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Towards Closing the Gap between Hexoses and NAcetlyhexosamines: Experimental and Computational Studies on the Collision-Induced Dissociation of Hexosamines Cheng-chau Chiu, Hai Thi Huynh, Shang-Ting Tsai, Hou-Yu Lin, Po-Jen Hsu, Huu Trong Phan, Arya Karumanthra, Hayden Thompson, Yu-Chi Lee, Jer-Lai Kuo, and Chi-Kung Ni J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b04143 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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The Journal of Physical Chemistry

Towards Closing the Gap between Hexoses and N-Acetlyhexosamines: Experimental and Computational Studies on the Collision-Induced Dissociation of Hexosamines

Cheng-chau Chiu,1† Hai Thi Huynh,1,2,3† Shang-Ting Tsai,1 Hou-Yu Lin,1,4 Po-Jen Hsu1, Huu Trong Phan,1,2,3 Arya Karumanthra,1,5 Hayden Thompson,1,6 Yu-Chi Lee,1 Jer-Lai Kuo,*1 Chi-Kung Ni*1,3

1

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, 10617, Taiwan

2

Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei, 11529, Taiwan

3

Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.

4

Department of Chemistry, National Taiwan University, Taipei, 10617, Taiwan.

5

Undergraduate Programme, Indian Institute of Science, Bangalore, 560012, India

6

Department of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom

*Corresponding †These

author’s email: [email protected], [email protected]

authors contributed equally to this work.

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Abstract Motivated by the fundamental difference in the reactivity of hexoses and Nacetylhexosamines under collision-induced dissociation (CID) mass spectrometry conditions, we have investigated the CID of two hexosamines, glucosamine (GlcN) and galactosamine (GalN), experimentally and computationally. Both hexosamines undergo ring-opening and then dissociate via the

0,2A

and the

0,3A

(0,3X) cross-ring cleavage

channels. The preference for the ring-opening is similar to the behavior of Nacetylhexosamines and explains, why the two anomers of the same sugar give the same mass spectrum. While the spectrum for GlcN is dominated by the intensities for both

0,2A

and the

0,3A (0,3X)

0,2A

signal, the signal

dissociation channels are comparable for GalN,

which allows GlcN and GalN to be distinguished easily. Calculations at MP2 level of theory indicate that this is related to the differences in the relative barrier heights for the 0,2A

and the

0,3A

(0,3X) cross-ring cleavage channels. This, in return, reflects the

circumstance that the

0,2A

cross-ring cleavage barriers are different for the two sugars,

while the barriers of all other dissociation channels are comparable. While the mechanisms of the cross-ring dissociation channels of hexoses are well described using retro-aldol reaction in the literature, this study proposes a new mechanism for the 0,3A (0,3X) cross-ring cleavage of hexosamines that involves the formation of an epoxy intermediate or a zwitterionic intermediate.

Keywords: Glucosamine, galactosamine, collision-induced dissociation, dissociation mechanism, MP2, mass spectrometry


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1.

Introduction Carbohydrates play important roles in various biochemical processes like cell

signaling1 or the immune system.2 A key to the improved understanding of these processes is the identification of the structures associated with the involved carbohydrates. However, due to the lack of a routine method, the structural determination of carbohydrates is still a challenging task.3 The difficulties arise, among others, from the circumstance that the monosaccharides, i.e., the building blocks for the carbohydrates, are often very similar molecules which only differ by the stereochemistry at a few stereocenters. Several concepts have been proposed for the structural characterization of carbohydrates, including methods based on liquid chromatography (LC),4 capillary electrophoresis spectroscopy,14

(CE),5,6 ion

spectroscopy,7–12

IR

mobility.15–19

rotational

spectroscopy,13

NMR

Due to its high sensitivity and the limited amount of

sample material from typical biological systems, mass spectrometry (MS) is also an often discussed approach.20–22 In the MS approach, single-stage mass spectrometry only provides limited information on carbohydrate structures. The combination of various fragmentation methods, including collision-induced dissociation (CID),23–26 higher collision energy dissociation,27 infrared multiphoton dissociation,28,29 ultraviolet dissociation,30 electronic excitation dissociation,31 electron capture dissociation,32 and electron transfer dissociation33 in tandem mass spectrometry reveals much more information on the carbohydrate structures. Our team has recently developed an MS-based approach referred to as the logically derived sequence (LODES) tandem mass spectrometry method to characterize the structure of oligosaccharides.34–37 In this approach, the sodium or lithium cation adduct of a sugar molecule is fragmentized with the CID method. The generated fragment ions are selected, according to a logically derived sequence, and fragmentized again. As the various monosaccharides and the different linkages between two monosaccharides have different characteristics in the fragmentation pattern, one can identify an oligosaccharide by comparing its mass spectra to a database of fragmentation patterns for known sugar structures. However, it is crucial to understand the molecular dissociation mechanisms leading to the observed fragmentation patterns in MS, if one wants to use the LODES approach to its full power. Quantum chemical calculations have been used to reveal the dissociation mechanisms of sugars.38–47 Most of these studies have concentrated on the theoretical investigation of carbohydrate decomposition in the context of renewable energy and mass spectrometry. In 3 ACS Paragon Plus Environment


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the present work, we use experiments and quantum chemical calculations to explore the dissociation mechanism of two sodiated hexosamines or “HexNs”: glucosamine (GlcN) and galactosamine (GalN). This work is a continuation of our previous studies on the CID of sodiated hexoses,42,44 as well as of sodiated N-acetylhexosamines (HexNAcs).46 While it could be shown that the dominating dissociation channel for hexoses is either the loss of an HO-CH=CH-OH fragment, referred to as the

0,2A

cross-ring dissociation according to the

conventional notation of Domon and Costello,48 or the loss of a water molecule via the H transfer from an OH group to another OH group,42,44 the main reaction channel for HexNAcs is the water elimination via the H transfer from a C atom to an OH group. 46 The fact that the mere presence of the NAc group in HexNAcs can induce a fundamental change to the dissociation pathway triggered our interest to understand the origin of the differences in the preferred reaction better. Therefore, we have decided to look at hexosamines, which can be understood as an “intermediate” class of saccharides between the previously investigated hexoses and HexNAcs. 2.

Methods and Models

2.1. Experimental Details The experimental details have been described in previous studies,42,44,46 thus only a brief description is given here. Glucosamine (99%) and galactosamine (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. The saccharides were dissolved in a 1:1 (v/v) methanol/water solution containing 210-4 M NaCl to produce 10-4 M solutions of the saccharides. The CID spectra were measured in the positive mode by using a LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA USA) equipped with an electrospray ionization (ESI) source and a Dionex Ultimate 3000 high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific Inc., Waltham, MA USA). The Dionex chromatography-mass spectrometry link was installed as an interface to control the Dionex chromatography system with Xcalibur (Thermo Fisher Scientific Inc., Waltham, MA USA). The separation of the two anomers of GlcN and GalN, respectively, via HPLC was performed using a Hypercarb (100 × 2.1 mm, particle size of 3 μm) column, operated at room temperature (25 °C). The volume of the sample solution injected into HPLC was 10 μL. The mobile phase for isocratic elution comprised 0.1% (v/v) aqueous formic acid with 4 ACS Paragon Plus Environment

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1×10−4 M NaCl. The mobile phase flow rate was set to 150 μL/min. The MS conditions were optimized using the built-in semiautomatic tuning procedure in Xcalibur. The ESI source was operated at a temperature of 280 °C with a sheath gas flow rate of 35 (arbitrary units), an auxiliary gas flow rate of 20 (arbitrary units) and a sweep gas flow rate of 15 (arbitrary units). The ion spray voltage was set to 4.50 kV and the transfer capillary temperature was 400 °C. A capillary voltage of 25.5 V and a tube lens voltage of 91 V were applied. Helium gas was used both as the buffer gas for the ion trap and as the collision gas in CID. The MSn experiments (n-stage MS) were carried out with a normalized collision energy of 25%, a Q value of 0.25, and 30 ms activation time. The amount of ions regulated by the automatic gain control was set to 1×105 for full scan mode and 1×104 for MSn mode, and the precursor ion isolation width was set to 1 u. In a separate experiment, a tandem quadrupole mass spectrometer (Bruker Daltonics Esquire 3000 plus ion trap mass spectrometer, Billerica, MA, USA) was used for low mass fragment measurement. The ESI source was operated at the following conditions: nebulizer 10 psi, dry gas 5.0 L/min, dry temperature 300 oC. The trap ion charge control (ICC) mode was set at 30000 (arb. units); max accumulation time was 300 ms; scan range was 15 -230 m/z, the number of averaged spectra was 7; start and end amplitudes were 100% and 550%, respectively; delay time 0 ms; mass width was 2 m/z. 2.2. Computational Details 2.2.1. Quantum Chemistry Calculations We have used four levels of theory for the calculations reported in this study. For preoptimization, we have used the semi-empirical DFTB3 method. 49 These calculations were carried out with the quantum-chemistry package GAMESS (2018 R1),50 using the 3OB (“Third-Order Parametrization for Organic and Biological Systems”) parameters. 51,52 In addition, we have performed calculations at non-spin polarized, hybrid DFT level using the B3LYP functional53–55 in combination with a 6-311+G(d,p) basis set.56–59 Apart from earlier benchmark studies showing that B3LYP yields reasonable energetics for minima structures of sugars,60 the main reason to use this functional is to ensure comparability to our earlier studies.42,44,46 The main results reported in this work were obtained from calculations at MP2 level using the same basis set as for the B3LYP calculations. To check the accuracy of the MP2 results, we have further re-evaluated the energies of the most important initial and transitions states (ISs and TSs) using the CCSD(T) method in 5 ACS Paragon Plus Environment


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combination with the jul-cc-pVDZ basis set.61 For this, a single point calculation at CCSD(T) level has been performed on the geometries optimized at MP2 level and are thus referred to as the “CCSD(T)//MP2” calculations. The B3LYP, MP2, and CCSD(T)//MP2 calculations have all been performed with the program Gaussian09.62 The TS structure search was mainly carried out using the Berny algorithm63 as implemented in Gaussian09. In some cases, we have used a self-implemented climbing image-nudged elastic band (CINEB) algorithm64 as developed by Henkelman et al.65 to get a pre-optimized TS geometry that is used for the TS calculation with the Berny algorithm. For all discussed geometries at both B3LYP and MP2 level, we have performed an analytical normal mode analysis within the harmonic approximation and made sure that the reported minima and TSs have exactly zero or one imaginary vibrational frequencies, respectively. All energies reported in this work are electronic energies corrected by the zero-point energies (ZPE). For the energies at CCSD(T)//MP2 level, we have used the ZPE values obtained self-consistently at MP2 level. 2.2.2. Screening of Minima Structures 2.2.2.1. Ring-Form For sodiated GlcN in ring-form, we have tried different approaches to generate a database with geometries optimized at B3LYP level. As we already had a structure database for sodiated glucose from our earlier work,44 we simply took these structures and replaced the OH group at C2 by an amino group to obtain the initial-guess structures for the geometry optimization. To identify structures that optimize to the same geometry, we have used our previously developed two-stage clustering algorithm, 66 which is based on an ultrafast shape recognition method.67 After removal of the duplicate structures and the classification of the structures according to the Cremer-Pople (CP) puckering index68 and the Na+ position, we obtained 80 and 71 distinct structures optimized at B3LYP level for sodiated α and β-GlcN, respectively. The above-described procedure to obtain a structure database is hereafter referred to as the method “A”. A more general way to obtain a structure database for sodiated and neutral sugars from scratch has been reported in our earlier works on hexoses42,44 and HexNAcs.46 Thus, we will, only briefly describe the procedure here and point out the small adjustments in the methodology that has been made for this work. We have started with 20000 randomly generated initial guess geometries for each neutral anomer under study. After preoptimization at DFTB3 level and the removal of structure duplicates, we have re-optimized the most stable 20% of the remaining structures at B3LYP level, resulting in 115, 127, 104, 6 ACS Paragon Plus Environment

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104 distinct geometries for neutral α-GlcN, β-GlcN, α-GalN, and β-GalN. The main reason for calculating the database for the neutral sugars is to identify the most stable neutral conformer, which is used for the calculation of the desodiation energy (see Section 2.2.5). Thus, it seems sufficient for us to only consider the most stable 20% of the geometries for optimization at B3LYP level. To generate the database for the sodiated structures, we have classified the DFTB3 geometries of the neutral sugars according to the puckering indexes. For each puckering index, we have taken the most stable conformer and randomly placed a Na+ in the vicinity of the N atom or one of the O atoms. These initial-guess structures have also been pre-optimized at DFTB3 level, before optimizing at B3LYP level. After removal of the structure duplicates, we have 124 and 132 distinct geometries for sodiated α- and βGalN, respectively. The above-described way to generate a structure database will be referred to as method “B”. In the case of GlcN, we have merged the geometries with the database obtained with method A, resulting in 174 and 193 structures for sodiated α- and βGlcN, respectively. For getting a structure database at MP2 level, we have simply taken the ten most stable geometries for the neutral and the sodiated form for each anomer as obtained by method B and reoptimized them at MP2 level. The main purpose of generating a structure database at MP2 level is to estimate the desodiation energy. For sodiated structures, we have further included a few minima geometries for optimization, which are associated with the energetically low lying TSs for the different dissociation channels. 2.2.2.2. Linear Form To screen the structures for GlcN and GalN in linear form, we have started with a randomly selected linear conformer for the sugar under study and optimized it at DFTB3 level. We have rotated each of the C-C, C-O, and C-N single bonds by a random angle between -180° and 180°. A Na+ ion is then randomly added in the vicinity of one of the O atoms or the N atom. Following this procedure, we have generated 2000 initial-guess structures for each sugar under study. If the resulting geometry could be described by the DFTB3 parameters, which means that the structure does not feature any unphysically small interatomic distances, it is optimized at DFTB3 level. After removing structure duplicates, the re-optimization at B3LYP level yielded 167 and 345 distinct structures for GlcN and GalN, respectively. To obtain the global minima of linear sugars at MP2 level, we have simply re-optimized the most stable 20% of the conformers obtained from the B3LYP calculations, resulting in 33 and 69 distinct conformers for sodiated, linear GlcN and GalN, 7 ACS Paragon Plus Environment


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respectively. Similar to the sugars in ring-form, we have also added local minima structures associated with the most favorable dissociation channels. 2.2.3. Screening of TS Structures The standard procedure for TS search at B3LYP level of theory starts with the prescreening of a structure database. We search for minima geometries that fulfill certain conditions to be a potential initial state (IS) geometry for a reaction of interest. These structures are then manually distorted along the guessed reaction path and used as an initial guess geometry for the TS optimization using the Berny algorithm. For the sugars in ring-form, we have considered the dehydration, the deamination, and the ring-opening reaction. For the dehydration and the deamination, we have only considered the transfer of an H atom from an OH or an NH2 group to an OH or an NH2, which are referred to as the D2O dehydration or ND3 deamination channels. This nomenclature has been introduced in our previous work on HexNAc,46 and reflects the molecule that would be eliminated from a deuterium-exchanged sugar molecule (H atoms in OH/NH2 functional groups are replaced by D atoms). Examples for these two reaction channels are depicted in Figure 1 (a) and (b). The counterparts to these reactions are the DHO dehydration and ND2H deamination channels, involving the transfer of an H atom from a C atom to an OH or an NH2 group, and have been omitted for HexNs in ring-form as our earlier work has shown that these reactions are kinetically hindered. 46 In reports on similar processes involving sugars in ring-form in literature, this dissociation channel does not play a role either,43,47 which is actually easy to understand: In the ring-form, there are no carbonyl groups, which would enhance the C-H acidity in α-position. Consequently, we think that we can safely neglect the DHO dehydration and the ND2H deamination here, as both reactions require increased C-H acidity to occur. A minima geometry was considered a potential initial state for the D2O dehydration or ND3 deamination channels, if i) the distance between the O/N atom of the H donating OH/NH2 group and the O/N of the H accepting OH/NH2 group is below 3 Å and ii) the Na+ ion is interacting with the H donating OH/NH2 group, with an O-Na or N-Na distance below 3 Å. The second condition was introduced as it is known that the presence of a Na+ ion can enhance the acidity of the interacting OH/NH2 groups.42,44,46 For the ring-opening proceeding via the H transfer from O1 to O5 followed by C1-O5 bond cleavage (Figure 1


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(c)), we only considered such minima structures as initial states in which the H at O1 is pointing towards O5.

Figure 1. Potential mechanisms of the different dissociation channels shown at the example of β-GlcN (a-d), linear GlcN (e-g) and linear GalN (h): (a) examples for D2O dehydration, (b) examples for ND3 deamination, (c) “classical” 0,2A cross-ring cleavage via ring-opening 9 ACS Paragon Plus Environment


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followed by the retro-aldol mechanism, (d) alternative direct 0,2A cross-ring cleavage without preceding ring-opening, (e) one-step mechanism and (f) conventional two-step mechanism for 0,3A cross-ring cleavage, (g) two-step epoxy mechanism the for 0,3A crossring cleavage of GlcN and (h) 1,2-zwitterionic mechanism for 0,3A cross-ring cleavage of GalN. The arrows in red denote the movement of the electron pairs. The red cross over the reaction arrow in (e) and (f) indicates that we were not able or did not try to locate a TS structure for these mechanisms. Note that “0,3A ion” typically refers to the fragment arising from the atoms C4, C5, and C6. In (e) to (h), the Na+ ion can also bind to the N-containing side, as indicated by the Na+ in parenthesis, resulting in the so-called “0,3X ion”. Further note that it is difficult to include all structural aspects in these schematic representations in two dimensions. For details, the interested reader is referred to the Cartesian coordinates provided in the SI.

For linear sugars, we have considered not only the D2O dehydration or ND3 deamination channels but also the DHO dehydration or ND2H deamination channels. For the former, the criteria for a minima structure being considered a potential IS geometry are the same as in the case of the analog reactions from sugars in ring-form. For the latter, we only imposed a condition: The H being transferred should be 3 Å or closer to the O or N atom of the accepting OH or NH2 group, irrespective of the Na+ position. In addition, there are the 0,2A and the 0,3A (or 0,3X, depending on which fragment the Na+ binds to) cross-ring cleavage reactions for the linear HexNs. In the case of the 0,2A cross-ring cleavage, which is well known in literature to, after initial ring-opening, follow a retro-aldol mechanism involving the H transfer from O3 to O1 and the simultaneous C2-C3 bond cleavage (see Figure 1 (c)),42,44,47,69–72 the conditions require an initial state to have the Na+ ion close to O3 and feature a distance of 2.9 Å or shorter between O1 and the H at O3. We have also considered an alternative mechanism for the

0,2A

cross-ring cleavage as shown in Figure 1

(d), but as the discussion in Section 3.2.1 will show, this mechanism is only of minor importance. For the C-C cleavage step following initial ring-opening in the

0,3A

cross-ring

cleavage, we started with the two mechanisms shown in Figure 1 (e) and (f). For the onestep mechanism,42 which proceeds via the concerted H transfers from O4 to O1 as well as from C2 to C1 and the C3-C4 bond cleavage, we only considered such ISs in which the H at O4 is pointing at O1. For the two-step

0,3A

cross-ring cleavage mechanism, also known

in literature to follow a retro-aldol mechanism,69 which starts with an H being transferred from the amino group to O1, we only did an extensive screening of initial state structures for the first step and only considered such minima geometries in which the amino H atom is pointing at O1. As the results in Sections 3.2.2.2 and 3.2.2.4 will show, this step is


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associated with a high barrier, so we have omitted to look at the second step of this mechanism. In some cases, where the procedure described above fails to yield an optimized TS structure, we have optimized the geometry of the anticipated product state and performed a pre-optimization using the CI-NEB method and used the so obtained geometry as an initial guess for the TS search with the Berny algorithm. Note that in some cases the optimized TS does not necessarily correspond with the assumed reaction mechanism. Thus, we have performed an analysis of the intrinsic reaction coordinate (IRC) for the most stable TSs for each dissociation pathway to reveal the actual mechanism associated with a TS structure. Due to the higher computational costs of the MP2 calculations, we simply took the TSs optimized at B3LYP level and re-optimized those at MP2 level. 2.2.4. Nomenclature of Structures To identify the key intermediates and TSs for the reactions under study, we introduce a label of the form w-x-y(-z) for the most important molecular structures calculated at MP2 level. w can be either a, b or l which denotes the α-, the β-anomer, and the linear sugar, respectively, or a geometry derived from it. x denotes the sugar and can be GlcN and GalN. The most stable neutral and sodiated conformer for each studied anomer are denoted as wx-neutral and w-x-m, respectively, where m stands for “minimum”. To label the initial state or transition state of a reaction, z can be i or t, respectively. y is used to specify the lowest lying pathway of a dissociation channel: d2o and dho denote the D2O and the DHO dehydration channel, nd3 and nd2h the ND3 and the ND2H deamination channels, ro the ring opening reaction, and 02 the C2-C3 cleavage step in the

0,2A

will show in Section 3.2.2, the most favorable mechanism for the

cross-ring cleavage. We 0,3A

(or

0,3X)

cross-ring

cleavage of both GlcN and GalN involves two-step mechanisms with an epoxy intermediate or a zwitterionic intermediate. The first and second step of these pathways will thus be denoted with labels y = 03e1 and 03e2 or y = 03z1 and 03z2, respectively. 2.2.5. Remarks on Energetics As done in our earlier works,42,44,46 we assume that all conformers of a given anomer or of the linear sugar are in thermal equilibrium. This means that the energy of the associated TS essentially determines the preference for a particular reaction pathway. To have a common energy reference, the reaction barriers reported in this study all refer to the global minima of the anomer/linear sugar under study. Similarly, we define the desodiation energy, 11 ACS Paragon Plus Environment


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i.e., the energy required to remove the Na+, as the difference between the energy of the global minima of the sodiated structures and the global minima of the neutral structure plus the energy of an isolated Na+ ion in gas phase. Note that the reported desodiation energies are not corrected for the basis set superposition error (BSSE), as the standard approach for it, the counterpoise correction (CPC) according to Boys and Bernardi,73 is not completely undisputed.74 Nevertheless, we have calculated the CPC for some cases to get a feeling for the dimension of the BSSE. The results indicate that the desodiation energies may be overestimated by up to 13 kJ/mol. A second remark on the energetics addresses the ZPE corrections and the comparison of the calculated results to isotope-marking experiments. The D2O dehydration and the DHO dehydration as well as the ND3 deamination and the ND2H deamination, introduced in Section 2.2.3 could only be distinguished from each other if one performs the experiments with deuterium-exchanged HexNs (D6-HexHs), in which the H atoms at OH and NH2 groups are replaced with D. To address the experiments using these heavier isotopologues of HexN, one in principle needs to account for the isotope-dependent ZPEs and their effect on the reaction barriers. Thus, we have, in addition to the results for unexchanged HexNs discussed below in the main text, evaluated the reaction barriers for D6-HexHs for the lowest-lying mechanism in each of the investigated dissociation channels in Section S3 of the SI. It can be shown that the effect is sufficiently small and can thus be neglected in the discussion below. 3.

Results and Discussions

3.1. Experimental Results As the  and anomers of each HexN, which only differ by the OH stereochemistry at C1, coexist in solution, we separated the two anomers by HPLC and measured the CID spectra right after the separation. Figure 2 (a), (b) and (c) show the HPLC chromatogram and the corresponding CID spectra for GlcN, respectively. Note that even if the anomers can be separated with HPLC, it is challenging to assign the peaks in the chromatogram to a specific anomer. The precursor ion is GlcNNa+, which is responsible for the signal at m/z 202. The dominant fragment ion is found at m/z 143, representing the

0,2A

cross-ring

dissociation. The other major fragment ions are m/z 112 and 113 with the former being slightly stronger in intensity than the latter. m/z 113 is associated with the

0,3A

cross-ring

dissociation accompanied by the loss of an OH-CH2-C(NH2)=CH-OH fragment. The m/z 12 ACS Paragon Plus Environment

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112 signal indicates that the Na+ ion remains with the molecular fragment mentioned above, resulting in the detection of the so-called 0,3X ion. The minor fragment ions include m/z 172, 184, and 185, representing the elimination of CH2O, H2O, and NH3, respectively. The assignments of these fragments were supported by the CID spectra of the isotope-labeled compound [1-18O]GlcN, as illustrated in Figure 2 (d). Another major dissociation channel, sodium ion elimination, which cannot be observed due to the low mass cutoff of the linear ion trap mass spectrometer, was observed using a tandem quadrupole mass spectrometer, as shown in Section S1 of the SI. The sodium ion is the only ion observed in the range from m/z 15 to 100. Unlike glucose, galactose, and mannose, which feature the dehydration and the

0,2A

cross-ring dissociation as the major dissociation channels,42,44 the dissociation of GlcN is dominated by the cross-ring dissociation while the elimination of water is rather insignificant. A further difference to the hexoses is the finding that the difference between the CID spectra of the two GlcN anomers is small, as illustrated in Figure 2(b) and (c). The small difference between the CID spectra of the  and anomer of GlcN, which is similar to the small difference between the CID spectra of the  and anomer of GlcNAc,46 makes the determination of the anomeric configuration of GlcN difficult. A last interesting aspect is the ratio between the

0,3A

(m/z 113) and

0,3X

(m/z 112) signals. This

issue is not so often discussed in the context of hexoses42 (or disaccharides of hexoses43,47, as the signals are generally much weaker. In addition, in the case of hexoses, the two formed fragments would have the same mass. However, isotope marking experiments have shown for hexoses that the

0,3A

peak is larger than the 0,3X peak,42 different to what we

observe in our experiments with HexNs. Similar dissociation properties as GlcN were found for GalN, as illustrated in Figure 3 (a)-(c). Although the difference between the CID spectra of the anomers of GlcN or of GalN is small, the difference between the CID spectra of GlcN and GalN is significant. The CID spectra of GalN features a large ion abundance resulting from

0,3A

(m/z 143) and

0,3X/0,3A

cross-ring dissociation. The relative ion abundances resulting from

0,2A

cross-ring cleavage (m/z 112 and 113) can be used to differentiate

GlcN and GalN.



Page 14 of 43

Figure 2. (a) Chromatogram of GlcN; CID spectra of (b) the first peak in the chromatogram, (c) the second peak in the chromatogram, (d) [1-18O]GlcN.


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Figure 3. (a) Chromatogram of GalN; CID spectra of (b) the first peak in the chromatogram, (c) the second peak in the chromatogram.

3.2. Computational Results 3.2.1. Results at DFT-B3LYP Level We have started the computational investigation with computing the TS at B3LYPlevel of theory. The corresponding results are documented in Table 1. To investigate the reactions for GlcN in ring-form, we initially only considered the geometries from the database obtained with method A as potential IS geometries for the reactions of interest, i.e., ring-opening, dehydration as well as deamination. The calculations predict that, for both α15 ACS Paragon Plus Environment


Page 16 of 43

and β-GlcN, the deamination step is more favorable than the dehydration and the ringopening process. In the case of the α-anomer, all three reactions under study have similar barriers, with differences of less than 5 kJ/mol. At variance, the deamination of β-GlcN is, by 19 kJ/mol and 30 kJ/mol, clearly favored over the ring-opening and the dehydration, respectively. In addition, the barriers for all three dissociation channels are lower than the calculated desodiation energies, irrespective of the anomeric configuration. As these computational results fundamentally contradict with the absence of a strong signal at m/z 185 (loss of ammonia) in the experimental mass spectra, we have suspected that the conformational space covered by the structure database obtained with method A is not large enough. Therefore, we re-performed the TS search with the structure database obtained with method B. However, we did not find any reaction channel that is more favorable than those discussed above. This suggests that the structure database A already covers the crucial parts of the conformational space for the two anomers of GlcN. As the experimental spectrum is dominated by the signal at m/z 143, we have also considered the possibility that the 0,2A cross-ring cleavage fragment is not formed via the “classical” crossring cleavage mechanism, consisting of the ring-opening step followed by a retro-aldol step as shown in Figure 1 (c). If there is an alternative mechanism for the

0,2A

cross-ring

cleavage that has a noticeably lower barrier than the values calculated for the deamination step, the contradiction between the experiment and the calculations at B3LYP level would be resolved. We have considered a direct

0,2A

cross-ring cleavage mechanism, in which,

while the sugar is in ring-form, the O3 hydrogen is transferred to the ring oxygen O5, thus triggering the simultaneous cleavage of the O5-C1 bond and of the C2-C3 bond (see Figure 1 (d)). However, for β-GlcN, the barrier associated with this mechanism is not only higher than the barrier for the deamination, by 48 kJ/mol, but also by 29 kJ/mol higher than the ring-opening barrier. For α-GlcN, the barrier of this alternative mechanism is 1 kJ/mol higher than the deamination barrier. Thus, it seems that there is no alternative

0,2A

cross-

ring cleavage mechanism that could explain the experimental observations. While keeping the apparent contradiction between the experiment and the calculations at B3LYP level for GlcN in mind, we have also performed the TS search for GalN. Here we have directly used the structure database B to search for potential IS geometries. The results that we have obtained here are shown in Table 1. The results are somewhat different from what has been found for GlcN. In the case of α-GalN, the most favorable reaction is the ring-opening process with a barrier of 180 kJ/mol. The barrier for the competing 16 ACS Paragon Plus Environment

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dehydration and deamination are by 31 kJ/mol and 7 kJ/mol higher. So far, for the αanomer of GalN, the B3LYP results seem to be at least not contradicting with the experiments, which indicates that the

0,2A

and

0,3A/0,3X

cross-ring cleavage, both reactions

requiring the ring-opening step as visible in Figure 1 (c), (e), (f), are the preferred dissociation pathways. Similar to the α-anomer, the most favorable reaction channel for βGalN, as predicted by the B3LYP calculations, is also the ring-opening reaction. However, the associated barrier is only slightly lower than the dehydration barrier and the deamination barrier, by 3 kJ/mol and 9 kJ/mol, respectively. Based on these results, one should at least expect some signal at m/z 184 in the mass spectra, which corresponds to the loss of water. The very weak water-loss signal in experiment, together with the other discrepancies between B3LYP calculations and the experiment discussed above, let us suspect that the B3LYP functional over-estimates the barriers for the ring-opening process in comparison to the barriers of the competing reactions. Therefore, we have re-evaluated the reaction barriers for GlcN and GalN in ring-form using the MP2 method, which is generally accepted to provide more reliable energetics. Table 1. Lowest barriers in kJ/mol for the studied dissociation channels of GlcN and GalN in ring-form as well as the desodiation energies calculated at B3LYP level and MP2 level. Where appropriate, the direction of the involved H transfer is shown below the energies.

Deamination Dehydration

B3LYP GlcN α 185 O1→N 190 O4→O1

β 162 O1→N 192a O3→O1 O6→O1 181

GalN α 187 O1→N 211 O4→O6

β 178 O1→N 172 O4→O1

MP2 GlcN α 216 O1→N 238 O4→O1

β 194 O1→N 220 O3→O1

GalN α 215 O1→N 245 O4→O3

β 211 O1→N 212 O4→O1

Ring-opening 188 180 169 191 179 185 174 Desodiation energy 210 203 201 217 198 190 188 206 a At B3LYP level, the two dehydration channels have essentially degenerated TSs, differing less than 1

kJ/mol.

3.2.2. Results at MP2 Level 3.2.2.1. Glucosamine in Ring-Form In the following, we will be discussing the most likely reaction channels for GlcN in ring-form, re-evaluated at MP2 level of theory. As illustrated by the direct comparison in Table 1, the MP2 results feature some striking differences to the results obtained at B3LYP 17 ACS Paragon Plus Environment


Page 18 of 43

level. An overview of the most dominating pathways in each of the considered dissociation channels is provided in Figure 4. The geometries of the most important minima and TSs are shown in Figure 5; an accompanying discussion of the geometries is provided in Section S2.1 of the SI.

Figure 4. Energy diagram for (a) α-GlcN, (b) β-GlcN, and (c) linear GlcN illustrating the energies of the low lying TS for each reaction channel (thin bars) as well as the associated initial states from which the reaction starts (thick bars directly below each thin bar). In each sub-figure, the energy of the indicated sugar in its most stable conformer is set to 0. Where calculated, the desodiation energy is represented by a gray dashed line. The (overlapping) blue dotted lines in (c) mark the energies of the ring-opening TSs for α- and β-GlcN referred to the global minima of linear GlcN. In (c) “0,3A(e1)” marks the TS for the first step of the epoxy mechanism for the 0,3A cross-ring cleavage. “0,3A(e2)” refers to the second step. The thick bars in the same color (e.g., the one marked as l-GlcN_03e2_i) show the energy of the intermediate from which the second step starts. The labels above and below the thick bars indicate the puckering index and the position of the Na+ ion. The label of type “x→y” at the TSs for the dehydration and deamination channels are used as a shorthand notation for the H atom transfer and correspond to “Cx→Oy” and “Ox→Oy” or 18 ACS Paragon Plus Environment

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“Cx→N” and “Ox→N” as used in the main text to distinguish the various pathways. Geometries that are shown in Figure 5 and Figure 6 are labeled with the name of the structure.

As illustrated in Table 1, the desodiation energies are calculated to be 198 kJ/mol and 190 kJ/mol for α- and β-GlcN, respectively, i.e., by slightly more than 10 kJ/mol lower than the corresponding values at B3LYP level. While the results at B3LYP level have predicted that any of the considered dissociation channels features at least one reaction pathway that has a barrier that is lower than the desodiation energy, MP2 calculations yield a different picture: While the barriers for all other dissociation channels are higher than the desodiation energy, ring-opening barriers for the α- and the β-anomer are calculated to be by 7 kJ/mol and 11 kJ/mol, respectively, lower than the desodiation energy. However, recall that the desodiation energy is subject to the BSSE that is comparable to the energy difference between the ring-opening barrier and the desodiation energy, as mentioned in Section 2.2.5. The ring-opening barriers are also much lower, by 15 kJ/mol or more, than the barriers for the competing deamination and dehydration, respectively. Interestingly, the barrier height for the ring-opening does not change substantially, by less than 3 kJ/mol, upon changing the computational methods from B3LYP to MP2. The situation is not quite the same for the ND3 deamination and the D2O dehydration channels. In the case of β-GlcN, the barriers of those channels increase by around 30 kJ/mol. Same can be stated for the deamination of αGlcN, whereas the impact of switching the computational methods is even larger for the dehydration α-GlcN, where an increase of 48 kJ/mol is seen. In other words, the differences between the MP2 and B3LYP results seem to be strongly depending on the type of the reaction. While the B3LYP calculations yield dehydration and deamination barriers that are significantly lower than the values at MP2 level, the agreement between the MP2 and B3LYP results for the ring-opening barriers is relatively good. These changes in the reaction barriers are not likely the direct result of changes in the reaction mechanisms: The deamination of both GlcN anomers proceeds via the same pathways as calculated at B3LYP level, namely the H transfer from O1 to the amino group accompanied by a rearrangement of the six-membered ring into a five-membered ring as visible from Figure 5. Also, the dehydration reactions of α-and -GlcN are calculated to follow the same mechanism as predicted by the B3LYP results, i.e., the H transfers from O4 to O1 for α-GlcN and from O3 to O1 for -GlcN. However, note that the B3LYP results indicated the presence of a second, quasi-degenerated dehydration channel for -GlcN involving the H transfer from 19 ACS Paragon Plus Environment


Page 20 of 43

O6 to O1. At MP2 level, this channel is calculated to be slightly less favored that than the O3→O1 dehydration channel.

Summing up the results discussed above, one can state that, according to the results obtained with MP2 calculations, the ring-opening should be the dominating reaction for both α- and β-GlcN. As the ring-opening leads to the loss of the stereo-information at C1, this finding can potentially explain why the experimental CID spectra of both anomers are nearly identical. Furthermore, unlike the B3LYP results, the MP2 results for the sugars in ring-form do not contradict with the experimental observation that the

0,2A

cross-ring

dissociation is the dominant reaction channel. Recall here that the “classical” 0,2A cross-ring dissociation as shown in Figure 1 (c) requires the ring-opening step. In the next section, we will discuss how GlcN will continue to react once its six-membered ring has been opened.

Figure 5 Geometries of the global minima, TSs with the lowest barriers for each dissociation channel of α- and β-GlcN and the corresponding minima structures. Reaction barriers in kJ/mol referred to the most stable structure are shown in red, interatomic distances of interest in Angstrom are shown in black. Color coding for atoms: O: red; N: blue; C: gray; H: white; Na: purple.


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3.2.2.2. Linear Glucosamine In this section, we will go through the possible reaction channels for linear GlcN. The most important results are documented in Table 2 and Figure 4. The IS and TS geometries of the most crucial reaction pathways are shown in Figure 6 and discussed in Section S2.2 of the SI. Due to the experiences from our earlier study on N-acetylglucosamine (GlcNAc),46 we have not only looked at the D2O dehydration and ND3 deamination channels, as done for GlcN in ring-form. We have also considered the DHO dehydration and ND2H deamination channels, in other words, reactions in which an H bound to a C atom is transferred to an OH or the NH2 group. As the data in Table 2 shows, the deamination is not a favored reaction for linear GlcN. While the ND3 deamination channel, which favors the H transfer from O3, features a barrier of 270 kJ/mol, the competing ND2H channel, preferentially involving an H transfer from C3, has a barrier that is only slightly lower, 263 kJ/mol. The values are not only higher than the deamination barriers for GlcN in ring-form as discussed in Section 3.2.2.1 but also higher than the competing dehydration process in linear form, which will be discussed below. We speculate that the elevated deamination barrier for linear GlcN is related to the carbonyl group in vicinal position to the amino group. A carbonyl group can stabilize a negative charge in α-position to itself via resonance. At variance, a surplus of positive change in α-position, i.e., where the amino group is located, is unfavorable, as the carbonyl C atom, C1, already features a positive partial charge. As N is more electronegative than a C atom, the heterolytic C-N bond scission in the deamination step is expected to be accompanied by the formation of a positive partial charge at C2. We speculate that this positive partial charge in α-position to the carbonyl group destabilizes the deamination TS structures. Compared to the deamination process, the dehydration of linear GlcN is more likely to happen. Here, the most preferred D2O dehydration channel proceeds via the H transfer from O5 to O6 and involves a barrier of 234 kJ/mol. The barrier for the most favorable DHO dehydration channel is by only 6 kJ/mol higher. Similar to what we have reported for GlcNAc in an earlier work, 46 the (single step) DHO dehydration proceeds via the H transfer from C2, i.e., the site with enhanced C-H acidity due to the adjacent carbonyl group, to O3. However, there are also some differences: Note that the clear preference of the DHO dehydration over the D2O dehydration channel reported for GlcNAc based on B3LYP calculations46 is not observed here. In addition, the most favorable dissociation channel for GlcNAc, a multi-step DHO elimination mechanism, 46 is 21 ACS Paragon Plus Environment


Page 22 of 43

not possible for GlcN, as it involves the transfer of the H at C2 to the acetyl group that does not exist in GlcN. The most likely dissociation pathway, according to the CID experiment, is the C-C cleavage leading to the

0,2A

cross-ring cleavage, for which we have only considered the

classical retro-aldol mechanism as shown in Figure 1 (c). This mechanism indeed has the lowest barrier of all reaction channels considered for linear GlcN, 154 kJ/mol. However, note the (overlapping) dotted lines in Figure 4 (c) that marks the energies of the ringopening TSs a-GlcN_ro_t and b-GlcN_ro_t, relative to the global minima of linear GlcN, l-GlcN_m. It is visible that the energetically highest lying TS along the complete reaction from GlcN in ring-form to

0,2A

cross-ring cleavage is actually associated with the ring-

opening step. The C-C cleavage in the

0,3A

cross-ring dissociation is an interesting case: We have

tried to find the TS geometries associated with the one-step mechanism (Figure 1 (e)) and the two-step mechanism (Figure 1 (f)). However, following the procedure described in Section 2.2.3, we have not been able to identify any TS structure for the one-step mechanism as discussed for hexoses.44 The analysis of the IRC for the so obtained TS structures indicated that they actually are associated with an alternative two-step mechanism in which the H atom at C2 moves to C1 first in a first step, leading to the formation of an intermediate with an epoxy group at C1 and C2. In the second step, the H at O4 is transferred to the epoxy O atom to open the epoxy-ring, while the C3-C4 bond breaks simultaneously (Figure 1 (g) and Figure 6). To distinguish this two-step mechanism from the “conventional” two-step retro-aldol mechanism shown in Figure 1 (f), we will refer to this mechanism as the “epoxy mechanism”. The initial H transfer from C2 to C1 has a barrier of 175 kJ/mol, which is slightly higher than the barrier of the subsequent step involving the concerted H transfer and C-C cleavage with 163 kJ/mol. As visible from Figure 4 (c), the TS of the first step is, within the error of the computational method, degenerated with the ring-opening TSs for GlcN in ring-form, a-GlcN_ro_t and bGlcN_ro_t. An interesting feature in the structure of l-GlcN-03e2_t, TS of the second step in the epoxy mechanism, is the fact that the Na+ is located between the two molecular fragments being formed, which means that the Na+ could potentially be found with either fragment, resulting in the detection of both the and the

0,3X

0,3A

(arising from atoms C4, C5, and C6)

ion (arising from atoms C1, C2, and C3) as seen in the experiment. For the

conventional two-step mechanism, we find that its first step involving the simultaneous H 22 ACS Paragon Plus Environment

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transfers from the amino group to O1 and from C2 to C1 has a barrier as high as 263 kJ/mol. In other words, the conventional mechanism, which was discussed as the most favorable 0,3A

cross-ring cleavage mechanism for hexoses44 or gentiobiose, a disaccharide of

glucose,43 can be ruled out as the preferred

0,3A

cross-ring cleavage mechanism for GlcN.

Therefore, we have omitted to calculate the barrier for the second step in the conventional two-step mechanism, the C3-C4 bond cleavage accompanied by the H transfer from O4 to the N atom. Summing up the computational results for GlcN at MP2 level, we can state that the dominating dissociation channel is the

0,2A

cross-ring reaction, followed by the

0,3A (0,3X)

cross-ring reaction via the epoxy mechanism. Given the fact that, when starting from GlcN in ring-form, both processes will pass through the same ring-opening TS, the competition between the two cross-ring cleavage channels is determined by the “0,2 0,3 gap”, the energy difference of 21 kJ/mol between the lower lying TS l-GlcN_02_t, which is associated with the

0,2A

cross-ring cleavage, and l-GlcN_03e1_t, TS of the first step in the

epoxy mechanism for the

0,3A

cross-ring. This is consistent with the experimental

observation that the mass spectrum features the m/z 143 peak as the main signal and the m/z 112 and 113 peaks as minor signals. The fact that barriers for the dehydration and the deamination process are calculated to be 59 kJ/mol or more less favorable than the crossring reactions is also in agreement with the absence of any strong signal associated with the loss of water (m/z 184) or ammonia (m/z 185).



Page 24 of 43

Figure 6. Geometries of the global minima, TSs with the lowest barriers for each dissociation channel of linear GlcN and the corresponding minima structures. Reaction barriers in kJ/mol referred to the most stable structure are shown in red, interatomic distances of interest in Angstrom are shown in black. Color coding for atoms: O: red; N: blue; C: gray; H: white; Na: purple. 3.2.2.3. Galactosamine in Ring-Form Similar to the discussion on GlcN, we have also summarized the MP2 energetics for the reactions of GalN in ring-form in Table 1 and provide an overview of the dominating pathways in Figure 7. Figure 8 shows the geometries of the most stable TS structures for each dissociation channel and the corresponding IS as well as the most stable conformers of both neutral and sodiated GalN. The structures are discussed in detail in Section S2.3 of the SI. For both anomers, we see the same trend in the barriers for the different dissociation channels as already observed for GlcN. The lowest barrier is associated with the ringopening reaction, which is required for the cross-ring cleavage, followed by the ND3 deamination and D2O dehydration. The most favorable deamination channels for both GalN anomers share the same mechanism: An H atom transfers from O1 to the neighboring amino group forming the ammonia molecule being eliminated. The elimination of the ammonia further triggers the simultaneous rearrangement of the six-membered ring into a five-membered ring with a bond between C2 and O5 instead of the bond between C1 and O5 (see Figure 8). At variance, the mechanism of the most favorable dehydration pathway 24 ACS Paragon Plus Environment

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is different for the two anomers. The most likely dehydration mechanism of α-GalN involves the transfer of an H from O4 to O3, whereas, for β-GalN, the water molecule being eliminated originates from the OH group at O1 and an H coming from O4.

Table 2. Lowest barriers in kJ/mol of the studied dissociation channels of linear GlcN and GalN calculated at MP2 level. Where appropriate, the direction of the involved H transfer is indicated. Dissociation channels

GlcN Barrier 270 263 234 240 154a

GalN Barrier 272 261 251 247 184b

H transfer H transfer ND3 deamination O3→N O3→N ND2H deamination C3→N C3→N D2O dehydration O5→O6 O4→O5 DHO dehydration C2→O3 C2→O3 C-C scission in 0,2A cross-ring C-C scission in 0,3A cross-ring epoxy / 1,2-zwitterionic 175c, 163 192b, 174 conventional 263, n/a 273, n/a a Energetically highest lying TS in the overall process from α-GlcN or β-GlcN to 0,2A cross-ring cleavage actually associated the ring opening-step. b Highest lying TS in the overall process from α-GalN to 0,2A or 0,3A cross-ring cleavage actually associated with the ring opening-step. The reactions starting from β-GalN have the barriers as indicated in the table above as rate limiting barriers. c The reactions starting from both α- and β-GlcN have the barriers as indicated in the table above as rate limiting barriers.



Page 26 of 43

Figure 7. Energy diagram for (a) α-GalN, (b) β-GalN, and (c) linear GalN illustrating energies of the lowest lying TS for each reaction channel (thin bars) as well as the associated initial states from which the reaction starts (thick bars directly below each thin bar). In each sub-figure, the energy of the indicated sugar in its most stable conformer is set to 0. Where calculated, the desodiation energy is represented by a gray dashed line. The blue dotted lines in (c) mark the energies of the ring-opening TSs for α- and β-GalN referred to the global minima of linear GalN. In (c) “0,3A(z1)” marks the TS for the first step of the 1,2-zwitterionic mechanism for the 0,3A cross-ring cleavage. “0,3A(z2)” refers to the second step. The thick bars in the same color (e.g., the one marked as l-GalN_03z2_i) shows the energy of the intermediate from which the second step starts. The labels above and below the thick bars indicate the puckering index and the position of the Na+ ion. The label of type “x→y” at the TSs for the dehydration and deamination channels are used as a shorthand notation for the H atom transfer and correspond to “Cx→Oy” and “Ox→Oy” or “Cx→N” and “Ox→N” as used in the main text to distinguish the various pathways. Geometries that are shown in Figure 8 and Figure 9 are labeled with the name of the structure. The color coding for the TSs is the same as in Figure 4 if not marked explicitly.


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As shown in Table 1, the desodiation energies of α- and β-GalN are 188 kJ/mol and 206 kJ/mol, respectively. The ring-opening barriers are 185 kJ/mol and 174 kJ/mol, i.e., 3 kJ/mol and 32 kJ/mol lower than the desodiation energies for α- and β-GalN, respectively. At variance, the competing dissociation channels, deamination, and dehydration, possess barriers higher than the desodiation energy. In the case of α-GalN, the difference between the desodiation energy and the barriers for deamination and dehydration are, 27 kJ/mol and 57 kJ/mol, respectively. For β-GalN, the corresponding values are 5 kJ/mol and 6 kJ/mol. Again here, keep in mind that the desodiation energy may be overestimated by up to 13 kJ/mol due to the BSSE. Note that the ring-opening barriers are, for both anomers, calculated to be much lower, by 30 kJ/mol or more, than the barriers of the competing dehydration and deamination, similar to what has been discussed for GlcN. Thus, the MP2 results suggest that any dissociation reaction observed in the experiment should occur from the linear form of GalN. This finding would also explain the similarity between the mass spectra measured for αand β-GalN.

Figure 8. Geometries of the global minima, TSs with the lowest barriers for each dissociation channel of α and β-GalN and the corresponding minima structures. Reaction barriers in kJ/mol referred to the most stable structure are shown in red, interatomic distances of interest in Angstrom are shown in black. Color coding for atoms: O: red; N: blue; C: gray; H: white; Na: purple.



Page 28 of 43

3.2.2.4. Linear Galactosamine Similar to the discussion on linear GlcN in Section 3.2.2.2, the most important results on the reaction channels of linear GalN are shown in Table 2 and Figure 7. Figure 9 illustrates the geometries of the most stable TS structures of each considered dissociation channel and their corresponding local minima as well as the structure of the global minima of linear GalN. The corresponding discussion is provided in Section S2.4 of the SI. A look at Table 2 shows that the reaction mechanisms and trends in the relative height of the different barriers are comparable to what has been discussed for GlcN. The two most striking differences are, on the one hand, the reaction mechanism of the D2O dehydration and, on the other hand, the barrier for the

0,2A

cross-ring dissociation. Both will be

addressed in more detail below. Similar to GlcN, the ND3 deamination channel proceeds via the H transfer from O3 to the amino group (and the simultaneous rearrangement of the H atom at C3 to C2). Also for GalN, this dissociation channel becomes relatively unfavorable in the linear form with a high barrier of 272 kJ/mol. The D2O dehydration channel becomes more preferable than the ND3 deamination upon ring-opining as the barrier of 251 kJ/mol shows. Unlike GlcN, the D2O dehydration proceeds via the H transfer from O4 to O5, instead of from O5 to O6. The dehydration and deamination channels involving an H atom from a C atom, i.e., the ND2H deamination and the DHO dehydration, feature comparable barriers to their counterparts proceeding via the H transfer from an OH/NH2 site. With values of 261 kJ/mol for the ND2H deamination and 247 kJ/mol for the DHO dehydration, their barriers are lower by only 11 kJ/mol or less. Following the retro-aldol mechanism for the C-C scission in the

0,2A

cross-ring

cleavage, we have been able to identify a TS structure for GalN which is associated with a barrier of 184 kJ/mol, which is much lower, by 64 kJ/mol or more, than the dehydration and deamination channels discussed above. This rationalizes the absence of any strong signal associated with the loss of water or ammonia in the experimental mass spectra for GalN as shown in Figure 3. For the C-C cleavage in the

0,3A(0,3X)

cross-ring cleavage, we find

similar results as mentioned for GlcN. Also here, we have not been able to find any hints for the existence of a one-step mechanism as shown in Figure 1 (e). The most preferable 0,3

A(0,3X) cross-ring cleavage mechanism seems to be comparable to the epoxy mechanism

discussed for GlcN. However, in the case of GalN, we were not able to locate the epoxy 28 ACS Paragon Plus Environment

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intermediate. We assume that the intermediate here is a zwitterionic structure with an alkoxy group at C1 and an iminium group at C2, similar to the corresponding TS structure l-GalN_03z1_t shown in Figure 9. We speculate that the formation of the zwitterionic structure instead of the epoxy structure is related to the position of the Na+, which is close to O1 and thus hinders the formation of the epoxy structure by stabilizing the positive charge of the alkoxy group. The following C3-C4 cleavage step is again comparable to the situation for GlcN. This mechanism, which we refer to as the 1,2-zwitterionic mechanism (see Figure 1 (h)), starts with the initial H rearrangement from C2 to C1 featuring a barrier of 192 kJ/mol, followed by a second step with a lower barrier, comparable to GlcN. The second step is the H transfer from O4 to O1, accompanied by the C3-C4 bond breaking, which has a barrier of only 174 kJ/mol. The associated TS, l-GalN_03z2_t, also has the Na+ between the forming molecular fragments. Thus, also this reaction could lead to both the

0,3A

and the

0,3X

ion, similar to what has been discussed for GlcN. A further similarity

between GlcN and GalN is that, also for GalN, the conventional two-step

0,3A

cross-ring

cleavage can be ruled out as a reaction pathway of importance, as the barrier of 273 kJ/mol associated with its initial step shows. Again here, we should compare the energies of the TSs of the cross-ring cleavage channels and of the ring-opening TSs marked in Figure 7 (c), as done for GlcN. If we consider a reaction starting from α-GalN, we find a situation comparable to the reaction of GlcN. The ring-opening TS a-GalN_ro_t is less stable than the

0,2A

cross-ring cleavage TS l-GalN_02_t, but of comparable energy as the TS

associated with the first step of the

0,3A

cross-ring cleavage, l-GalN_03z1_t. However, if

we consider the reaction starting from the β-anomer of GalN, the overall processes are determined by the barriers associated with l-Gal_02_t and l-GalN_03z1_t, respectively, as the ring-opening TS of β-GalN, b-GalN_ro_t, is lower in absolute energy. Following the same logic as discussed for GlcN, it is again the 0,2 0,3 gap, here the energy difference between l-GalN_02_t and l-GalN_03z1_t, that determines the preference for the one or the other cross-ring cleavage channel. As visible in Figure 3, the major reaction channels for GalN include not only the 0,2A cross-ring cleavage (m/z 143) but also, unlike GlcN, the

0,3A(0,3X)

cross-ring cleavage (m/z 112 and 113). This feature is, at least

qualitatively, reflected by the MP2 calculations: The 0,2 0,3 gap is 9 kJ/mol in the case of GalN, but more than twice as large in the case of GlcN, 21 kJ/mol. Based on this results, one would expect that the signal associated with the compared to the

0,3A

0,2A

cross-ring cleavage should be,

signals, more dominant in the mass spectrum of GlcN and less 29 ACS Paragon Plus Environment


Page 30 of 43

dominant in the spectrum of GalN. As visible from Table 2, the difference in the 0,2 0,3 gap mainly originates from the differences in the

0,2A

cross-ring cleavage barrier. A further

comparison of the absolute electronic energies, irrespective with or without ZPE correction, reveals that the difference in the 0,2A cross-ring cleavage barrier mainly is a consequence of the different stability of the TS structures l-GlcN_02_t and l-GalN_02_t (see Figure 6 and Figure 9) with the former being by 15 kJ/mol more stable than the latter. The origin of the difference in the TS stability is not very clear, as the TS structures are not very different; The Na+ is in both structures coordinated by O3, O4, and O5. However, due to the difference in the stereochemistry at O4, the conformation of the carbon chain is different, which in return leads to differences in the H bonds. Whether this really is the origin of the difference in the TS stability remains to be explored.

Figure 9. Geometries of the global minima, TSs with the lowest barriers for each dissociation channel of linear GalN and the corresponding minima structures. Reaction barriers in kJ/mol referred to the most stable structure are shown in red, interatomic distances of interest in Angstrom are shown in black. The text above each arrow indicates the dissociation channel. Color coding for atoms: O: red; N: blue; C: gray; H: white; Na: purple. 3.2.3. Calculated Rate Constants Although the above-reported reaction barriers already provide a relatively good idea of how favorable each dissociation channel is, it still is a somewhat abstract concept, as the barriers are not directly observable in the experiment. Thus, we have calculated the RRKMrate constants k as a function of the excitation energy Eex as described in Section S4 of the 30 ACS Paragon Plus Environment

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SI. In the case of perfect matching between experiment and theory, the ratio between the signal intensities of the peaks observed in the mass spectra should directly correspond to the ratio between the corresponding rate constants. However, one should also be aware of the fact that the deviation between rate constants from experiment and theory can easily be several order of magnitudes: A deviation of less than one order of magnitude from experimental rate constants is generally regarded as very accurate computational results. 75 The calculated rate constants are collected in Figure 10, and it is visible that over the range of considered excitation energies between 200 kJ/mol and 1500 kJ/mol, the ringopening indeed is the dominating reaction pathway for HexNs in ring-form (Figure 10 (a), (b), (d), and (e)). At Er = 1500 kJ/mol, the rate constants for the competing reactions can be as high as about 40% of the rate constant for the dominating ring-opening reaction. As we do not have a very clear idea to which excitation energy the experimental situation actually corresponds, it is somewhat hard to judge how good the agreement between experiment and theory is, but at least the rough trends do not contradict.



Page 32 of 43

Figure 10. Calculated RRKM rate constants k for the reactions of (a) α-GlcN, (b) β-GlcN, (c) linear GlcN, (d) α-GalN, (e) β-GalN, and (f) linear GalN as a function of the excitation energy Eex. Each sub-figure consist of four diagrams: left top are rate constants calculated self-consistently at MP2 level, left bottom shows the ratio between the rate constant and kmax, the rate constant of the most favorable reaction at a given temperature. The diagrams on the right side show the corresponding results obtained at CCSD(T)//MP2 level. Small arrows in the respective colors mark overlapping curves that are hard to recognize. The most interesting aspect about the rate constants for the reactions of linear HexNs (Figure 10 (c) and (f)), is the relative value for the

0,2A

and

0,3A(0,3X)

cross-ring channels.

Also here, we see a qualitative agreement with the experiment: The ratio between the rate constants for the two cross-ring channels (k0,3A / k0,2A) is lower for GlcN and higher for GalN. This is in line with the intensities for the signals at m/z 112 and 113 relative to the signal intensity for m/z 143, which are smaller for GlcN(Figure 2) and larger for GalN(Figure 3). In addition, we wanted to check how trustworthy the rate constants derived from MP2 calculations are. Thus, we have re-evaluated the reaction barriers at CCSD(T)//MP2 level, which are documented in Section S5 of the SI. Although the reaction barrier, in particular for the dehydration and deamination processes, can differ by as much as 21 kJ/mol from the corresponding values at MP2 level, the general trends are preserved. For the HexNs in ringform, ring-opening is also predicted by the CCSD(T)//MP2 results to be the dominant dissociation channel. However, the CCSD(T)//MP2 rate constants for the competing pathways are generally closer to the ring-opening rate constants than in the MP2 results. This effect is most prominent for β-GlcN, for which the deamination rate constant at Eex=1500 kJ/mol is about 90 % of the ring-opening rate constant, which is somewhat large compared to the experiment in which the signal at m/z 185 associated with the deamination is rather weak (Figure 2). A second interesting aspect about the rate constants at CCSD(T)//MP2 level are the rate constants for the cross-ring dissociation channels of linear GalN (Figure 10 (f)). The values for the

and

0,3A

cross-ring channels are very similar,

cross-ring channel being slightly more favorable. At variance, the

0,2A

cross-

ring channel for GlcN is, also at CCSD(T)//MP2 level, more favorable than the

0,3A

cross-

with the

0,3A

0,2A

ring dissociation (Figure 10 (c)), which qualitatively agrees with the experiment. 4.

Conclusions In this work, we have explored the behavior of two Na+ tagged hexosamines,

glucosamine and galactosamine, under CID conditions. Our experimental results deliver the characteristic dissociation patterns, which extends the reference database for the LODES 32 ACS Paragon Plus Environment

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approach mentioned in the introduction. Although the identification of the anomeric configuration remains challenging for both hexosamines, the two hexosamines can be distinguished easily by observing the relative intensity between the MS signals associated with the

0,2A

(m/z 143) and

0,3A(0,3X)

cross-ring cleavage (m/z 112 and 113). The

calculations in this study shows that this difference in the mass spectra is associated with the difference in the values for the “0,2 0,3 gap”, i.e., the difference in the rate-limiting barriers for the

0,2A

and

0,3A

cross-ring cleavage, which in return is mainly a result of the

different stability of the TS structures associated with the

0,2A

cross-ring cleavage.

Although there are other MS-based methods that aim at identifying monosaccharides including their chirality, e.g., via the investigation of (transition) metal complexes formed with the sugar under study,76–81 the fixed ligand kinetic method,82,83 or the comparison of the reaction rates of lithiated sugars and water in a quadrupole ion trap,84 the present work provides, together with some earlier studies, an easier applicable method that can, not only be used on isolated sugar samples, but is also suitable for the identification of monosaccharides, which are produced from the CID of oligosaccharides in mass spectrometer and captured in an ion-trap. A further aspect that can be seen from the computational calculations at MP2 level is that hexosamines are, in their reactivity, more like HexNAcs than hexoses. Both HexNs and HexNAcs share the feature that essentially all information on the anomeric configuration is lost under CID conditions, as the ring-opening is the dominating reaction for those sugars in ring-form. Also, the behavior of the linear forms of GlcN and GlcNAc are comparable, as both feature an energetically low-lying

0,2A

cross-ring dissociation channel. The main

difference between GlcN and GlcNAc is also easy to understand: The dehydration channel that dominates the dissociation of GlcNAc requires the H transfer to the NAc group that is not present in GlcN. At the end, there is a technical aspect regarding the computational results that is worth to be pointed out: The circumstance that calculations at B3LYP level of theory could not reproduce the experimental observation as in earlier works42,44,46 raises some questions about the quantum chemical methods. In light on the results of this study, it seems reasonable to put some efforts into investigating how the different computational methods affect the transition state calculations for sugars.



Page 34 of 43

Acknowledgments The authors thank Dr. Jien-Lian Chen and Dr. Jake A. Tan for valuable discussions. Parts of this work were financially supported by the Thematic Research Project of Academia Sinica (AS-iMATE-107-32 and AS-TP-107-M08), Taiwan. Computational resources were supported in part by the National Center for High Performance Computing of Taiwan. CCC is grateful for a Distinguished Postdoctoral Scholars Fellowship of Academia Sinica, and the IAMS Junior Fellowship of the Institute of Atomic and Molecular Sciences. PJH is supported by Postdoctoral Scholars Fellowship of Academia Sinica. HTH and HTP would like to thank the Taiwan International Graduate Program (TIGP) for Ph. D scholarships. AK and HT thank the IAMS-International Internship Program.

Supporting Information (SI): Mass spectra in low mass range, Discussion of the initial state and transition state geometries for the most important reaction pathways, Cartesian coordinates for the discussed geometries


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