-deuterated b-O-4 Type C modelsc an access by using NaBD 4 (or LiAlD 4 )a st he reducing agent. [60,61] As with the Adler method, phenolicm odels can be accessed by the integration of ab enzyl-protected group on the appropriate position of the starting material. The benzyl group can be readily removedb yh ydrogenolysis under mild conditions (Pd/C,1atm H 2 ). The Nakasubo methodology produces b-O-4 Type Cm odel compound mixtures of diastereomers. Ester aldol reactions have at ransition state predetermined anti selectivity when the ester group employed is not sterically bulky [62] (approximately 5:1 anti/syn ratios is observed when ethylesters are used). [45] Ad evelopment of the Nakasubo method employing sterically bulky esters such as tert-butyl-aryloxyesters was able to overcome this transition state predetermined anti selectivity as the stericb ulk of the tert-butyl-esters made the transition state leading to the syn andt he anti products more equal, allowing for a1 :1 anti/syn ratio to be achieved with some substrates. [45] Prior to reduction, the anti/syn mixtureso f11 are often separable via column chromatography (this somewhat dependso nt he substitution pattern of the aromatic rings and the type of ester group used);i ndeed, Bolm and co-workersr eportedt he preparation of ar ange of diastereomerically pure b-O-4 Type Cm odel compounds by using tert-butyl-aryloxyesters and subsequent careful silica gel chromatography. [45] Alternatively,a nanti-enriched fraction of esters 11 can, in somec ases, be recrystallized to give ap ure anti product. Pure syn b-O-4 Type Cm odel compounds have also been prepared via the hydroboration of (Z)-a-(2-methoxyphenoxy),3,4-dimethoxycinnamic acid, although yields throughout this synthesis are unfortunately poor. [63,64] The syntheticr outes to b-O-4 Type Ba nd Cm odels outlined above cover the most frequently used methods;h owever, other less frequently used methodologies including, for example, an approach utilizingb romoketoesters as intermediates have also been developed. Details of theser outes can be found elsewhere. [64][65][66][67][68] The two main routes described here (Nakasubo and Adler methods) to access b-O-4 Type Cm odel compounds have both advantages and disadvantages and are thusu sed intermittently betweend ifferent research groups based on available equipment and materials, experience, and the type of desired substitution patterns on the aromatic rings. From ap ractical standpoint, the advantages of the Adler method are that it does not involvet he use of particularlya ir-or moisture-sensitive reagents and does not requiret he use of cryogenic temperatures as the Nakasubo methodd oes. Therefore, as omewhat better-equipped laboratory and am ore highly trained chemist is required to carry out the synthesis via the Nakasubo method. The Adler method also has the advantage of having a point of divergencei nt he sequence, allowing access to b-O-4 Type Bm odel compounds to which the Nakasubo method does not give access. The disadvantages of the Adler methoda re the lack of availability or prohibitive cost of the variousa cetophenone derivatives and the fact that the required bromination reactionc an be troublesome with some substrates. Starting-material availability is less of an issue with the Nakasubo method. The Nakasubo method produces good-to-excellent yields over aw ide varietyo f substrates with little substituent effect issues being encountered. In our hands the Adler methods is preferred for the synthesis of basic G-G b-O-4 Type C model compounds, while for other aromatic substitution patterns the Nakasubo methodi sp referred.

Modified b-O-4 modelc ompounds
The b-O-4 linkingm otif is often subjected to reaction conditions that result in alterations to its structure during lignin processing and is also frequently the target of selectivem odification strategies to either produce lignin with specific functionalities or that can facilitate depolymerization. Modificationp rotocols for the model systemsd escribed above have been developed to assist in the study of these modified ligninsa nd to facilitater eactivity and depolymerization studies. Below,w ewill discussafew such examples that give modified modelc ompoundsi nh igh yield.
Duringe xtraction procedures aimed at retaining the core b-O-4 linking motif structure, protective modification is often carried out. Under acidic conditions in alcohol solvents the hydroxy group at the a-position of the b-O-4 linking motif is readilyc onverted to its corresponding ether,S cheme 2a, sometimes noted as b-O-4-aOR (see Figure 1) Model compounds with such am odification to the b-O-4 linking motif structure of both the b-O-4 Type Ba nd Cc an be accessed via reactionu nder acidic conditions (cat. HCl) in the desired alcoholo r% 1:1m ixtures of 1,4-dioxane and the desired alcohol at mild temperatures (60-80 8C). Moderate-to-high yields of the a-alkoxylated product (65-84 %) can be obtained for linear alcohols, with ethanol, resulting in compound 12, and butanol being the most commonly used. [69][70][71][72] Am ore recently developed protective modification approachd eveloped by Luterbacher and co-workers, uses aldehydes to form a cyclic acetal with the 1,3-diol in the backboneo ft he b-O-4 linking motif.T his approachr educes undesirable reactions such as linkage cleavage and/orr epolymerization from occurring during lignin extraction. 1,3-Diol-protected model compounds can be accessed via reactiono fab-O-4 Type Cm odel with HCl and an aldehyde of choice in 1,4-dioxane as solvent at 80 8C, Scheme 2b. [73][74][75] Ac ommonly encountered modification of lignin that has been appliedt oc orresponding b-O-4 Type Cm odel compounds is acetylation,S cheme 2c.T his modification is usually carriedo ut to aid the solubility of lignin as it has been found that acetylation enhances lignin solubility in many organic sol-vents. [76] Commonly used acetylation procedures for both lignin and model compounds alike utilize acetic anhydridea nd an amine base (pyridine or 1-methylimidazole) reacting at room temperature for 16-24 ht op roduce the desired peracetylated products (e.g., 14)i nq uantitative/nearq uantitative yields. [23,24,77,78] An importantm odification technique primarilyt argeted towards lignin degradation and functionalization is selective oxidation.T his approach is based on an appreciation that oxidation of either the a or the g alcohols of the b-O-4 linking motif results in ad ecrease in the bond dissociation energy of the CÀOb ond in the motif by % 10 kcal mol À1 and opens up opportunities for new chemical transformations to be applied. This has resulted in alarge number of approaches being developed to achieve selective oxidation. [79] Accessing benzylically oxidized b-O-4 Type Cm odelsi sb y far the most explored area;i ndeed, compounds of general structure 7 (Scheme 1a)o btained as an intermediate during the Adler method gives direct accesst ob enzylically oxidized b-O-4 Type Cm odels. When starting from the b-O-4 Type C-1 model stoichiometrica pproaches utilizing2 ,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) [58] and 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivatives [80] are often also quite convenient to achieve selective benzylic oxidation (Scheme 3). More elegant and green catalytic versionso ft hese approaches that utilize molecular oxygen as the terminal oxidant have also been used. [58,80] Photocatalytic and mechanochemicala pproaches have also been developed for this transformation. [57,81] Am odification of the catalytic DDQ approachh as also been developed to facilitatet he benzylic oxidation of the a,g-diol-protected b-O-4 linking motif such as compound 13. [75] The b-O-4 Type C-1 model has also successfully been converted via primary oxidation to its aldehyde 16 using as elective TEMPO/(diacetoxyiodo)benzene( DAIB) approach [82] or to its carboxylic acid derivative 17 employing a4 -acetamido-TEMPO-mediated electrochemical procedure. [83] Alternatively, methods for the production of aldehyde 18 or carboxylic acid derivatives 19 of a-etherified b-O-4 Type Cm odels such as 12 have been developed. [70,72,84] The benzylic alkoxy group prevents degradation via ar etro-aldol pathway and thus improves the stability of 18 in particular.

b-5 Type model compounds
The b-5 linkingm otif is one of the primary linking motifs in lignin,m aking up 9-12 %o fh igh-G-content lignins. Due to the lower abundance of this linking motif comparedt ot he b-O-4 linkingm otif, the use of model compounds for studying its chemistry has been less well developed. Nevertheless, many examples of model compounds of the b-5 linking motifc an be found in the literature of varying levels of complexity and resemblance to the native structure as outlinedi nF igure 4. The relative stereochemistry of the b-5 linking motif hasb een shown to be trans ( Figure 4), with cis being presenti nn egligible quantities, if at all. [43] Ac omputational study utilizing model substrates was used to determine that this stereochemistry is derived fromt he ring-closing reactionf ollowing the radicald imerization which forms the b-5 bond. Thisr ing closing is believed to be under thermodynamic rather than kinetic control,a llowing the more stable trans relationship of the substituentst of orm. [85] Thus, typically, b-5 linking motif model compounds are synthesized and used in the trans form. 2,3-Dihydrobenzofurana nd its 2-methyl derivative (b-5-Type A) are the simplest model systemso ft he b-5 linkingm otif. This types of model compounds lack most of the functionality present in lignin but are cheap, commerciallya vailable compounds. Therefore, b-5-Type Am odel compounds are often utilized, even as general models to represent aromatic-aliphatic ether linking motifs found in lignin. [86,87] Twom ain approaches have been taken to achievet he synthesis of the more complex b-5 model compounds (b-5-Types B, C, and D). These are oxidative phenolc oupling (b-5 Method 1), utilizing either am etal (b-5 Method1a) or an enzyme( b-5 Method1b) to carry out the required single-electron oxidation or an acid-catalyzed rearrangement of chalcone epoxides (b-5 Method 2). Both approaches will be outlined in more detail below.
The type of models that can be obtained via oxidative phenolc oupling (b-5 Method1)d epend on the startingm aterial used. Models of the b-5-Type Bc an be synthesized via the radicald imerizationo fi soeugenol (20)( Scheme 4). First reported as early as the 1900s, this reaction has been developedi n subsequentd ecades and used by many researchers. [88][89][90][91][92][93] A common b-5 Method 1a approachi st ou se as ingle-electron oxidant such as FeCl 3 .A sa na lternative, ceric ammonium nitrate (CAN) has recently been reported to produce better yields (30 %y ield using FeCl 3 ,8 1% using CAN). [91,93] Enzymatic methodologies (b-5 Method1b) have been developed for this reaction, initially using oxygen-laccasee nzymes. [88,94] Subsequently,h orseradish peroxidase (HRP) enzymes have been found to be excellent catalysts for this transformation with yields of 99 %b eing achieved. [94][95][96] Methylation of the phenolic compound b-5 Type B-1 can be used to access its non-phenolic analogues in high yield via standard phenolm ethylation procedures. [93,97] Pd/C reductions of the alkene in b-5 Type B-1 or its methylated derivativeu nder H 2 can give access to their propylc hain-containing analogues (Figure 4, b-5 Type B modelsR 3 = propyl). [98] An advantage of b-5 Type Bm odel compounds is that they can be accessed in just af ew steps, with each one giving good-to-excellent yields. There are, however, significant drawbacks to the use of b-5 Type Bm odels. The lack of any functionality on the g-carbon atom of the b-5 core leads to significantly different reactivity compared to the native b-5 linking motif. In this respect, b-5 Type C( containing esters) and b-5 Type D( containing the native hydroxyl) model compounds are an improvement as they incorporate functionalitya tt he g position.
Ferulate ester dimerization gives access to b-5 Type Cm odel compounds that contain additional ester groups at the g-positions when compared with the b-5 Type Bm odels (Scheme 5). The approacht ot he synthesis of these compounds is similar to that of the isoeugenold imers described above,w ith both b-5 Method 1a,( chemical) [85,[99][100][101] and b-5 Method 1b (enzymatic) [102][103][104][105] dimerization procedures being employed. Scheme4.Chemical (b-5 Method 1a)a nd enzymatic(b-5 Method 1b)a pproaches to the synthesis of the b-5-linked isoeugenold imer b-5 Type B-1. [93,95] ChemSusChem 2020, 13,4238 -4265 www.chemsuschem.org The best resultsi na ccessing model compounds of the b-5 Type C-1 is b-5 Method1au sing Ag 2 O. Reactions carriedo ut in am ixed acetone-benzene solvents ystem produce yields of 40 %. [106] Other,m ore practical,s olvents such as DCM have been employed giving similary ields. [85] In our experience, the best yields from this reaction, which mustb ec arried out in the absence of light, are obtained with very dry and degassed solvents, making it ac hallenge to scale up. Enzymatic dimerization (b-5 Method1b) using HRP hasp roved quite easily scalable as it is carriedo ut in aqueous conditions and has been used frequently to carry out this conversion with yields in the range of 30-50 %. [103][104][105]107] From ap ractical perspective, b-5 Method1bh as significant advantages over b-5 Method 1a; that is, less use of organic solvents, no necessity to go through extensive drying and degassing procedures, the generation of less waste, and ease of scale-up lead to ap reference for the use of this method. Access to b-5 models with S-S substitution patterns is not possible due to the lack of af ree 5-position on the aromatic ring. However, b-5 Type Cm odels with H-H substitutionp atterns have been accessed via the general methods b-5 Method 1a [108,109] and b-5 Method1b [110] using methyl p-hydroxycinnamate as starting material. The synthesis of mixed G-Sm odelsh as also been accomplished using, for example, b-5 Method1ba nd am ixture of methyl ferulate (21)a nd methyl sinapate. This method, however,s uffers from poor yields of the desired product (24 %) due to the competing consumption of the starting materials in homodimerization reactions. [111] As with b-5 Type Bm odels, b-5 Type Cm odels can be methylatedt oa ccess their non-phenolic analoguesu sing methyl iodide and ab ase;h owever, b-5 Type Cc ompounds are susceptible to ring-opening reactions under basic conditions. [56] In both phenolica nd non-phenolic models, the doubleb ond can be readily reduced under standard conditions with Pd/C. [100] Access to b-5 Type D-1 and b-5 Type D-2 models can be ach-ieved via LiAlH 4 or diisobutylaluminium hydride (DIBAL-H) reductiono ft he ester groups of the appropriate b-5 Type C models. [100,112] An alternative route to these b-5 Type Dm odels is to startf rom coniferyl alcohol, which can also undergo b-5 Method1ad imerization with Ag 2 Ot og ive compound b-5 Type D-1 directly (Scheme 6) in up to 50 %y ield. Hydrogenation of the double bond then gives access to compound b-5 Type D-2. [24,113] Coniferyl alcohol is much more expensive than ferulic acida nd therefore the ferulate-based methods are usually preferred, especiallyf or larger-scale preparations.
The presenceo ff unctionalg roups on the propyl sidechain of the 2,3-dihydrobenzofuran ring of the b-5 linking motif can be of great significance in their usefulness as model substrates. An example of this can be seen in the use of the non-phenolic derivativeo ft he b-5 Type D-2 model.I nt he study of lignin acidolysis, the side chain provedt ob ei nert to the reaction conditions and so the study of the reactivity of the b-5 core was not complicated by side reactions. [56] However,i nt he study of lignin oxidation, the sidechain proved to be reactive under the conditions being studied, complicating the studyo f the b-5 core. [114] This highlights the importance of choosing the correct model system and giving due consideration to side chainsa nd their potentialasc omplicating factors.
An alternative route (b-5 Method 2) for the synthesis of b-5 Type Dl ignin model compounds where R 3 = Hh as been reported in the literature and is shown in Scheme 7. This methodology has the advantage of being ablet op rovide access to b-5 Type D-3 model compounds, which have no side chain on the 2,3-dihydrobenzofuran ring. [115,116] This route was initially reported by Brunowa nd Lundquist [115] and was subsequently furtherd eveloped. [116] AC laisen-Schmidt condensation between an acetophenone derivative 21 and ap henolic benzaldehyde 22 is used to form the intermediate 23.T he phenol group in 23 is then protected prior to epoxidation to the chalcone epoxide 24.L ewis acid-catalyzedr earrangement of the chalconee poxidel eads to ad iastereomeric mixture of 25 anti and 25 cis. Treatment of 25 with HCl, forms the desired trans b-5 model compound as the major product. The syn product is also formed but only in small quantities ( % 2%). This is aversatile methodology that can also be appliedt ot he synthesis of the phenolica nalogue of b-5 Type D-3;h owever,t his approach is rarely used due to the number of synthetic steps involved when compared to the single-step dimerizationp rocedure discussed previously in this section. [117]

b-b-Type model compounds
The b-b linking motif ( Figure 5) is unusual as during lignification in planta it can only form via monolignol dimerizationr eactions rather than through chain elongation. As shown in Ta ble 1, the b-b linking motif is found to make up between 3-12 %o ft he linking motifs in lignin. As eries of model compounds that is used to study this linking motif is shown in Figure 5a nd these compounds are obtained either synthetically or by extraction from naturals ources.
Several approaches have been taken to synthesize the core unit of the b-b linking motif as this type of compounds is also of interest for its potentialb iological properties, including antitumor, antiviral, immunosuppressant, and anti-inflammatory effects. [118] However,t ypically,m any of these syntheses focus on obtaining isomers of the core unit with differentc onfiguration Scheme7.Synthetic route towards b-5 type model compounds(b-5 Method 2) developed by Lundquist and co-workers. [115,116]  of the benzylic carbon atoms compared to the native b-b lignin unit shown in Figure 5. [16,119] Therefore, only model compounds with the matching stereochemistry are discussed. Nonetheless, treatment of lignin during the extraction process or during degradationp rocedures (acidolysis for example) can result in the epimerizationo ft he benzylic carbon atoms of the b-b linking motif, resulting in different relative configurations. [56] Sesamin (26)i so ften au seful model compound for studying the softwood b-b linkingm otif as it is found in sesame oil in 0.1-0.5 wt %a nd can be readily isolated through,f or example, columnc hromatography. [56,[120][121][122] The downside of sesamin as am odel compound is that it contains am ethylenedioxy group on both aromatic rings:amotif not found in native lignin. Eudesmin( 27)a nd yangambin (28)c an be considereda sG -G (softwood) and S-S (hardwood) "internal" modelsw here the phenolsa re connected to the rest of the lignin polymer chain. Chemical biomimeticd imerization is ap opular approach to the formation of the b-b linking motif as it allows for the construction of the complex core in as imple one-step reaction. An interesting but not very practical synthesis reported in 1982, starting from ferulic acid (31), utilized ad imerization reaction using iron(III) chloridet op roduce the dilactone 32 (Scheme 8). [123] This dilactone could be methylated to produce 33 or acetylated to produce 34.L iAlH 4 reduction of 33 and 34 gave the tetraols 35 and 36,r espectively.A cidic treatment of these tetraol compounds yielded the desired eudesmin (43 % yield) from compound 33 and pinoresinol (24 %y ield) from compound 34.This synthesis strategy is relatively long and suffers from an extremely low-yielding initial dimerizationstep.
Dimerizations tartingf rom coniferyl or sinapyl alcohol as opposed to their carboxylic acidd erivatives was initially investigated in the 1950s by Freudenberg and Hübner [124] and has since been further developed. [125] This approachs implifies the route as the resinol structure is formed directly and so access to the desired phenolic pinoresinol/syringoresinol structures is achieved in one step. The reactions are, however,l ow yielding when pinoresinols tructures are targeted.Asimple methylation step can be employed to access the eudesmin/yangambin structures (Scheme 9). [126] The synthesis of b-b linking motif model compounds containing syringyl-type aromatic groups are higher yielding than those containing the guaiacyl ones due, in part, to the 5-position being "protected" against radical coupling reactions. Syringaresinol (30)c an be synthesized starting from sinapyl alcohol (3)c hemically using stoichiometric copper(II) sulfate in the presence of light and air with yields of 67 %b eing obtained following purification by crystallization. [127] Enzymatic dimerization can be carried out startingf rom 3 using al accase from Trametesv ersicolor,g iving 93 %y ield, or from the substantially cheaper 2,6-dimethoxy-4-allylphenol (37)i naone-pot twoenzyme conversion (Scheme 10). [128] The latter route involves the conversiono f31 initially to sinapyl alcoholv ia an eugenol oxidase( EUGO), ar eactiont hat generates hydrogen peroxide; this hydrogen peroxide is then consumed by HRP in the dimerization of 3 to 30,g iving an 81 %y ield over the two steps. [129] Some alternative approaches that do not involved imerization have been used for the synthesis of resinol structures. [130,131] In the example showni nS cheme 11 am ethodology utilizing Si-based carbonyl ylides (38)i se mployed. The ylide reacts with an appropriate alkene 39 via a3+ 2c ycloaddition, yieldingc ompound 40,w hich contains the b-b linking motif core structure. They ield of 40 is, however,l ow at 18 %a nd is formed along with the other potentiali somers. [131] This low yield limits the widespread application of this methodology but potentially enables as ynthetic route to access specific asymmetrical b-b modelc ompounds.
The synthesis of dibenzodioxocin models is relatively underexplored compared to b-O-4, b-5, and b-b models. [133,[135][136][137] There are, however,t wo reported syntheticr outes that can be used to access these structures. The structures of dibenzodioxocin model compounds that can be accessed through these two routes are shown in Figure7.T he synthesis of both starts with the formationo fa5 -5 bond (Scheme 12). This first unit 42 is synthesized via the radical coupling of 41,w hich itself is synthesized from isoeugenol 20,mediated by K 3 Fe(CN) 6 . The route from the 5-5-linked dimer to the dibenzodioxocin model described in Scheme13u ses a2bromoacetophenone derivative( 43)t of orm the baryl ether 44.T he model with the g-carbinoli ncorporated (dibenzodioxocin-2) can be accessed via the use of the Adler method using formaldehyde with base to make b-O-4 Type Cl inking motifs, as shown in Scheme 1a. [46] In this case  followed by ab enzyld eprotection step gives the free phenol intermediates 46 and 47.Anintramolecular cyclization reaction is initiated using trimethylsilyl bromide (TMSBr) to form ab enzylic bromide. Aqueous NaHCO 3 then generates aq uinonem ethide to which the phenol adds to give the desired products. When this synthetic route is used to access dibenzodioxocin-1, it gives a5 0% yield. However,w hen accessing dibenzodioxocin-2, this route suffers from low yield (8%) due to the extremely low-yielding final ring-closing step. [135,138] There is probably still room for improvement with regard to this final step as previous work did not seem to have carriedout further optimization. Aphenol methylation procedure hasbeen developed for dibenzodioxocin-1 to study an etherified model of the dibenzodioxocin linking motif. [136] An oxidative coupling approacht of orm the key dibenzodioxicinr ing in dibenzodioxocin-2 has proved more successful (Scheme 14). In one step, 42 is oxidatively coupled with 2 using Ag 2 O, giving dibenzodioxocin-2 in reasonable 53 %y ield. The alternative HRP/hydrogen peroxide-mediated coupling, however,g ave only a3%y ield. Due to its lown umber of synthetic steps and relatively high yield, this final approachi st he Scheme12. Synthesis of the 5-5 linking motif core of the dibenzodioxocin model compound.
The 4-O-5 linking motif is not formed in the initial stages of the lignification process but ratherv ia the coupling of lignin oligomers or dimers. [23] As am inor motif it constitutes approximately 2% of the linking motifs in lignin. [3] Model compounds that have been used to study the 4-O-5 motif range from the widely used simple and commercially available diphenyl ether [140][141][142] to substituted diphenyl ethers synthesized via Cucatalyzed arylation of phenols (49)w ith aryl halides ( 48), [140,143] to products of radicald imerization reactions. [107,144,145] Examples of the models that have been used are shown in Figure 8. Linking motif models of the type 4-O-5 Type Ac ontain ether-linked aryl groups;h owever,t hese lack the aromatic substitution patterns seen in lignin.L inking motif modelso ft he type 4-O-5 Type Bw ith lignin-like substitution patterns on the aromatic rings offer ac loser match to the native 4-O-5 linkage.
4-O-5 Type Bm odel synthesis is generally carriedo ut via Ag 2 O-mediated or peroxidase-catalyzed radicald imerizationr eactions (Scheme 15). Selectivity towards 4-O-5-coupled products and the prevention of oligomer formation are the primary issues in these reactions. The direct oxidation of vanillin with Ag 2 Or esultsi nacomplex product mixture of oligomeric and polymericp roducts. [144] However,w hen vanillyl alcohol (50)i s used a4 -O-5 dimer is produced in which one of the alcohol groups is oxidized to the aldehyde. This is thought to occur via the formation of vanillin in situ, which then couples selectively at the 5-positon with the 4-O radicalo fv anillyla lcohol, producing the mixed dimer,4 -O-5 Type B-1, in 30 %y ield. [144] This product is particularly useful as it can be modified through subsequent reactions to produce further derivatives for analysis of naturall ignins. [22,144] Alternatively,4 -O-5 Type B-1 has been used as as tartingm aterial for the production of more complex models ystems (see Scheme19). Enzymatic strategies such as the use of peroxidase enzymesf or the 4-O-5 dimerization of 4-propyl guaiacol (51)h ave been reported but suffer from poor yields. The dimerization of 4-propyl guaiacol provides the 4-O-5 dimer as the minor component (8 %) in the product mixture whereas the 5-5-linked primary product 42 is obtained in a5 6% yield, as discussed in the previouss ection. [107] As imilar enzymatic dimerizationr eactionh as been reportedw ith ap henolic G-G b-O-4 Type C, giving only 2.9 %o f the 4-O-5 coupled dimer. [23]

Multi-Linking Motif Lignin Model Compounds
Numerous higher-order lignin model compounds have been synthesized, made up of combinations of differentl inking motifs. Here, such modelsa re classifieda sc ontaining between 2a nd (approximately) 7l inking motifs in ad efined order. These can be relatively well characterized but still consist of complex mixtures of stereoisomers. The nomenclature for these multi-linking motif compounds generally refers to the Scheme14. Radicaloxidative approachest ot he synthesiso ft he dibenzodioxocin model compoundv ia enzymatic and chemical means. [135,139] Scheme15. Exampleso fa pproaches taken towards the synthesis of 4-O-5 linking motif-typemodel compounds. number of aromatic units that are in the oligomer rather than the number of linking motifs. Twos ynthetic strategies can be distinguished: 1) stepwise addition of linkingm otifs and 2) oxidative coupling reactions, which are separately discussed below.
As imilara pproachi st os ynthesize dimeric or trimeric sequences of linking motifs that are then subjected to radicald imerization reactions. This has been used to generate tetramers [147,148] with three linking motifs-(b-O-4) (5-5) (b-O-4) and (b-5) (5-5) (b-5)-and hexamers [149] with 5l inking motifs- These two related approaches can be very successful as they allow the swift building up of multi-linking motif model compounds in moderate-to-good overall yields. The radicaln ature of the coupling reactioni nt he second approachi sadrawback as it limits the types of linking motifs that can be formed and also limits the functional group compatibility of the reaction.
Others have taken the approacho fs ynthesizing as pecific linkingm otif or as eries of linking motifs and then combining them in discrete non-dimerization reactions to generate the desired oligomeric product. Each linking motif is combined with af unctional group or masked functional group, which can be used in subsequent steps to build-up the desired oligomer.T his approach is somewhat more versatile as it does not necessarily result in symmetrical modelc ompounds.T his approach has been used to synthesize trimers [58,[150][151][152][153][154] [56] is shown in Scheme18i nwhich a( b-O-4) (b-5) model compound of general structure 58 is synthesized.T he approach essentially follows the b-5 Method1aa pproach of dimerizing 21 to generate the b-5 Type C-1 as outlined in Scheme 5. This was followed by methylation or tert-butyldimethylsilyle ther (TBS) phenol protection and an oxidative cleavage step to install an aldehyde group.T his allowed the application of the Nakasubo methodo fb-O-4 synthesis as outlined in Scheme 1b,f ollowed by reduction and, if necessary,T BS removal to give access to the desired (b-O-4) (b-5) model compound.

Enzymatic coupling
The other general methodo fm ulti-linking motif model compound synthesis is to use HRP to synthesis al arge number of products (including dimers and oligomers) in as ingle reaction. [22,23] The advantages of this approach are that it can generate alarge number of model compounds in asingle reaction, which often have highly realisticf eatures when compared with native lignin. The disadvantages are that the purification of each individual compound from the generated mixture is quite challenging, and the compounds are generally isolated in poor yield. Despite these drawbacks, the methodology has been used to remarkable effect in generating 4-O-5-and 5-5-linked model oligomers, primarily trimers, comprised of 5-5 or 4-O-5 model compounds linked with b-5, b-b,a nd b-O-4 linking motifs (Scheme 19, only b-5a nd b-b are shown). These model compounds were generated from the appropriate 4-O-5 (62) or 5-5 (63)c ontaining startingc ompounds that also contained a4 -hydroxycinnamyla lcoholm otif. These startingc ompounds can then undergo dehydrogenative coupling with (excess) 2 to generate ar ange of new linkingm otifs. Scheme 19 shows only as election of the most interesting products from these reactions. Also formed were b-O-4-, b-5-, and b-b-linked homodimerization products of 2.A ll of these products contributet o the complexity of the product mixture, complicating the isolation of products and contributing to the relativelyp oor yields. Nevertheless, compounds generated via this methodology provedi nvaluable in the detailed study of multi-linking motifs in native lignin, using 2D HSQC NMR techniquesa nd detailed studies of the lignin biosynthesis pathways. [22,23]

Biomimetic synthetic lignins
The synthesis of model lignin polymers takes the final step in the hierarchy of complexity in relation to the complex polymeric substance that is lignin. Work has been carried out to synthesize model lignin polymers using biomimetic approaches that attemptt or eplicate the stepwise combinatorial radicalc oupling of monolignols that occurs during lignification, so called dehydrogenative polymerization (DHP). Depending on the exact conditions used these model polymers can be highly realistic, with as imilar complexity to lignin in planta or isolated native lignin, including replicating the two-and threedimensionals tructure, an attribute that cannotb ea chieved by individual linking motif model compounds and is unlikely to be fully achieved by oligomeric models. Nevertheless, their complexity reintroduces some of the characterizationc hallenges presentf or plant-derived lignins. Additionally,t he nature of combinatorial coupling meansi ti si mpossible to accurately control the linking motif distribution.
The most common approacht op repareD HP lignins (or DHPs) involves using HRP and hydrogen peroxide to polymerize mixtures of monolignols in buffered solutions as am imic for the biosynthesis of lignin in nature. [155][156][157][158][159][160] The advantages of this approach are that it generates the desired complex polymeri no ne step, and it should,i nt heory,i ntegrate all the knownl ignin linking motifs. Twom ain methods for the polymerization exists the so-called "zulauf"a nd the "zutropf" methods. The zulauf method involves the bulk polymerization of monolignols and leads to an overabundance of dimerization products compared to naturall ignin. The zutropf method, on the other hand, involves the slow addition of monolignol and hydrogen peroxide solutions to HRP,f avoring ane nd-wise polymerization process, reducing the proportion of dimerization products. This results in higherm olecular weightD HPs compared to the zulauf method. [161] DHPs produced using either of these methods, however,h ave lower molecular weights than in planta lignin and so an extension of the zutropf method has been developed were ac ellulosicd ialysis tube containing the HRP is placed in af lask containing the hydrogen peroxide and monolignols olution.T he use of dialysis tubing isolatest he HRP and growing polymer molecules from the bulk of the mono-a nd oligolignols, resulting in ar elatively high concentration of polymer radicals, which thus favorsp olymer-monolignol over monolignol-monolignol couplingr eactions. This method allowsf or the production of DHPs with molecular weights more akin to that of native lignin. [162] DHP lignins have found extensive use in studyingb iological depolymerization processes, [163,164] particularly due to the ability to 14 C label them; [165][166][167][168] in verifying the ability of non-canonical monolignols to participate on lignification; [169][170][171][172] andi ns tudying selectivedepolymerization processes. [173,174]

Non-biomimeticsynthetic lignins
Non-biomimetic approaches have also been thoroughlyi nvestigated, resulting in numerous literature methodologies for the synthesis of many kinds of these model polymers. Early lignin model polymers often lacked some aspects of individual linking motif structures [175] while others consist entirely of as ingle linking motif, usually b-O-4. [176][177][178] Twog eneral approaches for complete b-O-4-based lignin model compounds are outlined in Scheme20. In (a) ab rominated polymer precursor is synthesized (68), whichc an polymerize under mildly basic conditions. Af inal reduction step allows access to an exclusively b-O-4containing model lignin polymer ( 69). [176] In (b) ab ifunctional polymerp recursor is prepared with one end containing an ester andt he other end an aldehyde (70). This compound can then be polymerized by treatment with lithium diisopropylamide, av ariation of the Nakasubo methodo fb-O-4 synthesis outlined in Scheme1b. Final reduction of this polymer yields an exclusively b-O-4-containing modell ignin polymer (71). [58,[177][178][179] In more recent years, this methodology has been further developed by Lancefield and Westwood [104] (Scheme 21) for the synthesis of model lignin polymers that contain b-O-4, b-b, b-5, and 5-5 motifs. This wasa chieved via the synthesis of linking motif models with functional groups,w hicha llow them to participate in an adapted Nakasubo methodo fb-O-4 synthesis. Following ar eduction step, am odel lignin polymer with complete compositionalc ontrol can be accessed, making them highly realistic models for lignin.

Concluding Considerations for Using Lignin Model Compounds for Reactivity Studies
Many of the above models have ag reat value in aiding lignin structurale lucidation by,f or example aiding in identifying signals in 2D-HSQC NMR analysiso rb yc omparison of depolyme-rization mixture with those from models ystems. This hasa llowed for the identification of the structuralm otifs within lignin and also of the bonds between lignin and other biomacromolecules, and new structures are still being identified and confirmed to this day. [22,23,[180][181][182][183][184][185] Here, the complexity of the lignin biopolymer needs to be sufficiently matched by the complexity of the model compounds to ensure ag ood signal overlap is observedi n2 D-HSQC-NMR spectra.T herefore,c omplexity is often desired despite the synthetic effort. [22,23] 2D-HSQC NMR spectroscopy is indeed the preferred methodt o analyze lignin as wella sl argers ynthetic modelc ompounds as it circumventsp roblems with signal overlap in conventional 1 H and 13 CNMR spectra and thus gives very detailed structural information. Also, when selectivec hemical modifications are performed, 2D-HSQC NMR spectroscopy is the preferred method of analysist oi dentify structural changes. [72,114,[186][187][188] Problems with the quantification of 2D-HSQC NMR spectra can also now be circumvented by the development of specific pulse sequences. [189][190][191] For more specific information on the size of extracted lignins as well as larger model compounds researchers turn to size-exclusion chromatography,a lthough the molecular weightd ata obtained highly dependd on the systema nd standards used in combination with the mobile phase. [104,192,193] Other techniques can be applieds uch as MALDI, [194] but also DOSY NMR. [179,195] Identification and quantification of specific functional groups in lignin such as ketones,a lcohols/phenols, and carboxylic acids such as titrationsc an be performed using titrations, [196] FTIR spectroscopy, [20] or specific reagents in combination with 31 Pa nd 19 FNMR spectroscopy [197,198] The application of smaller (typically dimeric) model compounds differs significantly from those of larger oligomeric and polymericm odel compounds. Already simple dimeric model compounds can be applied to excellent effect in the development of new catalytic methodologies for the breakdown of specific linking motifs,i dentify potentiald epolymerization products, or to elucidate the effect of chemical treatment on the lignin structure. This is due to the simplified structural characterization and quantification of reactionp roducts. Due to the simplerp roduct mixtures 1D NMR [24,72,80,114,187,199] andm ass spectroscopy methods such as LCMS or GCMSa re available that give much greater detail on the chemical structures. [56,188] Once reactionp roducts have been identified, GC-FID and HPLC but also 1 HNMR spectroscopy can be used to monitorr eactions over time to give detailedr eaction profiles and pathways. [50,56,73,187] Based on our experience in the field we provide some basic guidelines on what to consider when selecting model compounds for lignin reactivity studies. This should then allow one to select the appropriate model compound and how to access it using the overview provided in this Review.T he primary considerations are related to 1) the feedstock used and 2) the stage or depth of the study in relation to the synthetic effort required to access specific highly complexl ignin model compounds. These two consid-erations will be discussed below.T his sectionc onstitutes studies specifically involving lignin linking motifsf or which model compounds are described in this manuscript.

Lignocellulose/ligninfeedstock
The target lignin feedstock of as tudy can significantly influence the choice of model compound for reactivity studies. The plant origin of the lignocellulosicb iomass can guide model compound selection to represent the lignin structure. The origin determines the ratios of the various aromatic units (S/G/ H) as well as the types of linking motifs presenti nt he native lignin. These can differ significantly not only between botanical speciesb ut also between the segments of the plant chosen as well as the growth environment and stage of development. Therefore, determining these beforehand can be beneficial while more generic information such as provided in Ta ble 1o f this manuscript can be helpful. This can direct specific substitution patterns of the aromatic rings of the selected model compounds. For example, in as tudy targeting softwoods that contain almost exclusively G-type aromatics the corresponding Gbased lignin model compounds can be selected whereas for lignin originating from hardwood ands pecific grasses S-type aromatic substitution patterns might be ab etter match (Figure 9). Examples are b-O-4 type Cm odel compounds 76/77 and 78/79 representing such motifs.A lthough the b-O-4 motif is the most abundant in nearly all native plant material, its relative abundance varies and in some speciess uch as grasses gesterified b-O-4 motifsc an be relativelyabundant. The distribution of the linking motifs is often also relatedt oa romatics ubstitutionp attern due to the nature of lignin biosynthesis. [9] An- other factor to bear in mind is that the distribution of b-O-4 diastereomers in lignin is under chemical control.I ng eneral, this means for softwood G-type lignins the syn/anti ratio is % 1:1a nd in hardwood S-type lignins the ratio is closer to % 1:3, with Su nits favoring the formationo fanti isomers. [16,43] Also, typically, G-type ligninsa re relatively more abundant in b-5l inking motifs whereas S-type lignins are relatively more abundanti nb-b linking motifs. [200] If as tudy is focusing on the effect of ac hemical conversion methodology on the lignin structure, this can thus guide model-compound selection to structures 80/81 or 28/30.T here are specific analytical methods to determine the S/G/H ratio and relative abundanceo f linking motifs. For example, whole-cell NMR methods can be used to determine both but require millinga nd solvation of the source material and access to cryo-probe-equipped NMR instruments and/ors ignificantly extended NMR measurement times. [201,202] Alternatively FT-Raman, [203] FTIR spectroscopy [204] and pyr-GC [205][206][207] can be used to determine S/G/H ratios with propercalibration.
The considerations above are important when dealing with lignin as part of the biomass material, for example, when converting lignin as part of the lignocellulosem aterialb yf ractionation methodologies of the lignin-first type. [208][209][210][211] However, in many cases research is done on specific fractionated lignin streams. Here, the fractionation methodology can significantly alter the structure of the lignin extracted from the originalb iomass source. For example, specificf ractionso fl ignin are often extracted, which results in different H/G/S ratios comparedt o the native lignin material. This effect is often minor compared to the effect of the processing on the native linking motifs in lignin. Indeed, the b-O-4 linking motif is the most common motif found in natural lignin and is, therefore, the main focus of study.H owever,o ft he linking motifs discussed in this Review,i ti s, apart from the a-O-4, also the most labile. Thus, one should consider that the b-O-4 motif is also affected by most lignin extractions trategies. In technical ligninso btained from current large-scale lignocellulosic biorefineries such as the Kraft process used in the paper industry,t he amount of native b-O-4 linking motifs is typically low duet ot he harsh conditions leading to its breakdown and/orm odification. Other linking motifs, primarily CÀC-containing motifs obtained via recondensation processes, become mored ominant. [3,52,212] Therefore, one should appreciate that selectingt he b-O-4 linking motif as being representative of the primary linkingm otif in all lignins is not correct.I fo ne wishest os tudy effective catalytic methodology to depolymerize technical lignins using b-O-4 model compounds is not going to be helpful. [213,214] In such cases CÀC-linkeda romatic dimers with alkyl or 5-5 linking motifs might be more suitable. [215] Such models are not the focus of this Review as the actual identityo ft he linking motifs in such technical lignins is highly diverse and for al arge part unknown. [52] More analysiso ft echnical lignins is required to guide the synthesis of an ew generation of model compounds for use in the valorization of these recalcitrant lignins. For structural determinationo ft hese newly formed linking motifs under pulpingp rocess conditions, reactivity studies using model compounds representing the native lignin structure, as discussed in this Review; are highly beneficial. [24,52] Overall,t his means that analysis of the lignin materialb yf or example NMR or IR spectroscopy can be extremelyu seful to providei nsight into the structure of lignin and how relevant the use of model compounds representing the native linking motif content can be. [20,52]

Technology development levels
As econd consideration for the choice of model compound is at what stage of development the study currently is and what insight is being targeted. In relationt os tudies that involve chemicalr eactions on lignin, be they novel depolymerization pathways or specific modification methodologies, or studying structurale ffects from ab iomass processing and fractionation perspective, there is ab alance to be struck between the desired synthetice ffort and the depth of the insightt hat can be gained. Here, we would like to distinguish three levels of development that link to the appropriate type of model compound selection ( Figure 10). These levels are the result of our experience in studying lignin reactivity in relation to the catalytic breakdown of its structurea sw ella ss elective chemical modification. Increasing levels represent an increasei nt he synthetic effort required to access the appropriate model compoundsw hile each level results in different levels of information regardingl ignin reactivity and how this can be used to develop lignin conversion methodologies.
The first level is the development of novel (catalytic) conversion methodology.I nt his case, screening of different conditions and catalysti sl ikely the focus. Thus, high-throughput is often desired or at least as wift answer to whether lignin-like CÀOo rC ÀCb onds can be broken. Relativelys imple model compounds are likely more suitable in this case owing to the minimal synthetic effort required to access them. Additionally, relativelym ore straightforwarda nalysis is possible, and reactions are likely to proceed without the intrusion of many possible complicating side reactions. This can be especially useful when substrates and products can be readily analyzed by GC (especially GC-MS), which gives easy access to quantitative data to compare and assess development directions. An example of ar elatively easy to access model compound is the b-O-4 Type Am odel 82.I ns uch studies, the choice for ar elevant aromatic substitution pattern is typically also considered less critical as the focus is primarily on activation of the b-O-4 CÀO bond. [216][217][218][219][220][221] Alternatively,ab-O-4 Type Bm odel such as 83 can be used if more information on the reaction product is desired [42,[222][223][224][225][226][227][228][229][230] or if the breaking of the b-O-4 CÀOb ond is envisioned to be facilitated by the oxidation of the benzylic alcohol in the a-position. [17,[231][232][233][234][235][236] For the breaking of CÀOb onds in general,s ometimes even more simple commerciallya vailable modelc ompounds such as benzyl phenylether are used for initial catalystdevelopment. [237,238] The second level involves more detailed reactiond evelopment. Here, the actualf easibility of the intended reactiono n lignin,t argeting as pecific lignin chemical motif, can be tested. This can be accompanied by detailed mechanistici nsighta nd identification of major reaction products as well as potential side products that might arise from the reactionw hen applied to lignin. At this point in methodology development, it would be advisablet oh ave the appropriate lignin-linking motif fully represented, including all functional groups and preferably also to have the correct substitution patterns on the aromatic rings. Returning to the example regarding insight into the reactivity of the b-O-4 linking motif, for this stage,a na ppropriate model compound mayb eab-O-4 Type Cm odel such as 76.H ere, all b-O-4 linking motif functionalities found in native lignin are present.T his allows for the monitoring of the fate of all fragments released upon the application of ad eveloping methodology that leads to the breakdown of this motif. This will lead to aromaticp roducts with similar chemical structures to those expected from the breakdown of lignin. This typically comes from cleavage of the b-O-4 CÀOa ryl bond releasing a phenolic fragment, [222,239,240] but b-O-4 Type Cc an also offer insight into CaÀCb bond cleavage that would not be observed in b-O-4 Type Aa nd Type Bm odel compounds. [82,[241][242][243] Additionally,m odification of the lignin model compounds to represent the chemical modificationo fl ignin from as pecific source can be taken into account at this stage. For example, b-O-4 Type Cm odel compounds that have ak etone at the a-position such as 85 are readilya ccessible and represent the b-O-4 linking motif in lignin that has been selectively oxidized att he benzylic position. [58,69,80] Additionally,m odifications arise from fractionation, such as organosolv extraction with alcohols, which incorporates alkoxyg roups at the a-position.M odels for these modified structures,s uch as compounds 12 and 84,c an be accessed. [70,72,84] It is during this stage of reactiond evelopment that the effects of model compound stereochemistry may become apparent upon detailed analysis of reactions. Working with, for example, diastereomeric mixtures of b-O-4 Type Cm odelsi ti s, in principle, possible to observe reactivity differences between diastereomersi fanalysisofc hanges in the diastereomeric ratios during the course of reactions is carried out. Such findings mayp rompt more detailed mechanistic studies, in which case model compounds synthesized via the routes discussed previously giving diastereomerically or enantiomerically pure model compounds can be employed. Studies at this level thusa lready give very good insight into the reactivity of the specific linkingm otif and what type of products are expected.
The third level seeks to emulate lignin reactions and products but also avoid the complete heterogeneityo fl ignin itself. Such as tudy can focuso nt he phenolic nature of products obtained through CÀOb ond cleavagem ethodologies, using b-O-4T ype Cm odel compounds such as 77.L argerm odel structures of lignin,s uch as 58-HH,w hich combinem ultiple linking motifsi ns equence and be used to directly predict and identify products from reactions with real lignin. The focus herei st wofold, firstly to determine the fate of linkagem otifs other than those upon which the methodology was developed. Indications of the reactivity of previously unstudied linkagem otifs can be obtainedb yu sing some of the simpler model systems discussed previously;h owever,b yu sing am odel such as 58-HH the second objective can also be achieved, whichi sg etting reaction products that exactly match those present in a lignin depolymerization mixture such ap ossible dimeric structures obtained from the reactivity differenceso fvarious linking motifs. Here, for example, it can be shown how other linking motifsm ight affect the reactivity of neighboring linking motifs as well as how these linking motifs influence the applied cata-lyst. Additionally,o ligomeric modelc ompounds can be used for reproducing challenges of workingw ith lignin itself regarding solubility,p roduct recovery,a nd analysis. [22,23,50,56,58,243,244] In the case of research into the catalytic breakdown of lignin, there are limitations to the use of dimeric model compounds such as b-O-4 Type Cm odel compound 76 ( Figure 11). As lignin is ap olymer,c ompound 76 cannot give the exact reaction products that would be observed from the degradation of the lignin structure as the 4-O-position is methylated, therefore, typically,aphenolic compound such as b-O-4 Type C model compound 77 (Figure 11)w ould be required. If, for example,i nt he sequence of al ignin chain a b-O-4 linking motif is flanked by two more b-O-4 linkingm otifs and aC ÀOb ondcleavage methodology is appliedt ot his sequence, ultimately, exclusively phenolicp roducts will result.I nt his sense, CÀO bond cleavage of the b-O-4 linking motif of compound 77 results in the formation of compounds that can be an exact match with those that come from lignin itself, which is not the case for the non-phenolic compound 76 (Figure 11 A). It is important to note that, as lignin depolymerization is in most cases, unlikely to be an exclusively step-wise process involving phenolic end groups,c ompound 76 is still av aluablem odel for studying the reactivity of internal (non-phenolic) lignin units during depolymerization processes. An additional advantage of using phenolic models sucha s77 is that the stabilityo f the lignin degradationp roducts can be assessed under the conditions at which they will be formed during lignin depolymerization to ensure repolymerization is not occurring. If a CÀCb ond-cleavages trategy is being utilized, phenolic models are also useful in determiningt he exact products that will be derived from lignin, but this is only the case if the b and g carbon atoms following the cleavage of the linking motif are themselves removed to reveal ap henol,a si ss een in Figure 11 B. [82] If this is not the case, tetrameric b-O-4 linking motif model compounds would be required [151] or authentic standards of suspected products need to be synthesized for authentication andc alibration purposes. [243] For the identification and quantification of lignin depolymerization products derived from other linking motifs, the b-5 for example, trimeric b-O-4,b-5 model compounds such as 58-HH (Figure 11 C) can be used. [56] This is especially useful when dealingwith minor products in highly complex depolymerization mixtures. The use of larger oligomeric model systems are also of use in this respect and can also provide great insighta nd confirmation regarding the structures within lignin and also structural modificationsi n lignin. [22,23,58,114] Figure 11. Selection of model compounds and their relation to actual lignin depolymerization reaction products, contrasting phenolic and non-phenolic model compounds using methodology developedbya )Stahl and co-workers, [50] b) Bolm andco-workers, [56] and c) Barta andco-workers. [82] Studies at all of thesel evels provide different insight. However,c ombining information from all levels offers the most powerful approach. Initial reactivity testing with new precious catalytic material can likely better be performed at the first level at which the analysiso fs tartingm ateriala nd products are not the limiting factor.O nt he other hand, furtherd evelopment towards understanding actual reactivity on lignin requires research at the second level preferably combining results from model compounds representing different lignin linking motifs presenti nt he source material. Finally,i dentification of reaction products can be greatlyf acilitated by research at the third level, aiding product identification and simulation of the behavior of the oligomeric/polymeric material. This is not necessarily the order at which such studies should takep lace. Feedbackf rom each study as well as the studies on lignin itself might pose research questions that can be answered by studies at each of these three levels. Findingaright balance between model compound reactions at the right level andr elating model compound structures appropriatelyt ot he source materialo finterests hould as aw hole yield insight that can bring lignin valorization technology to the next level.