The Unexpected Dissociation Mechanism of Sodiated N

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A: Kinetics, Dynamics, Photochemistry, and Excited States

The Unexpected Dissociation Mechanism of Sodiated N-Acetylglucosamine and N-Acetylgalactosamine Cheng-Chau Chiu, Shang-Ting Tsai, Po-Jen Hsu, HaiThi Huynh, Jien-Lian Chen, HuuTrong Phan, Shih-Pei Huang, Hou-Yu Lin, Jer-Lai Kuo, and Chi-Kung Ni J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b00934 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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The Journal of Physical Chemistry

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The Unexpected Dissociation Mechanism of Sodiated

2

N-Acetylglucosamine and N-Acetylgalactosamine

3 4 5 6 7 8 9 10

Cheng-chau Chiu1, Shang-Ting Tsai1, Po-Jen Hsu1, Hai Thi Huynh1,2,3, Jien-Lian

11

Chen1, Huu Trong Phan1,2,3, Shih-Pei Huang1, Hou-Yu Lin1,4, Jer-Lai Kuo*1 and Chi-

12

Kung Ni*1,3

13 14 15 16 17 18 19

1Institute

of Atomic and Molecular Sciences, Academia Sinica, P. O. Box 23-166,

Taipei 10617, Taiwan 2Molecular

Science and Technology, Taiwan International Graduate Program,

Academia Sinica, Taipei, 10617, Taiwan

20

3Department

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

21

4Department

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

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*Email address:[email protected]; [email protected]

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Abstract

2

The mechanism for the collision-induced dissociation (CID) of two sodiated N-

3

acetylhexosamines

4

acetylgalactosamine (GalNAc) was studied using quantum chemistry calculations

5

and resonance excitation in a low-pressure linear ion trap. Experimental results show

6

that the major dissociation channel of isotope labeled [1-18O, D5]-HexNAc is the

7

dehydration by eliminating HDO, where OD comes from the OD group at C3.

8

Dissociation channels of minor importance include the

9

No difference has been observed between the CID spectra of the - and -anomers of

10

the same HexNAc. At variance, the CID spectra of GlcNAc and GalNAc showed

11

some differences, which can be used to distinguish the two structures. It was observed

12

in CID experiments involving disaccharides with a HexNAc at the non-reducing end,

13

that a HexNAc shows a larger dissociation branching ratio for the glycosidic bond

14

cleavage than the -anomer. This finding can be exploited for the rapid identification

15

of the anomeric configuration at the glycosidic bond of HexNAc-R' (R'=sugar)

16

structures. The experimental observations indicating that the dissociation mechanisms

17

of HexNAcs are significantly different from those of hexoses were explained by

18

quantum chemistry calculations. Calculations show that ring opening is the major

19

channel for HexNAcs in ring form. After ring opening, dehydration has the lowest

20

barrier. In contrast, the glycosidic bond cleavage becomes the major channel for

21

HexNAcs at the non-reducing end of a disaccharide. This reaction has a lower barrier

22

for HexNAcs as compared to the barrier the corresponding -anomers, consistent

23

with the higher branching ratio for HexNAcs observed in experiment.

(HexNAc),

N-acetylglucosamine

0,2A

(GlcNAc)

and

N-

cross-ring dissociation.


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Introduction

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Carbohydrates, nucleic acids, and proteins are three information containing

3

macromolecules in creating life. They all are involved in many important processes in

4

biological systems. In order to rationalize their functions, it is necessary to understand

5

the structures of such molecules. The structures of these macromolecules are very

6

complicated due to the complex sequence of the monomers in them. The invention of

7

the methods to determine the sequence of nucleic acids and of proteins has made a

8

huge impact on science, whereas the structural identification of carbohydrates remains

9

difficult until today.1

10

The structures of carbohydrates can be determined by using infrared (IR)

11

spectroscopy,2 microwave spectroscopy,3 nuclear magnetic resonance spectroscopy,4

12

and mass spectrometry (MS).5 MS has a high sensitivity and is thus also applicable

13

for small quantities of samples, e.g., oligosaccharides extracted from biological

14

samples. In the MS approach, single-stage mass spectrometry is not likely to reveal

15

the structures of oligosaccharides because of the large number of isomers which only

16

feature subtle differences between them. To determine the structures of

17

oligosaccharides, it is required to have additional information obtained from further

18

analytical methods, e.g., IR spectroscopy6–12, ion mobility,13–15 reactions,16,17 or

19

fragments produced from multi-stage MS. Collision induced dissociation (CID),18–20

20

higher collision energy dissociation,21 infrared multiphoton dissociation,22,23

21

ultraviolet dissociation,24,25 electronic excitation dissociation,26 electron capture

22

dissociation,27,28 and electron transfer dissociation29,30 are techniques used in multi-

23

stage MS to generate fragments. Among these techniques, CID is the most commonly

24

used method and often applied in the structural determination of oligosaccharides.

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Although it has been used for many years, CID could, until very recently31,32, only be 3 ACS Paragon Plus Environment


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used to determine some aspects of the structure of oligosaccharides, e.g., only the

2

linkage positions, but not the anomeric configuration.19,20 Differentiating different

3

anomeric configurations is important in carbohydrate structural identification31,32 and

4

determination of enzyme mechanism.33,34 The complete structural determination of

5

oligosaccharides remains challenging.

6

Understanding the dissociation mechanisms in CID makes the fragment analysis

7

easier and provides the possibility to extend the structural assignment capability of

8

CID.35–45 However, most of the studies on CID mechanisms have been focusing on

9

the glucose and glucose disaccharides. Only few studies have been emphasizing on

10

the dissociation mechanism in CID of sugars other than hexoses. Glucose, galactose,

11

mannose (denoted as the three common hexoses) as well as N-acetylglucosamine

12

(GlcNAc), and N-acetylgalactosamine (GalNAc) are five common monosaccharides

13

often encountered in biological systems. Recently, ab initio quantum chemistry

14

calculations were used to investigate the dissociation mechanism of the three common

15

hexose sodium adducts.41,42 The study of these hexoses marked with a sodium cation

16

(“sodiated hexoses”) helped the development of a new MS approach to determine the

17

structures of oligosaccharides, the logically derived sequence (LODES) tandem mass

18

spectrometry,46,47 as well as to determine the retaining or inverting mechanisms of

19

carbohydrate-active enzyme.34 In this study, we investigated the collision-induced

20

dissociation of sodiated GlcNAc, and GalNAc (together denoted as HexNAc). The

21

results show that the dissociation mechanisms of these two common HexNAcs are

22

significantly different from that of the three common hexoses. These results will be

23

useful for the structural determination of oligosaccharides containing HexNAc.

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II. Experimental method

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The detailed description of the experimental method has been reported in 4 ACS Paragon Plus Environment

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previous studies,46,47 thus only a brief description is given here. N-Acetyl-D-

2

glucosamine (GlcNAc, ~98%) and N-Acetyl-D-galactosamine (GalNAc, 95%) were

3

purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA); Methyl 2-acetamido-2-

4

deoxy--D-glucopyranoside (-GlcNAc-OMe, 98%), Methyl 2-acetamido-2-deoxy-

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-D-glucopyranoside (-GlcNAc-OMe, 98%), Methyl 2-acetamido-2-deoxy--D-

6

galactopyranoside (-GalNAc-OMe, 98%), Methyl 2-acetamido-2-deoxy--D-

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galactopyranoside (-GalNAc-OMe, 98%), Benzyl 2-acetamido-2-deoxy--D-

8

glucopyranoside

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glucopyranoside (-GalNAc-OBn, 98%), -GalNAc-(1→3)-Gal (>95%), -

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GalNAc-(1→3)-Gal (95%), -Gal-(13)-GlcNAc (90%), -Gal-(14)-GlcNAc

11

(98%) and -Gal-(16)-GlcNAc (98%) were purchased from Carbosynth, Ltd.

12

(Berkshire, UK); N-[1,2-13C2]acetyl-D-glucosamine ([1,2-13C2]GlcNAc, 99%), N-

13

acetyl-D-[15N]glucosamine ([15N]GlcNAc, 98%), N-acetyl-D-[6,6'-2H2]glucosamine

14

([6,6’-D2]GlcNAc, 98%) and N-[Me-2H3]acetyl-D-glucosamine ([CD3]GlcNAc, 98%)

15

were obtained from Omicron Biochemicals, Inc. (South Bend, USA). All saccharides

16

mentioned above were used without further purification. The chemical structures of

17

all purchased isotope labeled saccharides as well as of those that have been generated

18

in the experiment are shown in Scheme 1. The saccharides were added to a 1:1 (v/v)

19

methanol/water solution containing 210-4 M NaCl to produce 10-4 M solutions of the

20

saccharides.

(-GalNAc-OBn,

98%),

Benzyl

2-acetamido-2-deoxy--D-



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Scheme 1. The schematic representation of (a) [1,2-13C2]GlcNAc, (b) [15N]GlcNAc, (c) [1-18O]GlcNAc, (d) [D5]GlcNAc, (e) [1-18O, D5]GlcNAc, (f) [6,6’-D2]GlcNAc, (g) [CD3]GlcNAc, (h) [1,2,4,6-D4]GlcNAc, (i) [1,2,3,6-D4]GlcNAc, (j) [1,2,3,4-

5 6 7

D4]GlcNAc, (k) [D8]Gal--(13)-GlcNAc, (l) [D8]Gal--(14)-GlcNAc, (m) [D8]Gal--(16)-GlcNAc, (n) [1-18O]GalNAc, (o) [D5]GalNAc, and (p) [1-18O, D5]GalNAc.

8 9

The CID spectra were measured in the positive mode by using a LTQ XL linear

10

ion trap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA USA)

11

equipped with an electrospray ionization source and a Dionex Ultimate 3000 high-

12

performance liquid chromatography (HPLC) system (Thermo Fisher Scientific Inc.,

13

Waltham, MA USA). The Dionex chromatography mass spectrometry link was

14

installed as an interface to control the Dionex chromatography system with Xcalibur

15

(Thermo Fisher Scientific Inc., Waltham, MA USA).

16

The separation of the two anomers of GlcNAc and GalNAc, respectively, via 6 ACS Paragon Plus Environment

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HPLC was performed using a Hypercarb (100 × 2.1 mm) column with a particle size

2

of 3 μm, operated at room temperature (25 °C). The mobile phase comprised (A)

3

0.1% (v/v) aqueous formic acid with 1×10−4 M NaCl, and (B) HPLC-grade

4

acetonitrile. The mobile phase for isocratic elution was a mixture of A:B = 90:10 (v/v).

5

The mobile phase flow rate was set to 300 μL/min and the volume of the sample

6

injected was 10 μL. The column eluate was directly infused into the electrospray

7

ionization (ESI) source without any post-column addition. The MS conditions were

8

optimized using the built-in semiautomatic tuning procedure in Xcalibur. The ESI

9

source was operated at a temperature of 280 °C with sheath gas flow rate 35 (arbitrary

10

units), auxiliary gas flow rate 20 (arbitrary units) and sweep gas flow rate 15

11

(arbitrary units). The ion spray voltage was set to 4.50 kV, and the transfer capillary

12

temperature was 400 °C. A capillary voltage of 25.5 V and a tube lens voltage of 91 V

13

were applied. Helium gas was used both as the buffer gas for the ion trap and as the

14

collision gas in CID. The MSn experiments (n-stage MS) were carried out with a

15

normalized collision energy of 25%, a Q value of 0.25, and 30 ms activation time.

16

The amount of ions regulated by the automatic gain control was set to 1×105 for full

17

scan mode and 1×104 for MSn mode, and the precursor ion isolation width was set to

18

1 u. For other compounds aside GlcNAc and GalNAc, the measurements were

19

performed using the same mass spectrometer under similar conditions, but without the

20

HPLC step. For deuterated compounds, solvent is a mixture of D2O and CH3OD.

21

III. Calculation method

22

We used a multi-stage structural sampling algorithm to construct the conformer

23

databases for neutral and sodiated HexNAcs. In the first step, we performed a random

24

sampling for neutral GlcNAc and GalNAc in ring form by optimizing their geometries

25

using the computationally cheap, classical GLYCAM force field (FF)48 as 7 ACS Paragon Plus Environment


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implemented the program Amber.49 The distinct neutral conformers were obtained by

2

screening out the duplicated structures with our previously developed two-stage

3

clustering algorithm.50 The Cremer-Pople (CP) puckering index was used to identify

4

the conformations of the neutral HexNAcs.51 The final geometries and energies

5

reported in this work were obtained by a further optimization step at non-spin

6

polarized, hybrid DFT level using the B3LYP functional.52–54 The B3LYP

7

calculations were performed with Gaussian 0955 using a 6-311+G(d,p) basis set.56–59

8

We have chosen to use the B3LYP functional for the present work in order to ensure

9

the comparability with earlier works from our group on the CID of other sugar

10

molecules.41,42 The B3LYP functional has been shown to be able to reproduce the

11

relative energies of glucose and mannose computed at domain-based local pair natural

12

orbital-CCSD(T) (DLPNO-CCSD(T)) level of theory with an average deviation of

13

slightly larger than 1 kcal/mol,60 which was considered to be sufficient for the

14

purposes of our study.

15

In the second stage, we selected the most stable conformers of neutral GlcNAc

16

and GalNAc for each puckering index to generate the database for the sodiated

17

monosaccharides. Starting from each of the selected conformers, we have generated

18

initial structure guesses for sodiated HexNAc, by placing a sodium ion in the vicinity

19

of O1, O3, O4, O6, the acetyl O, or the nitrogen atom at the NAc group. Around each

20

O or N atom, we considered 20 equally spaced sodium positions by varying the polar

21

angle and the azimuthal angle with respect to the O or the N atom. Here, we have

22

considered two different values for the polar angle and ten for the azimuthal angle. In

23

other words, we derive, from each of the selected neutral HexNAc conformers, 120

24

initial structure guesses for the corresponding sodiated HexNAc. Although we did not

25

explicitly consider sodium positions around the ring oxygen O0, there is a sufficient 8 ACS Paragon Plus Environment

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sampling of structures with the Na+ in the vicinity of O0. As O1 and O6 are close

2

enough to O0, one automatically samples structures with Na+ close to O0, when

3

considering the sodium positions around O1 and O6. The so obtained initial guesses

4

were then also optimized at B3LYP level, and the duplicated structures were removed

5

by the same structure screening technique as used for the neutral HexNAcs. For

6

simplicity, the detailed procedure of each sampling step is described in the SI for the

7

interested reader. The number of conformers in each database is listed in Table 1.

8

To consider sodiated GlcNAc in the linear form we used a slightly different

9

approach as done for the conformers in ring form. As the GLYCAM FF used for the

10

conformers in ring form was parametrized for sugars in ring form, we decided to use

11

the semi-empirical DFTB3 method61 for the pre-optimization of the GlcNAc

12

conformers in linear form. These calculations were carried out with the quantum-

13

chemistry package GAMESS (2018 R1),62 using the 3OB (“Third-Order

14

Parametrization for Organic and Biological Systems”) parameters.63,64 To obtain the

15

conformer database for linear GlcNAc, we started from a randomly chosen geometry

16

for GlcNAc in linear form and rotated every C-C and C-O single bond as well as the

17

C2-N bond by a random value between -180° and 180°. The C-N bond in the NAc

18

group was not altered as it should be more rigid due to the interaction between the

19

carbonyl group and the lone pair at the N atom. In addition, we randomly placed a

20

sodium ion in the vicinity of a randomly chosen O or N atom. If the so obtained

21

geometry does not feature any unphysically small interatomic distances, we used this

22

geometry as an initial guess for the geometry optimization. Here, we considered any

23

structure as unphysical when the DFTB3 parameters could not be used to calculate its

24

energy and thus resulting in an error termination of the DFTB3 calculation. By

25

following the steps described above, we performed about 1000 optimizations at 9 ACS Paragon Plus Environment


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DFTB3 level and then used the shape screening technique to remove the duplicate

2

structures again, and obtained 173 distinct optimized geometries for sodiated GlcNAc.

3

These geometries have been re-optimized at B3LYP level and re-screened again, after

4

removal of the duplicates we finally obtained 172 distinct structures optimized at

5

B3LYP level.

6

The transition state (TS) structures and energies of the various dissociation

7

channels were calculated at B3LYP level using the Berny algorithm.65 When

8

investigating the transition states for the reactions of interest, we did not consider

9

every possible reaction for every conformer, as various reaction pathways can already

10

be ruled out by analyzing the molecular geometry: For a hydrogen transfer step as

11

occurring in nearly every reaction under study, the H-donor group has to be close to

12

the acceptor group, otherwise, the reaction barrier will be high as the TS is expected

13

to involve the formation of a (nearly) unbound H atom. Therefore, we performed a

14

pre-screening of the conformers, and only considered a subset of conformers as

15

potential initial geometries for certain reactions. As we know from earlier studies41,42

16

that the presence of a Na+ ion in the vicinity of an OH group can enhance the acidity

17

of the OH group, we only considered such geometries as initial state structures for the

18

ring opening step in which the Na+ ion is close to the H atom donating OH group at

19

O1, with an O1-Na distance not exceeding 3 Å. Originally, we have also considered

20

an alternative ring-opening mechanism, in which the Na+ interacts with the ring

21

oxygen O0 to trigger the ring-opening. In this mechanism, O0 becomes a Na-alkoxy

22

group after ring-opening while at O1 becomes an oxonium group. Test calculations at

23

the example of α-GlcNAc have indicated a relatively high barrier of 246 kJ/mol for

24

this mechanism, while the barrier for the ring-opening in which Na+ interacting with

25

O1 can be as low as 176 kJ/mol. Therefore, we did not further consider that 10 ACS Paragon Plus Environment

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1

alternative ring-opening mechanism. For the dehydration via H atom transfer from an

2

OH group to another OH group, we followed a similar argument and only considered

3

such geometries which fulfill the two following conditions: i) the distance between

4

the O atom of the H donating OH group and the O of the accepting OH group is

5

below 3 Å and ii) the Na+ ion is interacting with the H donating OH group, with an O-

6

Na distance below 3 Å. For the H atom transferred from a C atom, we only looked at

7

such structures in which the donating H atom is 3 Å or closer to the O atom of the

8

accepting OH group, irrespective of the location of the sodium atom. For GlcNAc in

9

linear form we have considered a further reaction, C2-C3 bond cleavage which is the

10

second step in the so called cross-ring cleavage. As it proceeds via the transfer of the

11

H atom at O3 to O1 and the simultaneous scission of the C2-C3 bond, the initial

12

geometries that we have taken into consideration here feature the Na+ ion close to O3

13

and an O-H distance between O1 and the H atom at O3 of 2.9 Å or shorter.

14

IV. Results and Discussion

15

A. Experimental measurements

16

As the  and anomers of each HexNAc, which only differ by the OH

17

stereochemistry at the C1 carbon (i.e. the anomeric configuration), do coexist in

18

solution, we separated the two anomers by HPLC and measured the CID spectra right

19

after the separation. Figure 1(a), 1(b) and 1(c) show the HPLC chromatogram and the

20

corresponding CID spectra for GlcNAc, respectively. The major fragment ion is

21

found at m/z 226, representing the dehydration reaction. The minor fragment ions

22

include m/z 124 and 143, representing the 0,2A cross-ring dissociation (loss of neutral

23

m=120 u, HOCH2-(CHOH)2-CHO or neutral m=101 u, HO-CH=CH-NHCOCH3,

24

respectively), and m/z 148, representing the loss of a neutral fragment m = 96 u,

25

C3H2-NHCOCH3. The assignments of these fragments were based on the CID spectra

11

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of isotope-labelled compounds described below. Unlike glucose, galactose, and

2

mannose which feature large differences between the CID spectra of the two anomers,

3

so the relative intensities of the cross-ring dissociation and dehydration signals can be

4

used to identify the anomeric configuration of a sample, the difference between the

5

CID spectra of the two GlcNAc anomers is small, as illustrated in Figures 1(b) and

6

1(c). Thus, the determination of the anomeric configuration of GlcNAc is not as easy.

7 8

Figure 1. (a) Chromatogram of GlcNAc; CID spectra of (b) the first peak in 12 ACS Paragon Plus Environment

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1 2

chromatogram, (c) the second peak in chromatogram, (d) [1,2-13C2]GlcNAc, (e) [15N]GlcNAc, (f) [1-18O]GlcNAc, (g) [D5]GlcNAc, (h) [1-18O, D5]GlcNAc, (i) [6,6’-

3 4 5 6 7 8

D2]GlcNAc, (j) [CD3]GlcNAc, (k) [D8]Gal--(13)-GlcNAc [1,2,4,6-D4]GlcNAc fragments, (l) [D8]Gal--(14)-GlcNAc [1,2,3,6-D4]GlcNAcfragments, (m) [D8]Gal--(16)-GlcNAc [1,2,3,4-D4]GlcNAc fragments, (n) [D8]Gal--(13)GlcNAcfragments, (o) [D8]Gal--(14)-GlcNAcfragments, (p) [D8]Gal-(16)-GlcNAcfragments. To illustrate the position of the isotope labeling we show the structures of the isotope labeled species in Scheme 1.

9

The cross-ring dissociation was, in the case of the three common hexose sodium

10

adducts, explained by a retro-aldol reaction,35,41,42,66–69 as illustrated Scheme 2(c). The

11

same mechanism can be used to explain the cross-ring dissociation of GlcNAc. The

12

CID spectra of isotope substituted GlcNAc, [1,2-13C2]GlcNAc, [15N]GlcNAc, and [1-

13

18O]GlcNAc,

14

ring dissociation of GlcNAc, as illustrated in Scheme 2(d). In contrast to the cross-

15

ring dissociation, the dehydration mechanism for GlcNAc, was found to be very

16

different from the dehydration mechanism for the three common hexose sodium

17

adducts. The CID spectra of the isotope substituted GlcNAc, [1-18O]GlcNAc,

18

[D5]GlcNAc, and [1-18O, D5]GlcNAc, as illustrated in Figure 1, indicates that the

19

dehydration leads to the elimination of H216O, DH16O, and DH16O, respectively. If

20

GlcNAc would follow the dehydration mechanism of the three common hexoses, as

21

illustrated in Scheme 2(a), one would expect H218O, D216O, and D218O, respectively,

22

as elimination product.

illustrated in Figure 1, supports the retro-aldol mechanism for the cross-



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1 2 3 4

Scheme 2. Comparison of the dehydration of (a) -glucose, (b) -GlcNAc and the cross-ring dissociation mechanisms of (c) -glucose and (d) -GlcNAc. In (d) we also show the three-step dehydration mechanism after ring-opening of -GlcNAc.

5 6

To further investigate the dehydration mechanism, we studied the CID spectra of

7

other isotope substituted GlcNAc, namely [6,6’-D2]GlcNAc and [CD3]GlcNAc

8

(deuterated methyl group). Figures 1(i) and 1(j) show that no D2O or DHO

9

elimination was found, indicating that neither the H atoms connecting to the C6 atom

10

and nor those at the methyl group are involved in the dehydration. The CID spectra of

11

the isotope substituted disaccharides [D8]Gal--(13)-GlcNAc, [D8]Gal--(14)-

12

GlcNAc, and [D8]Gal--(16)-GlcNAc, in which all H atoms in the OH and NH

13

groups are replaced by D atoms, are displayed in Figures 1(n), 1(o), and 1(p). They

14

show that the dehydration of DHO remains large for the disaccharides with 14 and

15

16 linkages. In contrast, the dehydration of DHO for the disaccharide with 13

16

linkage disappears. Instead, a small signal associated with D2O elimination was found 14 ACS Paragon Plus Environment

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1

for the disaccharide with 13 linkage. The decrease of the signal associated with

2

DHO elimination for the disaccharide with 13 linkage indicates that the hydroxyl

3

group connecting to C3 is involved in the dehydration of GlcNAc.

4

A concrete evidence for the hydroxyl group connected to C3 being involved in

5

the dehydration is provided by the CID spectra of the fragments obtained from the

6

isotope substituted disaccharides, [D8]Gal--(13)-GlcNAc, [D8]Gal--(14)-

7

GlcNAc, and [D8]Gal--(16)-GlcNAc. One of the major dissociation channels for

8

Gal-GlcNAc disaccharides is the glycosidic bond cleavage, generating GlcNAc. For

9

[D8]Gal--(13)-GlcNAc,

[D8]Gal--(14)-GlcNAc,

and

[D8]Gal--(16)-

10

GlcNAc, the generation of GlcNAc mainly involves the transfer of a D atom of

11

galactose to the O atom of the glycosidic bond, followed by the C-O bond cleavage.

12

However, H atom transfer from galactose to GlcNAc was observed as a minor

13

channel. We took advantage of this minor channel and generated [1,2,4,6-D4]GlcNAc,

14

[1,2,3,6-D4]GlcNAc, and [1,2,3,4-D4]GlcNAc from [D8]Gal--(13)-GlcNAc,

15

[D8]Gal--(14)-GlcNAc, and [D8]Gal--(16)-GlcNAc, respectively. Figure 1(k),

16

1(l), and 1(m) shows that the CID of [1,2,3,6-D4]GlcNAc and [1,2,3,4-D4]GlcNAc

17

results in DHO elimination, but [1,2,4,6-D4]GlcNAc mainly results in H2O

18

elimination. The results support that the hydroxyl group connecting to C3 is involved

19

in the dehydration of GlcNAc.

20

Figure 2 shows the CID spectra of GlcNAc-OMe and GlcNAc-OBn, where Me

21

and Bn represent CH3 and CH2C6H5. As the H atom in the hydroxyl group at position

22

1 was substituted by OMe or OBn in these compounds, the ring opening reaction,

23

which is the first step leading to the cross-ring dissociation, becomes minor channel

24

compared with B/Y-type of dissociation (i.e., the cleavage of C(1)-O(1) glycosidic

25

bond). For these molecules, the MS signal associated with the water elimination is 15 ACS Paragon Plus Environment


Page 16 of 45

1

very weak. Instead, the major dissociation channels were found to lead to the

2

elimination of CH3OH and C6H5CH2OH. The results suggest that ring opening and

3

water elimination are correlated: Only those compounds from which the ring-opening

4

can take place can undergo water elimination.

5 6 7 8 9

Figure 2. CID spectra of (a) -GlcNAc-OMe, (b) -GlcNAc-OMe, (d) -GlcNAcOBn, (e) -GlcNAc-OBn, (g) -GalNAc-OMe, (h) -GalNAc-OMe, (i) -GalNAc(13)-Gal, (k) -GalNAc-(13)-Gal at normalized collision energy 30%. (c), (f), and (i): Branching ratio of C1-O1 cleavage calculated according to equation 1. The

10 11

larger branching ratio observed for the glycosidic bond cleavage in the  form suggests that the relative intensity of glycosidic bond cleavage can be used to 16 ACS Paragon Plus Environment

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1 2

determine the anomeric configuration of the glycosidic bond connecting to GlcNAc and GalNAc.

3 4

The largest intensities observed in the CID spectra of GlcNAc-OMe, and

5

GlcNAc-OBn result from ion m/z 226, representing the elimination of CH3OH and

6

C6H5CH2OH, respectively. Some dissociation channels were not observed in the CID

7

spectra, e.g., desodiation because of the low-mass cutoff of the ion trap. We

8

calculated the branching ratio, r, of CH3OH (or C6H5CH2OH) elimination (i.e., C1-O1

9

cleavage) as a function of the normalized collision energy (NEC) via equation (1).

10

𝑟 = 𝐼(𝑝𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟 𝑖𝑜𝑛,

𝐼(𝑚/𝑧 = 226, 𝑁𝐶𝐸 = 𝑥%) 𝑁𝐶𝐸 = 0%) ― 𝐼(𝑝𝑟𝑒𝑐𝑢𝑟𝑠𝑜𝑟 𝑖𝑜𝑛, 𝑁𝐶𝐸 = 𝑥%)

(1)

11

I(precursor ion, NCE=0%) and I(precursor ion, NCE=x%) represent the intensity of

12

the precursor ion signal at normalized collision energies 0% and x%, respectively.

13

I(m/z=226, NCE=x%) represents intensity of the signal at m/z=226 at a normalized

14

collision energy of x%. The results for the CH3OH (or C6H5CH2OH) elimination

15

branching ratios are shown in Figure 2. The branching ratios of CH3OH (or

16

C6H5CH2OH) elimination from the  form of the considered monosaccharides are two

17

to three times larger than the corresponding values for the  form. The branching

18

ratios suggest that the glycosidic bond in  form is much easier to break than the bond

19

in monosaccharides in  form. The difference can be used to identify the anomeric

20

configuration of the glycosidic bond in disaccharides. For example, the comparison of

21

disaccharides -GalNAc-(13)-Gal and -GalNAc-(13)-Gal, as illustrated in

22

Figure 2(j) and (k), shows that the branching ratio for the glycosidic bond cleavage

23

(m/z 226) from the  form is much larger than that from the  form. This is different

24

from the common hexose sodium adducts, for which the branching ratio for the

25

glycosidic bond cleavage from the  form is much smaller than that from the  17 ACS Paragon Plus Environment


1

Page 18 of 45

form.33,41,42,70

2

The CID spectra of GalNAc are shown in Figure 3. Although the major

3

dissociation channel of GalNAc is similar to that of GlcNAc, i.e., dehydration by

4

eliminating DH16O from [D5]GalNAc and [1-18O, D5]GalNAc, the minor dissociation

5

channels are quite different from that of GlcNAc. For example, the intensity of the ion

6

with m/z 196 in the CID spectra of GalNAc is much larger than the intensities of the

7

ions with m/z 143 and 148. However, the intensity of the ion with m/z 196 in the CID

8

spectra of GlcNAc is much smaller than the intensities of the ions with m/z 143 and

9

148. These minor channels can be used to differentiate GlcNAc and GalNAc.

10 18 ACS Paragon Plus Environment

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1 2 3 4

Figure 3. (a) Chromatogram of GalNAc; CID spectra of (b) the first peak in chromatogram, (c) the second peak in chromatogram, (d) [1-18O]GalNAc, (e) [D5]GalNAc, and (f) [1-18O, D5]GalNAc. To illustrate the position of the isotope labeling we show the structures of the isotope labeled species in Scheme 1.

5 6

B. Ab initio quantum chemistry calculations

7

α- and β-GlcNAc in ring form. Starting from the stable conformers obtained via

8

the procedure described in Section III, we investigated the possible reaction pathways

9

for α- and β-GlcNAc in ring form. We considered the ring opening reaction as well as

10

the dehydration reactions. The ring opening occurs via the transfer of an H atom from

11

O1 to the ring oxygen (O0) and the simultaneous cleavage of the C1-O0 bond. For the

12

dehydration, we considered the reactions in which an H atom of an OH group or the

13

amide group is transferred to another OH group which then leaves the GluNAc

14

molecule as water. These dehydrations would yield D2O if one would use

15

[D5]GlcNAc and are thus referred to as the D2O dehydration channels. In addition, we

16

also considered the reactions in which an H atom attached to a carbon atom transfers

17

to an OH group. These reaction pathways would lead to the formation of DHO if

18

[D5]GlcNAc was used and are therefore referred to as the DHO dehydration channels.

19

The key intermediates and TSs for the reactions under study are shown in Figure

20

4. These molecular structures are named by a label of the form w-x-y(-z). For the

21

sugars in ring form w can be either a or b representing the α- and the β-anomere,

22

respectively. x denotes the sugar and can be GlcNAc and GalNAc. The most stable

23

neutral and sodiated conformer for each studied anomere are denoted as w-x-neutral

24

and w-x-m, respectively, where m stands for “minimum”. To label the initial state,

25

the transition state and (where available) the product of a reaction, z can be i, t, or p, 19 ACS Paragon Plus Environment


Page 20 of 45

1

respectively. y is used to specify the reaction: d2o, dho and ro denote the D2O

2

dehydration channel, the DHO dehydration channel and the ring opening reaction

3

with the lowest TS energy, respectively.

4 20 ACS Paragon Plus Environment

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1 2 3 4 5

Figure 4. Geometries of the key intermediates and transition states involved in the lowest lying pathways of the investigated reaction channels (DHO dehydration, D2O dehydration, ring-opening and desodiation) for GlcNAc in ring form. Atomic distances of interest in Å are shown. The red numbers are the energies in kJ/mol referred to the global minima of each anomere (a-GlcNAc_m and b-GlcNAc_m).

6 7

For reference, we also determined the desodiation energy, i.e., the energy that is

8

required to remove the Na+ ion from the sugar molecules. The desodiation energy is

9

defined as the difference between the energies of the most stable conformer of the

10

sodiated sugar and the sum of the energies of the neutral sugar in its most stable

11

conformer and of an isolated Na+ ion. For both sodiated anomeres, the global minima

12

shown in Figure 4 (a-GlcNAc-m and b-GlcNAc-m), share some structural

13

similarities: The Na+ ion is coordinated by O3 and O4, which in return form hydrogen

14

bonds with the acetyl O atom and O6, respectively. After the removal of the Na+ ion,

15

the most stable geometry of α-GlcNAc (a-GlcNAc-neutral) features a hydrogen bond

16

between O3 and the acetyl O, at variance β-GlcNAc is characterized by hydrogen

17

bonds between O2 and the acetyl O as well as between O4 and O6 (b-GlcNAc-

18

neutral, see Figure 4). The desodiation energies for α- and β-GlcNAc were calculated

19

to be 221 kJ/mol and 218 kJ/mol, respectively, which are around 20 kJ/mol higher

20

than the calculated values of 198 kJ/mol and 194 kJ/mol for α- and β-glucose.42

21

The calculated energies of the most stable TS and the associated initial states are

22

summarized in Figures 5. For the following discussion, we assume that the minima

23

structures for each anomere are in thermal equilibrium. Thus, the preference of a

24

certain reaction channel is solemnly determined by the transition state. We further

25

assume that the effects from the partition function on the reaction rate can be

26

neglected, i.e. we will only need to compare the electronic energies of the TS

27

structures, referred to the global minimum, to find the most favorable reaction path. 21 ACS Paragon Plus Environment


Page 22 of 45

1

The main findings, for both α- and β-GlcNAc, are i) the main reaction channel is the

2

ring-opening reaction with barriers of 176 kJ/mol for α-GlcNAc and 148 kJ/mol for β-

3

GlcNAc, ii) dehydration will mainly occur via the transfer of an H atom from an OH

4

group to another OH group. In other words, the dehydration via the D2O channels

5

seems to be preferred over the DHO channels. The dehydration reaction which has the

6

lowest barrier is the H atom transfer from O4 to O1 (denoted as O4O1) for α-

7

GlcNAc (182 kJ/mol) and O6O1 for β-GlcNAc (168 kJ/mol). However, both of

8

them are larger than the barrier of the ring-opening reaction. These results are in line

9

with the experimental finding that the chemistry of GlcNAc indeed is fundamentally

10

different from the chemistry of glucose, despite the fact that the two species only

11

differ in the presence of the NAc group. Recall that for α-glucose, the dehydration via

12

the O2→O1 H transfer channel is more favorable than the ring opening reaction

13

which is required for the cross-ring cleavage. Compared to α-glucose, both the ring

14

opening and the dehydration of β-glucose have higher barriers. For the latter, the ring-

15

opening was reported to be slightly more preferable than the dehydration which

16

mainly occurs via the O2→O1 and O3→O1 channels.42 A discussion of the minima

17

and TS geometries involved in the dominating reactions and the corresponding

18

coordinates are provided in the SI for the interested reader.


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1 2 3 4 5 6 7 8

Figure 5: Energy diagram 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. The gray dashed line represents the desodiation energy for each species, except in (c), where the desodiation energies for both α- and β-GlcNAc (ring form) are shown. The light-red dashed lines in (a) and (b) represents the energy of the most stable GlcNAc in linear

9 10 11 12

form, which is more stable than -GlcNAc and -GlcNAc by 0.9 and 7.5 kJ/mol, respectively. In (c) “C2→Ac” marks the TS for the first step of the three-step dehydration mechanism. “Ac→O3” refers to the third step of the three-step dehydration. The thin bar represents the TS, while the wide bar shows the energy of 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7

Page 24 of 45

the intermediate from which the third 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 DHO and D2O dehydration channels are used as a shorthand notation for the H atom transfer and correspond to “Cx→Oy” and “Ox→Oy” used in the main text to distinguish the various pathways in the DHO and D2O dehydration channels. Geometries that are shown in Figures 4 and 6 are labeled with the name of the structure.

8 9

A closer look at the molecular structures shows that the change in the reactivity

10

induced by the NAc group can be easily understood. As the OH group at O2 in

11

glucose is replaced by the less acidic NAc group in GlcNAc, one of the main

12

dehydration channels for glucose is quasi-disabled for GlcNAc, as the barriers of 195

13

kJ/mol (α-GlcNAc) and 258 kJ/mol (β-GlcNAc) for the H transfer from NH to O1

14

indicate. Thus, the most preferred dehydration pathway in ring form is O4→O1 for α-

15

GlcNAc and O6→O1 for β-GlcNAc, respectively. Also, the ring opening mechanism

16

is affected: For glucose, the ring opening which involves the H atom transfer from O1

17

to O0, features TS geometries in which the Na+ ion is coordinated to O1 and O2. Such

18

a coordination is unfavored for GlcNAc, which can be rationalized with the steric

19

hindrance in the vicinity of the N atom of the NAc group, which formally replaces the

20

OH group of glucose at O2. The most favored ring-opening TS for GlcNAc (a-

21

GlcNAc-ro-ts and b-GlcNAc-ro-ts, in Figure 4) features the Na+ ion bonded, among

22

others, to O1 as well as to the acetyl O atom. Given these differences in the preferred

23

coordination of the Na+, it would be more surprising if the reactivity of glucose is

24

comparable to GlcNAc.

25

The DHO channels, which have reported to be very unfavorable for glucose,41

26

have been calculated to be very unlikely for GlcNAc in ring form as well, as shown

27

by barriers of around 250 kJ/mol or higher (Figures 5(a) and (b)). The finding that the 24 ACS Paragon Plus Environment

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1

dehydration via the D2O channels is more preferable than the DHO channel is what

2

we have expected before we conducted the experiments with [D5]GlcNAc. After all,

3

there is, with one exception, no obvious reason for the presence of enhanced C-H

4

acidity that would lead to a DHO dehydration channel with a low barrier. The only

5

exception is the methyl group in the NAc group: The corresponding H atoms are in α-

6

position to a carbonyl group and are therefore expected to be more acidic than other

7

C-H protons. This effect can be further enhanced by the presence of a Lewis acidic

8

center (e.g. Na+) close to the carbonyl O atom. The lowest dehydration pathway via a

9

H atom transfer from the methyl group to any OH group is the H atom transfer to the

10

OH group at O1. However, this reaction features barriers of 340 kJ/mol or higher. In

11

other words, the H atom transfer from the methyl group is not the most likely pathway

12

in the DHO channels and can thus be ruled out as the main reaction pathway. This

13

result is consistent with the experimental observation that the CID of [D3]GlcNAc

14

mainly leads to the elimination of H2O and not DHO. We speculate that the high

15

barrier is related to the fact that the dangling bond at C1 is not sufficiently saturated

16

once the C1-O1 bond is broken.

17

The fact that the ring-opening has the lowest barrier and therefore is the most

18

important reaction for GlcNAc in ring form shows that any decomposition of GlcNAc

19

observed in the experiment should occur from the linear form of GlcNAc, which will

20

be discussed below. Neither the dehydration via the D2O channels nor via the DHO

21

channels of GlcNAc in ring form as discussed above plays a significant role. Note that

22

the ring opening reaction of both anomers of GlcNAc yield the same product. With

23

this in mind, we can easily rationalized why CID cannot be used to characterize the

24

anomic configuration in the same way as discussed for the three common hexoses.42

25

The finding that the dehydration must occur after ring-opening can be verified by 25 ACS Paragon Plus Environment


Page 26 of 45

1

comparison with the experimental observations for the decomposition of GlcNAc

2

derivatives for which the ring-opening channel is strongly suppressed: e.g. GlcNAc-

3

OMe and GlcNAc-OBn. In other words, GlcNAc derivatives, in which the H atom at

4

O1 is replaced by a methyl group or CH2C6H5, so the ring-opening becomes

5

significantly less likely to occur. Recall that the experiments show that the signals

6

associated with the elimination of water becomes very weak, while MeOH and

7

C6H5CH2OH eliminations become the major channel for GlcNAc-OMe and GlcNAc-

8

OBn, respectively.

9

Figures 5(a) and (b) show that if ring-opening cannot occur, dehydration via

10

O4O1 followed by C1-O1 cleavage and O6O1 followed by C1-O1 cleavage have

11

the lowest barrier for -GlcNAc and -GlcNAc, respectively. These barriers are

12

lower than the corresponding desodiation energies by 39 and 50 kJ/mol, respectively,

13

indicating C1-O1 cleavage in -GlcNAc is much easier to occur than the

14

corresponding reaction for -GlcNAc. This agrees with the experimental observation

15

that the branching ratio for the cleavage of C1-O1 bond in -GlcNAc is much larger

16

than the corresponding value for -GlcNAc, as illustrated in Figure 2.

17

GlcNAc in Linear Form. Once the GlcNAc undergoes the ring-opening reaction,

18

there is no difference between the α- and β-form anymore, if the barriers for the

19

rotation around the C1-C2 single bond is sufficiently low. To check, whether this is

20

really the case, we have calculated the rotation barrier for the C1-C2 bond in the

21

structure a-GlcNAc-ro-p shown in Figure 4, which is found to be only 68 kJ/mol

22

referred to a-GlcNAc-ro-p. For the sake of simplicity, we assume that all rotamers of

23

linear GlcNAc are in thermal equilibrium under the reaction conditions of interest.

24

The barriers reported below all refer to the most stable conformer of sodiated linear

25

GlcNAc. Following the approach applied for the reactions of GlcNAc in ring form, 26 ACS Paragon Plus Environment

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1

we identified the conformers that have the required geometries for the reactions of

2

interest. We should mention here that for the DHO dehydration channel, we mainly

3

focused on the C2→O3 channel, as C2 is, after ring opening, in α-position to a

4

carbonyl group, which enhances the acidity of its H atoms. The H transfer from C1,

5

C3, C4, C5, C6 as well as from the methyl group to any OH group, has only been

6

tested at selected geometries.

7

The calculation of the transitions states for the reactions mentioned above

8

delivers some surprising results: For linear GlcNAc, the dehydration via H atom

9

transfer from C2 to O3, i.e. a DHO channel has the lowest barrier of all dehydration

10

reactions. With only 192 kJ/mol, it is not only far lower than any other DHO channel,

11

it is also more favorable than any D2O channel which features reaction barriers of 217

12

kJ/mol or higher. We speculate that the preference of the C2→O3 channel is related

13

to the circumstance that the dangling bonds at C2 and C3 can form a C=C double

14

bond to stabilize the TS structure. However, the barrier of the C2→O3 dehydration

15

channel is significantly higher than the activation energy required for the C2-C3 bond

16

cleavage (resulting in cross-ring cleavage), which is only 147 kJ/mol. Recall that the

17

products of the cross-ring cleavage are hardly visible in the experiment, indicating

18

that the C2→O3 channel is most likely not the dominating dehydration channel for

19

GlcNAc.

20

So far, we have only discussed dehydration mechanisms that consist of a single

21

reaction step. Our inability to find a single-step mechanism for dehydration that is

22

more favorable than the cross-ring cleavage led to the question whether the

23

dehydration is actually a multi-step process. Thus, we have considered a mechanism

24

in which the H atom connecting to C2 is first transferred to the O atom in the NAc

25

group (denoted as C2→Ac). This formally leads to the formation of a negative charge 27 ACS Paragon Plus Environment


Page 28 of 45

1

at C2, and a positive charge at the O atom of the NAc group. While the negative

2

charge can be stabilized by the neighboring carbonyl group at C1, the positive charge

3

is stabilized by the electron lone pair of the amino group. After a rearrangement of the

4

Na+ position, the H atom at the NAc group can be transferred to the OH group at O3,

5

which leaves the sugar molecule as water (denoted as Ac→O3). We tested several

6

initial state geometries, the lowest barrier for the initial C2→Ac step is 111 kJ/mol.

7

As it is known from earlier studies that the barriers associated with Na+ migration are

8

very low compared the to the dehydration barriers, we have not investigated the Na+

9

migration step explicitly. The energy for the TS of the third step, Ac→O3, is 105

10

kJ/mol referred to the most stable linear conformer of GlcNAc. The barriers of these

11

three steps are all lower than the barrier for the cross-ring cleavage. In other words,

12

this three-step mechanism, which represents a mechanism that would lead to the

13

elimination of DHO from [D5]GlcNAc, is significantly easier accessible than the

14

competing cross-ring cleavage. With this three-step mechanism, the computational

15

results fully agree with the experimental observation. The key intermediates and TSs

16

for the reactions discussed above are summarized in Figure 6. Following the logic

17

applied to the structures in ring form, the molecular structures discussed here all have

18

a label of the form l-GlcNAc-y(-z), where l stands for “linear”. Aside of d2o and dho,

19

y can also be cr, 2a and a3 here, representing the cross-ring cleavage as well as the

20

C2→Ac and Ac→O3 H transfer steps in the three-step dehydration mechanism. A

21

discussion the most important minima and TS geometries for the reactions of linear

22

GlcNAc and the corresponding coordinates are provided in the SI.


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1 2 3 4 5 6

Figure 6: Geometries of the most minima and transition states involved in the lowest lying pathways of the investigated reaction channels (DHO dehydration, D2O dehydration, cross-ring cleavage, and three-step dehydration) for GlcNAc in linear form. The red numbers are the energies in kJ/mol referred to the global minima for GlcNAc in linear form (l-GlcNAc_m).

7 8

α- and β-GalNAc in Ring Form. As the experiments have shown that GalNAc

9

behaves similarly to GlcNAc, we have only studied the D2O dehydration channels and

10

the desodiation process for GalNAc in ring form computationally. The computational

11

procedure for this task is the same as described above for the investigation of the

12

corresponding process of GlcNAc. The desodiation energy for α- and β-GalNAc has

13

been calculated to 242 kJ/mol and 225 kJ/mol, respectively. For β-GalNAc, the most 29 ACS Paragon Plus Environment


Page 30 of 45

1

favorable dehydration pathway occurs via the H transfer from O3 to the OH group at

2

O1 (O3O1), which is associated with a barrier of 159 kJ/mol. This result is

3

consistent with our earlier calculations on β-galactose, which also features the

4

O3→O1 channel as the most favorable dehydration channel. Compared to β-GalNAc,

5

the dehydration of α-GalNAc is kinetically generally less accessible, with the lowest

6

dehydration barrier calculated to be 212 kJ/mol. The most interesting finding here is

7

that this barrier is associated with the H transfer from the N atom to the OH group at

8

O1 (N2→O1). The lowest lying pathway for α-GalNAc, which involves the H

9

transfer from an OH group is the O4→O6 channel with a barrier of 221 kJ/mol, i.e., 9

10

kJ/mol higher. As mentioned in the discussion on GlcNAc, the H atom at the amino

11

group is expected to be less acidic than the H atoms in OH groups, thus the current

12

result is somewhat surprising. However, it seems to be in line with what we have

13

reported for α-galactose42 in our previous study: The dehydration of α-galactose

14

clearly favors the O2→O1 channel, as illustrated by the difference of 55 kJ/mol

15

between the barriers of the O2→O1 (166 kJ/mol) pathway and of the O4→O6 (221

16

kJ/mol) pathway, the second lowest dehydration pathway for α-galactose. This means

17

that the stereochemistry of α-galactose clearly favors the dehydration via the O2→O1

18

channel. This effect is retained for α-GalNAc making the N2→O1 pathway the most

19

favorable dehydration pathway. However, the reduced acidity of the amino group

20

compared to an OH group results in the circumstance that the N2→O1 channel is only

21

favored by 9 kJ/mol over the O4→O6 pathway.

22

Similarly to what has been done in the discussion for GlcNAc, we can also

23

compare the calculated results for GalNAc in ring form to the experiments using

24

GalNAc derivatives, for which the ring opening channel has been suppressed. The

25

experiments indeed suggest that the decomposition mainly occurs via the C1-O0 bond 30 ACS Paragon Plus Environment

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1

cleavage following the H atom transfer from an OH group. Comparable to GlcNAc,

2

the experiments also show that the branching ratio for the C1-O0 bond cleavage for

3

the β form is larger than then value for the α form. This is also in line with the

4

differences between the D2O dehydration barriers and the desodiation energies at

5

B3LYP level. For β-GalNAc, (desodiation energy)-(dehydration barrier) is 66 kJ/mol,

6

while the corresponding value is only 30 kJ/mol for α-GalNAc.

7

V. Conclusion

8

In this work, we have used isotope marking experiments alongside with quantum

9

chemical calculations to investigate the decomposition pathways of the hexose

10

derivatives GalNAc and in particular GlcNAc under CID conditions. We could show

11

that while the mechanisms for the ring-opening for sodiated HexNAcs are similar to

12

that of sodiated hexoses, the dominating dehydration mechanism shows some

13

fundamental differences.

14

The ring-opening, which proceeds via the H atom transfer from O1 to O0,

15

followed by C1-O0 bond cleavage, has been shown to be the major reaction channel

16

for the studied HexNAcs in ring form, irrespective of the anomeric configuration.

17

After ring-opening (and the associated loss of the stereo-information of the C1 atom),

18

dehydration, which involves the transfer of the H atom from C2 to the acetyl O atom,

19

followed by the transfer of the same H atom from the acetyl O atom to O3 atom and

20

the cleavage of the C3-O3 bond, has been calculated to feature the lowest barrier. This

21

mechanism for the dehydration following a ring-opening step explains, on the one

22

hand, why the elimination of HDO is the dominating reaction in the CID of

23

[D5]GlcNAc. On the other hand, it rationalizes why no difference between the CID

24

spectra of α-HexNAc and β-HexNAc can be observed, making the identification of

25

the anomeric configuration of HexNAc monomers based on CID impossible. In 31 ACS Paragon Plus Environment


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contrast, for HexNAc derivatives which cannot undergo ring-opening, e.g.,

2

disaccharides with a HexNAc located at the non-reducing end, the C1-O1 glycosidic

3

bond cleavage becomes the major reaction channel. As the C1-O1 cleavage from the

4

 form has a barrier lower than the same reaction from the  form, the branching ratio

5

for the glycosidic bond cleavage can be used to determine the anomeric configuration

6

of the glycosidic bond.

7

The results of these studies on the mechanisms will be helpful for further

8

development of the structural determination of oligosaccharides using CID

9

fragmentation.

10

Supporting Information

11

Detailed information regarding the numbers of conformers considered in

12

calculations, structurally distinct conformers plotted in Mercator projections of the

13

Cremer-Pople puckering sphere, two-stage clustering algorithm for screening out

14

duplicate conformers, structure search from neutral to sodiated HexNAc, transition

15

states and geometries along the major reaction pathways of GlcNAc, and the energy

16

ranges of conformers and transition states covered by the calculations is in supporting

17

information.

18

Acknowledgements

19

Part of this work was financially supported by the Thematic Research Project

20

(AS-TP-107-M08) of Academia Sinica, Taiwan. Computational resources were

21

supported in part by the National Center for High Performance Computing of Taiwan.

22

CCC is grateful for a Distinguished Postdoctoral Scholars Fellowship of Academia

23

Sinica, and the IAMS Junior Fellowship of the Institute of Atomic and Molecular

24

Sciences. PJH is supported by Postdoctoral Scholars Fellowship of Academia Sinica. 32 ACS Paragon Plus Environment

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1

HTH and HTP would like to thank Taiwan International Graduate Program (TIGP)

2

for Ph. D scholarships.

3



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Table 1. Numbers of conformers obtained and used at various calculation stages. α-GlcNAc

β-GlcNAc

Initial neutral conformers (GLYCAM)

19997

Distinct neutral conformers (GLYCAM) Distinct neutral conformers (B3LYP/6-311+G(d,p)) # of puckering index of neutral conformers

α-GalNAc

β-GalNAc

19988

19993

19991

3863

4562

3396

3726

1111

1098

917

1030

17

19

15

18

Initial sodiated conformers (DFTB)

linear GlcNAc

1053

Distinct sodiated conformers (B3LYP/6-311+G(d,p))

151

152

172

145

157

Transition state

92

146

239

86

139

2 3


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The Unexpected Dissociation Mechanism of Sodiated N

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