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For Peer ReviewTOP–DOWN LIGNOMIC MALDI-TOF-TANDEM MASS SPECTROMETRY ANALYSIS OF LIGNIN OLIGOMERS

EXTRACTED FROM SAUDI DATE PALM WOOD (SDPW)

Journal: Rapid Communications in Mass Spectrometry

Manuscript ID RCM-18-0263.R2

Wiley - Manuscript type: Research Article

Date Submitted by the Author: n/a

Complete List of Authors: Albishi, Tasahil; Memorial Unversity, BiochemistryMikhael, Abanoub; Memorial University of NewFoundland, ChemistryShahidi, Fereiidoon; Memorial University, BiochemistryFRIDGEN, TRAVIS; MEMORIAL UNIVERSITY, ChemistryDELMAS, MICHEL; University of Toulouse, Chemical Engineering Laboratory Banoub, Joe; Memorial University of Newfoundland Faculty of Humanities and Social Sciences, Chemsitry; Governmnet of Canada, Fisheries and Oceans Canada

Keywords:MALDI-TOF-MS, High-energy CID-MS/MS, Saudi Date Palm Wood Lignin oligomers, Top Down Lignomic Analysis, Retro Diels Alder Reaction, Distonic Cations

Abstract:

Rationale: We report for the first time the top-down lignomic analysis of the virgin released lignin (VRL) oligomers obtained from the Saudi date palm wood (SDPW), using a MALDI-TOF/TOF instrument. In addition, we are proposing new CID-MS/MS fragmentation routes for this series of unreported VRLs. Methods: We have used MALDI-TOF-MS direct analysis of the lignin oligomers mixture without any chromatographic pre-separation. High-energy CID-MS/MS analyses were used to confirm the precursor ions structures. Results: Six lignin oligomer protonated molecules were identified as: [C19H24O8+ H]+ composed of H(8-O-4)G; [C50H52O19+H]+ composed of H(8-O-4)H(8-O-4’)S(8-O-4”)S(8-O-4’”)G; [C58H54O18+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G; [C58H54O19+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)S(8-O-4’”)S(8-O-4””)G; [C61H68O25+H]+ composed of H(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G and [C61H68O26+H]+ composed of C(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G units. Two distonic cations were identified as [C39H43O15+ H]+• and [C40H43O16+H]+• deriving from two tetrameric lignin oligomers. The high-energy tandem mass spectrometry analyses allowed the confirmation of the proposed structures of this series of lignin oligomers. Conclusion: To our knowledge, this is the first elucidation of the

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unknown lignin structure of the Saudi seedling date palm wood that was accomplished using top-down lignomic strategy that was never published. The complex high-energy CID-MS/MS fragmentations presented herein are novel and have never been described before.

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TOP–DOWN LIGNOMIC MALDI-TOF-TANDEM MASS SPECTROMETRY

ANALYSIS OF LIGNIN OLIGOMERS EXTRACTED FROM DATE PALM WOOD

Tasahil Albishi,a Abanoub Mikhael ,b Fereidoon Shahidi,a Travis Fridgen,b Michel Delmas,c

Joseph Banouba,b, d*

a Department of Biochemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada

b Department of Chemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada

cUniversity of ToulouseInp-EnsiacetChemical Engineering Laboratory4, Allée Emile Monso31432, Toulouse, France

d Science Branch, Special Projects, Fisheries and Oceans Canada, St John’s, NL, A1C 5X1, Canada

These two authors (graduate students) have contributed equally in this research and have been

listed in alphabetically order

*Correspondence to: Joseph Banoub, Science Branch, Special Projects, Fisheries and Oceans Canada, St John’s, NL, A1C 5X1, Canada and Department of Chemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada. E-mail: [email protected]

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Abstract

Rationale: We report for the first time the top-down lignomic analysis of the virgin released

lignin (VRL) oligomers obtained from the Saudi date palm wood (SDPW), using a MALDI-

TOF/TOF instrument. In addition, we are proposing new CID-MS/MS fragmentation routes for

this series of unreported VRLs.

Methods: We have used MALDI-TOF-MS direct analysis of the lignin oligomers mixture

without any chromatographic pre-separation. High-energy CID-MS/MS analyses of the precursor

ions were used to confirm the molecular structures.

Results: Six lignin oligomer protonated molecules were identified as: [C19H24O8+ H]+ composed

of H(8-O-4)G; [C50H52O19+H]+ composed of H(8-O-4)H(8-O-4’)S(8-O-4”)S(8-O-4’”)G;

[C58H54O18+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G;

[C58H54O19+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)S(8-O-4’”)S(8-O-4””)G;

[C61H68O25+H]+ composed of H(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G and

[C61H68O26+H]+ composed of C(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G units. Two

distonic cations were identified as [C39H43O15+ H]+• and [C40H43O16+H]+• deriving from two

tetrameric lignin oligomers. The high-energy tandem mass spectrometry analyses allowed the

confirmation of the proposed structures of this series of lignin oligomers.

Conclusion: To our knowledge this is the first elucidation of the unknown lignin structure of the

Saudi seedling date palm wood that was accomplished using top–down lignomic strategy that

was never published. The complex high-energy CID-MS/MS fragmentations presented herein

are novel and have never been described before.

INTRODUCTION

Lignin is the second most abundant biopolymer in nature after cellulose; it is a product of

enzymatic oxidative polymerization of three monomeric aromatic compounds (monolignols):

coniferyl (H), sinapyl (S), and p-coumaryl (G) alcohols. It is found in all vascular plants, mostly

between the cells, as well as within the cells and in the vegetable cell walls (CWs).[1] For almost

a century, the structure of lignin was designated as a complex polymer composed of irregular

branched units.[2-4] Presently, lignin is viewed as a promising commercial source of a wide range

of aromatic compounds, which can be used as an alternative to fossil hydrocarbons. [5-7]

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There is still much debate on whether any lignin extract adequately represents the native

structure, and it has been proposed, that the several different chemicals, enzymatic and

mechanical extraction methods, are accountable for the major structural divergence that occurs

after extraction and isolation. [4,7] Recently, it was established that either strong acid or basic

depolymerisation extraction method can cause cleavage of ester and ether bonds, creating

reactive species. These latter species alternately react further, to yield more complex and

rearranged condensed lignin polymer/oligomer structures. [7,8]

Traditionally, it has been repetitively suggested that structural analysis of lignin should

be based on pure samples, However, preparing pure samples of unchanged lignin is not an easy

endeavor.[9A,B] For this reason, the structural determination of lignin is indeed a more challenging

task than that with other biopolymers. [9A,B]As a consequence it appears that the only logic step to

determine the natural structure of lignin is to isolate it from the vegetal matrix, without causing

any structural change. [10A,B;11]

Lately, we have proposed a new paradigm, which indicated that the intact natural lignin

oligomers present in the lignocellulosic biomass, were not actually either one and/or series of

similar biomolecules, like an individual cellulose fiber; instead, they were composed of a series

of different length linear related biosynthesized oligomers.[11] These oligomers may be formed

either from homo-oligomers repeating units and/or could be hetero-oligomers formed by mixed

units.[11] These have never been completely described in their natural unprocessed form. In

addition, it is proposed that lignins present in the lignocellulosic biomass are attached by either

ether and/or ester covalent links, in a crisscross manner, to both cellulose and hemicellulose

fibers, forming a glycolignin network. [11] So far, shorter oligomers of lignin can be extremely

useful in providing the blueprints on how the full lignin polymer is constructed.

It is well known that mass spectrometric analyses are the only promising methods

offering novel possibilities for the sequencing lignin oligomers and for interpreting the plant

‘lignome’. [11,12] Please note that the lignome was defined to represent the ensemble of all

biosynthetic phenolics, metabolites and (neo)lignan biosynthetic pathways and their derivatives,

as well as the lignin oligomers. [11,13]

In this manuscript, we present the structural elucidation of series of lignin biooligomers

which were extracted by “La Compagnie Industrielle de la Matière Végétale” (CIMV)solvolysis

technique. [14,15] This last technique appears to be the finest technique for lignin separation and

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this is due to its capabilities of being efficient and allowing the choice of simple organic solvents

such as acetic acid/formic acid/water combination. The structural identification of the virgin

released lignins (VRL) was done by using MALDI-Assisted Laser/Desorption Ionization time-

of-flight mass spectrometry (MALDI-TOF-MS) in conjunction with tandem mass spectrometry

using high energy collision dissociation CID-TOF/TOF-MS/MS (MS/MS in-space) instrument.

EXPERIMENTAL

Samples

The samples date palm wood (Phoenix dactylifera) examined were collected manually

from the Salman Alfarsi garden, Almadinah, Saudi Arabia. All the samples were frozen and

dried for 7 days at -48°C and 30 x 10-3 mbar (Freezone 6, model 77530, Labconco Co., Kansas

City, MO). The dried samples were then grounded, vacuum packed and stored in a freezer at -

20ºC.

Lignin Oligomers Isolation

The Saudi Date Palm Wood (SDPW) lignin was extracted using the CIMV procedure which

selectively separates the cellulose, hemicellulose and lignin, and allows the destructing of the

vegetable matter at atmospheric pressure (Lignin yield 17%). [14,15] The catalyst-solvent system

used was a mixture of formic acid/acetic acid/water (30/50/20) which produced after

precipitation with water and filtered the Saudi Date Palm Wood (SDPW) lignin. Approximately

0.1mg of the purified lignin was dissolved in 1mL dioxan/ methanol/chloroform (1:1:1) for MS

analysis.

MALDI-TOF-MS Analysis

Applied Biosystems/MDS SCIEX 4800 MALDI TOF/TOF™ Analyzer (MDS Sciex, 71 Four

Valley Dr., Concord, Ontario, Canada L4K 4V8) was used in this experiment for the analysis of

the Saudi Date Palm Wood (SDPW) lignin. In this analysis, 1 mg of the lignin sample was

dissolved in 1 mL of the dioxane/ methanol/chloroform (1:1:1) and 2,5- dihydroxy benzoic acid

(DHB) was used as matrix for the analysis. The MS data was acquired in the mass range 100 to

2000 m/z in the positive ion mode. The mass spectra instrument was equipped with Nd: YAG

200-Hz laser. The accelerating potential was 25KV. The MALDI plate was prepared by spotting

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1 μL of a 20mg/mL solution of DHB (dissolved in acetone, 0.1% trifluoroacetic acid) and then

dried at room temperature (the use of acetone allows a good homogeneity of the matrix in the

spot). Then, an aliquot of 1 μL of sample was spotted on the top of the dried matrix and allowed

to dry before the MALDI-TOF-MS experiments. For MS analysis, mass spectra were the sum of

400 laser shots and acquired in reflectron mode. For high-energy CID-MS/MS analysis, mass

spectra were the sum of 600 laser shots, collision energy of 1 keV, nitrogen as the collision gas

to induce high energy CID-fragmentation. The following standards were used to calibrate the

mass spectrometer: des-Arg1-Bradykinin, [C44H61N11O10], M.Wt. 904.0245 from Enzo Life

sciences, Inc (Farmingdale, NY 11735, USA); Angiotensin 1, [C62H89N17O14], M.Wt. 1296.4779,

from Tocris Bioscience (614 McKinley Place N.E., Minneapolis, Minnesota 55413) USA. The

differences between the calculated m/z and the observed m/z, for all CID-MS/MS analyses were

around 5-10 ppm.

Solid state 13C-NMR of the Saudi Date Palm Lignin

The 13C-NMR spectrum was obtained at 298 K using a Bruker Avance II 600 spectrometer,

equipped with a SB Bruker 4mm MAS double-tuned probe operating at 150.97 MHz for 13C.

Chemical shifts (δ) were referenced to tetramethylsilane (TMS) using adamantane as an

intermediate standard for 13C. The samples were spun at 15 kHz. Cross-polarization (CPMAS)

spectra were collected with a Hartmann-Hahn match at 62.5 kHz and 100 kHz 1H decoupling,

with a contact time of 2 ms, a recycle delay of 3 s and 1 k scans.

RESULTS AND DISCUSSION

In lignomics research, no explicit sequencing methods exist to establish the primary

structure of complex and simple lignin oligomers. Researchers are required to synthesize

authentic compounds as standards to enable verification and comparison with the MS/MS

scheme obtained by an unknown compound. [11,13]

It is imperative to mention that until today, no innovative sequencing of any natural

lignin oligomers using MS/MS method have been ever discussed to unravel unknown new

structures form newly extracted virgin lignins.[11] By analogy with proteomics, a new concept of

top–down lignomic strategy employing an MS/MS strategy, was introduced by Banoub et al.[11]

The top–down lignomic strategy was described as the identification by MS and MS/MS analysis

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of the native extracted lignins (VRL) obtained directly from the destructing of the vegetable

matter. The top–down lignomic strategy will permit the identification of all the components of

the intact non-modified virgin lignins (VRL) being analyzed.[11] Moreover, the top– down

approach enables direct measurement of the intact mass of the heterogeneous lignin oligomers as

well as product ions information relating to the original oligomer lignin sequences. [11]

Matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry has been used

in various structural lignin research. [16-19] Notwithstanding, the unquestionable advantages of

MALDI, this method is not currently very much used for the structural analyses of lignin for the

lack of ionization efficiency of the lignin biopolymer achieved using conventional matrices. [16-19]

In this manuscript, we have performed the direct analysis of the lignin oligomers mixture

without any chromatographic pre-separation and the determination of the chemical formulae was

completed by using some basic rules in MS and MS/MS explained by Thomas De Vijlder et al.

[20], The chemical structures were calculated from the MALDI-TOF-MS according to the

heteroatom, differences between calculated and observed masses in ppm, and isotopic

distributions of the molecular ions. [21,22] The level of unsaturation or the double bond equivalent

(DBE) for either of the characterized molecules and/or precursor ions, was measured to calculate

the number of unsaturation such as double/triple bonds and/or ring systems.[20]

The solid state 13C-NMR spectrum (Figure 1) was measured to pin-point the various

diagnostic functional groups in the proposed chemical structures of the SDPW oligomers.

Therefore, we assigned the signals in the range of 54-56 ppm to the methoxyl groups in different

lignin oligomers. The resonance region varying between 112 and 158 ppm can be assigned either

to the aromatic carbons and/or any conjugated aliphatic C=C such as -C=C-COOH [23]. The

signals in the region of 185.0 to 187.1 ppm were assigned to ketone functional groups that appear

in some of our proposed chemical structure. The signal at 171.3 ppm was attributed to an

aromatic carboxyl group [23]. The signals in the range of 77-65 ppm were assigned to different

primary and secondary alcohols [25]. Finally, the signals between 97.8 to at 88.0 ppm can be

assigned to C-8 (beta carbon) and alkyne carbons arising from double eliminations occurring on

the C7-C8 chain present in one of the proposed precursor ions described below.[24, 25] It is

essential to understand that this 13C-NMR spectrum verifies that even the mild CIMV extraction

process, can perhaps affect the original structure of the lignin oligomers. Consequently, this

alkyne group that links two aromatic rings can be easily formed by the elimination of water

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molecule followed by the loss of methanol molecule which is very common MS/MS

fragmentation pathway discussed in this manuscript.

The conventional scanning MADLI-TOF-MS analysis of the SDPW showed a series of

protonated molecules inter alia at 381.1541; 752.2699; 780.2611; 957.3162; 1039.3382;

1055.3391; 1201.4098 and 1217.4123 (Table 1, Figure 2A and 2B).

Figure 2A and 2B

The generation of MALDI-TOF-MS scan (+ ion mode) and various high energy

dissociation tandem mass spectrometry (CID-MS/MS) analyses will provide series of protonated

molecules and MS/MS diagnostic product ions (Table 1), which will serve as a tool for obtaining

high-quality mass spectra of VRL of seedling date palm wood (Phoenix dactylifera) suitable for

structural studies of the analyte biopolymer.

The following parts represent our thoughts and deductions for the elucidation of the

molecular structure of this series of VRL oligomers which according to our MALDI-TOF-MS,

were composed of H:G:S:C in a ratio of ca 12:16:10:1 residues. It is to be noted that recently

Ralph and coworkers reported the evidence of a catechyl lignin homopolymer (C lignin) derived

solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. This group

previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl

(G/S) lignins, and occasionally both. Therefore, our presented work is the first report of a lignin

series containing H/G/S and C units [26,27]. Consequently, we have identified six VRL protonated

molecules and two distonic VRL cations as follows (Figure 3A , Figure 3B and Table 1).

Figure 3A and 3B

Table 1

We begin with the identification of the chemical structure of the protonated molecules at

m/z 381.1541, tentatively assigned as [C19H24O8 + H]+ which was composed of the H(8-O-4‵)G

dilignol having the structure 1 (Figure 3A , Figure 4 and Table 1).

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The product ion scan of m/z 381.1541 gave three major diagnostic product ions inter alia

at m/z 201.0538; 219.0643; and 362.1350 which were assigned respectively as [C12H8O3 + H]+ ;

[C12H10O4 + H]+ and [C19H21O7 + H]+• (Figure 4).

Figure 4

It is important to notice the formation of the primary distonic protonated radical product ion at

m/z 362.1350 (distonic cation) which possessed an even m/z. [28,29] Please bear in mind that the

formation of molecular radical ions has been documented to occur for MALDI-TOF-MS

analysis.[30] .Furthermore, this distonic cation can be formed by the remote charge fragmentation

mechanism [31,32], occurring by loss of a molecule of water and hydrogen radical from the

precursor ion m/z 381.1541. The consecutive eliminations from the precursor ion at 381.1541 of

two methanol molecules along with retro-Diels Alder reaction (RDA) [33,34] occurring in both H

and G units (loss of hydroxyacetylene and methoxyacetylene) lead to the formation of the

primary product ion at m/z 219.0643. Bear in mind that the formation of secondary product ion at

m/z 201.0538 was created by the primary product ion m/z 362.1350. The high energy CID-

MS/MS of the precursor ion m/z 381.1541 and the remaining MS/MS fragmentations are shown

in Scheme 1.

Scheme 1

The distonic cation at m/z 752.2699 was assigned as [C39H43O15 + H]+• and existed as the

lignin tetramer H(8-O-4)G(8-O-4’)G(8-O-4”)G having the structure 2 (Figure 3A, Figure 5 and

table 1).

The product ion scan of the distonic cation 2 at m/z 752.2699 afforded the product ion at

m/z 709.2467, which was formed by the loss of one ethyne molecule C2H2 eliminated by a retro-

Diels Alder reaction (RDA)[33,34] and one hydroxyl radical, and it was assigned as

[C37H40O14+H]+ .

Figure 5

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It is interesting to note the successive formation of the product ions at m/z 711.2601; 713.2766;

and 715.2911 (Scheme 2A). These latter product ions were assigned as [C37H42O14+H]+,

[C37H44O14+H]+ and [C37H46O14+H]+ and appeared to be formed by hydrogenation caused by the

DHB matrix. This type of DHB matrix hydrogenation have been previously reported by

others.[35,36] The product ion at m/z 709.2467 can lose a molecule of carbon monoxide and

subjected to another two retro-Diels Alder reactions to afford the ion at m/z 599.2092 (Scheme

2A). [ 33,34] It is important to understand that the order of these eliminations could occur

simultaneously or in a stepwise fashion and this was not studied further

Scheme 2A

Moreover, the precursor distonic ion 2 can undergoes a concerted retro-Diels-Alder reaction by

cleavages of the C1-C2 and C5-C6 aromatic bonds of the fourth unit G and by cleavages of the

C1-C2 and C3-C4 aromatic bonds of the third unit G, along with successive losses of carbon

monoxide, hydroxyl group and two formaldehyde molecules to afford the product ion at m/z

495.1979 (Scheme 2B). The exact order of these MS/MS gas-phase eliminations has not been

established. This latter secondary product ion at m/z 495.1979 can either lose a molecule of

hydrogen to afford the secondary product ion at m/z 493.1833 and/or it can be subjected to

another retro-Diels-Alder reaction by loss of a molecule of ethyne to afford the ion at m/z

469.1831 (Scheme 2B). In addition, the product ion at m/z 495.1979 can also be subjected to

cleavage of the 8-O-4 bond between the upper two units to afford the secondary product ion at

m/z 429.1887 (Scheme 2B).

Scheme 2B

The presence of carboxylic acid group was deduced by finding the product ion at m/z 707.2667

assigned as [ C38H42O13 + H]+ which was formed by the loss of carbon dioxide and hydrogen

radical from the precursor ion at m/z 752.2699 (Scheme 2B). Also, the precursor distonic cation

2 produced the primary product ion at m/z 735.2591 assigned as [C39H42O14+H]+ by the loss of a

hydroxyl radical (Supplementary Material, SM1).[28,29] This latter primary product ion eliminates

a molecule of carbon dioxide to from the secondary product ion at m/z 691.2709 (Supplementary

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Material, SM1). Additionally, this latter secondary product can eliminates simultaneously, a

molecule of carbon monoxide following aromatic ring contraction and a molecule of water, to

afford the tertiary product ion at m/z 645.2655 (Supplementary Material SM1). The precursor

distonic[28,29] cation 2 afforded the primary product ions at m/z 675.2389 and 668.2049 by the

respective consecutive losses of either a molecule of carbon dioxide, a hydrogen radical and a

molecule of methanol, or two molecules of acetylene by RDA[33,34]rearrangements and a

molecule of methanol (Supplementary Material SM1). For the sake of brevity, additional

information on the remaining low m/z product ions: at m/z 278. 0773; 184.0724; 163.0384

created by the precursor ion at m/z 752.2699 are shown in Scheme 2A and 2B and the

Supplementary Material in Scheme SM1.

The distonic cation at m/z 780.2611 was assigned as [C40H43O16 + H]+• and structure 3

which was composed of a tetrameric lignin oligomer H(8-O-4)G(8-O-4’)S(8-O-4”)G (Figure 3A,

Figure 6 and Table 1).

Figure 6

The high energy collision dissociation of the precursor distonic cation 3 at m/z 780.2611

afforded a major product (base peak) at m/z 737.2399 which was created by loss of a molecule of

acetylene (RDA) [33,34] and a hydroxyl radical and it was assigned as [C38H40O15+H]+.

Furthermore, cleavage of S(8-O-4”)G bond of the precursor distonic cation 3 creates the

protonated molecule at m/z 163.0388 assigned as [C9H6O3 + H]+ (Scheme 3A). Similarly, the loss

of the lower end H unit from the precursor distonic cation 3, afforded the distonic product radical

ion at m/z 184.0724 which was tentatively assigned as [C9H11O4 + H]+•. Finally, the presence of

the carboxylic acid group was confirmed by the loss of carbon dioxide and hydrogen radical

from the precursor distonic cation at m/z 780.2611 to afford the product ion at m/z 735.2591

assigned as [C39H42O14 +H]+ (Scheme 3A).

Scheme 3A

Another CID-MS/MS fragmentation of the precursor distonic cation at m/z 780.2611 can also

occur by the simultaneous losses of a molecule of carbon monoxide following aromatic ring

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contraction of the first H residue, an acetylene molecule by RDA mechanism and a hydroxyl

radical to afford the primary product ion at m/z 597.1931 assigned as [C31H32O12+H]+ (Scheme

3B). This latter primary product ion loses a molecule of carbon dioxide and two molecules of

formaldehyde to afford the secondary product ion at m/z 493.1821 assigned as [C28H28O8+H]+

(Scheme 3B). Similarly, the precursor distonic cation at m/ z 780.2611 can lose instantaneously

either hydrogen radical and a molecule of methoxyacetylene or 3 molecules of methoxyacetylene

by RDA rearrangements, a molecule of methanol and a molecule of carbon dioxide to afford

respectively, the secondary product ions at m/z 723.2236 and 536.11636. These were assigned as

[C37H38O15+H]+ and [C29H27O10+H]+• (Scheme 3B).

Scheme 3B

The protonated molecule at m/z 957.3162 was assigned as [C50H52O19+H]+ and as

structure 4. It was composed of a pentameric lignin oligomer formed of 5 aromatic rings,

namely: H(8-O-4)H(8-O-4’)S(8-O-4”)S(8-O-4’”)G (Figure 3A, Figure 7 and Table 1).

Figure 7

` The product ion scan of the precursor ion 4 at m/z 957.3162 afforded the product ion at

m/z 939.3014, created by the loss of a water molecule assigned as [C50H50O18+H]+ (Scheme 4A).

Moreover, the product ion at m/z 939.3014 can lose a molecule of formaldehyde to form the

secondary product ion at m/z 909.2912 assigned as [C49H48O17+H]+ (Scheme 4A). In addition,

the precursor ion 4 at m/z 957.3162 can also lose simultaneously a hydrogen radical, a molecule

of carbon monoxide through aromatic ring contraction and a molecule of water to afford the

primary product ion at m/z 910.2988 assigned as [C49H49O17+H]+• (Scheme 4A). It is noteworthy

to mention, that the precursor protonated molecule 4 produced the dimeric product ion at m/z

347.1471 assigned as the [C19H22O6+H]+, which was created by the cleavage of the S(8-O-4)S

bond (Scheme 4A). This latter primary product ion affords the secondary product ion at m/z

319.1522 by loss of a molecule of carbon monoxide following ring contraction (Scheme 4A).

Scheme 4A

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The primary product ion at m/z 939.3014 can fragment further to create the secondary product

ions at m/z 493.1825; 478.1601; 405.1309 and 278.0772. This series of secondary product ions

are shown in the Supplementary Part SM2A. Similarly, the primary product ion at m/z 910.2988

underwent two separate RDA rearrangements by the loss of either a molecule of

methoxyacetylene and a molecule of water or two molecules of methoxyacetylene to afford

respectively the secondary product ions at m/z 836.2662 and 798.2475. These were assigned as

[C46H43O15+H]+• and [C43H41O15+H]+•, respectively (Supplementary Part SM2B). This latter

secondary product ion can lose two molecules of water and two molecules of formaldehyde to

afford the tertiary product ion at m/z 702.2051 (Supplementary Part SM2B). This latter tertiary

product ion afforded the quaternary product ion at m/z 658.2144 by the loss of a molecule of

carbon dioxide which confirm the presence of carboxylic acid group (Supplementary Part

SM2B).

The precursor ion 4 can also CID fragments by the loss of two consecutive molecules of

methanol to affords the secondary product ion at m/z 893.2615 assigned as the [C48H44O17 + H]+

(Scheme 4B). Further consecutive losses of this product ion by four molecules of formaldehyde

and five molecules of water afford the secondary product ion at m/z 683.1655, which was

assigned as [C44H27O8]+ (Scheme 4B). Moreover, the cleavage of the 8-O-4 bond of the first H

residue of this latter secondary product ion, along with the cleavage of C1-C7 of the upper

terminal G unit, affords the tetrameric product ion at m/z 502.1368, assigned as [C32H21O6+ H] +•

(Scheme 4B).

Scheme 4B

The lower m/z values product ions obtained by the product ion scan of 4 are explained in the

Supplementary Part SM2C. Henceforth, the product ion at m/z 184.0727 was formed from the

precursor ion 4 at m/z 957.3162 by double cleavages of the S(8-O-4”)S bond and C4-O aryl ether

bond of the top end G unit, followed by the loss of two molecules of formaldehyde. The product

ion at m/z 172.0725 was formed from the precursor ion 4 by cleavage of C1-C7 of the fourth S

unit followed by RDA which eliminates the pent-2-en-4-ynoic acid (C5H4O2, 96Da)

(Supplementary Part SM2C).

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The protonated molecule at m/z 1039.3382 was assigned as [C58H54O18+H]+ having

structure 5. This protonated molecule was composed of a hexameric lignin oligomer composed

of H(8-O-4)H(8-O-4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G (Figure 3B, Figure 8 and Table 1).

Figure 8

The product ion scan of the protonated molecule at m/z 1039.3382 afforded the product

ion at m/z 1011.3371 by loss of a molecule of carbon monoxide followed by aromatic ring

contraction of the first H residue (or vice versa). This product ion was as [C57H54O17+H]+ and

was composed of a hexameric lignin oligomer namely: cylcopentadienyl(7-O-4)H(8-O-4’)H(8-

O-4”)G(8-O-4’”)S(8-O-4””)G (Scheme 5a). The product ion at m/z 1011.3371 affords the

secondary product ion at m/z 975.3177 by loss of two molecules of water and it was assigned as

[C57H51O15]+ (Scheme 5A). This last product ion further CID fragmented by consecutive losses

of four molecules of formaldehyde, three molecules of ethyne eliminated by the retro-Diels

Alder mechanism and one molecule of carbon monoxide to afford the secondary product ion at

m/z 749.2343, assigned as[C46H37O10]+ (Scheme 5A). Once more, the order of these MS/MS

concerted losses was not established and is beyond the scope of this study. Finally, the presence

of the terminal carboxylic acid group was confirmed by the loss of carbon dioxide molecule from

the precursor ion at m/z 1039.3382 to afford the product ion at m/z 995.3432 assigned as

[C57H54O16 + H]+ (Scheme 5A).

Scheme 5A

Another CID-MS/MS fragmentation of the primary product ion at m/z 1011.3371 can also occur

by loss of a hydrogen molecule, to afford the secondary product ion at m/z 1009.3213 (Scheme

5B). This last ion loses consecutively 4 molecules of formaldehyde and four molecules of water

to afford the tertiary product ion at m/z 853.2593, assigned as [C53H41O11]+ (Scheme 5B).

Scheme 5B

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Similarly, the primary product ion at m/z 1011.3371 can also create the secondary product ion

at m/z 430.1392 by cleavage of the H(8-O-4”)G bond of the cyclopentadienyl(7-O-4)H(8-O-

4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G hexamer. This secondary trilignol ion product at m/z

430.1392 was assigned as [C26H21O6]+• and was composed of the trimer cylcopentadienyl(7-O-

4)H(8-O-4’)H (Scheme 5C).

Moreover, The cleavage of the cylopentadienyl(7-O-4) bond of the primary product ion at m/z

1011.3371 affords the pentameric product ion at m/z 893.2962 assigned as [C49H48O16+H]+,

composed of the tetrameric H(8-O-4)H(8-O-4’)G(8-O-4”)S(8-O-4’”)G oligomer (Scheme 5C).

Finally, this primary product ion at m/z 1011.3371 can undergo two different RDA reactions as

shown in Scheme 5C to afford the secondary product ions at m/z 851.2865 and 663.2196.

Scheme 5C

Additionally, The product ion scan of the protonated molecule at m/z 1039.3382 afforded the

primary product ion at m/z 1013.3171 by loss of molecule of acetylene by RDA mechanism

(Supplementary Material SM3A).[33,34] This primary product ion was assigned as

[C56H52O18+H]+. This latter product ion loses a molecule of carbon monoxide by an aromatic

ring contraction of the first H residue, to afford the secondary product ion at m/z 985.3219

assigned to possess the [C55H52O17+H]+ molecular formula (Supplementary Material SM3A).

Additional information on and the remaining low values m/z product ions are shown in the

Supplementary Material as Scheme SM3B.

The protonated molecule at m/z 1055.3391 was assigned as [C58H54O19+H]+ and was

composed of the hexameric unit formed by H(8-O-4)H(8-O-4’)H(8-O-4”)S(8-O-4’”)S(8-O-

4””)G. This protonated molecule was assigned as structure 6 (Figure 3B, Figure 9 and Table 1)

Figure 9

The product ion scan of this protonated molecule at m/z 1055.3391 afforded the primary

product ion at m/z 1027.3319 by loss of a molecule of carbon monoxide by ring contraction of

the first H residue of this hexameric unit (Scheme 6A). This last product ion at m/z 1027.3319

loses by one concerted mechanism two molecules of water, two molecules of methanol and two

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molecules of formaldehyde, not necessary in that order, to afford the secondary product ion at

m/z 867.2398 assigned as [C53H38O12 + H]+ (Scheme 6A). The cleavage of the precursor

protonated molecule between the contiguous two sinapyl residues affords the secondary product

ion at m/z 495.1832 assigned as [C24H30O11+H]+ (Scheme 6A). This latter secondary product

ion afforded tertiary product ion at m/z 469.1673 assigned as [C22H28O11+H]+ by the loss of

ethyne through RDA mechanism (Scheme 6A). [33,34]

Scheme 6A

Furthermore, the protonated molecule at m/z 1055.3391 can lose an ethyne molecule by RDA

mechanism to afford the primary product ion at m/z 1029.3109 assigned as [C56H52O19 + H]+or a

methane molecule, which can occur on either one of the S or G residues, to afford the primary

cation at m/z 1039.2958 assigned as [C57H51O19]+ (Supplementary information SM4A).

Moreover, The primary product ion at m/z 1027.3319 can lose an ethyne molecule by RDA

mechanism and five molecules of formaldehyde to afford the secondary product ion at m/z

851.2649 assigned as [C50H42O13+H]+ (Supplementary information SM4B). This latter secondary

product ion at m/z 851.2649 can lose carbon dioxide molecule to form the tertiary product ion at

m/z 806.2685 assigned as [C49H42O12+H]+ (Supplementary information SM4B).

The cleavage of C1-C7 bond of the fourth unit S in the primary product ion at m/z 1027.3319

lead to the formation of dilignol secondary product ion at m/z 495.1832 assigned as [C24H30O11 +

H ]+ (Supplementary information SM4B). This latter secondary product ion at m/z 495.1832

fragments by the cleavage of C-4-O aryl ether bond of the G unit and C1-C7 bond of the S unit

to afford four lower m/z tertiary products ions at m/z 269.1004, 172.0721, 116.0466 and

104.0619 (Supplementary information SM4B).

The presence of the carboxylic group in the precursor protonated molecule 6 was confirmed by

the loss of carbon dioxide molecule and hydrogen radical to afford the primary product ion at m/z

1011.3378 assigned as [ C57H54O17 + H]+ (Scheme 6B). This latter primary product ion afforded

the secondary product ion at m/z 719.1866 assigned as [C44H30O10 + H]+ by the loss of two

molecules of methanol, two molecules of formaldehyde and three molecules of

methoxyacetylene by RDA mechanism (not necessarily in that order) (Scheme 6B). Moreover,

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The cleavage of the H(8-O-4”)S bond of the precursor protonated molecule 6 lead to the

formation of the primary product ion at m/z 643.1979 assigned as [C32H34O14 + H]+(Scheme 6B).

Scheme 6B.

The protonated molecule at m/z 1201.4098 was assigned as [C61H68O25+H]+, and it was

composed of a hexameric oligomer H(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G and

was attributed as structure 7 (Figure 3B, Figure 10 and Table 1).

The product ion scan of the precursor ion at m/z 1201.4098 afforded the product ion at

m/z 1169.3803 by the loss of a molecule of methanol. This latter ion eliminated a molecule of

formaldehyde to afford the secondary product ion at m/z 1139.3692 (Figure 9).

Figure 10

Concerted loss a molecule of methanol, two molecules of formaldehyde and three molecules of

water from the precursor ion at m/z 1201.4098, lead to the formation of the primary product ion

at m/z 1055.3276, which was assigned as [C58H54O19+H]+ (Scheme 7A). Finally, once more the

presence of the terminal carboxylic acid group was confirmed by the loss of carbon dioxide and

Hydrogen molecule from the precursor ion at m/z 1201.4098 to afford the product ion at m/z

1155.4029 assigned as [C60H66O23 + H]+ (Scheme 7A).

Scheme 7A

Moreover, The secondary product ion at m/z 1139.3692 can lose hydrogen radical, two

molecules of water and one molecule of carbon monoxide to afford the distonic tertiary product

ion at m/z 1074.3444 assigned as [C58H57O20+H]+• (Supplementary information SM5A). This

latter tertiary product ion can lose a molecule of methanol to afford the quaternary product ion at

m/z 1042.3201 assigned as [C57H53O19+H]+• (Supplementary information SM5A). This latter

quaternary product ion afforded the quinary product ion at m/z 942.3012 assigned as

[C53H49O16+H]+• by the loss of a molecule of carbon dioxide, a molecule of formaldehyde and a

molecule of ethyne by RDA mechanism (Supplementary information SM5A). [33,34]

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The cleavage of the precursor ion at m/z 1201.4098 between the two contiguous S residues

affords the product ion at m/z 493.1678 which was assigned as [C24H28O11+H]+ (Scheme 7B). In

addition, the cleavage of the precursor ion at m/z 1201.4098 between the contiguous two G

residues affords the primary distonic radical cation product ion at m/z 824.2849 assigned as

[C42H48O17]+• (Scheme 7B). This last product ion loses either one or two molecules of hydrogen

to afford respectively, the secondary and tertiary product ions at m/z 822.2679 and 820.2523

assigned as [C42H46O17]+• and [C42H44O17] +• respectively (Scheme 7B). Furthermore, the primary

product ion at m/z 824.2849 loses a molecule of ethyne by RDA mechanism to create the

secondary product ion at m/z 798.2684, assigned as [C40H46O17]+• (Scheme 7B). [33,34] Finally, the

precursor ion at m/z 1201.4098 is subjected to a cleavage of the C-4-O aryl ether bond of the

third unit G, followed by the consecutive losses of six molecules of formaldehyde, four

molecules of water and three molecules of methanol (once more, not necessary in that order) to

create the product ion at m/z 479.1253 assigned as [C33H19O9] + (Scheme 7B).

Scheme 7B

Additionally, The secondary product ion at m/z 798.2684 can lose hydroxyl radical, a molecule

of hydrogen and a molecule of water to afford the tertiary product ion at m/z 761.2403 assigned

as [C41H41O15]+. This later tertiary product ion afforded the quaternary product ion at m/z

643.2122 assigned as [C36H35O11]+ by the loss of a molecule of carbon monoxide and three

molecules of formaldehyde. This latter quaternary product ion can loss two molecules of

formaldehyde to afford the quinary product ion at m/z 583.1915 assigned as [C34H31O9]+

(Supplementary information SM5B).

Another CID-MS/MS fragmentation of the protonated molecule 7 can occur by the loss of

methyl radical and ethyne molecule by RDA mechanism to afford the secondary distonic product

ion at m/z 1160.3641 assigned as [C58H63O25 + H ]+• or it can lose one molecule of methanol,

two molecules of formaldehyde and three molecules of water to afford the primary product ion at

m/z 1055.3276 assigned as [C58H54O19+H]+ (Supplementary information SM5C).

This latter primary product ion at m/z 1055.3276 can lose a molecule of water and a molecule of

methanol to afford the secondary product ion at m/z 1005.2899 assigned as [C57H48O17+H]+.

This latter secondary product ion afforded the tertiary product ion at m/z 873.2845 by loss of a

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molecule of carbon monoxide by aromatic ring contraction of the first H residue along with loss

of a molecule of carbon dioxide and two molecules of formaldehyde (Supplementary information

SM5C).

Likewise, the protonated molecule at m/z 1201.4098 can fragments by loss of hydrogen radical,

one molecule of water and one molecule of methanol to afford the primary distonic product ion

at m/z 1150.3601 assigned as [C60H61O23+H]+• or the loss of ethyne molecule by RDA[33,34] to

afford the primary product ion at m/z 1175.3908 assigned as [C59H66O25+H]+ (Supplementary

information SM5D).This latter primary product ion at m/z 1175.3908 afforded the secondary

product ion at m/z 1143.3630 assigned as [C58H62O24+H]+ by the loss of a molecule of methanol.

(Supplementary information SM5D).

Lastly, the protonated molecule at m/z 1217.4123 was assigned as [C61H68O26+H]+ and

was composed of the hexameric oligomer formed of C(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-

4’”)S(8-O-4””)G. This protonated molecule was assigned the structure 8 (Figure 3B, Figure 11

and Table 1).

Figure 11

The product ion scan of the precursor ion at m/z 1217.4123 afforded the primary product

ion at m/z 1187.3901 by the loss of a molecule of formaldehyde, assigned as [C60H66O25+H]+

(Scheme 8A). Similarly, loss of a molecule of methanol from this precursor ion gives the

primary product ion at m/z 1185.3744 assigned as [C60H64O25 + H]+. This latter product ion loses

one molecule of formaldehyde to afford the secondary product ion at m/z 1155.3636 assigned as

[C59H62O24 + H]+ (Scheme 8A).

Moreover, the secondary product ion at m/z 1155.3636 afforded the tertiary product ion at m/z

1055.3124 assigned as [C54H54O22+H]+ by the loss of a molecule of water, a molecule of

formaldehyde and a molecule of ethyne by RDA (Supplementary information SM6A). This

latter tertiary product ion can lose a molecule of carbon dioxide to afford the quaternary product

ion at m/z 1011.3266 assigned as [C53H54O20+H]+. This latter quaternary product ion afforded the

distonic[28,29] quinary product ion at m/z 934.2991 assigned as [C51H49O17+H]+• by the loss of

hydrogen radical, a molecule of carbon monoxide, a molecule of water and molecule of

formaldehyde (Supplementary information SM6A). Moreover, The cleavage of C7-C8 bond of

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the third unit G in the secondary product ion at m/z 1155.3636 lead to the formation of trilignol

tertiary product ion at m/z 496.133 assigned as [C26H24O10]+• (Supplementary information

SM6A)..

Scheme 8A

Similarly, the precursor ion at m/z 1217.4123 can experience a cleavage of G(8-O-4’)G bond to

form the tetramer distonic cation at m/z 824.2839 assigned as [C42H48O17]+•. This latter product

ion undergoes an oxidation by losing two molecules of hydrogen to afford the secondary product

ion at m/z 820.2532 (Scheme 8B). Finally, the cleavage of the C-4-O aryl ether bond of the third

unit G in the precursor ion at m/z 1217.4427, affords the diliginol product ion at m/z 479.1253,

which was assigned as [C33H19O4]+ (Scheme 8B).

Additionally, The primary distonic cation product ion at m/z 824.2839 afforded the secondary

product ion at m/z 796.2881 assigned as [C41H48O16]+• by the loss of a molecule of carbon

monoxide (Supplementary information SM6B). This latter secondary product ion can lose a

hydroxyl radical and a molecule of formaldehyde to afford the tertiary product ion at m/z

749.2760 assigned as [C40H45O14]+. This latter tertiary product ion afforded the quaternary

product ion at m/z 675.2401 assigned as [C37H39O12]+ by the loss of a molecule of water, a

molecule of formaldehyde and a molecule of ethyne by RDA[33,34] mechanism. This latter

quaternary product ion afforded the quinary product ion at m/z 559.1927 assigned as [C32H31O9]+

by the loss of three molecules of formaldehyde and a molecule of ethyne by

RDA[33,34]mechanism (Supplementary information SM6B).

Scheme 8B.

CONCLUSION

In the presented work, we have commenced the first structural investigation of the VRL

that showed that the Saudi date palm wood (Phoenix dactylifera) is a rich source of lignin.

To sum it up, analyses of the VRL of the Saudi date palm wood (Phoenix dactylifera) by

MALDI-TOF-MS (+ ion mode) afforded inter-alia six protonated molecules and two distonic

cations. Although, the elucidation of the construction of this series of molecular ions was

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undeniably challenging to interpret, we managed to obtain excellent blueprints of the different

structures of the VRL oligomers of the SDPW.

The high-energy tandem mass spectrometry analyses of these precursor ions gave

extremely intricate spectra and allowed us to confirm the complicated proposed structures of this

series of lignin oligomers. For that reason, we can say that the VRL of the Saudi date palm wood

(SPW) is composed of units H, G, S and C in different proportions. Furthermore, the MALD-

TOF-MS and high-energy CID-TOF/TOF-MS/MS analyses allowed the identification of the

following six lignin protonated oligomers: HG dimer, HHSSG pentamer, HHHGSG hexamer,

HHHSSG hexamer, HGGSSG hexamer and CGGSSG hexamer. In addition, we noticed the

formation of two distonic cations HGGG and HGSG.

It is important to note that in this manuscript we have described series of various types of

rapid MS/MS fragmentations involved concerted series of RDA rearrangements,[33,34]aromatic

ring contractions and multiple eliminations of formaldehyde, methanol and water.[37] These

MS/MS fragmentations appear to contradict the gas-phase fragmentation behavior described by

Morreel [13,38] and coworkers for the major lignin standards, using APCI-MS/MS with a QIT

instrument.

Once more, we attribute our type of MALDI-TOF/TOF-MS/MS fragmentations, as being

conducted at higher collision energies “in space” and as such, more energetic, than the ones

reported by Morrel and coworkers, which were conducted with lower energy CID-MS/MS in an

IT-MS instrument, operating in MS/MS “in time”. Conversely, be aware that we have also

obtained identical CID-MS/MS results than those performed by MALDI-TOF-MS, when the

analyses of the lignin oligomers were achieved with atmospheric pressure photoionization

measured with an QqTOF-MS/MS instrument and with electrospray ionization measured with an

extra-high resolution Orbitrap MS/MS instrument.[39] Needless to say, that these last two

instruments also operate with low-energies collision dissociation. For these reasons, it will be

imprudent to use the nomenclature described by Morrell and coworkers, as the basis of an

MS/MS fragmentation rule for identifying novel lignin oligomeric structures.

Considering the extremely lability of this series of VRL oligomers in the gas-phase, one

can hardly imagine what happens to them, while being purified by further chemical

manipulation. It is imperative to repeat that the isolation of lignin in its unaltered form, is a

highly unlikely process, due to the relatively harsh extraction conditions and other chemical

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modifications used for releasing and purifying the lignin.[11] For these reasons and as mentioned

before, there is still much dispute on whether any lignin extract adequately represents the native

lignin structure. Consequently, the harsh extraction condition required to release lignin from

lignocellulosic cellular material results in the degradation of the lignin polymeric structure itself.

For comparison sake, these extraction and purification conditions could be viewed as using a

“wrecking ball to break a crystal glass”. The reactivity of the released fragments may lead to

more complex reactive species that can further rearrange and condense to more artefactual

altered polymeric structure. [11]

On the basis of the total absence of structural information on the Saudi Date Palm Wood

(SDPW) lignin in the literature and the complexity of this series of lignin oligomers , it is evident

that the top-down lignomic new sequencing approach, allowed us to reveal this series of novel

oligomers structures, that did not concur with the current knowledge of any lignin structures

proposed [40,41]

To our knowledge, this is the first report on the structure of the lignin

biomolecules extracted from Saudi date palm wood (Phoenix dactylifera) samples.

Acknowledgment

We would like to thank the following; The Umm-El-Qurra University, Kingdom of Saudi Arabia

and the Saudi Cultural Bureau for a graduate fellowship to T. Albishi; the Chemistry

Department, Memorial University of Newfoundland for a graduate fellowship to A. F. Mikhael.

The Department of Fisheries and Oceans for their open Laboratories policy, and La Compagnie

Industrielle de la Matière Végétale (CIMV) France for financial assistance.

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structural analysis of lignins and application of novel tandem mass spectrometric strategies to

determine lignin sequencing. J. Mass Spectrom., 2015; 50: 5-48.

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Substances and Coal, Vol.1, Hofrichter, M., Steinbüchel, A. (Eds). Wiley Blackwell: Michigan,

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Inze´, D., Messens, E., Ralph, J., Boerjan, W., Mass spectrometry-based fragmentation as an

identification tool in lignomics, Anal. Chem., 2010, 82, 8095-8105.

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Preparation Conditions in the Study of Lignin by MALDI Mass Spectrometry, J. Anal. Chem.,

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20. Vijlder, T.D., Valkenborg, D., Lemière, F., Romijn, E.P., Laukens, K., & Cuyckens, F., A

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generation to assist in the identification of unknowns in effect-directed analysis, Anal. Chim.

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molecular structure of wheat straw lignin polymer by atmospheric pressure photoionization

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24. Terashima, N., Hafren, J., Westermark, U., VanderHart, D.L., Nondestructive analysis of lignin

structure by NMR spectroscopy of specifically 13C-enriched lignins. Part. 1.Solid state study of

ginkgo wood. Holzforschung, 2002, 56, 43–50.

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of Chemistry, 2004, 90-91.

26. Tobimatsu, Y., Chen, F., Nakashima, J., Escamilla-Treviño, L.L., Jackson, L., Dixon, R.A.,

Ralph, J., Coexistence but Independent Biosynthesis of Catechyl and Guaiacyl/Syringyl Lignin

Polymers in Seed Coats, Plant Cell, 2013, 25, 2587–2600.

27. Chen, F., Tobimatsu, Y., Jackson, L., Nakashima, J., Ralph, J., Dixon, R.A., Novel seed coat

lignins in the Cactaceae: structure, distribution and implications for the evolution of lignin

diversity, Plant J., 2013, 73, 201–211.

28. Sack, T.M., Cerny, R.L., Gross, M.L., Reactions of cyclic cation radicals with nucleophiles: a

new route to distonic ions., J. Am. Chem. Soc., 1985; 107(15): 4562–4564.

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investigations of gas-phase radical cation chemistry : The two step cycloaddition of the benzene

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30. Zenobi, R., Knochenmuss, R., Ion formation in MALDI mass spectrometry, Mass Spectrom.

Rev.,1998, 17(5), 337-366.

31. Wysocki, V.H., Ross, M.M., Charge-remote fragmentation of gas-phase ions: mechanistic and

energetic considerations in the dissociation of long-chain functionalized alkanes and alkenes. Int.

J. Mass Spectrom. Ion Processes. 1999; 104(3): 179-211.

32. Gross, M. L., Charge-remote fragmentation: an account of research on mechanisms and

applications, Int. J. Mass Spectrom, 2000; 200: 611-624.

33. Pandey, R., Chandra, P., Srivastva, M., Arya, K.R., Shukla, P.K., Kumar, B. A rapid analytical

method for characterization and simultaneous quantitative determination of phytoconstituents in

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34. Longo, E., Rossetti, F., Scampicchio, M., Boselli, E., Isotopic Exchange HPLC-HRMS/MS

Applied to Cyclic Proanthocyanidins in Wine and Cranberries, J. Am. Soc. Mass Spectrom.,

2018; 29: 663-674.

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spectrometric characterization of 2,5-dihydroxybenzoic acid-induced reductive hydrogenation of

oligonucleotides on cytosine residues, J. Mass Spectrom., 2000; 35: 1025-1034.

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https://www.sciencedirect.com/science/article/pii/S0040402001880856#!

https://www.sciencedirect.com/science/journal/00404020

https://www.sciencedirect.com/science/journal/00404020/42/22

http://pubs.rsc.org/EN/results?searchtext=Author%3ARenu%20Pandey

http://pubs.rsc.org/EN/results?searchtext=Author%3APreeti%20Chandra

http://pubs.rsc.org/EN/results?searchtext=Author%3AMukesh%20Srivastva

http://pubs.rsc.org/EN/results?searchtext=Author%3AK.%20R.%20Arya

http://pubs.rsc.org/EN/results?searchtext=Author%3APraveen%20K.%20Shukla

http://pubs.rsc.org/EN/results?searchtext=Author%3ABrijesh%20Kumar

For Peer Review

25

36. Tajiri, M., Takeuchi, T., & Wada, Y. Distinct features of matrix-assisted 6 μm infrared laser

desorption/ionization mass spectrometry in biomolecular analysis. Anal. Chem., 2009; 81(16):

6750-6755.

37. Demarque, D.P., Crotti, A.E., Vessecchi, R., et al., Fragmentation reactions using electrospray

ionization mass spectrometry: an important tool for the structural elucidation and

characterization of synthetic and natural products, Nat. Prod. Rep., 2016, 33(3), 432–455.

38. Morreel, K., Dima, O., Kim, H., Lu, F., Niculaes, C., Vanholme, R., Dauwe, R., Goeminne, G.,

Inze´, D., Messens, E., Ralph, J., Boerjan, W., Mass Spectrometry-Based Sequencing of Lignin

Oligomers, Plant Physiol., 2010; 153: 1464-1478.

39. Abanoub Mikhael, Tasahil Albishi, Michel Delmas, Joseph Banoub. Unpublished Results to be

submitted to Rapid Commun. Mass Spectrom.

40. Chakar, F.S., Ragauskas, A.J., Review of current and future softwood kraft lignin process

chemistry. Ind. Crops and prod., 2004; 20(2): 131–141.

41. Grabber, J., How Do Lignin Composition, Structure, and Cross-Linking Affect Degradability? A

Review of Cell Wall Model Studies., Crop Sci., 2005; 45(3): 820–831.

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Table 1. Identification of lignin Oligomers Peaks in Mass Spectrum of extracted from SDPW Wood using MALDI-TOF-MS

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LIST OF LEGENDS

TOP–DOWN LIGNOMIC MALDI-TOF-TANDEM MASS SPECTROMETRY ANALYSIS OF

LIGNIN OLIGOMERS EXTRACTED FROM DATE PALM WOOD

FIGURES

Figure 1: Solid-State CP/MAS 13C NMR spectrum of the extracted Saudi date palm wood lignin

Figure 2A. MALDI-TOF-MS (+ ion mode) of the extracted VRL Saudi Date Palm Wood recorded from m/z 100 to m/z 1000.

Figure 2B. MALDI-TOF-MS (+ ion mode) of the extracted VRL Saudi Date Palm Wood recorded from m/z 1000 to m/z 2000.

Figure 3A: Structures of the identified VRL oligomers extracted from for the Saudi Date Palm Wood

Figure 3B: Structures of the identified VRL oligomers extracted from for the Saudi Date Palm Wood

Figure 4. The high-energy CID-MS/MS of the protonated molecule [C19H24O8 + H]+ at m/z 381.1541

Figure 5. The high-energy CID-MS/MS of the distonic [C39H43O15 + H]+• at m/z 752.2699.

Figure 6. The high-energy CID-MS/MS of the distonic cation [C40H43O16 + H]+• at m/z 780.2611.

Figure 7. The high-energy CID-MS/MS of the protonated molecule [C50H52O19 + H] + at m/z 957.3162.


Figure 9. The high-energy CID-MS/MS of the protonated molecule [C58H54O19 + H] + at m/z 1055.3391

Figure 10. The high-energy CID-MS/MS of the protonated molecule C61H68O25 + H] + at m/z 1201.4098.


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SCHEMES

Scheme 1. Tentative fragmentation pattern of the product ion scan of the protonated molecule 1 at m/z 381.1541.

Scheme 2A. Tentative fragmentation pattern of the product ion scan of the distonic cation 2 at m/z 752.2699.

Scheme 2B. Tentative fragmentation pattern of the product ion scan of the distonic cation 2 at m/z 752.2699.

Scheme 3A. Tentative fragmentation pattern of the product ion scan of the distonic cation 3 at m/z 780.2661.

Scheme 3B. Tentative fragmentation pattern of the product ion scan of the diatonic cation 3 at m/z 780.2661.

Scheme 4A. Tentative fragmentation pattern of the product ion scan of the protonated molecule 4 at m/z 957.3162.

Scheme 4B. Tentative fragmentation pattern of the product ion scan of the protonated molecule 4 at m/z 957.3162.



Scheme 5C. Tentative fragmentation pattern of the product ion scan of the protonated molecule 5 at m/z 1039.3382.







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[ C50H52O19 + H ]+

Calculated (m/z): 957.3181Observed (m/z) : 957.3162

OMe

OHO

OMe

O

HHO

OMe

O

HO

HO

COOH

HO

HO

O

[ C39H43O15 + H ]+•


[ C40H43O16 + H ]+•


HO

OMe

O

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+


(4)

(2) (3)

(1)

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

COOH

OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O


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123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer ReviewOMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H ]+


OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+


OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

COOH

HO

MeO

+ HHO

HO

[ C58H54O18 + H ]+


(7)(6)

(5) [ C61H68O26 + H ]+


(8)

OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

OH


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Figure 5. The high-energy CID-MS/MS of the protonated molecule [C39H43O15 + H]+• at m/z 752.2699

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For Peer ReviewFigure 10. The high-energy CID-MS/MS of the protonated molecule C61H68O25 + H] + at m/z 1201.4098

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For Peer ReviewFigure 11. The high-energy CID-MS/MS of the protonated molecule [C61H68O26 + H] + at m/z 1217.4123

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SCHEMES

O

OH

HO

+ H

[ C12H10O3 + H ]+

m/z 203.0697

OMe

O

O

+ H

OH

HO

OMe

OHO

O

HO

OH

[ C18H14O4 + H ]+

m/z 295.0952[ C19H21O7 + H ]+•

m/z 362.1350

OH

O

HO

HO

+ H

OH

O

HO

+ H

[ C12H8O3 + H ]+

m/z 201.0538

[ C12H10O4 + H ]+

m/z 219.0643

- H2 O

HO

OMe

O

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+

m/z 381.1541

- 3 x H2O- CH3OH - 2 x CH 3O

HOMe

OH

--

OMe-- CH3OH

HO

HO

OH

-

- 2 x CH3OHOMe

OH

-

-

- H2O

OH

HO

HO+ H

[ C5H6O3 + H ]+

m/z 115.0387

OH

O

HO+ H

- H2

OH

O

HO+ H

[ C5H4O3 + H ]+

m/z 113.0231

OH

HO

HO+ H

Not Observed

- H2

- 2 x H2O

- CH3OH

- 2 x CH3OHOMeOH

--

RDA

+ H

- H •

Scheme 1. Tentative fragmentation pattern of the product ion scan of the protonated molecule 1 at m/z 381.1541

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OMe

OHO

OMeO

H+

HO

OMeO

HO

O

HO

COOH

HO

HO

OMe

OHO

OMeO

H+

HO

OMeO

HO

O

COOH

HO

HO

Hydrogenation due to matrix DHBgives higher product ions at m/z

711, 713, 715

- •OH

- C2H2

OHO

OMeO

H+

HO

OMeO

HO

COOH

HO

HO

[ C31H34O12 + H ]+

m/z 599.2092

- CO

- C2H2

OMe

-

[ C39H43O15 + H ]+•

m/z 752.2699[ C37H40O14 + H ]+

m/z 709.2467

OMe

OH

COOH

H+

OH

COOH

H+- CH2O

Not observed

- H2

[ C9H6O3 + H ]+

m/z 163.0384

H+

OH

HO

O

HO

[ C9H11O4 + H ]+•

m/z 184. 0724

- •OH

- CO

- 2 x C2H2

OMe

-

OHHO

OMeO

HO

HO

HO

- CH2O

- 2 H2

Not Observed

OHHO

O

HO

HO

HO

[ C14H14O6 ]+•

m/z 278.0773

RDA

Scheme 2A. Tentative fragmentation pattern of the product ion scan of the protonated molecule 2 at m/z 752.2699

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OMe

OHO

OMe

O

H+HO

OMe

O

HO

O

HO

COOH

HO

HO

- H2

- C2H2

HO

O

H+

HO

O

HO

HO

HO

[ C24H28O7 + H ]+

m/z 429.1887

OMe

OHO

OMe

O

H+

HO

OMe

O

HO

O

COOH

HO

HO

[ C37H40O14 + H ]+

m/z 709.2467

MeO

O

H+

HO

OMe

O

HO

O

HO- H2

MeO

O

H+

HO

OMe

O

HO

O

HO

Not Observed[ C20H20O8 + H ]+

m/z 389.1209

OHO

O

H+

HO

O

HO

HO

HO

[ C28H30O8 + H ]+

m/z 495.1979

[ C39H43O15 + H] +•

m/z 752.2699

COOH

-

- CO- 2 x CH2O

OMe-

OHO

O

H+

HO

O

HO

HO

HO

[ C26H28O8 + H ]+

m/z 469.1831

OHO

O

H+

HO

O

HO

HO

HO

[ C28H28O8 + H ]+

m/z 493.1833

RDA

OMe

OHO

OMeO

H+HO

OMeO

HO

O

HO

HO

HO

[ C38H42O13 + H ]+

m/z 707.2667

- CO2

( RDA)

- •OH

- H•

- •OH

- C2H2

Scheme 2B. Tentative fragmentation pattern of the product ion scan of the protonated molecule 2 at m/z 752.2699

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OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

OH

HO

HO

O

H+

OMe

OH

H+

OH

COOH

H+

[ C9H6O3 + H ] +

m/z 163.0388

[ C40H43O16 + H ]+•

m/z 780.2611

- CH2O

OMe

OHO

OMe

O

H+HO

OMe

O

CHO

HO

HO

MeO

O

- •OH

- C2H2

Hydrogenation byDHB gives m/z 739

Not observed

OH

HO

OH

HO

HO

OH

HO

Hydrogenationdue to MatrixDHB

[ C4H6O2 ]+•

m/z 86.0362

Not observed

Not Observed

RDA- CH3OH

- CO2

- H•

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

COOH

COOH

- H2COOH

Not Observed

Hydrogenation byDHB

OH

HO

HO

O

H+

[ C9H11O4 + H ] +•

m/z 184.0724

[ C39H42O14 + H ]+

m/z 735.2613

[ C38H40O15 + H ]+

m/z 737.2399


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OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

[ C40H43O16 + H ]+•

m/z 780.2611

- H•

OMe

-

OMe

OHO

OMe

O

H+HO

O

HO

HO

HO

HO

MeO

O

[ C37H38O15 + H ]+

m/z 723.2236

OMe

OHO

OH+

HO

O

CHO

HO

HO

MeO

- CO- C2H2

OMe

- 3 x

[ C31H32O12 + H ] +

m/z 597.1931

- CO2

- 2 x CH2O

OHO

O

H+HO

O

CHO

HO

HO

[ C28H28O8 + H] +

m/z 493.1821

- CO2

COOH COOH

COOH

- CH3OH

OHO

O

H+

O

HO

HO

HO

HO

MeO

O[ C29H27O10 + H ]+•

m/z 536.1636

- •OH


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OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

OH

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

OH

HO

MeO

O

[ 421 ]

- CO2

OMe

OHO

OMe

OH

HO

H+- CO

OMe

OHO

OMe

HO

H+

[ C18H22O5 + H ]+

m/z 319.1522

- CH2O

- H2O- CO

- H2O - CH2O

[ C49H49O17 + H ]+•

m/z 910.2988[ C50H52O19 + H ]+

m/z 957.3162

[ C50H50O18 + H ]+

m/z 939.3014[ C49H48O17 + H ]+

m/z 909.2912

[ C19H22O6 + H ]+

m/z 347.1471

- H2O

- CH2O

- H•


Page 48 of 114



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For Peer Review

OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

[ C50H52O19 + H ]+

m/z 957.3162

OMe

O

OMeO

H+

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

OMe

O

O

O

O

CO

O

O

OMe

O

O

O

O

O

H+- 2 X CH3OH - 4 x CH2O

- 5 x H2O

- 4 x CH2O

- 5 x H2O

- 2 x CH3OH

[ C48H44O17 + H ]+

m/z 893.2615

[ C44H27O8 ]+

m/z 683.1655

[ C32H21O6 + H] +•

m/z 502.1368


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For Peer Review

OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

COOH

HO

MeO

+ H

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.3371

OMe

O

OMeO

MeO

O

O

O

O

HO

CO

MeO

[ C46H37O10]+

m/z 749.2343

HO

HO

HO

[ C58H54O18 + H ]+

m/z 1039.3382

HO

HO

- CO -2 x H2O- CO

- 4 x CH2O- 3 x C2H2

O

O

O

O

O

O

HO

HO

[ C57H51O15 ]+

m/z 975.3177

- CO

-2 x H2O

- 2 x CO

- 4 x CH2O

- 3 x C2H2

- 2 x H2O- CO

- 2 x H2O

- 4 x CH2O

- 3 x C2H2

- CO2

OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

HO

MeO

+ HHO

HO

[ C57H54O16 + H]+

m/z 995.3432

HO

HO

HO

HO

( Loss of H2 givesproduct ion at m/z 973)


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OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.3371

HO

HO

- H2

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO

+ H

HO

HO

[ C57H52O17 + H ]+

m/z 1009.3213

- 4 X CH2O

- 2 X H2O

O

O

O

O

O

O

HO

CO

O

HO

HO

[ C53H41O11 ]+

m/z 853.2593

- H2

- 4 X CH2O

- 2 X H2O


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OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.3371

HO

HO

O

MeO

O

O

O

O

HO

MeO+ H

HO

HO

[ C39H34O10 + H ]+

m/z 663.2196

OH

O

O

O

HO

HO

[ C26H22O6 ]+•

m/z 430.1392

OMe

OHO

OMeO

MeO

O

O

O

COOH

HO

MeO

+ H

HO

HO

[ C47H46O15 + H ]+

m/z 851.2856

OMe

OHO

OMeO

MeO

O

O

OH

O

COOH

HO

MeO+ H

HO

HO

[ C49H48O16 + H ]+

m/z 893.2962

RDA

RDA

Scheme 5C. Tentative fragmentation pattern of the product ion scan of the protonated molecule 5 at m/z 1039.3382

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OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+

m/z 1055.3391

495

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C57H54O18 + H ]+

m/z 1027.3319

OMe

O

OMe

OMeO

O

O

O

O

HO

COOH

+ H

[ C53H38O12 + H ]+

m/z 867.2398

OMe

OHO

OMe

OMeO

HO

COOH

HO

HO

+ H

[ C24H30O11 + H ]+

m/z 495.1832

- CO

- 2 X CH2O

- 2 X CH3OH

- 2 X H2O

- CO - 2 X CH2O

- 2 X CH3OH - 2 X H2O

- C2H2

OMe

OHO

OMe

OMeO

HO

COOH

HO

HO

+ H

[ C22H28O11 + H ]+

m/z 469.1673


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For Peer Review

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+

m/z 1055.3391

- CO2

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

HO

HO

MeO

+ H

[ C57H54O17 + H ]+

m/z 1011.3378

[647]

Notobserved

- 2 H2

OMe

OHO

OMe

OMeO

HO

OMe

OH

COOH

HO

HO

MeO

+ H

[ C32H34O14 + H ]+

m/z 643.1979

O

O

O

O

O

O

O

HO

HO

HO

+ H

OMe

- 3 x

- 2 x CH2O

- 2 x CH3OH

[ C44H30O10 + H ]+

m/z 719.1866


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OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMeO

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H]+

m/z 1201.4098

OMe

OHO

OMeO

MeO

HO

OMeO

OMeO

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C59H62O23 + H ]+

m/z 1139.3692

- CH3OH

OMeO

HO

OMeO

MeO

HO

OMeO

OMeO

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C60H64O24 + H ]+

m/z 1169.3803

OMe

- CH2O

- CH3OH- CH2O

OMe

OHO

OMeO

MeO

HO

OMeO

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

- CH2O

- 3 x H2O

[ C58H54O19 + H ]+

m/z 1055.3276

- CO2

- H2

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMeO

HO

HO

OMeO

HO

HO

OH

HO

HO

HO

MeO

+ H

[ C60H66O23 + H ]+

m/z 1155.4029

- CH3OH- 2 x CH2O

- 3 x H2O


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OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H]+

m/z 1201.4098

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H48O17 ]+•

m/z 824.2849

OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H44O17 ]+•

m/z 820.2523

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H46O17 ]+•

m/z 822.2679

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C40H46O17 ]+•

m/z 798.2684

- C2H2

- H2

OMeO

HO

OMeO

MeO

HO

COOH

HO

HO

+ H

[ C24H28O11 + H ]+

m/z 493.1678

[495]- H2 [827]

- 6 x CH2O

- 4 x H2O- 3 x CH3OH

O

O

O

CO

[ C33H19O4]+

m/z 479.1253[ 842 ]

- H2O

- H2

- H2O

- C2H2

- 2 x H2

Not Observed Not Observed

Not Observed


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OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

- CH2O

OMe

OHO

OMe

OMeO

HO

OMe

O

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

- CH2O - CH3OH

- CH3OH

- CH3OH

- CH2O

OH

[ C61H68O26 + H ]+

m/z 1217.4123

OH

+ H

[ C60H64O25 + H ]+

m/z 1185.3744

OMe

OHO

OMe

OMeO

HO

OMe

O

OMe

O

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

OH

+ H

[ C59H62O24 + H ]+

m/z 1155.3636

OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

OH

[ C60H66O25 + H ]+

m/z 1187.3901


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OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ HOMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H48O17 ]+•

m/z 824.2849

OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H44O17 ]+•

m/z 820.2523

[827]- 6 x CH2O


O

O

O

CO

[ C33H19O4]+

m/z 479.1253

[ 842 ]

- H2O

- 2 x H2

Not Observed

Not Observed

OH

[ C61H68O26 + H ]+

m/z 1217.4123


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1

TOP–DOWN LIGNOMIC MALDI-TOF-TANDEM MASS SPECTROMETRY

ANALYSIS OF LIGNIN OLIGOMERS EXTRACTED FROM DATE PALM WOOD

Tasahil Albishi,a Abanoub Mikhael ,b Fereidoon Shahidi,a Travis Fridgen,b Michel Delmas,c

Joseph Banouba,b, d*

a Department of Biochemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada

b Department of Chemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada

cUniversity of ToulouseInp-EnsiacetChemical Engineering Laboratory4, Allée Emile Monso31432, Toulouse, France

d Science Branch, Special Projects, Fisheries and Oceans Canada, St John’s, NL, A1C 5X1, Canada

These two authors (graduate students) have contributed equally in this research and have been

listed in alphabetically order

*Correspondence to: Joseph Banoub, Science Branch, Special Projects, Fisheries and Oceans Canada, St John’s, NL, A1C 5X1, Canada and Department of Chemistry, Memorial University of Newfoundland, St John’s, Newfoundland, A1C 5X1, Canada. E-mail: [email protected]

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Abstract

Rationale: We report for the first time the top-down lignomic analysis of the virgin released

lignin (VRL) oligomers obtained from the Saudi date palm wood (SDPW), using a MALDI-

TOF/TOF instrument. In addition, we are proposing new CID-MS/MS fragmentation routes for

this series of unreported VRLs.

Methods: We have used MALDI-TOF-MS direct analysis of the lignin oligomers mixture

without any chromatographic pre-separation. High-energy CID-MS/MS analyses were used to

confirm the precursor ions structures.

Results: Six lignin oligomer protonated molecules were identified as: [C19H24O8+ H]+ composed

of H(8-O-4)G; [C50H52O19+H]+ composed of H(8-O-4)H(8-O-4’)S(8-O-4”)S(8-O-4’”)G;

[C58H54O18+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G;

[C58H54O19+H]+ composed of H(8-O-4)H(8-O-4’)H(8-O-4”)S(8-O-4’”)S(8-O-4””)G;

[C61H68O25+H]+ composed of H(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G and

[C61H68O26+H]+ composed of C(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G units. Two

distonic cations were identified as [C39H43O15+ H]+• and [C40H43O16+H]+• deriving from two

tetrameric lignin oligomers. The high-energy tandem mass spectrometry analyses allowed the

confirmation of the proposed structures of this series of lignin oligomers.

Conclusion: To our knowledge, this is the first elucidation of the unknown lignin structure of the

Saudi seedling date palm wood that was accomplished using top-down lignomic strategy that

was never published. The complex high-energy CID-MS/MS fragmentations presented herein

are novel and have never been described before.

INTRODUCTION

Lignin is the second most abundant biopolymer in nature after cellulose; it is a product of

enzymatic oxidative polymerization of three monomeric aromatic compounds (monolignols):

coniferyl (H), sinapyl (S), and p-coumaryl (G) alcohols. It is found in all vascular plants, mostly

between the cells, as well as within the cells and in the vegetable cell walls (CWs).[1] For almost

a century, the structure of lignin was designated as a complex polymer composed of irregular

branched units.[2-4] Presently, lignin is viewed as a promising commercial source of a wide range

of aromatic compounds, which can be used as an alternative to fossil hydrocarbons. [5-7]

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There is still much debate on whether any lignin extract adequately represents the native

structure, and it has been proposed, that the several different chemicals, enzymatic and

mechanical extraction methods, are accountable for the major structural divergence that occurs

after extraction and isolation. [4,7] Recently, it was established that either strong acid or basic

depolymerization extraction method can cause cleavage of ester and ether bonds, creating

reactive species. These latter species alternately react further, to yield more complex and

rearranged condensed lignin polymer/oligomer structures. [7,8]

Traditionally, it has been repetitively suggested that structural analysis of lignin should

be based on pure samples, However, preparing pure samples of unchanged lignin is not an easy

endeavor.[9A,B] For this reason, the structural determination of lignin is indeed a more challenging

task than that with other biopolymers. [9A,B]As a consequence it appears that the only logic step to

determine the natural structure of lignin is to isolate it from the vegetal matrix, without causing

any structural change. [10A,B;11]

Lately, we have proposed a new paradigm, which indicated that the intact natural lignin

oligomers present in the lignocellulosic biomass, were not actually either one and/or series of

similar biomolecules, like an individual cellulose fiber; instead, they were composed of a series

of different length linear related biosynthesized oligomers.[11] These oligomers may be formed

either from homo-oligomers repeating units and/or could be hetero-oligomers formed by mixed

units.[11] These have never been completely described in their unprocessed natural form. In

addition, it is proposed that lignins present in the lignocellulosic biomass are attached by either

ether and/or ester covalent links, in a crisscross manner, to both cellulose and hemicellulose

fibers, forming a glycolignin network. [11] So far, shorter oligomers of lignin can be extremely

useful in providing the blueprints on how the full lignin polymer is constructed.

It is well known that mass spectrometric analyses are the only promising methods

offering novel possibilities for the sequencing lignin oligomers and for interpreting the plant

‘lignome’. [11,12] Please note that the lignome was defined to represent the ensemble of all

biosynthetic phenolics, metabolites and (neo)lignan biosynthetic pathways and their derivatives,

as well as the lignin oligomers. [11,13]

In this manuscript, we present the structural elucidation of series of lignin bio-oligomers

which were extracted by “La Compagnie Industrielle de la Matière Végétale” (CIMV)solvolysis

technique. [14,15] This last technique appears to be the finest technique for lignin separation, and

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4

this is due to its capabilities of being efficient and allowing the choice of simple organic solvents

such as acetic acid/formic acid/water combination. The structural identification of the virgin

released lignins (VRL) was done by using MALDI-Assisted Laser/Desorption Ionization time-

of-flight mass spectrometry (MALDI-TOF-MS) in conjunction with tandem mass spectrometry

using high energy collision dissociation CID-TOF/TOF-MS/MS (MS/MS in-space) instrument.

EXPERIMENTAL

Samples

The samples date palm wood (Phoenix dactylifera) examined were collected manually

from the Salman Alfarsi garden, Almadinah, Saudi Arabia. All the samples were frozen and

dried for 7 days at -48°C and 30 x 10-3 mbar (Freezone 6, model 77530, Labconco Co., Kansas

City, MO). The dried samples were then grounded, vacuum packed and stored in a freezer at -

20ºC.

Lignin Oligomers Isolation

The Saudi Date Palm Wood (SDPW) lignin was extracted using the CIMV procedure which

selectively separates the cellulose, hemicellulose and lignin, and allows the destructing of the

vegetable matter at atmospheric pressure (Lignin yield 17%). [14,15] The catalyst-solvent system

used was a mixture of formic acid/acetic acid/water (30/50/20) which produced after

precipitation with water and filtered the Saudi Date Palm Wood (SDPW) lignin. Approximately

0.1mg of the purified lignin was dissolved in 1mL dioxane / methanol/chloroform (1:1:1) for MS

analysis.

MALDI-TOF-MS Analysis

Applied Biosystems/MDS SCIEX 4800 MALDI TOF/TOF™ Analyzer (MDS Sciex, 71 Four

Valley Dr., Concord, Ontario, Canada L4K 4V8) was used in this experiment for the analysis of

the Saudi Date Palm Wood (SDPW) lignin. In this analysis, 1 mg of the lignin sample was

dissolved in 1 mL of the dioxane/ methanol/chloroform (1:1:1) and 2,5- dihydroxy benzoic acid

(DHB) was used as matrix for the analysis. The MS data was acquired in the mass range 100 to

2000 m/z in the positive ion mode. The mass spectra instrument was equipped with Nd: YAG

200-Hz laser. The accelerating potential was 25KV. The MALDI plate was prepared by spotting

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1 μL of a 20mg/mL solution of DHB (dissolved in acetone, 0.1% trifluoroacetic acid) and then

dried at room temperature (the use of acetone allows a good homogeneity of the matrix in the

spot). Then, an aliquot of 1 μL of sample was spotted on the top of the dried matrix and allowed

to dry before the MALDI-TOF-MS experiments. For MS analysis, mass spectra were the sum of

400 laser shots and acquired in reflectron mode. For high-energy CID-MS/MS analysis, mass

spectra were the sum of 600 laser shots, collision energy of 1 keV, nitrogen as the collision gas

to induce high energy CID-fragmentation. The following standards were used to calibrate the

mass spectrometer: des-Arg1-Bradykinin, [C44H61N11O10], M.Wt. 904.0245 from Enzo Life

sciences, Inc (Farmingdale, NY 11735, USA); Angiotensin 1, [C62H89N17O14], M.Wt. 1296.4779,

from Tocris Bioscience (614 McKinley Place N.E., Minneapolis, Minnesota 55413) USA.

Solid state 13C-NMR of the Saudi Date Palm Lignin

The 13C-NMR spectrum was obtained at 298 K using a Bruker Avance II 600 spectrometer,

equipped with a SB Bruker 3.2 mm MAS triple-tuned probe operating at 150.97 MHz for 13C.

Chemical shifts (δ) were referenced to tetramethylsilane (TMS) using adamantane as an

intermediate standard for 13C. The samples were spun at 20 kHz. Cross-polarization (CPMAS)

spectra were collected with a Hartmann-Hahn match at 62.5 kHz and 100 kHz 1H decoupling,

with a contact time of 2 ms, a recycle delay of 2s and 2 k scans.

RESULTS AND DISCUSSION

In lignomics research, no explicit sequencing methods exist to establish the primary

structure of complex and simple lignin oligomers. Researchers are required to synthesize

authentic compounds as standards to enable verification and comparison with the MS/MS

scheme obtained by an unknown compound. [11,13]

It is imperative to mention that until today, no innovative sequencing of any natural

lignin oligomers using MS/MS method has been ever discussed to unravel unknown new

structures form newly extracted virgin lignins.[11] By analogy with proteomics, a new concept of

top-down lignomic strategy employing an MS/MS strategy, was introduced by Banoub et al.[11]

The top-down lignomic strategy was described as the identification by MS and MS/MS analysis

of the native extracted lignins (VRL) obtained directly from the destructing of the vegetable

matter. The top–down lignomic strategy will permit the identification of all the components of

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the intact non-modified virgin lignins (VRL) being analyzed.[11] Moreover, the top– down

approach enables direct measurement of the intact mass of the heterogeneous lignin oligomers as

well as product ions information relating to the original oligomer lignin sequences. [11]

Matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry has been used

in various structural lignin research. [16-19] Notwithstanding, the unquestionable advantages of

MALDI, this method is not currently very much used for the structural analyses of lignin for the

lack of ionization efficiency of the lignin biopolymer achieved using conventional matrices. [16-19]

In this manuscript, we have performed the direct analysis of the lignin oligomers mixture

without any chromatographic pre-separation and the determination of the precursor ions

chemical formulae was accomplished by using some basic rules in MS and MS/MS explained

by Thomas De Vijlder et al. [20], The chemical structures were calculated from the MALDI-TOF-

MS according to the heteroatom, isotopic distributions of the precursor ions[21,22], and the

possible combinations of the different lignin units The level of unsaturation or the double bond

equivalent (DBE) for either of the characterized molecules and/or precursor ions, was calculated

to determine the number of unsaturation such as double/triple bonds and/or ring systems.[20]

The solid state 13C-NMR spectrum (Figure 1) was measured to pinpoint the various

diagnostic functional groups in the proposed chemical structures of the SDPW oligomers.

Therefore, we assigned the signal at 56 ppm to the methoxyl groups in different lignin oligomers.

The resonance region varying between 115 and 161 ppm can be assigned either to the aromatic

carbons and/or any conjugated aliphatic C=C such as -C=C-COOH [23]. The signal at 182ppm

was assigned to ketone functional groups that appear in some of our proposed chemical structure.

The signal at 171ppm was attributed to the carboxyl group carbon [23]. The signals in the range

of 62-74 ppm were assigned to different primary and secondary alcohols [25]. Finally, the signals

between 83 to at 104 ppm can be assigned to C-8 (beta carbon) and alkyne carbons arising from

double eliminations occurring on the C7-C8 of the chain present in one of the proposed precursor

ions described below.[24, 25] It is essential to understand that this 13C-NMR spectrum verifies that

even the mild CIMV extraction process, can perhaps affect the original structure of the lignin

oligomers. Consequently, this alkyne group that links two aromatic rings can be easily formed by

the elimination of water molecule followed by the loss of methanol molecule which is very

common in MS/MS fragmentation pathway discussed in this manuscript.

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The conventional scanning MADLI-TOF-MS analysis of the SDPW showed a series of

protonated molecules inter alia at 381.1; 752.2; 780.2; 957.3; 1039.3; 1055.3; 1201.4 and 1217.4

(Table 1, Figure 2A and 2B).

Figure 2A and 2B

The generation of MALDI-TOF-MS scan (+ ion mode) and various high energy

dissociation tandem mass spectrometry (CID-MS/MS) analyses will provide series of protonated

molecules and MS/MS diagnostic product ions (Table 1), which will serve as a tool for obtaining

high-quality mass spectra of VRL of seedling date palm wood (Phoenix dactylifera) suitable for

structural studies of the analyte biopolymer.

According to the MALDI-TOF-MS study, we deduced that the SDPW lignin was

composed of H:G:S:C in a ratio of ca 12:16:10:1 residues. It is to be noted that recently Ralph

and coworkers reported the evidence of a catechyl lignin homopolymer (C lignin) derived solely

from caffeyl alcohol in the seed coats of several monocot and dicot plants. This group previously

identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins,

and occasionally both[26,27]. Therefore, our presented work is the first report of a lignin series

containing H/G/S and C units. Consequently, we have identified six VRL protonated molecules

and two distonic VRL cations as follows (Figure 3A , Figure 3B and Table 1).

Figure 3A and 3B

Table 1

We begin with the identification of the chemical structure of the protonated molecules at

m/z 381.1, tentatively assigned as [C19H24O8 + H]+ which was composed of the H(8-O-4‵)G

dilignol having the structure 1 (Figure 3A , Figure 4 and Table 1).

The product ion scan of m/z 381.1 gave three major diagnostic product ions inter alia at

m/z 201.05; 219.06; and 362.13 which were assigned respectively as [C12H8O3 + H]+ ; [C12H10O4

+ H]+ and [C19H21O7 + H]+• (Figure 4).

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Figure 4

It is important to notice the formation of the primary distonic protonated radical product ion at

m/z 362.1 (distonic cation) which possessed an even m/z. [28,29] Please bear in mind that the

formation of molecular radical ions has been documented to occur for MALDI-TOF-MS

analysis.[30] .Furthermore, this distonic cation can be formed by the remote charge fragmentation

mechanism [31,32], occurring by the loss of a molecule of water and hydrogen radical from the

precursor ion m/z 381.1. The consecutive eliminations from the precursor ion at 381.1 of two

methanol molecules along with retro-Diels Alder reaction (RDA) [33,34] occurring in both H and

G units (loss of hydroxyacetylene and methoxyacetylene) lead to the formation of the primary

product ion at m/z 219.06. Bear in mind that the formation of secondary product ion at m/z

201.05 was created from the primary product ion m/z 362.13. The high energy CID-MS/MS of

the precursor ion m/z 381.1 and the remaining MS/MS fragmentations are shown in Scheme 1.

Scheme 1

The distonic cation at m/z 752.2 was assigned as [C39H43O15 + H]+• and existed as the

lignin tetramer H(8-O-4)G(8-O-4’)G(8-O-4”)G having the structure 2 (Figure 3A, Figure 5 and

table 1).

The product ion scan of the distonic cation 2 at m/z 752.2 afforded the product ion at m/z

709.24, which was formed by the loss of one ethyne molecule C2H2 eliminated by a retro-Diels

Alder reaction (RDA)[33,34] and one hydroxyl radical, and it was assigned as [C37H40O14+H]+ .

Figure 5

It is interesting to note the successive formation of the product ions at m/z 711.26; 713.27; and

715.29 (Scheme 2A). These latter product ions were assigned as [C37H42O14+H]+,

[C37H44O14+H]+ and [C37H46O14+H]+ and appeared to be formed by hydrogenation caused by the

DHB matrix. This type of DHB matrix hydrogenation has been previously reported by

others.[35,36] The product ion at m/z 709.24 can lose a molecule of carbon monoxide and subjected

to another two retro-Diels Alder reactions to afford the ion at m/z 599.20 (Scheme 2A). [ 33,34] It is

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important to understand that the order of these eliminations could occur simultaneously or in a

stepwise fashion and this was not studied further

Scheme 2A

Moreover, the precursor distonic ion 2 can undergoes a concerted retro-Diels-Alder reaction by

cleavages of the C1-C2 and C5-C6 aromatic bonds of the fourth unit G and by cleavages of the

C1-C2 and C3-C4 aromatic bonds of the third unit G, along with successive losses of carbon

monoxide, hydroxyl radical and two formaldehyde molecules to afford the product ion at m/z

495.19 (Scheme 2B). The exact order of these MS/MS gas-phase eliminations has not been

established. This latter secondary product ion at m/z 495.19 can either lose a molecule of

hydrogen to afford the secondary product ion at m/z 493.18 and/or it can be subjected to another

retro-Diels-Alder reaction by the loss of a molecule of ethyne to afford the ion at m/z 469.18

(Scheme 2B). In addition, the product ion at m/z 495.19 can also be subjected to cleavage of the

8-O-4 bond between the upper two units to afford the secondary product ion at m/z 429.18

(Scheme 2B).

Scheme 2B

The presence of carboxylic acid group was deduced by finding the product ion at m/z 707.26

assigned as [ C38H42O13 + H]+ which was formed by the loss of carbon dioxide and hydrogen

radical from the precursor ion at m/z 752.2 (Scheme 2B). Also, the precursor distonic cation[28,29]

2 produced the primary product ion at m/z 735.25 assigned as [C39H42O14+H]+ by the loss of a

hydroxyl radical (Supplementary Material, SM1). This latter primary product ion eliminates a

molecule of carbon dioxide to from the secondary product ion at m/z 691.27 (Supplementary

Material, SM1). Additionally, this latter secondary product can eliminate simultaneously, a

molecule of carbon monoxide following aromatic ring contraction and a molecule of water, to

afford the tertiary product ion at m/z 645.26 (Supplementary Material SM1). The precursor

distonic[28,29] cation 2 afforded the primary product ions at m/z 675.23 and 668.20 by the

respective consecutive losses of either a molecule of carbon dioxide, a hydrogen radical and a

molecule of methanol, or two molecules of acetylene by RDA[33,34]rearrangements and a

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molecule of methanol (Supplementary Material SM1). For the sake of brevity, additional

information on the remaining m/z product ions: at m/z 621.19, 575.18, 278. 07; 184.07; 163.03

created by the precursor ion at m/z 752.2 are shown in Scheme 2A and 2B and the

Supplementary Material in Scheme SM1.

The distonic cation at m/z 780.2 was assigned as [C40H43O16 + H]+• which was composed

of a tetrameric lignin oligomer H(8-O-4)G(8-O-4’)S(8-O-4”)G having structure 3 (Figure 3A,

Figure 6 and Table 1).

Figure 6

The high energy collision dissociation of the precursor distonic cation 3 at m/z 780.2

afforded a major product (base peak) at m/z 737.23 which was created by the loss of a molecule

of acetylene (RDA) [33,34] and a hydroxyl radical, and it was assigned as [C38H40O15+H]+.

Furthermore, cleavage of S(8-O-4‵‵)G bond of the precursor distonic cation 3 creates the

protonated molecule at m/z 163.03 assigned as [C9H6O3 + H]+ (Scheme 3A). Similarly, the loss

of the lower end H unit from the precursor distonic cation 3, afforded the distonic product radical

ion at m/z 184.07 which was tentatively assigned as [C9H11O4 + H]+•. Finally, the presence of the

carboxylic acid group was confirmed by the loss of carbon dioxide and hydrogen radical from

the precursor distonic cation at m/z 780.2 to afford the product ion at m/z 735.20 assigned as

[C39H42O14 +H]+ (Scheme 3A).

Scheme 3A

Another CID-MS/MS fragmentation of the precursor distonic cation at m/z 780.2 can also occur

by the simultaneous losses of a molecule of carbon monoxide following aromatic ring

contraction of the first H residue, one acetylene molecule and two methoxyacetylene molecules

by RDA mechanism, and a hydroxyl radical to afford the primary product ion at m/z 597.19

assigned as [C31H32O12+H]+ (Scheme 3B). This latter primary product ion loses a molecule of

carbon dioxide and two molecules of formaldehyde to afford the secondary product ion at m/z

493.18 assigned as [C28H28O8+H]+ (Scheme 3B). Similarly, the precursor distonic cation at m/ z

780.2 can lose instantaneously either hydrogen radical and a molecule of methoxyacetylene or

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three molecules of methoxyacetylene by RDA rearrangements, a molecule of methanol and a

molecule of carbon dioxide to afford the secondary product ions at m/z 723.22 and 536.16

assigned as [C37H38O15+H]+ and [C29H27O10+H]+• respectively (Scheme 3B).

Scheme 3B

The protonated molecule at m/z 957.3 was assigned as [C50H52O19+H]+ having structure

4. It was composed of a pentameric lignin oligomer composed of five aromatic rings, namely:

H(8-O-4)H(8-O-4’)S(8-O-4”)S(8-O-4’”)G (Figure 3A, Figure 7 and Table 1).

Figure 7

` The product ion scan of the precursor ion 4 at m/z 957.3 afforded the product ion at m/z

939.30 which wascreated by the loss of a water molecule and assigned as [C50H50O18+H]+

(Scheme 4A). Moreover, the product ion at m/z 939.30 can lose a molecule of formaldehyde to

form the secondary product ion at m/z 909.29 assigned as [C49H48O17+H]+ (Scheme 4A). In

addition, the precursor ion 4 at m/z 957.3 can also lose simultaneously a hydrogen radical, a

molecule of carbon monoxide through aromatic ring contraction and a molecule of water to

afford the primary product ion at m/z 910.26 assigned as [C49H49O17+H]+• (Scheme 4A). It is

noteworthy to mention, that the precursor protonated molecule 4 produced the dimeric product

ion at m/z 347.14 assigned as the [C19H22O6+H]+, which was created by the cleavage of the S(8-

O-4‵‵)S bond (Scheme 4A). This latter primary product ion affords the secondary product ion at

m/z 319.15 by loss of a molecule of carbon monoxide following ring contraction (Scheme 4A).

Scheme 4A

The primary product ion at m/z 939.30 can fragment further to create the secondary product ions

at m/z 493.18; 478.16; 405.13 and 278.07. This series of secondary product ions are shown in the

Supplementary Part SM2A. Similarly, the primary product ion at m/z 910.26 underwent two

separate RDA rearrangements by the loss of either a molecule of methoxyacetylene and a

molecule of water or two molecules of methoxyacetylene to afford the secondary product ions at

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m/z 836.26 and 798.24assigned as [C46H43O15+H]+• and [C43H41O15+H]+•, respectively

(Supplementary Part SM2B). This latter secondary product ion can lose two molecules of water

and two molecules of formaldehyde to afford the tertiary product ion at m/z 702.20

(Supplementary Part SM2B). This latter tertiary product ion afforded the quaternary product ion

at m/z 658.21 by the loss of a molecule of carbon dioxide which confirms the presence of

carboxylic acid group (Supplementary Part SM2B).

The precursor ion 4 can also CID fragments by the loss of two consecutive molecules of

methanol to affords the secondary product ion at m/z 893.26 assigned as the [C48H44O17 + H]+

(Scheme 4B). Further consecutive losses of four molecules of formaldehyde and five molecules

of water from the latter primary product ion afford the secondary product ion at m/z 683.16,

which was assigned as [C44H27O8]+ (Scheme 4B). Moreover, the cleavage of the 8-O-4 bond of

the first H residue of this latter secondary product ion, along with the cleavage of C1-C7 of the

upper terminal G unit, affords the tetrameric product ion at m/z 502.13, assigned as [C32H21O6+

H] +• (Scheme 4B).

Scheme 4B

The lower m/z values product ions obtained by the product ion scan of 4 are explained in the

Supplementary Part SM2C. Henceforth, the product ion at m/z 184.07 was formed from the

precursor ion 4 at m/z 957.3 by double cleavages of the S(8-O-4‵‵)S bond and C4-O aryl ether

bond of the top end G unit, followed by the loss of two molecules of formaldehyde. The product

ion at m/z 172.07 was formed from the precursor ion 4 by cleavage of C1-C7 of the fourth S unit

followed by RDA which eliminates the pent-2-en-4-ynoic acid (C5H4O2, 96Da) (Supplementary

Part SM2C).

The protonated molecule at m/z 1039.3 was assigned as [C58H54O18+H]+ having structure

5. This protonated molecule was composed of a hexameric lignin oligomer composed of H(8-O-

4)H(8-O-4’)H(8-O-4”)G(8-O-4’”)S(8-O-4””)G (Figure 3B, Figure 8 and Table 1).

Figure 8

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The product ion scan of the protonated molecule at m/z 1039.3 afforded the product ion at

m/z 1011.33 by the loss of a molecule of carbon monoxide followed by aromatic ring

contraction of the first H residue (or vice versa). This product ion was as [C57H54O17+H]+ and

was composed of a hexameric lignin oligomer namely: cylcopentadienyl(7-O-4)H(8-O-4’)H(8-

O-4”)G(8-O-4’”)S(8-O-4””)G (Scheme 5a). The product ion at m/z 1011.33 affords the

secondary product ion at m/z 975.31 by loss of two molecules of water and it was assigned as

[C57H51O15]+ (Scheme 5A). This last product ion further CID fragmented by consecutive losses

of four molecules of formaldehyde, three molecules of ethyne eliminated by the retro-Diels

Alder mechanism and one molecule of carbon monoxide to afford the tertiary product ion at m/z

749.2343, assigned as[C46H37O10]+ (Scheme 5A). Once more, the order of these MS/MS

concerted losses was not established and is beyond the scope of this study. Finally, the presence

of the terminal carboxylic acid group was confirmed by the loss of carbon dioxide molecule from

the precursor ion at m/z 1039.3 to afford the product ion at m/z 995.29assigned as [C57H54O16 +

H]+ (Scheme 5A).

Scheme 5A

Another CID-MS/MS fragmentation of the primary product ion at m/z 1011.33 can also occur by

loss of a hydrogen molecule, to afford the secondary product ion at m/z 1009.32 (Scheme 5B).

This last ion loses consecutively four molecules of formaldehyde and four molecules of water to

afford the tertiary product ion at m/z 853.25, assigned as [C53H41O11]+ (Scheme 5B).

Scheme 5B

Similarly, the primary product ion at m/z 1011.33 can also create the secondary product ion at

m/z 430.13 by cleavage of the H(8-O-4‵‵)G bond of the cyclopentadienyl(7-O-4)H(8-O-4’)H(8-

O-4”)G(8-O-4’”)S(8-O-4””)G hexamer. This secondary trilignol ion product at m/z 430.13 was

assigned as [C26H21O6]+• and was composed of the trimer cylcopentadienyl(7-O-4)H(8-O-4’)H

(Scheme 5C).

Moreover, The cleavage of the cylopentadienyl(7-O-4) bond of the primary product ion at m/z

1011.33 affords the pentameric product ion at m/z 893.29 assigned as [C49H48O16+H]+, composed

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of H(8-O-4)H(8-O-4’)G(8-O-4”)S(8-O-4’”)G oligomer (Scheme 5C). Finally, this primary

product ion at m/z 1011.33 can undergo two different RDA reactions as shown in Scheme 5C to

afford the secondary product ions at m/z 851.28 and 663.21.

Scheme 5C

Additionally, The product ion scan of the protonated molecule at m/z 1039.33 afforded the

primary product ion at m/z 1013.3171 by the loss of molecule of acetylene by RDA mechanism

(Supplementary Material SM3A).[33,34] This primary product ion was assigned as

[C56H52O18+H]+. This latter product ion loses a molecule of carbon monoxide by an aromatic

ring contraction of the first H residue, to afford the secondary product ion at m/z 985.32 assigned

as [C55H52O17+H]+ (Supplementary Material SM3A). Additional information on and the

remaining low values m/z product ions are shown in the Supplementary Material as Scheme

SM3B.

The protonated molecule at m/z 1055.3 was assigned as [C58H54O19+H]+ and was

composed of the hexameric unit formed by H(8-O-4)H(8-O-4’)H(8-O-4”)S(8-O-4’”)S(8-O-

4””)G having structure 6 (Figure 3B, Figure 9 and Table 1)

Figure 9

The product ion scan of this protonated molecule at m/z 1055.3 afforded the primary

product ion at m/z 1027.33 by the loss of a molecule of carbon monoxide by ring contraction of

the first H residue of this hexameric unit (Scheme 6A). This last product ion at m/z 1027.33 loses

by one concerted mechanism two molecules of water, two molecules of methanol and two

molecules of formaldehyde, not necessarily in that order, to afford the secondary product ion at

m/z 867.23 assigned as [C53H38O12 + H]+ (Scheme 6A). The cleavage of the precursor

protonated molecule between the contiguous two sinapyl residues affords the secondary product

ion at m/z 495.18 assigned as [C24H30O11+H]+ (Scheme 6A). This latter secondary product ion

afforded tertiary product ion at m/z 469.16 assigned as [C22H28O11+H]+ by the loss of ethyne

through RDA mechanism (Scheme 6A). [33,34]

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Scheme 6A

Furthermore, the protonated molecule at m/z 1055.3 can lose an ethyne molecule by RDA

mechanism to afford the primary product ion at m/z 1029.31 assigned as [C56H52O19 + H]+or a

methane molecule, which can occur on either one of the S or G residues, to afford the primary

cation at m/z 1039.29 assigned as [C57H51O19]+ (Supplementary information SM4A).

Moreover, The primary product ion at m/z 1027.33 can lose an ethyne molecule by RDA

mechanism and five molecules of formaldehyde to afford the secondary product ion at m/z

851.26 assigned as [C50H42O13+H]+ (Supplementary information SM4B). This latter secondary

product ion at m/z 851.26 can lose carbon dioxide molecule to form the tertiary product ion at

m/z 806.26 assigned as [C49H42O12+H]+ (Supplementary information SM4B).

The cleavage of C1-C7 bond of the fourth unit S in the primary product ion at m/z 1027.33 leads

to the formation of dilignol secondary product ion at m/z 495.18 assigned as [C24H30O11 + H ]+

(Supplementary information SM4B). This latter secondary product ion at m/z 495.18 fragments

by the cleavage of C-4-O aryl ether bond of the G unit and C1-C7 bond of the S unit to afford

four lower m/z tertiary products ions at m/z 269.10, 172.07, 116.04 and 104.06 (Supplementary

information SM4B).

The presence of the carboxylic group in the precursor protonated molecule 6 was confirmed by

the loss of carbon dioxide molecule and hydrogen radical to afford the primary product ion at m/z

1011.33 assigned as [ C57H54O17 + H]+ (Scheme 6B). This latter primary product ion afforded the

secondary product ion at m/z 719.18 assigned as [C44H30O10 + H]+ by the loss of two molecules

of methanol, two molecules of formaldehyde and three molecules of methoxyacetylene by RDA

mechanism (not necessarily in that order) (Scheme 6B). Moreover, The cleavage of the H(8-O-

4‵‵)S bond of the precursor protonated molecule 6 lead to the formation of the primary product

ion at m/z 643.19 assigned as [C32H34O14 + H]+(Scheme 6B).

Scheme 6B.

The protonated molecule at m/z 1201.4 was assigned as [C61H68O25+H]+, and it was

composed of a hexameric oligomer H(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-4””)G and

was attributed to structure 7 (Figure 3B, Figure 10 and Table 1).

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The product ion scan of the precursor ion at m/z 1201.4 afforded the product ion at m/z

1169.38 by the loss of a molecule of methanol. This latter ion eliminated a molecule of

formaldehyde to afford the secondary product ion at m/z 1139.36 (Figure 9).

Figure 10

Concerted loss a molecule of methanol, two molecules of formaldehyde and three molecules of

water from the precursor ion at m/z 1201.4, lead to the formation of the primary product ion at

m/z 1055.32, which was assigned as [C58H54O19+H]+ (Scheme 7A). Finally, once more the

presence of the terminal carboxylic acid group was confirmed by the loss of carbon dioxide and

hydrogen molecule from the precursor ion at m/z 1201.4 to afford the product ion at m/z 1155.40

assigned as [C60H66O23 + H]+ (Scheme 7A).

Scheme 7A

Moreover, The secondary product ion at m/z 1139.36 can lose hydrogen radical, two molecules

of water and one molecule of carbon monoxide to afford the distonic tertiary product ion at m/z

1074.34 assigned as [C58H57O20+H]+• (Supplementary information SM5A). This latter tertiary

product ion can lose a molecule of methanol to afford the quaternary product ion at m/z 1042.32

assigned as [C57H53O19+H]+• (Supplementary information SM5A). This latter quaternary product

ion afforded the quinary product ion at m/z 942.30 assigned as [C53H49O16+H]+• by the loss of a

molecule of carbon dioxide, a molecule of formaldehyde and a molecule of ethyne by RDA

mechanism (Supplementary information SM5A). [33,34]

The cleavage of the precursor ion at m/z 1201.4 between the two contiguous S residues affords

the product ion at m/z 493.16 which was assigned as [C24H28O11+H]+ (Scheme 7B). In addition,

the cleavage of the precursor ion at m/z 1201.4 between the contiguous two G residues affords

the primary distonic radical cation product ion at m/z 824.28 assigned as [C42H48O17]+• (Scheme

7B). This last product ion loses either one or two molecules of hydrogen to afford the secondary

and tertiary product ions at m/z 822.26 and 820.25 assigned as [C42H46O17]+• and [C42H44O17] +•

respectively (Scheme 7B). Furthermore, the primary product ion at m/z 824.28 loses a molecule

of ethyne by RDA mechanism to create the secondary product ion at m/z 798.26, assigned as

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[C40H46O17]+• (Scheme 7B). [33,34] Finally, the precursor ion at m/z 1201.40 is subjected to a

cleavage of the C-4-O aryl ether bond of the third unit G, followed by the consecutive losses of

six molecules of formaldehyde, four molecules of water and three molecules of methanol (once

more, not necessarily in that order) to create the product ion at m/z 479.12 assigned as

[C33H19O4] + (Scheme 7B).

Scheme 7B

Additionally, The secondary product ion at m/z 798.26 can lose hydroxyl radical, a molecule of

hydrogen and a molecule of water to afford the tertiary product ion at m/z 761.24 assigned as

[C41H41O15]+. This later tertiary product ion afforded the quaternary product ion at m/z 643.21

assigned as [C36H35O11]+ by the loss of a molecule of carbon monoxide and three molecules of

formaldehyde. This latter quaternary product ion can loss two molecules of formaldehyde to

afford the quinary product ion at m/z 583.19 assigned as [C34H31O9]+ (Supplementary

information SM5B).

Another CID-MS/MS fragmentation of the protonated molecule 7 can occur by the loss of

methyl radical and ethyne molecule by RDA mechanism to afford the secondary distonic product

ion at m/z 1160.36 assigned as [C58H63O25 + H ]+• or it can lose one molecule of methanol, two

molecules of formaldehyde and three molecules of water to afford the primary product ion at m/z

1055.32 assigned as [C58H54O19+H]+ (Supplementary information SM5C).

This latter primary product ion at m/z 1055.32 can lose a molecule of water and a molecule of

methanol to afford the secondary product ion at m/z 1005.28 assigned as [C57H48O17+H]+. This

latter secondary product ion afforded the tertiary product ion at m/z 873.28 by loss of a molecule

of carbon monoxide by aromatic ring contraction of the first H residue along with loss of a

molecule of carbon dioxide and two molecules of formaldehyde (Supplementary information

SM5C).

Likewise, the protonated molecule at m/z 1201.4 can fragment by loss of hydrogen radical, one

molecule of water and one molecule of methanol to afford the primary distonic product ion at

m/z 1150.36 assigned as [C60H61O23+H]+• or the loss of ethyne molecule by RDA[33,34] to afford

the primary product ion at m/z 1175.39 assigned as [C59H66O25+H]+ (Supplementary information

SM5D).This latter primary product ion at m/z 1175.39 afforded the secondary product ion at m/z

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1143.36 assigned as [C58H62O24+H]+ by the loss of a molecule of methanol. (Supplementary

information SM5D).

Lastly, the protonated molecule at m/z 1217.4 was assigned as [C61H68O26+H]+ and was

composed of the hexameric oligomer formed of C(8-O-4)G(8-O-4’)G(8-O-4”)S(8-O-4’”)S(8-O-

4””)G having structure 8 (Figure 3B, Figure 11 and Table 1).

Figure 11

The product ion scan of the precursor ion at m/z 1217.4 afforded the primary product ion

at m/z 1187.39 by the loss of a molecule of formaldehyde, assigned as [C60H66O25+H]+ (Scheme

8A). Similarly, loss of a molecule of methanol from this precursor ion gives the primary product

ion at m/z 1185.37 assigned as [C60H64O25 + H]+. This latter product ion loses one molecule of

formaldehyde to afford the secondary product ion at m/z 1155.36 assigned as [C59H62O24 + H]+

(Scheme 8A).

Moreover, the secondary product ion at m/z 1155.36 afforded the tertiary product ion at m/z

1055.31 assigned as [C54H54O22+H]+ by the loss of a molecule of water, a molecule of

formaldehyde and two molecules of ethyne by RDA (Supplementary information SM6A). This

latter tertiary product ion can lose a molecule of carbon dioxide to afford the quaternary product

ion at m/z 1011.32 assigned as [C53H54O20+H]+. This latter quaternary product ion afforded the

distonic[28,29] quinary product ion at m/z 934.29 assigned as [C51H49O17+H]+• by the loss of

hydrogen radical, a molecule of carbon monoxide, a molecule of water and molecule of

formaldehyde (Supplementary information SM6A). Moreover, The cleavage of C7-C8 bond of

the third unit G in the secondary product ion at m/z 1155.36 lead to the formation of trilignol

tertiary product ion at m/z 496.13 assigned as [C26H24O10]+• (Supplementary information

SM6A)..

Scheme 8A

Similarly, the precursor ion at m/z 1217.41 can experience a cleavage of G(8-O-4’)G bond to

form the tetramer distonic cation at m/z 824.28 assigned as [C42H48O17]+•. This latter product ion

undergoes oxidation by losing two molecules of hydrogen to afford the secondary product ion at

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m/z 820.25 (Scheme 8B). Finally, the cleavage of the C-4-O aryl ether bond of the third unit G in

the precursor ion at m/z 1217.44, affords the diliginol product ion at m/z 479.12, which was

assigned as [C33H19O4]+ (Scheme 8B).

Additionally, The primary distonic cation product ion at m/z 824.28 afforded the secondary

product ion at m/z 796.28 assigned as [C41H48O16]+• by the loss of a molecule of carbon

monoxide (Supplementary information SM6B). This latter secondary product ion can lose a

hydroxyl radical and a molecule of formaldehyde to afford the tertiary product ion at m/z 749.27

assigned as [C40H45O14]+. This latter tertiary product ion afforded the quaternary product ion at

m/z 675.24 assigned as [C37H39O12]+ by the loss of a molecule of water, a molecule of

formaldehyde and a molecule of ethyne by RDA[33,34] mechanism. This latter quaternary product

ion afforded the quinary product ion at m/z 559.19 assigned as [C32H31O9]+ by the loss of three

molecules of formaldehyde and a molecule of ethyne by RDA[33,34]mechanism (Supplementary

information SM6B).

Scheme 8B.

CONCLUSION

In the presented work, we have commenced the first structural investigation of the VRL

that showed that the Saudi date palm wood (Phoenix dactylifera) is a rich source of lignin.

To sum it up, analyses of the VRL of the Saudi date palm wood (Phoenix dactylifera) by

MALDI-TOF-MS (+ ion mode) afforded inter-alia six protonated molecules and two distonic

cations. Althoughthe elucidation of this series of precursor ions was undeniably challenging to

interpret, we managed to obtain excellent blueprints of the different structures of the VRL

oligomers of the SDPW.

The high-energy tandem mass spectrometry analyses of these precursor ions gave

extremely intricate spectra and allowed us to confirm the complicated proposed structures of this

series of lignin oligomers. For that reason, we can say that the VRL of the Saudi date palm wood

(SPW) is composed of units H, G, S and C in different proportions. Furthermore, the MALD-

TOF-MS and high-energy CID-TOF/TOF-MS/MS analyses allowed the identification of the

following six lignin protonated oligomers: HG dimer, HHSSG pentamer, HHHGSG hexamer,

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HHHSSG hexamer, HGGSSG hexamer and CGGSSG hexamer. In addition, we noticed the

formation of two distonic cations HGGG and HGSG.

It is important to note that in this manuscript we have described series of various types of

rapid MS/MS fragmentations involved concerted series of RDA rearrangements,[33,34]aromatic

ring contractions and multiple eliminations of formaldehyde, methanol and water.[37] These

MS/MS fragmentations appear to contradict the gas-phase fragmentation behavior described by

Morreel [13,38] and coworkers for the major lignin standards, using APCI-MS/MS with a QIT

instrument.

Once more, we attribute our type of MALDI-TOF/TOF-MS/MS fragmentations, as being

conducted at higher collision energies “in space” and as such, more energetic, than the ones

reported by Morrel and coworkers, which were conducted with lower energy CID-MS/MS in an

IT-MS instrument, operating in MS/MS “in time”. Conversely, be aware that we had also

obtained identical CID-MS/MS results than those performed by MALDI-TOF-MS, when the

analyses of the lignin oligomers were achieved with atmospheric pressure photoionization

measured with an QqTOF-MS/MS instrument and with electrospray ionization measured with an

extra-high resolution Orbitrap MS/MS instrument.[39] Needless to say, that these last two

instruments also operate with low-energies collision dissociation. For these reasons, it will be

imprudent to use the nomenclature described by Morrell and coworkers, as the basis of an

MS/MS fragmentation rule for identifying novel lignin oligomeric structures.

Considering the extremely lability of this series of VRL oligomers in the gas-phase, one

can hardly imagine what happens to them, while being purified by further chemical

manipulation. It is imperative to repeat that the isolation of lignin in its unaltered form, is a

highly unlikely process, due to the relatively harsh extraction conditions and other chemical

modifications used for releasing and purifying the lignin.[11] For these reasons and as mentioned

before, there is still much dispute on whether any lignin extract adequately represents the native

lignin structure. Consequently, the harsh extraction condition required to release lignin from

lignocellulosic cellular material results in the degradation of the lignin polymeric structure itself.

For comparison sake, these extraction and purification conditions could be viewed as using a

“wrecking ball to break a crystal glass”. The reactivity of the released fragments may lead to

more complex reactive species that can further rearrange and condense to the more artefactual

altered polymeric structure. [11]

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On the basis of the total absence of structural information on the Saudi Date Palm Wood

(SDPW) lignin in the literature and the complexity of this series of lignin oligomers , it is evident

that the top-down lignomic new sequencing approach, allowed us to reveal this series of novel

oligomers structures, that did not concur with the current knowledge of any lignin structures

proposed [40,41]

To our knowledge, this is the first report on the structure of the lignin

biomolecules extracted from Saudi date palm wood (Phoenix dactylifera) samples.

Acknowledgment

We would like to thank the following; The Umm-El-Qurra University, Kingdom of Saudi Arabia

and the Saudi Cultural Bureau for a graduate fellowship to T. Albishi; the Chemistry

Department, Memorial University of Newfoundland for a graduate fellowship to A. F. Mikhael.

The Department of Fisheries and Oceans for their open Laboratories policy, and La Compagnie

Industrielle de la Matière Végétale (CIMV) France for financial assistance.

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https://www.sciencedirect.com/science/article/pii/S0040402001880856#!

https://www.sciencedirect.com/science/journal/00404020

https://www.sciencedirect.com/science/journal/00404020/42/22

http://pubs.rsc.org/EN/results?searchtext=Author%3ARenu%20Pandey

http://pubs.rsc.org/EN/results?searchtext=Author%3APreeti%20Chandra

http://pubs.rsc.org/EN/results?searchtext=Author%3AMukesh%20Srivastva

http://pubs.rsc.org/EN/results?searchtext=Author%3AK.%20R.%20Arya

http://pubs.rsc.org/EN/results?searchtext=Author%3APraveen%20K.%20Shukla

http://pubs.rsc.org/EN/results?searchtext=Author%3ABrijesh%20Kumar

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39. Abanoub Mikhael, Tasahil Albishi, Michel Delmas, Joseph Banoub. Unpublished Results to be

submitted to Rapid Commun. Mass Spectrom.

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chemistry. Ind. Crops and prod., 2004; 20(2): 131–141.

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Review of Cell Wall Model Studies., Crop Sci., 2005; 45(3): 820–831.

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Table 1. Identification of lignin Oligomers Peaks in Mass Spectrum of extracted from SDPW Wood using MALDI-TOF-MS

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LIST OF LEGENDS

TOP–DOWN LIGNOMIC MALDI-TOF-TANDEM MASS SPECTROMETRY ANALYSIS OF

LIGNIN OLIGOMERS EXTRACTED FROM DATE PALM WOOD

FIGURES







Figure 5. The high-energy CID-MS/MS of the distonic [C39H43O15 + H]+• at m/z 752.2

Figure 6. The high-energy CID-MS/MS of the distonic cation [C40H43O16 + H]+• at m/z 780.2




Figure 10. The high-energy CID-MS/MS of the protonated molecule C61H68O25 + H] + at m/z 1201.4


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SCHEMES


Scheme 2A. Tentative fragmentation pattern of the product ion scan of the distonic cation 2 at m/z 752.2

Scheme 2B. Tentative fragmentation pattern of the product ion scan of the distonic cation 2 at m/z 752.2

Scheme 3A. Tentative fragmentation pattern of the product ion scan of the distonic cation 3 at m/z 780.2

Scheme 3B. Tentative fragmentation pattern of the product ion scan of the diatonic cation 3 at m/z 780.2












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[ C50H52O19 + H ]+

m/z 957.3

OMe

OHO

OMe

O

HHO

OMe

O

HO

HO

COOH

HO

HO

O

[ C39H43O15 + H ]+•

m/z 752.2

[ C40H43O16 + H ]+•

m/z 780.2

HO

OMe

O

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+

m/z 381.1

(4)

(2) (3)

(1)

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

COOH

OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O


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For Peer ReviewOMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H ]+

m/z 1201.4

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+

m/z 1055.3

OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

COOH

HO

MeO

+ HHO

HO

[ C58H54O18 + H ]+

m/z 1039.3

(7)(6)

(5)[ C61H68O26 + H ]+

m/z 1217.4(8)

OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

OH


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For Peer ReviewFigure 11. The high-energy CID-MS/MS of the protonated molecule [C61H68O26 + H] + at m/z 1217.4

Page 99 of 114



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For Peer Review

SCHEMES

O

OH

HO

+ H

[ C12H10O3 + H ]+

m/z 203.06

OMe

O

O

+ H

OH

HO

OMe

OHO

O

HO

OH

[ C18H14O4 + H ]+

m/z 295.09[ C19H21O7 + H ]+•

m/z 362.13

OH

O

HO

HO

+ H

OH

O

HO

+ H

[ C12H8O3 + H ]+

m/z 201.05

[ C12H10O4 + H ]+

m/z 219.06

- H2 O

HO

OMe

O

HO

HO

OH

HO

+ H

OH

[ C19H24O8 + H ]+

m/z 381.1

- 3 x H2O- CH3OH - 2 x CH 3O

HOMe

OH

--

OMe-- CH3OH

HO

HO

OH

-

- 2 x CH3OHOMe

OH

-

-

- H2O

OH

HO

HO+ H

[ C5H6O3 + H ]+

m/z 115.03

OH

O

HO+ H

- H2

OH

O

HO+ H

[ C5H4O3 + H ]+

m/z 113.02

OH

HO

HO+ H

Not Observed

- H2

- CH3OH

- 2 x CH3OHOMeOH

--

RDA

+ H

- H •

- H2O

- OH•


Page 100 of 114



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For Peer ReviewOMe

OHO

OMeO

H+

HO

OMeO

HO

O

HO

COOH

HO

HO

OMe

OHO

OMeO

H+

HO

OMeO

HO

O

COOH

HO

HO

Hydrogenation due to matrix DHBgives higher product ions at m/z

711, 713, 715

- •OH

- C2H2

OHO

OMeO

H+

HO

OMeO

HO

COOH

HO

HO

[ C31H34O12 + H ]+

m/z 599.20

- CO

- C2H2

OMe

-

[ C39H43O15 + H ]+•

m/z 752.2[ C37H40O14 + H ]+

m/z 709.24

OMe

OH

COOH

H+

OH

COOH

H+- CH2O

Not observed

- H2

[ C9H6O3 + H ]+

m/z 163.03

H+

OH

HO

O

HO

[ C9H11O4 + H ]+•

m/z 184. 07

- •OH

- CO

- 2 x C2H2

OMe

-

OHHO

OMeO

HO

HO

HO

- CH2O

- 2 H2

Not Observed

OHHO

O

HO

HO

HO

[ C14H14O6 ]+•

m/z 278.07

RDA


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For Peer Review

OMe

OHO

OMe

O

H+HO

OMe

O

HO

O

HO

COOH

HO

HO

- H2

- C2H2

HO

O

H+

HO

O

HO

HO

HO

[ C24H28O7 + H ]+

m/z 429.18

OMe

OHO

OMe

O

H+

HO

OMe

O

HO

O

COOH

HO

HO

[ C37H40O14 + H ]+

m/z 709.24

MeO

O

H+

HO

OMe

O

HO

O

HO- H2

MeO

O

H+

HO

OMe

O

HO

O

HO

Not Observed[ C20H20O8 + H ]+

m/z 389.12

OHO

O

H+

HO

O

HO

HO

HO

[ C28H30O8 + H ]+

m/z 495.19

[ C39H43O15 + H] +•

m/z 752.2

COOH

-

- CO- 2 x CH2O

OMe-

OHO

O

H+

HO

O

HO

HO

HO

[ C26H28O8 + H ]+

m/z 469.18

OHO

O

H+

HO

O

HO

HO

HO

[ C28H28O8 + H ]+

m/z 493.18

RDA

OMe

OHO

OMeO

H+HO

OMeO

HO

O

HO

HO

HO

[ C38H42O13 + H ]+

m/z 707.26

- CO2

( RDA)

- •OH

- H•

- •OH

- C2H2


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For Peer Review

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

OH

HO

HO

O

H+

OMe

OH

H+

OH

COOH

H+

[ C9H6O3 + H ] +

m/z 163.03

[ C40H43O16 + H ]+•

m/z 780.2

- CH2O

OMe

OHO

OMe

O

H+HO

OMe

O

CHO

HO

HO

MeO

O

- •OH

- C2H2

Hydrogenation byDHB gives m/z 739

Not observed

OH

HO

OH

HO

HO

OH

HO

Hydrogenationdue to MatrixDHB

[ C4H6O2 ]+•

m/z 86.03

Not observed

Not Observed

RDA- CH3OH

- CO2

- H•

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

COOH

COOH

- H2COOH

Not Observed

Hydrogenation byDHB

OH

HO

HO

O

H+

[ C9H11O4 + H ] +•

m/z 184.07

[ C39H42O14 + H ]+

m/z 735.20

[ C38H40O15 + H ]+

m/z 737.23


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For Peer Review

OMe

OHO

OMe

O

H+HO

OMe

O

HO

HO

HO

HO

MeO

O

[ C40H43O16 + H ]+•

m/z 780.2

- H•

OMe

-

OMe

OHO

OMe

O

H+HO

O

HO

HO

HO

HO

MeO

O

[ C37H38O15 + H ]+

m/z 723.22

OMe

OHO

OH+

HO

O

CHO

HO

HO

MeO

- CO- C2H2

OMe

- 3 x

[ C31H32O12 + H ] +

m/z 597.19

- CO2

- 2 x CH2O

OHO

O

H+HO

O

CHO

HO

HO

[ C28H28O8 + H] +

m/z 493.18

- CO2

COOH COOH

COOH

- CH3OH

OHO

O

H+

O

HO

HO

HO

HO

MeO

O[ C29H27O10 + H ]+•

m/z 536.16

- •OH

OMe

- 2 x


Page 104 of 114



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For Peer Review

OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

OH

HO

MeO

MeO

O

OMe

OHO

OMeO

H+HO

OMeO

O

COOH

HO

O

OH

HO

MeO

O

- CO2

OMe

OHO

OMe

OH

HO

H+- CO

OMe

OHO

OMe

HO

H+

[ C18H22O5 + H ]+

m/z 319.15

- CH2O

- H2O- CO

- H2O - CH2O

[ C49H49O17 + H ]+•

m/z 910.26[ C50H52O19 + H ]+

m/z 957.3

[ C50H50O18 + H ]+

m/z 939.30[ C49H48O17 + H ]+

m/z 909.29

[ C19H22O6 + H ]+

m/z 347.14

- H2O

- CH2O

- H•

OMe

OHO

OMeOH

COOH

HO

MeO

+ H

Not Observed


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For Peer Review

OMe

OHO

OMeO

H+HO

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

[ C50H52O19 + H ]+

m/z 957.3

OMe

O

OMeO

H+

OMeO

HO

O

COOH

HO

HO

OH

HO

MeO

MeO

O

OMe

O

O

O

O

CO

O

O

OMe

O

O

O

O

O

H+- 2 X CH3OH - 4 x CH2O

- 5 x H2O

- 4 x CH2O

- 5 x H2O

- 2 x CH3OH

[ C48H44O17 + H ]+

m/z 893.26

[ C44H27O8 ]+

m/z 683.16

[ C32H21O6 + H] +•

m/z 502.13


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For Peer Review

OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

COOH

HO

MeO

+ H

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.33

OMe

O

OMeO

MeO

O

O

O

O

HO

CO

MeO

[ C46H37O10]+

m/z 749.23

HO

HO

HO

[ C58H54O18 + H ]+

m/z 1039.3

HO

HO

- CO -2 x H2O- CO

- 4 x CH2O- 3 x C2H2

O

O

O

O

O

O

HO

HO

[ C57H51O15 ]+

m/z 975.31

- CO

-2 x H2O

- 2 x CO

- 4 x CH2O

- 3 x C2H2

- 2 x H2O- CO

- 2 x H2O

- 4 x CH2O

- 3 x C2H2

- CO2

OMe

OHO

OMeO

MeO

O

O

O

O

O

HO

HO

MeO

+ HHO

HO

[ C57H54O16 + H]+

m/z 995.29

HO

HO

HO

HO

( Loss of H2 givesproduct ion at m/z 973)


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For Peer Review

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.33

HO

HO

- H2

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO

+ H

HO

HO

[ C57H52O17 + H ]+

m/z 1009.32

- 4 X CH2O

- 2 X H2O

O

O

O

O

O

O

HO

CO

O

HO

HO

[ C53H41O11 ]+

m/z 853.25

- H2

- 4 X CH2O

- 2 X H2O


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For Peer Review

OMe

OHO

OMeO

MeO

O

O

O

O

HO

COOH

HO

MeO+ H

[ C57H54O17 + H ]+

m/z 1011.33

HO

HO

O

MeO

O

O

O

O

HO

MeO+ H

HO

HO

[ C39H34O10 + H ]+

m/z 663.21

OH

O

O

O

HO

HO

[ C26H22O6 ]+•

m/z 430.13

OMe

OHO

OMeO

MeO

O

O

O

COOH

HO

MeO

+ H

HO

HO

[ C47H46O15 + H ]+

m/z 851.28

OMe

OHO

OMeO

MeO

O

O

OH

O

COOH

HO

MeO+ H

HO

HO

[ C49H48O16 + H ]+

m/z 893.29

RDA

RDA


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For Peer Review

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+

m/z 1055.3

495

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C57H54O18 + H ]+

m/z 1027.33

OMe

O

OMe

OMeO

O

O

O

O

HO

COOH

+ H

[ C53H38O12 + H ]+

m/z 867.23

OMe

OHO

OMe

OMeO

HO

COOH

HO

HO

+ H

[ C24H30O11 + H ]+

m/z 495.18

- CO

- 2 X CH2O

- 2 X CH3OH

- 2 X H2O

- CO - 2 X CH2O

- 2 X CH3OH - 2 X H2O

- C2H2

OMe

OHO

OMe

OMeO

HO

COOH

HO

HO

+ H

[ C22H28O11 + H ]+

m/z 469.16


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For Peer Review

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

[ C58H54O19 + H ]+

m/z 1055.3

- CO2

OMe

OHO

OMe

OMeO

HO

OMe

O

O

O

O

O

HO

HO

HO

MeO

+ H

[ C57H54O17 + H ]+

m/z 1011.33

[647]

Notobserved

- 2 H2

OMe

OHO

OMe

OMeO

HO

OMe

OH

COOH

HO

HO

MeO

+ H

[ C32H34O14 + H ]+

m/z 643.19

O

O

O

O

O

O

O

HO

HO

HO

+ H

OMe

- 3 x

- 2 x CH2O

- 2 x CH3OH

[ C44H30O10 + H ]+

m/z 719.18


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For Peer Review

OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMeO

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H]+

m/z 1201.4

OMe

OHO

OMeO

MeO

HO

OMeO

OMeO

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C59H62O23 + H ]+

m/z 1139.36

- CH3OH

OMeO

HO

OMeO

MeO

HO

OMeO

OMeO

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C60H64O24 + H ]+

m/z 1169.38

OMe

- CH2O

- CH3OH- CH2O

OMe

OHO

OMeO

MeO

HO

OMeO

O

O

O

O

HO

COOH

HO

HO

MeO

+ H

- CH2O

- 3 x H2O

[ C58H54O19 + H ]+

m/z 1055.32

- CO2

- H2

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMeO

HO

HO

OMeO

HO

HO

OH

HO

HO

HO

MeO

+ H

[ C60H66O23 + H ]+

m/z 1155.40

- CH3OH- 2 x CH2O

- 3 x H2O


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For Peer Review

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

[ C61H68O25 + H]+

m/z 1201.4

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H48O17]+•

m/z 824.28

OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H44O17 ]+•

m/z 820.25

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H46O17 ]+•

m/z 822.26

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C40H46O17 ]+•

m/z 798.26

- C2H2

- H2

OMeO

HO

OMeO

MeO

HO

COOH

HO

HO

+ H

[ C24H28O11 + H ]+

m/z 493.16

[495]- H2 [827]

- 6 x CH2O


O

O

O

CO

[ C33H19O4]+

m/z 479.12[ 842 ]

- H2O

- H2

- H2O

- C2H2

- 2 x H2

Not Observed Not Observed

Not Observed


Page 113 of 114



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For Peer Review

OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

- CH2O

OMe

OHO

OMe

OMeO

HO

OMe

O

OMe

O

HO

HO

OMe

O

HO

HO

OH

HO

COOH

HO

HO

MeO

- CH2O - CH3OH

- CH3OH

- CH3OH

- CH2O

OH

[ C61H68O26 + H ]+

m/z 1217.4

OH

+ H

[ C60H64O25 + H ]+

m/z 1185.37

OMe

OHO

OMe

OMeO

HO

OMe

O

OMe

O

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

OH

+ H

[ C59H62O24 + H ]+

m/z 1155.36

OMe

OHO

OMe

OMeO

HO

OMe

O

HO

OMe

O

HO

HO

O

HO

HO

OH

HO

COOH

HO

HO

MeO

+ H

OH

[ C60H66O25 + H ]+

m/z 1187.39


Page 114 of 114



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For Peer Review

OMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

HO

OMeO

HO

HO

OH

HO

COOH

HO

HO

MeO

+ HOMe

OHO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H48O17 ]+•

m/z 824.28

OMeO

HO

OMeO

MeO

HO

OMeO

HO

OMe

O

HO

CO

HO

HO

MeO

[ C42H44O17 ]+•

m/z 820.25

[827]- 6 x CH2O


O

O

O

CO

[ C33H19O4]+

m/z 479.12

[ 842 ]

- H2O

- 2 x H2

Not Observed

Not Observed

OH

[ C61H68O26 + H ]+

m/z 1217.4


Page 115 of 114



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