Uracil-5-yl O-Sulfamate: An Illusive Radiosensitizer. Pitfalls in Modeling the Radiosensitizing Derivatives of Nucleobases

Article abstract of DOI:10.1021/acs.jpcb.0c03844

Efficient radiotherapy requires the concomitant use of ionizing radiation (IR) and a radiosensitizer. In the present work uracil-5-yl O-sulfamate (SU) is tested against its radiosensitizing potential. The compound possesses appropriate dissociative electron attachment (DEA) characteristics calculated at the M06-2X/6-31++G(d,p) level. Crossed electron-molecular beam experiments in the gas phase demonstrate that SU undergoes efficient DEA processes, and the single C-O or S-O bond dissociations account for the majority of fragments induced by electron attachment. Most DEAs proceed already for electrons with kinetic energies of ～0 eV, which is supported by the exothermic thresholds calculated at the M06-2X/aug-cc-pVTZ level. However, in water solution under reductive conditions and physiological pH, SU does not undergo radiolysis, which demonstrates the crucial influence of aqueous environment on the radiosensitizing properties of modified nucleosides.

Full text of DOI:10.1021/acs.jpcb.0c03844

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules
Uracil-5-yl O-Sulfamate – an Illusive Radiosensitizer. On the Pitfalls
in Modeling the Radiosensitizing Derivatives of Nucleobases
Paulina Spisz, Magdalena Zdrowowicz, Witold Kozak, Lidia Chomicz-Ma#ka, Karina
Falkiewicz, Samanta Makurat, Artur Sikorski, Dariusz Wyrzykowski, Janusz Rak, Eugene
Arthur-Baidoo, Patrick Ziegler, Mateus Salomão Rodrigues Costa, and Stephan Paul Denifl
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.0c03844 • Publication Date (Web): 15 Jun 2020
Downloaded from pubs.acs.org on June 16, 2020
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Uracil-5-yl O-Sulfamate – an Illusive
Radiosensitizer. On the Pitfalls in Modeling the
Radiosensitizing Derivatives of Nucleobases
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Paulina Spisz,1 Magdalena Zdrowowicz,1 Witold Kozak,1 Lidia Chomicz-Mańka,1 Karina
Falkiewicz,1 Samanta Makurat,1 Artur Sikorski,2 Dariusz Wyrzykowski,3 Janusz Rak,1* Eugene
Arthur-Baidoo4, Patrick Ziegler,4 Mateus Salomao Rodrigues Costa,4 and Stephan Denifl4*
1
Group of Biological Sensitizers, Physical Chemistry Department, Faculty of Chemistry,
University of Gdańsk, 80-308 Gdańsk, Poland
2
Group of Crystallochemistry, Physical Chemistry Department, Faculty of Chemistry,
University of Gdańsk, 80-308 Gdańsk, Poland
3
Group of Physicochemistry and Complex Compounds, General and Inorganic Chemistry
Department, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
4
Institut für Ionenphysik und Angewandte Physik and Center for Biomolecular Sciences
Innsbruck, Leopold-Franzens Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck,
Austria
*To whom correspondence should be addressed.
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ABSTRACT
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Efficient radiotherapy requires the concomitant use of ionizing radiation (IR) and a
radiosensitizer. In the present work uracil-5-yl O-sulfamate (SU) is tested against its
radiosensitizing potential. The compound possesses appropriate dissociative electron
attachment (DEA) characteristics calculated at the M06-2X/6-31++G(d,p) level. Crossed
electron-molecule beam experiments in the gas phase demonstrate that SU undergoes efficient
DEA processes and the single C–O or S–O bond dissociations account for the majority of
fragments induced by electron attachment. Most DEAs proceed already for electrons with
kinetic energies of ~0 eV, which is supported by the exothermic thresholds calculated at the
M06-2X/aug-cc-pVTZ level. However, in water solution under reductive conditions and
physiological pH SU does not undergo radiolysis which demonstrates crucial influence of
aqueous environment on the radiosensitizing properties of modified nucleosides.
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1. INTRODUCTION
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Radiotherapy is one of the most common modalities in anti-cancer treatment. Indeed,
around eighty percent of cancerous patients are exposed to ionizing radiation (IR) at certain
stages of their therapy.1 This modality usually employs sparsely ionizing radiation, i.e. X-ray
photons or high energy electrons delivered by linear accelerators (Linacs).2 Still much less
common, although already available clinically, are heavy-particle or proton beam therapies.
Although such modalities seem to be less affected by tumor hypoxia, large cost related to the
construction and usage of heavy particle facilities makes a serious limitation to their widespread
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usage.2
X-rays and beams of electrons produced by Linacs generate hydroxyl radicals and
secondary electrons (water radiolysis) when passing through water, which constitutes c. 70%
of human body.2 As indicated by recent studies carried out by the Mostafavi group ultrashort-
lived prehydrated3 or conduction band4 electrons may partially account for DNA damage
induced by secondary electrons in cellular environments.
However, an efficient radiotherapy should be associated with the concomitant use of IR
and a radiosensitizing agent. Several classes of small-molecule radiosensitizers have been
proposed and tested in the clinic so far.5 Nevertheless, as suggested by on-going clinical trials,
the number of tested radiosensitizers is relatively low.6 As far as the clinically allowed
radiosensitizers are concerned, the situation is even worse. For instance, no chemicals working
as radiosensitizers are used in clinical practice against gastrointestinal cancers7 and nimorazole
– 4-[2-(5-nitroimidazol-1-yl)ethyl]morpholine – is a rare example of approved radiosensitizing
molecule for the treatment of head and neck cancers in Denmark.8
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Uridine analogs which incorporate to DNA belong to a class of radiosensitizers
comprising the most thoroughly studied 5-bromo- and 5-iodo-2’-deoxyuyridine.9 It is believed
that radiosensitizing properties of these molecules, while constituting a part of DNA, are related
to the electron-attachment induced dissociation of the C5–X bond that releases a halide anion
leaves behind a reactive uracil-5-yl radical in the biopolymer molecule.10 Secondary reactions
beginning with hydrogen atom transfer between the uracil-5-yl radical and an adjacent sugar
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molecule lead ultimately to a single strand break which may result in cell death if not repaired.9
It is worth emphasizing that in this mode of action the radiosensitizing uridines utilize solvated
electrons, which are one of most abundant products of water radiolysis.11 It has long been
demonstrated that solvated electrons bind to nucleobases, nucleotides and DNA almost at
diffusion controlled rate.12 However, no strand breaks are produced as a result of electron
attachment to the native DNA, which was proved experimentally13 and justified theoretically.14
Only specific chemical modifications to the DNA monomeric units make them prone to
dissociative electron attachment (DEA).15 The mechanism that utilizes electrons unreactive
towards native DNA and is operative under hypoxia when the damaging properties of hydroxyl
radical are significantly impaired, prompted us to propose several new uridine radiosensitizers.
To this end, 5-selenocyanato-2’-deoxyuridine (SeCNdU),16 5-trifluoromethanesulfonyl-2’-
deoxyuridine (OTfdU),16 5-iodo-4-thio-2’-deoxyuridine (ISdU)17 or 5-thiocyanato-2’-
deoxyuridine (SCNdU)18 can be mentioned as representative examples. In the heart of our
approach lies the quantum chemically calculated DEA profile obtained for a verified
nucleoside/nucleobase. Consequently, we seek for derivatives for which releasing of an anion
(a leaving group) triggered by electron attachment is associated with a sufficiently large
thermodynamic stimulus, that makes the whole damage process spontaneous, and with a tiny
activation barrier preventing the protonation of the formed anion in an aqueous solution. The
latter process, fast enough to be competitive with DEA, is probably responsible for the lack of
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DNA strand breaks in the IR irradiated aqueous solution, as opposed to a plasmid DNA
bombarded with low energy electrons (LEEs) under ultra-high vacuum,19 where strand breaks
are induced by electrons with energies well below the ionization threshold of the biomolecule.
The body of data concerning the radiosensitizing nucleobases/nucleosides suggests that an
effective radiosensitizer has to be decomposed efficiently due to electron attachment.15,20-24
The dissociation channels that can be opened by the attachment of LEEs to a molecule in the
gas phase account for the damaging potential that inhabits electron-molecule interactions. At
least some of the channels, observed in the gas phase, are expected to be also operative in an
aqueous solution. Hence, the analysis of ion yields induced by electron attachment in the gas
phase should help one to interpret and comprehend the reactivity triggered by hydrated
electrons in solution. The decomposition of a potential radiosensitizer by low energy electrons
(LEEs) can be studied in the gas phase using a crossed electron-molecule beam technique,
which utilizes mass spectrometry for the analysis of fragment anions formed as a result of
DEA.25,26
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In the current work, we report for the first time on the physicochemical characteristics
of uracil-5-yl O-sulfamate (SU, NH2SO3C4H4N2O2; molecular mass 207 g ‧ mol-1) – a potential
radiosensitizer. Since favorable DEA characteristics have been calculated for this compound at
the DFT level, it has been chemically synthesized and its molecular structure has been
confirmed crystallographically. Moreover, its propensity to electron-induced decomposition
has been determined in both the gas phase and water solution. The measured energy onsets of
LEE triggered dissociation in the gas phase have been supported by the quantum-chemical
calculations of the thermodynamic thresholds for the occurrence of the observed anions. On the
other hand, the outcome of radiolysis of water solutions containing the studied compound has
been interpreted in terms of measured pKa and the DEA profile calculated at the G2MP2 level.
2.
EXPERIMENTAL AND COMPUTATIONAL METHODS
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2.1. Synthesis. To a stirred solution of chlorosulfonyl isocyanate (3.05 mL, 7.02 mmol) in dry
dichloromethane (DCM) (12 mL) at 40 °C, a mixture of formic acid (1.36 mL, 7.21 mmol) and
N,N-dimethylacetamide (DMA) (0.029 mL, 0.075 mmol) was added. After ca. 3 h of vigorous
stirring, a suspension of 5-hydroxyuracil (100 mg, 0.78 mmol) in DMA (5 mL) was added. The
mixture was stirred at ambient temperature for 24 h and then poured into water (20 mL). After
3 h, the formed precipitate was filtered off. Uracil-5-yl O-sulfamate was obtained as a white
solid (54 mg) in a 33.4% yield. 5-Hydroxyuracil, chlorosulfonyl isocyanate, DMA, formic acid
and anhydrous DCM were commercially available from Sigma-Aldrich.
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1H NMR (Figure S1 in Supporting Information), δ: 11.45 (s, 1H), 11.00 (m, 1H), 7.93
(s, 2H), 7.52 (d, 1H). HRMS (Figure S2 in Supporting Information), m/z: [M–H]− calcd. for
C4H5N3O5S 207.1646, found 206.0003; UV spectrum (water; Figure S3 in Supporting
Information), λmax: 268 nm.
The 1H NMR spectrum was recorded on a Bruker AVANCE III, 500 MHz spectrometer.
Chemical shifts are reported in ppm relative to the residual signal of DMSO-d6 (2.49 ppm). The
MS measurements were done with use of TripleTOF 5600+ (SCIEX, Germany) and the UV
spectrum was recorded on a Dionex UltiMate 3000 System with diode array detector.
2.2. XRD Measurements and Refinements. A good-quality single-crystal of uracil-5-yl O-
sulfamate has been selected for the X-ray diffraction experiments at T = 295(2) K (Table 1). It
was mounted with epoxy glue at the tip of glass capillary. The diffraction data were collected
on an Oxford Diffraction Gemini R ULTRA Ruby CCD diffractometer with MoKα (λ =
0.71073 Å) radiation. The lattice parameters were obtained by least-squares fit to the optimized
setting angles of the reflections collected by means of CrysAlis CCD.27 The data were reduced
using CrysAlis RED software27 and applying multi-scan absorption corrections (empirical
absorption correction using spherical harmonics, implemented in the SCALE3 ABSPACK
scaling algorithm). The structural resolution procedure was carried out using the SHELX
package.28 The structure was solved with direct methods that carried out refinements by full-
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matrix least-squares on F2 using the SHELXL-2017/1 program.28 A hydrogen atom bound to
the aromatic carbon atom was placed geometrically and refined using a riding model with C–
H = 0.93 Å and Uiso(H) = 1.2Ueq(C). All hydrogen atoms bound to nitrogen atoms were placed
geometrically and refined freely with Uiso(H) = 1.2Ueq(N). All interactions were calculated
using the PLATON program.29 The ORTEPII,30 PLUTO-78,31 and Mercury32 programs were
used to prepare the molecular graphics.
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Full crystallographic details of title compound have been deposited in the Cambridge
Crystallographic Data Center (deposition No. CCDC 1997918) and they may be obtained from
www: http://www.ccdc.cam.ac.uk, e-mail: deposit@ccdc.cam.ac.uk or The Director, CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK.
2.3. Crossed Electron-Molecule Beam Setup. The anion efficiency curves for mass-selected
fragment anions were obtained with a high-resolution crossed electron-molecule beam
apparatus, which consists of a hemispherical electron monochromator (HEM) combined with a
quadrupole mass analyser. The experiment is described in detail in reference.33 The sample
molecule were placed in a copper oven installed in a vacuum chamber. The oven was resistively
heated up to 428 K to achieve sufficient sublimation of the sample. The formed neutral effusive
beam was introduced into the interaction region of the monochromator via a capillary (ɸ = 1
mm) attached to the oven. In the interaction region, the molecular beam perpendicularly crossed
a well-defined electron beam. The electron beam was formed in the HEM, which was operating
at the electron energy resolution of 100 meV and electron currents of 5-30 nA ensuring a
reasonable balance between electron energy resolution and ion intensity. The chamber pressure
was about 1.6·10−11 atm ensuring single-collision conditions. The anions formed in the
interaction region were extracted by a weak electrostatic field into the entrance of a quadrupole
mass analyser and detected using a channel electron multiplier via a single-pulse counting
mode. The electron energy resolution was determined using the well-known s-wave electron
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attachment to CCl4, which leads to the formation of Cl− at 0 eV. This reaction was also used to
calibrate the energy scale.
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2.4. Radiolysis. A water solution of SU at the concentration of 0.1 mM, in the presence of 30
mM t-BuOH – scavenger of hydroxyl radicals, •OH, and 10 mM phosphate buffer (pH = 7.0)
was prepared. The mixture was saturated with argon for c. 3 min. After that all samples were
irradiated with the dose of 140 Gy. The samples containing SU and BrU (both at the
concentration of 0.1 mM) were prepared using the same procedure. All radiolysis experiments
were performed in a Cellrad X-ray cabinet (Faxitron X-ray Corporation). All samples were
prepared at least in triplicate.
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After X-ray irradiations, samples were analyzed with the RP-HPLC method. The C18
column (Wakopak Handy ODS, 4.6 × 150 mm, 5 μm in particle size and 100 Å in pore size),
isocratic elution with 0.1% HCOOH and flow rate 1 mL/min for the separation of analytes were
used. The HPLC analyses were carried out using a Dionex UltiMate 3000 System with a Diode
Array Detector, which was set at 260 nm. All samples were analyzed at least in triplicate.
2.5. Potentiometric Titrations. Potentiometric titrations were performed at 298.15 K, using a
Cerko Lab System microtitration unit fitted with 5-mL Hamilton’s syringe, pH combined
electrode (Hydromet ERH-13-6) calibrated according to IUPAC recommendations34 and a self-
made measuring cell (30 mL) equipped with a magnetic stirrer. The temperature was controlled
using the Lauda E100 circulation thermostat. The composition of the titrand solution was as
follows: 1 mM uracil-5-yl O-sulfamate and 2.55 mM HCl. The solutions were
potentiometrically titrated with the standardized 24 mM NaOH solution in the pH range from
2.5 to 11.5. The experiment consisted of injecting of 0.02 mL of the titrant at 2-min intervals
into the reaction cell, which initially contained 5.0 mL of the titrand solution. The dissociation
constants were refined by least-squares calculations using the Hyperquad2008 (ver. 5.2.19)
computer program.35
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2.6. Quantum Chemical Calculations. The thermodynamic thresholds for various dissociation
pathways of SU in the gas phase were calculated for its most stable conformer. These thresholds
were obtained as the difference between the Gibbs free energies, ΔG, of the products and
substrate in their ground states, as it was performed before,36 with eq. (1a)
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퐺 = 퐺
퐺
푠푢푏푠푡푟푎푡푒
∆
-
(1a)
푝푟표푑푢푐푡푠
The optimized reactants were subjected to the frequency calculations with respect to both
standard state (298.15 K, 1 atm) and the experimental conditions (430.15 K, 1.6 × 10−11 atm).
The pressure correction to the G value for the experimental pressure was calculated with eq.
(1b):37
11
-
-
퐺1.6 10
= 퐺1atm,푇 + 푇 푆
∙
(1b)
atm, 푇
푡푟푎푛푠;1.6 10 11atm,푇
푇 푆
- ∙
푡푟푎푛푠;1atm,푇
∙
∙
where Gp,T and Strans;p,T are the free enthalpy and the translational entropy, both at pressure p
and temperature T.
In these calculations, the M06-2X38 functional combined with the aug-cc-pVTZ39,40
basis set has been used. Such methodology was shown to be successful for this kind of
calculations,25giving results comparable to those obtained with the G4 scheme.41
The mechanisms of the electron-attachment induced degradation of SU were analyzed
computationally, primarily at the density functional theory (DFT) level, with the use of the
M06-2X38 and B3LYP42 hybrid functionals, and the 6-31++G(d,p) basis set.43,44 The
Polarization Continuum Model (PCM)45 was used to mimic aqueous reaction environment.
Similar methodology was successfully employed in our previous reports regarding the electron-
attachment induced degradation of uracil derivatives.16,46,47 In order to verify the DFT
estimates, we also employed the composite G2MP248 method of chemical accuracy. Gas phase
G2MP2 calculations were then PCM corrected to mimic aqueous solution. Discussed in the
current report Gibbs free energies (G) in water were calculated as in eq. 2:
G= Ggas + GMP2 water – GMP2 gas
(2)
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where Ggas stands for the G2MP2 gas phase Gibbs free energy, while GMP2 water and GMP2 gas are
Gibbs free energies obtained in water solution (PCM) and gas phase, respectively, at the all-
electron MP2/6-31G(d)49 level.
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All calculations were carried out with the Gaussian 1650 or Gaussian 0951 suite of
programs.
3.
RESULTS AND DISCUSSION
3.1. Uracil-5-yl O-sulfamate: a Potential Radiosensitizer. One of our previous works
concerned the combination of computational and radiolytic studies on 5-
trifluoromethanesulfonyl-2’-deoxyuridine (OTfdU).16 A favorable DEA profile calculated for
OTfdU was confirmed by the extent of its radiolytic decomposition in water solution, which
was similar to that measured for BrdU under the same experimental conditions. Considering
that, similarly to aryl triflates, aryl O-sulfamates (SU) are perceived as good electrophiles,52 we
decided to check the radiosensitizing properties of uracil-5-yl O-sulfamate. It is known that C–
O bonds in aryl O-sulfamates are less reactive than such bonds in aryl triflates (cf. reactivity of
O-sulfamates versus that of O-triflates in Suzuki53 or Kumada54 reactions) and thereby, we
expected the electron induced O–S bond cleavage rather than the C–O one. This conclusion
was actually confirmed by the DEA profile of SU calculated in aqueous solution, which shows
that the O–S bond breaks almost barrierless (see Figure 1).
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Figure 1. DEA profile calculated for uracil-5-yl O-sulfamate at the M06-2X/6-31++G(d,p) level in an aqueous
solution.
Encouraged by the favorable DFT characteristics, we synthesized uracil-5-yl O-
sulfamate. The compound was obtained via a reaction of 5-hydroxyuracil with sulfamoyl
chloride (H2NSO2Cl), obtained in situ from chlorosulfonyl isocyanate and formic acid in the
presence of catalytic amount of DMA (Figure 2).55
O
O
HO
O
NH
Cl
Cl
O
HCOOH
DMA
DCM
O
O
S
NH
N
H
O
S
S
H2N
O
O
DMA
O
N
C
O
NH2
N
O
H
Figure 2. Synthesis of uracil-5-yl O-sulfamate.
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Besides NMR and HRMS characteristics (see Methods) the X-ray analysis confirmed
the expected structure of the synthesized derivative. Single-crystal X-ray diffraction
measurements show that uracil-5-yl O-sulfamate crystallized in the monoclinic P21/n space
group with one molecule in the asymmetric unit (Figure 3 and Table S1).
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Figure 3. Molecular structure of uracil-5-yl O-sulfamate, showing the atom-labeling scheme (displacement
ellipsoids are drawn at the 25% probability level and H atoms are shown as small spheres of arbitrary radius).
In the crystal of title compound the molecules are linked via N1–H1···O7 and N3–
H3···O8 hydrogen bonds to form sheets of asymmetric ribbons along [1 0 1] direction (Table
S2, Figure 4), similar to those observed in the crystal of form II of 5-fluorouracil.56 The
neighboring, anti-parallel ribbons are connected through N13–H13A···O8, N13–H13B···O12
and C6–H6···O12 hydrogen bonds to form 3D framework.
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Figure 4. Crystal packing of uracil-5-yl O-sulfamate viewed along the a-axis (hydrogen bonds are represented
by dashed lines).
3.2. Dissociative Electron Attachment to Uracil-5-yl O-sulfamate in the Gas Phase. Using
the experimental setup described above, we have studied the formation of anionic fragments
upon electron interaction with the synthesized potential radiosensitizer. In DEA, the resonant
capture of an electron (e−) by a neutral molecule (MX) results in the formation of a transient
negative ion (TNI), which subsequently relaxes by spontaneous emission of the excess electron
or by dissociation, which leads to release of a fragment anion and neutral fragment(s) as shown
in the following reaction:
e− + MX → (MX)−* → M + X−
(3)
where (MX)−* is TNI and M, X− represent the neutral and fragment anion respectively.
DEA to molecules may result in the simple bond cleavage, like indicated in reaction (3), or in
multiple bond cleavage including complex re-arrangement involving the entire molecule. In the
present study, we found 12 fragment anions upon DEA to SU in the gas phase that are discussed
in the next sections. The results for the observed anions, summarized in Table 1, comprise the
maxima of peak positions, experimentally obtained thresholds and calculated thermodynamic
thresholds. For the sake of clarity, we divide the registered DEA reactions into four main
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pathways leading to, (a) fragment anions from the C–O bond cleavage, (b) fragment anions
from the S–O bond cleavage, (c) fragment anions from the uracil-5-yl side group and (d)
fragment anions from the sulfamate side group. For all measured anion efficiency curves shown
in Figures 5-10, we show both the draw data (black line) and cumulative multiple Gaussian fits
(red line). We just note that the overall intensity of the anion yields was about three orders of
magnitude lower to that reported for OTfU,25 which can be explained substantially lower vapour
pressure of the uracil-5-yl O-sulfamate.
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49
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59
60
Table 1. Summary of the resonance positions, experimental thresholds and calculated thermodynamic thresholds
for the fragment anions formed upon electron attachment to uracil-5-yl O-sulfamate.
Threshold (eV)
Maxima of peak positions (eV)
1.58 × 10−11 atm
Mass Anion
m/z
Exp.
Calc.
1.
2.
3.
4.
430.15 K 298.15 K 430.15 K
127
126
99
C4H3N2O3
~0
0.2
1.5
-
~0
~0
~0
~0
~0
~0
−0.36
−0.25
−0.66
−0.12
−0.89
-
−0.90
−0.78
−1.70
−0.65
−1.41
-
C4H2N2O3
C3H3N2O2
NH2SO3
NHSO3
~0
0.1
~0
~0
0.1
~0
0.1
0.8
0.3
0.2
0.3
-
0.2
-
1.2
-
-
-
-
-
96
1.3
-
95
86
C2H2N2O2
NH2SO2
0.9
-
80
~0
~0
~0
−0.79
−1.76
0.09
−1.33
−2.29
−0.45
SO3
SO2
64
62
48
42
16
1.1
0.6
5.0
~0
5.8
0.8
5.4
0.4
8.3
-
-
-
-
-
-
-
NSO
NH2O2
OCN
NH2
~0.3
~4.5
~0
−0.98
3.84
-
−2.01
3.31
-
-
1.3
11.6
4.9
~4
2.63
2.11
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O
~4
2.86
2.40
5
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3.2.1. Anions from the Cleavage of the C5–O Bond. The C–O bond cleavage in SU upon
DEA proceeds via reaction (4) and (5):
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e− + NH SO OU (NH SO OU)*−
(U-yl)⦁ + NH SO
(4)
(5)
−
→
→
→
2
2
2
2
2
3
−
e− + NH SO OU (NH SO OU)*−
(U) + NHSO
→
2
2
2
2
3
This single bond cleavage splits the molecule into two parts, the sulfamate and the uracil-5-yl
moiety. We note that in the DEA experiment only the negatively charged reaction product is
detected, i.e. we assigned the anion yield at m/z 96 to (NH2SO3)−. The detection of the latter
anion implies a rise of the corresponding neutral radical (U-yl)⦁. Similarly, to our previous study
on OTfU,25 the reaction pathway for the formation of the (U-yl)− anion is not observed here.
We, therefore, can infer that the uracil-yl anion is unstable and undergoes subsequent
dissociation into other anions with smaller mass, which we will discuss later. Figure 5 presents
the anion efficiency curve of the anion at m/z 96, which shows a sharp resonance at 0 eV, a
lower resonance at 0.25 eV and a broad resonance at about 1.31 eV. We also observed anion
yield at m/z 95 and assigned it to (NHSO3)− formed by the additional loss of a single hydrogen
atom in the sulfamate anion, which occurs via exothermic reaction (5) with a predicted
theoretical thermodynamic threshold of −1.41 eV. The ion yields for (NH2SO3)− and (NHSO3)−
are similar except that the former anion shows an additional peak close 1 eV (see Figure 5).
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Figure 5. Anion efficiency curve as a function of electron energy for the fragment anions NH2SO3− at m/z 96 and
NHSO3− at m/z 95 upon electron attachment to uracil-5-yl O-sulfamate.
3.2.2. Anions from the S–O Bond Cleavage and the Loss of a Hydrogen Atom. The single
bond cleavage of the S–O bond results in the formation of two complementary fragment anions
as shown in Figure 6. The anion formation proceeds via reactions (6) - (8):
⦁
e− + NH SO OU (NH SO OU)*−
(UO)− + NH SO
(6)
(7a)
(7b)
(8)
→
→
→
2
2
2
2
2
2
e− + NH SO OU (NH SO OU)*−
(UO)⦁ + NH SO
2
−
→
2
2
2
2
2
e− + NH2SO2OU → (NH2SO2OU)*− → (UNH2) + SO3
e− + NH SO OU (NH SO OU)*− (UO–H)− + NH SHO
2
−
→
→
2
2
2
2
2
We observed a fragment anion at m/z 127, which we assign to (UO)−/(SU–NH2SO2)−. The
⦁
corresponding neutral radical is NH2SO2 with mass of 80 u. The anion efficiency curves for
the formation of the fragment anions via reaction (6) and (7) are shown in Figure 6. The anion
efficiency curve for the (UO)− anion at m/z 127 shows an intense resonance close to 0 eV. In
addition to the peak at 0 eV, we registered two resonances between 0.3 and 3 eV with a broad
resonance peaking around 1.5 eV. The experimental threshold observed at 0 eV is in agreement
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with the calculated one of −0.90 eV (Table 1). On the other hand, we observed another fragment
5
6
7
8
−
−
anion at m/z 80, which we assign to either of the two isobaric species NH2SO2 or SO3 formed
via the exothermic reactions (7a) or (7b), respectively. The formation of each anion begins at
the experimental threshold of 0 eV, which is in line with the theoretically determined
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−
−
thermodynamic threshold of −1.33 eV (NH2SO2 ) and −2.29 eV for SO3 (Table 1).
Figure 6. Anion efficiency curves as functions of electron energies for fragment anions observed at m/z 126, 127
and 80 formed from the single S–O bond cleavage upon electron attachment to uracil-5-yl O-sulfamate.
Furthermore, we observed as the most abundant reaction channel the fragment anion at m/z
126, which we assigned to (UO–H)−/(SU–NH2SO2–H)− formed due to the loss of hydrogen
atom in the (UO)− anion. The predicted thermodynamic threshold of −0.78 eV (Table 1) at the
most favourable site, N3–H,25 is exothermic which remains in accordance with the
experimentally determined value of 0 eV. We observed four resonance positions for this anion,
dominated by a sharp peak at 0 eV, followed by two weak resonances at 0.1 and 0.2 eV and a
broad resonance at 1.1 eV.
3.2.3. Fragment Anions from Single and Multiple Bond Cleavage in the Sulfamate Group.
In addition to the afore-mentioned fragment anions, we have observed anions formed due to
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single and multiple bond cleavages in the sulfamate side group. Five different fragment anions
5
6
were recorded, which proceeded via the following reaction pathways:
7
8
9
−
e− + NH SO OU (NH SO OU)*−
(UONH ) + SO
2
(9)
(10)
→
→
→
→
→
→
2
2
2
2
2
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60
e− + NH SO OU (NH SO OU)*−
(UO)⦁ + NSO− + H O
→
2
2
2
2
2
e− + NH SO OU (NH SO OU)*−
(UOS)⦁ + NH O ¯
(11)
→
2
2
2
2
2
2
e− + NH SO OU (NH SO OU)*−
(SU–O)⦁ + O−
(SU–NH )⦁ + NH
2
(12a)
(12b)
→
2
2
2
2
e− + NH SO OU (NH SO OU)*−
−
→
2
2
2
2
2
Figures 7 and 8 show the anion yield curves for the anions at m/z 64 and 48, and at m/z 62
−
respectively. The anion at m/z 64 is assigned to SO2 , which arises from multiple bond
cleavages and rearrangement in the sulfamate side group of the parent molecule via reaction (9)
with UONH2 as the neutral fragment. We observed for this anion two major resonances peaks
close to 1.1 eV and a broad resonance at higher energy around 5.8 eV. The calculated
thermodynamic threshold of −0.45 eV matches with the experimental threshold of 0 eV. On the
−
other hand, we also observed the anion at m/z 48 which we assign to NH2O2 . Unlike most of
the other anions, no peak was observed at low electron energies. The anion is only formed
above a threshold of about 4.5 eV. Our calculations predict an endothermic reaction with a
threshold of 3.31 eV (Table 1), which agrees with the absence of a peak at low energies. The
dissociation of the sulfamate group through multiple-bond cleavage and rearrangement showed
by reaction (10) leads to the NSO− anion at m/z 62. This reaction is accompanied by the release
of H2O and UO⦁ as the counterpart radical. The anion was detected as the second most abundant
fragment anion. The calculated exothermic thermochemical threshold was found to be −2.01
eV (Table 1) in agreement with the experimental threshold of about 0 eV. As shown in Figure
8, two narrow closely spaced resonances with relatively high intensity were found with maxima
at 0.6 and 0.8 eV.
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Figure 7. Anion efficiency curves as functions of electron energies for the fragment anions formed at m/z 64 and
48 upon electron attachment to uracil-5-yl O-sulfamate.
Figure 8. Anion efficiency curve of the fragment anion observed at m/z 62 as a function of electron energy upon
electron attachment to uracil-5-yl O-sulfamate.
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Figure 9 shows the yield curve of the anion at m/z 16. We note that two isobaric fragment
5
6
−
anions, O− formed via reaction (12a) and NH2 via reaction (12b) have the same nominal m/z.
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9
It was not possible to separate these isobaric anions with the quadrupole mass spectrometer.
Our M06-2X/aug-cc-pVTZ calculations indicate that, both anions are possible to form with a
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−
thermodynamic threshold of 2.11 eV for NH2 (S–NH2 bond cleavage) and 2.40 eV for O− (S–
O bond cleavage), respectively (Table 1). Therefore, the threshold values cannot be used to
assign the peaks shown in Figure 9 to the exact sites of bond cleavage. However, a comparison
with results from previous DEA studies for compounds containing oxygen atoms and/or amino
groups may allow a tentative assignment.
Figure 9. Anion efficiency curve as a function of electron energy for the fragment anion observed at m/z 16
upon electron attachment to uracil-5-yl-sulfamate.
In the previous study Denifl et al.57 reported a resonance at 4.5 eV in the anion efficiency curve
of O− upon electron attachment to uracil in the gas phase. The peak showed a similar onset of
about 4 eV as obtained here. Besides, the results from the DEA study by Alizadeh et al.58 on
alanine anhydride with a peak at 4.4 eV is not far from the aforementioned results on O−. The
resonance contributions of the two isobaric anions O− and NH2 were distinguished upon
−
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electron attachment to the amino acid valine59 using a high-resolution VG-ZAB mass
spectrometer. It was reported that the O− anion is formed at electron energies of 4.4 and 8.3 eV
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60
−
whilst the NH2 is observed at 6 eV. Moreover, the recently published work by Ameixa et al.
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on the formation of fragment anions upon electron attachment to benzaldehyde showed the
formation of O− via the cleavage of C=O bond in a characteristic resonance between 8 and 9
eV. In our present study, the second resonance position of 8.3 eV is in the same energy range.
Hence, by analogy we can infer that our experimentally found peaks at 4.9 and 8.3 eV can be
ascribed to oxygen anion. The presented data also indicate a third resonance at around 11.6 eV
−
at m/z 16, where we omitted an assignment to either O− or NH2 .
3.2.4. Fragment Anions from Single and Multiple Bond Cleavage in the Uracil-5-yl
moiety. Single and multiple bond cleavages in the uracil-5-yl group result in the formation of
three other anionic species via the following reactions:
⦁
e− + NH OSO U (NH OSO U)*−
(C H N O )− + NH SO + CO
(13)
(14)
(15)
→
→
→
→
2
2
2
2
3
3
2
2
2
2
e− + NH OSO U (NH OSO U)*−
(C H N O )− + NH OSO C H
2 2 2 2 2 2 2
→
2
2
2
2
e− + NH OSO U (NH OSO U)*−
(OCN)− + NH OSO C NH O
2 2 3 3
→
2
2
2
2
We show the anion yield curves for the anions occurring via reaction (13) - (15) in Figure 10.
−
The formation of C3H3N2O2 at m/z 99 is formed above the experimental threshold of 0 eV.
The suggested fragmentation reaction (13) leads to a ring-opening of the uracil-5-yl moiety
⦁
with the formation of NH2SO2 and C=O as radical and neutral fragments. The respective
thermodynamic threshold for this channel was predicted to be exothermic (−1.70 eV). We also
observed (C2H2N2O2)− at m/z 86 with an experimental threshold of 0 eV, formed by the loss of
C–H and C=O in the dissociation of the uracil-5-yl radical. We report for this anion three
resonances at 0.1 eV, 0.3 and 0.9 eV. The calculated thermodynamic threshold for the formation
of this anion was found to be 3.31 eV (p = 1 atm T = 198.15 K) indicating an endothermic
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reaction pathway, which disagrees with the experimental thresholds obtained. Even though we
investigated certain reactions computationally, we could not account for this difference
regardless of the pressure and temperature. Thus, we conclude that the large disagreement
indicates that other reaction pathways will probably occur.
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Figure 10. Anion efficiency curves as functions of electron energies for anions at m/z 99, m/z 86 and m/z 42
upon electron attachment to uracil-5-yl O-sulfamate.
In the case of m/z 42 represented by reaction (15), the formation of OCN− may proceed via
different reaction channels depending on the site of bond cleavage (Figure S4 in Supporting
Information). Due to the variety of possible reaction channel, we omitted further calculation on
the threshold of OCN−. We observed that the experimental findings are similar to the results
obtained for the formation of NCO− from DEA to the potential radiosensitizer hydroxyurea
(which has the structural formula CH4N2O2 or OH–NH–CO–NH2)61 using the same
experimental setup. It is worth noting that the involved carbon atom has similar bonding
environment (HN–CO–NH) to that in the current molecule. Three peaks were found at positions
close to 0, 0.4, and 1.2 eV, respectively. The resonance positions for NCO− are similar for SU
and hydroxyurea, except for an additional peak at 0.1 eV that was reported for the latter
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compound. We further note, that (C2H2N2O2)− could be an intermediate reaction product in the
dissociation pathway leading to OCN− by the loss of two hydrogen atoms from (C2H2N2O2)−
followed by subsequent C–N bond cleavage. OCN− was the second most abundant fragment
anion after the dehydrogenated parent anion in DEA to the nucleobases uracil and thymine.62 It
was shown for these compounds, that OCN− forms in a sequential dissociation process with initial
H-loss from one of the nitrogen sites of the nucleobase anion.62,63 Since we do not observe the
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dehydrogenated parent anion of uracil-5-yl O-sulfamate within the detection limit of the
apparatus, we may rule out this reaction pathway found for nucleobases.
3.3. Radiolysis of SU under Reductive Conditions. The results described in the previous
section demonstrate that several dissociative channels are triggered by LEE attachment to SU.
To define the potential of the studied compound as a radiosensitizer, steady state radiolysis was
also performed. Water solutions containing SU, free hydroxyl radical scavenger (t-BuOH) and
phosphate buffer were irradiated with the dose of 140 Gy. To avoid scavenging of electrons by
oxygen, all samples were deoxygenated before exposure to IR. Irradiated and non-irradiated
samples were analyzed using the HPLC methods (Figure 11). To our surprise, no product of
electron induced degradation of SU was observed. It is worth noting that the change in solution
pH (5.6, 8.0 – 10 mM phosphate buffer and 4.0, 4.9 – 10 mM formate buffer) and the use of
higher radiation dose (280 Gy) did not affect the radiolysis process. None of the examined
radiolysis conditions led to the appearance of any radioproducts from SU.
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Figure 11. HPLC traces for a solution of uracil-5-yl O-sulfamate before (black) and after irradiation
(orange) with a dose of 140 Gy.
3.4. Rationalization of the Results of Radiolysis Observed for SU Solutions. Striking
difference between the complex DEA picture and lack of SU reactivity in the radiolyzed
samples may result from a number of reasons. To continue our quest for searching of efficient
radiosensitizing nucleosides, we have to understand why so far successful DFT model turned
out to be abortive in case of SU which, in spite of favorable computational characteristics,
turned out to have no radiosensitizing properties.
In order to eliminate possible experimental errors we carried out X-ray irradiations of
solutions containing both SU and BrU (Figure 12). In this way the radiolysis of both substances
was carried out under identical conditions. As indicated by Figure 12 for the dose of 140 Gy,
BrU decomposes with the yield equal to 15.83 ± 0.56% - the similar decay (15.78 ± 0.93%)
was observed in anther independent experiment, while practically no decomposition of SU is
observed.
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Figure 12. HPLC traces for a solution containing uracil-5-yl O-sulfamate and 5-bromouracil before (black
chromatogram) and after irradiation with a dose of 140 Gy (orange chromatogram).
The results depicted in Figure 12 suggest that SU is not only unreactive to solvated electrons
but also does not bind them. Indeed, the presence of equimolar amounts of SU and BrU in the
solution does not affect the decomposition yield of BrU (see above).
In our first attempt to explain this observation, we assumed that in water yet before
irradiation SU primarily exists as an anion originating from the deprotonation of the NH2 group.
In fact, the formation of such anion could prevent attachment of an electron due to repulsion
between the negative charge of electron and the molecular anion which, in turn, would explain
the results of radiolysis depicted in Figures 11 and 12. The DEA process to the described above
anion is depicted in Scheme S1 (SI) and discussed in Supporting Information. In order to verify
the above mentioned assumption, the acidic dissociation constants of SU were obtained by
taking into account two equilibria depicted in Figure 13. This model has provided very good
fitting of the calculated data to the experimental ones (Figure S5 in SI). The potentiometric
titration reveals that SU can be considered as a weak acid which dissociates in two steps shown
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in Figure 13. The calculated dissociation constants are equal to: pKa1 = 7.52 (± 0.06) and pKa2
= 9.64 (± 0.04), while the relative concentrations of the species existing in SU solution as a
function of pH, obtained employing these pKa values and the HySS program,64 are depicted in
Figure 14.
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O
O
O
O
H
H
N
N S O
H
O
S O
H
pKa1 = 7.52
N
N
H
+ H3O+
O
O
+ H2O
N
H
N
H
O
LH- (2)
LH2 (1)
O
O
O
O
H
H
N
H
N
S O
S O
O
N
N
pKa2 = 9.64
+ H3O+
O
+ H2O
N
O
N
O
H
H
LH- (2)
L2- (3)
Figure 13. The proton dissociation scheme for uracil-5-yl O-sulfamate.
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(3)
(1)
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(2)
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(1)
H2L
(2)
(3)
HL-
L2-
4
6
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12
pH
Figure 14. The concentration distribution of species as a function of pH in the uracil-5-yl O-sulfamate solution.
Hence, it is clear that our tentative hypothesis about existence of anionic forms of SU before
irradiation does not hold. Indeed, at pH = 7 most of SU (around 80%, see Figure 13) is in the
neutral, H2L form. Nevertheless, we carried out additional radiolytic experiments in solutions
of various pH and determined the yield of SU decomposition. Regardless of pH (4 < pH < 8)
the results of irradiation were always the same – SU did not decompose, even at larger doses of
X-rays (up to 280 Gy). On the other hand, it is well known that nucleobases bind solvated
electrons with an almost diffusion controlled rate.12 Thus, the same yield of BrU decomposition
observed for a solution containing exclusively BrU and equimolecular amounts of BrU and SU
only seemingly suggests that solvated electrons do not attach to SU. Indeed, the adiabatic
electron affinities calculated for both molecular systems are pretty similar (2.31 and 2.43 eV,
1for BrU and SU, respectively, G2MP2 free energy level). Hence, the yield of BrU
decomposition in solutions containing both BrU and SU indicates that some electrons are
temporarily captured by SU and then transferred to BrU, where they are ultimately consumed
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in the DEA reaction. In consequence, these results suggest that SU does not undergo electron-
induced degradation in water.
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9
All the mentioned above facts imply that the discrepancy between experimental picture and
theoretical predictions may lie in our computational model. In order to check this hypothesis,
we calculated the DEA profiles for SU using also the G2MP2 method having chemical accuracy
(errors below 1 kcal/mol). To make sure that a possible discrepancy between the M06-2X level
and more accurate approach originates from the DFT inaccuracies for the SU DEA profile, the
G2MP2 calculations were repeated for two other uracil derivatives, which represent a close
correspondence between the theoretical and actual radiolytic behavior (Figure 15, SCNU and
BrU).
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B
A
O
O
O
O
O
C
S
N
Br
S
NH
NH
NH
H2N
O
B
A
A
NH
O
NH
O
NH
O
BrU
SCNU
SU
Figure 15. Structures of uracil derivatives along with their abbreviated names: uracil-5-yl O-sulfamate (SU), 5-
thiocyanateuracil (SCNU) and 5-bromouracil (BrU). Arrows indicate bonds possible to break during DEA process.
Two DEA possible paths are marked: path A (the bond cleavage between uracil and its substituent) and path B
(the bond cleavage within the substituent).
Table 2 summarizes computational characteristics obtained at the DFT and G2MP2
levels. G2MP2, as oppose to the M06-2X method, seems to support the results of radiolytic
experiments where no electron-induced degradation of SU was found. Namely, the crucial
activation barrier, i.e. breaking of the O–S bond (path B; Figure 15), is significantly
underestimated at the M06-2X level, as it has risen from 2.3 to 7.5 kcal/mol (G2MP2). Thus,
the activation barrier calculated with high accuracy method explains the experimental
observations. Indeed, as indicated in our recent studies on radiosensitizing properties of 5-iodo-
17 and 5-bromo-4-thio-2’-deoxyuridine,18 the activation energy of c. 7 kcal/mol is sufficient to
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completely quench the electron attachment induced release of the halide anion from the
mentioned above thiouridines. Hence, the activation barriers of 11.1 and 7.5 kcal/mol for the
dissociation of the C–O and O–S bonds, respectively, obtained at the G2MP2 level, justify the
observed stability of the SU anion in the radiolytic experiments. These dissociation paths are
opened in the gas phase even for 0 eV electrons (see section 3.2.1 and 3.2.2) since electron
attachment (AEA = 56.0 kcal/mol at the G2MP2 free energy level, in solution) delivers the
amount of energy that several times overcomes the calculated kinetic barriers. The discussed
dissociations are exothermic, both in the gas and aqueous phase, but in the latter environment
the energy released due to electron attachment is, unlike in the gas phase, swiftly dissipated to
the solvent. Therefore, in a liquid phase, the formed SU anions are kinetically stable, while the
medium barriers do not constitute any obstacle for the respective bond cleavage in the gas phase
at low pressure.
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It is worth noticing that the discrepancy between the DFT and G2MP2 models does not
exist for the two other derivatives: 5-thiocyanatouracil and 5-bromouracil (see Figure 15). For
both systems the DFT data (M06-2X for SCNU and B3LYP for BrU) are in good agreement
with the radiolytic results. The G2MP2 estimates reveal that the crucial activation barriers
change only slightly for the degradation of BrU (from 2.5 kcal/mol at the B3LYP level to 1.8
kcal/mol at G2MP2) and SCNU (from 8.7 kcal/mol at the M06-2X level to 7.9 kcal/mol at
G2MP2 for path A, and from 4.1 kcal/mol at M06-2X to 1.6 kcal/mol at G2MP2 for path B;
see Table 2). A similar conclusion can be drawn for the thermodynamic data. The free energies
of dissociation for all bonds except S–O differ no more than several kcal/mol (see Table 2).
Only the thermodynamic stimulus associated with the S–O bond cleavage in the SU anion is
overestimated at the M06-2X and B3LYP level by as much as 22 and 25.2 kcal/mol,
respectively. The inaccurate estimation of activation barriers and thermodynamics, calculated
for the S–O bond scission, seem to be reminiscent of the semiempirical nature of DFT
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