The effects of intramolecular hydrogen bonding on the reactivity of phenoxyl radicals in model systems

Article abstract of DOI:10.1016/j.ijms.2015.06.008

The effects of hydrogen bonding and spin density at the oxygen atom on the gas-phase reactivity of phenoxyl radicals were investigated experimentally and theoretically in model systems and the dipeptide LysTyr. Gas-phase ion-molecule reactions were carried out between radical cations of several aromatic nitrogen bases with the neutrals nitric oxide and n-propyl thiol. Reactivity of radical cations 4-6 correlated with the spin density. The possibility of hydrogen bonding was explored in compounds which allowed four-, five-, and six-membered rings to be formed between the protonated nitrogen and the phenoxyl oxygen, while possessing similar spin density at the oxygen atom. The N⁺-H?O? bond length was calculated to decrease in the series (1-3), consistent with the theoretical calculations finding weak hydrogen bonding in 2 and strong hydrogen bonding in 3. This coincided with the decrease in reaction rates of 1-3 with both nitric oxide and n-propyl thiol. DFT calculations found that the lowest energy structure of the distonic radical cation of the dipeptide [LysTyr(O)?]⁺ has a short hydrogen bond between the protonated Lys side chain and the phenoxyl oxygen, 1.70 ?, which is consistent with its low reactivity.

Full text of DOI:10.1016/j.ijms.2015.06.008

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International Journal of Mass Spectrometry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
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The effects of intramolecular hydrogen bonding on the reactivity
of phenoxyl radicals in model systems
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Michael Lesslie, Andrii Piatkivskyi, John Lawler, Travis Helgren, Sandra Osburn ,
Richard A.J. O’Hair , Victor Ryzhov
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Department of Chemistry and Biochemistry, and Center for Biochemical and Biophysical Sciences, Northern Illinois University, DeKalb, IL 60115, USA
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a r t i c l e i n f o
a b s t r a c t
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Article history:
The effects of hydrogen bonding and spin density at the oxygen atom on the gas-phase reactivity of phe-
noxyl radicals were investigated experimentally and theoretically in model systems and the dipeptide
LysTyr. Gas-phase ion-molecule reactions were carried out between radical cations of several aromatic
nitrogen bases with the neutrals nitric oxide and n-propyl thiol. Reactivity of radical cations 4–6 cor-
related with the spin density. The possibility of hydrogen bonding was explored in compounds which
allowed four-, five-, and six-membered ring to be formed between the protonated nitrogen and the
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Received 31 March 2015
Received in revised form 12 June 2015
Accepted 16 June 2015
Available online xxx
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Keywords:
Radical ions
Tyrosyl radicals
Ion-molecule reactions
Hydrogen bonding
Density functional theory calculations
+
•
phenoxyl oxygen, while possessing similar spin density at the oxygen atom. The N -H· · ·O bond length
was calculated to decrease in the series (1–3), consistent with the theoretical calculations finding weak
hydrogen bonding in 2 and strong hydrogen bonding in 3. This coincided with the decrease in reaction
rates of 1–3 with both nitric oxide and n-propyl thiol. DFT calculations found that the lowest energy
•
+
structure of the distonic radical cation of the dipeptide [LysTyr(O )] has a short hydrogen bond between
the protonated Lys side chain and the phenoxyl oxygen, 1.70 A˚ , which is consistent with its low reactivity.
©
2015 Published by Elsevier B.V.
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1. Introduction
spectroscopy, which revealed structural information about local
protein environments, it is widely accepted that tyrosyl radicals
are often stabilized by hydrogen bonding [14–23]. Hydrogen bond-
ing between a phenolic hydrogen and a properly oriented basic
group such as histidine residue (Scheme 1, B [19]) modulates the
formation and chemical behavior of the ensuing tyrosyl radical
by changing redox potential of the tyrosine/tyrosyl radical pair
[19,24–29].
There has been considerable interest in developing model sys-
tems to better understand the effects of hydrogen bonding on the
properties of tyrosyl radicals [17,30–33]. Most of these systems
incorporate both a phenol and a basic nitrogen atom in a close
proximity to facilitate the formation of hydrogen-bonded phenoxyl
radical, as shown for C in Scheme 1 [31]. Varying the substituents
in the phenyl ring and the chemical surroundings of the nitro-
gen was shown to affect both redox potentials and the EPR signals
[17,27,28,34,35].
Despite the success of solution-based approaches, there are sev-
eral advantages of using mass spectrometry-based approaches to
examine the fundamental gas-phase chemistry of relevant distonic
ion model systems [36]. Apart from significantly reducing the time
and sample quantity required for these studies, they shut down the
possibility of radical self-termination reactions due to the coulom-
bic repulsion of the charge sites. In addition, they reduce the overall
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Free radicals play key roles in protein chemistry due in part
to their ability to transform substrates within the active sites of
enzymes [1,2]. Radical sites in proteins are normally found on reac-
tive amino acid side chains, of which, tyrosine phenoxyl radicals
are among the most prominent (Scheme 1, A). These tyrosyl rad-
icals are thought to be important intermediates in the action of
the enzyme Class I Ribonucleotide Reductase (RNR) of Escherichia
coli [3–5], production of oxygen in photosystem II [6–8], and the
oxidation of peroxides in cytochrome C oxidases [9]. They have
also been implicated in radical-induced protein damage, and are
the precursors of various post-translational modifications (3,3 -
dityrosine, 3-nitrotyrosine, and tyrosine-cytosine cross-linking)
[10–13].
With the abundance of recent experimental data obtained
via X-ray crystallography, high-frequency EPR, and ENDOR
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∗ Corresponding author. Tel.: +1 815 753 6955.
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E-mail address: ryzhov@niu.edu (V. Ryzhov).
ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, School
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of Chemistry and Bio21 Institute of Molecular Science and Biotechnology, The Uni-
versity of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia.
http://dx.doi.org/10.1016/j.ijms.2015.06.008
1
387-3806/© 2015 Published by Elsevier B.V.
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formation of Tyr-based radical cations. This method has been suc-
82
cessful in generating radical cations of iodotyrosine-containing
peptides through photo-irradiation by Julian and co-workers
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[
44,45]. They postulated that the initial phenyl carbon radical
85
can quickly rearrange into the oxygen-based phenoxyl radical
species [45,46]. However, because the tyrosyl radical easily loses
its side chain in the gas phase, resulting in the captodatively sta-
bilized glycine radical, forming and studying the oxygen-based
radical cation of tyrosine is a challenge [47–49]. Siu and co-
workers were able to form small amounts of the tyrosyl radical
through collision-induced dissociation (CID) of [Cu(Tyr)2]2+ com-
plex [47]. However, these ions dissociated rapidly to yield the
p-hydroxybenzyl and p-cresol radical cations, which indicated the
dissociation of the ␣–␤ bond and loss of the side chain [47]. Simi-
larly, radical generation at tyrosine residues in peptides is known to
result in characteristic side chain losses under mild CID conditions
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Scheme 1.
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[
48,50].
In this study, we circumvent this problem by using model nitro-
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gen bases 1–3 (Scheme 3) that possess a phenoxyl radical site but
lack facile elimination channels as seen with the loss of the Tyr side
chain. Choosing the position of the nitrogen atom in the molecule
allows us to explore the possibility and vary the extent of hydrogen
bonding in the resulting radical cation. The effects of spin den-
sity at the oxygen atom on these reactions are also investigated
using compounds 4–6. We utilize gas-phase ion-molecule reac-
tions (IMRs) to probe the reactivity of these radical species and
density functional theory (DFT) calculations to complement the
experimental data. We also examine the chemistry of the radical
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Scheme 2.
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complexity of the system by limiting the chemistry of intramolec-
ular interactions and avoiding complications from intermolecular
hydrogen bonding with solvent molecules. Notwithstanding the
recent renaissance of mass spectrometry-based studies of amino
acid and peptide radical ions, the effects of hydrogen bonding on
radical reactivity have not been widely studied. A rare example
ascribed the differences between the gas-phase reactivity of dis-
tonic radical cations of cysteine and homocysteine to hydrogen
bonding effects arising from the difference in the distances between
the N-terminal hydrogen atom and the sulfur radical (Scheme 2)
[37].
Mass spectrometry has been used to study gas-phase chemistry
of tyrosyl radicals [38–40]. Siu and co-workers initially demon-
strated the ability to form radical cations of peptides using the
ternary copper (II) complex dissociation method in peptides con-
taining tyrosine and an assisting basic amino acid [39]. This method
was later utilized to form radicals and study a wide variety of
tyrosine-containing peptides [40–43]. Covalent chemical mod-
ification of the tyrosine side chain and subsequent homolytic
cleavage of a labile bond has been another productive route to
•
+
cation of the dipeptide, [LysTyr(O )] .
2. Experimental
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2.1. Materials
All chemicals and reagents were used as received with-
out any further purification. All model compounds, or their
precursors, including 2-hydroxypyridine, 8-hydroxyquinoline,
4-methoxypyridine, 2-methoxypyridine, 6-methoxyquinoline, sal-
icylaldehyde, and propylamine, were purchased from Sigma-
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Aldrich (Milwaukee, WI). The remaining reagents, CuSO , 2,2 :6 ,2 -
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terpyridine, n-propyl thiol, potassium carbonate, dimethylformide
(DMF), dimethylsulfate, diethylether, and sodium sulfate, were
Scheme 3.
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purchased from Sigma-Aldrich (Milwaukee, WI). The dipeptide
LysTyr was synthesized by, and purchased from Selleckchem
Stock solutions of the synthesized or purchased model com-
pound precursors were prepared as 1 mM in methanol. The
methoxy derivatives of species 1, 2, 4–6 were diluted to 10 ␮M in
methanol with 1% acetic acid. Compound 3 was mixed at a 1:1 ratio
with a solution of 1 mM CuSO4 and 1 mM 2,2 :6 ,2 -terpyridine in
1:1 water/methanol, allowed to react for 30 min, and diluted to a
final concentration of 10 ␮M in methanol.
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2Q3 4 (Houston, TX). Premixed gas containing 1% nitric oxide in laser
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grade helium and pure nitric oxide were purchased from AirGas
(Chicago, IL). Methanol was purchased from Fisher (Pittsburgh, PA).
Water was purified (18 Mꢀ) in-house.
ꢀ
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All samples were subsequently introduced into the electrospray
ionization (ESI) source of the mass spectrometer at a flow rate
1
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2.2. Synthesis
−
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of 5 ␮L min . The nebulizer gas, needle voltage, and temperature
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2-Propyliminomethyl-phenol. This compound was synthesized
through a standard Schiff base procedure [51–53]. Ethanol (5 mL)
was added to a reaction vessel. Salicylaldehyde (1 mmol) and
propylamine (1 mmol) were then added directly to the solvent.
The mixture was allowed to stir for 30 min, during which time a
precipitate formed. The title compound was isolated via filtration
as a solid yellow powder. The sample was used without purifica-
tion.
◦
were adjusted to approximately 12 PSI, 4.0 kV, and 250 C. All other
tunable instrument parameters were optimized to give maximum
yield of the desired ions.
The formation of the radical ions of interest was achieved
through successive isolation of the parent ion, CID, and a second
isolation of the radical cation. Under these conditions, methoxy
compounds underwent facile methyl radical loss to yield the rad-
ical cation of interest. The radical cation of compound 3 was
formed using the copper complex dissociation method, as pre-
viously described [38,39]. The isolation window used in these
experiments ranged from 0.7 to 2.0 m/z. The exact value was deter-
mined during the experiment as the width that resulted in the
maximum abundance of the desired ion while excluding all other
ions. The CID amplitude ranged from 0.40 to 0.75 V and was exper-
imentally set to the value that yielded the greatest intensity of the
desired fragment ion.
The methoxy compounds were synthesized in a similar manner
as previously described [54,55].
2-Methoxypyridine. 2-Hydroxypyridine (2 mmol) and sodium
hydride (4 mmol) were sealed in a reaction vessel. The mixture was
purged with nitrogen and DMF (5 mL) was added. The resulting
◦
mixture was heated to 50 C for 30 min. The reaction mixture was
then allowed to cool to room temperature. Finally, dimethylsulfate
(4 mmol) was added dropwise to the reaction mixture. The result-
ing solution was allowed to stir at room temperature overnight.
The reaction mixture was then poured over water (20 mL) and the
aqueous mixture was extracted with diethylether (2× 20 mL). The
combined organic extract was rinsed with brine (50 mL) and dried
over Na SO . Title compound was isolated as a colorless oil upon
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2.4. Ion-molecule reactions
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The isolated radical cations were subjected to IMRs with the
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solvent removal under reduced pressure. The sample was used
without further purification.
neutral species nitric oxide and n-propyl thiol. Reactions were
achieved by introducing nitric oxide into the trap via the helium
line from a 1% nitric oxide in helium premixed cylinder. The internal
helium regulator was unmodified from normal usage. The n-propyl
thiol was introduced to the trap through a leak valve, as previ-
ously described [37]. In both cases, a scan delay was employed
and determined the reaction time. The exponential decay of the
reactant radical cation was found by monitoring the relative ion
intensity over a series of delay times (0–5000 ms). Each time point
was acquired for a minimum of 1 min. The relative rate constants
were determined in comparison to that of the 4-hydroxypyridine
radical cation, which was run each day that an experiment was
performed.
Verification of relative reactivity for slower reacting radical
cations was achieved by reacting the isolated radical with nitric
oxide introduced through a leak valve, which was able to supply a
higher partial pressure of the gas. For these experiments, the reac-
tion time was determined based on a scan delay of 1000 ms before
the final product ion scan.
5-Methoxyquinoline. DMF (2 mL) and dimethylsulfate
(0.6 mmol) were added to a mixture of 5-hydroxyquinoline
(0.3 mmol) and potassium carbonate (0.7 mmol). The resulting
suspension was allowed to stir at room temperature overnight.
The mixture was then poured over water (10 mL) and the aqueous
mixture as extracted with diethylether (2× 10 mL). The combined
organic extract was rinsed with brine (30 mL) and dried over
Na SO . The solvent was removed under reduced pressure to
2
4
afford the title compound as a deep purple solid. The sample was
used without further purification.
8-Methoxyquinoline. DMF (2 mL) and dimethylsulfate
(0.6 mmol) were added to a mixture of 8-hydroxyquinoline
(0.3 mmol) and potassium carbonate (0.7 mmol). The resulting
suspension was allowed to stir at room temperature overnight in a
sealed vessel. The mixture was then poured over water (10 mL) and
the aqueous mixture as extracted with diethylether (2× 10 mL).
The combined organic extract was rinsed with brine (30 mL)
and dried over Na SO . The solvent was removed under reduced
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pressure to afford the title compound as an off-white solid. The
sample was used without further purification.
2.5. Density functional theory calculations
229
Caution! Dimethyl sulfate is a confirmed carcinogen, muta-
gen and is readily absorbed through the skin or respiratory
tract. Additionally, the compound is extremely flammable. Users
should be trained in the handling of air sensitive reagents
and should familiarize themselves with the corresponding MSD
sheet.
Optimization of the geometries for each of the structures was
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done using the Gaussian 09 suite of programs [57]. Initial opti-
mizations were performed using the hybrid B3LYP functional and
the 3-21G* basis set. All minima located at this level of theory
were then re-optimized using the same functional and the 6-
3
11++G(d,p) basis set. Zero-point corrected energies and Natural
Bond Order (NBO) were computed at the same level of the-
ory.
Critical point and bond path calculations were performed using
B3LYP/6-311++G(d,p) wavefunctions and the AIMAll program [58],
which implements the Quantum Theory of Atoms In Molecules
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2.3. Mass spectrometry
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Mass spectrometry experiments were carried out using a Bruker
Esquire 3000 quadrupole ion trap mass spectrometer (Bruker
Daltonics, Bremen, Germany) modified to conduct ion-molecule
reactions as described previously [56].
(QTAIM) theory developed by Bader and co-workers [59–61].
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3. Results and discussion
3.1. Formation of radical cations
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The radical cations of species 1, 2, and 4–6 were generated via
homolytic cleavage of the methyl group (loss of 15 Da) from the
appropriate methylated precursor ion (i.e., Eq. (1) and Fig. 1a).
CH
3
O
O
CID
+
CH3
N
H
N
H
2
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(1)
2
2
2
2
2
2
2
2
2
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This method was chosen as it specifies the initial location of the
radical on the oxygen atom, resulting in exclusively structures 1,
2, and 4–6. The high yield of the radical cation and the minimal
amount of competing fragmentation channels allowed for suffi-
cient ion production to carry out IMRs and determine relative rate
constants.
The radical cation of 2-propyliminomethyl-phenol (3) was
produced through the dissociation of the copper ternary metal
complex (Eq. (2)) [38,39].
[Cu (terpy)(M)] → [Cu (terpy)]+ + [M]•+
II
2+
I
(2)
2
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2
2
2
2
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Using copper complex dissociation, the initial radical location is
often debated. In solution, these types of compounds have been
shown to coordinate Cu(II) through the oxygen atom [31]. In addi-
tion, a compound similar to the radical cation of model 3 was
previously produced in solution electrochemically (Scheme 1, C;
R , R = H, R = C H7), and was assigned the oxygen-based radi-
cal structure [31]. To supplement these findings, DFT calculations
were performed to search for alternative structures of the 2-
propyliminomethyl-phenol radical cation (3). They all consistently
converged on a structure with the nitrogen protonated and the
radical located on the phenoxyl oxygen.
1
2
3
3
2
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3.2. DFT analysis of model compounds
2
2
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The radical cations, 2-hydroxypyridine (1), 8-hydroxyquinoline
(2), and 2-propyliminomethylphenol (3), have 4-, 5-, and
+
6-membered ring structures, respectively, containing the N -
•
H· · ·O motif. To further compare the compounds, DFT calculations
+
•
were used to determine the N -H· · ·O bond length and elec-
tron spin density in the lowest energy structures (Fig. 2).
+
•
As expected, N -H· · ·O bond lengths decreased as the num-
ber of ring members increased, ranging from ∼2.5 A˚ for
2-hydroxypyridine (1), to ∼2.3 A˚ for 8-hydroxyquinoline (2), and
∼1.9 A˚ for 2-propyliminomethylphenol (3). The electron spin den-
sity, which can be used as a measure of radical delocalization,
was found to be 0.37 for 2-hydroxypyridine (1), 0.36 for 8-
hydroxyquinoline (2), and 0.34 for 2-propyliminomethyl-phenol
(3). The similarity of electron spin density in the three compounds
(1–3) narrows the comparison of their reactivity to the effect of the
potential hydrogen bond length and angle.
Natural Bond Order analysis and Quantum Theory Atom-In-
Molecules calculations were also performed on these systems.
QTAIM calculations find regions of inflection of electron density,
and characterize the regions as “critical points” based on the effect
of the inflection. Metaphorically, a bond critical point is a transi-
tion point between two nuclear attractors, just as a conventional
transition state is a stationary point between reactants and prod-
ucts. The bond path can be thought of as the path of maximum
electron density between atomic nuclei, with a bond critical point
+
Fig. 1. (a) CID of the protonated 4-methoxypyridine ([M+H] ) at m/z 110. The result-
ing ion at m/z 95 is the radical cation from homolytic cleavage of the RO–CH3
bond, corresponding to the 4-hydroxypyridine radical cation (4). No attempt was
made to identify other fragmentation products in this spectrum. (b) IMR of the 4-
•
+
hydroxypyridine radical cation (4), [M] , with nitric oxide. This reaction produced
+
the radical combination product, [M+NO] , at m/z 125. Reaction time 75 ms. (c) IMR
of the 4-hydroxypyridine radical cation (4), [M]•+, with n-propyl thiol. This reaction
+
results in HAT, yielding [M+H] at m/z 96. Reaction time 250 ms.
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Fig. 2. DFT-calculated (B3LYP 6-311++G(d,p)) lowest energy structures of model compounds: 2-hydroxyquinoline (1), 8-hydroxyquinoline (2), 2-propyliminomehtylphenol
3), 4-hydroxyquinoline (4), 5-hydroxyquinoline (5) and 6-hydroxyquinoline (6). Bond lengths are in Å and spin densities are indicated in italics.
(
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
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as the position where an entity traveling that path away from one
nucleus finds equilibrium between that nucleus and another. In the
cases of the molecules examined here, bond paths deviated from
geometric paths (the conventional ones thought of as bonds, used
to define molecular geometries), by less than 0.002 A˚ .
with a spin density at the oxygen atom of 0.61, was found to be
the most reactive towards NO of all the compounds tested. There-
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•
fore, all other rates are reported as percent relative to the reactivity
of 4. Radical cation 6 (spin density of 0.43) displayed a rate of 7%,
and species 5 (spin density of 0.34) was even less reactive at 2%.
Since reactivity of the radical cations correlates with the spin den-
sity at the oxygen atom, a subset of model compounds (1–3) with
approximately the same spin density (∼0.35) were used to inves-
NBO calculations did not find stabilization for compound 1
−1
above a threshold of 1 kJ mol and QTAIM did not find a critical
point for a hydrogen bond. For compound 2, NBO calculations indi-
−
1
+
•
cated that this radical cation was stabilized by 2.4 kJ mol over a
molecule of identical structure but without a hydrogen bond. How-
ever, no critical point was found in the QTAIM calculation. Finally,
for compound 3, NBO calculations found a strong hydrogen bond
tigate the effects of intramolecular N -H· · ·O distance on oxygen
radical reactivity.
Previous studies in solution by Ingold and co-workers have eval-
uated the reactivity of phenols with various reactive radical species
and found that the degree of intermolecular hydrogen bonding
of the phenol to the solvent generally decreases the reaction
rate [64–66]. All of these studies investigated radicals react-
ing with hydrogen-bonded compounds. For the instances where
intramolecular hydrogen bonding within the radical-containing
species affects its reactivity, there are very little data. Our previous
study explained the differences between the gas-phase reactivity
of distonic radical cations of cysteine and homocysteine by the dif-
ference in the distances between the N-terminal hydrogen atom
and the sulfur radical (Scheme 2) [37]. Sulfur, however, is not as
efficient at forming strong hydrogen bonds as F, O, and N.
Model compounds 1–3 were reacted with nitric oxide (Eq. (3)).
−
1
providing a stabilization of 46 kJ mol . The QTAIM calculations for
this compound show a clear critical point of the correct curvature.
The electron spin density on the oxygen atom was also calcu-
lated for radical cations 4–6 (Scheme 3). They were found to be 0.61
for 4, 0.34 for 5, and 0.43 for 6.
3
12
3.3. Gas-phase reactivity of model compounds
3
3
3
13
14
15
To evaluate reactivity, model radical cations were reacted with
nitric oxide yielding radical combination, which results in an addi-
tion of 30 Da (Eq. (3) and Fig. 1b):
•
+
•
+ NO → [M + NO]+
3
16
M
(3)
+
•
These data were compared with the calculated N -H· · ·O distance
(
Table 1). The rate constants are listed relative to reactivity of 4-
3
3
3
3
3
3
17
18
19
20
21
22
This was the only reaction product observed in all cases. Reactiv-
ity of radicals in general, and phenoxyl radicals in particular, is
known to be dependent on spin density at the reactive site [62,63].
To test how spin densities affect gas-phase reactivity, compounds
4–6, where spin densities at the oxygen atom were calculated to
vary substantially, were reacted with nitric oxide. Compound 4,
hydroxypyridine (4). As shown in the table, species 1 and 2 are
much less reactive towards NO than 4 (5% and 0.7%, respectively).
For the slowest reacting compound, 2-propyliminomethyl-
phenol (3), the partial pressure of the stock 1% nitric oxide in helium
was not sufficient to bring the reaction rate into the quantifiable
Table 1
Kinetics and thermodynamics for the reactions of model compounds 1–4 with nitric oxide and n-propyl thiol. Rate constants are displayed as percents relative to the rate of
the 4-hydroxypyridine radical cation (4) with each neutral.
Radical cation species
H-bond length (Å)a
Nitric oxide ꢁH of
Nitric oxide rate constant
relative to 1 (%)
n-Propyl thiol rate constant
relative to 1 (%)
reaction (kJ mol )a
−
1
1
2
3
4
(2-Hydroxypyridine)
2.45
2.26
1.87
–
−102.7
−64.4
5
0.7
<0.3
100
90
9
(8-Hydroxyquinoline)
(2-Propyliminomethyl-phenol)
(4-Hydroxypyridine)
b
<3b
100
−61.4
−163.5
a
Calculated at the B3LYP 6-311++G(d,p) level of theory.
Reactivity observed, but product yield was below quantifiable range.
b
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M. Lesslie et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
6
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
54
55
56
57
58
59
60
61
62
63
64
65
66
67
range. To circumvent this issue, the partial pressure in the trap
was increased with additional nitric oxide via a leak valve [37].
This allowed for the determination of the upper limit of the rate
constant, <0.3% relative to compound 4.
As expected, there was an evident decrease in reactivity with
+
•
decreasing N -H· · ·O distance. In the case of the 2-hydroxypyridine
radical cation (1) this distance was calculated to be 2.45 A˚ and
the geometry of the ion is unfavorable because N H and C
O
bonds point away from one another. This is consistent with both
NBO and QTAIM calculations that found no hydrogen bond in 1.
+
•
The 8-hydroxyquinoline radical cation (2) has a shorter N -H· · ·O
distance of 2.26 A˚ with a 5-membered ring structure resulting
in more favorable hydrogen bonding angle. NBO analysis found
minor stabilization of 2.4 kJ mol for this interaction, which sug-
6Q8 5 gests the possibility of a weak hydrogen bond. This correlates
−1
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
with a factor of seven decrease in rate constants going from 1 to
2. The slowest reacting compound, 2-propyliminomethyl-phenol
(3), had the shortest hydrogen bond of 1.87 A˚ which is part of a
six-membered ring enabling almost optimal orbital interaction for
hydrogen bonding, confirmed by both NBO and QTAIM. The corre-
sponding decrease in reaction rate constants from 2 to 3 is at least
a factor of two.
•
+
Fig. 3. Structure of the [LysTyr(O )] radical cation in the gas phase calculated at the
B3LYP 6-311++G(d,p) level of theory. The structure represents the lowest energy
conformer with a hydrogen bond from the Lys side chain amine to the phenoxyl
radical of 1.7 A˚ . The spin density at the oxygen atom is indicated in italics.
The nearly constant spin density in compounds 1–3 (∼0.35) and
3.4. Hydrogen bonding of phenoxyl radicals in peptides
415
the lack of steric hindrance at the oxygen atom (Fig. 2) suggest
+
•
that the N -H· · ·O may affect the radical reactivity in the subset
1–3. These results mirror our previous findings involving Cys and
Hcy radical cations. In both amino acids the spin density at the
sulfur atom was calculated to be nearly 1.0. However, the decreased
The degree of hydrogen bonding to the phenoxyl radical of tyro-
sine oxidation sites in proteins varies greatly across enzymes [68].
Even within RNRs, the mouse variety possesses a hydrogen bond
to the radical, while the E. coli version does not [69]. The redox-
active phenol in E. coli class I RNR does possess a hydrogen bond
to a neighboring aspartate residue, but upon oxidation the hydro-
gen bond is broken [69]. It has been proposed that state-specific
changes in the hydrogen bonding to redox-active tyrosines, like
those in E. coli, may be an example of broader kinetic modulation
of radical chemistry [70]. Therefore, the effect of hydrogen bonding
on the reactivity of phenoxyl radicals is important to understanding
oxidation mechanisms in enzyme catalysis.
While efforts to form the radical cation of the amino acid tyro-
sine through dissociation of copper ternary complexes has not often
been successful in generating suitable yields of the radical [47],
the tyrosine-based phenoxyl radical in peptides with neighboring
basic amino acid residues can be formed [39,40]. To compare the
results of our model compounds, the radical cation of the dipeptide
LysTyr was generated. This type of radical was previously assigned
the structure with protonated Lys side chain and the Tyr phe-
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
•
+
length of the S · · ·H-N hydrogen bond in Hcy (Scheme 2) resulted
in a greater than a two-fold decrease in radical reactivity.
In addition, we computationally explored the thermodynam-
•
ics of the radical ion recombination reaction with NO (Eq. (3)).
The calculated enthalpies of this reaction for the radical cation
species 1–4 are given in Table 1. The corresponding reaction
free energies as well as the product geometries are shown in
Figure S1. The recombination reaction for the “control” radical
−
1
cation 4 was calculated to be exothermic by 164 kJ mol . For
the 2-hydroxypyridine radical cation (1), ꢁH was found to be
−
1
−103 kJ mol , while for the 8-hydroxyquinoline radical cation (2),
−
1
the corresponding enthalpy change was −64 kJ mol . The radical
cation of 2-propyliminomethyl-phenol (3) had the lowest enthalpy
change at −61 kJ mol . A typical O NO bond dissociation energy
−1
−1
(BDE) is in the range of 45–75 kJ mol in substituted phenols [67],
where electron-withdrawing substituents stabilize the radical less
resulting in higher BDE values. In compound 4 the protonated
imino substituent is highly electron-withdrawing. However, the
BDE value falls in the middle of the typical O-NO range, possi-
bly due to extra stabilization of the phenoxyl radical by the short
hydrogen bond. These calculations suggest that both the exother-
•
noxyl radical [39,40]. It is well known that Tyr(O ) radical cations
undergo facile cleavage of the C␣–C␤ bond and loss of the side chain
during CID [46,50]. Approximately 5% of this loss is seen in the
CID of the LysTyr radical cation (Figure S2). The presence of Tyr
side-chain cleavage indicates the existence of the oxygen-based
radical isomer. However, the low yield of this fragment suggests
that either hydrogen bonding is stabilizing the phenoxyl radical or
other isomers of the radical cation of LysTyr are formed. To address
+
•
micity of reaction (3) and the N -H· · ·O interaction correlate with
•
the gas-phase reactivity towards NO in the series 1–4.
To further investigate the reactivity trends, the radical cations
1–4 were also subjected to reactions with n-propyl thiol. Such reac-
tions proceeded to give hydrogen atom transfer (HAT) (Eq. (4) and
Fig. 1c).
•
+
these questions, studies involving ion spectroscopy of [LysTyr]
are underway.
The DFT analysis of gas-phase structures of [LysTyr(O )]+
•
revealed that the lowest-energy structure has a hydrogen bond
(
1.70 A˚ ) from the Lys side-chain amine to the phenoxyl oxygen, in
M +
addition to another hydrogen bond (1.65 A˚ ) between the Lys side-
+
HS
M+H]+
[
+
S
4
09
(4)
chain amine and the amide oxygen (Fig. 3). Structures that lack one,
or both, of these hydrogen bonds are all higher in energy (Figure
S3).
In an effort to compare the dipeptide radical cation to the
model compounds, IMRs with both nitric oxide and n-propyl
thiol were attempted. Under the same conditions used to react
the model compounds, only minor unidentifiable products were
formed. The calculated hydrogen bond length in the lowest-energy
4
4
4
4
4
10
11
12
13
14
The neutral n-propyl thiol was introduced through a leak valve, and
rate constants were determined relative to the reactivity of com-
pound 4 (Table 1). The decrease in reactivity of n-propyl thiol with
+
•
compounds 1 > 2 > 3 correlates with the relative N -H· · ·O length
and geometry.
Please cite this article in press as: M. Lesslie, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.06.008

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M. Lesslie et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
7
structure of [LysTyr(O )]+ is 1.70 A˚ , which is shorter than 1.87 A˚ in
•
4
4
4
58
59
60
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531
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539
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541
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4. Conclusions
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
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The gas-phase reactivity of radical cations containing a pro-
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•
[
motif and the rate of ion-molecule reactions of these radical species
with nitric oxide and n-propyl thiol. The reactivity decreases
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gen bonding was found by theoretical calculations, to the 5- and
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•
the N -H· · ·O distance decreases.
The precise roles that hydrogen bonding exerts on bimolecular
reactivity (i.e., steric hindrance, distortion of the SOMO orbital, etc.)
will be further explored by using DFT calculations to fully exam-
ine the potential energy diagrams associated with small model
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vent–substrate hydrogen bonding on radical reactivity and the
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the radical microenvironment.
One of the main advantages of gas-phase studies is the ability
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This work was supported by the Department of Chemistry and
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[
[
[
00
5
01
Appendix A. Supplementary data
5
5
02
03
Supplementary data associated with this article can be found, in
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Please cite this article in press as: M. Lesslie, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.06.008