Gas-Phase Intercluster Thiyl-Radical Induced C–H Bond Homolysis Selectively Forms Sugar C2-Radical Cations of Methyl D-Glucopyranoside: Isotopic Labeling Studies and Cleavage Reactions

Article abstract of DOI:10.1007/s13361-017-1667-2

A suite of isotopologues of methyl D-glucopyranosides is used in conjunction with multistage mass spectrometry experiments to determine the radical site and cleavage reactions of sugar radical cations formed via a recently developed ‘bio-inspired’ method. In the first stage of CID (MS²), collision-induced dissociation (CID) of a protonated noncovalent complex between the sugar and S-nitrosocysteamine, [H₃NCH₂CH₂SNO + M]⁺, unleashes a thiyl radical via bond homolysis to give the noncovalent radical cation, [H₃NCH₂CH₂S^? + M]⁺. CID (MS³) of this radical cation complex results in dissociation of the noncovalent complex to generate the sugar radical cation. Replacement of all exchangeable OH and NH protons with deuterons reveals that the sugar radical cation is formed in a process involving abstraction of a hydrogen atom from a C–H bond of the sugar coupled with proton transfer to the sugar, to form [M – H^? + D⁺]. Investigation of this process using individual C-D labeled sugars reveals that the main site of H/D abstraction is the C2 position, since only the C2-deuterium labeled sugar yields a dominant [M – D^? + H⁺] product ion. The fragmentation reactions of the distonic sugar radical cation, [M – H^?+ H⁺], were studied by another stage of CID (MS⁴). ¹³C-labeling studies revealed that a series of three related fragment ions each contain the C1–C3 atoms; these arise from cross-ring cleavage reactions of the sugar. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-017-1667-2

B American Society for Mass Spectrometry, 2017
J. Am. Soc. Mass Spectrom. (2017) 28:1425Y1431
DOI: 10.1007/s13361-017-1667-2
FOCUS: BIO-ION CHEMISTRY: INTERACTIONS OF BIOLOGICAL IONS
WITH IONS, MOLECULES, SURFACES, ELECTRONS, AND LIGHT : RESEARCH ARTICLE
Gas-Phase Intercluster Thiyl-Radical Induced C–H Bond
Homolysis Selectively Forms Sugar C2-Radical Cations
of Methyl D-Glucopyranoside: Isotopic Labeling Studies
and Cleavage Reactions
Sandra Osburn,1,2 Gaetano Speciale,1 Spencer J. Williams,1 Richard A. J. O’Hair1,2
1School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010,
Australia
2ARC Center of Excellence for Free Radical Chemistry and Biotechnology, The University of Melbourne, Victoria, 3010, Australia
Abstract. A suite of isotopologues of methyl D-glucopyranosides is used in conjunc-
tion with multistage mass spectrometry experiments to determine the radical site and
cleavage reactions of sugar radical cations formed via a recently developed ‘bio-
inspired’ method. In the first stage of CID (MS2), collision-induced dissociation (CID)
of a protonated noncovalent complex between the sugar and S-nitrosocysteamine,
[H3NCH2CH2SNO + M]+, unleashes a thiyl radical via bond homolysis to give the
noncovalent radical cation, [H3NCH2CH2S• + M]+. CID (MS3) of this radical cation
complex results in dissociation of the noncovalent complex to generate the sugar
radical cation. Replacement of all exchangeable OH and NH protons with deuterons
reveals that the sugar radical cation is formed in a process involving abstraction of a
hydrogen atom from a C–H bond of the sugar coupled with proton transfer to the sugar, to form [M – H• + D+].
Investigation of this process using individual C-D labeled sugars reveals that the main site of H/D abstraction is
the C2 position, since only the C2-deuterium labeled sugar yields a dominant [M – D• + H+] product ion. The
fragmentation reactions of the distonic sugar radical cation, [M – H•+ H+], were studied by another stage of CID
(MS4). 13C-labeling studies revealed that a series of three related fragment ions each contain the C1–C3 atoms;
these arise from cross-ring cleavage reactions of the sugar.
Keywords: Noncovalent complex, S-nitrosocysteamine, Carbohydrate, Collision-induced dissociation, Radical
chemistry
Received: 18 January 2017/Revised: 22 March 2017/Accepted: 22 March 2017/Published Online: 4 May 2017
induced dissociation (CID) of their even electron ions [1, 2].
However, relatively few studies have investigated the use of
Introduction
lectrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry are
routinely used to sequence oligosaccharides by collision-
gas-phase radical ion chemistry involving odd electron species
to direct the fragmentation of sugars. Early pioneering electron
ionization (EI) studies by Finan and other workers highlighted
the rich chemistry of sugar radical cations, which included
cross-ring cleavage reactions [3–7]. Unfortunately the genera-
tion of traditional EI mass spectra is restricted by volatility
issues to monosaccharides and small oligosaccharides, which
can in some cases be overcome by derivatization strategies [8].
To overcome such limitations, several approaches that exploit
the gas-phase chemistry of ions formed using ESI have been
developed that unleash radical sites on sugars. These include:
the use of electron capture dissociation (ECD) and electron
transfer dissociation (ETD), which result in radical-directed
E
Dedicated to Professor Scott McLuckey on the occasion of his ASMS award
and in recognition of his important contributions to gas-phase ion chemistry and
service to the American Society of Mass Spectrometry. One of us (R.A.J.O.)
fondly remembers sabbatical visits with Scott at Oak Ridge National Labora-
tories and at Purdue.
Electronic supplementary material The online version of this article (doi:10.
1007/s13361-017-1667-2) contains supplementary material, which is available
to authorized users.
Correspondence to: Richard O’Hair; e-mail: rohair@unimelb.edu.au

1426
S. Osburn et al.: Fragmentation of Sugar Radical Cations in the Gas Phase
as received. Deuterium-labeled [(2-2H)-, (3-2H)-, (4-2H)-, (5-
cleavage of oligosaccharides [9–11]; formation of radical ions
by electron detachment dissociation (EDD) and the related
negative electron transfer dissociation (NETD) method [12–
16], or by photo-activated electron detachment [17–20]; the use
of CID of a ternary metal complex containing the carbohydrate
to form radical anions [21]; and an extension of Beauchamp’s
free radical initiated peptide sequencing (FRIPS) method [22]
to oligosaccharides [23, 24].
2H)-, and (6,6'-2H2)-] D-glucose (98 atom-%), 13C-labeled [(1-
13C)-, (2-13C)-, (3-13C)-, (4-13C)-, (5-13C)-, (6-13C)-] D-
glucose (99 atom-%), and doubly labeled (1-2H, 1-13C)-D-
glucose (98,99 atom-%) were purchased from Omicron Bio-
chemicals (South Bend, IN, USA).
Preparation of Labeled Methyl D-Glucopyranosides
Inspired by Nature’s use of enzyme radical chemistry to
biocatalytically transform carbohydrate-based substrates [25–31],
we have previously described an approach that utilizes a charged
noncovalent complex between a sugar and a radical to generate a
charged sugar radical [32, 33]. The method (Scheme 1) involves:
(1) transfer to the gas phase of a charged noncovalent complex
containing a sugar and a radical precursor (an S-nitrosoamine); (2)
formation of the radical cation noncovalent complex by
unleashing the radical site from the precursor in preference to
dissociation of the noncovalent complex [step (a) of Scheme 1];
and (3) transfer of both the radical and charge sites to the sugar
upon dissociation of the radical cation non-covalent complex [step
(b) of Scheme 1]. A wide range of noncovalent complexes were
found to produce varying amounts of radical cations of mono- and
oligosaccharides; however, the sites of the hydrogen atom transfer
(HAT) from the sugar and the mechanistic details of the radical
fragmentation reactions were not examined. Here we use a series
of isotopically labeled methyl D-glucopyranosides (shown in
Scheme 2) to identify: (1) the site(s) of hydrogen atom abstraction;
and (2) which bonds within the sugar are cleaved upon CID of the
radical cation. Based on previous studies that revealed that the best
S-nitrosoamines to form the highest yield of sugar radical cations
are those with a short linker X [33], S-nitrosocysteamine was
chosen for this work.
A solution of isotopically labeled D-glucose (3.0 mg, 0.017
mmol) in dry MeOH (0.5 mL) was treated with 3 M HCl in dry
MeOH (0.3 mL) (prepared by addition of the appropriate
amount of acetyl chloride to dry methanol), and heated to
reflux overnight. The reaction was cooled to room temperature,
then was adjusted to neutral pH by the addition of a small
quantity of Dowex 1X8 (HO− form), filtered and the solvent
evaporated. Methanol-d4 was used in place of methanol for the
synthesis of the anomeric methyl-labeled isotopologue. Anal-
1
ysis of the products by H NMR spectroscopy revealed a
typically 4:1 α/β ratio of pyranose anomers.
Preparation of S-Nitrosocysteamine
A mixture of tert-butyl nitrite (1.5 equivalents) and a 1 mM
solution of the cysteamine (in 1:1 methanol:water with 1%
acetic acid) was allowed to react for 10 min at room tempera-
ture. The reaction mixture was then diluted 100-fold using 50/
50 methanol:water with 1% acetic acid.
Mass Spectrometry
Solutions containing the sugar and S-nitrosocysteamine were
made by mixing two equivalents of the sugar with one equivalent
of S-nitrosocysteamine in 1:1 methanol:water with 1% acetic acid
to give a final concentration of 1 μM. The labeled noncovalent
complex of 2 was formed in a similar fashion, except that 1 was
mixed with S-nitrosocysteamine in D2O and 1% acetic acid-d4
for 15 min, and this solution was then subjected to ESI.
ESI mass spectra were collected using a Thermo Scientific
LTQ-FT-ICR hybrid mass spectrometer (Bremen, Germany),
consisting of a linear ion trap coupled to a 7T FT-ICR mass
spectrometer. Analyte solutions were injected onto the ion trap
Materials and Methods
Materials
Methyl α-D-glucopyranoside, cysteamine, methanol-d4 (>99.8
atom-% and tert-butyl nitrite were purchased from Sigma
Aldrich Chemical Co. (Castle Hill, NSW, Australia) and used
non-covalent complexes
(a)
(b)
OH
OH
O
OH
CID MS2
O
CID MS3
−
H
+ H+
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
OCH3
OCH3
S
OCH3
H
N
H
N
H
X
H
X
O
S
N
radical cation
H
H
radical precursor
Scheme 1. Sequential CID steps used to form radical cations of methyl α-D-glucopyranoside. X = a short linker. For this work, X =
CH2CH2

S. Osburn et al.: Fragmentation of Sugar Radical Cations in the Gas Phase
1427
OH
O
O
D
OH
O
O
HO
HO
D
O
HO
HO
D
D
O
HO
D
O
HO
OCH3
OCH3
-Glc-OD labelled
OCH3
(
1
) Me-
α
-Glc-unlabelled
MW = 194
(
2
) Me-
α
(
3
) Me-
α
-Glc-C1-D1 labelled
MW = 196
MW = 198
OH
OH
O
OH
D
D
O
O
HO
HO
HO
HO
HO
HO
HO
OCH3
HO
OCH3
HO
OCH3
D
(
4
7
) Me-
α
-Glc-D2 labelled
(
5
) Me-
α
-Glc-D3 labelled
(6) Me-α-Glc-D4 labelled
MW = 195
MW = 195
MW = 195
OH
O
OH
OH
O
D
HO
HO
2C
O
HO
HO
HO
HO
D
HO
OCH3
HO
HO
OCD3
OCH3
(
) Me-
α
-Glc-D5 labelled
(
8
) Me-
α
-Glc-D2-6 labelled
(9) Me-α-Glc-CD3 labelled
MW = 195
MW = 195
MW = 197
OH
O
OH
O
OH
O
HO
HO
HO
HO
HO
HO
HO
OCH3
HO
OCH3
HO
OCH3
(
10) Me-
α
-Glc-C1 labelled
MW = 195
(
11) Me-
α
-Glc-C2 labelled
MW = 195
(12) Me-α-Glc-C3 labelled
MW = 195
OH
O
OH
O
OH
O
HO
HO
HO
HO
HO
HO
HO
OCH3
HO
OCH3
HO
OCH3
(
13) Me-
α
-Glc-C4 labelled
MW = 195
(
14) Me-
α
-Glc-C5 labelled
MW = 195
(
15) Me-α-Glc-C6 labelled
MW = 195
Scheme 2. Structures and MWs of methyl α-D-glucopyranoside isotopologues. A red dot denotes the site of a 13C labeled atom
mass spectrometer using a syringe pump with a flow rate of 5.0
μL min–1. The instrument settings were optimized for maxi-
mum [H3NCH2CH2SNO + M]+ signal intensity by using the
auto-tune routine within the Tune program. The spray needle
voltage, nitrogen sheath gas flow rate, and the capillary
temperature were maintained at +5 kV, 5 arbitrary units,
and 200 °C, respectively. Helium gas was used as the colli-
sion gas for CID experiments, and all MSn experiments were
carried out in the linear ion trap by mass selecting the pre-
cursor ion and subjecting it to collisional activation. The
normalized collision energy (RCE), which determines the
amplitude of the rf energy applied to the end-cap electrodes,
was set between 16% and 18% (arbitrary units) depending on
the precursor ion. The activation Q was set at 0.25, and the
activation time was 30 ms.
between protonated S-nitrosocysteamine and various
isotopically-labeled methyl D-glucopyranosides. MS/MS
of the unlabeled noncovalent complex 1 (Figure 1a) gave
rise to a radical cation of the complex (Equation 1, m/z
271) as well as protonated S-nitrosocysteamine (Equa-
tion 2, m/z 107) and the radical cation of cysteamine
(Equation 3, m/z 77). Isolation of the radical cation
complex, followed by a second stage of CID (Figure 1b),
gave the sugar radical cation at m/z 194 (Equation 4), as
well as fragment ions at m/z 177, 176, and 158. A minor
competing loss of the cysteamine radical cation is also
observed (Equation 5, m/z 77). Hydrogen atom abstrac-
tion (Equation 6) and formation of the protonated sugar
(Equation 7) are not competitive fragmentation pathways.
The fragment ions at m/z 176 and 158 are likely to occur
from loss of one or two molecules of H2O from the
radical cation, the ion at m/z 177 can occur from loss
of HO• form the radical cation or from H2O loss from a
completely dissociating [M + H]+ (initially formed via
Equation 7). Isolation of the radical cation of the sugar
Results and Discussion
A series of multistage mass spectrometry experiments
were carried out on the noncovalent complexes formed

1428
S. Osburn et al.: Fragmentation of Sugar Radical Cations in the Gas Phase
Figure 1. MSn CID mass spectra of: (a) [1 + H3NCH2CH2SNO]+, MS2; (b) [1 + H3NCH2CH2S•]+, MS3; (c) [1 – H• + H+], MS4. X =
CH2CH2. The spectra were collected using an LTQ-FT-ICR hybrid mass spectrometer. The RCE value was 18%
glucopyranose) = 188 kcal/mol [34] with GB(EtNH2) = 210
kcal/mol [35]] and that sugars readily form hydrogen bonded
ammonium ion complexes [M + NH4]+ (and not [M + H +
NH3]+) [36], we assume that complexes of the type (Scheme 1)
play a key role in the formation of the sugar radical cation,
which requires both hydrogen atom abstraction from the sugar
and proton transfer to the resultant sugar radical. To separate
out these two distinct intra-cluster reactions, we performed a
deuterium labeling experiment in which all of the labile hydro-
gens of the noncovalent complex were exchanged for deuteri-
um (Supporting Information, Supplementary Figure S1). In the
second stage of CID (MS3), the peak corresponding to the
radical cation of the sugar (m/z 194) shifted to m/z 199 (Sup-
plementary Figure S1b), confirming that abstraction of a hy-
drogen from one of the C–H bonds of the sugar coupled with
D+ transfer had occurred. This suggests that the radical is
located on a carbon atom and the charge is located on an
oxygen atom (i.e., formation of a distonic ion [37]). This
observation is consistent with the lower bond dissociation
energy of the C–H versus O–H bonds in sugars [23], and that
the only sites for protonation in sugars are the O atoms [34, 38].
Thus the structure of the radical cation formed in our method is
different to the initially formed canonical structures of radical
cations of sugars generated via EI/MS [8].
Although the preceding experiment is silent on the site of
protonation, by using the regiospecifically deuterium labeled
precursors 3–9 (note: 3 contains both 13C and 2H) we reasoned
that we could determine the site(s) of H atom abstraction from
C–H bond(s). Surprisingly, HAT was found to occur almost
exclusively from the C2 position (Supplementary Figure S3),
whereas essentially no HAT occurs from the C1, 3, 4, 5, or 6
positions (Supplementary Figures S2, S4–S7). How do these
results compare to previous studies? Radical reactions of sugars
have been extensively examined in solution, and the regiose-
lectivity of these reactions has been found to be determined by
a range of factors, including: (1) the nature/reactivity of the
radical species that abstracts a hydrogen atom; (2) relative
stabilities of sugar radicals formed by hydrogen atom
followed by another stage of CID (Figure 1c) proceeded
through the loss of 1 and 2 water molecules (m/z 176
and 158, respectively) as well as by fragmentation of the
sugar ring to give the peaks at m/z 102, 103, and 104.
þ
½H3NCH2CH2SNO þ MŠ
ð1Þ
→½H3NCH2CH2S• þ MŠþ þ NO•
→½H3NCH2CH2SNOŠþ þ M
ð2Þ
ð3Þ
ð4Þ
→½H3NCH2CH2S•Šþ þ NO• þ M
þ
½H3NCH2CH2S• þ MŠ
→½M–H• þ HþŠ þ H2NCH2CH2SH
→½H3NCH2CH2S•Šþ þ M
ð5Þ
ð6Þ
ð7Þ
→½H3NCH2CH2SHŠþ þ ½M–HŠ
•
→½M þ HŠþ þ H2NCH2CH2S•
Site of Hydrogen Atom Abstraction
Based on the fact that monosaccharides have lower gas-phase
basicities (GBs) than primary amines [compare GB(α-D-

S. Osburn et al.: Fragmentation of Sugar Radical Cations in the Gas Phase
1429
abstraction; (3) stereochemical effects; and (4) steric effects of
alcohol protecting groups [39, 40]. Owing to the absence of
protecting groups, this last effect is not significant in this work.
Regarding (1), Bhot^ hydroxyl radicals, HO•, are nonselective
suggestion of likely intermediates in the fragmentation reac-
tions (A and B in Scheme 3) and the assignment of product ion
formulae for the fragment ions m/z 102 (C), m/z 103 (D), and
m/z 104 (E) formed in the CID spectra of the sugar distonic ion,
[M – H• + H+]. All CID spectra are given in the Supporting
Information, and Scheme 3 provides a summary of how the m/z
values for these three fragment ions either remain the same or
change as a function of the site(s) of the labels in the sugars.
The CD3 sugar, 9 (Scheme 2), gives product ions that are
shifted by 3 Da, highlighting that the anomeric methoxy group
is maintained in these fragment ions. Use of the 13C labeled
sugars 10–15 clearly identifies that the product ions each
contain the C1, C2, and C3 carbons of the starting sugar. Thus
fragment ions in the CID spectra of [M – H• + H+] are all shifted
by 1 Da for 10, 11, and 12, but not for 13, 14, and 15. The
deuterium labeled sugars 3–8 (note: 3 contains both 13C and
2H) provide fragments with m/z values consistent with the
structures shown in Scheme 3. Finally, more than one bond
must be cleaved to form fragment ions C–E and thus the
mechanisms for their formation are likely to be complicated.
Ignoring H-atom shifts, it seems likely that the initially formed
distonic radical cation A undergoes β cleavage [43] of either
the C1–O5 and/or the C3–C4 bond to give the ring opened ions
B1 and B2. Subsequent fragmentation reactions of B1 and B2
ultimately induce cleavage of the C3–C4 and C1–O5 bonds,
respectively.
•–
[41], whereas the resonance stabilized radical anion SO4
produces radicals at the C1, C2, C5, and C6 positions of α-D-
glucose [42]. With respects to (2), a previous study utilizing
DFT calculations has revealed that the C–H bond dissociation
energies (BDEs) of methyl α-D-glucopyranoside fall into a
narrow range, with the C1 position having the lowest BDE
[20]. While C1 hydrogen atom abstraction often occurs in
solution [27, 28, 34–37], other C–H bonds can also be abstract-
ed [39–42]. Abundant H atom abstraction from the C2 position
in our experiments are consistent with a previous study that
suggests the resultant C-based radical is stabilized by SOMO-
σ* interactions between the unpaired electron at C2 and the
eclipsing C1–OMe bond [42]. The observed regioselectivity
for HAT is likely further controlled by hydrogen bonding
within the noncovalent complex.
Origin of the m/z 102, 103, and 104 Fragment Ions
in the CID Spectrum of the Radical Cation of Methyl
D-Glucopyranoside
The use of the isotopically-labeled sugars allows not only the
site of H atom abstraction to be established but allows the
OH
OH
O
OH
O
+ H+
+ H+
+ H+
CID MS4
O
HO
HO
HO
HO
HO
HO
and
/or
OCH3
β
cleavage
OCH3
OCH3
HO
HO
B1
HO
A
B2
further fragmentation
C4H8O3 + H+
C4H7O3 + H+
C4H6O3 + H+
+ H+
+ H+
+ H+
O
OCH3
H
O
OCH3
OH OCH3
H
H
H
H
H
O
OH
OH
E
C
D
m/z
observed
m/z
observed
m/z
observed
labelling
ion
ion
ion
none
C1-D1
D2
D3
D4
C1
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
:
m/z 102
m/z 104
m/z 102
m/z 103
m/z 102
m/z 102
m/z 102
m/z 105
m/z 103
m/z 103
m/z 103
m/z 102
m/z 102
m/z 102
D1
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
:
:
:
:
:
:
:
:
m/z 103
m/z 105
m/z 103
m/z 104
m/z 103
m/z 103
m/z 103
m/z 106
m/z 104
m/z 104
m/z 104
m/z 103
m/z 103
m/z 103
E1
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
E14
E15
:
m/z 104
m/z 106
m/z 104
m/z 105
m/z 104
m/z 104
m/z 104
m/z 107
m/z 105
m/z 105
m/z 105
m/z 104
m/z 104
m/z 104
:
:
:
:
:
:
:
:
:
:
:
:
:
:
D5
D2-6
CD3
C1
C2
C3
C4
C5
C6
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
Scheme 3. Possible structures of key intermediates, A and B1, B2, and fragment ions, C–E, formed by CID of the radical cation;
m/z values of fragment ions are given for the sugar isotopologues used as precursors (see Scheme 2 for structures and
Supplementary Figures for MS4 spectra)

1430
S. Osburn et al.: Fragmentation of Sugar Radical Cations in the Gas Phase
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Conclusions
This study emphasizes the power of experimental studies uti-
lizing systematic isotope labeling to unravel fragmentation
reactions of natural products [44]. The use of a suite of sys-
tematically stable-isotope labeled methyl glucosides has
allowed the establishment of: (1) C2 as the site of the H atom
abstraction within the noncovalent radical cation complex,
[H3NCH2CH2S• + M]+, formed by S–NO bond homolysis
(Equation 1); and (2) the bonds that are cleaved to form the
fragment ions C, D, and E involve cross-ring cleavage of the
sugar to release a fragment ion derived from the sugar C1–C3
atoms. A unique feature of this system, which uses intra-cluster
proton and hydrogen transfer reactions facilitated by N-
nitrosocysteamine to generate the distonic sugar radical is the
highly regiospecific HAT from the sugar C2 position, and
results in a simple series of fragment ions through one major
fragmentation channel.
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Acknowledgements
R.A.J.O. thanks the Australian Research Council (ARC) for
financial support via the ARC Center of Excellence in Free
Radical Chemistry and Biotechnology (CE0561607). S.J.W. is
an ARC Future Fellow (FT130100103) and thanks the ARC
for financial support (DP160100597).
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