Why 6-Iodouridine Cannot Be Used as a Radiosensitizer of DNA Damage? Computational and Experimental Studies

Abstract

Previous density functional theory (DFT) studies on 6-brominated pyrimidine nucleosides suggest that 6-iodo-2'-deoxyuridine (6IdU) should act as a better radiosensitizer than its 5-iodosubstituted 2'-deoxyuridine analogue. In this work, we show that 6IdU is unstable in an aqueous solution. Indeed, a complete disappearance of the 6IdU signal was observed during its isolation by reversed-phase high-performance liquid chromatography (RP-HPLC). As indicated by the thermodynamic characteristics for the SN1-type hydrolysis of 6IdU obtained at the CAM-B3LYP/DGDZVP++ level and the polarizable continuum model (PCM) of water, 6-iodouracil (6IU) was already released quantitatively at ambient temperatures. The simulation of the hydrolysis kinetics demonstrated that a thermodynamic equilibrium was reached within seconds for the title compound. To assess the reliability of the calculations carried out, we synthesized 6-iodouridine (6IUrd), which was, unlike 6IdU, sufficiently stable in an aqueous solution at room temperature. The activation barrier for the N-glycosidic bond dissociation in 6IUrd was estimated experimentally using an Arrhenius plot. The stabilities in water calculated for 6IdU, 6IUrd, and 5-iodo-2'-deoxyuridine (5IdU) could be explained by the electronic and steric effects of the 2'-hydroxy group present in the ribose moiety. Our studies highlight the issue of the hydrolytic stability of potentially radiosensitizing nucleotides which, besides having favorable dissociative electron attachment (DEA) characteristics, must be stable in water to have any practical application.

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Why 6‑Iodouridine Cannot Be Used as a Radiosensitizer of DNA
Damage? Computational and Experimental Studies
Karina Falkiewicz, Witold Kozak, Magdalena Zdrowowicz, Paulina Spisz, Lidia Chomicz-Manka,
Mieczyslaw Torchala, and Janusz Rak*
Cite This: J. Phys. Chem. B 2023, 127, 2565−2574
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sı Supporting Information
ABSTRACT: Previous density functional theory (DFT) studies
on 6-brominated pyrimidine nucleosides suggest that 6-iodo-2′-
deoxyuridine (6IdU) should act as a better radiosensitizer than its
5-iodosubstituted 2′-deoxyuridine analogue. In this work, we show
that 6IdU is unstable in an aqueous solution. Indeed, a complete
disappearance of the 6IdU signal was observed during its isolation
by reversed-phase high-performance liquid chromatography (RP-
HPLC). As indicated by the thermodynamic characteristics for the
SN1-type hydrolysis of 6IdU obtained at the CAM-B3LYP/
DGDZVP++ level and the polarizable continuum model (PCM)
of water, 6-iodouracil (6IU) was already released quantitatively at
ambient temperatures. The simulation of the hydrolysis kinetics
demonstrated that a thermodynamic equilibrium was reached
within seconds for the title compound. To assess the reliability of the calculations carried out, we synthesized 6-iodouridine (6IUrd),
which was, unlike 6IdU, suciently stable in an aqueous solution at room temperature. The activation barrier for the N-glycosidic
bond dissociation in 6IUrd was estimated experimentally using an Arrhenius plot. The stabilities in water calculated for 6IdU, 6IUrd,
and 5-iodo-2′-deoxyuridine (5IdU) could be explained by the electronic and steric eects of the 2′-hydroxy group present in the
ribose moiety. Our studies highlight the issue of the hydrolytic stability of potentially radiosensitizing nucleotides which, besides
having favorable dissociative electron attachment (DEA) characteristics, must be stable in water to have any practical application.
1. INTRODUCTION
Radiotherapy involves damaging genomic DNA by exposing
living cells to ionizing radiation.
1,2
Direct eects, where
ionizing photons reach the DNA molecules, are negligible for
sparsely ionizing radiation, for example, using X-rays, which is
commonly employed in radiotherapy.
3
Since water is the main
constituent of most cells, its radiolysis (producing mainly
hydroxyl radicals and hydrated electrons) accounts for most of
the secondary eects related to interactions between ionizing
radiation and living matter.
4
Hypoxic cells a hallmark of solid
tumors
5
are about 3 times more resistant to ionizing radiation
than normoxic ones.
6
To overcome the state of hypoxia which
is disadvantageous from a radiotherapy standpoint, modified
nucleosides (MNs) with radiosensitizing properties could be
introduced into anticancer therapy.
7
Note that some MNs can
easily penetrate cell membranes, be phosphorylated in the
cytoplasm, and then processed by DNA polymerases,
ultimately leading to their incorporation into the newly
biosynthesized DNA.
3,8,9
Such modified DNA then becomes
sensitive to hydrated electrons, one of the most abundant
radiolysis products of water, which after their attachment may
lead to a single-strand break (SSB).
The most thoroughly studied radiosensitizing nucleoside
analogues employing the above-mentioned mechanism of
DNA sensitization are 5-bromo- (5BrdU) and 5-iodo-2′-
deoxyuridine (5IdU).
10−12
The sensitizing eects of these
pyrimidines are related to an ecient dissociative electron
attachment (DEA) as a consequence of interactions between
MNs and hydrated electrons.
13
Indeed, hydrated electron
attachment may initiate the DEA process, which will produce
reactive and genotoxic nucleoside radicals. If these reactions
take place inside the DNA, the reactive species resulting from
DEA can lead to DNA strand breaks and finally to cell death.
14
Recently, several new uracil derivatives and 2′-deoxyuridines
with radiosensitizing properties have been proposed by our
group.
15−18
Most radiosensitizing nucleosides are 5-substituted pyrimi-
dines (5-Pyrs).
8,9,19
However, we have previously postulated
Received: January 24, 2023
Revised: February 14, 2023
Published: March 9, 2023
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that 6-substituted uridine or cytidine analogs could work as
DNA radiosensitizers more eciently than 5-Pyrs.
20,21
In fact,
our computational studies have demonstrated that the uridine-
6-yl radical, forming with a low activation barrier DEA to the 6-
substituted pyrimidine nucleosides, can abstract a hydrogen
atom not only from the sugar moiety of an adjacent nucleoside
in a DNA strand but also from its own 2′-deoxyribose.
20,21
Thus, the latter process should increase the amount of strand
breaks in DNA labeled with 6-substituted pyrimidines relative
to DNA labeled with 5-substituted ones.
In this work, we have attempted to experimentally follow the
concept indicated in our previous theoretical investiga-
tions,
20,21
i.e., we have tried to synthesize 6-iodo-2′-
deoxyuridine (6IdU) and carry out radiolytic studies on its
aqueous solution coupled with the liquid chromatography−
mass spectrometry (LC−MS) identification of radioproducts.
However, it was observed that the presence of the iodine atom
at the C6 position of 2′-deoxyuridine made the compound
susceptible to hydrolysis already at ambient temperatures.
Hence, this observation suggests that 6IdU cannot work as a
radiosensitizer since any radiosensitizer has to be relatively
stable in water to be transported to the cell cytoplasm and then
incorporated into the cellular DNA. To explain the high
observed propensity of 6IdU to hydrolysis, in addition to the
attempted synthesis of 6-iodo-2′-deoxyuridine, we synthesized
6-iodouridine (6IUrd) and compared its hydrolytic behavior
and that of 5-iodo-2′-deoxyuridine (5IdU) to that of 6IdU (see
Figure 1 for the structures of the studied species). The
mechanism of hydrolysis of the studied compounds was then
described at the density functional theory (DFT) level. The
thermodynamic stimuli and activation barriers were calculated
for the SN1 substitution of the sugar moiety at the C1′position
with a water molecule. To validate the assumed level of theory,
the obtained computational barrier was compared to that
determined experimentally with the use of an Arrhenius plot
for a relatively stable 6IUrd nucleoside. The surprising ease of
hydrolysis of 6IdU compared to 6IUrd could be explained in
terms of both electronic factors concerning uracil and the lack
of a hydroxyl group at the C2′position in the deoxyribose
moiety.
2. METHODS
2.1. Synthesis. 2.1.1. Synthesis of 2′,3′-O-Isopropylide-
neuridine 1(See Figure 2). Sulfuric acid (H2SO4, 0.65 mL)
was added dropwise to a stirred suspension of uridine (1 g,
4.13 mmol) in acetone (50 mL) at room temperature (RT),
and the resulting mixture was stirred for 3 h. Then, the
reaction was neutralized with Et3N and evaporated. The raw
product was purified by preparative column chromatography
using CHCl3/MeOH 15:1 as an eluent to produce the desired
compound 1(1.05 g) in a 90.2% yield.
2.1.2. Synthesis of 2′,3′-O-Isopropylidene-5′-O-methox-
ymethyluridine 2(See Figure 2). Dimethoxymethane (13.33
mL, 150.5 mmol) and methanesulfonic acid (0.133 mL, 2.05
mmol) were added to a suspension of 1(0.5 g, 1.76 mmol) in
acetone (6.5 mL), and the resulting mixture was stirred
overnight. The stirred mixture was then poured into a 25%
aqueous solution of NH4OH and extracted with CHCl3. The
raw product was purified by preparative column chromatog-
raphy using CH2Cl2/MeOH 80:1 as an eluent to produce the
desired compound 2(385 mg) in a 66.7% yield.
2.1.3. Synthesis of 6-Iodo-2′,3′-O-isopropylidene-5′-O-
methoxymethyluridine 3(See Figure 2). A solution of 2
(300 mg, 0.91 mmol) in anhydrous tetrahydrofuran (THF, 3.5
mL) was added dropwise to a stirred solution of lithium
diisopropylamide (LDA, 0.95 mL, 2.00 mmol, 2 M solution in
THF) in anhydrous THF (3.7 mL) at −78 °C. After stirring
for 1 h, iodine (232 mg, 0.91 mmol) in anhydrous THF (2.9
mL) was added and the mixture was stirred for an additional 5
h at −78 °C. Then, AcOH (0.45 mL) was added to quench the
reaction. After bringing to RT, the raw product was extracted
with AcOEt and the organic layer was washed with an aqueous
solution consisting of NaHCO3and brine. The product was
purified by preparative column chromatography using hexane/
AcOEt 7:6 as an eluent to produce the desired compound 3
(140 mg) in a 31.7% yield.
2.1.4. Synthesis of 6-Iodouridine 4(See Figure 2). A stirred
suspension of 3(100 mg, 0.22 mmol) in water (0.77 mL) was
treated with a 50% aqueous solution of trifluoroacetic acid
(TFA, 1.15 mL) at 0 °C. Then, the reaction mixture was
stirred for 48 h at RT. Following evaporation, the product was
purified by preparative column chromatography using CHCl3/
EtOH 10:1 as an eluent to produce the desired compound 4
Figure 1. Chemical structure of 5-iodo-2′-deoxyuridine, 6-iodo-2′-
deoxyuridine, and 6-iodouridine.
Figure 2. Synthesis of 6-iodouridine 4.
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(48 mg) in a 58.9% yield. Proton nuclear magnetic resonance
(1H NMR, Bruker AVANCE III, 500 MHz, DMSO), δ:13C
NMR (125 MHz, DMSO), δ: 162.5, 147.6, 116.1, 115.9,
102.6, 84.9, 72.0, 69.9, 62.3 (Figure S1). High-resolution mass
spectrometry (HRMS) (TripleTOF 5600+, SCIEX), m/z: [M
−H]−calcd for C9H11IN2O6368.9588, found 368.8943;
ultraviolet (UV) spectrum (water), λmax: 270 nm.
2.2. Kinetic Measurements and Calculations of
Kinetic Profiles. The vials with the reaction mixture (50
μL) containing 6-iodouridine in a phosphate buer (0.1 M, pH
= 7.0) were placed in an Eppendorf thermocycler for specified
periods of time (see time points in Figure S8) at the following
temperatures: 60, 65, 70, 75, and 80 °C. After heating, the
solutions were analyzed by high-performance liquid chroma-
tography (HPLC). The HPLC separations were performed on
a Dionex UltiMate 3000 System with a diode array detector,
which was set at 260 nm for monitoring the euents. The
analytes were separated on a Wakopak Handy ODS column
(4.6 mm ×150 mm) using an isocratic elution with 0.1%
formic acid in deionized water. The flow rate was set at 1 mL
min−1. Each solution was injected into the HPLC instrument
using an autosampler. The experiment was carried out in
duplicate. The identification of the thermal analysis products
was performed by mass spectrometry (LC−MS and LC−MS/
MS experiments). The MS and MS/MS spectra are presented
in Supporting Information Figures S2−S7. The LC−MS
conditions were as follows: a TripleTOF 5600+ (SCIEX)
mass spectrometer (operating in a negative mode) coupled
with a Nexera X2 ultrahigh-performance liquid chromatog-
raphy (UHPLC) system; a Kinetex column (Phenomenex,
C18, 2.1 mm ×150 mm); a flow rate of 0.3 mL min−1; an
isocratic elution with 0.1% formic acid in deionized water. All
MS and MS/MS analyses were performed using a spray voltage
of −4.5 kV and a source temperature of 300 °C.
The logarithmic values of the concentrations of 6IUrd at
time zero and at dierent time intervals were used to establish
the degradation plots. The degradation kinetic parameters (the
degradation rate constants) at 60, 65, 70, 75, and 80 °C were
derived from the plots. The predicted kinetics for the
degradation of 6IUrd at 25 °C was extrapolated from the
Arrhenius plot.
2.3. Quantum Chemical Calculations. The mechanisms
of hydrolysis of the three considered derivatives were studied
using the DFT. We applied the CAM-B3LYP functional
22
combined with the DGDZVP++ basis set.
23
The polarization
continuum model
24
(PCM) was used to mimic an aqueous
reaction environment. All geometries were optimized and
subjected to the frequency calculations for the standard state
(T= 298 K, p= 1 atm). The analysis of harmonic frequencies
confirmed that the stationary geometries were localized (all
force constants were positive for minima; all but one were
positive for the first-order transition states). The intrinsic
reaction coordinate (IRC)
25
procedure was used to verify that
the transition state connected the proper minima.
The changes in Gibbs free energy (ΔG) for particular
reactions were calculated as the dierence between the Gibbs
free energy of the products and substrates. The activation
barriers (ΔG*) were calculated as the dierence between the
Gibbs free energies of the transition state and the substrate. All
DFT computations were performed with the use of Gaussian
09.
26
2.4. Calculations of Equilibrium Concentrations and
Kinetics. The equilibrium concentrations of 5IdU, 6IdU, and
6IUrd at 25 °C were calculated using the following set of
equilibrium (see eqs 1−3and the Equilibrium Concentrations
Section in the Supporting Information)
++
nucleoside base sugarV
(1)
+ +
+ +
sugar 2 H O sugar (OH) H O
2 3
V
(2)
+ +base H O base (H) HO
2V
(3)
where Kdiss,Ka, and Kbstand for the equilibrium constants for
nucleoside dissociation eq 1 and cationic eq 2 and anionic eq 3
hydrolysis, respectively.
The cationic (Ka) and anionic (Kb) hydrolysis constants
(Table 1) were estimated employing the experimental values of
acid dissociation constants (Kref) for dimethoxycarbene
(MeO)2CHOH (pKa
27
=−5.7) and uracil (pKa
28
= 9.45),
respectively, using eq 4.
29
=K K G
RT
p p 2.303
a/ b ref
(4)
where pKa/b and pKref stand for the negative decimal logarithm
of Ka/Kband Kref, respectively, while ΔΔGindicates the
change in Gibbs free energy for the reactions defined in Table
1.
These equilibrium constants were, in turn, used to calculate
some of the rate constants eq 5 that were required to simulate
the overall kinetics of hydrolysis using eq 5
=Kk
k
1
1
(5)
where k1and k−1are the rate constants for forward and reverse
reactions, respectively, while Kis the respective equilibrium
constant.
The assumed elemental reactions running in the solution of
a nucleoside eqs 1−3led to the following set of ordinary
dierential equations eqs 6−15
[ ] = [ ][ ] [ ]
+
t
k k
d Nuc
d
2 R A 1 Nuc
(6)
[ ] = [ ][ ] [ ][ ]
[ ][ ] + [ ]
+
+ +
+
t
k k k
k
d H O
d3 R H O 4 R(OH) H O 7
H O OH 8 H O
3
2
2
3
3 2
2
(7)
Table 1. pKand KValues for the Given and Ref Compounds along with the Respective ΔΔG[kcal mol−1]
reaction ΔΔGpK K
5IU−+ U →5IU + U−4.1 pKb= 7.53 Kb= 3.0 ×10−8
6IU−+ U →6IU + U−7.3 pKb= 9.91 Kb= 1.2 ×10−10
deoxyribose++ (MeO)2CHOH →deoxyribose + (MeO)2CHOH+9.1 pKa=−12.36 Ka= 2.3 ×1012
ribose++ (MeO)2CHOH →ribose + (MeO)2CHOH+15.5 pKa=−17.09 Ka= 1.2 ×1017
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[ ] = [ ][ ] [ ][ ] [ ]
[ ] + [ ]
+
t
k k k
k
d OH
d5 A H O 6 A(H) OH 7 H O
OH 8 H O
2 3
2
2
(8)
[ ] = [ ][ ] + [ ] [ ][ ] +
[ ][ ]
+
+ +
+
t
k k k k
d R
d2 R A 1 Nuc 3 R H O 4
R(OH) H O
2
2
3
(9)
[ ] = [ ][ ] [ ][ ]
+ +
t
k k
d R(OH)
d3 R H O 4 R(OH) H O
2
2
3
(10)
[ ] = [ ][ ] + [ ] [ ][ ] +
[ ][ ]
+
t
k k k k
d A
d2 R A 1 Nuc 5 A H O 6
A(H) OH
2
(11)
[ ] = [ ][ ] [ ][ ]
t
k k
d A(H)
d5 A H O 6 A(H) OH
2
(12)
[ ] = [ ][ ] + [ ][ ] +
[ ][ ] [ ][ ] + [ ]
[ ] [ ]
+ + +
t
k k k
k k
k
d H O
d5 A H O 6 A(H) OH 4
R(OH) H O 3 R H O 7 H O
OH 8 H O
2
2
3 2
2
3
2
2
(13)
Additional Equations for 6IUrd Only
[ ] = [ ][ ] + [ ][ ]
t
k k
d hydrNuc
d10 hydrNuc I 9 Nuc OH
(14)
[ ] = [ ][ ] + [ ][ ]
t
k k
d I
d10 hydrNuc I 9 Nuc OH
(15)
The abbreviations used in this system of kinetic equations
were the following: Nuc�nucleoside (substrate), A(H)�
nucleic base, R(OH)�sugar moiety, A−�anionic form of
nucleic base, R+�cationic form of sugar moiety, and
hydrNuc�6-hydroxyuridine (6OHUrd). The system of dier-
ential equations, shown in eqs 6−15, was solved with the use of
the Octave program.
30,31
The initial concentration of a
nucleoside was assumed to be 10−3M, and the initial
concentrations of the remaining reactants were set to 0 M at
time = 0. Due to the small concentrations of reactants, we
assumed a constant concentration of water of 55.5 M over the
course of the studied processes.
32
3. RESULTS AND DISCUSSION
One of the basic reactions that nucleosides may undergo in an
aqueous solution is their hydrolysis resulting in the detachment
of a nucleobase (see Figure 3).
According to Kochetkov,
33
2′-deoxynucleosides are hydro-
lyzed faster than ribonucleosides (about 100−1000 times).
Indeed, the half-life of hydrolysis, τ1/2, for 2′-deoxyuridine
(dU) amounts to 104 min, while uridine (Urd) is hydrolyzed
only to a small extent even in the 3 times longer period under
the same experimental conditions (5% hydrolysis in 5 h).
33
Moreover, the identity of a nucleobase plays an important role
in the stability of the N-glycosidic bond. Actually, the
hydrolysis of purine nucleosides is faster than that of
pyrimidine ones.
33
The rate of hydrolysis is also determined
by the type of substituent present in the nucleobase moiety. In
fact, the degree of cleavage of the N-glycosidic bond for 2′-
deoxycitidine (dC) is 76%, for dU�3%, and for 5XdU, where
X = F, Cl, or Br, 13−17%, for a reaction occurring in 5%
trichloroacetic acid for 30 min at 100 °C.
33
Schroeder et al.
34
demonstrated that the experimental value of the activation
Gibbs free energy, ΔG*, for the heterolytic dissociation of the
C1′−N1 glycosidic bond in dU amounted to 30.5 kcal mol−1.
In turn, Przybylski et al.
35
carried out computational studies for
this reaction using the B3LYP/6-31+G(d,p) level of theory
and obtained a value of ΔG*similar to that found
experimentally, i.e., 28.7 kcal mol−1.
To explain the observed hydrolytic instability of 6IdU, we
initially decided to use four functionals, including the B3LYP
one, as recommended by Przybylski et al.
35
and Chen et al.;
36
we then calculated the free energy of hydrolysis for dU and
three other derivatives considered in the current work (see
Table 2). The B3LYP functional is a classical choice and has
been widely used in studies of the glycosidic bond cleavage in
nucleic acid-type systems.
37−39
To describe this type of bond
dissociation, Chen et al.
36
also employed other DFT
functionals, including long-range corrected hybrid functionals
CAM-B3LYP
22
and wB97XD.
40
These authors obtained the
best accuracy at the CAM-B3LYP level (the lowest mean
absolute error among the considered functionals). As indicated
by the results gathered in Table 2, the B3LYP and CAM-
B3LYP functionals led to the most similar values compared to
the experimental estimate of ΔG*for dU. On the other hand,
calculations for 6IUrd proved that CAM-B3LYP provided the
value that was most compatible with the experimental
activation energy, as shown in the current study (see Table 2
and the Probable Mechanism of Hydrolysis Section). There-
fore, we settled to use CAM-B3LYP for the ultimate estimates
of the kinetic characteristics of the nucleosides considered in
this study.
3.1. Probable Mechanism of Hydrolysis. It has been
reported in the literature that attempts to obtain 6-aryl-2′-
deoxyuridines through a 6-iodo-2′-deoxyuridine (6IdU)
derivative (both hydroxyls protected with the 1,1,3,3-
tetraisopropoxydisiloxanylidene (TIPDS) group) had failed.
41
Indeed, treating the TIPDS-protected 6IdU with tetrabuty-
lammonium fluoride (TBAF) or ammonium fluoride (NH4F)
Figure 3. Hydrolysis of a nucleoside.
33
Table 2. Activation Free Energy [kcal mol−1] (ΔG*) of the
Heterolytic Dissociation of the C1′−N1 Glycosidic Bond
(Figure 3) for the Selected DFT Functionals
a
system
functional dU 5IdU 6IdU 6IUrd
B3LYP 28.2 24.3 11.9 17.9
CAM-B3LYP 34.2 30.5 18.0 24.1
ωB97XD 37.2 33.5 21.5 28.7
experimental 30.5 26.8
a
All calculations were conducted using the PCM solvation model and
the DGDZVP++ basis set. The CAM-B3LYP results are highlighted
in bold.
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produced 6-iodouracil and other degradation products.
38
The
above-mentioned findings suggest the low stability of 6IdU.
Nevertheless, we attempted to synthesize 6IdU to verify
whether or not we could capture the moment of its
degradation. First, dU was treated with two equivalents of t-
butyldimethylsilyl chloride (TBDMSCl) in the presence of
imidazole. In the next step, the TBDMS-protected dU was
introduced into a mixture of LDA and iodine at −78 °C
yielding 2′,5′-di-t-butyldimethylsilyl-6-iodo-2′-deoxyuridine. In
the last step, the deprotection of the 2′and 5′-OH groups was
performed with TBAF. The reaction was monitored by thin-
layer chromatography (TLC), which showed the appearance of
the desired product. We attempted to isolate this product with
RP-HPLC (in a gradient of acetonitrile�ACN�and water)
but registered only 6IU as a degradation product of 6IdU.
Obviously, the interaction of 6IdU with water led to the swift
conversion of the synthesized nucleoside into the 6-substituted
base (6IU). To describe the probable mechanism of this
degradation, we decided to synthesize 6IUrd which, according
to the kinetic data, shown in Table 2, should have been
suciently stable in water to allow for its kinetic stability to be
assessed (ΔG*= 24.1 kcal mol−1for 6IUrd vs ΔG*= 18.0 kcal
mol−1for 6IdU at the CAM-B3LYP level). The 6IUrd, 4, was
synthesized through a modification of procedures available in
the literature
42,43
and the protocol of its synthesis is described
in detail in Section 2 (also see Figure 2). The synthesized
nucleoside was then subjected to kinetic studies. The
measurements were carried out at elevated temperatures so
that the reaction proceeded at convenient times (a significant
Figure 4. High-performance liquid chromatography (HPLC) traces of the thermal degradation of 6-iodouridine over temperatures ranging from 60
to 80 °C after 120 min of heating.
Figure 5. Arrhenius plot for 6IUrd hydrolysis proceeding in the 60−
80 °C range.
Table 3. Thermodynamic and Activation Barriers [kcal
mol−1] of 6OHUrd Formation (Figures S9−S11)
substrates products ΔGΔG*
6IUrd, OH−6OHUrd, I−−72.3 11.8
6IUrd, H2O 6OHUrd, HI −11.7 43.1
6IUrd, 2H2O 6OHUrd, I−, H3O+−10.4 39.2
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degradation at relatively short times). After specific periods of
time had elapsed, the reaction was “frozen” by a rapid lowering
of the temperature to an ambient one. Such a procedure
allowed the activation barrier of hydrolysis to be determined
experimentally. Figure 4 shows the HPLC traces for the
reaction mixture after its 120 min conditioning at several
elevated temperatures. Here, we also observed small amounts
of 6-hydroxyuridine (6OHUrd; see Figure 4) in addition to the
formation of 6-iodouracil. Both products were identified by
LC−MS.
The above-described kinetic measurements revealed a
dependence of the 6IUrd concentration vs time at several
temperatures, which allowed the kinetic constants for each
considered temperature to be estimated (see Figure S8). In
turn, these estimated rate constants allowed us to construct an
Arrhenius plot (Figure 5) and, finally, to calculate the
activation energy at 298 K. Derived in such a way, the
experimental activation energy was equal to 26.8 kcal mol−1
and remained in good agreement with the ΔG*value of 24.1
kcal mol−1(see Table 1) calculated at the CAM-B3LYP level.
As indicated in Figure 4, the hydrolysis of 6IUrd led to two
stable products: 6IU (product of the hydrolysis of the N-
glycosidic bond, Figure 3) and 6OHUrd. Three possible
mechanisms yielding 6OHUrd were considered (Figures S9−
S11). All of the analyzed reactions were thermodynamically
allowed (Table 3;ΔG< 0).
However, with one water molecule, the system ended up
with HI in addition to 6OHUrd, and the value of the activation
barrier reached 43.1 kcal mol−1(Table 3). In the model
comprising two water molecules, the predicted barrier of 39.2
kcal mol−1(Table 3) is somewhat lower than that calculated
for a single water molecule but was still too high to allow the
reaction to be completed over the experimental times (τ1/2 for
an activation energy of the order of 40 kcal mol−1at 25 °C is
equal to ca. 7 ×109years). Finally, the activation barrier for
the nucleophilic substitution involving the hydroxyl anion
rather than water molecule(s) amounted to only 11.8 kcal
mol−1(Table 3). Thus, although the concentration of OH−in
the studied 6IUrd solution was below 10−7M, the direct attack
of OH−on the 6IU moiety (SN2 mechanism) was probably
responsible for the formation of 6OHUrd as a side-product.
A general mechanism for nucleoside hydrolysis which
describes the hydrolysis of 6IdU, 5IdU, and 6IUrd specifically
is depicted in Figure 6. The main reaction (I) consisted of a
heterolytic dissociation of the C1′−N1 glycosidic bond, which
led to the carbocation of the sugar moiety and carbanion of the
substituted uracil. The subsequent steps involved the
hydrolyses of the cationic (II) and anionic (III) products of
Figure 6. Elemental reactions for the studied mechanism of hydrolysis of the three considered derivatives.
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the C1′−N dissociation. Moreover, 6-hydroxyuridine was
formed through the 6IUrd hydrolysis (V).
3.2. Simulation of Kinetic Profiles. The equations that
enabled the equilibrium concentrations in the reaction mixture
Figure 7. Concentration−time plots for 5IdU (A), 6IdU (B), and 6IUrd (C). Nuc�nucleoside, A�base, R�sugar moiety, A−and R+�anionic
and cationic forms of the base and sugar moieties.
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to be calculated are derived in the Equilibrium Concentrations
section in the Supporting Information, while the equilibrium
characteristics are summarized for 5IdU, 6IdU, and 6IUrd in
Table S2. However, one should note that these data do not
contain any information about the time which elapsed from the
dissolution of a nucleoside in water and the moment these
concentrations occurred. It is worth emphasizing that these
times were measures of the stability of the studied compounds
and would help decide about the possible usage of a nucleoside
as a radiosensitizer. Hence, to determine the time profiles of
hydrolysis, we performed kinetic simulations by integrating a
system of ordinary dierential eqs 6−15 and using rate
constants obtained as indicated in Section 2.4 for each of the
three studied nucleosides (see Table S1).
Figure 7 shows the concentration profiles plotted against the
time of hydrolysis reaction. For all studied systems, a decrease
in the concentration of a nucleoside (Nuc) and a proportional
increase in the concentration of a base (A) and sugar product
(R) were observed.
The equilibrium concentration of 6IU assumed a signifi-
cantly lower value than the initial concentration of 6IUrd as
was also indicated by the experimental data (see Figure 4). In
the case of 5IdU, the equilibrium was attained before the
complete hydrolysis of the substrate. Our kinetic simulations
led to τ1/2 being equal to 13.8 h and 1.4 s for 6IUrd and 6IdU,
respectively, while for 5IdU, τ1/2 was not defined. One could,
however, determine the time after which the equilibrium
concentrations were reached (42% of the substrate decom-
poses) to be ca. 126 years. The data obtained thus explain the
observed stabilities of 6IUrd and, in particular 5IdU, and the
significant instability of 6IdU in an aqueous solution. To
confirm the accuracy of the kinetic simulations performed here,
the calculated equilibrium concentrations shown in Table 4
were compared with those obtained through the kinetic
calculations. As demonstrated by these data, both concen-
trations were in excellent agreement.
At first glance, such a profound influence of subtle structural
dierences between the studied systems seems surprising, but
it could be explained by steric and electronic eects. Indeed,
since the hydration energies were almost identical for the
studied nucleosides, the lower stability of 6IdU relative to
5IdU was associated with the larger steric hindrance in the 6-
substituted derivative (the repulsion between the iodine atoms
and the sugar residue was much smaller in 5IdU); in turn, the
lower stability of the ribose cation relative to the 2′-
deoxyribose one made the half-life of 6IdU significantly
shorter than that of 6IUrd. Indeed, the free energy of
nucleoside dissociation (eq I in Figure 6) was ca. 6.4 kcal
mol−1lower at the CAM-B3LYP/PCM level for 6IdU than for
6IUrd. The presence of the OH group in the 2′position of the
ribose moiety, exerting a negative inductive eect, was
probably responsible for the lower stability of their respective
carbocations.
4. SUMMARY
This paper presents a combination of theoretical and
experimental studies devoted to understanding the aqueous
stabilities of specific modified nucleosides with potentially
radiosensitizing properties. This investigation was prompted by
our previous findings that suggested that 6-substituted uracils
were better radiosensitizers than their 5-substituted analogues.
Our attempts to synthesize 6IdU were unsuccessful, which
was ascribed to the low stability of the compound in water (the
synthesized nucleoside was isolated and purified with RP-
HPLC using an ACN:water mobile phase).
The experimental activation energy and as a consequence,
the half-life and the calculated free activation energy remained
in good agreement for 6IUrd. The half-life of the latter
determined by kinetic simulations amounted to 13.8 h (see
Figure 7). On the other hand, predicted τ1/2’s of 1.4 s and 126
years were estimated for 6IdU and 5IdU, respectively. The
above-mentioned dierences in τ1/2 were likely due to
dissimilarities between steric and electronic eects of the
considered molecules.
Since slight structural dierences between nucleosides led to
huge variations in their stabilities in water, to design a working
radiosensitizer, one has to estimate the aqueous stability of
considered nucleosides alongside performing calculations of
their DEA profiles
3
before the actual synthesis. Therefore, the
proposed kinetic model should be employed in future studies
devoted to the rational engineering of new radiosensitizers.
■ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.3c00548.
1H NMR spectrum; high-resolution mass spectra of 6-
iodouridine, 6-hydroxyuridine, and 6-iodouracil; reac-
tion profiles for 6OHUrd formation from 6IUrd and
OH−, single water molecule and two water molecules;
calculations for equilibrium concentrations and table
Table 4. Equilibrium Concentrations [mol dm−3] (Equilibrium; for Details, See the Equilibrium Concentration section in the
Supporting Information) and the Corresponding Values Resulting from the Integration of Eqs 6−15 (Kinetics)
5IdU 6IdU 6IUrd
reagent equilibrium kinetics
a
equilibrium kinetics
a
equilibrium kinetics
a
nucleoside [Nuc] 5.8 ×10−45.8 ×10−43.6 ×10−10 9.0 ×10−10 2.4 ×10−10 4.1 ×10−9
base [A] 4.1 ×10−44.3 ×10−47.5 ×10−47.5 ×10−47.5 ×10−47.5 ×10−4
base anion [A−] 1.2 ×10−51.1 ×10−52.5 ×10−42.5 ×10−42.5 ×10−42.5 ×10−4
sugar moiety [R] 4.2 ×10−44.2 ×10−41.0 ×10−31.0 ×10−31.0 ×10−31.0 ×10−3
sugar moiety cation [R+] 2.2 ×10−21 2.3 ×10−21 1.1 ×10−19 1.1 ×10−19 2.1 ×10−24 2.1 ×10−24
[OH−] 8.5 ×10−10 7.8 ×10−10 4.0 ×10−11 4.0 ×10−11 4.0 ×10−11 4.0 ×10−11
[H3O+] 1.2 ×10−51.3 ×10−52.5 ×10−42.5 ×10−42.5 ×10−42.5 ×10−4
6OHUrd [hydrNuc] 1.6 ×10−4
iodide anion [I−] 1.6 ×10−4
a
Small dierences between the equilibrium and kinetic concentrations of some regents were due to the finite integration time of the kinetic
equations.
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2572

with their values; and system of dierential equations for
the kinetic profiles in the Octave program (PDF)
■AUTHOR INFORMATION
Corresponding Author
Janusz Rak −Laboratory of Biological Sensitizers, Department
of Physical Chemistry, Faculty of Chemistry, University of
Gdansk, 80-308 Gdansk, Poland; orcid.org/0000-0003-
3036-0536; Email: janusz.rak@ug.edu.pl
Authors
Karina Falkiewicz −Laboratory of Biological Sensitizers,
Department of Physical Chemistry, Faculty of Chemistry,
University of Gdansk, 80-308 Gdansk, Poland
Witold Kozak −Laboratory of Biological Sensitizers,
Department of Physical Chemistry, Faculty of Chemistry,
University of Gdansk, 80-308 Gdansk, Poland; orcid.org/
0000-0003-3253-5555
Magdalena Zdrowowicz −Laboratory of Biological
Sensitizers, Department of Physical Chemistry, Faculty of
Chemistry, University of Gdansk, 80-308 Gdansk, Poland
Paulina Spisz −Laboratory of Biological Sensitizers,
Department of Physical Chemistry, Faculty of Chemistry,
University of Gdansk, 80-308 Gdansk, Poland; Laboratory of
Intermolecular Interactions, Department of Bioinorganic
Chemistry, Faculty of Chemistry, University of Gdansk, 80-
308 Gdansk, Poland
Lidia Chomicz-Manka −Laboratory of Biological Sensitizers,
Department of Physical Chemistry, Faculty of Chemistry,
University of Gdansk, 80-308 Gdansk, Poland
Mieczyslaw Torchala −Laboratory of Biological Sensitizers,
Department of Physical Chemistry, Faculty of Chemistry,
University of Gdansk, 80-308 Gdansk, Poland; orcid.org/
0000-0002-4542-9156
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.3c00548
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was supported by the Polish National Science
Center (NCN) under grant no. UMO-2014/14/A/ST4/
00405. The calculations were carried out using resources
provided by the Wroclaw Centre for Networking and
Supercomputing (https://wcss.pl), grant No. 209.
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