Molecular structure and composition elucidation of an industrial humin and its fractions.

Humins, (side-)products of the acid-catalysed dehydration of carbohydrates, will be produced in substantial quantities with the development of industrial biorefining processes. Most structural knowledge about such humins is based on synthetic model humins prepared at lab-scale from typical carbohydrate(-derived) compounds. Here, we report the first extensive characterisation study of an industrial humin. The soluble humin was generated from pilot plant-scale methanolic cyclodehydration of D-fructose to 5-methoxymethyl-2-furfural (MMF), as part of the Avantium YXY® process to produce FDCA. Purification of the industrial humin followed by fractionation allowed isolation of a water-insoluble, high molecular weight fraction (WIPIH) and a water-soluble, low-to-middle molecular weight soluble fraction (WES). Characterisation by elemental analysis, thermogravimetry, IR and NMR spectroscopy and size exclusion chromatography provided a detailed picture of the humin structure in both fractions. Aided by a comprehensive NMR spectral library of furanic model compounds, we identified the main furanic building blocks and inter-unit linkages and propose a structure for this industrial humin sample. The WIPIH and WES fractions were found to be composed of furanic rings interconnected by short aliphatic chains containing a wide range of functionalities including alcohols, ethers, carboxylic acids, esters, aldehydes and ketones. The low level of crosslinking and high functional group content of the industrial humin differ from the more extensively studied, (highly over-)condensed synthetic model humins, towards which they can be considered intermediates. The structural and compositional insights into the nature of an actual industrial humin open up a broad spectrum of valorisation opportunities.

CRISPR/Cas9 suppression of OsAT10, a rice BAHD acyltransferase, reduces p-coumaric acid incorporation into arabinoxylan without increasing saccharification.

Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 represents the major enzyme responsible for the addition of p-CA to arabinoxylan in rice plants. We found no change in the digestibility of rice husk lacking AX-associated p-CA, suggesting that the changes in digestibility seen in OsAT10 overexpressing plants were solely due to compensatory decreases in AX-associated FA.

Dynamic Trapping as a Selective Route to Renewable Phthalide from Biomass-Derived Furfuryl Alcohol.

A novel route for the production of the versatile chemical building block phthalide from biorenewable furfuryl alcohol and acrylate esters is presented. Two challenges that limit sustainable aromatics production via Diels-Alder (DA) aromatisation-an unfavourable equilibrium position and undesired regioselectivity when using asymmetric addends-were addressed using a dynamic kinetic trapping strategy. Activated acrylates were used to speed up the forward and reverse DA reactions, allowing for one of the four DA adducts to undergo a selective intramolecular lactonisation reaction in the presence of a weak base. The adduct is removed from the equilibrium pool, pulling the system completely to the product with a fixed, desired regiochemistry. A single 1,2-regioisomeric lactone product was formed in up to 86 % yield and the acrylate activating agent liberated for reuse. The lactone was aromatised to give phthalide in almost quantitative yield in the presence of Ac2 O and a catalytic amount of strong acid, or in 79 % using only catalytic acid.

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

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.

Copper-Mediated Conversion of Complex Ethers to Esters: Enabling Biopolymer Depolymerisation under Mild Conditions.

Selective processing of the ß-O-4 unit in lignin is essential for the efficient depolymerisation of this biopolymer and therefore its successful integration into a biorefinery set-up. An approach is described in which this unit is modified to incorporate a carboxylic ester with the goal of enabling the use of mild depolymerisation conditions. Inspired by preliminary results using a Cu/TEMPO/O2 system, a protocol was developed that gave the desired ß-O-4-containing ester in high yield using certain dimeric model compounds. The optimised reaction conditions were then applied to an oligomeric lignin model system. Extensive 2D NMR analysis demonstrated that analogous chemistry could be achieved with the oligomeric substrate. Mild depolymerisation of the ester-containing oligomer delivered the expected aryl acid monomer.

Linkage Abundance and Molecular Weight Characteristics of Technical Lignins by Attenuated Total Reflection-FTIR Spectroscopy Combined with Multivariate Analysis.

Lignin is an attractive material for the production of renewable chemicals, materials and energy. However, utilization is hampered by its highly complex and variable chemical structure, which requires an extensive suite of analytical instruments to characterize. Here, we demonstrate that straightforward attenuated total reflection (ATR)-FTIR analysis combined with principle component analysis (PCA) and partial least squares (PLS) modelling can provide remarkable insight into the structure of technical lignins, giving quantitative results that are comparable to standard gel-permeation chromatography (GPC) and 2D heteronuclear single quantum coherence (HSQC) NMR methods. First, a calibration set of 54 different technical (fractionated) lignin samples, covering kraft, soda and organosolv processes, were prepared and analyzed using traditional GPC and NMR methods, as well as by readily accessible ATR-FTIR spectroscopy. PLS models correlating the ATR-FTIR spectra of the broad set of lignins with GPC and NMR measurements were found to have excellent coefficients of determination (R2 Cal.>0.85) for molecular weight (Mn , Mw ) and inter-unit abundances (ß-O-4, ß-5 and ß-ß), with low relative errors (6.2-14 %) as estimated from cross-validation results. PLS analysis of a second set of 28 samples containing exclusively (fractionated) kraft lignins showed further improved prediction ability, with relative errors of 3.8-13 %, and the resulting model could predict the structural characteristics of an independent validation set of lignins with good accuracy. The results highlight the potential utility of this methodology for streamlining and expediting the often complex and time consuming technical lignin characterization process.

Selective Primary Alcohol Oxidation of Lignin Streams from Butanol-Pretreated Agricultural Waste Biomass.

Chemically modified lignins are important for the generation of biomass-derived materials and as precursors to renewable aromatic monomers. A butanol-based organosolv pretreatment has been used to convert an abundant agricultural waste product, rice husks, into a cellulose pulp and three additional product streams. One of these streams, a butanol-modified lignin, was oxidized at the γ position to give a carboxylic acid functionalized material. Subsequent coupling of the acid with aniline aided lignin characterization and served as an example of the flexibility of this approach for grafting side chains onto a lignin core structure. The pretreatment was scaled up for use on a multi-kilogram scale, a development that enabled the isolation of an anomeric mixture of butoxylated xylose in high purity. The robust and scalable butanosolv pretreatment has been developed further and demonstrates considerable potential for the processing of rice husks.

Identification of a diagnostic structural motif reveals a new reaction intermediate and condensation pathway in kraft lignin formation.

Kraft lignin, the main by-product of the pulping industry, is an abundant, yet highly underutilized renewable aromatic polymer. During kraft pulping, the lignin undergoes extensive structural modification, with many labile native bonds being replaced by new, more recalcitrant ones. Currently little is known about the nature of those bonds and linkages in kraft lignin, information that is essential for its efficient valorization to renewable fuels, materials or chemicals. Here, we provide detailed new insights into the structure of softwood kraft lignin, identifying and quantifying the major native as well as kraft pulping-derived units as a function of molecular weight. De novo synthetic kraft lignins, generated from (isotope labelled) dimeric and advanced polymeric models, provided key mechanistic understanding of kraft lignin formation, revealing different process dependent reaction pathways to be operating. The discovery of a novel kraft-derived lactone condensation product proved diagnostic for the identification of a previously unknown homovanillin based condensation pathway. The lactone marker is found in various different soft- and hardwood kraft lignins, suggesting the general pertinence of this new condensation mechanism for kraft pulping. These novel structural and mechanistic insights will aid the development of future biomass and lignin valorization technologies.

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.

Revealing the fate of the phenylcoumaran linkage during lignin oxidation reactions.

The fate of most lignin linkages, other than the ß-O-4, under selective oxidation conditions is largely unknown. In this work we use advanced ß-5 lignin model compounds to identify the fate of phenylcoumaran units in a softwood lignin during oxidation with DDQ. By using model compounds combined with detailed characterisation of the oxidised lignin polymer using HSQC and HMBC NMR we show that phenylcoumarones are a major product, and therefore constitute a novel non-native ß-5 linkage in oxidised lignins. Additionally, the reactivity of these units in lignin led us to further investigate their connectivity in lignin, showing that they are found as both phenolic and etherified units. The findings and approach developed here will help improve the efficiency of selective oxidative lignin depolymerisation processes, particularly those aimed at the upgrading of softwood lignin in which phenylcoumarans are a major linkage.

Bacterial Catabolism of ß-Hydroxypropiovanillone and ß-Hydroxypropiosyringone Produced in the Reductive Cleavage of Arylglycerol-ß-Aryl Ether in Lignin.

Sphingobium sp. strain SYK-6 converts four stereoisomers of arylglycerol-ß-guaiacyl ether into achiral ß-hydroxypropiovanillone (HPV) via three stereospecific reaction steps. Here, we determined the HPV catabolic pathway and characterized the HPV catabolic genes involved in the first two steps of the pathway. In SYK-6 cells, HPV was oxidized to vanilloyl acetic acid (VAA) via vanilloyl acetaldehyde (VAL). The resulting VAA was further converted into vanillate through the activation of VAA by coenzyme A. A syringyl-type HPV analog, ß-hydroxypropiosyringone (HPS), was also catabolized via the same pathway. SLG_12830 (hpvZ), which belongs to the glucose-methanol-choline oxidoreductase family, was isolated as the HPV-converting enzyme gene. An hpvZ mutant completely lost the ability to convert HPV and HPS, indicating that hpvZ is essential for the conversion of both the substrates. HpvZ produced in Escherichia coli oxidized both HPV and HPS and other 3-phenyl-1-propanol derivatives. HpvZ localized to both the cytoplasm and membrane of SYK-6 and used ubiquinone derivatives as electron acceptors. Thirteen gene products of the 23 aldehyde dehydrogenase (ALDH) genes in SYK-6 were able to oxidize VAL into VAA. Mutant analyses suggested that multiple ALDH genes, including SLG_20400, contribute to the conversion of VAL. We examined whether the genes encoding feruloyl-CoA synthetase (ferA) and feruloyl-CoA hydratase/lyase (ferB and ferB2) are involved in the conversion of VAA. Only FerA exhibited activity toward VAA; however, disruption of ferA did not affect VAA conversion. These results indicate that another enzyme system is involved in VAA conversion.IMPORTANCE Cleavage of the ß-aryl ether linkage is the most essential process in lignin biodegradation. Although the bacterial ß-aryl ether cleavage pathway and catabolic genes have been well documented, there have been no reports regarding the catabolism of HPV or HPS, the products of cleavage of ß-aryl ether compounds. HPV and HPS have also been found to be obtained from lignin by chemoselective catalytic oxidation by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of the ß-aryl ether with zinc. Therefore, value-added chemicals are expected to be produced from these compounds. In this study, we determined the SYK-6 catabolic pathways for HPV and HPS and identified the catabolic genes involved in the first two steps of the pathways. Since SYK-6 catabolizes HPV through 2-pyrone-4,6-dicarboxylate, which is a building block for functional polymers, characterization of HPV catabolism is important not only for understanding the bacterial lignin catabolic system but also for lignin utilization.

Fractionation and DOSY NMR as Analytical Tools: From Model Polymers to a Technical Lignin.

One key challenge hindering the valorization of lignin is its structural complexity. Artificial lignin-like materials provide a stepping stone between the simplicity of model compounds and the complexity of lignin. Here, we report an optimized synthesis of an all-G ß-O-4 polymer 1 designed to model softwood lignin. After acetylation, the polymer Ac-1(V) was fractionated using a protocol that involved only volatile organic solvents, which left no insoluble residue. Using diffusion ordered spectroscopy NMR in combination with gel permeation chromatography, it was revealed that this fractionated material behaved like a flexible linear polymer in solution (average α > 0.5). Acetylated kraft lignin was subsequently processed using the same fractionation protocol. By comparison with the model polymer, we propose that the acetylated kraft lignin is composed of two classes of materials that exhibit contrasting physical properties. One is comparable to the acetylated all-G ß-O-4 polymer Ac-1, and the second has a significantly different macromolecular structure.

The synthesis and analysis of lignin-bound Hibbert ketone structures in technical lignins.

Understanding the structure of technical lignins resulting from acid-catalysed treatment of lignocellulosic biomass is important for their future applications. Here we report an investigation into the fate of lignin under acidic aqueous organosolv conditions. In particular we examine in detail the formation and reactivity of non-native Hibbert ketone structures found in isolated organosolv lignins from both Douglas fir and beech woods. Through the use of model compounds combined with HSQC, HMBC and HSQC-TOCSY NMR experiments we demonstrate that, depending on the lignin source, both S and G lignin-bound Hibbert ketone units can be present. We also show that these units can serve as a source of novel mono-aromatic compounds following an additional lignin depolymerisation reaction.

Metal Triflates for the Production of Aromatics from Lignin.

The depolymerization of lignin into valuable aromatic chemicals is one of the key goals towards establishing economically viable biorefineries. In this contribution we present a simple approach for converting lignin to aromatic monomers in high yields under mild reaction conditions. The methodology relies on the use of catalytic amounts of easy-to-handle metal triflates (M(OTf)x ). Initially, we evaluated the reactivity of a broad range of metal triflates using simple lignin model compounds. More advanced lignin model compounds were also used to study the reactivity of different lignin linkages. The product aromatic monomers were either phenolic C2-acetals obtained by stabilization of the aldehyde cleavage products by reaction with ethylene glycol or methyl aromatics obtained by catalytic decarbonylation. Notably, when the method was ultimately tested on lignin, especially Fe(OTf)3 proved very effective and the phenolic C2-acetal products were obtained in an excellent, 19.3±3.2 wt % yield.

Total synthesis of dehaloperophoramidine using a highly diastereoselective Hosomi-Sakurai reaction.

The synthesis of dehaloperophoramidine, a non-halogenated derivative of the marine natural product perophoramidine, and its biological activity towards HCT116, HT29 and LoVo colorectal carcinoma cells is reported. A [3,3]-Claisen rearrangement and an epoxide opening/allylsilylation reaction installed the contiguous all-carbon quaternary stereocentres with the required relative stereochemistry.

Tandem Catalytic Depolymerization of Lignin by Water-Tolerant Lewis Acids and Rhodium Complexes.

Lignin is an attractive renewable feedstock for aromatic bulk and fine chemicals production, provided that suitable depolymerization procedures are developed. Here, we describe a tandem catalysis strategy for ether linkage cleavage within lignin, involving ether hydrolysis by water-tolerant Lewis acids followed by aldehyde decarbonylation by a Rh complex. In situ decarbonylation of the reactive aldehydes limits loss of monomers by recondensation, a major issue in acid-catalyzed lignin depolymerization. Rate of hydrolysis and decarbonylation were matched using lignin model compounds, allowing the method to be successfully applied to softwood, hardwood, and herbaceous dioxasolv lignins, as well as poplar sawdust, to give the anticipated decarbonylation products and, rather surprisingly, 4-(1-propenyl)phenols. Promisingly, product selectivity can be tuned by variation of the Lewis-acid strength and lignin source.

Advanced Model Compounds for Understanding Acid-Catalyzed Lignin Depolymerization: Identification of Renewable Aromatics and a Lignin-Derived Solvent.

The development of fundamentally new approaches for lignin depolymerization is challenged by the complexity of this aromatic biopolymer. While overly simplified model compounds often lack relevance to the chemistry of lignin, the direct use of lignin streams poses significant analytical challenges to methodology development. Ideally, new methods should be tested on model compounds that are complex enough to mirror the structural diversity in lignin but still of sufficiently low molecular weight to enable facile analysis. In this contribution, we present a new class of advanced (ß-O-4)-(ß-5) dilinkage models that are highly realistic representations of a lignin fragment. Together with selected ß-O-4, ß-5, and ß-ß structures, these compounds provide a detailed understanding of the reactivity of various types of lignin linkages in acid catalysis in conjunction with stabilization of reactive intermediates using ethylene glycol. The use of these new models has allowed for identification of novel reaction pathways and intermediates and led to the characterization of new dimeric products in subsequent lignin depolymerization studies. The excellent correlation between model and lignin experiments highlights the relevance of this new class of model compounds for broader use in catalysis studies. Only by understanding the reactivity of the linkages in lignin at this level of detail can fully optimized lignin depolymerization strategies be developed.

The use of residual dipolar coupling for conformational analysis of structurally related natural products.

Determining the conformational preferences of molecules in solution remains a considerable challenge. Recently, the use of residual dipolar coupling (RDC) analysis has emerged as a key method to address this. Whilst to date the majority of the applications have focused on biomolecules including proteins and DNA, the use of RDCs for studying small molecules is gaining popularity. Having said that, the method continues to develop, and here, we describe an early case study of the quantification of conformer populations in small molecules using RDC analysis. Having been inspired to study conformational preferences by unexpected differences in the NMR spectra and the reactivity of related natural products, we showed that the use of more established techniques was unsatisfactory in explaining the experimental observations. The use of RDCs provided an improved understanding that, following use of methods to quantify conformer populations using RDCs, culminated in a rationalisation of the contrasting diastereoselectivities observed in a ketone reduction reaction.

Isolation of functionalized phenolic monomers through selective oxidation and C-O bond cleavage of the ß-O-4 linkages in lignin.

Functionalized phenolic monomers have been generated and isolated from an organosolv lignin through a two-step depolymerization process. Chemoselective catalytic oxidation of ß-O-4 linkages promoted by the DDQ/tBuONO/O2 system was achieved in model compounds, including polymeric models and in real lignin. The oxidized ß-O-4 linkages were then cleaved on reaction with zinc. Compared to many existing methods, this protocol, which can be achieved in one pot, is highly selective, giving rise to a simple mixture of products that can be readily purified to give pure compounds. The functionality present in these products makes them potentially valuable building blocks.