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(-)ESI/CAD MSn Procedure for Sequencing Lignin Oligomers Based on a Study of Synthetic Model Compounds with #-O-4 and 5-5 Linkages Huaming Sheng, Weijuan Tang, Jinshan Gao, James Steven Riedeman, Guannan Li, Tiffany Mae Jarrell, Matthew R. Hurt, Linan Yang, Priya Murria, Xin Ma, John Joseph Nash, and Hilkka I. Kenttamaa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01911 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017
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Analytical Chemistry
(-)ESI/CAD MSn Procedure for Sequencing Lignin Oligomers Based on a Study of Synthetic Model Compounds with β-O-4 and 5-5 Linkages Huaming Sheng,a Weijuan Tang,b Jinshan Gao,b James S. Riedeman,b Guannan Li,b Tiffany M. Jarrell,c Matthew R. Hurt,b Linan Yang,b Priya Murria,b Xin Ma,b John J. Nashb* and Hilkka I. Kenttämaab* aMerck
& Co., Inc., Process Research, 126 E Lincoln Ave RY800-C262, Rahway, NJ 07065; bDepartment of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907; cMerck Animal Health, 2 Giralda Farms, Madison, NJ 07940-1026
KEYWORDS: Lignin, β-O-4 linkage, 5-5 linkage, sequencing, mechanism, biomass, tandem mass spectrometry, ESI ABSTRACT: Seven synthesized G-lignin oligomer model compounds (ranging in size from dimers to an octamer) with 5-5 and/or β-O-4 linkages, and three synthesized S-lignin model compounds (a dimer, trimer and tetramer) with β-O-4 linkages, were evaporated and deprotonated using negative-ion mode ESI in a linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer. The collision-activated dissociation (CAD) fragmentation patterns (obtained in MS2 and MS3 experiments, respectively) for the negative ions were studied in order to develop a procedure for sequencing unknown lignin oligomers. Based on the observed fragmentation patterns, the measured elemental compositions of the most abundant fragment ions, and quantum chemical calculations, the most important reaction pathways and likely mechanisms were delineated. Many of these reactions occur via charge-remote fragmentation mechanisms. Deprotonated compounds with only β-O-4 linkages, or both 5-5 and β-O-4 linkages, showed major 1,2eliminations of neutral compounds containing one, two or three aromatic rings. The most likely mechanisms for these reactions are charge-remote Maccoll and retro-ene eliminations resulting in the cleavage of a β-O-4 linkage. Facile losses of H2O and CH2O were also observed for all deprotonated model compounds, which involves a previously published charge-driven mechanism. Characteristic “ion groups” and “key ions” were identified that, when combined with their CAD products (MS3 experiments), can be used to sequence unknown oligomers.
Introduction
HO HO
Lignocellulosic biomass is the most abundant renewable energy resource in nature.1-6 While conversion of cellulose to fuels and valuable chemicals has received much attention,1-6 this is not the case for lignin. Lignin has significant potential as a feedstock for the sustainable production of aromatic chemicals currently obtained from crude oil. Catalysts are being developed for the generation of useful chemicals from lignin degradation products.7-12 Unfortunately, these efforts are hindered by the lack of analytical methods for the unambiguous determination of the structures of lignin degradation products. Multiple-stage tandem mass spectrometry (MSn) can provide structural information for individual molecules directly in complex mixtures.13,14 Collision-activated dissociation (CAD) is the most common method used to examine the structures of ionized analytes in MSn experiments.15 Indeed, some reports have appeared in the literature on the determination of the structures of lignin degradation products by using MSn and CAD.16-22 For example, our group has developed an HPLC/MSn method for the identification of small deprotonated lignin degradation products, such as those found in organosolv lignin, ionized by negative ion-mode electrospray ionization.23-26 Major obstacles still remain. One is the high diversity of lignin linkages. The most common lignin linkages are β-O-4, 5-5, β-5, ββ, β-1 and 4-O-5 (Figure 1), of which the β-O-4 and 5-5-linkages
α
γ 1
β O 4
HO
HO
5 4
β
O
β
HO
O
1
O O
O
O
O O
β-O-4
O O
β-1
O
O β
O
O
β-5
β O
ββ
O O
5 OH
5-5
5 HO
O
O
4 O 5 O
OH
4-O-5
Figur e 1. Common lignin linkages (highlighted in red) in nature. dominate.1 CAD of deprotonated synthetic linear lignin dimers, trimers and tetramers with β-O-4, β-5, and β-β linkages has been examined.27-30 These studies yielded invaluable information on sequencing of oligomers containing these three linkages. However, all of the model compounds that contained only β-O-4 linkages had a coniferyl alcohol end group with an allyl alcohol moiety, which may influence the fragmentation patterns of the deprotonated molecules. The allyl alcohol moiety is unlikely to be present in lignin degradation products, the primary interest here. Further, larger (synthetic) oligomers than tetramers with known structures need to be examined in order to test the validity of conclusions made based on small oligomers. Moreover, information on oligomers containing 5-5 linkages is
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critically needed for two reasons. First, this linkage is the second most common in lignin. Second, this linkage is one of the two linkages that cause branching in the otherwise linear oligomers21,22 and hence may have a major influence on the fragmentation reactions of deprotonated oligomers. Finally, although several reasonable fragmentation mechanisms were proposed in these previous studies, no supporting evidence, such as quantum chemical calculations, was provided. Generalizations that enable the development of reliable sequencing processes require more detailed knowledge on the reaction mechanisms. One reason for the lack of knowledge on MS fragmentation reactions of deprotonated lignin oligomers with known structures is the scarcity of commercially available lignin model compounds. Unlike oligosaccharides, polypeptides and oligonucleotides, the only commercially available lignin oligomers are dimers. Hence, synthesis of lignin oligomers with different linkages is needed to improve the understanding of the fragmentation pathways and mechanisms for deprotonated lignin oligomers and to develop reliable sequencing approaches. In this study, seven lignin model compounds (ranging from dimers to an octamer; Figure 2) with 5-5 and/or β-O-4 linkages and three S-lignin model compounds (a dimer, trimer and tetramer) with β-O-4 linkages were synthesized and the CAD behavior of their deprotonated molecules was studied by MSn experiments using a linear quadrupole ion trap coupled to a high-resolution Fourier-transform ion cyclotron resonance mass spectrometer. The elemental compositions of all abundant ions were determined. The mechanisms of the gas-phase fragmentations were probed by using quantum chemical calculations, which revealed that several mechanisms proposed in the literature may be incorrect. Most importantly, while almost all literature mechanisms are charge-driven, our calculations indicate that in many cases, charge-remote fragmentations take place. The results obtained here enabled the development of a general sequencing protocol for lignin oligomers with β-O-4 and 5-5 linkages. 5 5
O
O
OH
OH O
HO
O
O
OH OH
5
1
OH OH O
4
OH HO 5 5
O
O
β
HO
OH
OHHO
O
OH OH HO
2
O
5
6
O
OH OH
O
OH
5
O
OH
A' B'
O
OH HO
O O
5
5
A
O
O O
OH OH
OH
D'
O
HO
OH
O
HO
OH
C'
Experimental Section Materials Detailed synthetic procedures as well as 1H and 13C NMR spectroscopic data for 1-7 (all mixtures of stereoisomers with (R,S) or (S,R) absolute stereochemistry for the α- and β-carbons in each β-O-4 linkage; see Figure 1) are given in Supporting Information (SI). Synthetic procedures for the S-lignin model compounds are also given in SI. High-performance HPLC/MSgrade water, methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). All chemicals were used as received. Sample Preparation Stock solutions of all analytes were prepared at a final concentration of 0.1 mM in methanol. For HPLC/MS analysis, all analytes were dissolved in acetonitrile to achieve a final volume of 1 mL and an analyte concentration of 0.01 mM. Experiments All experiments were performed using a Thermo Scientific linear quadrupole ion trap (LQIT)/Fourier transform ion cyclotron resonance (FT-ICR; 7 T magnet) mass spectrometer. The mass spectrometer was equipped with an ESI source that was operated in the negative-ion mode. The ESI conditions were set as follows: 3.5 kV spray voltage, 20 (arbitrary units) flow of sheath gas, 10 (arbitrary units) flow of auxiliary gas, and a 275°C transfer capillary temperature. The LQIT/FT-ICR mass spectrometer was
operated using the LTQ Tune Plus interface. The automated tuning feature of the instrument was used to optimize the measurements for low mass ions (m/z 50 to m/z 500). Automated gain control was used to ensure a stable ion signal. A nominal pressure of 0.6 × 10−5 Torr, as read by an ion gauge, was maintained in the higher pressure LQIT vacuum manifold and 2.0 × 10−10 Torr in the FT-ICR vacuum manifold, as read by an ion gauge. In collision-activated dissociation (CAD) experiments, the advanced scan features of the LTQ Tune Plus interface were used to isolate the ions by using an m/z-window of 2 units. At a q value of 0.25, the ions were subjected to CAD by using helium as the collision gas for an activation time of 30 ms. “Normalized collision energies” were varied from 15 up to 30. A lower q value of 0.15 – 0.20 was also used to observe the low mass fragment ions. The elemental compositions of all major fragment ions were verified by using high-resolution measurements in the FT-ICR. Xcalibur 2.0 software was used for processing of all data. All mass spectra acquired are an average of at least 50 mass spectra.
OH
B O
O
O
7
OH OH
O
HO HO
O O
O
O
HO
3
HO
O
HO
OH
O
O
OH
O
O
O
O
O
O
OH
HO
HO
1 4
O
O
O
α
O
HO
O
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O
C
O
O
D
Quantum Chemical Calculations
O OH
4
HO HO
OH
Figure 2. Synthesized G-lignin model compounds 1-7. The 55 and β-O-4 linkage is highlighted in red.
Geometries for all ground states and transition states were computed by using density functional theory (DFT) with the M06-2X functional31 and the 6-311++G(d,p) basis set.32 All M06-2X geometries were verified to be local minima (or transition states) by computation of analytic vibrational frequencies, and these (unscaled) frequencies were used to compute zero–point vibrational energies (ZPVE) and 298 K thermal (H298 – E0) and free energies (G298 – E0) contributions for all species. DFT calculations were carried out with the Gaussian 09 electronic structure program
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Analytical Chemistry
suite.33 IRC runs were performed to connect the transition states to reactants and products.
Results and Discussion Selection of the ionization method is discussed first. The types of fragmentations observed are then introduced by comparing the fragmentation patterns of the two simplest deprotonated Glignin model compounds (1 and 5), which contain one β-O-4 or one 5-5 linkage, respectively. The identification of two major and one minor fragmentation pathways and their likely mechanisms follows. The fragmentation patterns of deprotonated Glignin compounds 2 and 6 are then rationalized, and the concepts of “key ions” and “ion groups” are introduced. Because the fragmentation patterns of the more complicated deprotonated G-lignin oligomers, including 3, 4 and 7 (Tables S1 and S2), as well as all deprotonated S-lignin model compounds follow the same trends, the data measured for these ions and discussion of the data are provided in Supporting Information (SI). For all of the deprotonated lignin oligomers discussed below, the aromatic ring in the monomer unit bearing the negative charge is called the A ring and is denoted as A-. The rings in the other monomers are called B, C and D (see Figures 1 and 2). For compounds with a 5-5 linkage, the rings in the monomers on one side of the 5-5-linkage are called A, B, C and D, and the rings on the other side of the 5-5-linkage, A’, B’, C’ or D’ (Figure 2). The sequence of the deprotonated oligomers is given as a list of the symbols of the rings in adjacent monomer units. For example, the sequence of tetramer 7 with a deprotonated A ring is denoted as A-BCD. Since compounds 2-4 with a 5-5 linkage are symmetrical, the negative charge is designated to the A’ ring. For example, the sequence of octamer 4 with deprotonated A’ ring is denoted as D’C’B’A’-ABCD. The sequences of fragment ions are denoted in the same way.
related deprotonated compounds has been reported to readily cleave upon CAD, leading to loss of H2O and CH2O or a neutral aromatic compound.27 As shown in Figure 3, similar observations were made for deprotonated 5. Losses of both H2O and CH2O or guaiacol (N1; Figure 3) yield the most abundant fragment ions (the mechanisms are discussed in the next section). However, the CAD mass spectrum of deprotonated 1 shows only methyl radical loss (Figure S6 in SI). CAD of the fragment ion (an MS3 experiment) results in another methyl radical loss (in competition with a loss of a water molecule and a loss of a hydroxyl radical; Figure S6), after which no further fragmentation was observed. This finding is consistent with previous CAD studies on deprotonated guaiacol and 2-methoxy-4methylphenol,23 which indicated that phenoxy-methyl bonds cleave readily in a homolytic manner in these types of ions. In sharp contrast, the carbon-carbon bond between the two aromatic rings (5-5 linkage) in 1 cannot be cleaved by CAD under these conditions. Therefore, CAD of deprotonated model compounds 2-4 with both 5-5 and β-O-4 linkages is expected to involve cleavages of the carbon-oxygen bonds and β-O-4 linkages but not the 5-5 linkage. O
O O O
O
A
O
O
-48 (H2O + CH2O) OH OH
Path I
5 [M-H]
OH O
Path II
O
-124 (N1)
O
O
O
-30 (CH2O)
A
m/z 123 B Ion Group I
m/z -271 AB Ion Group II
m/z- 319 A BC
-178 (N1')
B
O
Path I
Minor O
B
A
-18 (H2O)
m/z -301 AB Ion Group II
O
O
O
A
B
Path III
O
B
O
OH OH
m/z 195 A Ion Group I
Path I
O A O
m/z 165 B Ion Group I
Desorption and Ionization An ideal desorption/ionization method for degraded lignin (or any mixture of organic compounds) generates ions that contain the entire intact analyte molecule (e.g., protonated or deprotonated molecules, metal cation adducts) without fragmentation. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the most commonly used ionization methods for thermally labile, nonvolatile analytes. However, APCI has been reported to induce fragmentation to ionized lignin model compounds under both positive and negative ion-modes.23 Because of this and the promising results obtained23,24 previously using negative ion-mode electrospray ionization (ESI) with NaOH dopant, this method was chosen to evaporate and ionize the model compounds. This method leads to deprotonation of a phenol functionality for each model compound (formation of ([M-H]- ions) without fragmentation (Figures S4 and S5 in SI). No doubly deprotonated molecules ([M2H]2-) were observed although formation of [M-2H]2- ions from compounds with two phenol or two carboxylic acid functional groups has been reported for ESI.34,35
Figure 3. CAD mass spectrum and proposed fragmentation pathways for deprotonated 5 (key ions are shown in red boxes; the product from pathway 3 is shown in purple).
CAD Mass Spectra of Deprotonated 1 and 5 Among the seven G-lignin model compounds, compounds 1 and
5 are the simplest – dimers with only one 5-5 or β-O-4 linkage, respectively, connecting the monomers. The β-O-4 linkage in
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Major Fragmentation Pathways
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Two major fragmentation pathways were found for all deprotonated compounds with β-O-4 linkages. Elimination of H2O and CH2O (pathway 1) leads to the most abundant fragment ions in the CAD MS2 spectra of all the deprotonated compounds studied (2-7; Figure 3 and others; Figures S1, S2 and S3) with the exception of 1. The structures shown here for these and other fragment ions are supported by MS3 experiments wherein the fragment ions were isolated and subjected to CAD (data in SI). H2O and CH2O have been proposed27,36 to be lost from the hydroxyl group at the α-position and the hydroxymethyl group at the γ-position of the β-O-4 linkage, respectively, in the chargebearing monomer in related deprotonated compounds via the charge-driven mechanism shown on top in Figure 4. Indeed, our quantum chemical calculations predict a low barrier for the charge-driven loss of H2O and CH2O via this mechanism (28.8 kcal mol-1; Figure 4). This barrier is substantially lower than that calculated for the charge-remote elimination of H2O and CH2O (63.2 kcal mol-1; Figure 3).
After initial charge-driven losses of H2O and CH2O from the charge-bearing unit of the deprotonated lignin oligomers, the [M-H-H2O-CH2O]- fragment ions (with the exception of those formed from the dimers) continue to lose H2O and CH2O when isolated and subjected to CAD in MS3 experiments (Figures S7-S11). These reactions cannot be charge-driven since they occur too far away from the charge-bearing unit. Therefore, these reactions are likely to occur via charge-remote H2O and CH2O losses as shown in Figure 4 (bottom right). The second major fragmentation pathway involves the loss of neutral guaiacol (N1; 124 Da) or a guaiacol derivative with a 1,3-dihydroxy-2-(2-methoxyphenol)propyl group (N2; 320 Da) or a combination of two such groups (N3; 516 Da; Figure 5) at position 4 (pathway 2). These neutral molecules and the corresponding pathways are colored in green, blue or red, respectively, in Figure 5 and other Figures. Their structures were assigned based on the examination of the structures of the precur-
Charge-remote [M-H-N1] ion formation: OH
-
Charge-driven [M-H-H2O-CH2O] ion formation:
O OH OH
α OH O
HO
H O
β
γ
O O
O
-18 (H2O)
O
Path 1
O
H
O
O
Path 2
O O
O
O
O
O
O
-124 (N1)
O
O
O
O
O
OH OH
H
Pathway 1
-
Charge-remote [M-H-N2] ion formation: -
H OH O
Pathway 2
O
-48 (H2O+CH2O)
O
O
HO OH OH
O
O
Path 1
H O
O O
O
O
O O
O O
O
O
O
O
-30 (CH2O) Path 1
Concerted charge-remote [M-H-H2O-CH2O] ion formation:
OH OH
OH OH O
O
O
-320 (N2)
O
O
Path 2 OH OH
H
O
H
H
O
O
-
O
O
H
-
O
Charge-remote [M-H-N3] ion formation:
O O
HO H
O
OH OH
O
OH OH O
O H
O
HO
63.2
O O
HO O
OH O
OH OH O
O
HO O
O
O
O
O OH OH
OH OH
O
O
-516 (N3)
O
HO
Path 2
O OH OH
H OH O O O
O
H
H
O
28.8
∆G -1 (kcal mol )
O
O
O
O
25.6
OH OH
OH OH O
O
O H
O
O
H
O
HO
11.9
13.9 1.7 0.0
0.9
(b)
(c)
(d)
(e)
(a)
OH OH
OH OH O
O
0.0
H O
(a)
O O
O
(b)
(c)
H O
HO
O O
O
(d)
20,30
Figure 4. Charge-driven (top; published previously ) and charge-remote (bottom) fragmentation mechanisms for pathway 1. Below the mechanisms are potential energy surfaces calculated (M06-2X/6-311++G(d,p)//M06-2X/6-311++G(d,p)) for simple model compounds (for an ion on left, for a neutral molecule on right). Bottom left: (a) Lowest-energy conformation; (b) lowest-energy conformation with the two O-H bonds properly aligned (syn) for H2O loss; (c) H2O loss transition state; (d) H2O loss product coordinated with a H2O molecule; (e) product resulting from loss of H2O and CH2O. Bottom right: (a) Lowest-energy conformation; (b) lowest-energy conformation with the two O-H bonds properly aligned (syn) for H2O loss; (c) transition state for (simultaneous) loss of H2O and CH2O; (d) product resulting from loss of H2O and CH2O.
60.3
∆G -1 (kcal mol )
OH OH
49.7
O
O
+
+ OH OH
OH OH
O
O HO O
0.0 -6.4 (a)
(b)
(c)
0.0
(a)
-1.5
(b)
(c)
Figure 5. Top: Charge-remote Maccoll fragmentation mechanisms for pathway 2. Below the mechanisms are potential energy surfaces calculated (M06-2X/6-311++G(d,p)//M06-2X/6311++G(d,p)) for simple model compounds (for an ion on left, for a neutral molecule on right). Bottom left and right: (a) Lowest-energy conformation (with C-H and C-O bonds properly aligned (eclipsed) for fragmentation); (b) fragmentation transition state; (c) products resulting from fragmentation.
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Analytical Chemistry OH OH
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O H O
O
OH OH O
+
O
O O
O
Scheme 1. Retro-ene elimination. sor and fragment ions by MS2 and MS3 (see SI). Based on quantum chemical calculations, these fragmentations occur via a charge-remote 1,2-elimination mechanism, likely either the Maccoll (Figure 5) or retro-ene elimination (Scheme 1). Both mechanisms cleave the β-O-4 linkage and lead to the formation of a double bond between the α− and β−carbons in the β-O-4 linkage of the product ions. As shown in Figure 4, the calculated free energy barrier for such a 1,2-elimination via the Maccoll mechanism for a simple model compound is 60.3 kcal mol-1. The analogous value for the retro-ene elimination is 56.8 kcal mol-1. Somewhat lower barriers were calculated for 1,2-eliminations occurring on a β-O-4 linkage next to the charged site (e.g., in a deprotonated β-O-4 dimer; 49.7 kcal mol-1 and 51.4 kcal mol-1 for Maccoll (Figure 5) and retro-ene (Scheme 1), respectively).All of these barriers are substantially higher than that calculated for the charge-driven H2O and CH2O losses (28.8 kcal mol-1; Figure 4). This explains why the latter losses dominate for the deprotonated compounds. However, loss of H2O and CH2O is not nearly as dominant for the fragment ions of the deprotonated compounds (Figures S7-S12), which is explained by the larger barrier for the charge-remote mechanism. The MS3 spectra of [M-H-H2O-CH2O]- and [M-H-H2O]- fragment ions derived from compounds 2-7 (Figures S7 –S12) show similar patterns of N1, N2 and N3 losses as the MS2 spectra of the deprotonated molecules, indicating that charge-remote fragmentations are responsible for losses of these neutral molecules.
hereafter and their m/z-values are highlighted by red boxes in Figures 3, 6 and 7, and S1-S3. The term “ion groups” is introduced here for a group of fragment ions that contain the same number of benzene rings. Each ion group contains a key ion and its H2O and/or CH2O elimination products (formed through pathway 1). The ion groups are given symbols I–VI based on the number of benzene rings they contain; ions in group I contain only one benzene ring, ions in group II contain two benzene rings, and so on (see Figures 3, 6 and 7). Consideration of key ions and ion groups facilitates sequencing of unknown lignin oligomers, as described below. In lignin oligomers containing only β-O-4 or both β-O-4 and 55 linkages, the total number of linkages (including both β-O-4 and 5-5 linkages) equals the number of ion groups minus one. As shown in Figure 6 and Table S2, CAD of the deprotonated β-O-4 trimer 6 (m/z 515, with the sequence A-BC) yields three ion groups: trimeric ions (A-BC, ion group III), dimeric ions (A-B and B-C, ion group II) and monomeric ions (A- or B-, ion group I). Hence, it can be concluded that this ion contains two linkages. Besides losing H2O and CH2O, the deprotonated triOH OH O C
O
-48 (H2O + CH2O) m/z 271 O Path 1 BC Ion Group II
-196 (N1')
O
B
O
O
B
O
OH OH
C O
-
-124 (N1)
th
Pa
2 Path 2
OH OH
m/z 165 A or B
-30 (CH2O) Path 1
O
-124 (N1)
-320 (N2)
OH OH
O
m/z 195
Ion Group I
-
-
A or B Ion Group I
A BC Ion Group III
-
[M-H] m/z 515 A BC
BC Ion Group II
m/z 497 m/z 467 -
Path 1
OH OH 6
m/z 319
Path 2
-18 (H2O) -48 (H2O + CH2O)
O
O
A
Path 3
O
O
B
O
O
A
OH OH
m/z 391
-18 (H2O) -48 (H2O + CH2O)
m/z 373 m/z 343 -
Path 1
AB Ion Group II
-
AB Ion Group II
A minor fragmentation pathway (pathway 3) was observed to involve elimination of a neutral aromatic fragment N1’ or an analog (the smallest of these molecules is likely to have the structure shown in Figure 3; a calculated charge-driven mechanism is shown in Scheme S1). These eliminations always occur from the charge-bearing ring, which makes them useful in the identification of the mass of this ring. This fragmentation was found to produce small amounts of fragment ions for the deprotonated lignin dimer 5, trimer 6 and tetramer 7 (Figures 3, 6 and S1; products from pathway 3 are highlighted in purple). In sharp contrast, deprotonated compounds containing the 5-5 linkage (2–4) only show trace amounts of fragment ions formed via this pathway (Figure 7, Figures S2 and S3). CAD Mass Spectra of Deprotonated Compounds 2 and 6: “Key Ions” And “Ion Groups” Compound 2 is a tetramer and the simplest G-lignin model compound with both β-O-4 and 5-5 linkages while compound 6 is a trimer containing only β-O-4 linkages. Elucidation of the special features of the CAD mass spectra of these two deprotonated compounds demonstrates the differences in the fragmentation patterns of compounds with and without 5-5 linkages as well as the utility of “key ions” and “ion groups”. Fragment ions resulting only from pathways 2 and 3 without accompanying/additional H2O or CH2O losses are essential to the sequencing of β-O-4 lignins. These ions are called “key ions”
Figure 6. CAD mass spectrum and proposed fragmentation pathways for deprotonated 6 (m/z-values of key ions are in red boxes; products from pathway 3 are highlighted in purple). mer 6 can either lose N1 (pathway 2) to form a dimeric ion AB (m/z 391; ion group II) or N1’ (pathway 3) to form a dimeric ion B-C (m/z 319; ion group II). The B-C ion undergoes 1,2elimination to expel N1 (pathway 2) to form the monomeric ion of m/z 195 (ion group I). Ion m/z 195 can also be formed from deprotonated 6 ([M-H]-) through 1,2-elimination of N2. “Key ions” are identified as m/z 391 (A-B), m/z 319 (B-C), and
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m/z 195 (A- or B-). The sequence of 6 (A-BC) can be deduced from these three fragment ions as the ion of m/z 319 is formed via elimination of the A ring, m/z 391 via the elimination of the C ring, and m/z 195 via elimination of either B and C rings simultaneously or first A ring and then C ring. As shown in Figure 7 and Table S1, CAD of the deprotonated tetramer 2 containing both β-O-4 and 5-5 linkages yields four ion groups: tetrameric ions (B’A’-AB, ion group IV), trimeric ions (B’A’-A, ion group III), dimeric ions (A’-A, ion group II) and monomeric ions (B’-, ion group I). Hence, the number of total linkages can be concluded to be three. -124 (N1)
m/z 483,-465 ' B'A A Ion Group III
O HO
Path 1
O O
O B'
Path 2
-18 (H2O) -30 (CH2O)
Path 1
O
m/z-589 ' B'A AB Ion Group IV
A'
A
-124 (N1)
O
O B'
HO
OH
HO
OH
-48 (H2O + CH2O)
O HO A'
O O
O OH
Path 2 OH
m/z 513
O
O A
'-
B'A A Ion Group III
-N1' analog
-
[M-H] m/z -637
B'
-
B' Ion Group I
'
B'A AB
+ O HO
O
Path 2
-124 (N1)
A'
O O
A O
O O HO
O
A'
B
HO
O
HO
496 Da
A
-30 (CH2O) HO
OH
Path 1 HO
OH
m/z 389
m/z 359 -
'
-30 (CH2O)
Path 1 A A Ion Group II
It should be noted that ions of m/z 465 were found to be formed in two different ways, either via the route M-H-N1-H2O-CH2O or route M-H-H2O-CH2O-N1, which was confirmed by MS3 experiments (Figure S20). Other fragment ions that are formed via more than one route can be found in the CAD mass spectra of deprotonated hexamer 3 and octamer 4 (Figures S2 and S3). The m/z values of such fragment ions are given in bold in Table S1. All of their formation pathways were confirmed by MS3 experiments.
m/z 123
Path 3 Minor
HO
and III. Determination of the mass differences between deprotonated 2 and these two ions reveals the masses of the two eliminated end units B and B’. The mass of the central AA’ unit is revealed by the m/z-value of the ion formed upon elimination of the two N1 units. The sequence of tetramer 2 can thus be deduced based on this information.
A Summary of Sequencing of Deprotonated Lignin Model Compounds with Both β-O-4 and 5-5 Linkages
O
B
2 HO
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m/z-329
'
A A Ion Group II
'-
A A Ion Group II
Unbranched deprotonated lignin oligomers with both β-O-4 and 5-5 linkages can be sequenced based on their CAD MS2 and MS3 spectra. First, the number of linkages (β-O-4 and/or 5-5 linkages) can be determined from the number of ion groups. For larger lignin molecules, MS3 experiments are needed to accomplish this. If the compound only contains β-O-4 linkage(s), then the total number of β-O-4 linkages is equal to the number of ion groups minus one. If the compound contains both β-O-4 and 55 linkages, the number of β-O-4 linkages is equal to the number of ion groups minus the number of 5-5 linkages minus one. Second, the monomer sequence can be determined by consideration of the m/z-values of the key ions for each ion group. For example, as shown in Figure 8, Figures S1 and S23, and Table S2, deprotonated tetramer 7 shows key ions of m/z 587 ([M-HN1]-, A-BC), m/z 515 ([M-H-N1’]-, B-CD), m/z 391 ([M-HN2]-, A-B), m/z 319 ([M-H- 2N1’]-, C-D) and m/z 195 ([M-H2N1’-N1]-, A-) formed upon losses of N1, N2, and/or N1’. The mass of the aromatic unit furthest away from the charged site (e.g., D for 7) is equal to the mass of the lost N1 molecule. The mass of this unit plus the one next to it (C and D) is equal to the mass of the lost N2 molecule. The mass of the neutral molecule N1’ lost in pathway 3 reveals the mass of the charged A ring and the mass of the following N1’ loss reveals the mass of the B ring. The mass of the C unit is equal to the mass difference between N2 and N1.
Figure 7. CAD mass spectrum and proposed fragmentation pathways for deprotonated 2 (m/z-values of key ions are in red boxes; products from pathway 3 are highlighted in purple). Only one fragment ion in group I (B’-, m/z 123) was observed and it was detected only after lowering the low mass limit of the instrument (Figure S22). This ion is formed via pathway 3. The lack of formation of monomeric ions via pathway 2 indicates that the compound contains a 5-5 linkage as this linkage is stable toward CAD under the conditions employed here. Two consecutive N1 losses via pathway 2 result in the formation of key ions (m/z 513 and m/z 389, respectively) in ion groups II
Finally, the existence of a 5-5 linkage in an unknown ligninrelated molecule can be verified by examining CAD of the dimeric fragment ions (an MS3 experiment). If no further fragmentations are observed for some of these ions, the deprotonated molecule contains a 5-5 linkage. Conclusions In this study, seven G-lignin model compounds with β-O-4 and/or 5-5 linkages and three S-lignin model compounds with β-O-4 linkages were synthesized and the collision-activated dissociation (CAD) patterns of their deprotonated molecules were studied in MS2 and MS3 experiments in order to facilitate sequencing of unknown lignin oligomers. Quantum chemical
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calculations were used to gain a deeper understanding of the fragmentation mechanisms. Examination of the model compounds with a 5-5 linkage demonstrated that the 5-5 linkage cannot be cleaved under the conditions employed here. Two major and one minor fragmentation pathways were discovered for deprotonated compounds with only β-O-4 linkages or both 5-5 and β-O-4 linkages. The most informative fragmentations involve losses of neutral molecules containing one, two or three aromatic rings (N1, N2 and N3, respectively) via charge-remote Maccoll or retro-ene elimination mechanisms and a minor
fragmentation involving elimination of a neutral molecule (N1’) or analog. Consideration of key ions and ion groups enables sequencing of deprotonated lignin oligomers in three steps: (1) the number of ion groups in CAD mass spectra reveals the number of linkages, (2) calculation of the mass differences between the deprotonated molecules and key ions in each ion group reveals the identity of the lignin monomers and their sequence, and (3) utilization of MS3 experiments to verify the fragmentation behavior of fragment ions in ion group III reveals the presence of 5-5 linkages.
No. of β-O-4 Linkages = No. of Ion Groups - No. of 5-5 linkages - 1 OH OH
Molecule only contains β-O-4 linkages
Molecule contains 5−5 linkages
O O
Sequenced
A
O B
O
D
O
O
C
No. of Ion Groups
CAD
Four (I, II, III, IV) (Tetramer)
O
No. of β-O-4 Linkages = No. of Ion Groups - 1
No
O
OH OH
Yes
OH OH
7 [M-H] - 711 m/z A BCD
MS2 Spectrum
No. of Linkages = No. of Ion Groups - 1
Can all the dimer ions be fragmented to monomer ions?
OH OH O A O
m/z 195
-
A Ion Group I
Sequence of key ions provides further information on the sequence of the oligomer
OH OH
O
CAD (MS3) of key ions
D
O
O
C
O
O
B
O
O
A
O OH OH
( β-O-4 and/ or 5-5 linkages)
OH OH
m/z 391
CD Ion Group II
AB Ion Group II
-
OH OH
OH OH O
O B
Three linkages
m/z 319
-
O
Identify "key ions" in three ion groups (I, II, III)
O D
O
O A
O
C
O B O
O C O
O OH OH
OH OH
OH OH
m/z- 587
m/z- 515
A BC Ion Group III
B CD Ion Group III
Figure 8. Sequencing flow chart for deprotonated tetramer 7 (m/z-values for key ions are in red boxes).
ASSOCIATED CONTENT
Notes
Supporting Information. Additional MS2 and MS3 spectra of ionized lignin model compounds, synthesis procedures, NMR data related to synthesis of compounds, and Tables of Cartesian coordinates, electronic energies, zero-point vibrational energies and 298 K thermal contributions for all computed structures are provided in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org/.
AUTHOR INFORMATION Corresponding Author
The authors declare no competing financial interest.
ACKNOWLEDGEMENT This work was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0000997. A reviewer is thanked for proposing the mechanism shown in Scheme S1.
*(H. I. K) Telephone: (765) 494-0882; fax: (765) 494-0239; E-mail:
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(21) (22) (23)
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(26) Marcum, C. L.; Jarrell, T. M.; Zhu, H.; Owen, B. C.; Haupert, L.; Easton, M.; Hosseinaei, O.; Bozell, J.; Nash, J. J.; Kenttamaa, H.I. ChemSusChem 2016, 9, 3513. (27) Morreel, K.; Kim, H.; Lu, F.; Dima, O.; Akiyama, T.; Vanholme, R.; Niculaes, C.; Goeminne, G.; Inze, D.; Messens, E.; Ralph, J.; Boerjan, W. Anal. Chem. 2010, 82, 8095-8105. (28) Morreel, K.; Dima, O.; Kim, H.; Lu, F.; Niculaes, C.; Vanholme, R.; Dauwe, R.; Goeminne, G.; Inze, D.; Messens, E.; Ralph, J.; Boerjan, W. Plant Physiol. 2010, 153, 1464-1478. (29) Morreel, K.; Ralph, J.; Kim, H.; Lu, F.; Goeminne, G.; Ralph, S.; Messens, E.; Boerjan, W. Plant Physiol. 2004, 136, 3537-3549. (30) Evtuguin, D. V.; Amado, F. M. L. Macromol. Biosci. 2003, 3, 339-343. (31) Zhao, Y.; Truhlar, D. G. Theor. Chem. Acc. 2008, 120, 215-241. (32) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem. Phys. 1980, 72, 650-654. (33) Gaussian 09, Revision E.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.; Gaussian, Inc., Wallingford CT, 2009. (34) Michelsen, P.; Jergil, B.; Odham, G. Rapid Commun. Mass Spectrom. 1995, 9, 1109-1114. (35) Gallart-Ayala, H.; Moyano, E.; Galceran, M. T. Rapid Commun. Mass Spectrom. 2007, 21, 4039-4048. (36) Bowie, J. H. Mass Spectrom. Rev. 1990, 9, 349-379.
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Table of Contents Picture: O
O
HO
O
O
O O
O
HO
OH OH
HO
O OH
O OH
O
O
HO HO
O
O
O O
HO HO
OH OH
Sequencing
CAD
MS2 and MS3 Spectra Interpretation
Key Ions Ion Groups
9
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