Structural Basis for the Formation and Regulation of LigninâXylan

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Structural basis for the Formation and Regulation of Lignin-Xylan Bonds in Birch Nicola Giummarella, and Martin Lawoko ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00911 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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ACS Sustainable Chemistry & Engineering

Structural basis for the Formation and Regulation of Lignin-Xylan Bonds in Birch Nicola Giummarella and Martin Lawoko* Wallenberg Wood Science Center (WWSC), Department of Fiber and Polymer Technology, School of Chemistry, Royal Institute of Technology, KTH, Teknikringen 56, 100 44 Stockholm, Sweden. *Corresponding author: [email protected] KEYWORDS: Lignin Carbohydrate Complexes (LCCs), Mild Quantitative Fractionation, 2D HSQC NMR, Thioacidolysis-GC MS/31P NMR, Phenyl glycosides, Benzyl ethers, Gamma esters.

ABSTRACT

The covalent connectivity between lignin and polysaccharides forming the so-called lignin carbohydrate complexes (LCCs) is important to obtain fundamental knowledge on wood formation and may shed light on molecular aspects of wood processing. Although widely studied, unequivocal proofs of their existence in native state biomass still lack mainly due to harsh pre-analytical fractionation conditions that could cause artifacts. In the present study, we applied a mild protocol for quantitative LCCs fractionation and performed detailed structural

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studies using 2D HSQC NMR, 31P NMR and thioacidolysis in combination with GC MS and GC FID. The detailed structural analysis of LCCs including both lignin and carbohydrate skeleton unveiled insights into the role of molecular structure of xylan on the type of lignin carbohydrate (LC) bonds formed. More specifically, it is shown that xylan LCC differs in the degree of substitution of hydroxyl functionality on xylan skeleton by the presence of acetyl- or 4-Omethylglucuronic acid. The highly substituted xylan had a lower prevalence of phenyl glycosidic- and benzyl ether LC bond types than the lowly substituted. In addition, structural differences in the lignin part of LCCs are observed. Based on the results, it is suggested that acetylation on xylan regulates the type and frequency of LC bond.

INTRODUCTION One of the fundamental questions in wood chemistry is if covalent bonds between lignin and the carbohydrate, the so-called lignin carbohydrate complexes (LCCs) exist in native state. The critic is that the pre-analytical fractionation practices which have been considered to be harsh may cause serious structural modifications to the native polymers. This dogma can only be overcome if the fractionation is convincingly mild enough. Nevertheless, three main types of covalent lignin carbohydrate bonds have been suggested in wood; namely benzyl ethers, benzyl esters and phenylglycoside.1 The direct extraction of LCCs from wood by hot water at rather harsh conditions (autohydrolysis conditions at 140 °C), was first demonstrated by Traynard in 1953.2 Later Bjorkman,3 introduced lignin and LCCs isolation procedures from ball milled wood to obtain somewhat higher yields of both pure lignin and LCCs. The ball milling is to date the single most widely adopted starting point for preparation of both lignin and LCCs in close to native state for characterization. What differentiates several studies is the next step after ball


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milling. These include hot water extraction, alkaline extractions and organic solvent extractions. An excellent summary on developmental studies in LCCs fractionation and characterization until 1990s can be found in the book by Watanabe and Koshijima.4 After the 1990s, LCCs studies have taken a quantitative turn. The first quantitative fractionation from ball milled wood was reported by Lawoko and co-workers.5 This protocol however was only quantitative for softwoods, applied alkaline dissolution steps and involved several steps. Later, Du et al.6 reported a quantitative protocol for LCCs fractionation with fewer steps and involved the complete dissolution of ball milled wood in quaternary ammonium base with dimethylsulfoxide (DMSO) solvent. The protocol was universal (applicable to both softwoods and hardwoods). However, the basic condition has recently been shown to cause de-acetylation of the partially acetylated hydroxyl groups present on native hemicelluloses.7 It is thus likely that some deesterification of lignin carbohydrate esters bonds occurs. Wang et al.8 developed a solvent system LiCl/DMSO capable of directly dissolving ball milled wood. This dissolution protocol has been expanded to include precipitations steps followed by subsequent enzymatic hydrolyses to enrich LCC substrates.9 Although the last-named procedure is mild and provides useful information on LC linkages, when combined with classical 2D NMR spectroscopic analyses, detailed structure of individual LCC fractions, including both carbohydrate and lignin skeleton is lost. For this purpose, the fractionation of whole LCC at equally mild conditions would be beneficial. Traditionally, wet chemistry is applied for the analysis of LCC isolates, and in an excellent review Balakshin et al.9 summarize these techniques. To date 2D HSQC NMR is the best analytical tool for inter-unit linkages in LCC7,10,11 and lignin12,13 structures. However, due to the inability to detect some of the lignin inter-unit linkages, 2D NMR can be complimented with


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degradation techniques such as thioacidolysis14 or derivatization for reductive cleavage (DFRC).15 Though not quantitative due to incomplete degradation, the products can be studied by 31P NMR16 and/or GC MS, GC FID to obtain qualitative data. In the present work we apply a mild quantitative protocol for the isolation of LCCs from hardwood and use the abovementioned analytical techniques to unravel detailed structure of LCC in ball milled birch, which was found to be beneficial in understanding molecular aspects of Lignin Carbohydrates bond formation. MATERIALS AND METHODS Materials and chemicals All chemicals used were of analytical grade and purchased from Sigma Aldrich. LCCs fractionation LCC fractionation was performed as recently described by Giummarella et al.17 with slight modification in the final step. Briefly, 200 ml of deionized water was added to 20 g of ball milled acetone extracted birch wood and stirred at 80°C for 4 h. The insoluble residue was separated from the solution by centrifugation. The supernatant was fractionated using polyaromatic resin (Amberlite® XAD4) into a permeated fraction (Fraction 1-F1) and a retained fraction (Fraction 2-F2). The water insoluble residue was air-dried from acetone, then pre-swollen in [Amim]Cl and then stirred overnight at 70 °C after addition of DMSO. After cooling, deionized water was added (80:20 vDMSO/vWater) and the precipitate (Fraction 3-F3) was separated by centrifugation, washed three times and freeze dried. The first wash of F3 was added to the supernatant and then three times its volume of absolute ethanol was added and left to stand overnight at 4 °C. The formed precipitated (Fraction 4-F4) was separated by centrifugation,


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dialyzed and freeze dried. Finally, to the remaining solution, three times its volume of water was added and a precipitate (Fraction 5-F5) was obtained and treated in a similar way to fraction 4. The final step to obtain F5 deviated from the recently described procedure.17 Carbohydrate, 4-O-methyl glucuronic acid and lignin-quantitative analysis Carbohydrates were hydrolyzed to monosugars according to Effland18 and sugar analysis was carried on by HPAEC PAD as described by Davis.19 Klason lignin was determined as described by Effland18 and acid soluble lignin according to Tappi method 250.20 The content of 4-Omethyl glucuronic acid was determined by GC FID after methanolysis and subsequent acetylation. Erythritol was used as internal standard and 0.66 as detector response factor.21 The monomeric products of thioacidolysis were quantified by GC FID using tetracosane as internal standard subsequent to silylation. The Raney-nickel desulphurated products were analyzed by GC MS14 after acetylation. Phenolic content of LCCs fractions Dissolution of LCC samples in 1-allyl-3-methylimidazoliumchloride [Amim]Cl, derivatization to phosphite derivatives and subsequent 31P NMR were done according to the protocol used for dissolution of lignocelluloses samples.22 Thioacidolysis, Raney-nickel desulphuration and 31P NMR Thioacidolysis and desulphurization of thioacidolysis products was done according to literature.23 Quantitative

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P NMR analysis and signal assignments were done according to

previous work.24,25


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Size Exclusion Chromatography in DMSO+0.5% LiBr Size exclusion chromatography (SEC) of LCCs fractions was performed on a SEC-curity 1260 system (Polymer Standards Services, Mainz, Germany) coupled to a UV detector and a RI detector. Pullulan standards of 708 kDa, 344 kDa, 47.1 kDa, 21.1 kDa, 9.6 kDa, 6kDa, 1.08 kDa, 342 Da were used for standard calibration. Preparation of the samples, separation and experimental set-up was the same as reported by Duval et al.26 2D HSQC NMR analysis For 2D NMR, 100 mg of the initial LCCs fractions were dissolved in 750 µl of deuterated DMSO-d6 or CDCl3 in the case of acetylated samples derivatized at same condition reported by Ralph et al.27 NMR spectra were recorded and processed as described.17 The unsubstituted carbon 2 of aromatic groups was used as internal standard for quantification as elsewhere.28 The central DMSO (δC/δH=39.5/2.5 ppm) and chloroform (δC/δH=77.3/7.2) ppm were used as internal reference. RESULTS AND DISCUSSION The unequivocal proof for existence of native lignin carbohydrate bonds has been prevented by harsh pre-analytical fractionation conditions. A weakness in all “native” LCC studies, including the present work, is the necessity to perform some mechanical pretreatment (milling) which is a prerequisite for dissolution of the otherwise inaccessible woody structure. The effect of the ball milling on the native structure remains a concern requiring future attention. New methods that avoid milling are therefore an important focus of our future investigations.


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Protocol for LCC fractionation In the endeavor to develop a global protocol, we sought to improve on existing fractionation methodologies. Herein, a protocol for the mild quantitative fractionation of milled birch wood into fractions containing structurally different LCCs (Figure 1) is reported. It is worth noting that the usage of the word “mild” in this report is relative to the conditions used in previous reports. The protocol is global as we have demonstrated that it works for both softwood17 and herein hardwood, albeit a small difference; namely that in the final step to obtain F5 fraction, water is used as anti-solvent. The strength of the protocol, in regard to fractionation, lies in its mildness (pH-neutrality, low temperatures) and quantitative nature (Table 1, 93-97% total recovery, 90% lignin recovery). In addition, the bulk of the hemicelluloses are separated at an earlier stage (F1 and F2, Figure 1), which serves an analytical advantage; namely that it enhances the signals assigned to LC bonds in the obtained fractions, when subjected to NMR studies.

Figure 1. Fractionation scheme for lignin carbohydrate complexes (LCCs). BMW = Ball Milled Wood The residue after water extraction, which contains the bulk of the wood polymers, enriched in LCC and cellulose, is pre-swollen in an ionic liquid [Amim]Cl and subsequently dissolved completely with DMSO as co-solvent. A selective precipitation of various fractions (F3, F4 and F5) is achieved by sequential anti-solvent additions (Figure 1). The mass balance, lignin and


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sugar compositions are reported in Table 1. The bulk of material is in the F3 fraction. Regarding the carbohydrate composition, the F1 and F2 fractions are enriched in xylan, the F3 in glucan and the F4 in both xylan and glucan. Lastly the F5 was enriched in lignin; however the bulk of the lignin was in the F3 and F4 fraction, which together accounted for about 70% of the total lignin. The detailed values of Klason and acid soluble lignin content of the fractions are reported in Supplementary information (Table S1). To get some insight on whether lignin in the fractions was linked to the carbohydrate, 96% dioxane was used to extract the original ball milled wood (BMW) and the fractions F1-F5. This solvent system is known to extract lignin, classically referred to as milled wood lignin (MWL)3 or dioxane lignin. About 33% of the lignin in BMW was soluble according to UV measurement at 280 nm using an extinction coefficient ε=13.1.29 The insoluble lignin was probably linked to the carbohydrates in LCCs. In fact the crude dioxane lignin fraction also contains some LCCs.3 From the evaluation of the quantities of dioxane lignin in the fractions (Table 1, Column 2), it is suggested that a major portion of the lignin in each fraction is somewhere linked to carbohydrates.


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Table 1. Composition and mass balance of precipitated fractions. Lignin Mass balance

Content

4OM GA

Dioxane 96% soluble

Total*

% of wood in fr.

% of lignin fr. in soluble fr.

% of wood lignin in fr.

A

Gal

G

X

M

% on X

BMW

100

33

100

0.6

1.3

60.6

34.9

2.6

4.8

F1

10.5±1

9.5

4±0.5

1.0±0.1 2.9±0.2

9.7±0.3

79.2±0.1

7.3±0.4

2.3

F2

10±1

48.5

6 ± 0.5

1.7±0.1 3.1±0.1

12.3±0.7

75.3±2

7.6±1.1

1.8

F3

66±1

6

55.5 ± 2

0.5±0.2 0.8±0.1

77.6±2

19.4±2.6

1.6±0.2

4.1

F4

9±2

49.7

16.5 ± 2

0.8±0.1 2.0±0.2

41.3

52.4±0.5

3.5±0.2

4.9

F5

2

92

9 ±1

27.8

47.4

6.4

1.6

Sugar analysis by HPAEC/PAD %

8.3

10.1

A=Arabinose; Gal=Galactose; G=Glucose; X=Xylose; M=Mannose; 4OMGA=4-O-methyl Glucuronic Acid; fr. = fraction; *=Klason lignin + Acid soluble lignin.

Size Exclusion Chromatography of Fractions To further investigate lignin-carbohydrate connectivity, size exclusion chromatography (SEC) with a dual detector system consisting of a differential refractive index (DRI) and a UV detector set at 280 nm was applied. The traces are provided in Supplementary information (Figure S1). The DRI is a universal concentration detector whereas the UV measures the absorbance of lignin at the set wavelength. Using the known concentration of the carbohydrate and lignin in each fraction as a gauge, the elution profiles observed in the chromatograms can be used to deduce possible LC linkages. Superimposed DRI and UV signals either implies that the hydrodynamic


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volumes of the carbohydrates and lignin are equal or that the lignin is chemically linked to the carbohydrates. For the fractions F1 and F2, the major RI signal, representing a high concentration of solutes, was superimposed with a UV signal, yet lignin was a minor component in the fractions. Unless their hydrodynamic volumes are similar in unbound state, this indicates LC linkages. In the case of the F3, solubility was poor and the dissolved fraction contained lignin and xylan in equal amounts. The SEC detector signals, where the bulk eluted was superimposed, also indicating LC linkages. Similar observations were noted for the F4 fraction. The lignin-rich F5 fraction showed a unimodal distribution for both detectors and the signals were also superimposed but here LCC could not be deduced since the concentration of carbohydrates was low. Overall, the SEC analysis suggested xylan was an important carbohydrate in LCCs. Analysis of Molecular Structures The linkage analysis by 2D HSQC NMR was performed directly on the fractions and in some cases also on acetylated samples. The assignments of LC linkages were made according to the literature.7,10,30,31,32 The inter-monolignol linkages were assigned according to previous work12,13,33 and for quantification, the CH correlation of the C2 of the aromatic ring was used as internal standard in accordance with Sette et al.28 This position on the aromatic ring of milled wood lignins is not substituted hence representative of the number of aromatic rings in lignin. In the case of xylan structure, the degree of acetylation on carbon 2 and 3 (C2, C3) was determined by integrating the areas of the cross peaks appearing at δC/δH=73.1/4.48 ppm and δC/δH=74.6/4.78 ppm respectively and comparing with that of the sum of anomeric C1 signals of the xylan which appears at δC/δH=101.4/4.23 ppm and δC/δH=98.9/4.68. Of the two anomeric signals, the former is a result of correlation when the adjacent C2 hydroxyls are not acetylated


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and the latter is the resultant shift when C2 is acetylated. The anomeric signals were chosen as internal reference due to less overlap in this region. Thioacidolysis in combination with GC FID was used to quantify the monomers and reflects the content of non-condensed βO4 structures in lignin.14 Here, it is worth noting that incomplete cleavage of aryl ether linkages (76%) occurs during thioacidolysis and therefore a factor of 0.76 is considered when quantifying the monomers.14 However, we further caution that reported values could be an underestimation as the effect of thioacidolysis on benzyl ether type LC bonds has not been carefully studied. Thioacidolysis GC MS studies were applied on selected acetylated samples following Raneynickel desulphuration to get information on dimeric condensed products14,34 which reflect on the amount of condensed βO4 structures. Dioxane soluble fractions The 2D NMR spectra (Figure 3) showed cross peaks assigned to both carbohydrate and lignin structures and the assignments are reported in the Supplementary information (Tables S4-S7). Interestingly, the spectrum of dioxane lignin (Figure 3a) showed that pectin was the most abundant carbohydrate in this fraction as manifested in the cross peaks assigned to C1H1 in polygalacturonic acid appearing at δC/δH: 98.3/4.89. This assignment was made based on 2D HSQC studies on a commercial polygalacturonic acid. This observation is interesting from a topochemistry view point. It is reported in the literature that the concentration of pectin is high in the middle lamella, and that it is covalently bonded to lignin that is rich in p-hydroxyphenyl type monolignol.24,35 Thus we further studied all fractions by

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P NMR and confirmed that the

dioxane lignin and the F5 fraction had a fourfold higher content of p-hydroxyphenyl content than the other samples where it was detected (Table S2, Supplementary information). The similarity between dioxane lignin and F5 is expected since around 92% of the lignin in F5 is dioxane


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soluble. This fraction is therefore enriched in middle lamella LCC, although co-mixture with cell wall lignin cannot be excluded. Cross signal from C4H4 in 4-O-methylglucuronic acid (4OMGA) appeared at δC/δH:81.8/2.96. Glucan and xylan were also present. The C2 and C3 hydroxyls on xylan are partially acetylated. Partial acetylation is consistent with the literature on native xylan.36,37 Lignin carbohydrate bonds of the gamma ester type38 were identified and assigned according to Balakshin.10 These signals appear at δC/δH:62-65/4.0-4.5, although quantification was not done due to the region being heavily overlapped. The main inter-monolignol linkage detected was the βO4 quantified to 65 % of the phenyl propane (C9) units for the non-acetylated samples and 10% higher when the sample was acetylated (Table 2). This difference is because the resolution of the βO4 signal is improved by acetylation (Figure 3d and Figure S4 a, c, d, Supplementary information). Spirodienone (SD) and resinol (ββ) structures were also present (Figure 2). The syringyl to guaiacyl (S/G) ratio was 1.8 (Table S8, Supplementary information, Column 3) confirming the expected predominance of S-type lignin in hardwood.


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Table 2. Quantification of inter-unit linkages in lignin and between lignin and carbohydrate.

Dioxane lignin

F1

F2

F3

F4

F5

Solubility in DMSO-d6 (at 140 mg/mL)

Full

Full

Full

Poor/good

Full

Full

Main sugars detected in solution

X,G

X

X

X,G*

X,G

X,G

Acetylation C2 xylan

D

14

5

21

37

D

Acetylation C3 xylan

D

22

7

24

34

D

Lignin Linkage (% of C9 unit)

LC linkages (Relative % of C9 unit)

Err

O

Ac

O

O

O

Ac

O

Ac

O

Ac

βO4

0.2

65

75

55

55

70

65

64

71

55

50

β5

0.1

ND

ND

ND

ND

ND

ND

ND

ND

2.6

2.6

ββ

1

8

10

ND

9.5

ND

ND

9.5

10

9.5

7.7

SD

0.1

2.7

2.8

ND

ND

ND

ND

ND

ND

ND

ND

Phenyl Glycoside (PG)

ND

39.6

28.5

ND

ND

4.8

ND

Benzyl Ether type 1 (BE1)

ND

ND

ND

ND

ND

ND

D

Benzyl Ether type 2 (BE2)

ND

ND

ND

ND

ND

ND

3

γ Ester

D

ND

6,3

ND

ND

ND

3

Values are expressed as % of C9-units; Err=Error margin; X= Xylose; G=Glucose; O=Original sample; Ac and * =Values for acetylated samples; ND= Not detected; D= Detected but not quantified due to heavily overlapped region; PG, BE1, BE2 and γ Ester structures can be found in Figure 2.


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The F1 to F5 fractions The 2D NMR studies of these fractions gave valuable information on molecular structures and the covalent connectivity between lignin and carbohydrates. Titration of the spectral data (Figure 3 and Figure S4, Supplementary Information) produced the results in Table 2, from which the following important observations are made: •

Differences in the levels of C2-C3 acetylation of hydroxyls in xylan. The highest levels of C2-C3 acetylation were found in the F4 fraction and the lowest in the F2 and F1 fractions. In fact, the xylan with higher degree of acetylation also had higher content of 4-O-methylglucuronic acid (Table 1, last column-4OMGA). Thus, two types of native xylan differing in the degree of acetyl and 4OMGA substitution are found in Birch.

•

Differences in relative amounts of lignin inter-unit linkages between the fractions. In all the fractions the βO4 structure was predominant, but the highest levels were found in the F3 and F4 fractions. The 2D NMR analysis gives the total amount of βO4 structures but does not distinguish between condensed and non-condensed type βO4. Thus, to determine the proportions of the different βO4, we quantified the monomers produced from their cleavage during thioacidolysis by GC FID following silylation (Figure S3, Supplementary information). For this study only the F3, F4 and F5 fractions were analyzed and the results are reported in Table S8 (Supplementary information, Column 1). When compared with the 2D NMR data, it can be concluded that 85-90% of βO4 in the studied fractions were non-condensed. It should be noted here that the F3 sample was poorly soluble for the 2D NMR study but solubility was greatly enhanced when the sample was acetylated. The structures that could not be assigned by the 2D NMR studies such as 4-O-5 and 5-5 structures that are not involved


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in dibenzodioxin structures were studied by GC MS of the Raney-nickel desulphurated products of thioacidolysis. From GC MS chromatograms (Figure S2, Supplementary information) relative amounts of the dimers were reported (Table S8, Supplementary information, last 5 columns) confirming the presence of 4-O-5 type linkages in the studied fractions apart from F3. Relative to the other inter-unit linkages also studied by 2D NMR, it is concluded that the amounts are quite significant. •

Differences in the monolignol composition manifested in the syringyl: guaiacyl (S/G ratios). The values of the S/G ratios obtained by 2D NMR fell into 3 categories: high (5.5) medium (around 3.3) and low (around 2.0). The high value of 5.5 is probably an artifact due to the low levels of lignin in the soluble fraction leading to an underestimation of guaiacyl content. These high values were observed in the poorly soluble F3 fraction (non-acetylated) and the F1 fraction with low lignin content. Upon acetylation of the F3, enhanced solubility was observed and a new S/G ratio determined at around 3.3. S/G ratios were also determined for the thioacidolysis monomers and were in agreement with 2D NMR data (Table S8, Supplementary information, Columns 2 and 3). Hence, two types of lignin differing in S/G ratios (i.e. one ratio of around 2 and the other of 3) are found in LCCs. The lower S/G ratio is found in the F5 fraction and is similar to that of dioxane lignin. This is expected since around 92% of the lignin in F5 originates from dioxane lignin. This suggests that in the middle lamella lignin portion of the syringyl units (S) is replaced by p-hydroxyphenyl units which were relatively more present in these two fractions as discussed earlier, lowering the S/G ratio.


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Differences in the relative amounts of LC bond types. Lignin carbohydrate bonds were detected in all fractions except the F3. In the F3 detection was eluded most probably due to the low detection limits as it contained the bulk of cellulose. The assumption that LCC exists in this fraction stems from the indirect evidence derived from size exclusion chromatography indicating that the fraction contained a soluble xylan LCC. Future work on F3 will focus on enrichment of LCC fraction to enable the LC linkage studies. The F1 and F2, although low in lignin content, had a high prevalence of phenyl glycosidic bonds to xylan which appeared at δC/δH:100.4/5.02 and were assigned according to literature.11 In general, the LC benzyl ether linkages to xylan (Figure 2) seemed rather scarce and were only detectable in the F5 fraction.32 The signals appear at δC/δH: 80.1-81.2/4.21-4.68 (BE1, Figure 2) and at δC/δH: 82.9/5.23 (BE2, Figure 2).


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Figure 2. Main lignin substructures (1-7) and lignin-carbohydrate complexes (PG, γ Ester, BE) identified in the 2D HSQC spectra of birch LCC fractions. A=Arabinose; Gal=Galactose; G=Glucose; X=Xylose; M=Mannose; Ar=Aryl group.


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Figure 3. 2D HSQC NMR spectra of birch LCCs fractions. X=Xylan; G=Glucose; GalA = Galacturonic acid; Ac=Acetylated; U= Uronic acid; t=Carbon terminal in reducing end (r) or non-reducing end (nr); C=Carbon; C2-Ac=Acetylated Carbon in position 2. Aromatic region: S=Syringyl unit; G=Guaiacyl unit. The number in subscript indicates the carbon either in the aromatic or sugar ring.


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Salient features of this study Several observations from the structural studies suggest that the presented quantitative LCC fractionation protocol is relatively milder than those reported in the literature. These gauging observations are the preservation of native structures in the isolates which include: -

acetyl groups partially substituting the hydroxyls at C2 and C3 positions in xylan

-

high prevalence of aryl ether bonds in the sample lignins

-

presence of pH sensitive uronic acid ester linkages

An interesting observation is the relationship between the degree of substitution on xylan hydroxyls by acetylation and the type and frequency of LC bonds. The xylan fraction that was relatively more highly substituted had very low prevalence of LC bonds, while the lower substituted fraction showed high frequency of phenyl glycosidic bonds. Although the mechanism of formation of phenylglycosides is not yet known, acetylation at adjacent C2 hydroxyl seems to be unfavorable for its formation. One hypothesis could be that acetylation at C2 on xylan sterically hinders the coupling of lignin at adjacent C1 hydroxyl. Another hypothesis, assuming the formation of phenyl glycosides is an enzymatic process, is that acetylation inhibits the coupling due to enzyme specificity issues. Further investigations are required to shed light on this subject. A higher substitution by acetylation would also reduce the probability of formation of benzyl ethers on xylan. This may explain why benzyl ethers were not detectable in these fractions. Here, it is important to note that benzyl ethers detected in the literature involve mainly C2 and C3 hydroxyls on xylan. Unlike softwood xylan which is not acetylated, the scarce presence of benzyl ether type linkage we observe in birch wood could be due the significant levels of acetylation on xylan. Our recent study on spruce showed that benzyl ethers to xylan are relatively more prevalent.17 On the other hand, benzyl esters have been proposed to be common


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in hardwoods, which could be in response to the reduced possibilities of ether and glycoside formation by acetylation. Acetylation in hemicelluloses in wood may thus function as regulators of the type and frequency of LC bonds as illustrated in the Figure 4.

Figure 4. Proposed regulatory mechanism of LC bond frequency in Xylan by acetylation at C2 and C3. Ac = Acetyl group.

ASSOCIATED CONTENT Supporting information Information as mentioned in the text: SEC chromatograms, lignin content, 31P NMR, GC MS and GC FID assignment, 2D HSQC spectra. This material is available free of charge via the Internet at http://pubs.acs.org Abbreviations BMW, Ball milled wood; 4OMGA, 4-O-methyl Glucuronic acid; MWL, Milled Wood Lignin; SEC, Size Exclusion Chromatography; [Amim]Cl, 1-allyl-3-methylimidazoliumchloride. Corresponding Author e-mail: [email protected]; Tel: +46 8 7908047, +46 73 4607647; Fax: +46 8 7908066


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ACKNOWLEDGMENT This work was supported by Knut and Alice Wallenberg Foundation gratefully acknowledged for financial support to Wallenberg Wood Science Center. REFERENCE

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For Table of Contents Use Only Title: Structural basis for the Formation and Regulation of Lignin-Xylan Carbohydrate Bonds in Birch Authors: Nicola Giummarella and Martin Lawoko Synopsis Detailed structural studies on mildly fractionated lignin carbohydrate complexes were insightful to the formation and regulation of lignin carbohydrate bond frequency in Birch. Content graphic


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Fractionation scheme for lignin carbohydrate complexes (LCCs). BMW = Ball Milled Wood 29x14mm (600 x 600 DPI)


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Main lignin substructures (1-7) and lignin-carbohydrate complexes (PG, γ-ester, BE) identified in the 2D HSQC spectra of birch LCC fractions. A=Arabinose; Gal=Galactose; G=Glucose; X=Xylose; M=Mannose. 199x199mm (300 x 300 DPI)



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2D HSQC NMR spectra of birch LCCs fractions. X=Xylan; G=Glucose; GalA = Galacturonic acid; Ac=Acetylated; U= Uronic acid; t=Carbon terminal in reducing end (r) or non-reducing end (nr); C=Carbon; C2-Ac=Acetylated Carbon in position 2. Aromatic region: S=Syringyl unit; G=Guaiacyl unit. The number in subscript indicates the carbon either in the aromatic or sugar ring. 175x182mm (300 x 300 DPI)


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Proposed regulatory mechanism of LC bond frequency in Xylan by acetylation at C2 and C3. Ac = Acetyl group. 57x46mm (300 x 300 DPI)



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Table of contents graphic 74x47mm (300 x 300 DPI)


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Structural Basis for the Formation and Regulation of LigninâXylan

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Structural Basis for the Formation and Regulation of LigninâXylan